DNA AND RNA
This classroom outline discusses deoxyribonucleic acid (DNA) and ribonucleic
acid (RNA). The narrative is arranged in an objective format and are generally
cognitive unless otherwise noted. The student at the end of this instructional
unit, is responsible for meeting these objectives by achieving a score of 70% or
better on all problem sets, case studies, major exams, quizzes, and other
assignments. It is the student’s responsibility to:
01
EXPLAIN WHY DNA MEDICALLY IMPORTANT
Deoxyribonucleic acid (DNA) is made up of nucleotides is specific sequences. From time to time mutations (faulty replication, movement, or repair) in the
base sequences causes a change in the DNA molecule. This mutation can be
transmitted to a daughter cell or if it is in a reproductive cell the offspring
will demonstrate the defect. The transmission of the defective gene cause by
this type of mutation is known as vertical transmission of inherited diseases. Viruses, ultraviolet light, ionizing radiation, and chemical can increase the
rate of mutation. This type of mutation can result in the appearance of a
disease or cancer. The transmission of a defective gene due to one of these
mutative factors is known as a horizontal transmission. If a portion of a
chromosome, such as chromosome 9, breaks away and attaches to another chromosome
(chromosome 22) the Philadelphia chromosome is formed and is known to be
associated with chronic myelogenous leukemia. When three copies of chromosome 21
comprise a cell, Down’s syndrome results. If a gene undergoes a repetitive
expansion where the repeat sequence of CAG nucleotides goes from 11 to 34 to 42
to 100, then Huntington’s disease expresses itself.
02
COMPARE THE RELATIONSHIP OF CHROMATIN, NUCLEAR
PROTEINS, AND DNA
Chromatin is a double-stranded DNA molecule that is associated with a nearly
equal mass of basic proteins called histones and a lesser amount of acidic
proteins known as nonhistone proteins and a small quantity of ribonucleic acid
(RNA). This mixture constitutes chromatin. Histones has a smaller molecular mass
than nonhistones. The nonhistone proteins may or may not be closely associated
with the chromosomes. The nonhistone proteins include enzymes associated with
the synthesis of DNA and RNA. Another nonhistone protein is high mobility group
(HMG) proteins that are associated with that segment of the chromatin that is
active in RNA synthesis.
The double stranded DNA molecule, when stretched out, greatly exceeds the
diameter of the cell. The DNA is able to condense into compact structure
with the aid of the histones. Associated with the chromatin are the
nucleosomes which are dense spherical particles about 10 nanometers (nM) in
diameter. Nucleosomes are made up of DNA wound about histone molecules.
03
DISCUSS THE HISTONES
The histones, as basic proteins, are a group of closely related molecules
consisting of five classes. The H1 type histones are loosely bound to
chromatin. This protein is considered to be an accessory protein and does not
form a major structural part of the nucleosome. It is found bound to an exterior
region of each nucleosome. Its function appears to be that of stabilizing the
solenoid structure of the chromosome and facilitating chromosome condensation in
mitosis and meiosis. H2A and H2B have a high content of the amino acid lysine
and combine to form dimers which in turn can combine into oligomeric complexes.
H3 and H4 histones are characterized by high levels of arginine. These types of histones are subject to covalent modification through acetylation, methylation,
phosphorylation, and ADP-ribosylation. Acetylation of the H3 and H4 histones
enhances transcription. The H2 histones can be modified by covalent linkages. The covalent modifications link the histones to the nuclear protein ubiquitin. The modification function is unclear. The histone molecules contain a C-terminal
end with an ordinary composition of amino acids that make up two-thirds of the
molecule. The N-terminal end with the remaining one-third of the molecule is
rich basic amino acids. The H3 and H4 histones forms tetramers of two H3 and two
H4 molecules. If two tetramers (and may be composed of H2A-H2B and H3-H4
tetramers) combine a histone octamer is formed. DNA can wrap around the histone
complexes like thread on a spool and it is protected from digestion by the DNA
nucleases.
04
DESCRIBE THE ROLE OF
NUCLEOSOMES IN CHROMATIN
Nucleosomes form the primary structural units of chromatin. Its primary function
is to condense DNA. When chromatin is extracted in a low ionic strength
solution, it will ‘unravel’ stretch out is a way that it resembles beads on a
necklace. Note the H1 histone is usually released and does not contribute to the
bead-like appearance. DNA appears as a thin fiber containing the 10 nM diameter
beads. The DNA strands between the nucleosomes are referred to as spacer DNA and
will be of about 30 base pairs but can be of variable length. As the chromosome
uncondenses, it passes through a thick fiber stage in which the nucleosomes are
packed in a spiral called a
solenoid. Note the following illustration of the
solenoid type structure.
DNA coiled around the histone protein core to form the nucleosome (expanded
view)
Solenoid-like structure (coiled view of above illustration)
A nucleosome is a disk shaped histone octamer with a segment of DNA, containing
between 160 and 200 base pairs, which will wound about two turns around the
octamer or core histone. This structure or particle is known as a nucleosome
core particle and is associated with one H1 histone molecule. This forms the
first high order structure. The octamer consists of two copies of H2A, H2B, H3,
and H4. In the solenoid form the DNA-Histone complex forms a helical secondary
structure and is a thick 30 nM diameter fiber. The helical nature of the 10 nM
fibers enables the formation of the 30 nM fiber. The solenoid, in its helical
configuration contains six to seven nucleosomes per turn which forms a
chromatosome and is the second high-ordered structure. The solenoids can be
rearranged into giant super-coiled loops which give structure to the chromosome.
This compacting into loops is a 100-fold phenomenon. Overall the DNA must be
compressed about 8,000-fold to produce a chromosome structure as observed in
metaphase.
The relationships of DNA and histones are the result of studies of chromosomes
in the metaphase interval of cell division. Chromatin fibers are found to
consist of 30,000 to 100,000 base-pair loops (also called divisions) that are
anchored to the supporting nuclear matrix of the nucleus. These loops or domains
are thought to some genetic function and will contain both coding and noncoding
regions of the gene.
