CYTOGENETICS
MODULE 1: HISTORY OF CYTOGENETICS
WALTER FLEMMING
 Attributed to him the beginning of human
cytogenetics
 Austrian cytologist and professor of anatomy, who
published the first illustrations of human
chromosomes in 1882.
 Chromatin- stainable portion of the nucleus
 First used the term mitosis
WALDEYER
 In 1888, he introduced the word chromosome
from the Greek words for “colored body”
 Several prominent scientists of the day began to
formulate the idea that determinants of heredity
were carried on chromosomes.
SUTTON
 After the rediscovery of Mendelian inheritance in
1900, Sutton (at the same time BOVERI) formally
developed a chromosome theory of inheritance.
 Combined the disciplines of cytology and genetics
when he referred to the study of chromosomes as
cytogenetics
 The study of cytogenetics continued, with an
emphasis placed by some on determining the
correct number of chromosomes, as well as the
sex chromosome configuration, in humans.
VON WINIWARTER
 In 1912, he concluded that men have 47
chromosomes and women have 48
PAINTER
 In 1923, he studies meiotic chromosomes derived
from the testicles of several men who had been
incarcerated, castrated, and ultimately hanged in
Texas State Insane Asylum
 He reported that the human diploid chromosome
number to be 48, (double the 24 bivalents he saw)
 Also proposed the X and Y sex chromosome
mechanism in man.
LEVITSKY
 Formulated the term karyotype to refer to the
ordered arrangement of chromosomes
HSU
 In 1952, he reported that rather than depending
on histologic sections, examination of
chromosomes could be facilitated if one studied
cells growth with tissue culture techniques
published by FISHER
 He then demonstrated the value of this method by
using it to examine human embryonic cell
cultures, from which he produced both mitotic
metaphase drawings and an ideogram of all 48
human chromosomes.
 It was found that the well-spread metaphase were
the result of an accident. Instead of being washed
in isotonic saline, the cultures had been washed in
hypotonic solution before fixation. The hypotonic
solution caused water to enter the cells via
osmosis, which swelled the cell membranes and
separated the chromosomes, making them easier
to visualize. This accident is the key that unlocked
the future of human cytogenetics.
HSU & POMERAT
 Within 1 year, realizing that the potential of the
event reported a “hypotonic shock” procedure.
FORD & HAMERTON
 In 1955, they modified the technique of Hsu &
Pomerat and had also worked out a method for
pretreating cells grown in culture with colchicine
so as to destroy the mitotic spindle apparatus and
thus accumulate dividing cells in the metaphase.
JOE HIN TIJIO
 American-born Indonesian, learned about these
procedures and worked with Hamertonand Ford to
further improve upon them.
 In November 1955, he was invited to Lund,
Sweden to work on human embryonic lung
fibrolast cultures in the laboratory with Levan
LEVAN
 A Spaniard who had learned the colchicine and
hypotonic method in Hsu’s Laboratory at the
Sloan-Kettering Institute in New York.
TIJIO & LEVAN
 Both opotimized the colchicine/hypotonic method
for these cells
 In January 1956, after carefully reviewing images
from decades of previously reported work, they
diplomatically reported that the human diploid
chromosome number appeared to be 46, not 48.
They referenced anecdotal data from a colleague
who had been studying liver mitoses from aborted
human embryos in the spring 1955, but
temporarily abandoned the research because the
workers were unable to find all the 48 human
chromosomes in their material, as a matter of
fact, the number 46 was repeatedly counted in
their slides.
WAARDENBURG
 In 1932, he made the suggestion that Down
syndrome could perhaps be the result of
chromosomal aberration.
LEJEUNE
 In 1958, studied the chromosomes of fibroblast
cultures from patients with Down syndrome
 In 1959, he and his colleagues described an extra
chromosome in each of these cells. The trisomy
was reported to involve one of the smallest pairs
of chromosomes and would eventually be referred
to as trisomy.
 He had proved Waardenburg’s hypothesis by
reporting the first example of a chromosomal
syndrome in man, and in December 1962, he
received one of the first Joseph Kennedy Jr.
Foundation International Awards for his work.
FORD et al.
 In 1959, he reported that all females with Turner
syndrome have 45 chromosomes, apparently with
a single X chromosomes and no Y.
JACOBS & STRONG
 Demonstrated that men with Klinefelter syndrome
have 47 chromosomes with the additional
chromosome belonging to the group that
contained the X chromosomes, with the additional
chromosome belonging to the group that
contained the X chromosome
JACOBS
 A female with sexual dysfunction has also shown
by him, to have 47 chromosomes and was
believed to have an XXX sex chromosome
complement.
MURRAY BARR
 In 1949 he was studying fatigue in repeatedly
stimulated neural cells of the cat.
 He observed a small stained body on the
periphery of some interphase nuclei, and his
records were detailed enough for him to realize
that this was present only in the nuclei of female
cats. This object, (referred to as sex chromatin
known as X chromatin or the Barr body) is actually
the inactivated X chromosome present in
nucleated cells of all normal female mammals but
absent in normal males.
LYON
 CYTOGENETICS
 In 1961, he developed the single active X
chromosome mechanism of X-dosage
compensation in mammals. And has been known
as Lyon hypothesis.
LEJEUNE ET AL.
 Report the chromosomal basis of Down syndrome
that other autosomal abnormalities were
discovered.
