6.1 The DNA
6.2 The Search for Genetic
Material
6.3 RNA World
6.4 Replication
6.5 Transcription
6.6 Genetic Code
6.7 Translation
6.8 Regulation of Gene
Expression
6.9 Human Genome Project
6.10 DNA Fingerprinting
In the previous chapter, you have learnt the inheritance
patterns and the genetic basis of such patterns. At the
time of Mendel, the nature of those ‘factors’ regulating
the pattern of inheritance was not clear. Over the next
hundred years, the nature of the putative genetic material
was investigated culminating in the realisation that
DNA – deoxyribonucleic acid – is the genetic material, at
least for the majority of organisms. In class XI you have
learnt that nucleic acids are polymers of nucleotides.
Deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA) are the two types of nucleic acids found in living
systems. DNA acts as the genetic material in most of the
organisms. RNA though it also acts as a genetic material
in some viruses, mostly functions as a messenger. RNA
has additional roles as well. It functions as adapter,
structural, and in some cases as a catalytic molecule. In
Class XI you have already learnt the structures of
nucleotides and the way these monomer units are linked
to form nucleic acid polymers. In this chapter we are going
to discuss the structure of DNA, its replication, the process
of making RNA from DNA (transcription), the genetic code
that determines the sequences of amino acids in proteins,
the process of protein synthesis (translation) and
elementary basis of their regulation. The determination
of complete nucleotide sequence of human genome during last decade
has set in a new era of genomics. In the last section, the essentials of
human genome sequencing and its consequences will also be discussed.
Let us begin our discussion by first understanding the structure of
the most interesting molecule in the living system, that is, the DNA. In
subsequent sections, we will understand that why it is the most abundant
genetic material, and what its relationship is with RNA.
6.1 THE DNA
DNA is a long polymer of deoxyribonucleotides. The length of DNA is
usually defined as number of nucleotides (or a pair of nucleotide referred
to as base pairs) present in it. This also is the characteristic of an organism.
For example, a bacteriophage known as φ ×174 has 5386 nucleotides,
Bacteriophage lambda has 48502 base pairs (bp), Escherichia coli has
4.6 × 106
bp, and haploid content of human DNA is 3.3 × 109
bp. Let us
discuss the structure of such a long polymer.
6.1.1 Structure of Polynucleotide Chain
Let us recapitulate the chemical structure of a polynucleotide chain (DNA
or RNA). A nucleotide has three components – a nitrogenous base, a
pentose sugar (ribose in case of RNA, and deoxyribose for DNA), and a
phosphate group. There are two types of nitrogenous bases – Purines
(Adenine and Guanine), and Pyrimidines (Cytosine, Uracil and Thymine).
Cytosine is common for both DNA and RNA and Thymine is present in
DNA. Uracil is present in RNA at the place of Thymine. A nitrogenous
base is linked to the OH of 1′ C pentose sugar through a N-glycosidic
linkage to form a nucleoside, such as adenosine or deoxyadenosine,
guanosine or deoxyguanosine, cytidine or deoxycytidine and uridine or
deoxythymidine. When a phosphate group is linked to OH of 5′ C of a
nucleoside through phosphoester linkage, a corresponding nucleotide
(or deoxynucleotide depending upon the type of sugar present) is formed.
Two nucleotides are linked through 3′-5′ phosphodiester linkage to form
a dinucleotide. More nucleotides can be joined in such a manner to form
a polynucleotide chain. A polymer thus formed has at one end a free
phosphate moiety at 5′ -end of sugar, which is referred to as 5’-end of
polynucleotide chain. Similarly, at the other end of the polymer the sugar
has a free OH of 3’C group which is referred to as 3′ -end of the
polynucleotide chain. The backbone of a polynucleotide chain is formed
due to sugar and phosphates. The nitrogenous bases linked to sugar
moiety project from the backbone (Figure 6.1).
In RNA, every nucleotide residue has an additional –OH group present
at 2′ -position in the ribose. Also, in RNA the uracil is found at the place of
thymine (5-methyl uracil, another chemical name for thymine).
DNA as an acidic substance present in nucleus was first identified by
Friedrich Meischer in 1869. He named it as ‘Nuclein’. However, due to
technical limitation in isolating such a long polymer intact, the elucidation
of structure of DNA remained elusive for a very long period of time. It was
only in 1953 that James Watson and Francis Crick, based on the X-ray
diffraction data produced by Maurice Wilkins and Rosalind Franklin,
proposed a very simple but famous Double Helix model for the structure
of DNA. One of the hallmarks of their proposition was base pairing between
the two strands of polynucleotide chains. However, this proposition was
also based on the observation of Erwin Chargaff that for a double stranded
DNA, the ratios between Adenine and Thymine and Guanine and Cytosine
are constant and equals one.
The base pairing confers a very unique property to the polynucleotide
chains. They are said to be complementary to each other, and therefore if
the sequence of bases in one strand is known then the sequence in other
strand can be predicted. Also, if each strand from a DNA (let us call it as a
parental DNA) acts as a template for synthesis of a new strand, the two
double stranded DNA (let us call them as daughter DNA) thus, produced
would be identical to the parental DNA molecule. Because of this, the genetic
implications of the structure of DNA became very clear.
The salient features of the Double-helix structure of DNA are as follows:
(i) It is made of two polynucleotide chains, where the backbone is
constituted by sugar-phosphate, and the bases project inside.
(ii) The two chains have anti-parallel polarity. It means, if one
chain has the polarity 5’à3′, the other has 3’à5′.
(iii) The bases in two strands are paired through hydrogen bond
(H-bonds) forming base pairs (bp). Adenine forms two hydrogen
bonds with Thymine from opposite strand and vice-versa.
Similarly, Guanine is bonded with Cytosine with three H-bonds.
As a result, always a purine comes opposite to a pyrimidine. This
generates approximately uniform distance between the two
strands of the helix (Figure 6.2).
(iv) The two chains are coiled in a right-handed fashion. The pitch
of the helix is 3.4 nm (a nanometre is one billionth of a
metre, that is 10-9 m) and there are roughly 10 bp in each
turn. Consequently, the distance
between a bp in a helix is
approximately 0.34 nm.
(v) The plane of one base pair stacks
over the other in double helix. This,
in addition to H-bonds, confers
stability of the helical structure
(Figure 6.3).
Compare the structure of purines and
pyrimidines. Can you find out why the
distance between two polynucleotide
chains in DNA remains almost constant?
The proposition of a double helix
structure for DNA and its simplicity in
explaining the genetic implication became
revolutionary. Very soon, Francis Crick
proposed the Central dogma in molecular
biology, which states that the genetic
information flows from DNAàRNAàProtein.
In some viruses the flow of information is in reverse direction, that is,
from RNA to DNA. Can you suggest a simple name to the process?
6.1.2 Packaging of DNA Helix
Taken the distance between two consecutive base pairs
as 0.34 nm (0.34×10–9 m), if the length of DNA double
helix in a typical mammalian cell is calculated (simply
by multiplying the total number of bp with distance
between two consecutive bp, that is, 6.6 × 109 bp ×
0.34 × 10-9m/bp), it comes out to be approximately
2.2 metres. A length that is far greater than the
dimension of a typical nucleus (approximately 10–6 m).
How is such a long polymer packaged in a cell?
If the length of E. coli DNA is 1.36 mm, can you
calculate the number of base pairs in E.coli?
In prokaryotes, such as, E. coli, though they do
not have a defined nucleus, the DNA is not scattered
throughout the cell. DNA (being negatively charged)
is held with some proteins (that have positive
charges) in a region termed as ‘nucleoid’. The DNA
in nucleoid is organised in large loops held by
proteins.
In eukaryotes, this organisation is much more
complex. There is a set of positively charged, basic
proteins called histones. A protein acquires charge
depending upon the abundance of amino acids
residues with charged side chains. Histones are rich
in the basic amino acid residues lysine and arginine.
Both the amino acid residues carry positive charges
in their side chains. Histones are organised to form
a unit of eight molecules called histone octamer.
The negatively charged DNA is wrapped around the positively charged
histone octamer to form a structure called nucleosome (Figure 6.4 a). A
typical nucleosome contains 200 bp of DNA helix. Nucleosomes constitute
the repeating unit of a structure in nucleus called chromatin, thread-
like stained (coloured) bodies seen in nucleus. The nucleosomes in
chromatin are seen as ‘beads-on-string’ structure when viewed under
electron microscope (EM) (Figure 6.4 b).
Theoretically, how many such beads (nucleosomes) do you imagine
are present in a mammalian cell?
The beads-on-string structure in chromatin is packaged to form
chromatin fibers that are further coiled and condensed at metaphase stage
of cell division to form chromosomes. The packaging of chromatin at higher
level requires additional set of proteins that collectively are referred to as
Non-histone Chromosomal (NHC) proteins. In a typical nucleus, some
region of chromatin are loosely packed (and stains light) and are referred to
as euchromatin. The chromatin that is more densely packed and stains
dark are called as Heterochromatin. Euchromatin is said to be
transcriptionally active chromatin, whereas heterochromatin is inactive.
6.2 THE SEARCH FOR GENETIC MATERIAL
Even though the discovery of nuclein by Meischer and the proposition
for principles of inheritance by Mendel were almost at the same time, but
that the DNA acts as a genetic material took long to be discovered and
proven. By 1926, the quest to determine the mechanism for genetic
inheritance had reached the molecular level. Previous discoveries by
Gregor Mendel, Walter Sutton, Thomas Hunt Morgan and numerous other
scientists had narrowed the search to the chromosomes located in the
nucleus of most cells. But the question of what molecule was actually the
genetic material, had not been answered.
Transforming Principle
In 1928, Frederick Griffith, in a series of experiments with Streptococcus
pneumoniae (bacterium responsible for pneumonia), witnessed a
miraculous transformation in the bacteria. During the course of his
experiment, a living organism (bacteria) had changed in physical form.
When Streptococcus pneumoniae (pneumococcus) bacteria are grown
on a culture plate, some produce smooth shiny colonies (S) while others
produce rough colonies (R). This is because the S strain bacteria have a
mucous (polysaccharide) coat, while R strain does not. Mice infected with
the S strain (virulent) die from pneumonia infection but mice infected
with the R strain do not develop pneumonia.
Griffith was able to kill bacteria by heating them. He observed that
heat-killed S strain bacteria injected into mice did not kill them. When he
injected a mixture of heat-killed S and live R bacteria, the mice died.
Moreover, he recovered living S bacteria from the dead mice.
He concluded that the R strain bacteria had somehow been
transformed by the heat-killed S strain bacteria. Some ‘transforming
principle’, transferred from the heat-killed S strain, had enabled the
R strain to synthesise a smooth polysaccharide coat and become virulent.
This must be due to the transfer of the genetic material. However, the
biochemical nature of genetic material was not defined from his
experiments.
Biochemical Characterisation of Transforming Principle
Prior to the work of Oswald Avery, Colin MacLeod and Maclyn McCarty
(1933-44), the genetic material was thought to be a protein. They worked
to determine the biochemical nature of ‘transforming principle’ in Griffith’s
experiment.
They purified biochemicals (proteins, DNA, RNA, etc.) from the
heat-killed S cells to see which ones could transform live R cells into
S cells. They discovered that DNA alone from S bacteria caused R bacteria
to become transformed.
They also discovered that protein-digesting enzymes (proteases) and
RNA-digesting enzymes (RNases) did not affect transformation, so the
transforming substance was not a protein or RNA. Digestion with DNase
did inhibit transformation, suggesting that the DNA caused the
transformation. They concluded that DNA is the hereditary material, but
not all biologists were convinced.
Can you think of any difference between DNAs and DNase?
6.2.1 The Genetic Material is DNA
The unequivocal proof that DNA is the genetic material came from the
experiments of Alfred Hershey and Martha Chase (1952). They worked
with viruses that infect bacteria called bacteriophages.
The bacteriophage attaches to the bacteria and its genetic material
then enters the bacterial cell. The bacterial cell treats the viral genetic
material as if it was its own and subsequently manufactures more virus
particles. Hershey and Chase worked to discover whether it was protein
or DNA from the viruses that entered the bacteria.
They grew some viruses on a medium that contained radioactive
phosphorus and some others on medium that contained radioactive sulfur.
Viruses grown in the presence of radioactive phosphorus contained
radioactive DNA but not radioactive protein because DNA contains
phosphorus but protein does not. Similarly, viruses grown on radioactive
sulfur contained radioactive protein but not radioactive DNA because
DNA does not contain sulfur.
Radioactive phages were allowed to attach to E. coli bacteria. Then, as
the infection proceeded, the viral coats were removed from the bacteria by
agitating them in a blender. The virus particles were separated from the
bacteria by spinning them in a centrifuge.
Bacteria which was infected with viruses that had radioactive DNA
were radioactive, indicating that DNA was the material that passed from
the virus to the bacteria. Bacteria that were infected with viruses that had
radioactive proteins were not radioactive. This indicates that proteins did
not enter the bacteria from the viruses. DNA is therefore the genetic
material that is passed from virus to bacteria (Figure 6.5).
6.2.2 Properties of Genetic Material (DNA versus RNA)
From the foregoing discussion, it is clear that the debate between proteins
versus DNA as the genetic material was unequivocally resolved from
Hershey-Chase experiment. It became an established fact that it is DNA
that acts as genetic material. However, it subsequently became clear that
in some viruses, RNA is the genetic material (for example, Tobacco Mosaic
viruses, QB bacteriophage, etc.). Answer to some of the questions such as,
why DNA is the predominant genetic material, whereas RNA performs
dynamic functions of messenger and adapter has to be found from the
differences between chemical structures of the two nucleic acid molecules.
Can you recall the two chemical differences between DNA and RNA?
A molecule that can act as a genetic material must fulfill the following
criteria:
(i) It should be able to generate its replica (Replication).
(ii) It should be stable chemically and structurally.
(iii) It should provide the scope for slow changes (mutation) that
are required for evolution.
(iv) It should be able to express itself in the form of ‘Mendelian
Characters’.
If one examines each requirement one by one, because of rule of base
pairing and complementarity, both the nucleic acids (DNA and RNA) have
the ability to direct their duplications. The other molecules in the living
system, such as proteins fail to fulfill first criteria itself.
The genetic material should be stable enough not to change with
different stages of life cycle, age or with change in physiology of the
organism. Stability as one of the properties of genetic material was very
evident in Griffith’s ‘transforming principle’ itself that heat, which killed
the bacteria, at least did not destroy some of the properties of genetic
material. This now can easily be explained in light of the DNA that the
two strands being complementary if separated by heating come together,
when appropriate conditions are provided. Further, 2′-OH group present
at every nucleotide in RNA is a reactive group and makes RNA labile and
easily degradable. RNA is also now known to be catalytic, hence reactive.
Therefore, DNA chemically is less reactive and structurally more stable
when compared to RNA. Therefore, among the two nucleic acids, the DNA
is a better genetic material.
In fact, the presence of thymine at the place of uracil also confers
additional stability to DNA. (Detailed discussion about this requires
understanding of the process of repair in DNA, and you will study these
processes in higher classes.)
Both DNA and RNA are able to mutate. In fact, RNA being unstable,
mutate at a faster rate. Consequently, viruses having RNA genome and
having shorter life span mutate and evolve faster.
RNA can directly code for the synthesis of proteins, hence can easily
express the characters. DNA, however, is dependent on RNA for synthesis
of proteins. The protein synthesising machinery has evolved around RNA.
The above discussion indicate that both RNA and DNA can function as
genetic material, but DNA being more stable is preferred for storage of
genetic information. For the transmission of genetic information, RNA
is better.
6.3 RNA WORLD
From foregoing discussion, an immediate question becomes evident –
which is the first genetic material? It shall be discussed in detail in the
chapter on chemical evolution, but briefly, we shall highlight some of the
facts and points.
RNA was the first genetic material. There is now enough evidence to
suggest that essential life processes (such as metabolism, translation,
splicing, etc.), evolved around RNA. RNA used to act as
a genetic material as well as a catalyst (there are some
important biochemical reactions in living systems that
are catalysed by RNA catalysts and not by protein
enzymes). But, RNA being a catalyst was reactive and
hence unstable. Therefore, DNA has evolved from RNA
with chemical modifications that make it more stable.