05
DISCUSS CHROMATIN, ITS FUNCTIONS, AND ITS REGIONS
Chromosomes contains genetic information in the form of DNA nucleotide
sequences. Each chromosome contains a single DNA duplex (double stranded
molecule) consisting of about 1.3
× 108
nucleotides. Chromatin contains active
regions called euchromatin which is less condensed will stain less densely. The
inactive regions or heterochromatin regions stains darker. Heterochromatin is of
two types. One type, constitutive heterochromatin, always remains condensed and
is inactive. This type is located near the chromosomal centromere and at the
ends of the chromosome, the telomeres. The other, facultative heterochromatin
may remain condensed and is inactive. It can become uncondensed and be actively
transcribes. When it uncondenses it is euchromatin. It appears that the
chromosome contains three levels of chromosome activity:
[ 1 ] repressed regions (no activity),
[ 2 ] potentially active regions,
[ 3 ] active regions.
It is interesting to note that one of the two female sex chromosomes will appear
as heterochromatin and is inactive transcriptionally. In the development
of the embryo the inactivation of the X chromosome takes place. The female
has a X chromosome donated by the father (XF) and by the mother (XM).
Inactivation is a random process and about one-half of the XF chromosomes and XM
chromosomes become heterochromatin. During gametogenesis, the inactive X
chromosome becomes transcriptionally active and it is designated as facultative
heterochromatin.
Chromosomes are associated with the functions of [ 1 ] storage and transmission
of genetic information, [ 2 ] expression of genetic information, [ 3 ]
maintaining genetic information, and [ 4 ] recombination of genetic information.
06
DEFINE AND DISCUSS THE GENE
Genes are the fundamental units of heredity and occupies a specific sites (loci)
on a chromosome. They are capable of reproducing an exact duplicate at each cell
division, and also direct the formation of enzymes or other proteins. The gene
as a functional unit consists of a distinct segment of a the DNA molecule. DNA
consists of the purine (adenine and guanine) and pyrimidine (cytosine and
thymine) bases in a specific sequence to code the arrangement of amino acids of
a specific peptide. Protein synthesis is mediated by molecules of messenger-RNA
formed on the chromosome with the gene acting as template. The RNA then passes
into the cytoplasm and becomes oriented on the ribosomes. The RNA molecule the
acts as template to organize a chain of amino acids to form a peptide. Genes
normally occur in pairs in autosomal cells except gametes, a consequence that
all chromosomes are paired except the sex chromosomes (X and Y) of the male.
Examples of some types of genes are:
[ 1 ] lethal gene: produces a genotype that is not compatible
with life.
[ 2 ] modifier gene: a nonallelic gene that controls or
changes the manifestation
of a gene by interfering
with its transcription.
[ 3 ] housekeeping gene: genes that are generally always
expressed and thought to
be involved in
routine cellular metabolism.
[ 4 ] operator gene a gene with the function of activating the
production of
messenger RNA by
one or more adjacent structural loci; part of the feedback
system for
determining the rate of production of an enzyme.
[ 5 ] H gene: This is also called the histocompatibility gene.
It elicits an immune
response and
thereby cause rejection of a homograft when tissue is
transplanted from
one individual to another; in humans, histocompatibility
gene's control HLA
antigens.
The human gene does contain many genes common to a number of animals, but the
organization of the genes may not be colinear among the different animal
chromosomes. It is estimated that about 10% of the human genome is
necessary to encode the approximate 100,000 genes thought to exist. The
function of the remaining 90% of the genome is not understood. Located in the
gene are active segments of DNA that encodes for amino acids. These active
areas are called
exons. There is interspersed among the exons, sequences
of DNA that are noncontibutary to genetic information needed to synthesize an
sequence of amino acids for a protein. These apparently inactive areas of
intervening sequences are known as
introns.
07
REVIEW CHROMOSOME ORGANIZATION
The chromosome is a highly organized molecule. At the ends of each chromosome
are telomeres. Telomeres are short, repetitive DNA sequences that consist of
TTAGGG nucleotides of variable numbers that facilitate DNA synthesis and
protects the chromosomal terminal from degradation. The teleomere requires the
presence of telemerase (a RNA-dependent DNA polymerase) that can the appropriate
nucleotides to the ends of chromosomes. The centromere is a protein-DNA complex
on the chromosome and interacts with the mitotic spindle. The fibrils of the
mitotic spindle attaches to the centromere and provides the force needed to
segregate the chromosomes into their respective daughter cells. The centromere
contains long sequences of A-T nucleotides and a significant repetitive sequence
of GGAAT nucleotides.
08
DESCRIBE THE PRIMARY STRUCTURE OF DNA AND RNA
Both DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are nucleic acids
that are made up of repeating units of nucleotides. The nucleotide is made up of
three chemical moieties: a nitrogen base, a five carbon sugar, and a phosphate
group. The nucleotide bases found in DNA are two purines (adenine and guanine)
and two pyrimidines (cytosine and thymine). In a few DNA molecules, an occasional
modified nucleotide may be found, such as 5-methyl cytosine. The nucleotide base
composition in RNA are two purines (adenine and guanine) and two pyrimidines
(cytosine and uracil). RNA may contain a rare thymine nucleotide, but it is
mostly replaced by uracil. RNA is characterized by the presence of modified
nucleotides such as thiouridine, 5-methylcytidine, dihydrouridine, pseudouridine,
1-methylinosine, 7-methylguanosine, 6-methyladenosine, and 6-O-methylguanosine.
These modified nucleotides are more commonly seen in messenger RNA (mRNA). RNA differs from
DNA in its sugar which attaches the phosphate group to the nitrogen base. RNA
has a ribose where DNA contains 2'-deoxyribose. DNA exists as a double stranded
molecule but RNA is a single stranded molecule and exists in several unique
configurations. These configurations are:
[ 1 ] mRNA is the most
heterogeneous in its size and stability. There are an numerous different mRNAs
in the mammalian cell with sizes that vary from 100 to more than 2500
nucleotides in length. All members of this class function as a messenger that
carries information from the gene to ribosome for synthesizing polypeptides.
These molecules have a variable half-life ranging from a few hours up to 24
hours. All eukaryotic mRNAs have a cap at the 5' end consisting of
7-methlyguanosine triphosphate that is linked to an adjacent
2'-O-methyl-ribonucleoside at the 5-hydroxyl group through the triphosphate end
of mRNA. This cap allow the protein synthesizing machinery to recognize the
right end of the mRNA to begin protein synthesis. The 3' end consists of a tail
of 100 to 250 adenosine residues (the poly-A tail) that maintains molecular
stability. mRNA in the eukaryotic cell is always associated with proteins. Refer
to the following simplified illustration of the structural features of mRNA.