PATAU ET AL.
 In April 9, 1960 edition of The Lancet they
described two similar infants with an extra “D
group” chromosome who had multiple anomalies
quite different from those seen in Down syndrome
EDWARDS ET AL.
 They described “A New Trisomic Syndrome” in an
infant girl with yet another constellation of
phenotypic abnormalities and a different
autosomal trisomy.
 The former became known as “Patau’s syndrome”
or “D trisomy” and the latter as Edward’s
syndrome or “E-trisomy”.
NOWELL & HUNGERFORD
 Reported the presence of the Philadephia
chromosome in chronic myelogenous leukemia,
demonstrating for the first time, an association
between chromosomes and cancer
LEJEUNE ET AL.
 In 1963 and 1964, reported that three infants with
the cri du chat (cat cry) syndrome of phenotypic
anomalies, which included severe mental
retardation and a characteristic kitten-like mewing
cry, had a deletion of the short arm of B-group
chromosome, designated as chromosome 5
JACOBS ET AL.
 Within 2 years, they described “aggressive
behavior, mental subnormality and the XYY male”
and the chromosomal instabilities associated with
Bloom syndrome and Fanconi anemia were
reported.
NOWELL
 In 1960, he observed that the kidney bean extract
phytohemagglutinin used to separate red and
white blood cells, stimulated lymphocytes to
divide. He introduced its use as a mitogen,
permitting a peripheral blood sample to be used
for chromosome analysis.
 Within nine years of the discovery of the number
of chromosomes in humans, only three autosomal
chromosomal abnormality associated with cancer,
and two chromosomes breakage disorders had
been described as recognizable chromosomal
syndromes.
TORBJORN CASPERSON
 In 1968, he observed that when plant
chromosomes were stained with fluorescent
quinacrine compounds, they did not fluoresce
uniformly, but rather produced a series of bright
and dull areas across the length of each
chromosome. Furthermore, each pair fluoresced
with a diferent pattern, so that previously
indistinguishable chromosomes could now be
recognized.
CASPERSSON AND COLLEAGUES
 Turned their attention from plants to the study of
human chromosomes. They hypothesized that the
quinacrine derivative quinacrine mustard (QM)
would preferentially bind to guanine residues and
that C-G rich regions of chromosomes should
therefore produce brighter “striations” as they
initially referred to them, whereas, A-T rich regions
would be dull. Although it ultimately turned out
that it is the A-T-rich regions that fluorescence
brightly and that ordinary quinacrine
dihydrochloride works as well as QM
 By 1971 he and his co workers had successfully
produced and reported a unique “banding”
pattern for each human chromosome pair.
DRETS & SHAW
 Described a method of producing similar
chromosomal banding patterns using an alkali and
saline pretreatment followed by staining with
Giemsa, a compund developed for identification in
blood smears of the protozoan that causes
malaria.
 What followed was a cascade of defined
chromosomal abnromalities and syndromed:
aneuploidies, deletions, microdeletions,
translocations, inversion, insertions, and
mosaicisms, plus and ever-increasing collection or
rearrangements and other cytogenetic anomalies
associated with neoplasia, and a seemingly
infinite number f patient - and family-specific
rearrangements.
Molecular cytogenetics- utilizes the techniques of
fluorescence in situ hybridization (FISH)
MODULE 2: CENTRAL DOGMA OF LIFE
Genome- the ENTIRE genetic information of an
organism
Gene- is a portion of the DNA that codes of a particular
trait.
 are functional units of genetic information that
reside on each of the 23 pairs of chromosomes.
 These units are linear sequences of nitrogenous
bases that code for protein molecules necessary
for the proper functioning of the body
- Example: Human genome project- gene cloning
(Understanding human disease)
STRUCTURE OF NUCLEIC ACID
DNA- Deoxyribonucleic acid
 James Watson and Francis Crick- elucidated
the molecular structure of DNA in 1953 using X-
ray diffraction data.
 Deoxyribonucleic acid (DNA) is the raw
material of inheritance and ultimately influences
all aspects of the structure and functioning of the
human body.
 Double-stranded helix (clockwise direction)-
The helix of the DNA is right handed means looks
like a barrel follows a clockwise direction
 A single molecule of DNA, along with associated
proteins, comprises a chromosome.
 Most DNA are found the nucleus of a cell- forms
chromosomes proteins histones that binds to DNA
 DNA is composed of repeating units- the
nucleotides
 Nucleotides are the monomers or building blocks
of nucleic acids
Each nucleotide cosnsists of:
 Pentose (five carbon sugar)- deoxyribose (DNA),
ribose (RNA) - a chemical group attached to the
DNA.
 Nitrogenous bases
 Phosphate group- attached to a 5 carbon of the
sugar both DNA & RNA
 DNA- hydrogen is attached to the carbon
 RNA- hydroxyl is attached to the carbon
 Directionality
 CYTOGENETICS
 The fifth (5') carbon (trunk end) of one  Chromatin consists of a single continuous
deoxyribose molecule and the third (3') carbon molecule of DNA complexed with histone and
(tail end) of the next deoxyribose are joined by a nonhistone proteins.
covalent phosphate linkage. a. Histones
 This gives each strand of the helix a chemical  They are relatively small basic proteins with a very
orientation with the two strands running opposite high proportion of positively-charged amino acids
or antiparallel to one another.  The (+) charge helps the histones bind DNA
Nitrogenous Bases tightly.
