CYTOGENETICS – BIOCHEMICAL BASIS OF HEREDITY

DR. TULIN | SUMMER TERM | BSMT – 1J

THE GENETIC MATERIAL CENTRAL DOGMA OF MOLECULAR BIOLOGY

● Four major characteristics:
○ Replication
○ Storage of Information
○ Expression of Information
○ Variation by mutation
REPLICATION
● One of the facets of the cell cycle/one of
the fundamental properties of all living
organism.
● Meiosis
○ Once genetic material of cells is
replicated, it doubles in amount,
thereby, it must be partitioned
equally into daughter cells so that
in the formation of gametes, this
partition are now haploid cells of
gametes, will join together to • How genetic information is transmitted:
constitute the complete form. o DNA replication → RNA
transcription →Protein translation
STORAGE OF INFORMATION • 3 types of RNA that are synthesized:
• Requires that the molecule will act as a o Messenger RNA (mRNA)
repository of genetic information that o Ribosomal RNA (rRNA)
may or may not be expressed by the cell o Transfer RNA (rRNA)
in which it resides. • Each RNA is a product of a specific gene
that directs the synthesis of a protein
VARIATION BY MUTATION • In translation, the chemical information
• The genetic material is also the source of found in the mRNA directs the
variation among organisms through the construction of different amino acids
process of mutation or a change in the that make up the protein
chemical composition of DNA. • The amino acids collectively when they
• Once this occurs, it may be reflected form a chain, are called polypeptides
during the translation into the specific which then subsequently fold into the
protein it will produce. The mutated gene protein
or DNA becomes an altered protein.
• If such mutation is present in gametes, it NUCLEIC ACID CHEMISTRY
may be present in the future generation • Nucleic acids are nucleotides
after fertilization. With time, that single o Nucleotides are building blocks of
mutation from a cell could be distributed nucleic acids
to the entire population. • Three essential components:
Nitrogenous ● Purine (9-membered
EXPRESSION OF INFORMATION Base ring)
• The basis of the process of information o Adenine
flow within the cell o Guanine

TRANSCRIBED BY: JHULIA CABAÑESAS 1

LECTURE #3 | CYTOGENETICS – BIOCHEMICAL BASIS OF HEREDITY

● Pyrimidine (6- o +2 phosphate groups

membered ring) ▪ Nucleoside triphosphate
o Cytosine (NTP) / adenosine
o Uracil – only triphosphate (ATP)
found in RNA
o Thymine
Pentose (5 ● Ribonucleic acid (RNA)
carbons) o Ribose
● Deoxyribonucleic acid
(DNA)
o 2-deoxyribose
Phosphate ●
Group
• Nucleoside: • Phosphodiester bond – formed between
o N base + pentose sugar two mononucleotides by linking the
• Nucleotide: phosphate groups
o N base + pentose sugar + • Each structure has a (C-3) end and a
phosphate group (C-5) end
• Nucleotide bonding: o C-3 end – hydroxyl group
o C1 – N base o C-5 end – phosphate group
o C2, C3, C5 – phosphate group • Terminologies:
▪ C5 most common o Dinucleotide – 2 nucleotides
o Trinucleotide – 3 nucleotides
o Oligonucleotides - <20
nucleotides
o Polynucleotides - >20 nucleotides

DEOXYRIBONUCLEIC ACID (DNA)

• Fully extended eukaryotic molecule = 3m
per genome
• Genetic information of an organism
• Packages in chromatin using histones
and organized into chromosomes
• Located in the nucleus in eukaryotes and
in the nucleoid region of the cytosol in
prokaryotes
• Each human cell contains 2 sets of 23
chromosomes which constitute the
• Nucleotides are also described by the human genome
term “Nucleoside monophosphate” • DNA is a double helix structure because
o +1 phosphate group the bases need to pair with one another.
▪ Deoxynucleoside Unpaired DNA is unstable.
diphosphate (dDNP) /
Deoxythymidine DNA is packaged into chromatin
diphosphate (dTDP) using histones, giving it a name
"beads on a string" appearance

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LECTURE #3 | CYTOGENETICS – BIOCHEMICAL BASIS OF HEREDITY

ORGANIZATION OF EUKARYOTIC CHROMOSOMES Stability Stable Subject to base

Degraded by hydrolysis
DNase Degraded by
RNase
Function Maintains Carries genetic
genetic information to
information in cytoplasm
nucleus

NUCLEIC ACID ENZYMES AND ASSOCIATED

FUNCTIONS
POLYMERASES
• DNA polymerases
• RNA polymerases
• IN VIVO FUNCTION:
o Polymerases join DNA or RNA
nucleotides together to form a
single-stranded daughter
molecule using a stretch of
single-stranded parent molecule
as a template. These enzymes
perform syntheses according to
RIBONUCLEIC ACID (RNA) base pair rules and proceed in
• Single stranded molecule the 5’ to 3’ direction.
• Ribose sugar containing a hydroxyl group
at the 2’ position REVERSE TRANSCRIPTASE
• Thymine is replaced by the methylated
● IN VIVO FUNCTION:
uracil
○ Mostly of viral origin, reverse
• Susceptible to alkaline hydrolysis
transcriptase catalyzes the
• Rapidly degraded by RNA – specific RNA to DNA synthesis of DNA from either an
enzymes
RNA or DNA template.