[ 2 ] rRNA (ribosomal RNA) is the
most stable of the RNAs and it exists in a complex with proteins of the
ribosome. This is a cytoplasmic nucleoprotein structure that forms the means by
which protein synthesis from mRNA templates can take place. The ribosome may be
made up of 50% RNA and 50% protein. The size of the ribosome, RNA and the other
large molecules are measured in Svedberg (S) units. The eukaryotic ribosome
consists of two subunits with a S value of 80. The ribosome is composed of two
unequal subunits, one with a S value of 40 and the other of 60. The 60S subunit
contains three RNA molecules, the first with 4800 nucleotides (28S), the second
with 160 nucleotides (5.8S), the third with 120 nucleotides (5S) and the
remainder of the subunit is made up of 50 proteins. The 40S subunit contains a
single 18S RNA with 1900 bases and 34 proteins. The 28S, 18S, and 5.8S rRNA
molecules are encoded by a 45S precursor molecule of 14,500 nucleotides.
Ribosome with its subunits provides sites for binding the proteins and mRNA.
[ 3 ] tRNA (transfer RNA) are
molecules that contain about 75 nucleotides with a molecular weight that ranges
from 25,000 to 30,000 (the S units would be from 4 to 5). There are an estimated
40 different types of tRNAs although some textbooks cite about 20. There is a
lot of diversity in the tRNA molecules but there are some common
characteristics.
A. All contain an amino acid receptor
region that terminates in a CCA sequence.
B. A TψCG tetranucleotide in the right-hand
loop which functions as a binding site
C. All contain many modified nucleotide bases.
D. The anticodon loop or arm located midway
in the molecule that positions the
tRNA in
the right sequence on the mRNA. It
contains three nucleotides
designated as
the anticodon that recognized the codon
of the template mRNA.
E. 75% of the tRNA contain an extra arm
and provides a basis for classifying the
tRNA and may be necessary for binding
to specific proteins and facilitating
codon
recognition.
F. The D arm with its 7 to 10 nucleotides
contains the nucleotide dihydrouridine.
G. The following illustration t is for an
asparagine tRNA.
[ 4 ] Small stable RNA. There is
significant number of discrete, small stable RNA entities in the eukaryotic
cell. Many exist as a ribonucleoprotein entity and may be found in the nucleus
or cytoplasm or both. They contain from 90 to 300 nucleotides and are involved
in mRNA processing and gene regulation.
09
DISCUSS DNA SYNTHESIS AND REPLICATION
There are two general functions for DNA. First, it must replicate itself
so that its information is transmitted exactly to the progeny cells. The second
function is that the information in the DNA must be expressed in a useful way.
It does this through the intermediatory role of RNA as templates to synthesize
proteins required by a living cell. The process of transferring information is
known as transcription which utilizes the four letter language of the nucleic
acids. Transcription occurs from a single stranded DNA (ssDNA) which must unwind
from its double-stranded DNA (dsDNA) structure. This produces the RNA molecule.
The flow of information from the RNA molecule to the production of the protein
molecule is called translation.
In the replication process, the two anti-parallel strands of the parent DNA
molecule must unwind and allow its two complementary strands to form the
template to synthesize a new DNA strand resulting in two new identical DNA
molecules. Each new DNA’s will contain one parent strand and one newly formed
strand. This type of replication is known as the semiconservative mechanism and
takes place in the 5' to 3' direction. As the original DNA molecule starts to
unwind at it point of replication origin, the two complimentary strands
initially appear ‘bubble-like’. As the DNA strand separates and the replication
process begins, the spreading apart of the DNA molecule (with it replication
process) is known as the replication fork. One strand of the unfolding DNA
molecule is called the leading strand and the other is the lagging strand.
Because there are no enzymes in the nucleus that can synthesize DNA in the 3' to
5' direction. As the replication process takes place in the eukaryotic
chromosomal DNA molecule, the leading strand, the duplicating process proceeds
continuously (called continuous DNA synthesis). The lagging strand is duplicated
simultaneously in fragments called Okazaki fragments and represents
semi-discontinuous DNA synthesis. An estimated 250 Okazaki fragments are
synthesized in sequence on the lagging strand at each replication fork of the
unfolding DNA molecule.
Okazaki fragments represent the relatively
short [100–1000 base pair (bp)] fragment of DNA that is later joined by DNA
ligase to allow for 3c’ —> 5c’ overall chain growth during replication.
There are several enzymes required to synthesize DNA and the same enzymes can
replicate on both strands of DNA.
[ 1 ] DNA polymerase α,
with a molecular weight of 155,000 and its three
associated subunits, is
considered to hold the more important role in DNA
replication. It is in a higher
concentration than the other polymerases. It
major function appears to be in
lagging strand synthesis. It can add
nucleotides to the 3' end of an
existing strand. One of the subunits of this
enzyme has a primase activity
to initiate the synthesis of a RNA primer. This
enzyme is bound to the inner
surface of the nuclear membrane. It is inhibited
by sulfhydryl reagents.
This may be comparable to DNA
polymerase I in the
E. coli system for DNA
replication.
[ 2 ] DNA polymerase β,
with a molecular weight of 43,000, is a small and stable
enzyme. It is an enzyme that checks
the integrity of the replicating DNA chains
and repairs mistakes. This process is
called proof reading activity. It adds the
replacement nucleotide to the 3' end.
[ 3 ] DNA polymerase γ,
with a molecular weight of 193,000 and containing
four oligomers, functions
as a DNA primer and a ribonucleotide template
for mitochondrial DNA
synthesis. This enzyme added nucleotides to the 3'
end of the developing DNA
molecule.
[ 4 ] DNA polymerase δ
(with a molecular weight of approximately 200,000) is
functional in leading
strand synthesis. Note. This may be comparable to
DNA polymerase III
in the E. coli system for DNA replication.
[ 5 ] DNA polymerase ε
(with a molecular weight of approximately 250,000) is
a DNA repair enzyme similar in
activity to that of DNA polymerase β.
This
may be comparable to
DNA polymerase II
in the E. coli system for DNA
replication.