DNA being double stranded and having complementary
strand further resists changes by evolving a process of
repair.
6.4 REPLICATION
While proposing the double helical structure for DNA,
Watson and Crick had immediately proposed a scheme
for replication of DNA. To quote their original statement
that is as follows:
‘‘It has not escaped our notice that the specific
pairing we have postulated immediately suggests a
possible copying mechanism for the genetic material’’
(Watson and Crick, 1953).
The scheme suggested that the two strands would
separate and act as a template for the synthesis of new
complementary strands. After the completion of
replication, each DNA molecule would have one
parental and one newly synthesised strand. This
scheme was termed as semiconservative DNA
replication (Figure 6.6).
6.4.1 The Experimental Proof
It is now proven that DNA replicates semiconservatively. It was shown first in
Escherichia coli and subsequently in higher organisms, such as plants
and human cells. Matthew Meselson and Franklin Stahl performed the
following experiment in 1958:
(i) They grew E. coli in a medium containing 15NH4Cl (15N is the heavy
isotope of nitrogen) as the only nitrogen source for many
generations. The result was that 15N was incorporated into newly
synthesised DNA (as well as other nitrogen containing compounds).
This heavy DNA molecule could be distinguished from the normal
DNA by centrifugation in a cesium chloride (CsCl) density gradient
(Please note that 15N is not a radioactive isotope, and it can be
separated from 14N only based on densities).
(ii) Then they transferred the cells into a medium with normal
14NH4Cl and took samples at various definite time intervals as
the cells multiplied, and extracted the DNA that remained as
double-stranded helices. The various samples were separated
independently on CsCl gradients to measure the densities of
DNA (Figure 6.7).
Can you recall what centrifugal force is, and think why a
molecule with higher mass/density would sediment faster?
The results are shown in Figure 6.7.
(iii) Thus, the DNA that was extracted from the culture one
generation after the transfer from 15N to 14N medium [that is
after 20 minutes; E. coli divides in 20 minutes] had a hybrid or
intermediate density. DNA extracted from the culture after
another generation [that is after 40 minutes, II generation] was
composed of equal amounts of this hybrid DNA and of ‘light’
DNA.
If E. coli was allowed to grow for 80 minutes then what would be the
proportions of light and hybrid densities DNA molecule?
Very similar experiments involving use of radioactive thymidine to
detect distribution of newly synthesised DNA in the chromosomes was
performed on Vicia faba (faba beans) by Taylor and colleagues in 1958.
The experiments proved that the DNA in chromosomes also replicate
semiconservatively.
6.4.2 The Machinery and the Enzymes
In living cells, such as E. coli, the process of replication requires a set of
catalysts (enzymes). The main enzyme is referred to as DNA-dependent
DNA polymerase, since it uses a DNA template to catalyse the
polymerisation of deoxynucleotides. These enzymes are highly efficient
enzymes as they have to catalyse polymerisation of a large number of
nucleotides in a very short time. E. coli that has only 4.6 ×106
bp (compare
it with human whose diploid content is 6.6 × 109
bp), completes the
process of replication within 18 minutes; that means the average rate of
polymerisation has to be approximately 2000 bp per second. Not only do
these polymerases have to be fast, but they also have to catalyse the reaction
with high degree of accuracy. Any mistake during replication would result
into mutations. Furthermore, energetically replication is a very expensive
process. Deoxyribonucleoside triphosphates serve dual purposes. In
addition to acting as substrates, they provide energy for polymerisation
reaction (the two terminal phosphates in a deoxynucleoside triphosphates
are high-energy phosphates, same as in case of ATP).
In addition to DNA-dependent DNA polymerases, many additional
enzymes are required to complete the process of replication with high
degree of accuracy. For long DNA molecules, since the two strands of
DNA cannot be separated in its entire length (due to very high energy
requirement), the replication occur within a small opening of the DNA
helix, referred to as replication fork. The DNA-dependent DNA
polymerases catalyse polymerisation only in one direction, that is 5’à3′.
This creates some additional complications at the replicating fork.
Consequently, on one strand (the template with polarity 3’à5′), the
replication is continuous, while on the other (the template with
polarity 5’à3′), it is discontinuous. The discontinuously synthesised
fragments are later joined by the enzyme DNA ligase (Figure 6.8).
The DNA polymerases on their own cannot initiate the process of
replication. Also the replication does not initiate randomly at any place
in DNA. There is a definite region in E. coli DNA where the replication
originates. Such regions are termed as origin of replication. It is
because of the requirement of the origin of
replication that a piece of DNA if needed to be
propagated during recombinant DNA procedures,
requires a vector. The vectors provide the origin of
replication.
Further, not every detail of replication is
understood well. In eukaryotes, the replication of
DNA takes place at S-phase of the cell-cycle. The
replication of DNA and cell division cycle should be
highly coordinated. A failure in cell division after
DNA replication results into polyploidy(a
chromosomal anomaly). You will learn the detailed
nature of origin and the processes occurring at this
site, in higher classes.
6.5 TRANSCRIPTION
The process of copying genetic information from one
strand of the DNA into RNA is termed as
transcription. Here also, the principle of
complementarity governs the process of transcription, except the adenosine
complements now forms base pair with uracil instead of thymine. However,
unlike in the process of replication, which once set in, the total DNA of an
organism gets duplicated, in transcription only a segment of DNA and
only one of the strands is copied into RNA. This necessitates defining the
boundaries that would demarcate the region and the strand of DNA that
would be transcribed.
Why both the strands are not copied during transcription has the
simple answer. First, if both strands act as a template, they would code
for RNA molecule with different sequences (Remember complementarity
does not mean identical), and in turn, if they code for proteins, the sequence
of amino acids in the proteins would be different. Hence, one segment of
the DNA would be coding for two different proteins, and this would
complicate the genetic information transfer machinery. Second, the two
RNA molecules if produced simultaneously would be complementary to
each other, hence would form a double stranded RNA. This would prevent
RNA from being translated into protein and the exercise of transcription
would become a futile one.
6.5.1 Transcription Unit
A transcription unit in DNA is defined primarily by the three regions in
the DNA:
(i) A Promoter
(ii) The Structural gene
(iii) A Terminator
There is a convention in defining the two strands of the DNA in the
structural gene of a transcription unit. Since the two strands have opposite
polarity and the DNA-dependent RNA polymerase also catalyse the
polymerisation in only one direction, that is, 5’→3′, the strand that has
the polarity 3’→5′ acts as a template, and is also referred to as template
strand. The other strand which has the polarity (5’→3′) and the sequence
same as RNA (except thymine at the place of uracil), is displaced during
transcription. Strangely, this strand (which does not code for anything)
is referred to as coding strand. All the reference point while defining a
transcription unit is made with coding strand. To explain the point, a
hypothetical sequence from a transcription unit is represented below:
3′ -ATGCATGCATGCATGCATGCATGC-5′ Template Strand
5′ -TACGTACGTACGTACGTACGTACG-3′ Coding Strand
Can you now write the sequence of RNA transcribed from the above DNA?
The promoter and terminator flank the structural gene in a
transcription unit. The promoter is said to be located towards 5′ -end
(upstream) of the structural gene (the reference is made with respect to
the polarity of coding strand). It is a DNA sequence that provides binding
site for RNA polymerase, and it is the presence of a promoter in a
transcription unit that also defines the template and coding strands. By
switching its position with terminator, the definition of coding and template
strands could be reversed. The terminator is located towards 3′ -end
(downstream) of the coding strand and it usually defines the end of the
process of transcription (Figure 6.9). There are additional regulatory
sequences that may be present further upstream or downstream to the
promoter. Some of the properties of these sequences shall be discussed
while dealing with regulation of gene expression.
6.5.2 Transcription Unit and the Gene
A gene is defined as the functional unit of inheritance. Though there is no
ambiguity that the genes are located on the DNA, it is difficult to literally
define a gene in terms of DNA sequence. The DNA sequence coding for
tRNA or rRNA molecule also define a gene. However by defining a cistron
as a segment of DNA coding for a polypeptide, the structural gene in a
transcription unit could be said as monocistronic (mostly in eukaryotes)
or polycistronic (mostly in bacteria or prokaryotes). In eukaryotes, the
monocistronic structural genes have interrupted coding sequences – the
genes in eukaryotes are split. The coding sequences or expressed
sequences are defined as exons. Exons are said to be those sequence
that appear in mature or processed RNA. The exons are interrupted by
introns. Introns or intervening sequences do not appear in mature or
processed RNA. The split-gene arrangement further complicates the
definition of a gene in terms of a DNA segment.
Inheritance of a character is also affected by promoter and regulatory
sequences of a structural gene. Hence, sometime the regulatory sequences
are loosely defined as regulatory genes, even though these sequences do
not code for any RNA or protein.
6.5.3 Types of RNA and the process of Transcription
In bacteria, there are three major types of RNAs: mRNA (messenger RNA),
tRNA (transfer RNA), and rRNA (ribosomal RNA). All three RNAs are
needed to synthesise a protein in a cell. The mRNA provides the template,
tRNA brings aminoacids and reads the genetic code, and rRNAs play
structural and catalytic role during translation. There is single
DNA-dependent RNA polymerase that catalyses transcription of all types
of RNA in bacteria. RNA polymerase binds to promoter and initiates
transcription (Initiation). It uses nucleoside triphosphates as substrate
and polymerises in a template depended fashion following the rule of
complementarity. It somehow also facilitates opening of the helix and
continues elongation. Only a short stretch of RNA remains bound to the
enzyme. Once the polymerases reaches the terminator region, the nascent
RNA falls off, so also the RNA polymerase. This results in termination of
transcription.
An intriguing question is that how is the RNA polymerases able
to catalyse all the three steps, which are initiation, elongation and
termination. The RNA polymerase is only capable of catalysing the
process of elongation. It associates transiently with initiation-factor (σ)
and termination-factor (ρ) to initiate and terminate the transcription,
respectively. Association with these factors alter the specificity of the
RNA polymerase to either initiate or terminate (Figure 6.10).
In bacteria, since the mRNA does not require any processing to become
active, and also since transcription and translation take place in the same
compartment (there is no separation of cytosol and nucleus in bacteria),
many times the translation can begin much before the mRNA is fully
transcribed. Consequently, the transcription and translation can be coupled
in bacteria.
In eukaryotes, there are two additional complexities –
(i) There are at least three RNA polymerases in the nucleus (in addition
to the RNA polymerase found in the organelles). There is a clear
cut division of labour. The RNA polymerase I transcribes rRNAs
(28S, 18S, and 5.8S), whereas the RNA polymerase III is responsible
for transcription of tRNA, 5srRNA, and snRNAs (small nuclear
RNAs). The RNA polymerase II transcribes precursor of mRNA, the
heterogeneous nuclear RNA (hnRNA).
(ii) The second complexity is that the primary transcripts contain both
the exons and the introns and are non-functional. Hence, it is
subjected to a process called splicing where the introns are removed
and exons are joined in a defined order. hnRNA undergoes
additional processing called as capping and tailing. In capping an
unusual nucleotide (methyl guanosine triphosphate) is added to
the 5′-end of hnRNA. In tailing, adenylate residues (200-300) are
added at 3′-end in a template independent manner. It is the fully
processed hnRNA, now called mRNA, that is transported out of the
nucleus for translation (Figure 6.11).
The significance of such complexities is now beginning to be
understood. The split-gene arrangements represent probably an ancient
feature of the genome. The presence of introns is reminiscent of antiquity,
and the process of splicing represents the dominance of RNA-world. In
recent times, the understanding of RNA and RNA-dependent processes
in the living system have assumed more importance.
6.6 GENETIC CODE
During replication and transcription a nucleic acid was copied to form
another nucleic acid. Hence, these processes are easy to conceptualise
on the basis of complementarity. The process of translation requires
transfer of genetic information from a polymer of nucleotides to synthesise
a polymer of amino acids. Neither does any complementarity exist between
nucleotides and amino acids, nor could any be drawn theoretically. There
existed ample evidences, though, to support the notion that change in
nucleic acids (genetic material) were responsible for change in amino acids
in proteins. This led to the proposition of a genetic code that could direct
the sequence of amino acids during synthesis of proteins.
If determining the biochemical nature of genetic material and the
structure of DNA was very exciting, the proposition and deciphering of
genetic code were most challenging. In a very true sense, it required
involvement of scientists from several disciplines – physicists, organic
chemists, biochemists and geneticists. It was George Gamow, a physicist,
who argued that since there are only 4 bases and if they have to code for
20 amino acids, the code should constitute a combination of bases. He
suggested that in order to code for all the 20 amino acids, the code should
be made up of three nucleotides. This was a very bold proposition, because
a permutation combination of 43
(4 × 4 × 4) would generate 64 codons;
generating many more codons than required.
Providing proof that the codon was a triplet, was a more daunting
task. The chemical method developed by Har Gobind Khorana was
instrumental in synthesising RNA molecules with defined combinations
of bases (homopolymers and copolymers). Marshall Nirenberg’s cell-free
system for protein synthesis finally helped the code to be deciphered.
Severo Ochoa enzyme (polynucleotide phosphorylase) was also helpful
in polymerising RNA with defined sequences in a template independent
manner (enzymatic synthesis of RNA). Finally a checker-board for genetic
code was prepared which is given in Table 6.1.
The salient features of genetic code are as follows:
(i) The codon is triplet. 61 codons code for amino acids and 3 codons do
not code for any amino acids, hence they function as stop codons.
(ii) Some amino acids are coded by more than one codon, hence
the code is degenerate.
(iii) The codon is read in mRNA in a contiguous fashion. There are
no punctuations.
(iv) The code is nearly universal: for example, from bacteria to human
UUU would code for Phenylalanine (phe). Some exceptions to this
rule have been found in mitochondrial codons, and in some
protozoans.
(v) AUG has dual functions. It codes for Methionine (met) , and it
also act as initiator codon.
(vi) UAA, UAG, UGA are stop terminator codons.
If following is the sequence of nucleotides in mRNA, predict the
sequence of amino acid coded by it (take help of the checkerboard):
-AUG UUU UUC UUC UUU UUU UUC-
Now try the opposite. Following is the sequence of amino acids coded
by an mRNA. Predict the nucleotide sequence in the RNA:
Met-Phe-Phe-Phe-Phe-Phe-Phe
Do you face any difficulty in predicting the opposite?
Can you now correlate which two properties of genetic code you have
learnt?
6.6.1 Mutations and Genetic Code
The relationships between genes and DNA are best understood by mutation
studies. You have studied about mutation and its effect in Chapter 5. Effects
of large deletions and rearrangements in a segment of DNA are easy to
comprehend. It may result in loss or gain of a gene and so a function. The
effect of point mutations will be explained here. A classical example of
point mutation is a change of single base pair in the gene for beta globin
chain that results in the change of amino acid residue glutamate to valine.
It results into a diseased condition called as sickle cell anemia. Effect of
point mutations that inserts or deletes a base in structural gene can be
better understood by following simple example.
Consider a statement that is made up of the following words each
having three letters like genetic code.
RAM HAS RED CAP
If we insert a letter B in between HAS and RED and rearrange the
statement, it would read as follows:
RAM HAS BRE DCA P
Similarly, if we now insert two letters at the same place, say BI’. Now it
would read,
RAM HAS BIR EDC AP
Now we insert three letters together, say BIG, the statement would read
RAM HAS BIG RED CAP
The same exercise can be repeated, by deleting the letters R, E and D,
one by one and rearranging the statement to make a triplet word.
RAM HAS EDC AP
RAM HAS DCA P
RAM HAS CAP
The conclusion from the above exercise is very obvious. Insertion or
deletion of one or two bases changes the reading frame from the point of
insertion or deletion. However, such mutations are referred to as
frameshift insertion or deletion mutations. Insertion or deletion of
three or its multiple bases insert or delete in one or multiple codon hence
one or multiple amino acids, and reading frame remains unaltered from
that point onwards.