Purine  Most abundant proteins in chromatin
 9 membered double winged structure They are of 2 types:
 Fused heterocyclic rings a. nucleosomal histones - small proteins responsible
 Adenine (A), Guanine (G) for
Pyrimidine folding the DNA into nucleosomes; compose of H2A,
 6 membered single winged structure H2B, H3 and H4
 Heterocyclic ring b.linker histones - H1 and H5
 Cytosine ( C ), Thymine (T), Uracil (U) b. Nucleosome “beads of strings”
Complementary Base Pairing  First and most basic level of chromosome
 Adenine- Thymine organization
 Guanine- Cytosine  are the unit particles of chromatin (DNA
 Two hydrogen bonds (2 in A-T) link the adenine associated with proteins).
and thymine pairs, whereas three (3 in G-C)  Each nucleosome contains a set of protein core
hydrogen bonds link the guanine and cytosine made of 8 histone molecules (2 copies each of the
pairs. 4 nucleosomal histones).
 Thus, G-C is more stronger because of the number  These form an octameric protein core around
of bonds present which the doublestranded DNA fragment (~146
 The complementarity of DNA strands is what bp) is wound.
allows the molecule to replicate faithfully.  A region of linker DNA (about 60 bp long)
Nucleoside: base + sugar covalently linked in an N- separates each nucleosome from the next.
glycosidic bond  Nucleosome core particle is released from
 C1’of sugar to N1 of pyrimidine chromatin through digestion of a linker DNA with a
 C1’of sugar to N9 of purine nuclease.
 Phosphate group/s: maybe 1, 2 or 3 phosphate/s Nucleases- break down DNA by cutting between the
N-glycosidic bonds nucleosome.
 the linkage between the bases and the sugar c. Solenoid
group  a 30 nm DNA structure containing 6 nucleosomes;
 In Purine- attached to the nitrogen and to the one  The solenoids are packed into DNA looped
carbon of the sugar group domains attached to a nonhistone protein matrix.
 In Pyrimidine- one nitrogen on its base attached to  The looped domains coil further to give rise to
the one carbon of the sugar group highly compacted units, the chromosomes
Phosphodiester bond Non-histone proteins
 Linking the phosphate as well as the sugar.  Proteins associated with DNA, apart from
Comparison of Double Helical DNA histones
Conformations  Less abundant than histones
Parameter A-form B-form Z- form  Acidic proteins- since they have a net
Helical Right-handed Right handed Left- handed negative charge
Sense clockwise counterclockwis
 Include proteins that play a role in the
e
Zigzag processes of DNA replication, repair,
arrangement transcription, and recombination
Average 10.7 10 12 CENTRAL DOGMA OF LIFE
base pairs/  DNA will replicate itself and will be transcribed to
helical RNA then translated to protein
turns
 DNA acts through an intermediary particle called
Degrees of 19 -1 because -9
inclination parallel to the ribonucleic acid
of base pair the helical A. DNA REPLICATION
from axis  DNA replicates for growth and renewal of cells.
normal to  Coping process in which a single DNA molecule
helical axis
becomes two identical molecules
Rise per 2.3 3.3 -3.4 3.8
base pair  DNA replication is semiconservative. Each
Pitch per 24.6 33.2 45.6/ 24.6 strand in the double helix acts as a template for
helical turn synthesis of a new, complementary strand.
Helical Approx. 26 Approx. 20 Approx. 18  Semiconservative because DNA molecule if it will
diameter be untwisted the two strands the parent strands
Major Short and Thinner & Flattened will be separated and will become a template for
groove wide with a longer for
Narrow and the same the synthesis of the daughter strand in order to be
deep number of complementary by the parent strand
base pairs,  The first step of the process involves breakage of
wide and the hydrogen bonds that hold the DNA strands
deep together.
Minor Wide and Narrow and Thin &
groove shallow deep elongated,
 New DNA is made by enzymes called DNA
Narrow & deep polymerases, which require a template and a
primer (starter) and synthesize DNA in the 5' to 3'
direction.
Packaging of DNA into Chromosomes
 CYTOGENETICS
 During DNA replication, one new strand (the
leading strand) is made as a continuous piece.
The other (the lagging strand) is made in small
pieces.
 DNA replication requires other enzymes in
addition to DNA polymerase, including DNA
primase, DNA helicase, DNA ligase, and
topoisomerase.
 DNA replication begins at fixed points in the
chromosome (called replication forks).
 DNA replication is a high-fidelity and high-
accuracy process.
 Error rate is very low because DNA polymerases
have proofreading functions.
 Replication rate is approx. 1000 bp/sec/replication
fork.
 The area of DNA at the active region of separation
is a Y-shaped structure referred to as a replication
fork.
Basic steps and Enzymes involved:
1. DNA unwinding
 catalyzed by ATP-requiring helicases
 Single-stranded binding proteins (SSB) coat the
separated strands of DNA near the replication
fork, keeping them from coming back together
into a double helix.
 Toposisomerases - assist DnaB in unwinding the
helix
2. Primer synthesis
 Primase catalyzes the formation of short RNA
segments called primers, required for the initiation
of DNA replication.
 Primosome is composed of primase plus several
auxillary proteins.
3. DNA synthesis
 catalyzed by large multienzyme complexes
referred to as DNA polymerases.