[ 6 ] Helicases
function to denature the DNA helix by utilizing energy obtained
from ATP. These enzymes will
slide along the DNA molecule slightly in
advance of the DNA polymerases.
Note after the DNA strand is unwound,
single stranded DNA binding
proteins will prevent the DNA molecule
re-annealing to it original
double helical structure.
[ 7 ] Single-stranded DNA protein (SSB) have a high affinity for the
single stranded
DNA molecule. This will
prevent the DNA helix from reforming once the
helicase enzyme has
separated the two strands.
[ 8 ] DNA primase
synthesizes short RNA primer molecules that facilitate DNA
synthesis along the
lagging strand. These primers extend in the 5' to 3'
direction and are
utilized by DNA polymerase δ.
[ 9 ] Topoisomerases
(I and II) are involved with the unwinding of the dsDNA
molecule.
A.
Topoisomerases I
releases the torque built into the helical structure of
DNA. It induces a single strand break into the superhelix. This allows
the unbroken strand to rotate (with the enzyme attached) about the
unbroken strand
B.
Topoisomerases II
(also called
DNA gyrase) plays a role in the
unwinding of replicating DNA. This enzyme introduces what is called
negative superhelices ahead of the replicating fork which will eliminate
the torque pressure occurring in the replication of the DNA molecule.
This enzyme can cleave both strands of DNA.
[ 10 ] DNA Ligase
seals the single strand nicks on the lagging strand of DNA
between the
Okazaki fragments of DNA.
The following illustration identifies the different components at the
replication fork of an original DNA molecule.
10
BRIEFLY DISCUSS HOW RNA IS SYNTHESIZED
The synthesis of any RNA molecule is a complex process that requires DNA, RNA
polymerase enzymes, and several associated proteins. The basic steps to
synthesis of a RNA molecule are
[ 1 ] initiation, [ 2 ] elongation, and [ 3 ] termination. The DNA molecule forms the
template for synthesizing RNA and the nucleotides in the RNA molecule are
complimentary to the strand of DNA being copied with the enzymatic action of
DNA-dependent RNA polymerase. The dsDNA must unwind and the strand that is
transcribed into the RNA molecule is called the template strand. The opposite
complimentary strand of DNA is known as the coding strand because it is exactly
like the newly formed RNA molecule with the exception of where the uracil bases
replacing thymine.
There are three distinct RNA polymerases in the eukaryotic cell:
[ 1 ]
RNA polymerase I
is found in the nucleolus and synthesizes the precursors
for 5.85S, 18S, and 28S rRNA.
[ 2 ]
RNA polymerase II
is found in the nucleoplasm and synthesizes
heterogenous
nuclear RNA (hnRNA) some of which will progress into
mRNA. This enzyme will also
synthesize other nuclear RNA molecules.
[ 3 ]
RNA polymerase III
is located in the nucleoplasm where it synthesizes
tRNA
and 5S rRNA.
The mitochondria contains a mitochondrial form of RNA polymerase that
synthesizes the mitochondrial forms of rRNA, tRNA, and mRNA.
The general mechanism of RNA synthesis proceeds as follows:
[ 1 ] In the initiation stage,
RNA polymerase
recognizes the correct strand of the dsDNA molecule and binds at the DNA’s promoter site, forming an open-promoter
complex, and the DNA molecule will separate about 17 nucleotides. The attachment
site is about 35 nucleotides above where the point where the actual
transcription will take place. A topoisomerase
will attach to facilitate the
unwinding of the DNA molecule. Helicases and single strand binding proteins are
not required as in the duplication of the DNA molecule. The first nucleotide
that is attached the developing RNA molecule is usually guanine (purine) but
in some cases may be adenine (purine) that contains three phosphate residues
to mark the 5' end of the RNA molecule.
[ 2 ] The next step is to elongate the RNA molecule, the elongation stage. The
RNA
polymerase
proceeds to move along the DNA template and as the strand unwinds,
nucleotides are added that are complimentary to the template. After moving down
the DNA strand, a second
RNA polymerase
molecule may attache and synthesize a
second RNA molecule. This process may continue with additional enzymes until the
required RNA molecules have been synthesized.
[ 3 ] The last step is to terminate the synthesis process. The termination signal
is encoded in the DNA molecule. There appears to be a termination protein
designated as the rho (ρ) factor. This protein acts as a
helicase
and causes
termination of the synthesis process and release of
RNA polymerase
and the RNA
molecule.
Comment. The synthesis of RNA is better understood in the prokaryotic cells than
in the eukaryotic cells.
The newly formed RNA molecule is larger than its
product. There is a post-transcriptional process in which nucleotides can be
removed, added, or modified to obtain the final product.
11
BRIEFLY DISCUSS THE FOLLOWING TERMS ASSOCIATED WITH DNA AND RNA
Activator: A transcription factor that stimulates transcription initiation
rates.
Anticodon: A unit of three nucleotides in the tRNA that is complimentary to
corresponding three nucleotides (called a codon) in the mRNA.
A-DNA: Is an right-handed helical configuration of base-paired duplex (double
stranded) DNA and is a more compact structure than B-DNA. It is characterized by
eleven base-pairs per turn. Because of the variation in its grooves (nearly
equal in depth) and more compact form, this type of DNA is less likely to enter
into protein-DNA interactions.
Allele: Any one of a series of two
ormore different genes that occupy the same locus on a specific chromosome.
If the same allele occupies the sam locus on the paired chromosomes, it is
homozygous. It is heterozygous when the alleles are different. A
capital letter usually indicates a dominant allele and a lower case letter if it
is recessive.
B-DNA: Is the predominate structural form of base-paired duplex DNA found in
cells. It has a right-handed helical configuration with a 3-dimensional
configuration that differs from A-DNA and Z-DNA. It is characterized by ten base
pairs per turn. The B-DNA contains a major deep groove and minor groove with the
major groove running next to the nitrogen and oxygen atoms of the nucleotide
bases. Proteins are able to inscribe into these grooves and from a direct
contact with specific nucleotide sequences.
Chromatin: Comprised primarily of DNA, it is complexed to proteins made up of
histones and other non-histone proteins.
Chromosome: That part of the cell nuclear material containing genetic material.
Codon: Three consecutive nucleotides in the mRNA that is complimentary to the tRNA codon (called an anticodon). Each codon specifies an amino acid during
protein synthesis.