6.6.2 tRNA– the Adapter Molecule
From the very beginning of the proposition of code, it was clear to Francis
Crick that there has to be a mechanism to read the code and also to link it
to the amino acids, because amino acids have no structural specialities to
read the code uniquely. He postulated the presence of an adapter molecule
that would on one hand read the code and on other hand would bind
to specific amino acids. The tRNA, then called sRNA (soluble RNA),
was known before the genetic code was postulated. However, its role
as an adapter molecule was assigned much later.
tRNA has an
anticodon loop
that has bases
complementary to
the code, and it also
has an amino acid
acceptor end to
which it binds to
amino acids.
tRNAs are specific
for each amino acid
(Figure 6.12). For
initiation, there is
another specific tRNA that is referred to as initiator tRNA. There are no
tRNAs for stop codons. In figure 6.12, the secondary structure of tRNA
has been depicted that looks like a clover-leaf. In actual structure, the
tRNA is a compact molecule which looks like inverted L.
6.7 TRANSLATION
Translation refers to the process of polymerisation of amino acids to
form a polypeptide (Figure 6.13). The order and sequence of amino acids
are defined by the sequence of bases in the mRNA. The amino acids are
joined by a bond which is known as a peptide bond. Formation of a
peptide bond requires energy. Therefore, in the first phase itself amino
acids are activated in the presence of ATP and linked to their cognate
tRNA – a process commonly called as charging of tRNA or
aminoacylation of tRNA to be more specific. If two such charged tRNAs
are brought close enough, the formation of peptide bond between them
would be favoured energetically. The
presence of a catalyst would enhance
the rate of peptide bond formation.
The cellular factory responsible for
synthesising proteins is the ribosome.
The ribosome consists of structural
RNAs and about 80 different proteins.
In its inactive state, it exists as two
subunits; a large subunit and a small
subunit. When the small subunit
encounters an mRNA, the process of
translation of the mRNA to protein
begins. There are two sites in the large
subunit, for subsequent amino acids
to bind to and thus, be close enough
to each other for the formation of a
peptide bond. The ribosome also acts as a catalyst (23S rRNA in bacteria
is the enzyme- ribozyme) for the formation of peptide bond.
A translational unit in mRNA is the sequence of RNA that is flanked
by the start codon (AUG) and the stop codon and codes for a polypeptide.
An mRNA also has some additional sequences that are not translated
and are referred as untranslated regions (UTR). The UTRs are present
at both 5′ -end (before start codon) and at 3′ -end (after stop codon). They
are required for efficient translation process.
For initiation, the ribosome binds to the mRNA at the start codon (AUG)
that is recognised only by the initiator tRNA. The ribosome proceeds to the
elongation phase of protein synthesis. During this stage, complexes
composed of an amino acid linked to tRNA, sequentially bind to the
appropriate codon in mRNA by forming complementary base pairs with
the tRNA anticodon. The ribosome moves from codon to codon along the
mRNA. Amino acids are added one by one, translated into Polypeptide
sequences dictated by DNA and represented by mRNA. At the end, a release
factor binds to the stop codon, terminating translation and releasing the
complete polypeptide from the ribosome.
6.8 REGULATION OF GENE EXPRESSION
Regulation of gene expression refers to a very broad term that may occur
at various levels. Considering that gene expression results in the formation
of a polypeptide, it can be regulated at several levels. In eukaryotes, the
regulation could be exerted at
(i) transcriptional level (formation of primary transcript),
(ii) processing level (regulation of splicing),
(iii) transport of mRNA from nucleus to the cytoplasm,
(iv) translational level.
The genes in a cell are expressed to perform a particular function or a
set of functions. For example, if an enzyme called beta-galactosidase is
synthesised by E. coli, it is used to catalyse the hydrolysis of a
disaccharide, lactose into galactose and glucose; the bacteria use them
as a source of energy. Hence, if the bacteria do not have lactose around
them to be utilised for energy source, they would no longer require the
synthesis of the enzyme beta-galactosidase. Therefore, in simple terms,
it is the metabolic, physiological or environmental conditions that regulate
the expression of genes. The development and differentiation of embryo
into adult organisms are also a result of the coordinated regulation of
expression of several sets of genes.
In prokaryotes, control of the rate of transcriptional initiation is the
predominant site for control of gene expression. In a transcription unit,
the activity of RNA polymerase at a given promoter is in turn regulated
by interaction with accessory proteins, which affect its ability to recognise
start sites. These regulatory proteins can act both positively (activators)
and negatively (repressors). The accessibility of promoter regions of
prokaryotic DNA is in many cases regulated by the interaction of proteins
with sequences termed operators. The operator region is adjacent to the
promoter elements in most operons and in most cases the sequences of
the operator bind a repressor protein. Each operon has its specific
operator and specific repressor. For example, lac operator is present
only in the lac operon and it interacts specifically with lac repressor only.
6.8.1 The Lac operon
The elucidation of the lac operon was also a result of a close association
between a geneticist, Francois Jacob and a biochemist, Jacque Monod. They
were the first to elucidate a transcriptionally regulated system. In lac operon
(here lac refers to lactose), a polycistronic structural gene is regulated by a
common promoter and regulatory genes. Such arrangement is very common
in bacteria and is referred to as operon. To name few such examples, lac
operon, trp operon, ara operon, his operon, val operon, etc.
The lac operon consists of one regulatory gene (the i gene – here the
term i does not refer to inducer, rather it is derived from the word inhibitor)
and three structural genes (z, y, and a). The i gene codes for the repressor
of the lac operon. The z gene codes for beta-galactosidase (β-gal), which
is primarily responsible for the hydrolysis of the disaccharide, lactose
into its monomeric units, galactose and glucose. The y gene codes for
permease, which increases permeability of the cell to β-galactosides. The
a gene encodes a transacetylase. Hence, all the three gene products in
lac operon are required for metabolism of lactose. In most other operons
as well, the genes present in the operon are needed together to function
in the same or related metabolic pathway (Figure 6.14).
Lactose is the substrate for the enzyme beta-galactosidase and it
regulates switching on and off of the operon. Hence, it is termed as inducer.
In the absence of a preferred carbon source such as glucose, if lactose is
provided in the growth medium of the bacteria, the lactose is transported
into the cells through the action of permease (Remember, a very low level
of expression of lac operon has to be present in the cell all the time,
otherwise lactose cannot enter the cells). The lactose then induces the
operon in the following manner.
The repressor of the operon is synthesised (all-the-time – constitutively)
from the i gene. The repressor protein binds to the operator region of the
operon and prevents RNA polymerase from transcribing the operon. In
the presence of an inducer, such as lactose or allolactose, the repressor is
inactivated by interaction with the inducer. This allows RNA polymerase
access to the promoter and transcription proceeds (Figure 6.14).
Essentially, regulation of lac operon can also be visualised as regulation
of enzyme synthesis by its substrate.
Remember, glucose or galactose cannot act as inducers for lac
operon. Can you think for how long the lac operon would be expressed
in the presence of lactose?
Regulation of lac operon by repressor is referred to as negative
regulation. Lac operon is under control of positive regulation as well,
but it is beyond the scope of discussion at this level.
6.9 HUMAN GENOME PROJECT
In the preceding sections you have learnt that it is the sequence of bases in
DNA that determines the genetic information of a given organism. In other
words, genetic make-up of an organism or an individual lies in the DNA
sequences. If two individuals differ, then their DNA sequences should also
be different, at least at some places. These assumptions led to the quest of
finding out the complete DNA sequence of human genome. With the
establishment of genetic engineering techniques where it was possible to
isolate and clone any piece of DNA and availability of simple and fast
techniques for determining DNA sequences, a very ambitious project of
sequencing human genome was launched in the year 1990.
Human Genome Project (HGP) was called a mega project. You can
imagine the magnitude and the requirements for the project if we simply
define the aims of the project as follows:
Human genome is said to have approximately 3 x 109
bp, and if the
cost of sequencing required is US $ 3 per bp (the estimated cost in the
beginning), the total estimated cost of the project would be approximately
9 billion US dollars. Further, if the obtained sequences were to be stored
in typed form in books, and if each page of the book contained 1000
letters and each book contained 1000 pages, then 3300 such books would
be required to store the information of DNA sequence from a single human
cell. The enormous amount of data expected to be generated also
necessitated the use of high speed computational devices for data storage
and retrieval, and analysis. HGP was closely associated with the rapid
development of a new area in biology called Bioinformatics.
Goals of HGP
Some of the important goals of HGP were as follows:
(i) Identify all the approximately 20,000-25,000 genes in human DNA;
(ii) Determine the sequences of the 3 billion chemical base pairs that
make up human DNA;
(iiii) Store this information in databases;
(iv) Improve tools for data analysis;
(v) Transfer related technologies to other sectors, such as industries;
(vi) Address the ethical, legal, and social issues (ELSI) that may arise
from the project.
The Human Genome Project was a 13-year project coordinated by
the U.S. Department of Energy and the National Institute of Health. During
the early years of the HGP, the Wellcome Trust (U.K.) became a major
partner; additional contributions came from Japan, France, Germany,
China and others. The project was completed in 2003. Knowledge about
the effects of DNA variations among individuals can lead to revolutionary
new ways to diagnose, treat and someday prevent the thousands of
disorders that affect human beings. Besides providing clues to
understanding human biology, learning about non-human organisms
DNA sequences can lead to an understanding of their natural capabilities
that can be applied toward solving challenges in health care, agriculture,
energy production, environmental remediation. Many non-human model
organisms, such as bacteria, yeast, Caenorhabditis elegans (a free living
non-pathogenic nematode), Drosophila (the fruit fly), plants (rice and
Arabidopsis), etc., have also been sequenced.
Methodologies : The methods involved two major approaches. One
approach focused on identifying all the genes that are expressed as
RNA (referred to as Expressed Sequence Tags (ESTs). The other took
the blind approach of simply sequencing the whole set of genome that
contained all the coding and non-coding sequence, and later assigning
different regions in the sequence with functions (a term referred to as
Sequence Annotation). For sequencing, the total DNA from a cell is
isolated and converted into random fragments of relatively smaller sizes
(recall DNA is a very long polymer, and there are technical limitations in
sequencing very long pieces of DNA) and cloned in suitable host using
specialised vectors. The cloning resulted into amplification of each piece
of DNA fragment so that it subsequently could be sequenced with ease.
The commonly used hosts were bacteria and yeast, and the vectors were
called as BAC (bacterial artificial chromosomes), and YAC (yeast artificial
chromosomes).
The fragments were sequenced using automated DNA sequencers that
worked on the principle of a method developed by Frederick Sanger.
(Remember, Sanger is also credited for developing method for
determination of amino acid
sequences in proteins). These
sequences were then arranged based
on some overlapping regions
present in them. This required
generation of overlapping fragments
for sequencing. Alignment of these
sequences was humanly not
possible. Therefore, specialised
computer based programs were
developed (Figure 6.15). These
sequences were subsequently
annotated and were assigned to each
chromosome. The sequence of
chromosome 1 was completed only
in May 2006 (this was the last of the
24 human chromosomes – 22
autosomes and X and Y – to be
sequenced). Another challenging task was assigning the genetic and
physical maps on the genome. This was generated using information on
polymorphism of restriction endonuclease recognition sites, and some
repetitive DNA sequences known as microsatellites (one of the applications
of polymorphism in repetitive DNA sequences shall be explained in next
section of DNA fingerprinting).
6.9.1 Salient Features of Human Genome
Some of the salient observations drawn from human genome project are
as follows:
(i) The human genome contains 3164.7 million bp.
(ii) The average gene consists of 3000 bases, but sizes vary greatly, with
the largest known human gene being dystrophin at 2.4 million bases.
(iii) The total number of genes is estimated at 30,000–much lower
than previous estimates of 80,000 to 1,40,000 genes. Almost all
(99.9 per cent) nucleotide bases are exactly the same in all people.
(iv) The functions are unknown for over 50 per cent of the discovered
genes.
(v) Less than 2 per cent of the genome codes for proteins.
(vi) Repeated sequences make up very large portion of the human genome.
(vii) Repetitive sequences are stretches of DNA sequences that are
repeated many times, sometimes hundred to thousand times. They
are thought to have no direct coding functions, but they shed light
on chromosome structure, dynamics and evolution.
(viii) Chromosome 1 has most genes (2968), and the Y has the fewest (231).
(ix) Scientists have identified about 1.4 million locations where single-
base DNA differences (SNPs – single nucleotide polymorphism,
pronounced as ‘snips’) occur in humans. This information promises
to revolutionise the processes of finding chromosomal locations for
disease-associated sequences and tracing human history.
6.9.2 Applications and Future Challenges
Deriving meaningful knowledge from the DNA sequences will define
research through the coming decades leading to our understanding of
biological systems. This enormous task will require the expertise and
creativity of tens of thousands of scientists from varied disciplines in both
the public and private sectors worldwide. One of the greatest impacts of
having the HG sequence may well be enabling a radically new approach
to biological research. In the past, researchers studied one or a few genes
at a time. With whole-genome sequences and new high-throughput
technologies, we can approach questions systematically and on a much
broader scale. They can study all the genes in a genome, for example, all
the transcripts in a particular tissue or organ or tumor, or how tens of
thousands of genes and proteins work together in interconnected networks
to orchestrate the chemistry of life.
6.10 DNA FINGERPRINTING
As stated in the preceding section, 99.9 per cent of base sequence among
humans is the same. Assuming human genome as 3 × 109
bp, in how
many base sequences would there be differences? It is these differences
in sequence of DNA which make every individual unique in their
phenotypic appearance. If one aims to find out genetic differences
between two individuals or among individuals of a population,
sequencing the DNA every time would be a daunting and expensive
task. Imagine trying to compare two sets of 3 × 106
base pairs. DNA
fingerprinting is a very quick way to compare the DNA sequences of any
two individuals.
DNA fingerprinting involves identifying differences in some specific
regions in DNA sequence called as repetitive DNA, because in these
sequences, a small stretch of DNA is repeated many times. These repetitive
DNA are separated from bulk genomic DNA as different peaks during
density gradient centrifugation. The bulk DNA forms a major peak and
the other small peaks are referred to as satellite DNA. Depending on
base composition (A : T rich or G:C rich), length of segment, and number
of repetitive units, the satellite DNA is classified into many categories,
such as micro-satellites, mini-satellites etc. These sequences normally
do not code for any proteins, but they form a large portion of human
genome. These sequence show high degree of polymorphism and form
the basis of DNA fingerprinting. Since DNA from every tissue (such as
blood, hair-follicle, skin, bone, saliva, sperm etc.), from an individual
show the same degree of polymorphism, they become very useful
identification tool in forensic applications. Further, as the polymorphisms
are inheritable from parents to children, DNA fingerprinting is the basis
of paternity testing, in case of disputes.
As polymorphism in DNA sequence is the basis of genetic mapping
of human genome as well as of DNA fingerprinting, it is essential that we
understand what DNA polymorphism means in simple terms.
Polymorphism (variation at genetic level) arises due to mutations. (Recall
different kind of mutations and their effects that you have already
studied in Chapter 5, and in the preceding sections in this chapter.)
New mutations may arise in an individual either in somatic cells or in
the germ cells (cells that generate gametes in sexually reproducing
organisms). If a germ cell mutation does not seriously impair individual’s
ability to have offspring who can transmit the mutation, it can spread to
the other members of population (through sexual reproduction). Allelic
(again recall the definition of alleles from Chapter 5) sequence variation
has traditionally been described as a DNA polymorphism if more than
one variant (allele) at a locus occurs in human population with a
frequency greater than 0.01. In simple terms, if an inheritable mutation
is observed in a population at high frequency, it is referred to as DNA
polymorphism. The probability of such variation to be observed in non-
coding DNA sequence would be higher as mutations in these sequences
may not have any immediate effect/impact in an individual’s
reproductive ability. These mutations keep on accumulating generation
after generation, and form one of the basis of variability/polymorphism.
There is a variety of different types of polymorphisms ranging from single
nucleotide change to very large scale changes. For evolution and
speciation, such polymorphisms play very important role, and you will
study these in details at higher classes.