 The pol III holoenzyme comprises at least 10
subunits.
 Replisome (DNA replicating machine) comprises 2
copies of the pol III holoenzyme, the primosome
and DNA unwinding proteins.
4. Joining DNA fragments (Okazaki fragments)
 catalyzed by DNA ligase.
5. Supercoiling control
 DNA topoisomerases prevent tangling of DNA
strands. Type I topoisomerase alter supercoiling by
producing transient single-strand breaks in DNA;
 type II topoisomerases produce transient
doublestrand breaks.
REPLICATION INITIATION
 for the preparing the double helix for the
complementary base pairing
 First step: formation of a “bubble” which
corresponds to the local unwinding of the DNA
helix at the origin of DNA replication (oriC). The
points at which the parental DNA is being
unwound, and the new DNA strands are being
synthesized, is called the replication fork.
Enzymes and Proteins Involved:
1. DnaA protein
 binds to four 9-bp sites within the oriC sequence.
The formation of the DnaA-DNA complex causes
the “melting” of the DNA duplex in three nearby
13-bp sequences (A-T rich)---- replication bubble.
2. DnaB protein
 a helicase that unwinds the helix.
3. DnaC protein
 forms a complex with DnaB
4. Toposisomerases
 assist DnaB in unwinding the helix
5. Single-stranded binding proteins (SSB)
 keep apart the single strands and protect ssDNA
segments from attack by nucleases.
DNA ELONGATION
 connecting the correct sequence of nucleotides
into a continuous new strand of DNA
Replication forks have leading and lagging strands:
1. Leading strand
 proceeds continuously in the 5’ to 3’ direction;
requires a single RNA primer for the DNA
polymerase to initiate DNA synthesis.
2. Lagging strand
 requires discontinuous 5’ to 3’ synthesis through
the formation of short Okazaki fragments; requires
multiple RNA primers for the DNA polymerase to
initiate synthesis of the Okazaki fragments.
REPLICATION TERMINATION
 replication ends when the replication forks will
meet at a termination site - the “ter” region.
 The ter region is composed of a pair of 20-bp
inverted repeat ter sequences separated by a 20-
bp segment.
 Each ter sequence prevents further progression of
one of the replication forks when a 36 kD ter
binding protein (TBP) is bound.
 The end result: single double-stranded molecule
becomes replicated into two copies with identical
sequences.
EDITING & PROOF-READING FUNCTON
 Before transcription
 An error-correcting mechanism that reduce the
mutation frequency of resulting from the
incorporation of incorrect nucleotides in DNA
replication.
 Exonuclease activity that breaks the
phosphodiester bonds in the sugar phosphate
backbone of the nucleic acid chain
 To correct the mismatch nucleotide
 If it will not be edited properly: result to mutation
(sickle cell anemi- first molecular disease.)
B. TRANSCRIPTION
 transferring the genetic information in DNA to RNA
base sequences.
 The DNA will unwind in a short region and the RNA
polymerase will catalyze the synthesis of an RNA
molecule
 Only 1 strand of the double strand will be
transcribed into an RNA molecule
 Transcription is the first step in gene expression. It
involves copying a gene's DNA sequence to make
an RNA molecule.
 Transcription is performed by enzymes called RNA
polymerases, which link nucleotides to form an
RNA strand (using a DNA strand as a template).
 CYTOGENETICS
 In eukaryotes, RNA molecules must be processed
after transcription: they are spliced and have a 5'
cap and poly-A tail put on their ends.
 Transcription is controlled separately for each
gene in your genome.
RNA polymerase
 Large, multisubunit complexes
 RNA polymerase Holoenzyme (active form)
 Eukaryotic RNA polymerases are even larger than
those in prokaryotes, and include more subunits in
the holoenzyme.
 the enzymes that copy DNA to RNA
Types:
RNA polymerase I
 Used in producing the transcript that becomes
pribosomal RNA
RNA polymerase II
 Is the workhorse eukaryotic polymerase
responsible for transcribing all protein-coding
genese and the genes for snRNAs used in RNA
processing
RNA polymerase III
 Is used in transcribing all tRNA genes and the 5S
component of rRNA
Classes of RNA/ RNA structures
Transfer RNA
tRNA
 To transport a covalently attached amino acid to
the ribosome and facilitate its proper
incorporation into a protein sequence.
 Carries a particular amino acid as well as three-
base recognition region that base-pairs with a
group of three adjacent bases in the mRNA
Structure:
Primary: Single strand of nucleic acid containing 60-
95 nucleotides.
Secondary: Double-stranded stems connected to
single-stranded loops. Stems are in the A-form
consisting of Hbonded base pairs G-C and A-U.
Tertiary: Distinctly globular in appearance, resembling
an L shape.
Messenger RNA
mRNA
 Carrier of the genetic code from DNA to the
ribosome.
 Template for polypeptide synthesis
 Relatively high proportion of the nucleotides
actually code for amino acids.
Structure:
 Single-stranded nucleic acid which is relatively
less structured than other RNAs; hairpin or stem-
loop structures provide a means of controlling
gene expression.
 Eukaryotic mRNAs: 5’ cap and 3’ polyA tail
Ribosomal RNA
rRNA
 Major component of the ribosome, comprising
about 2/3 of the gross weight and forming the
functional portion involved in protein synthesis.