DNA: It is a polymer of deoxyribonucleotides known as deoxyribonucleic acid. It
contains the genetic information of the cell.
Elongation: It is the lengthening of a macromolecule, in this case the addition
of nucleotides to form a DNA or RNA molecule.
Eukaryotic: Refers to a cell with a separate nucleus that contains almost all of
the cellular DNA. Eukaryotic cells are seen in yeasts and all higher organisms,
including mammals.
Exon: A portion of a DNA that codes for a section of the mature messenger RNA
from that coding part of the DNA, and is therefore expressed or translated into
protein at the ribosome.
Gene: An encoded sequence of DNA that determines an inherited trait.
Histones: Small basic proteins who function appears to be that of organizing the
DNA of chromosomes into nucleosomes.
hnRNA: Designated as heterogenous nuclear RNA, these are very large molecules of
heterogeneous size found in the nucleus. This is the primary RNA transcript of a
gene. With its high molecular weight, it never leaves the nucleus and is thought
to be the precursor of messenger RNA.
Initiation: It is the starting point for the replicating synthesis of
macromolecules on the DNA or RNA strand.
Introns: A non-coding portion of DNA that lies between two exons, is transcribed
into RNA, but does not appear in that RNA after maturation, and so is not
expressed (as protein) in protein synthesis. It is also known as an intervening
sequence.
Karyotype: A visual presentation of chromosomes (in the metaphase of cell
division) that make up the chromosomal composition of an individual. There are
44 (22 pairs) autosomes and two (one pair) sex chromosomes.
kb: An acronym that means kilobase pairs. It is made up of 10,000 nucleotides.
Lagging strand: That template part of the DNA molecule which is being copied
(replicated) in a discontinuous manner during DNA synthesis in small segments in
a 3' to 5' direction.
Leading strand: The template part of the DNA molecule that is being copied
(replicated) in a continuous manner in a 5' to 3' direction during DNA
synthesis.
mRNA: Messenger ribonucleic acid that forms the RNA transcript template for
protein synthesis in eukaryote cells.
Muton: The smallest chromosome
segment or unit that can undergo mutation.
Nucleoside: Comprised of a sugar (ribose or deoxyribose) that is connected to a
nitrogen base, such as adenine or cytosine.
Nucleosome: A repetitive subunit structure in chromosomes.
Nucleotide: A molecule comprised of a sugar (ribose or deoxyribose) attached to
one to three phosphate groups and a nitrogen base (such as adenine or cytosine).
Okazaki fragment: A relatively short (100–1000 bp) fragment of DNA generated
through discontinuous replication that is later joined by DNA ligase to allow
for 3' —> 5' overall chain growth of the lagging strand during replication.
Plasmid: A circular DNA molecule and functions alongside a chromosomal DNA and
provides an extra source of genetic material.
Polymerase chain reaction: Designated as PCR, this is a method that selects and
amplifies a specific DNA sequence when a mixture of DNA sequences are available.
Probe: A device or agent used to detect or explore a substance; e.g., a molecule
used to detect the presence of a specific fragment or sequence of DNA or RNA in
a mixture of sequences.
Promoter: This is the recognition site where the RNA polymerase will bind to the
DNA strand and is about 35 nucleotides distant from where transcription will
actually begin.
Pseudogene: A gene segment that is none functional.
Recombination: the presence of combinations of genotypes and perhaps phenotypes,
not present in either parent that results from crossing-over. It is an exchange
of genetic information between chromosomes occurring during meiosis.
Replication fork: Region of replication where the DNA is unwinding and synthesis
is bidirectional with one strand designated as the leading strand and the other
as the lagging strand.
Replicon: A replication unit. It is a segment of a chromosome (or of the DNA of
a chromosome or similar entity) that can replicate, with its own initiation and
termination codons, independently of the chromosome in which it may be located.
Ribosome: A large ribonucleoprotein complex that is essential for protein
synthesis in cells.
snRNA: Designated as small nuclear RNA, these ribonucleoprotein particles have
sizes ranging from 100 to 300 nucleotides and contain a significant number of
uracil bases. They are though to function in the maturation of tRNA and mRNA.
Termination: This is the cessation of the elongation process. Synthesis stops
and the macromolecule is released.
Transcription: Transfer of genetic code information from one kind of nucleic
acid to another, especially with reference to the process by which a base
sequence of messenger RNA is synthesized (by an RNA polymerase) on a template of
complementary DNA.
Transcription unit: The section of the DNA or RNA molecule that forms the
template beginning at the point where the polymerase enzyme attaches and extends
to the point where the transcription stops.
Z-DNA: This is a left-handed helix containing twelve nucleotides per turn. This
form is stabilized by the methylation of its bases and has been found to occur
in inactive genes.
12
DESCRIBE HOW DNA MOLECULE CAN BE DAMAGED AND AN APPROPRIATE REPAIR MECHANISM
DNA damage can be corrected through three general mechanisms.
1. One mechanism is a where a mismatched or misincorporated nucleotide that can
be repaired by the proof reading activity of
DNA polymerases. It is possible
that a cytosine could be inserted opposite an adenine. If the polymerase slips,
then an extra nucleotide or two can be inserted. Specific proteins can read the
new synthesized DNA molecule and if a mismatch is found, then a specific endonuclease
can ‘cut’ the faulty sequence out. Cellular enzymes (such as ligase,
polymerase, or single strand binding (SSB) proteins) can remove and replace the
strand.
2. Another process is base excision occurs when a single base is damaged. An
example is the depurination of DNA which spontaneously happens due the
thermoliability of the purine N-glycosidic bond and the nitrogen base is lost.
Specific enzymes can recognize the defective site and will replace the
appropriate purine directly. Cytosine and adenine then to spontaneously deaminate and form uracil and hypoxanthine respectfully.
N-glycosylases
can
recognize the deaminated nitrogen bases and remove the base from the DNA leaving
a defect in the chain. Apurinic
or
apyrimidinic endonuclease
can detect the
chain defect to remove the abasic sugar. The proper base is then replaced with a
repair
DNA polymerase
and a ligase.