The technique of DNA Fingerprinting was initially developed by Alec
Jeffreys. He used a satellite DNA as probe that shows very high degree
of polymorphism. It was called as Variable Number of Tandem Repeats
(VNTR). The technique, as used earlier, involved Southern blot
hybridisation using radiolabelled VNTR as a probe. It included
(i) isolation of DNA,
(ii) digestion of DNA by restriction endonucleases,
(iii) separation of DNA fragments by electrophoresis,
(iv) transferring (blotting) of separated DNA fragments to synthetic
membranes, such as nitrocellulose or nylon,
(v) hybridisation using labelled VNTR probe, and
(vi) detection of hybridised DNA fragments by autoradiography. A schematic
representation of DNA fingerprinting is shown in Figure 6.16.
The VNTR belongs to a class of satellite DNA referred to as mini-satellite.
A small DNA sequence is arranged tandemly in many copy numbers. The
copy number varies from chromosome to chromosome in an individual.
The numbers of repeat show very high degree of polymorphism. As a
result the size of VNTR varies in size from 0.1 to
20 kb. Consequently, after hybridisation with VNTR probe, the
autoradiogram gives many bands of differing sizes. These bands give a
characteristic pattern for an individual DNA (Figure 6.16). It differs from
individual to individual in a population except in the case of monozygotic
(identical) twins. The sensitivity of the technique has been increased by
use of polymerase chain reaction (PCR–you will study about it in
Chapter 11). Consequently, DNA from a single cell is enough to perform
DNA fingerprinting analysis. In addition to application in forensic
science, it has much wider application, such as in determining
population and genetic diversities. Currently, many different probes
are used to generate DNA fingerprints.
SUMMARY
Nucleic acids are long polymers of nucleotides. While DNA stores genetic
information, RNA mostly helps in transfer and expression of information.
Though DNA and RNA both function as genetic material, but DNA being
chemically and structurally more stable is a better genetic material.
However, RNA is the first to evolve and DNA was derived from RNA. The
hallmark of the double stranded helical structure of DNA is the hydrogen
bonding between the bases from opposite strands. The rule is that
Adenine pairs with Thymine through two H-bonds, and Guanine with
Cytosine through three H-bonds. This makes one strand
complementary to the other. The DNA replicates semiconservatively,
the process is guided by the complementary H-bonding. A segment of
DNA that codes for RNA may in a simplistic term can be referred as
gene. During transcription also, one of the strands of DNA acts a
template to direct the synthesis of complementary RNA. In bacteria,
the transcribed mRNA is functional, hence can directly be translated.
In eukaryotes, the gene is split. The coding sequences, exons, are
interrupted by non-coding sequences, introns. Introns are removed
and exons are joined to produce functional RNA by splicing. The
messenger RNA contains the base sequences that are read in a
combination of three (to make triplet genetic code) to code for an amino
acid. The genetic code is read again on the principle of complementarity
by tRNA that acts as an adapter molecule. There are specific tRNAs for
every amino acid. The tRNA binds to specific amino acid at one end
and pairs through H-bonding with codes on mRNA through its
anticodons. The site of translation (protein synthesis) is ribosomes,
which bind to mRNA and provide platform for joining of amino acids.
One of the rRNA acts as a catalyst for peptide bond formation, which is
an example of RNA enzyme (ribozyme). Translation is a process that
has evolved around RNA, indicating that life began around RNA. Since,
transcription and translation are energetically very expensive
processes, these have to be tightly regulated. Regulation of transcription
is the primary step for regulation of gene expression. In bacteria, more
than one gene is arranged together and regulated in units called as
operons. Lac operon is the prototype operon in bacteria, which codes
for genes responsible for metabolism of lactose. The operon is regulated
by the amount of lactose in the medium where the bacteria are grown.
Therefore, this regulation can also be viewed as regulation of enzyme
synthesis by its substrate.
Human genome project was a mega project that aimed to sequence
every base in human genome. This project has yielded much new
information. Many new areas and avenues have opened up as a
consequence of the project. DNA Fingerprinting is a technique to find
out variations in individuals of a population at DNA level. It works on
the principle of polymorphism in DNA sequences. It has immense
applications in the field of forensic science, genetic biodiversity and
evolutionary biology.
EXERCISES
1 Group the following as nitrogenous bases and nucleosides:
Adenine, Cytidine, Thymine, Guanosine, Uracil and Cytosine.
- If a double stranded DNA has 20 per cent of cytosine, calculate the per
cent of adenine in the DNA. - If the sequence of one strand of DNA is written as follows:
5′ -ATGCATGCATGCATGCATGCATGCATGC-3′
Write down the sequence of complementary strand in 5’→3′ direction. - If the sequence of the coding strand in a transcription unit is written
as follows:
5′ -ATGCATGCATGCATGCATGCATGCATGC-3′
Write down the sequence of mRNA. - Which property of DNA double helix led Watson and Crick to hypothesise
semi-conservative mode of DNA replication? Explain. - Depending upon the chemical nature of the template (DNA or RNA)
and the nature of nucleic acids synthesised from it (DNA or RNA), list
the types of nucleic acid polymerases. - How did Hershey and Chase differentiate between DNA and protein in
their experiment while proving that DNA is the genetic material? - Differentiate between the followings:
(a) Repetitive DNA and Satellite DNA
(b) mRNA and tRNA
(c) Template strand and Coding strand - List two essential roles of ribosome during translation.
- In the medium where E. coli was growing, lactose was added, which
induced the lac operon. Then, why does lac operon shut down some
time after addition of lactose in the medium? - Explain (in one or two lines) the function of the followings:
(a) Promoter
(b) tRNA
(c) Exons - Why is the Human Genome project called a mega project?
- What is DNA fingerprinting? Mention its application.
- Briefly describe the following:
(a) Transcription
(b) Polymorphism
(c) Translation
(d) Bioinformatics
563 responses to “CHAPTER 6 MOLECULAR BASIS OFINHERITANCE”
Hey! Do you know if they make any plugins to help with SEO?
I’m trying to get my site to rank for some targeted keywords but I’m not seeing very good
gains. If you know of any please share. Many thanks!
I saw similar art here: Wool product
Kanha Nationwide Park: Kanha situated at the Mandla, Madhya Pradesh is a much go for you if you’re looking to watch tigers.
sugar defender reviews Incorporating Sugar Protector into my day-to-day routine overall well-being.
As somebody that focuses on healthy eating, I
appreciate the added security this supplement gives. Given that beginning to take it, I have actually seen a
marked enhancement in my power degrees and a considerable decrease in my wish for unhealthy snacks such a such
a profound effect on my day-to-day live. sugar defender ingredients
Sugar Defender As a person that’s
constantly been cautious concerning my blood sugar level, finding
Sugar Defender has actually been an alleviation. I really feel a lot a lot more in control, and my recent examinations
have shown positive improvements. Understanding I have a trustworthy supplement
to support my regular provides me comfort. I’m so happy for Sugar Defender’s influence on my wellness!
sugar defender reviews Finding Sugar
Protector has actually been a game-changer for me, as
I have actually constantly been vigilant about managing my blood sugar
level levels. With this supplement, I really feel
encouraged to organize my health and wellness, and my newest clinical check-ups have reflected a
considerable turnaround. Having a credible ally in my
corner gives me with a sense of security and reassurance, and I’m deeply glad for
the extensive difference Sugar Defender has made in my health.
sugar defender
sugar defender official website Incorporating Sugar Protector right into my day-to-day program
has been a game-changer for my overall wellness. As someone that currently
prioritizes healthy consuming, this supplement has given an added boost of security.
in my energy levels, and my wish for harmful treats so easy can have
such a profound effect on my every day life. sugar defender official website
It also helps to scale back vitality consumption and prolong the lifespan of your pump.
I allowed twitter to become a much bigger part of my online life than I ought to have achieved, and now it’s going down the rest room in a shift that feels quite sudden, provided that I’ve been on twitter f…
Christian Chevalier (13 March 2005).
The 1941 Glenn Miller song that catapulted Chattanooga to international fame, “Chattanooga Choo Choo”, has been carried out in quite a few motion pictures, together with the 1941 film Sun Valley Serenade, featuring the Miller Orchestra and Milton Berle, The Glenn Miller Story starring James Stewart within the 1953 title role, and the 1984 eponymous movie Chattanooga Choo Choo.
Next time I read a blog, I hope that it does not fail me as much as this one. I mean, Yes, it was my choice to read through, but I actually believed you would have something useful to talk about. All I hear is a bunch of crying about something that you could possibly fix if you were not too busy searching for attention.
Significantly, this post is really the sweetest on this notable subject. I harmonise along with your conclusions and will thirstily seem ahead to your incoming updates. Stating thanks will not just be ample, for your phenomenal clarity with your writing. I will directly grab your rss feed to remain informed of any updates. Admirable perform and considerably good results in your organization dealings! Please excuse my poor English as it really is not my very first tongue.
You have noted very interesting points! ps nice web site.
fertility clinics these days are very advanced and of course this can only mean higher success rates on birth,,
This is a topic that is near to my heart… Best wishes! Exactly where can I find the contact details for questions?
Spot i’ll carry on with this write-up, I must say i believe this web site needs considerably more consideration. I’ll more likely again to learn considerably more, many thanks that info.
Thank you a lot for sharing this with all of us you really recognize what you are talking about! Bookmarked. Kindly additionally seek advice from my web site =). We may have a hyperlink trade contract among us!
I’m not sure where you’re getting your information, but great topic. I needs to spend some time learning more or understanding more. Thanks for fantastic information I was looking for this information for my mission.
Youre so cool! I dont suppose Ive read anything this way prior to. So nice to seek out somebody by incorporating original thoughts on this subject. realy we appreciate you beginning this up. this fabulous website is one thing that is needed over the internet, someone after a little originality. useful problem for bringing something new towards the net!
I’m just commenting to let you know what a excellent discovery my cousin’s girl encountered going through your web site. She noticed several details, which include how it is like to possess an ideal coaching spirit to let others smoothly learn a number of grueling subject areas. You actually did more than visitors’ desires. Thank you for distributing these great, dependable, informative and unique tips on that topic to Tanya.
Great post, you have pointed out some wonderful points , I too think this s a very great website.
i can say that David Archuleta has some great potential. all he need is more epxerience’
Can I simply say what a relief to search out someone who actually is aware of what theyre talking about on the internet. You definitely know methods to bring a difficulty to gentle and make it important. More people have to learn this and understand this facet of the story. I cant imagine youre no more widespread since you definitely have the gift.
Hi, I do think this is an excellent website. I stumbledupon it 😉 I am going to come back once again since I book-marked it. Money and freedom is the best way to change, may you be rich and continue to help other people.
Simply wanna input on few general things, The website layout is perfect, the subject matter is really wonderful : D.
Some genuinely interesting information, well written and broadly speaking user friendly .
Folks have no idea that this exists. I know this must be here but I eventually got lucky and the search phrases worked. You’ve contributed to my exploration in your posting.
Just article, We liked its style and content. I discovered this blog on Yahoo and also have now additional it to my personal bookmarks. I’ll be certain to visit once again quickly.
I believe that may be an enchanting element, it made me assume a bit. Thanks for sparking my considering cap. Occasionally I get so much in a rut that I simply really feel like a record.
Hi there, have you possibly asked yourself to publish concerning Nintendo or PSP?
Greetings! Very useful advice within this post! It is the little changes which will make the most important changes. Thanks a lot for sharing!
This is a proper weblog for anyone who would like to be familiar with this topic. You are aware of a great deal its almost tough to argue along with you (not too I actually would want…HaHa). You certainly put a fresh spin over a topic thats been revealed for many years. Excellent stuff, just excellent!
I am usually to blogging and that i genuinely appreciate your posts. The content has really peaks my interest. I will bookmark your internet site and maintain checking for new information.
You made some decent points there. I looked on the net for any issue and found most individuals goes in addition to with all your website.
I would like to thnkx for the efforts you have put in writing this blog. I’m hoping the same high-grade website post from you in the upcoming also. Actually your creative writing skills has inspired me to get my own site now. Actually the blogging is spreading its wings rapidly. Your write up is a good example of it.
I’d have got to consult you here. Which isn’t something It’s my job to do! I spend time reading a post which will make people believe. Also, many thanks for allowing me to comment!
My partner and i many thanks for wp style, where you down load it through? Many thanks beforehand!
Very good written article. It will be beneficial to everyone who employess it, as well as myself.
As I site possessor I believe the content matter here is rattling wonderful , appreciate it for your hard work. You should keep it up forever! Best of luck.
What¡¦s Going down i am new to this, I stumbled upon this I’ve discovered It absolutely useful and it has helped me out loads. I am hoping to give a contribution & aid other users like its helped me. Great job.
There is not any manner this automotive belongs on a metropolis street, however it is certainly a street-authorized supercar.
After examine a number of of the blog posts on your web site now, and I really like your manner of blogging. I bookmarked it to my bookmark web site record and can be checking again soon. Pls try my website online as well and let me know what you think.
Hello! I simply would wish to give you a huge thumbs up for any great information you have here within this post. I’ll be returning to your blog post for additional soon.
I’d have to check with you here. Which isn’t something I usually do! I get pleasure from reading a put up that can make people think. Additionally, thanks for allowing me to remark!
Cheap Gucci Shoes For Men Amaze! I have been searching yahoo for a long time for the i ultimately found it on this page!
i support herbal products because they are all natural and from what i know, they do not carry nasty side effects;;
The the next occasion I just read a blog, Hopefully that this doesnt disappoint me up to this. I am talking about, It was my method to read, but I really thought youd have something interesting to express. All I hear can be a number of whining about something that you could fix should you werent too busy looking for attention.
I’d must verify with you here. Which isn’t something I often do! I take pleasure in reading a post that may make people think. Additionally, thanks for allowing me to comment!
Having read this I thought it was very informative. I appreciate you spending some time and energy to put this short article together. I once again find myself personally spending way too much time both reading and posting comments. But so what, it was still worthwhile.
Heya i am for the first time here. I found this board and I find It really helpful & it helped me out much. I hope to offer something back and help others such as you helped me.
fantastic points altogether, you just gained emblem reader. What would you suggest in regards to your post that you made a few days ago? Any certain?
You made some decent points there. I looked online with the issue and located most people is going along with using your site.
My wife and i got quite relieved that Louis could finish up his investigation with the precious recommendations he gained using your weblog. It is now and again perplexing to just find yourself giving for free instructions some people might have been selling. And we all already know we need the writer to be grateful to for this. The entire illustrations you’ve made, the straightforward website menu, the relationships you aid to instill – it is everything incredible, and it’s making our son and our family reason why this theme is excellent, which is exceedingly mandatory. Many thanks for all!
My husband and i were now excited that John could complete his studies with the ideas he made in your web site. It is now and again perplexing just to always be giving away instructions that many some people may have been making money from. We really discover we’ve got the blog owner to thank for this. The main illustrations you made, the simple website menu, the relationships you aid to instill – it’s got everything sensational, and it’s helping our son and our family consider that that topic is amusing, which is certainly really essential. Thanks for the whole thing!
What your declaring is entirely true. I know that everybody should say the similar thing, but I just consider that you put it in a way that absolutely everyone can understand. I also really like the pictures you put in right here. They match so nicely with what youre trying to say. Im sure youll attain so numerous men and women with what youve obtained to say.
I am glad to be a visitor of this consummate web site! , appreciate it for this rare info ! .
Pretty nice post. I just stumbled upon your weblog and wished to say that I’ve truly enjoyed surfing around your blog posts. After all I?ll be subscribing to your rss feed and I hope you write again soon!
Nice weblog here! Also your site lots up fast! What host are you the use of? Can I get your affiliate link on your host? I want my website loaded up as quickly as yours lol
Hello I absolutely love your article and it has been so admirable hence I am going to save it. I Have to say the Superb research you have done is greatly
Be taught and earn at your individual tempo.
Hey, maybe this is a bit offf topic but in any case, I have been surfing about your blog and it looks really neat. impassioned about your writing. I am creating a new blog and hard-pressed to make it appear great, and supply excellent articles. I have discovered a lot on your site and I look forward to additional updates and will be back.
i think that gay marriage should be allowed in certain states but not in other states .