DNA LIGASES
● IN VIVO FUNCTION:
○ Joins DNA fragments formed by
discontinuous synthesis in DNA
replication or by DNA repair
pathways.
COMPARISON OF KEY FEATURES OF DNA & RNA
Feature DNA RNA NUCLEASES, DNases, & RNases
Sugar deoxyribose Ribose ● IN VIVO FUNCTION:
○ Nucleases “digest” nucleic acid
Base Thymine- Uracil-adenine
molecules by breaking
pairs adenine Cytosine-
phosphodiester bonds.
Cytosine- guanine
guanine
Structure Double- Single-
stranded stranded
Alpha helix Random

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LECTURE #3 | CYTOGENETICS – BIOCHEMICAL BASIS OF HEREDITY

ENDONUCLEASES & EXONUCLEASES • Since it can synthesize the strand in a

● IN VIVO FUNCTION: continuous manner, it is called a
○ Endonucleases digest nucleic “leading strand”
acids from the middle of the • Since DNA is anti-parallel, the other
molecule whereas exonucleases strand is called the “lagging strand”
begin at a free end and may because the DNA polymerase can
require a 3’ or 5’ end. Nucleases only synthesize the direction 5’-3’.
may have a single-stranded, • The fragments it creates are called
double-stranded, DNA, or RNA the "Okazaki fragments”
specificity. Some polymerases
also have nuclease activity.

RESTRICTION ENDONUCLEASES
● IN VIVO FUNCTION:
○ Bacterial endonucleases that
recognize specific short DNA base
pair sequences and cleave the
4. *opposite strand* Primase comes in to
DNA molecule only at the
put a primer then DNA polymerase
recognition site.
subsequently adds the DNA bases,
complementary to the parent strand or
PROPERTIES OF BACTERIAL DNA POLYMERASES I,
template strand, to create the growing
II, and III
strand
PROPERTIES I II III
5. Primers are removed through the
Initiation of chain synthesis - - - enzyme “endonuclease”, DNA EXONUCLEASE
polymerase comes in to replenish the
5’-3’ polymerization + + +
removed primers
3’-5’ exonuclease activity + + +
• Primers are removed because
5’-3’ exonuclease activity + - -
they don’t contain genetic
Molecules of 400 ? 15
information
polymerase/cell
6. DNA Ligase seals off everything together

DNA REPLICATION
DETAILED DNA REPLICATION
● Each strand has a 5’ end → 3’ end. The
opposite strand has a 5’ end → 3’ end.
DNA is anti-parallel. 3 --> 5

• Topoisomerase – reduce the

1. An enzyme comes in called “DNA tension from the parental DNA as
helicase”, it unzips the paired DNA, the helicase unzips the molecule
creating the replication fork • Single-strand binding protein –
2. Arrival of the enzyme called “primase”, prevents the template strand to
adds RNA bases, creating the primer coil back to each other
3. The primer is in place, it now calls
another enzyme called “DNA
polymerase”, it only runs in 5’ → 3’, it adds
bases to create a growing chain

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LECTURE #3 | CYTOGENETICS – BIOCHEMICAL BASIS OF HEREDITY

SEMICONSERVATIVE REPLICATION everything in the DNA should be

translated into protein
• remove those that don’t
contain the coding part / part
of the DNA that can be
translated into a protein
• 2 parts of the mRNA strand
created from the DNA:
o Exons – coding part
CONTINUOUS AND DISCONTINUOUS SYNTHESIS o Introns – non-coding
• Antiparallel strands: part (removed)
o 5’ → 3’ direction • Post-transcriptional
o 3’ → 5’ direction modifications
• DNA polymerase III synthesizes’ DNA in 5. Remove introns so that exons are the
only the 5’ → 3’ direction, so it creates a only ones left
leading strand and a lagging strand • Cannot be immediately
• Leading strand – template for continuous translated into protein
DNA synthesis because it cannot leave the
• Lagging strand – discontinuous DNA nucleus, it needs the mRNA to
synthesis, thereby, creating the Okazaki carry its message for it to be
fragments translated into protein
• DNA can only stay in the
RNA TRANSCRIPTION & PROTEIN TRANSLATION nucleus, once it leaves the
• Once a specific gene is activated it nucleus, it will be denatured /
initiates the entire molecule to produce a destroyed
specific protein corresponding to that 6. mRNA that carried the information
gene from the DNA will go outside the
o Gene is the brain of it all, it nucleus and will be sensed by the
decides what would be done. The rRNA / ribosomes
protein is the functional 7. Ribosomes will bind to the mRNA
component, executes what the
gene or the information found on
DNA is given
1. When a gene is activated, an enzyme
called “RNA polymerase” attaches to
the start of the gene
2. RNA polymerase moves along the
DNA, making a strand of messenger
RNA, out of free bases in the nucleus
• Messenger RNA – it carries the
message from the DNA for it to 8. with the use of the tRNA, it can be
be translated to proteins translated into a protein
3. The DNA code determines the order in • tRNA carries amino acids, that
which the free bases are added to particular amino acids
the messenger RNA, this process is depends on the specific
called “transcription” codon it carries once it
4. mRNA leaves the nucleus but it needs matches the specific bases on
to be processed first because not the growing mRNA strand