3. The third corrective mechanism is nucleotide excision repair. This is a
repair mechanism that allows for the replacement of up to thirty bases in a
damaged DNA molecule. DNA damage can be the result of ultra-violet light
exposure which causes the formation of cyclobutane pyrimidine-pyrimidine dimers. Smoking cause formation of benzo[α]pyrene-guanine adducts (the addition of a
product to the nucleotide). Another cause of DNA damage comes via radiation,
chemotherapy, or industrial chemicals. These can cause base modification, strand
breaks, cross-linking between opposite strands, or cross-linking between the DNA
and a protein. To repair this type of defect requires that the phosphodiester
bonds on the defective strand be hydrolyzed followed by a special excision
nuclease (also called an exinuclease) remove the defective strand. A special
polymerase will replace the missing base pairs and a
DNA ligase
will join the
existing strands.
13
DESCRIBE HOW DNA SYNTHESIS OCCURS DURING THE CELL CYCLE
There is specified time during the mitotic phase of mammalian cell that divides
on a regular basis. A mammalian cell takes about 24 hours to go through the
mitotic cycle. The stage in which the cells is resting or mitotically inactive
is the GO phase. Note: ‘G’ means gap. Once the cell is stimulated to begin
mitosis, it enters the G1 phase where is will persist for about twelve hours as
the production of cyclins increases. Cyclins are proteins which activate other
enzymes necessary for the progression to the S phase and duplication of nucleus
and cytoplasm. It is in the G1 phase that active complexes are formed and
substrates assembled. The cell then enters the S phase where the synthesis of
DNA is initiated and rapid protein synthesis takes place. In this phase is found
the largest quantities of DNA polymerase as well as substrates for DNA
synthesis. In the S phase nuclear DNA is duplicated only once forming a doubled
amount of DNA. DNA is unable to replicate again. The mitotically active cell
will persist in this phase for about six to eight hours. The cell then passes
into the G2 phase which represents a separation between the cessation of DNA
synthesis and actual mitosis. In this phase the cell may continue RNA synthesis. The cell is preparing for the formation of two daughter cells with supplementing
of cytoplasm and cytoplasmic structures. The last phase, the M or ‘mitosis’
interval, the cell divides, giving each of the daughter cells equal amounts of
DNA, RNA, nuclear material, cytoplasm, and cytoplasmic organelles.
14 LIST
AND BRIEFLY DESCRIBE COMPOUNDS THAT CAN INHIBIT DNA AND RNA SYNTHESIS
Cancer cells replicate DNA and do other things that cells do but on a more rapid
scale. These abnormal cells may be nonfunctional or very active producing
products that are detrimental to the well-being of the organism. Because of this
behavior, many cancer drugs are synthesized to target either DNA or RNA
synthesis. Since cancer cells have a greater metabolic activity, they will
utilize more substrate and therapy strategies use this phenomenon to treat
cancerous disorders. The cancer cells will take up more of the drugs than normal
cells.
An alkylating agent is one that highly reactive in a physiological condition and
will donate an alkyl or carbonium ion to a biologically important macromolecule. The reaction involves the guanine molecule and it will also cross link it to
another guanine molecule on the opposite strand of DNA. DNA, RNA, and proteins
can undergo cross-linking. This reaction will inactive the molecule and cause
cell division to cease. It cytotoxic activity affects both abnormal and normal
cells. These agents can be both carcinogenic and mutagenic.
[ 1 ]
Doxorubicin
alters DNA structure and function by intercalating between the
bases in the DNA molecule. This will untwist the molecule and render it
nonfunctional. This drug has been used to treat leukemias, breast cancers,
ovarian carcinoma, and cancer of the GI tract.
[ 2 ]
Hydroxyurea
is more effective against DNA synthesis than against RNA. It
inhibits the incorporation of thymidine into DNA. It is used to treat melanoma,
chronic granulocytic leukemia, ovarian carcinoma, and squamous cell cancers of
the head and neck.
[ 3 ]
Uracil mustard
is an alkylating and cross-linking agent related to the
nitrogen mustard gas of World War I. It is used to treat chronic lymphocytic
leukemia, Hodgkin’s disease, chronic myelogenous leukemia, lymphosarcoma,
lymphoblastoma, reticulum cell sarcoma, ovarian carcinoma, mycosis fungoides.
[ 4 ]
Cyclophosphamide
is also an alkylating and cross linking agent and is
related to the nitrogen mustard gases. It is used as a therapy strategy for
disorders such as multiple myeloma, Hodgkin’s disease, Burkitt’s lymphoma,
monocytic leukemia, acute lymphoblastic leukemia, neuroblastoma, and
adenocarcinoma of the ovary.
[ 5 ]
Cysplatin
functions as an alkylating agent and interfers with DNA
replication by forming complexes with DNA. It does alkylate the guanine
structure and disrupts hydrogen bonding. It is used to treat solid tumors.
Specific interventions include testicular tumors, bladder cancer, and ovarian
cancer.
[ 6 ]
Etoposide
is a semisynthetic derivative of podophyllotoxin and acts as a
mitotic inhibitor at the G2 phase of the cell cycle. It inhibits
topoisomerase
II
which prevents unwinding of DNA coils preventing replication. It has been
used to treat refractory testicular cancers.
[ 7 ]
CeeNU
(Lomustine)
chemical name is 1-(2-chloro-ethyl)-3-cyclehexyl-1-mitrosourea. It
is used to treat certain neoplastic diseases such as brain tumors and Hodgkin's
disease. Its mode of action is to alkylate DNA and RNA. It will also
inhibit a number of enzyme processes by its carbamoylation (transfer of the
radical NH2―CO―) of the amino acids in the proteins.
This is a very toxic compound and can produce adverse side effects.
Bas-line blood and pulmonary studies should be performed prior to administering
this drug. CeeNU can cause bone marrow suppression, liver disfunction, and it is
a renal toxin. It can also cause stomatitis, alopecia, optic atrophy, and
visual disturbances. It has carcinogenic and mutagenic properties.
It can impair fertility. It is administered as a single oral dose every
six weeks.
NOTE: For more informatiom about these toxic drugs / medications, refer to
a PDR manual.