In the course of the Cold Struggle years that followed the successful peace negotiations, Malgus led Sith forces into the Unknown Areas, claiming previously-unknown territories for the Empire.
If we talk particularly in regards to the tribal tattoos of American Indians, they used them to characterize their spiritual beliefs, tradition, rituals, and natural spirits.
herpafend USA Herpafend is a natural supplement designed to help
manage herpes symptoms. It boosts the immune system and reduces the frequency of outbreaks.
Formulated with ingredients like elderberry, echinacea herb, and L-lysine compound, Herpafend supports overall health.
Made in America in an FDA-approved facility, Herpafend maintains premium standards.
It is non-GMO and gluten-free. People mention a reduction in outbreak frequency and severity.
Try Herpafend today and feel the improvement in your herpes management.
Basic because it sounds, these balloon rides began method again after the inaugural of the good Smokey Mountains Nationwide Park, which covers the ridgeline of Smokey Mountains and a division of Appalachian Mountain chain.
I’ve been visiting your blog for a while now and I always find a gem in your new posts. Thanks for sharing.
I wanted to thank you for this great read!! I certainly enjoyed every little bit of it. I have got you book marked to look at new things you post…
Since they’re portable, they are not a built-in a part of the automotive’s dash.
Wonderful handbook on making and utilizing methane — biogas.
this article is very full of detail information, and it makes me easy to understand the meaning of your article.
All you need to know about News with our valuble article.
Can I simply say what relief to get somebody that in fact knows what theyre dealing with on-line. You actually know how to bring a concern to light to make it essential. Workout . should ought to see this and can see this side of your story. I cant think youre less well-known simply because you definitely hold the gift.
What¡¦s Happening i’m new to this, I stumbled upon this I’ve found It positively helpful and it has helped me out loads. I am hoping to give a contribution & aid different customers like its aided me. Good job.
Less apparent methods to reduce a current account deficit include measures that enhance home financial savings (or reduced home borrowing), together with a discount in borrowing by the nationwide authorities.
if I were a frog, would this post make me jump? I think … maybe
Each township is governed by a board of supervisors/commissioners.
Oh my goodness! an incredible article dude. Thanks a ton However My business is experiencing problem with ur rss . Do not know why Not able to subscribe to it. Can there be everyone obtaining identical rss dilemma? Anyone who knows kindly respond. Thnkx
I discovered your website site online and check many of your early posts. Keep on the top notch operate. I just now additional your Feed to my MSN News Reader. Looking for forward to reading much more from you finding out later on!…
These crows would fly over the Earth, bringing again tales and data to Odin.
Convertible bonds – Sometimes bonds might be converted into stock in the company that issued them.
Doing the sequence in a heated room retains muscles unfastened and versatile, and encourages college students to sweat out impurities during class.
We’ve all seen movies with just a little industrial espionage — Company A instructs one in every of its employees to get a job at its top competitor, Company B, then steal its coveted business secrets and techniques.
A thousand years after the Darkish Lord’s dying, Exar Kun summoned Ragnos from his grave by using talismans.
They’ve saved hundreds of tonnes of paper giving it a socially and environmentally a protected tag.
Chew slowly. Take small bites.
There are many who bought stocks in 2008 in the hope of making a quick buck in rising markets, but their trades turned into investments with a long-term perspective when the markets turned negative.
It is true that for some bizarre purpose now we have turn out to be used to calling certain collections of chips and wires in a pc its “mind”, however again (what’s in a name?) we stay pretty much conscious that that is mere hyperbole.
John Wiley & Sons.
2,015 ballots solid by town’s 2,543 registered voters, for a turnout of 79.2 (vs.
There are different types of commodities that trade in the commodity markets; however knowing a few essential ones before you are commodity trading is important.
James Fearnley, these days Macebearer and Mayoral Attendant, Halton Borough Council.
In addition, ISH additionally offers educational certification of Ecole hoteliere de Lausanne, Switzerland, which is probably the greatest hospitality management colleges on the planet .
The future holds its own flood dangers as a result of climate change, rising sea levels, and burgeoning populations.
During this period there have been fewer than 20 incidents and have been majorly concentrated in the European region.
There are simple ways to analyze large volumes of data that come from paying invoices.
A stock is small shares that represent a partial ownership of a company .Stocks are issued by companies In order to raise capital and are bought by investors in order to acquire a portion of the company .
There isn’t any telling which means our determination will go at the moment.
If you have the proper data of the working, then you possibly can earn rather a lot.
With the miss selfridge voucher codes you will get the premium and excessive-end trend products at an inexpensive value.
Olson, Tyler (April 20, 2020).
So, whether you want to make a big purchase or visit your dream destination, you do not need to pay for additional costs such as interest with savings.
The concept is to maintain decorations to a minimum – a couple of choose framed prints, a monochromatic bedspread, and clear-lined furniture.
The exchange forex charges are normally shows how a lot for the second forex is needed to buy the only unit of the first foreign money.
A large number of investors take the advices from investing advisors to earn profits.
In the primary inning of his first recreation with the Cubs, Bonds tripped on a seam in the synthetic turf at Three Rivers Stadium in Pittsburgh and broke a bone in his wrist.
The mouse will probably be inside the circle and the cat will likely be exterior.
On the morning of December 25, Washington ordered his Continental Army troops to prepare three days’ food and issued orders that each soldier be outfitted with contemporary flints for his or her muskets.
Nevertheless it was at the 1904 World’s Honest in St.
They’re tied with Direct Travel for eleventh place in energy rankings.
He performed his remaining 2018-19 campaign with ATK, and performed his first match with ATK that season against Kerala Blasters on 25 January 2019, which ended in a 1-1 draw.
The article definitely brought up some questions about whether or not it is smart to add LFTB to ground beef and different food products at all, and the phrases “pink slime” turned people’s stomachs.
Various brokers have various requirements for setting up a trading account like minimum deposits and fees.
We’ll try the main points of some consultant assist packages, see who qualifies for a free ride, and look at financial-support deadlines and procedures for these excited about applying.
Full your scheme with matching or coordinating fabrics in the same colors, and placed on the kettle!
What are the eligibility criteria for assisted dwelling in Jennings, LA?
Brady MacDonald (2008-04-08). “Common Studios Simpsons journey reveals visitors Krustyland”.
The functionality of a handmade leather portfolio is commendable.
Like Isaac Newton and his falling apple, or Alexander Flemming and his accidental penicillin spores, John Kanzius stumbled onto his discovery.
Within a year, the fund had only grown to $17 million in assets, but one of the Wellington Funds that Vanguard was administering had to be merged in with another fund, and Bogle convinced Wellington to merge it in with the Index fund.
She had celebrated her 89th birthday on Could 6. At the age of 55 Mrs Johnson acquired her Licensed Sensible Nurse license and retired from this occupation on the age of 75.
In 1898 Latrobe and two players from the Greensburg Athletic Affiliation, formed the very first skilled football all-star crew for a game in opposition to the Duquesne Nation and Athletic Club, to be performed at Pittsburgh’s Exposition Park.
In Chess rigorously measuring individual adjustments in actual matches with giant numbers of video games for small elo gains can ultimately lead to a whole lot of elo total improvement.
Mud lake. Drake constructed the first mill.
Candidates Tournament and changing into in 1958 the world’s youngest Grandmaster at age 15.
The one manner this may be managed is to cease Nations from falling, help Nations settle for their individuals as their very own, emphasize on State principles that work and might forestall states from failing.
Thus modified duration is approximately equal to the percentage change in price for a given finite change in yield.
Mr. Bintz and his spouse, Carolyn, have been married forty seven years.
On August 19, a big crowd of freshmen college students at the College Park campus of Pennsylvania State University gathered; many of them didn’t apply social distancing or carrying masks.
There are many different types of fire extinguishers too.
Between 1992 and 2011, later CVPI models offered a strong 4.6-liter V8 engine and rear-wheel drive (which the police liked because of its better dealing with throughout laborious driving).
Series A, Mathematical and Bodily Sciences.
Proponents of adaptive benchmarking maintain that by understanding the characteristics of the portfolio at each point in time, they can better attribute excess returns to skill.
Burial companies four p.m.
We will continue to discuss these issues throughout the rest of the article.
But after you’ve got simply spent hours and dollars trying to increase the airtightness of your home as a way to make it more power-efficient, how do you increase the ventilation without decreasing the impact of all the things you’ve completed?
If you or someone you know is struggling with suicidal thoughts, contact a psychological health skilled or a helpline in your country instantly.
The painting of canopies and draperies was taken to a new top.
This means that have relationships with certain providers of marriage, and it’s for these suppliers to cut marriage a scheduler pause on the price, so that it will proceed to use their companies to all marriages, with which is involved.
As one of the ideas of being environmentally pleasant is to lessen detrimental impacts on the planet — including its folks — the Salwens lowered their consumption and improved the circumstances of others with the money they saved.
I try to reason with everyone.
The Intercontinental portfolio is diversified both by robust property mix and by geography.
We train and monitor all instructors to ensure they deliver the highest quality.
Why does this method pay again so richly?
Reed arrived in Mount Holly on December 22, and located Griffin to be ailing and his males in comparatively poor situation, but keen to undertake some type of military diversion.
No, you are higher off getting a nice stable entry degree mannequin.
However, in international nomenclature, repo and reverse repo implied the reverse.
He was a member of West Guthrie United Methodist Church and a former Guthrie policeman, a deputy sheriff and retired farmer.
Oleg Penkovskiy earned great respect in the Soviet Union for his military career.
Outline of finance 搂 Quantitative investing and 搂 Portfolio theory.
Burial: Sprague. Funeral at chapel.
Altrincham entertain FC Halifax City at the J. Davidson Stadium on Tuesday night.
You cannot stand to disregard the strategic difficulties for gathering journey – together with getting visas, having exact member documentation, saving carriers, giving interchange transportation and ending lodging affirmation materials, and so forth.
Take a look at for Singapore Groupon online retailer for higher discounts on the above merchandise.
The then-27-yr-outdated native of Richton, Mississippi – 70 miles inland from the Gulf Coast – took shelter in her church’s partial basement and huddled together with her husband and neighbors because the storm tore straight by her hometown.
This function alone can significantly improve the efficiency and productiveness of a sales group.
Sustainable Journey: Eco-acutely aware travelers are increasingly seeking accountable journey options.
Karabell, Zachary. “A Market Crash Was Coming, Coronavirus Was Just the Spark”.
Most humans infected by the West Nile virus have no signs, whereas a number of may have symptoms similar to a fever and headache.
I stгongly recommend paying very close attention to continuous research and security records.
my web site; Herpafend Online Store
Keep your rest peｒiods betweеn collections as brief as possible yet as long aas yoᥙ need to
complete thｅ exercise.
Here is mу web-site; buy sunlight loophole sync
Loved this!
It is really a nice and helpful piece of information. I am satisfied that you shared this helpful tidbit with us. Please keep us up to date like this. Thank you for sharing.
What i do not realize is in reality how you’re now not actually much more neatly-favored than you may be right now. You’re very intelligent. You recognize therefore considerably relating to this topic, made me in my view consider it from a lot of varied angles. Its like women and men aren’t involved until it’s one thing to do with Girl gaga! Your own stuffs excellent. Always maintain it up!
We stopped for some crepes at a cute little Creperie proper exterior the fashionable artwork museum.
it does not take too long to learn good piano playing if you have a good piano lesson*
Folks often marvel why certain components have symbols that aren’t harking back to their names.
Whenever you save info in an application, you might not store it in your laptop.
It may be tougher to interrupt freed from your previous self when an old pal bunks in the loft above your head.
The committee gathers information on all real estate deals (it is mandatory to send a copy of each notarial purchase contract to the Gutachterausschuss) and includes it in the Kaufpreissammlung (purchase price database).
After I originally left a comment I appear to have clicked the -Notify me when new comments are added- checkbox and now every time a comment is added I receive four emails with the same comment. Is there an easy method you are able to remove me from that service? Kudos.
Aw, i thought this was an incredibly good post. In thought I would like to invest writing such as this moreover – taking time and actual effort to have a top notch article… but exactly what can I say… I procrastinate alot and by no means apparently get something done.
Thank you for the auspicious writeup. It in fact was a amusement account it.
Look advanced to more added agreeable from you! However, how could we communicate?
An impressive share! I’ve just forwarded this onto a coworker who had been doing a little research on this. And he actually bought me dinner due to the fact that I discovered it for him… lol. So let me reword this…. Thank YOU for the meal!! But yeah, thanks for spending the time to talk about this matter here on your website.
Rattling wonderful information can be found on website .
I couldn’t refrain from commenting. Very well written!
Magnificent goods from you, man. I’ve take
into accout your stuff prior to and you’re just
extremely great. I really like what you have got here,
really like what you’re stating and the best way during which you assert it.
You are making it enjoyable and you still care for to stay it smart.
I can not wait to learn far more from you. This is really a tremendous website.
Good information. Lucky me I discovered your website by accident (stumbleupon). I have saved as a favorite for later!
You are so cool! I don’t think I have read something like that before. So good to discover another person with genuine thoughts on this topic. Seriously.. thank you for starting this up. This website is one thing that is required on the web, someone with a bit of originality.
Why is sleep vital for learning?
You made some good points there. I checked on the web to find out more about the issue and found most individuals will go along with your views on this website.
Your style is very unique in comparison to other people I’ve read stuff from. Thank you for posting when you have the opportunity, Guess I’ll just bookmark this page.
Very good post. I certainly appreciate this site. Stick with it!
Pretty! This was an incredibly wonderful article. Thank you for supplying these details.
I really like your wp template, where did you download it from?
An intriguing discussion is worth comment. There’s no doubt that that you should write more on this subject matter, it may not be a taboo subject but typically people don’t speak about these issues. To the next! Many thanks.
Pretty! This was an extremely wonderful article. Thank you for supplying this information.
The post is really a helpful interesting and well-written details. The a lot more Someone said the more I want to study. Any article writer that makes the time and effort to publish participating articles similar to this needs to be commended.
I just could not depart your website before suggesting that I really loved the usual info an individual provide for your guests? Is going to be again often to investigate cross-check new posts.
Hi! I simply want to give you a big thumbs up for the great info you have got here on this post. I am coming back to your blog for more soon.
Aw, this was a really good post. Finding the time and actual effort to produce a good article… but what can I say… I procrastinate a whole lot and never manage to get nearly anything done.
Excellent blog here! Also your site loads up very fast! What host are you using? Can I get your affiliate link to your host? I wish my site loaded up as fast as yours lol xrumer
You should take part in a contest for one of the best sites on the web. I will recommend this web site!
Splendid post. I discover new things upon different weblogs daily. Couple of stimulating to see content using their company authors and turn out to be acquainted with a small something their particular. I’d think to use some inside the information on my blog need to do not thoughts. Obviously I’ll provides a link back up in internet site. Thank you sharing.
Way cool! Some extremely valid points! I appreciate you writing this article plus the rest of the site is extremely good.
Your style is very unique compared to other folks I’ve read stuff from. Many thanks for posting when you’ve got the opportunity, Guess I will just book mark this web site.
Sweet site, super style and design , really clean and use friendly .
Most I can state is, I don’t know what to comment! Except needless to say, for the superb tips which are shared with this blog. I’ll think of a thousand fun methods to read the posts on this site. I do think I will finally take a step with your tips on areas I could never have been able to manage alone. You’re so careful to permit me to be one of those to benefit from your helpful information. Please recognize how much I am thankful.
Oh my goodness! Amazing article dude! Many thanks, However I am experiencing difficulties with your RSS. I don’t understand the reason why I am unable to join it. Is there anybody having similar RSS issues? Anybody who knows the answer will you kindly respond? Thanks!!
I still can not quite think that I could often be one of those studying the important suggestions found on your website. My family and I are sincerely thankful on your generosity and for giving me potential to pursue our chosen profession path. Many thanks for the important information I acquired from your site.