Structure:
 Highly structured nucleic acid containing an array
of double-stranded stems and single-stranded
loops;
 Secondary structure: also contains interior loops,
bulge loops and multibranched loops in its
secondary structure.
RNA transcription
 the synthesis of RNA from a DNA template
Mechanism of RNA Synthesis
 Synthesis is in the 5’ to 3’ direction.
 Pre-existing primer is not needed.
 No editing during synthesis; higher error rate and
lower fidelity.
 Coding (+) strand is the template.
 Noncoding (-) strand has the same sequence as
the transcript (except Thymine is replaced with
 Uracil).
 Key controlling point: the regulation of initiation
Promoter
 where RNA polymerase binds; acts in a site-
dependent and orientation dependent manner.
 bind with the RNA polymerase
 dictate the enzyme which strand is the template
strand and where the transcription begins.
Prokaryotic promoter:
Pribnow box -10 region TATAAT consensus
-35 region TTGACA sequences
Eukaryotic promoter:
Hogness box or TATA box located ~30 bp away from
the initiation site.
Other promoter sites: CCAAT, sp1, NF-1
INITIATION
STEPS:
 Binding of RNA polymerase to DNA and migration
to the promoter, the initiation site (σ subunit of
RNA polymerase responsible for promoter
recognition).
 Formation of a closed-promoter complex.
 Formation of an open-promoter complex (when
RNA polymerase unwinds ~12bp of DNA).
 Initiation site binds ATP and GTP preferentially so
the first ribonucleotide is a purine (A or G).
 the RNA polymerase bind to the promoter and it
will start to unwind the DNA strands.
ELONGATION
STEPS:
 Incorporation of ribonucleotides added to the first
nucleotide.
 During transcription of first 10 nucleotides, the σ
subunit dissociates from the transcription
complex.
 Core enzyme moves along the duplex DNA and
simultaneously unwinds the DNA for the synthesis
of incoming nucleotides and rewinds the template
behind the 3’ end of the growing RNA chain.
TERMINATION
1. rho factor-independent termination (intrinsic)
 2 structural features found at the 3’ end of genes:
2 symmetrical GC-rich segments that can
potentially form a stem-loop structure a
downstream run of 4-8 A residues
 the signal for the termination depends on the
nucleotide sequence of the template
 Requires a termination process which is
associated in the polymerase complex
STEPS:
 RNA polymerase pauses or slows down as it
reaches the first GC-rich segments (G-C base pairs
more difficult to unwind).
 Time delay leads to complementary base pairing
of the GC-rich segments.
 Complex of RNA polymerase + DNA template +
RNA is weakened leads to dissociation when A-rich
segment is
2. ρ factor-dependent termination
 Presence of a termination protein associated with
the polymerase complex.
STEPS:
 CYTOGENETICS
 factor recognizes a site on the RNA transcript and  50S subunit of the ribosome has 2 binding sites
binds to it. for tRNA:
 Binding of ρ (rho) factor unwinds the RNA-DNA  P or peptidyl site = binds a tRNA with a peptide
duplex. chain
 Moves along the transcript towards the 3’ end  A or aminoacyl site = binds an aminoacyl-tRNA
causing the release of the DNA template and RNA
polymerase also dissociate. 4. Chain termination
 Stop signals: UAA, UAG, & UGA for termination
 RNA processing converts the original RNA  GTP & release factors are required.
transcript into messenger RNA
 The 5’ end is altered by the addition of a modifief MODULE 3: CHROMOSOME
guanosine in an uncommon 5’-5’ linkage this Chromosomes
terminal is called the gap  Consists of 2 sister chromatids
 The 3’ end is usually modified by the addition of a  Consists of an extremely elaborate complex, made
sequence called the poly-A tail up of supercoils of DNA
 Certain regions internal to the transcript (introns)  Consists of two sister chromatids
are removed by splicing.  Areas of chromosomes: centromere, telomere, and
nucleolar organizing regions
C. TRANSLATION CENTROMERE
 The synthesis of a polypeptide under the direction  Visible on metapahase chromosomes
of an mRNA molecule.  is a constriction which is visible on the metaphase
 tRNA molecules “translating” chromosome where the 2 sister chromatids are
 The mRNA molecule is translated in joined together.
nonoverlapping groups of three bases called  essential to the survival of the chromosome
codons. during cell division
Location:
Genetic Code: code used to translate the genetic  Near the middle in metacentric chromosomes
message from mRNA to protein.  Centromere in the middle, equal arms
 Near one end in an acrocentric chromosomes
Features of the Code:  Centromere very close to one end, unequal length
 triplet-a sequence of 3 bases (codon) specifies  Between the middle end in submetacentric
one amino acid chromosomes
 nonoverlapping-no bases are shared between  Centromere is somewhat up the center, leaning
consecutive codons upwars, short arm and long arm
 commaless-no intervening sequences exist Function:
between codons  Essential to the chromosome survival during cell
 degenerate-more than one triplet can code for the  Interaction with the mitotic spindle during cell
same amino acid division
 universal-the same code is used by all organisms  Basis of human chromosome nomenclature
(prokaryotes, eukaryotes, viruses) with few Components:
exceptions  Heterochromatic regions consist of alpha satetlite
 has start and stop signals DNA
TELOMERES
Steps in protein synthesis: Location:
1. Amino acid activation  The physical end of chromosomes
 catalyzed by aminoacyl-tRNA synthetases Function:
a. formation of aminoacyl-AMP intermediate ATP +  Act as protective caps to chromosome ends
amino acid aminoacyl-AMP + PPi  Prevents end-to-end fusion of chromosomes and
b. formation of aminoacyl-tRNA aminoacyl-AMP + tRNA DNA degradation
aminoacyl-tRNA + AMP  Synapsis during meiosis
 Aminoacyl-tRNA synthetases are Mg++-requiring  Protect the ends of chromosomes from nucleases
and highly specific for both amino acid and tRNA.  Tumor-suppressor mechanism as it decreases in
There is one synthetase for each amino acid length with age, exposure to UV
2. Chain initiation  Provides non-sticy ends
 Eukaryotes: Initiating AUG and internal AUG = Component:
methionine.  Heterochromatin with tandem repeats of the
 Eukaryotic initiation factors (eIF) nitrogenous base sequence TTAGGG over 3-20 kbp
 The initiator methionine is unmodified , although a NUCLEOLAR ORGANIZER REGIONS (NGO)
special initiator tRNA still brings it to the  in the human body there are 10 NOR’s however
ribosome. they are not active only a few.