Note... Antineoplastic agents are toxic and dangerous to use. Because they are
cell poisons, they also inhibit normal cells. Because the neoplastic cell is
much more active, it theoretically takes up more of the antineoplastic agent and
leaving the normal cells less affected. Patients who receive antineoplastic
agents must be closely monitored because of the various side effects produced. Such effects can be minimal or extremely severe and life threatening. The dosage
of antineoplastic medications are critical, being just enough to destroy the
cancer cell, but having a minimal lethal effect upon the normal cells. Bone
marrow and the gastrointestinal mucosal cells are most sensitive to these toxic
agents because of their high rate of metabolic activity. Patients are monitored
by looking for signs of bone marrow depression. This means that blood counts
will be conducted, sometimes as often as twice a week. If the blood test
indicates a precipitous fall in the WBC count (below 2000/μL) or platelet count
(below 100,000/μL), then the treatment may have to be discontinued. Other signs
that the physician monitors are:
[1] Developing allergic reaction to the drug.
[2] Untoward reactions that include:
A. Gastrointestinal signs: nausea, vomiting, anorexia, diarrhea, stomatitis,
enteritis, abdominal cramps, and intestinal ulcers.
B. Hepatic signs: jaundice (liver toxicity) and changes in liver enzymes levels.
C. Dermatological: dermatitis, erythema, alopecia, pruritus, and urticaria
(itching wheals).
D. Nervous system: depression, lethargy, confusion, dizziness, headache,
fatigue, fever, malaise (feeling of discomfort and uneasiness), and weakness.
E. Urogenital: renal failure, amenorrhea, and azoospermia.
15
SUMMARIZE THE GENETIC CODE
The genetic code is a triplet of nucleotide sequences of DNA that has been
transcribed into a specific nucleotide sequence of an RNA molecule that reads in
the 5' to 3' direction. There are 64 words that may be referred to as codons.
The genetic code as assigned to the nRNA are given in Table 1. A linear array of codons on a DNA strand that specifies the synthesis of various RNA molecules
constitutes a gene. The genetic code is describes as being:
[ 1 ]
degenerate: there are multiple codons that code for the same amino acid
[ 2 ]
unambiguous: a given codon designates a specific amino acid
[ 3 ]
nonoverlapping: codons do not overlap when molecules are being synthesized.
[ 4 ]
without punctuation: the message in the molecule is read in a continuous
sequence
[ 5 ] universal: means that the same code words are utilized in all living
organisms.
It has been found that this is really not the case. It has been found
that there is
variation with species and between different tissues in a species.
TABLE 1. Genetic Code
first second third
nucleotide nucleotide nucleotide
-----------------------------------------------------------------------------------------
U C A G
------------------------------------------------------------------------------------------
Phe Ser Tyr Cys U
U Phe Ser Tyr Cys C
Leu Ser Term Term1 A
Leu Ser Term Trp G
-----------------------------------------------------------------------------------------
Leu Pro His Arg U
C Leu Pro His Arg C
Leu Pro Gln Arg A
Leu Pro Gln Arg G
-----------------------------------------------------------------------------------------
Ile Thr Asn Ser U
A Ile Thr Asn Ser C
Ile2 Thr Lys Arg3 A
Met4 Thr Lys Arg5 G
-----------------------------------------------------------------------------------------
Val Ala Asp Gly U
G Val Ala Asp Gly C
Val Ala Glu Gly A
Val Ala Glu Gly G
------------------------------------------------------------------------------------------
U = Uridine Nucleotide C = Cytosine Nucleotide
A = Adenine Nucleotide G = Guanine Nucleotide
1 = UGA in the mammalian cells is also a codon for Trp
2 = AUA also codes for Met
3 = AGA will also serve as a chain terminator
4 = AUG which codes for Met will also serve as an initiator codon in the
mammalian cell
5 = AGG in the mammalian cells also serves as a chain terminator
NOTE: Termination codons are also
nonsense codons. These nonsense codons (UAG, UGA, UAA) are designated as
'stop' codons.
Only sixty one of the codons specify an amino acid. The other three codons that
do not code for any specific amino acid have been designated as nonsense codons. Two of these codons are used as termination signals. Examination of Table 1
suggests that for several of the condons the two more important members of the
codon are the first two nucleotides. If you look at the codon that begins with CU
then the third codon does not contribute to the determination of the amino acid
leucine. The same is true for serine, proline, threonine, valine, alanine, and
glycine. This relative nonspecificity of the third nucleotide of the codon is
commonly called wobble which helps to explain why one anticodon of tRNA can be
complimentary to more than one mRNA codon. Wobble seems to mean that the first
two nucleotide pairs are important to the positioning of the anticodon and codon.
It is used to explain why the tRNA molecule can allow its third nucleotide to be
a inosine, guanine, or uracil nucleotide in the anticodon.
Nitrogen base substitutions can occur which results in mutations. If a single
base changes, then it is a point mutation. If one purine replaces another purine
(for example cytosine for thymine) then it is a transitional mutation. It is
also applicable when one pyrimidine replaces another pyrimidine. The exchange
between a purine and pyrimidine or it reverse, then it is a transversional
mutation. If these types of mutations do not cause any change in the protein
product it is called a silent mutation. If you look at Table 1 and if a GUU
codon changes to GUC, the same amino acid (valine) is used to produce the
protein. If the change were from GUU to GAC, then the amino acid used to produce
the protein is alanine. This is designated as a missense mutation. If valine and
alanine are similar enough in their function in the protein, then the protein
will most likely be functional. If the change is from GUU to GAU, then if
aspartate may produce enough modification in the protein to render it
nonfunctional. If the mutation involves creating a terminal codon (such as UCA
for serine mutating to UAA) then the protein synthesis will stop and the protein
will be nonfunctional.
Frame shift mutations occurs when a single nucleotide inserts into the reading
frame of codons and causes the remainder of the message to be scrambled.
Consider the following example of a nucleotide sequence for a given set of
frames.
AGG/GCU/UAC/UCA/AGA
(arg) (ala) (tyr) (ser) (arg)
If a uracil nucleotide (U) is inserted as shown, the message will be changed
AGG/GCU/UAU/CUC/AAG/A
(arg) (ala) (tyr) (leu) (lys)
To correct this mutation defect requires that the added uracil nucleotide be
removed from the gene.
Note, if one base is deleted it will have a similar effect and it also true for
the addition or deletion of two bases.
16
BRIEFLY DESCRIBE HOW PROTEIN SYNTHESIS OCCURS
Protein synthesis, also called protein translation can be divided into four
stages. The first stage is the initiation stage requiring rRNA, tRNA, mRNA, GTP,
ATP, amino acids, and a minimum of ten eukaryotic initiation factors (eIF-#).