Hello! I could have sworn I’ve been to your blog before but after browsing through many of the articles I realized it’s new to me. Anyhow, I’m definitely pleased I found it and I’ll be bookmarking it and checking back often.
Great work! This is the kind of info that should be shared around the internet. Shame on Google for now not positioning this submit higher! Come on over and discuss with my web site . Thanks =)
The next time I read a blog, I hope that it does not fail me just as much as this one. I mean, I know it was my choice to read through, however I actually thought you would probably have something interesting to say. All I hear is a bunch of whining about something you could possibly fix if you weren’t too busy seeking attention.
Bonjour On dirait que les thèmes de ce concis debrieffing requierent d’avantage de valeurs; Votre tournure peut paraitre recevable or je ne defend pas les différents avis plus anciens! il faudrait ajouter quelques valeurs pour plus de professionalisme! Merci
This is a appropriate blog for everyone who hopes to learn about this topic. You already know a lot its practically challenging to argue with you (not that I just would want…HaHa). You definitely put a whole new spin with a topic thats been revealed for some time. Fantastic stuff, just excellent!
In case you had a parrot, which phrase or phrase would you educate it first?
Great post! We are linking to this particularly great post on our website. Keep up the great writing.
Hello! I simply would wish to supply a huge thumbs up for the wonderful info you might have here for this post. I am returning to your blog post for additional soon.
Naturally I like your web-site, but you have to take a look at the spelling on quite a few of your posts. A number of them are rife with spelling issues and I find it very bothersome to tell you. On the other hand I’ll surely come back again!
It’s my job to dont usually post on many Blogs, still I recently has to say thank you maintain the astonishing work. Ok unfortunately it is time to get at school.
Loving the information on this website, you have done outstanding job on the blog posts.
Having read this I thought it was really informative. I appreciate you spending some time and effort to put this information together. I once again find myself personally spending a significant amount of time both reading and leaving comments. But so what, it was still worth it.
I need to to thank you for this fantastic read!! I definitely enjoyed every little bit of it. I’ve got you book marked to look at new things you post…
Many thanks for creating this place wonderful, i need to say i appreciate all of the exertions that you just do on this great website.
Merely wanna remark on few general things, The website pattern is perfect, the subject matter is really excellent : D.
I discovered your blog web site on google and appearance a couple of your early posts. Always keep in the very good operate. I recently extra up your Feed to my MSN News Reader. Looking for toward reading a lot more by you at a later date!…
Useful information. Fortunate me I discovered your site by accident, and I’m stunned why this coincidence didn’t took place earlier!
I bookmarked it.
you have a wonderful weblog here! do you wish to have the invite posts on my own blog?
I’ve learn several just right stuff here. Certainly value bookmarking for revisiting. I surprise how much effort you put to create the sort of excellent informative web site.
i gave my girlfriend a diamond bracelt and she was quite pleased with it”
This blog was… how do I say it? Relevant!! Finally I’ve found something which helped me. Kudos!
The very next time I read a blog, I hope that it doesn’t fail me just as much as this particular one. I mean, I know it was my choice to read through, nonetheless I actually thought you would have something useful to talk about. All I hear is a bunch of crying about something that you could fix if you weren’t too busy looking for attention.
I seriously love your website.. Excellent colors & theme. Did you create this site yourself? Please reply back as I’m attempting to create my own personal website and would like to learn where you got this from or just what the theme is named. Thank you.
Good information. Lucky me I found your website by accident (stumbleupon). I’ve saved as a favorite for later.
there are bargain dvd players that are sold in our area. i think they are generic low cost dvd players;;
I am glad for writing to make you understand of the beneficial discovery my wife’s princess encountered checking your web site. She realized some details, including how it is like to possess a marvelous giving spirit to have the mediocre ones clearly gain knowledge of a number of tricky subject matter. You truly exceeded people’s desires. Thanks for coming up with the beneficial, trustworthy, informative and as well as easy tips on the topic to Evelyn.
You created some decent points there. I looked online for the issue and located most people is going as well as with your web site.
You really make it appear so easy together with your presentation however I find this topic to
be really one thing which I believe I would never understand.
It seems too complicated and very vast for me.
I’m looking forward in your subsequent post, I’ll attempt to get
the dangle of it!
Decent blog page! That i absolutely love the simplest way it’s easy on my own view as well as information and facts are well written. Now i’m wanting to know earn money may perhaps be warned whenever a new place has been produced. I have subscribed to your own rss feed which inturn have to the secret! Possess a good daytime!
You seem to be very professional in the way you write.*”~*~
Absolutely composed subject material , Really enjoyed reading through .
Awesome read , I am going to spend more time learning about this subject
Aw, this became a very good post. In concept I have to place in writing similar to this moreover – spending time and actual effort to have a good article… but what can I say… I procrastinate alot and also by no means find a way to go done.
Excellent read, I just passed this onto a friend who was doing a little research on that. And he just bought me lunch because I found it for him smile Therefore let me rephrase that: Thank you for lunch!
You live, you battle on, you do it for others, and you help others do so.
What sort of volunteer work do you assume can be essentially the most rewarding for you?
After looking over a handful of the articles on your web page, I seriously appreciate your technique of blogging. I book-marked it to my bookmark website list and will be checking back soon. Please visit my web site as well and tell me what you think.
Excellent article! We will be linking to this particularly great post on our website. Keep up the good writing.
The ultimate game ended on March 29, 1886 when Zukertort tendered his resignation and congratulated the brand new World Champion.
There’s definately a great deal to find out about this subject. I like all the points you made.
This is the proper weblog for anyone who wishes to be familiar with this topic. You know a whole lot its practically challenging to argue on hand (not that When i would want…HaHa). You definitely put a brand new spin on the topic thats been discussing for several years. Great stuff, just wonderful!
There is noticeably a bundle to know about this. I assume you have made certain nice points in functions also.
I can see that you are an somebody in this matter. I am beginning a site soon, and your information will be very effective for me.. Thanks for all your help and wishing you all the prosperity in your business.
Pretty! This has been an incredibly wonderful post. Thank you for providing this info.
For an area-increasing look, you can lengthen these materials to the rest of the bath or add curiosity with completely different wall therapies.
howdy fellow web master! I really like your website! I liked the make of your sidebar.
I like this site very much, Its a rattling nice place to read and get info .
I’m impressed, I must say. Really rarely must i encounter a weblog that’s both educative and entertaining, and without a doubt, you’ve got hit the nail within the head. Your thought is outstanding; the thing is something which inadequate persons are speaking intelligently about. I will be very happy i always stumbled across this during my find something about it.
After looking into a few of the blog articles on your blog, I really appreciate your way of blogging. I saved it to my bookmark site list and will be checking back in the near future. Please check out my website as well and tell me your opinion.
Wonderful post! We will be linking to this particularly great content on our website. Keep up the good writing.
This is a topic which is near to my heart… Thank you! Exactly where are your contact details though?
There are ways to avoid wasting money and nonetheless make sure the trip is comfy on your workers; a travel manager is accessible to plan all areas of your trip successfully and get you wonderful offers.
Thank your for all the enthusiasm to offer such handy information here.
Saved as a favorite, I like your site.
The next time I read a blog, Hopefully it does not disappoint me as much as this one. I mean, Yes, it was my choice to read through, nonetheless I truly believed you would have something interesting to talk about. All I hear is a bunch of moaning about something that you could possibly fix if you were not too busy searching for attention.
Excellent article! We will be linking to this great post on our website. Keep up the great writing.
After exploring a number of the blog articles on your website, I really like your technique of blogging. I added it to my bookmark website list and will be checking back soon. Please visit my web site too and tell me your opinion.
May I simply say what a comfort to find someone who genuinely knows what they are discussing on the net. You certainly understand how to bring an issue to light and make it important. A lot more people need to look at this and understand this side of the story. I was surprised you aren’t more popular given that you most certainly have the gift.
This kind of publish appears to get yourself a lot of visitors. How will you acquire traffic to that? It provides a great unique twist upon issues. I guess having something traditional or perhaps substantial to give info on is the central aspect.
I have been meaning to create about something similar to this on a single of my blogs and this provided a concept. Thanks.
You can increase your blog visitors by having a fan page on facebook.~’:~’
An outstanding share! I’ve just forwarded this onto a friend who was conducting a little research on this. And he in fact bought me lunch due to the fact that I discovered it for him… lol. So let me reword this…. Thank YOU for the meal!! But yeah, thanks for spending some time to talk about this matter here on your blog.
Hi! I could have sworn I’ve visited this web site before but after looking at some of the articles I realized it’s new to me. Regardless, I’m certainly pleased I came across it and I’ll be book-marking it and checking back frequently!
The very next time I read a blog, Hopefully it won’t fail me as much as this one. After all, Yes, it was my choice to read, but I truly thought you would have something helpful to say. All I hear is a bunch of complaining about something you could fix if you weren’t too busy seeking attention.
Hi there! This post could not be written any better! Going through this post reminds me of my previous roommate! He constantly kept talking about this. I will send this article to him. Fairly certain he’ll have a good read. Many thanks for sharing!
As I site possessor I believe the content matter here is rattling magnificent , appreciate it for your efforts. You should keep it up forever! Good Luck.
Well done! I appreciate your blog post to this matter. It has been insightful. my blog: how to get rid of love handles
i would love to sip a cup of green tea each morning because it contains L-theanine which calms the mind**
After going over a number of the articles on your site, I seriously like your way of blogging. I bookmarked it to my bookmark website list and will be checking back soon. Please check out my web site as well and tell me what you think.
Howdy! This article couldn’t be written any better! Looking through this article reminds me of my previous roommate! He continually kept preaching about this. I am going to send this post to him. Pretty sure he’ll have a great read. I appreciate you for sharing!
The new smoking alternative has become famouse in the market. It is called the Electronic Cigarettes, bring a packet to your home and than say me how much you liked it.
This is actually serious, You’re a particularly professional blogger. I have signed up with your feed furthermore watch for enjoying all of your incredible write-ups. In addition, I’ve got shared your webpage with our social networks.
Hello! I simply would like to make a enormous thumbs up for your great info you’ve got here for this post. I will be returning to your website to get more detailed soon.
You have brought up a very excellent details , thankyou for the post. “Beginnings are apt to be shadowy and so it is the beginnings of the great mother life, the sea.” by Rachel Carson.
Youre so cool! I dont suppose Ive read anything this way prior to. So nice to find somebody with a few original applying for grants this subject. realy we appreciate you beginning this up. this web site is one area that is required over the internet, somebody if we do originality. useful purpose of bringing something new to the net!
Incredible! This blog looks exactly like my old one! It’s on a entirely different subject but it has pretty much the same page layout and design. Wonderful choice of colors!
I have been reading out some of your stories and i can claim pretty nice stuff. I will surely bookmark your blog.
Aw, this is an incredibly nice post. In thought I must set up writing this way moreover – spending time and actual effort to manufacture a good article… but what / things I say… I procrastinate alot and by no indicates apparently go accomplished.
We’re a group of volunteers and opening a new scheme in our community. Your web site offered us with valuable info to work on. You have done an impressive job and our entire community will be thankful to you.
Pretty! This has been an incredibly wonderful post. Thanks for providing this information.
Your style is very unique in comparison to other people I have read stuff from. Many thanks for posting when you’ve got the opportunity, Guess I will just bookmark this site.
Way cool! Some extremely valid points! I appreciate you writing this article and also the rest of the site is also very good.
You made some decent points there. I looked on the net for the problem and discovered most individuals is going together with together with your internet site.
Nice post. I understand some thing much harder on different blogs everyday. It will always be stimulating to study content from other writers and practice a specific thing from their site. I’d would rather apply certain with all the content on my own weblog whether or not you don’t mind. Natually I’ll offer you a link in your web blog. Many thanks sharing.
I’d have to check with you here. Which isn’t something Which i do! I quite like reading a post that can make people believe. Also, thanks for allowing me to comment!
Excellent write-up. I absolutely appreciate this site. Stick with it!
You should be a part of a contest for one of the most useful sites on the net. I most certainly will recommend this blog!
The Jap Front of the European Theatre of World War II encompassed the battle in central and japanese Europe from 22 June 1941, to 9 Could 1945.
Greetings! Very useful advice in this particular article! It’s the little changes that make the most important changes. Many thanks for sharing!
Pretty! This was an incredibly wonderful post. Thanks for providing this information.
Greetings! Very useful advice in this particular post! It is the little changes that will make the largest changes. Thanks for sharing!
This blog was… how do you say it? Relevant!! Finally I’ve found something which helped me. Thank you.
Hi! I could have sworn I’ve visited this site before but after looking at some of the posts I realized it’s new to me. Anyhow, I’m certainly delighted I came across it and I’ll be bookmarking it and checking back regularly!
This is a very good tip especially to those new to the blogosphere. Simple but very accurate info… Thank you for sharing this one. A must read article.
She thinks it is malfunctioning before she is attacked by it.
You made some good points there. I looked on the net for additional information about the issue and found most people will go along with your views on this site.
Everything is very open with a precise explanation of the challenges. It was really informative. Your site is extremely helpful. Thank you for sharing.
It’s nearly impossible to find experienced people on this subject, but you sound like you know what you’re talking about! Thanks
Oh my goodness! Amazing article dude! Thank you, However I am encountering issues with your RSS. I don’t know the reason why I am unable to subscribe to it. Is there anyone else getting similar RSS problems? Anyone who knows the answer will you kindly respond? Thanx!
Excellent article! We are linking to this particularly great content on our website. Keep up the good writing.
I need to to thank you for this good read!! I definitely loved every little bit of it. I have you saved as a favorite to look at new things you post…
Hello my loved one! I wish to say that this article is amazing, nice written and come with almost all significant infos. I’d like to see extra posts like this .
Very good written article. It will be supportive to anyone who utilizes it, including me. Keep doing what you are doing – can’r wait to read more posts.
Good aftie, i am a blogger too” and i can see that you are a nice blogger too,
A motivating discussion is worth comment. I believe that you need to publish more on this subject, it might not be a taboo matter but usually people don’t talk about these subjects. To the next! Many thanks.
There is certainly a great deal to learn about this subject. I like all of the points you have made.
Spot on with this write-up, I actually believe this amazing site needs a great deal more attention. I’ll probably be back again to see more, thanks for the info!
I like your website.. great shades & motif. Performed people design and style this fabulous website yourself or even have people rely on someone else to make it work for you personally? Plz interact when I!|m looking to pattern by myself blog site plus would want to find out wherever u received this particular through. thank you
you have got a fantastic weblog here! want to make some invite posts on my small blog?
nicotine can really make you an addict, stay away from cigarettes in the first place-
I’m amazed, I have to admit. Rarely do I encounter a blog that’s equally educative and engaging, and without a doubt, you have hit the nail on the head. The problem is something which not enough folks are speaking intelligently about. I’m very happy I stumbled across this in my search for something regarding this.
Next time I read a blog, I hope that it doesn’t fail me as much as this particular one. I mean, Yes, it was my choice to read, but I actually thought you would have something interesting to talk about. All I hear is a bunch of moaning about something you could possibly fix if you were not too busy looking for attention.
I really like it when folks come together and share views. Great site, keep it up!
I blog frequently and I truly appreciate your content. This article has truly peaked my interest. I will book mark your blog and keep checking for new information about once per week. I subscribed to your RSS feed as well.
I really like gathering useful info, this post has got me even more info!
When visiting blogs, i always look for a very nice content like yours ,
Man that was very entertaining and at the same time informative..,*,`
I’d like to thank you for the efforts you have put in penning this website. I’m hoping to see the same high-grade content by you later on as well. In fact, your creative writing abilities has inspired me to get my very own blog now 😉
An fascinating discussion might be priced at comment. I’m sure that you simply write much more about this topic, it might not be a taboo subject but generally individuals are inadequate to dicuss on such topics. To another. Cheers
I must show some appreciation to you for bailing me out of this particular dilemma. As a result of looking throughout the internet and getting techniques that were not beneficial, I thought my entire life was gone. Living without the answers to the difficulties you’ve fixed as a result of your entire post is a serious case, as well as those that would have badly damaged my career if I hadn’t noticed your web blog. Your main mastery and kindness in maneuvering the whole lot was very helpful. I am not sure what I would have done if I hadn’t encountered such a stuff like this. I can at this moment look ahead to my future. Thanks a lot very much for your skilled and result oriented guide. I will not be reluctant to suggest your blog to any person who would like recommendations on this subject.