 No Shine-Dalgarno sequence present; at least 8 Location:
initiation factors  The satelite stalks of human acrocentric
3. Chain elongation chromosomes
 Recruitment of other elongation factors into the Function:
initiation complex begins the elongation phase of  Site of nucleolus production
polypeptides synthesis.  Site of ribosomal RNA genes and production of
rRNA
Three processes: Components:
- Bringing each new aminoacylated tRNA into line  Tandemly repeated sequences of ribosomal genes
- Forming the new peptide bond to elongate the rRNA
polypeptide
- Moving the ribosome to the next codon along the CENTROMERE
mRNA  Region of the eukaryotic chromosome that
 CYTOGENETICS
becomes visible during condensation  Contains active, early replicating genes and stains
 Serves as the point of assembly of the kinetochore lightly with GTG banding techniques.
 Hundreds of thousan of copies of a 170-bp DNA
sequence (alpha satelite) TYPES of DNA
Component: A. Unique sequence or single-copy DNA
 Heterochromatins with alpha-satellite DNA  Most common class of DNA
Function:  Consists of nucleotide sequence that are
 Constriction in a chromosome where a kinetochore represented only once in a haploid set
is attached  Genes that code for proteins are single-copy of
Location: DNA
 Center   75% of the genome
 Near one end  B. Repetitive or repeated sequence DNA
 Between the center and tip most part of a  Classified according to the number of repeats and
chromosome whether the repeats are tandem or interspered
Types: among unique sequence DNA
 Metacentric   First discovered with a cesium chloride density
 Submetacentric  gradient
 Acrocentric  Visualized as separated bands in the gradient
Microscopy:  25% of the genome
 Light Microscope
KINETOCHORE Satellite DNA
 Complex of DNA and proteins to which the spindle Alpha-satellite DNA
fibers attach  Repeat of a 171- basepair sequence organized in a
 The site at which the spindle fibers shorten tandem array up to a million bp or more
Kinetochore assembly is said to be epigentic- refers  Not transcribed, location: heterochromatin
to changes in gene expression that are not directly associated with the centromeres of chromosomes
specified by the DNA sequence  Associated with centromeres, its role in
Component: centromere function has not been determined
 Protein Minisatellites
Function:  Repeats that are 20-70 bp in length, with a total
 Consists of approximately 20 binding sites for the length of a few thousand bp
mitotic spindles. Microsatellites
Location:  Repeat units of , 3 or 4 bp in length is usually less
 At the centromere than a few hundred bp
Types:  Both micro and mini are useful markers for gene
 Inner  mapping and identity testing
 Outer Gene mapping- the process of determining or
Microscopy: mapping of genes in a chromosome
 Electron microscope Identity testing- methods to identify variation or
differences in proteins that differ among individuals.