The initiation stage requires the separation of the 80S ribosome into its 40S
and 60S subunits.
[ 1 ] Initiation factor eIF-3A binds to the 60S subunit and may prevent reassociation.
[
2 ] eIF-3 and eIF-1A binds to the 40S subunit to prevent reassociation of
subunits.
[ 3 ] The 40S preinitiation complex continues to develop by the binding of GTP
with eIF-2 to form a binary complex which will combine with met-tRNA to form a
ternary complex. This will bind the met-tRNA to the initiating codon (AUG).
[ 4 ] The ternary complex is completed when it consists of 40S subunit, eIF-2
bound to Met-tRNA with GTP, eIF-3, eIF-4c and other protein factors (not yet
determined). At this stage it can combine with mRNA to form the preinitiation
complex.
[ 5 ] A methyl-guanosyl triphosphate molecule (cap binding protein) is attached to
the 5' terminal of the mRNA. This activity is called capping and will facilitate
the binding of mRNA to the 40S complex as part of the initiation process. ATP,
eIF-4A and eIF-4B, and CBP (cap binding protein) are required to correctly
orient the mRNA molecule. eIF-4A and eIF-4B binds to the methyl-guanosyl
molecule. The complex is now ready to start the synthesis process at the
initiation codon (usually AUG).
[ 6 ] With the correct orientation of the capped mRNA, the 60S subunit will
reorient itself to the 40S subunit with the help of eIF-5. GTP is hydrolyzed to
GDP and Pi, eIF-2, eIF-1A and eIF-3 are released from the complex and a 80S
ribosome complex is formed that is ready to begin protein synthesis.
[ 7 ] Refer to
the following illustration, depicting the initiation of the synthesis of
proteins.
The next step in protein synthesis is the elongation step. This is a multistep
process that requires several elongation factors (eEF). This process can be
simplified to three steps:
1. Binding of the aminoacyl-tRNA.
A. When the 40S subunit and 60S subunit combine to form the 80S unit, the
initiating Met-tRNA is positioned in the P site (peptidyl site). The adjacent
codon to receive the incoming tRNA with its amino acid is designated as
the A
site (aminoacyl site). There is a third site adjacent to the P site, the E
site
(exit site). Located above the P and A sites in the 60S subunit is the
peptidyl
transferase enzyme that catalyzes the formation of a peptide bond
between the
amino acids positioned in the P and A sites.
2. Forming the peptide bond.
A. The incoming amino acid-tRNA is a ternary complex with GTP and
eEF-1α will
bind to the A site if the codon-anticodon interactions are
correct. The incoming
ternary complex will position adjacent to the P site
and with the amino acids of
each tRNA’s positioned together and the
peptidyl transferase facilitates the
peptide bond and forms a dipeptidyl-
tRNA. The energy released by the splitting
of a phosphate group from the
GTP nucleotide juxtapositions the tRNA on the
ribosome and the eEF-1α
dissociates.
B. The next step is to reposition the mRNA and the two tRNA’s to [1] eliminate
the initiator tRNA and [2] allow another elongation cycle to take place. A
translocase enzyme (eEF-2) in conjunction with GTP will cause mRNA to
shift one codon unit and these things take place:
a. The initiator tRNA is moved into the E site and the dipeptidyl-tRNA is
relocated in the P site.
b. The A site is now empty and ready to receive another incoming ternary
complex
of amino acid-tRNA, GTP, and eEF-1α.
c. The peptidyl transferase site remains intact and is ready to initiate the
formation of a peptide bond when the dipeptide-tRNA and amino
acid-tRNA are
juxtapositioned on the ribosome.
3. Removal of the tRNA from the P site and causing the mRNA to shift. This is
known as translocation.
A. The positional changes with the mRNA and movement of the tRNA’s from
the P
and A sites provide the energy to move any tRNA’s from the E site
into the
cytoplasm to combine again with their respective amino acid and
to possible
re-enter the elongation cycle.
4. Refer to the following
illustration depicting the elongation of the polypeptide chain.
Successive cycles of ternary complexes of amino acid-tRNA, GTP, and eEF-1α with
positioning into the A site followed with formation of the peptide bone then the
activity of GTP and eEF-2 to cause the mRNA shift and relocation of the tRNA’s
creates and elongated polypeptide.
The final step in the synthesis of the protein chain is termination. This takes
place when one of the terminating codons (UUA, UAG, or UGA) is recognized in the
A site. This nonsense codon prevents any amino acid-tRNA’s from binding and
stops the polymerization of the protein molecule. A releasing factor (a
non-ribosomal protein) in a complex with GTP will bind to the ribosome. In
conjunction with peptidyl transferase, hydrolysis of the peptide bond between
tRNA and the new protein is cleaved by the addition of a water molecule
releasing the protein. This also releases the tRNA from the P site and the 80S
ribosome dissociated into its 40S and 60S subunits. The ribosomal subunits are
recycled. T he GTP is hydrolyzed to GDP and Pi which allows for the releasing
protein dissociate from the ribosome and nucleotide. Refer to the
following illustration depicting the termination
of protein synthesis.
17
LIST SOME COMPOUNDS THAT INHIBIT PROTEIN SYNTHESIS
AND BRIEFLY DESCRIBE THEIR MODE OF ACTION
Streptomycin. This antibiotic will physically bind to the 30S subunit of
ribosome of the procaryote and block the initiation of protein synthesis and
stop the elongation process. It will also cause the misreading of mRNA
information. Streptomycin will be augmented by protein S12 and allow interference
with tRNA’s.
Neomycin. This is a aminoglycoside antibiotic. It interacts with the 30S subunit
but at a site different than streptomycin and cause mistranslations of the mRNA
messages.
Tetracycline. This molecule binds directly to the ribosome and prevents the
binding of aminoacyl-tRNA’s preventing elongation of the protein chain.
Puromycin. This molecule resembles an aminoacyl-tRNA and when it binds the
ribosomal A site where it terminates protein synthesis.
Erythromycin. This is a macrolide antibiotic and interferes with the
translocation sequence in the elongation process.
Ricin. A plant toxin from the castor bean, this N-glycosidase molecule cleaves
an adenosine from the RNA molecule which inactivates it.