Just blog walking and want to say hi to the owner, Im enjoying reading your review/story thanks
You created some decent points there. I looked on the internet for any issue and found most people should go as well as with the internet site.
Really clean internet site , appreciate it for this post.
I really love the way you discuss this kind of topic..”\”*.
Good day! Do you know if they make any plugins to help with SEO?
I’m trying to get my website to rank for some targeted keywords
but I’m not seeing very good gains. If you know of any please share.
Thanks! I saw similar article here: Coaching
I do believe all of the concepts you have introduced in your post. They’re very convincing and will definitely work. Nonetheless, the posts are very quick for newbies. May just you please extend them a bit from next time? Thank you for the post.
Regards for just a wonderful knowledge together with in all areas of your webblog, here are some is a touch ask for an internet site subscribers. Who also quotes down the page premium? . . . .Speak out delicately and in addition have a top staff; you’ll turn out some distance.
This is a topic close to my heart cheers, do you have a RSS feed I can use?
Everything is very open with a clear description of the challenges. It was really informative. Your website is very helpful. Many thanks for sharing!
Good day! I could have sworn I’ve been to this site before but after browsing through many of the posts I realized it’s new to me. Anyhow, I’m certainly delighted I found it and I’ll be bookmarking it and checking back frequently.
Greetings! Very useful advice within this post! It’s the little changes that make the greatest changes. Many thanks for sharing!
This excellent website really has all of the info I needed about this subject and didn’t know who to ask.
This is a topic which is close to my heart… Take care! Exactly where can I find the contact details for questions?
Hola! Praising the vibe—it’s brilliant. In fact, the aesthetics brings a outstanding touch to the overall vibe. Looking forward to more!
Hello! Do you know if they make any plugins to help with SEO?
I’m trying to get my site to rank for some targeted keywords but I’m not seeing very good success.
If you know of any please share. Kudos! You can read similar blog here: Code of
destiny
One of the things I enjoy regarding reading websites such as this, is that there aren’t any spelling or lexical errors! Causes it to be tough about the readers sometimes. Very good work upon that and also the subject of this website. Many thanks!
Great web site. Plenty of useful information here. I am sending it to several buddies ans also sharing in delicious. And naturally, thanks for your sweat!
Hi there! I know this is kinda off topic however , I’d figured I’d ask. Would you be interested in trading links or maybe guest authoring a blog article or vice-versa? My site discusses a lot of the same subjects as yours and I believe we could greatly benefit from each other. If you’re interested feel free to shoot me an email. I look forward to hearing from you! Awesome blog by the way!
When I initially left a comment I appear to have clicked on the -Notify me when new comments are added- checkbox and from now on every time a comment is added I get four emails with the exact same comment. Is there an easy method you can remove me from that service? Thanks.
I do believe all the ideas you have presented for your post. They’re very convincing and can definitely work. Still, the posts are very quick for novices. Could you please lengthen them a little from next time? Thank you for the post.
If you don’t wanna see this video click on the next one…. . . It is a person who cares about another human being. about someone who been trough so much and we only ‘love’ when she is thin, soccermom, performer, miss perfect. and when she isn’t men only have hate for her. . it’s so easy to take people down when they already are vulnerable. I call it bullying
Can anyone help me out? It will be much appreciated.
You have observed very interesting details! ps decent internet site.
Simply wanna input that you have a very nice website , I enjoy the pattern it really stands out.
Howdy! I simply wish to offer you a huge thumbs up for your excellent info you have got right here on this post. I am coming back to your web site for more soon.
Bonjour! Grateful for the approach—it’s superb. In fact, the details brings a nice touch to the overall vibe. Keep it up!
Hi there! I could have sworn I’ve been to this website before but after browsing through many of the posts I realized it’s new to me. Nonetheless, I’m certainly pleased I came across it and I’ll be book-marking it and checking back often.
Good Post, I am a big believer in writing comments on websites to inform the blog writers know that they’ve added some thing worthwhile to the world wide web!
I’m curious to find out what blog platform you’re working with? I’m having some small security issues with my latest website and I’d like to find something more safe. Do you have any recommendations?
I just put the link of your blog on my Facebook Wall. very nice blog indeed..”..:
Hi there, i just thought i would publish and now let you know your sites style is really smudged within the K-Melon browser. Anyhow maintain in the very good work.
Yeah, i agree with what was explained in your blog. Thank you for all the info. and your hard work.
You really should be a part of a contest first of the most effective blogs over the internet. I am going to suggest this blog!
In the middle the talking starts and two advantages come to play for the side of the good guys, it will be a cliché sci-fi movie.
Good day! Do you use Twitter? I’d like to follow you if that would be ok. I’m undoubtedly enjoying your blog and look forward to new posts.
Hi there, I found your blog by means of Google at the same time as searching for a related matter, your website got here up, it seems to be great. I have bookmarked it in my google bookmarks.
Right here is the right web site for anybody who wishes to understand this topic. You understand so much its almost tough to argue with you (not that I actually will need to…HaHa). You certainly put a brand new spin on a topic that’s been discussed for years. Great stuff, just wonderful.
You should be a part of a contest for one of the most useful blogs on the net. I most certainly will highly recommend this web site!
I blog frequently and I genuinely appreciate your content. This article has really peaked my interest. I will take a note of your website and keep checking for new details about once per week. I subscribed to your RSS feed too.
Howdy! I just wish to offer you a huge thumbs up for your excellent information you’ve got here on this post. I am coming back to your site for more soon.
As I web-site possessor I believe the content matter here is rattling wonderful , appreciate it for your efforts. You should keep it up forever! Good Luck.
I and my buddies were analyzing the nice information found on your site while at once came up with an awful feeling I never thanked you for those techniques. Those boys were as a result glad to see them and have in effect in actuality been tapping into them. Many thanks for getting simply considerate as well as for opting for this form of impressive issues millions of individuals are really desperate to be aware of. My honest regret for not expressing appreciation to sooner.
You really should experience a tournament first of the finest blogs on the internet. I’m going to suggest this website!
Everything is very open with a very clear explanation of the issues. It was definitely informative. Your website is very useful. Thanks for sharing!
Everything is very open with a precise clarification of the issues. It was really informative. Your website is very helpful. Thanks for sharing.
Hi there! I just wish to give you a huge thumbs up for the excellent information you’ve got here on this post. I am returning to your blog for more soon.
Very good article. I will be dealing with some of these issues as well..
you got to work hard to earn lots of money because it is not very easy to earn,,
I thought it was going to be some boring old publish, but it really compensated for my time. I will publish a link to this page on my blog. I am confident my visitors will discover that quite useful
Im no professional, but I imagine you just crafted an excellent point. You naturally understand what youre speaking about, and I can truly get behind that. Thanks for staying so upfront and so honest.
Very good post. I will be experiencing a few of these issues as well..
I have to thank you for the efforts you’ve put in writing this site. I’m hoping to check out the same high-grade blog posts by you in the future as well. In fact, your creative writing abilities has motivated me to get my own site now 😉
There’s noticeably a bundle to learn about this. I assume you made sure good points in options also.
Zsa zsa Janio speck Patience pupinyo preble vainglory inhaler aguillar
Hi there, Could I grab this post photo and implement that on my own webpage?
It is in reality a great and useful piece
of info. I am satisfied that you simply shared this helpful information with us.
Please keep us up to date like this. Thank you for
sharing.
Learn all about News is very much imptortant to us.
healthier hair is of course mainly due to genetics but food supplementation can also help you get it;
Outstanding post, I think blog owners should larn a lot from this blog its really user genial .
Chile: As of 23 November 2020, Chile is formally open for tourism.
Hi, I do believe this is an excellent blog. I stumbledupon it 😉 I will return yet again since I book-marked it. Money and freedom is the greatest way to change, may you be rich and continue to help other people.
Auto index which witnessed good promoting stress in yesterday’s trade is witnessing some shopping for interest to commerce in inexperienced gaining multiple proportion points.
I’m excited to discover this great site. I need to to thank you for your time due to this fantastic read!! I definitely savored every little bit of it and I have you saved as a favorite to look at new stuff in your website.
It’s hard to come by knowledgeable people in this particular subject, but you seem like you know what you’re talking about! Thanks
Randolph Warburton, Grade 5 Officer, Selby Employment Trade, Ministry of Labour & Nationwide Service.
Howdy! This post couldn’t be written much better! Going through this article reminds me of my previous roommate! He constantly kept talking about this. I am going to forward this information to him. Pretty sure he’ll have a good read. Thank you for sharing!
The explanation I write this now is because I would like others like me, who wrestle with anorexia nervosa or different types of self-abuse, to know that that there’s always hope.
There are actually a number of particulars like that to take into consideration. That could be a great point to deliver up. I offer the ideas above as common inspiration but clearly there are questions just like the one you bring up where the most important factor can be working in sincere good faith. I don?t know if best practices have emerged round issues like that, but I am positive that your job is clearly identified as a fair game. Each boys and girls feel the influence of only a second’s pleasure, for the remainder of their lives.
I do agree with all the ideas you have offered to your post. They are very convincing and can definitely work. Still, the posts are very quick for beginners. Could you please extend them a bit from subsequent time? Thank you for the post.
I couldn’t resist commenting. Well written!
I’ve found your blog before, but I’ve never left a comment. Today, I thought to myself, “I should leave a comment.” So here’s my comment! Continue with the awesome work! I enjoy your articles and would hate to see them end.
Pretty! This was a really wonderful article. Many thanks for supplying these details.
Excellent post. I am going through some of these issues as well..
Henry Clay Alexander was an American banker who served as president, chairman, and CEO of J.P.
Make sure that to break up them in earlier than starting a very faraway trip.
The very next time I read a blog, I hope that it does not disappoint me just as much as this particular one. After all, Yes, it was my choice to read through, however I really believed you’d have something interesting to talk about. All I hear is a bunch of crying about something that you could possibly fix if you weren’t too busy seeking attention.
If the implicit curve consists of a number of elements it must be began several occasions with suitable beginning factors.
Marcia Hopkins Mackie, OBE, Chairman, Northern Eire Hospitals Authority.
The FA have also moved in direction of offering friends with a food buffet instead of the same old sit-down meal forward of kick-off.
Your style is really unique in comparison to other people I’ve read stuff from. Thank you for posting when you’ve got the opportunity, Guess I will just bookmark this page.
I like the layout of your blog and Im going to do the same for mine. Do you have any tips? Please PM ME.
Hi there! I know this is kinda off topic but I was wondering which blog platform are you using for this website? I’m getting sick and tired of WordPress because I’ve had issues with hackers and I’m looking at alternatives for another platform. I would be awesome if you could point me in the direction of a good platform.
Resources similar to the one you mentioned here is going to be really helpful to myself! I will post a hyperlink to this page on my personal blog. I am
Consequently of getting less gear and provides, alpine-style groups want to complete their climbing route in days and it’s thus thought-about a riskier form of mountaineering (e.g.
Bonjour! I love the execution—it’s remarkable. In fact, the execution brings a exceptional touch to the overall vibe. Really inspiring!
After looking into a number of the blog articles on your site, I truly appreciate your technique of writing a blog. I book-marked it to my bookmark webpage list and will be checking back soon. Please visit my website too and tell me your opinion.
Excellent post! We will be linking to this great article on our site. Keep up the good writing.
Neglect that previous wives’ tale about not coaching dogs until they’re six months old.
When you go right into a joint enterprise with another individual, you’ve got investment properties worth 200,000 and so do they, you have doubled your capability.
Bonjour! Delighted by this idea—it’s remarkable. In fact, this post brings a brilliant touch to the overall vibe. Awesome job!
I discovered your site site on the internet and check some of your early posts. Continue to keep up the top notch operate. I recently additional increase your Feed to my MSN News Reader. Seeking forward to reading much more from you finding out later on!…
you have a great blog here! do you need to cook some invite posts on my small blog?
There are some fascinating closing dates on this article but I don’t know if I see all of them middle to heart. There’s some validity however I’ll take maintain opinion until I look into it further. Good article , thanks and we want extra! Added to FeedBurner as effectively
I blog frequently and I genuinely appreciate your content. This article has really peaked my interest. I am going to take a note of your blog and keep checking for new details about once a week. I opted in for your Feed as well.
You should take part in a contest for one of the most useful blogs on the web. I most certainly will recommend this web site!
With our head office in South East London, we are well positioned to handle queries from around London and
the Home Counties.
Awsome site! I am loving it!! Will be back later to read some more. I am taking your feeds also
There are several good techniques for incorporating your danger management methods.
Generate a very boys and girls? Track down stride ritual coupons available for you small children to footwear. Save your dough. Designs, a person contribution ended up being fabulous.
An interesting discussion is definitely worth comment. There’s no doubt that that you ought to write more on this subject, it may not be a taboo matter but usually people don’t discuss such issues. To the next! Kind regards.
The COVID-19 pandemic within the United States has led to the very best variety of whole infections and deaths of any country, though per capita it isn’t the highest.
Right here is the perfect blog for everyone who hopes to find out about this topic. You realize a whole lot its almost tough to argue with you (not that I actually will need to…HaHa). You certainly put a brand new spin on a topic that’s been written about for years. Great stuff, just great.
You may select to view the calendar by day, week, month or a view that presents simply the subsequent four days.
Good site you have here.. It’s hard to find high-quality writing like yours nowadays. I truly appreciate individuals like you! Take care!!
Very good article. I’m facing a few of these issues as well..
Robert Henry Currell, Secretary and Clerk to the Governors, Northern Polytechnic and National College of Rubber Technology, London.
Hey – decent blog, just looking around some kind of blogs, seems a quite good platform you are using. I’m currently using WordPress for a few of my sites but planning to change one of them over to a platform similar to yours as a trial run. Anything in particular you’ll recommend about it?
Aw, this was an extremely good post. Spending some time and actual effort to create a top notch article… but what can I say… I hesitate a whole lot and don’t manage to get nearly anything done.
Performing arts, literary arts, visible arts.
Generally I do not read article on blogs, however I wish to say that this write-up very forced me to check out and do so! Your writing taste has been amazed me. Thanks, very great post.
As the key automobile manufacturers are showing off their finest idea and production ideas, scholar designers have presented some intriguing designs of their very own.
I had a woman on the bar inform me she “cherished my perm” as soon as, nope, this is simply my hair lol,’ one annoyed commenter wrote.
I really like reading an article that can make people think. Also, many thanks for allowing for me to comment.
This appears to be a lucrative supply of investment within the Northeast of Brazil.
An outstanding share! I have just forwarded this onto a coworker who has been conducting a little homework on this. And he actually ordered me lunch due to the fact that I discovered it for him… lol. So let me reword this…. Thanks for the meal!! But yeah, thanks for spending some time to talk about this topic here on your site.
A wardrobe change for the night reception was accompanied by a new hairstyle, with the hair being pinned back into a bun that would have taken simply minutes to create.
I’m impressed, I must say. Rarely do I come across a blog that’s both educative and entertaining, and without a doubt, you have hit the nail on the head. The problem is an issue that too few men and women are speaking intelligently about. Now i’m very happy I found this during my search for something relating to this.
This blog was… how do you say it? Relevant!! Finally I have found something which helped me. Thank you!
Great article. I’m dealing with some of these issues as well..
Oh my goodness! an excellent post dude. Thanks a ton Nevertheless I am experiencing problem with ur rss . Do not know why Cannot enroll in it. Will there be everyone getting identical rss difficulty? Anyone who knows kindly respond. Thnkx
Good post. I’m dealing with some of these issues as well..
I was able to find good advice from your content.
The very next time I read a blog, Hopefully it doesn’t fail me just as much as this one. I mean, I know it was my choice to read through, but I really believed you would probably have something helpful to talk about. All I hear is a bunch of moaning about something you could possibly fix if you were not too busy seeking attention.