Chromatin
 Nucleosome (basic structural unit) Midle repetitive sequences (18S and 28S
 Complex aggregate of DNA and proteins ribosomal RNA’s)
 In the electron microscope, chromatin resembles a  Tandemly arranged on the short arms of the
regularly beaded thread formed into a coiled fiber, acrocentric chromosomes
known as the 30-nm chromatin fiber. Dispersed repetitive DNA (short or long)
 consists of single continuous molecule DNA SINES (short interspersed elements)
Types of chromatin:  90 to 500 bp
A. Heterochromatin  Alu sequence- high G-C content and Giemsa-light
 Regions of chromatin that are compact and bands of chromosomes
heavily stained in interphase
LINES (long interspersed elements)
 2 types of heterochromatin: facultative and
 7000 bp
constitutive chromatin
 Predominant member (L1)- high A-T content and
 Inactive, late replicating during the synthesis
Giemsa-dark bands of chromosomes
phase of mitosis, and contracted

HUMAN CHROMOSOME NOMENCLATURE

Chromosome Banding and Identification
Constitutive
Banding patterns
 simple repeats of nitrogenous bases that are
 Are specific for each chromosome pair, thus
located around the centromere of all
enabling the identification
chromosomes at the distal end of the Y
 Human somatic cells have 46 chromosomes, 44
chromosome
are autosomes, 2 are sex chromosomes
Facultative
Methods:
 are regions which are inactive. Regulates the gene
 Giemsa or G-Banding
function
 Quinacrine mustard or Q- Banding
 Reverse or R- Banding
B. Euchromatin
 Constitutive heterochromatin or C- Banding
 Visible only after chromosome condensation in
mitosis or meiosis
Chromosome Regions and Band designations
 Loosely organized, extended and uncoiled
 CYTOGENETICS
Numbering system (designating specific bands and  DNA synthesis occurs
regions)  DNA replicate itself
 The centromere divides a chromosome into a  Completed before G2 can begin
short or “p” arm and a long or “q” arm  Light- replicate early
 p10 and q10 (Centromeres organization)  Dark- replicate late
 Each arm ends in a terminus G2
 Each chromosome arm is divided into regions. This  last for 3 hours
division is based on landmark present on each  Cell prepares or undergoes cell division
chromosome  Completion of this stage represents the end of the
 Lighter or darker in staining intensity interphase
MITOSIS
Karyotype Description  end stage or final stage of the cell cycle
Rule: first item specified is the total number of  1-2 hours
chromosomes followed by a comma and the sex  Process of nuclear division that ensure that each
chromosomes in that order of the two daughter cells receive a diploid
Example: female karyotyoe- 46, XX complement of chromosome identical to the
Male karyotype- 46, XY diploid complement of the parent cell
 Short system for designating structural  Cell reproduce and will create 2 daughter cells
chromosome abnormalities. that are identical to one another and to the parent
cells
DESCRIPTION  Accompanied by cytokinesis

Cyclins and cyclin-dependent protein kinases

Cyclin-CDK complexes
 Control progression through the cell cycle
 Attach phosphate groups to the hydroxyl groups
present in the amino acid (serine, threonine,
tyrosine)
 The activities of the phosphorylated form of a
protein differ from those of the unphosphorylated
form
 Phosphorylation at a different sites may have
opposite effects.

MITOSIS (Basic Process)

 Each chromosome is a duplicated structure at the
beginning of nuclear division
 Each chromosome divides longitudinally into
identical halves that become separated from each
MODULE 4: CELL CYCLE other
CELL DIVISION  The separated chromosomes halves move in
 Traditional cytogenetic techniques opposite directions, and each becomes included in
 Cytogenetic abnormalities result from errors in cell one of the two daughter nuclei that are formed.
division
PROPHASE
Two types of cell division:
 chromosomes begin to coil, more condensed
 Mitosis - division of somatic cells
 Be visible as discrete structure
 Meiosis- division that occurs only in gametes
 Nuclei invisible but disappears as stages progress
CELL CYCLE
PROMETAPHASE
 The repeating pattern of cell growth followed by
division.  Short period
CELL CYCLE STAGES:  Nuclear membrane disappears and there are
 G1 Gap 1 spindle fibers
 S Synthesis  Chromosomes are attaching to the spindle fibers
 G2 Gap 2 at the kinetochore location
 M Mitosis METAPHASE
 The mitotic spindle is completed
FUNCTION:  Centrioles divide and moves to opposite poles
 Ensure that each chromosomal DNA molecule is  Chromosome line up to the equatorial plate, the
replicated once and only once per cycle. maximum state of contraction
 Ensure that the identical replicas of each ANAPHASE
chromosome are distributed equally to the  Centromeres divide
daughter cells.  Chromatids separate
 Average cell cycle last about 17-18 hours  Sister chromatids migrate to opposite poles
CELL CYCLE PROCESS TELOPHASE
G1  Final stage
 9 hours  The chromosome uncoil and become
 Chromosome exists as single chromatids indistinguishable and the nuclei reforms
 Cells are active  Nuclear membrane is reconstructed
 Where protein synthesis takes place  Usually followed by cytokinesis
G0 CYTOKINESIS
 Do not undergo further division  Cytoplasmic division
SYNTHESIS
 5 hours MEIOSIS
 CYTOGENETICS
 Mode of cell division in which cells are created  Chromosomes are individually distinguishable
that contain only one member of each pair of under light microscope only during cell division.
chromosomes present in the premeiotic cell Stimulant- phytohemaglutanin
 Result: four daughter cells, each genetically
different and each containing one haploid set of  Peripheral blood lymphocytes, tissue biopsies and
chromosomes amniotic fluid samples are routinely cultured
 The first meiotic division (meiosis I) is called the obtain cells.
reductional division- it divides the chromosome  Lymphocytes requires the addition of a mitotic
number in half stimulant.
 The second meiotic division- (meiosis II) is called  The most critical element is that living cells
the equational division- the chromosome capable of cell division be received by the
number remains the same in each cell before and laboratory.
after the second division.  Specimen containers must be sterile and must be
Prophase I labeled
 complicated stage  with the patient’s name.
LEPTOTENE
 the chromosomes begin to condense once SPECIMENS
leptotene takes place the cell is committed to A. Peripheral blood
meiosis  blood contain in a purple tub
ZYGOTENE  Sterile syringes or vacuum tubes containing
 chromosomes appear as threadlike structures preservative free sodium heparin
(synapsis)- necessary for the phenomenon on  Best sent 24 hours after collection
what we call crossing over  Room temp. Above 4 degree celsius
PACHYTENE B. Bone marrow aspirate
 synapsis will be completed  same requirement
 The chromosome continue to condense and will  First few millimeters, best sample
appear as thicker threads  Processed immediately to avoid cell death
 Crossing over takes place in this stage that results C. Amniotic fluid
in reshuffling or recombination of genetic material  15- 30 ml under sterile conditions
between homologs and create new combination  First few millimeters are contaminated with
genes and daughter cells maternal cells
D. Solid tissues
DIPLOTENE
 (skin biopsies, chorionic villi, products of
 Chromosomes will continue to shorten and thicken
conception, and stillbirth biopsies)
and the homologous chromosomes will begin to
 Culture media shall be integrated
repel each other.