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LECTURE #3 | CYTOGENETICS – BIOCHEMICAL BASIS OF HEREDITY

translation. AUG or methionine is

the start codon.
o stop codon – the point where it
releases the polypeptide chain for
it to fold together to become a
protein. UAG, UGA, UAA are stop
codons.
• eventually it will fold together to create a
protein

9. binds complementary and produces

amino acids becoming a polypeptide
chain STOP?
10. once it reaches a stack codon, it will
fold together and be translated into a
protein

DETAILED RNA TRANSCRIPTION

POST TRANSCRIPTIONAL MODIFICATIONS

• Spliceosome – identifies and splices
boundaries of introns
• 5' end (cap) - addition of 7-methyl
guanosine residues
o Aids in binding ribosome to mRNA
• 3’ end (tail) – addition of poly-A tail
o For stability and transport

DETAILED PROTEIN TRANSLATION

• conversion of mRNA coded genes into a
functional protein
• use of tRNA and ribosome, it creates
amino acids corresponding to bases that
the mRNA carried that are read in three
known as codon
o start codons – encountered by
tRNA for it to initiate the process of

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LECTURE #3 | CYTOGENETICS – BIOCHEMICAL BASIS OF HEREDITY

DNA MUTATIONS • Red blood cells of the heterozygote

POINT exhibit sickle cell trait but they do not
● single base pair substitutions exhibit the disease
○ sickle cell anemia o Heterozygous are carriers of the
defective gene which is
DELETION / INSERTION transmitted on average to 50% of
the offspring.
● subtraction/addition of amino acid
codons in multiples of three. Reading
frame is retained.
○ Becker muscular dystrophy

• Called point mutation because only one

DELETION / INSERTION WITH FRAMESHIFT
point is replaced
● Subtraction/addition of amino acid
codons in non-multiples of three. Results MECHANISMS OF DNA REPAIR
in a shift of the reading frame and a DIRECT REPAIR
completely different amino acid coding
● Repairs certain types of DNA damage in
sequence from the mutation on.
a single-step reaction.
○ Duchenne muscular dystrophy

MISMATCH REPAIR
AMPLIFICATION / TRINUCLEOTIDE REPEAT
● Check for errors made when DNA is
● Increase in the number of repeat
replicated. Any mispaired bases in the
sequences in microsatellite DNA. Results
daughter strand are removed and
in disruption of gene expression.
replaces with the correct match.
○ Fragile X syndrome

BASE EXCISION REPAIR

TRANSLOCATION
● Repairs small, nonhelix-deforming
● Interchromosomal exchange of large
adducts such as those produced by
chromosome segments. Results in a new
methylation, oxidation, reduction, or base
protein with different function.
fragmentation by ionizing radiation.
○ Chronic myelogenous leukemia

NUCLEOTIDE EXICSION REPAIR

SICKLE CELL ANEMIA
● Removes bulky DNA adducts such as
• Affected individuals:
thymine dimers and certain
o Erythrocytes become elongated
photoproducts as well as chemical
and curved because of the
adducts and cross-links.
polymerization of hemoglobin
o The variety of tissues are deprived
DOUBLE-STRAND BREAK REPAIR
of oxygen and suffers severe
damage. ● Repairs double-strand breaks that result
• Affects kidneys, muscles, joints, brain, GI from physiologic processes or from
tract, etc. ionizing radiation and oxidative insults.
• In 1949, James Mill and his colleagues
demonstrated that the disease is NUCLEIC ACID ANALYSIS: ELECTROPHORETIC
inherited as a Mendelian trait SEPARATION
o Unaffected genotype: HbA HbA • Physical method of separation of DNA
o Affected genotype: HbS HbS and RNA based on molecular weight and
o Heterozygous individuals: HbA length of the molecule.
HbS o Repeating sugar-phosphate
backbone of the nucleic acid

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LECTURE #3 | CYTOGENETICS – BIOCHEMICAL BASIS OF HEREDITY

results in a net negative charge

evenly distributed over these
linear molecules.
• Used to characterize the size of nucleic
fragment by separation.
• Variables that contribute to resolution:
o Thickness of the gel
o Length of the electrophoresis
o Time of the electrophoresis
o Applied voltage
• Medium commonly used: agarose
o Visualized by:
▪ staining with intercalating
dyes: ethidium bromide
▪ UV transilluminations

TRANSCRIBED BY: JHULIA CABAÑESAS 8