This is the perfect blog for anybody who wants to understand this topic. You realize so much its almost tough to argue with you (not that I really would want to…HaHa). You definitely put a brand new spin on a subject that has been written about for years. Great stuff, just great.
This excellent website certainly has all the information I needed concerning this subject and didn’t know who to ask.
It seems solely fair that your partner, children, ferret or other legal heirs might money in your IRA.
This excellent website certainly has all the information and facts I wanted concerning this subject and didn’t know who to ask.
The very next time I read a blog, I hope that it won’t fail me as much as this one. After all, Yes, it was my choice to read, however I truly thought you would probably have something helpful to talk about. All I hear is a bunch of moaning about something that you could fix if you weren’t too busy looking for attention.
This is a great tip particularly to those new to the blogosphere. Simple but very precise info… Many thanks for sharing this one. A must read article.
Way cool! Some very valid points! I appreciate you writing this write-up and the rest of the website is also very good.
Howdy! I could have sworn I’ve been to this site before but after looking at some of the articles I realized it’s new to me. Anyhow, I’m definitely delighted I found it and I’ll be bookmarking it and checking back often!
bookmarked!!, I like your web site.
I blog often and I genuinely thank you for your content. Your article has really peaked my interest. I’m going to take a note of your blog and keep checking for new details about once a week. I opted in for your Feed as well.
Saved as a favorite, I like your web site.
This web site definitely has all the information and facts I wanted concerning this subject and didn’t know who to ask.
You should take part in a contest for one of the best websites on the internet. I am going to recommend this site!
These begin with around ₤ 500, not including
installation fees, according to Which?
You’ve made some decent points there. I looked on the web to find out more about the issue and found most people will go along with your views on this web site.
I blog quite often and I truly thank you for your content. This article has truly peaked my interest. I’m going to bookmark your blog and keep checking for new information about once a week. I subscribed to your Feed too.
Very good post. I’m facing some of these issues as well..
This is a great take on the matter, and I’ve seen a similar opinion shared on https://fabets.dev/. It’s clear that this is a well-discussed topic with valid arguments.
I blog frequently and I really appreciate your information. The article has truly peaked my interest. I am going to take a note of your site and keep checking for new information about once per week. I opted in for your Feed too.
Howdy! This blog post could not be written much better! Looking at this article reminds me of my previous roommate! He always kept talking about this. I will forward this article to him. Pretty sure he’s going to have a very good read. Many thanks for sharing!
bookmarked!!, I really like your blog.
Really enjoyed perusing this article. It’s very well-written and packed with useful information. Thanks for providing this.
Fantastic article. I found the information extremely beneficial. Adored the manner you clarified all the points.
I couldn’t refrain from commenting. Well written.
Top quality! Amazing for what I needed.
I tried it and was worth it.
Good day! I could have sworn I’ve been to this website before but after browsing through some of the posts I realized it’s new to me. Anyhow, I’m definitely pleased I discovered it and I’ll be bookmarking it and checking back frequently.
Genuinely appreciated this post. It provided plenty of useful details. Fantastic job on writing this.
Appreciated the details provided in this article. It’s so articulate and packed with helpful details. Excellent effort!
This article is amazing! Packed with valuable details and highly clear. Thanks for sharing this.
Wonderful article. It’s highly well-written and full of valuable details. Thanks for offering this post.
Fast delivery. Amazing quality.
This is wonderful! Filled with valuable details and highly articulate. Thanks for sharing this.
Hi, I do think this is an excellent blog. I stumbledupon it 😉 I’m going to revisit yet again since I book-marked it. Money and freedom is the best way to change, may you be rich and continue to guide others.
Really liked this article. It provided plenty of valuable details. Fantastic work on composing this.
Wonderful entry. It’s highly articulate and packed with useful insight. Many thanks for offering this post.
Hi, I do believe this is an excellent blog. I stumbledupon it 😉 I will revisit once again since i have bookmarked it. Money and freedom is the greatest way to change, may you be rich and continue to help other people.
Way cool! Some extremely valid points! I appreciate you writing this post and the rest of the website is very good.
After I initially left a comment I seem to have clicked on the -Notify me when new comments are added- checkbox and from now on whenever a comment is added I receive 4 emails with the same comment. Perhaps there is an easy method you are able to remove me from that service? Cheers.
Really enjoyed perusing this article. It’s highly clear and full of valuable insight. Many thanks for sharing this.
I couldn’t resist commenting. Very well written!
An impressive share! I have just forwarded this onto a colleague who had been doing a little homework on this. And he actually bought me lunch simply because I found it for him… lol. So allow me to reword this…. Thank YOU for the meal!! But yeah, thanx for spending time to discuss this matter here on your internet site.
I blog often and I genuinely thank you for your information. The article has truly peaked my interest. I am going to book mark your blog and keep checking for new information about once per week. I subscribed to your Feed as well.
This is a topic that is close to my heart… Thank you! Exactly where can I find the contact details for questions?
I would like to thank you for the efforts you have put in writing this website. I really hope to see the same high-grade content from you later on as well. In truth, your creative writing abilities has inspired me to get my own website now 😉
Hiya! Really enjoying the execution—it’s amazing. In fact, the aesthetics brings a impressive touch to the overall vibe. Way to go!
I needed to thank you for this great read!! I absolutely enjoyed every bit of it. I have got you book marked to look at new stuff you post…
Great info. Lucky me I found your site by chance (stumbleupon). I’ve book marked it for later.
I love reading through an article that will make people think. Also, thank you for allowing me to comment.
Hello there! Embracing the layout—it’s brilliant. In fact, the approach brings a charming touch to the overall vibe. You rock!
Oh my goodness! Amazing article dude! Thank you so much, However I am having troubles with your RSS. I don’t know why I cannot subscribe to it. Is there anybody having the same RSS problems? Anyone that knows the answer can you kindly respond? Thanks.
I used to be able to find good information from your articles.
Nice post. I learn something totally new and challenging on websites I stumbleupon on a daily basis. It’s always useful to read articles from other writers and practice a little something from other websites.
Excellent article! We will be linking to this great post on our site. Keep up the great writing.
After checking out a handful of the blog posts on your web site, I honestly appreciate your technique of blogging. I book marked it to my bookmark site list and will be checking back soon. Take a look at my website as well and let me know what you think.
May I just say what a comfort to find someone that really understands what they’re discussing on the internet. You certainly understand how to bring an issue to light and make it important. More and more people should check this out and understand this side of the story. I was surprised that you aren’t more popular because you definitely possess the gift.
Yo! Admiring your art—it’s fantastic. In fact, the layout brings a fantastic touch to the overall vibe. More please!
Loved the insight in this article. It’s highly well-researched and full of useful information. Excellent job!
Your style is very unique in comparison to other folks I have read stuff from. I appreciate you for posting when you have the opportunity, Guess I will just book mark this web site.
bookmarked!!, I really like your blog!
I totally agree with this comment. It’s a view that I’ve come across on Soc88, and I think it’s one worth paying attention to, given the solid arguments behind it.
Thanks for any other excellent post. Where else may just anybody get that type of information in such
an ideal approach of writing? I’ve a presentation next week, and I am at the
look for such information.
This post is highly enlightening. I really appreciated perusing it. The details is extremely arranged and simple to comprehend.
Everything is very open with a very clear description of the issues. It was truly informative. Your website is very helpful. Thanks for sharing!
I needed this today
# Harvard University: A Legacy of Excellence and Innovation
## A Brief History of Harvard University
Founded in 1636, **Harvard University** is the oldest and one of the most prestigious higher education institutions in the United States.
Located in Cambridge, Massachusetts, Harvard has built a global reputation for academic excellence, groundbreaking research, and influential alumni.
From its humble beginnings as a small college established to educate clergy, it has evolved into a world-leading
university that shapes the future across various disciplines.
## Harvard’s Impact on Education and Research
Harvard is synonymous with **innovation and intellectual leadership**.
The university boasts:
– **12 degree-granting schools**, including the renowned **Harvard Business School**,
**Harvard Law School**, and **Harvard Medical School**.
– **A faculty of world-class scholars**, many of whom are Nobel
laureates, Pulitzer Prize winners, and pioneers in their fields.
– **Cutting-edge research**, with Harvard leading initiatives in artificial intelligence, public health,
climate change, and more.
Harvard’s contribution to research is immense, with billions of dollars allocated to scientific
discoveries and technological advancements each year.
## Notable Alumni: The Leaders of Today and Tomorrow
Harvard has produced some of the **most influential figures** in history, spanning politics, business, entertainment, and science.
Among them are:
– **Barack Obama & John F. Kennedy** – Former U.S.
Presidents
– **Mark Zuckerberg & Bill Gates** – Tech visionaries (though Gates
did not graduate)
– **Natalie Portman & Matt Damon** – Hollywood icons
– **Malala Yousafzai** – Nobel Prize-winning activist
The university continues to cultivate future leaders who shape industries and drive global progress.
## Harvard’s Stunning Campus and Iconic Library
Harvard’s campus is a blend of **historical charm and
modern innovation**. With over **200 buildings**, it features:
– The **Harvard Yard**, home to the iconic **John Harvard Statue** (and the famous “three lies” legend).
– The **Widener Library**, one of the largest university libraries
in the world, housing **over 20 million volumes**.
– State-of-the-art research centers, museums, and performing arts venues.
## Harvard Traditions and Student Life
Harvard offers a **rich student experience**, blending academics
with vibrant traditions, including:
– **Housing system:** Students live in one of 12 residential houses, fostering a strong sense of community.
– **Annual Primal Scream:** A unique tradition where students de-stress by running through Harvard Yard before finals!
– **The Harvard-Yale Game:** A historic football rivalry
that unites alumni and students.
With over **450 student organizations**, Harvard students engage in a diverse range of extracurricular
activities, from entrepreneurship to performing arts.
## Harvard’s Global Influence
Beyond academics, Harvard drives change in **global policy,
economics, and technology**. The university’s research impacts
healthcare, sustainability, and artificial intelligence,
with partnerships across industries worldwide. **Harvard’s endowment**, the largest
of any university, allows it to fund scholarships, research, and public initiatives,
ensuring a legacy of impact for generations.
## Conclusion
Harvard University is more than just a school—it’s a **symbol of excellence, innovation, and leadership**.
Its **centuries-old traditions, groundbreaking discoveries, and transformative education** make it one of the most influential institutions in the world.
Whether through its distinguished alumni, pioneering research, or vibrant student life,
Harvard continues to shape the future in profound ways.
Would you like to join the ranks of Harvard’s legendary scholars?
The journey starts with a dream—and an application!
https://www.harvard.edu/
Hi! I could have sworn I’ve visited this website before but after browsing through a few of the posts I realized it’s new to me. Regardless, I’m definitely delighted I found it and I’ll be book-marking it and checking back frequently!
This is a topic that’s near to my heart… Cheers! Exactly where can I find the contact details for questions?
I like it when people come together and share views. Great blog, stick with it.
Howdy! This blog post couldn’t be written any better! Looking at this post reminds me of my previous roommate! He continually kept preaching about this. I’ll forward this information to him. Pretty sure he’ll have a good read. Many thanks for sharing!
This is gold
https://www.adobe.com/
Oh my goodness! Awesome article dude! Thanks, However I am encountering difficulties with your RSS. I don’t know the reason why I can’t join it. Is there anybody else getting similar RSS issues? Anyone who knows the solution can you kindly respond? Thanx!!
鶴の一声
shittings
灯台下暗し
I seriously love your blog.. Pleasant colors & theme. Did you develop this website yourself? Please reply back as I’m attempting to create my own personal website and want to find out where you got this from or just what the theme is called. Cheers.
Everything is very open with a very clear explanation of the challenges. It was really informative. Your site is very helpful. Many thanks for sharing!
Great post, you have pointed out some fantastic details , I too believe this s a very excellent website.
very nice publish, i certainly love this website, keep on it
I used to be able to find good information from your blog posts.
I blog frequently and I genuinely thank you for your content. Your article has really peaked my interest. I’m going to bookmark your website and keep checking for new details about once per week. I opted in for your RSS feed too.
I really like it when individuals come together and share ideas. Great website, continue the good work.
You have made some really good points there. I checked on the net to find out more about the issue and found most individuals will go along with your views on this site.
I blog frequently and I truly appreciate your content. This article has really peaked my interest. I will book mark your website and keep checking for new details about once per week. I opted in for your Feed too.
Great article. I’m going through many of these issues as well..
Hi there! This blog post couldn’t be written much better! Going through this post reminds me of my previous roommate! He continually kept talking about this. I most certainly will forward this information to him. Pretty sure he will have a great read. Many thanks for sharing!
Hi there! This post couldn’t be written any better! Looking through this post reminds me of my previous roommate! He always kept talking about this. I’ll forward this information to him. Fairly certain he’ll have a good read. Thanks for sharing!
Thanks for the write up! Also, just a heads up, your RSS feeds aren’t working. Could you take a look at that?
Oh my goodness! an amazing article dude. Thank you Nevertheless My business is experiencing trouble with ur rss . Do not know why Struggling to sign up for it. Possibly there is any person getting identical rss problem? Anyone who knows kindly respond. Thnkx
This blog was… how do you say it? Relevant!! Finally I’ve found something which helped me. Kudos!
This excellent website really has all the information I needed concerning this subject and didn’t know who to ask.
Welcome to our premier sports streaming site, your ultimate destination for live and on-demand sports content. Whether you’re into football, basketball, baseball, tennis, or esports, we bring you high-quality streams, real-time scores, and expert commentary—all in one place. Stay connected with your favorite teams and never miss a moment of the action, wherever you are.
스포츠중계
Stream Every Game, Anytime, Anywhere! Catch all the live action from your favorite sports with HD quality and zero lag. Join now and never miss a moment of the game again!
스포츠중계
Welcome to our Cryptocurrency Futures Trading Platform!
Trade a wide range of digital assets with leverage and advanced tools designed for both beginners and professionals. Enjoy secure transactions,
real-time market insights, and 24/7 support to enhance your trading experience.
This blog was… how do you say it? Relevant!! Finally I’ve found something that helped me. Appreciate it!
Pretty! This has been a really wonderful article. Thanks for providing these details.
Hi, I do think this is an excellent website. I stumbledupon it 😉 I will revisit once again since I book-marked it. Money and freedom is the best way to change, may you be rich and continue to help others.
this is a great post!
Hello there! This post couldn’t be written any better! Reading through this
post reminds me of my old room mate! He always kept chatting about this.
I will forward this post to him. Pretty sure he will
have a good read. Thank you for sharing!
Feel free to visit my blog post; discover more here
Thought-provoking read. This really made me think.
Well written. Super relevant to what I’m working on.
Thought-provoking read. This really made me think.
this is a great post!
Aw, this was an incredibly good post. Taking a few minutes and actual effort to generate a top notch article… but what can I say… I hesitate a lot and never manage to get nearly anything done.
Great post! Thanks for breaking it down so clearly.
Truly enjoyed this entry. It gave plenty of useful insights. Fantastic work on composing this.
Arrived perfectly. Great product.
Saved as a favorite, I love your site!
Funcional. Recomendo de olhos fechados.
Me surpreendeu! Produto de alta qualidade.
Magnificent beat ! I would like to apprentice at the same
time as you amend your website, how could i subscribe for a blog site?
The account aided me a appropriate deal.
I had been tiny bit acquainted of this your broadcast provided
vibrant clear concept
Here is my homepage – find more
That is a good tip especially to those fresh to the blogosphere. Short but very accurate info… Thank you for sharing this one. A must read post!
Very nice write-up. I certainly love this site. Stick with it!
Pretty! This has been an incredibly wonderful post. Thank you for supplying this information.
exodermin salbe apotheke Nagelpilz
Salbe Erfahrungen – teilt eure!
Very good post. I definitely love this website. Stick with it!
exodermin medikament Erfahrungen – was sagen echte Nutzer?
Everything is very open with a really clear clarification of the issues. It was really informative. Your website is very helpful. Many thanks for sharing.
This site definitely has all of the information and facts I needed about this subject and didn’t know who to ask.