 The culture media l-glutamine serum, antibiotics
 Chliasma- thickening of chromosomes
DIAKINESIS
HIGH RESOLUTION STUDIES
 Stage where chromosome reach their greatest  Chromosomes are routinely examined during
contraction metaphase when they are at their most
 Last stage of prophase 1 contracted state.
 The following are the techniques used to prevent
condensation of chromosomes:
METAPHASE 1
 disappearance of the nuclear membrane Cell synchronization techniques
 Bivalents will line up at the equatorial plate with  randomly dividing cells can be synchronized with
their centromeres oriented towards the opposite knowledge of the average timing stages of the
pole. human cell cycle
ANAPHASE 1
 Each centromeres of each bivalent separate and  Cell are Blocked and released at an appropriate
migrate to the opposite poles time so that a large percentage of cells
TELOPHASE 1 accumulate the prophase or prometaphase at the
 Two haploid sets of chromosome reaching time of harvest
opposite poles and the cytoplasm will divide that Chemical elongation techniques
results to two cells containing 23 chromosome  Bromide can be added to cultures prior to harvest
each composed of 2 chromatins to achieve longer chromosomes
METAPHASE II  Bromide acts by intercalating between the basis of
 The 23 chromosome line up on the equatorial DNA, thus preventing or slowing its contraction
plate and the chromatids separate and move to  Results in the collection of long if not truly
the opposite pole prometaphase chromosomes
ANAPHASE II  Used routinely in blood or bone marrow culture
TELOPHASE II
 The result are 4 cells and each contain 23 CULTURE FAILURE
chromosomes consisting of a single chromatid  All culture failures must be investigated.
 There are many possible origins of culture failure.
MODULE 5,6,7: EXAMINING & ANALYZING It can be the result of
CHROMOSOMES: BASIC LABORATORY 1. improper specimen collection or transport,
PROCEDURES 2. improper laboratory technique,
 Actively dividing cells are required in studying 3. or the condition of the sample.
cytogenetic techniques.  There are general sources of failure that apply to
 Chromosomes are best examined during all sample types and specific ones that pertain to
metaphase. one or more of the sample types.
PRESERVATION OF CELLS
 CYTOGENETICS
 Cultured cells can be kept alive by
cryopreservation, the storage of cell in liquid
nitrogen.
 The freezing process is critical to cell survival. The
cells must be cooled slowly so that water is lost
before the cells freeze. The addition of 10%
glycerol or dimethyl sulfoxide (DMSO) to the
storage medium lowers the freezing points and
aids in this process.
 One milliliter aliquots of the sample in storage
medium are placed in cryogenic freezing tubes.
The samples are then slowly frozen under
controlled conditions at a rate of 1°C per minute
to a temperature of –0°C.
 The sample can then be rapidly frozen to about –
0°C. Alternately, the samples can be placed in a –
0°C freezer for 1– hours.
 After this initial freezing has been accomplished,
the cells are stored in liquid nitrogen at about –
90°C.
 Thawing of the sample is also critical. Rapid
thawing is necessary to prevent the formation of
ice crystals.
 Lymphocytes can be transformed so that they will
proliferate indefinitely in tissue culture by
exposing them to Epstein–arr virus (EBV).
 These immortalized lymphoblastoid cell lines do
not become senescent and can, therefore, be
maintained indefinitely in culture.
CHROMOSOME ANALYSIS
 These begin with the microscope, where selection
of appropriate metaphases begins the process.
Often represent abnormal clones.
 Under high power, the chromosome morphology
and degree of banding (resolution) are evaluated.
Then the number of chromosomes is counted, and
the sex chromosome constitution is typically
determined.
 An“identifier” of the cell is also noted. This is
typically the position of one or more chromosomes
at some reference point(s) and serves to verify,
should there be a need to relocate a cell, that the
correct metaphase has been found.
 Any other characteristics of the metaphase being
examined, such as a chromosome abnormality or
quality of the banding and chromosome
morphology are also noted.

 Once the appropriate number of mitotic cells has

been examined and analyzed, a representative
sample must be selected for imaging and ultimate
preparation of karyotypes. This will involve either
traditional photography and manual arrangement
of chromosomes (becoming increasingly rare) or
computerized image capture and automated
karyotyping.
 The final steps of the process typically involve a
clerical review of all relevant clinical, technical,
and clerical data, examination of the patient’s
chart and karyotypes by the laboratory director
(often preceded by the supervisor and/or other
senior laboratory personnel), and generation of
the formal clinical report.
 In addition to the appropriate physician and
patient demographic information, this should
include the number of metaphases that were
examined microscopically, the banding resolution
obtained for the specimen, the number of cells
analyzed in detail, the number of karyotypes
prepared, the patient’s karyotype, and a clinical
interpretation of the results, including, where
appropriate, recommendations for additional
testing and/or genetic counseling.