CYTOGENETICS

History and Application

❖ Genetic Concept
- started with the literature of greeks Bazopoulou-Kyrkanidou (1992)
- constant lineages of kings and gods (basis of perception and theories on
heredity and procreation)

❖ Historical Summary of Genetics As Presented in Klug and Kummings (2005)

a. Charles Darwin; Theory of Evolution
b. Gregor Mendel; the dawn of Modern Genetics
c. Present; Modern and Molecular Genetics

❖ Important Pioneers
● Johannes and Zacharias Sachariassen (1588-1631)- two Dutch eyeglass
makers, father and son; produced the first operational compound microscope
between the years 1591 and 1608
● William Harvey (1578-1657)- Theory of Epigenesis; all living things,
including man, originate from eggs
● Marcello Malphigi (1628-1964)- discovered the microscopic anatomy of both
animals and plants; completed the story of the circulation
● Robert Hooke (1628-1694)- described cork and other cells and introduced the
term cell and made the first drawing of cells
● Regnier de Graaf (1641-1673)- discovered follicles in the human ovary and
identified them, incorrectly, as eggs; Graafian follicles; both sexes must
transmit agents of heredity
● Nehemiah Grew (1628-1711)- worked with Hooke in London and described
bladders and pores; understanding of the reproductive parts of plants
● Anton van Leeuwenhoek- incorrectly given credit for the invention of the
microscope; studies organisms living in pond water He calls them
"Animalcules."
● Schleiden and Schwann- summarize the findings of many scientists and
conclude that all living organisms are made of cells; basis of the Cell Theory
of Biology

❖ Various Applications

➔ Chromosome Identification. ➔ Analysis of chromosome

➔ Aneuploidy Detection translocation
➔ Centromere Analysis ➔ Detection of unique
➔ Identification of Marker sequence
Chromosome ➔ Microdeletion investigation
➔ Whole Chromosome ➔ Analysis of gene
Analysis amplification
 CYTOGENETICS
Introduction to Cytology

❖ Cytology- study of cells: began when Robert Hooke coined the word cellulae to
describe empty cell walls of cork
- Theodor Schwann concluded, about two centuries later, that all animal tissues
are made of cells
- Louis Pasteur established beyond any reasonable doubt that “cells arise only
from other cells”

❖ Modern Cell Theory

- All organisms are composed of cells and its products; cells are the simplest
structural and functional unit of life and come come only on pre-existing cells

❖ Components of a Cell
➢ Organelles- Cellular machinery; 2 kinds: derived from membranes and
bacteria-like organelles. Light microscope reveals plasma membrane, nucleus,
and cytoplasm (fluid between nucleus and surface membrane)
➢ Plasma (cell) membrane- Surrounds cell, defines boundaries and is made up of
proteins and lipids
➢ Cytosol- intracellular fluid

❖ Organic Molecules of Cells

➢ Proteins- Most diverse and complex macromolecules in the cell; for structure,
function and information and is made up of linear arranged amino acids
Types:
1. Enzymes – catalyzes covalent bond breakage or formation
2. Structural – collagen, elastin, keratin, etc.
3. Motility – actin, myosin, tubulin, etc.
4. Regulatory – bind to DNA to switch genes on or off
5. Storage – ovalbumin, casein, etc.
6. Hormonal – insulin, nerve growth factor (NGF), etc.
7. Receptors – hormone and neurotransmitter receptors
8. Transport – carries small molecules or iron
9. Special purpose proteins – green fluorescent protein, etc

❖ Lipids- hydrophobic; for energy storage, membrane components, signal molecules

1. Triglycerides
2. Phospholipids
3. Waxes
4. sterols
❖ Carbohydrates- Major substrates of energy metabolism (ex: Sugars, storage
(glycogen, starch), Structural polymers (cellulose and chitin))

❖ Water Molecule- its polarity allows the bonding of hydrogen; Imbalance of H+ and
OH- give rise to “acids and bases”
- pH influence charges of amino acid groups on protein, causing a specific
activity
- Buffering systems maintain intracellular and extracellular pH

❖ Plasma Membrane
➢ Unit membrane- forms the border of the cell and many of its organelles; dark
parallel lines around the cell
➢ Plasma membrane- unit membrane at the surface; Controls passage of
materials in and out of cell

❖ Membrane Lipids (98% of plasma membrane molecules)

➢ Phospholipids- 75% of membrane; they’re Amphiphilic molecules arranged in
a bilayer. They drift laterally and this movement keeps the membrane fluid
➢ Cholesterol- 20% of membrane lipids; holds phospholipids still and can stiffen
membranes. Sterols stabilize the membrane.
➢ Glycolipids- 5% of the membrane lipids. They’re phospholipids with short
carbohydrate chains on extracellular face and contributing to glycocalyx

❖ Membrane Proteins
- 2% of the molecules in plasma membrane and 50% of its weight
➢ Transmembrane Protein- pass through the membrane; have hydrophilic
regions in contact with cytoplasm and extracellular fluid and hydrophobic
regions that pass back and forth through the lipid of the membrane. Most are
glycoproteins
➢ Peripheral Proteins- adheres the the face of the membrane; tethered to the
cytoskeleton

❖ Functions of membrane proteins:

● Receptors- binds to chemical messengers such as hormones; surface proteins
on plasma membrane of target cell
● Second-messenger system- Triggers changes within the cell that produce a
second messenger in the cytoplasm
● Enzymes- breaks down a chemical messenger and terminates its effect
● Ion channel- channel protein that’s always open
● Carrier/Pumps- Transmembrane proteins bind to glucose, electrolytes, and
other solutes and consume ATP in the process
● Channel proteins- Transmembrane proteins with pores that allow water and
dissolved ions to pass through membrane
 ● Cell-identity markers- a glycoprotein that distinguishes the body’s own cell
from foreign cells
● Cell adhesion molecules (CAM)- binds one cell to another; Cells do not grow
or survive normally unless they are mechanically linked to the extracellular
material
➢ Glycocalyx- Unique fuzzy coat external to the plasma membrane for protections,
immunity, defense transplant compatibility, etc
➢ Cytoplasm- Viscous fluid containing organelles; made up of filaments and fibers,
fluid, organelles and storage substances

❖ Cell Organelles- internal structures that carry out specialized metabolic processes
➢ Membranous organelles
➔ Nucleus- has a nuclear envelope, chromatin, DNA and nucleolus
➔ Mitochondria- double membrane; powerhouse of the cell
➔ Lysosome- for intracellular digestion and breakdown of waste; also
aids in cell renewal
➔ Peroxisome- Hydrogen Peroxide generated and degraded
➔ ER- cell membrane and exported material is made; rough ER makes
protein; smooth ER makes lipids
➔ Golgi complex- receives and modifies new materials
➔ Cytosol- water based gel; chemical reactions
➔ Cytoskeleton- filaments (actin, intermediate, and microtubules) for
movement and strength
➔ Vesicles- for material transport
➢ Non Membranous organelles
➔ Ribosomes
➔ Centrosomes
➔ Centrioles
➔ Basal bodies

● Microvilli- Serves to increase the cell's surface area. Gives 15 to 40 times more
absorptive surface area. They may appear dense and appear like a brush border
● Cilia- Hairlike processes 7–10 m long; Single, nonmotile primary cilium found on
nearly every cell that has an antenna for monitoring nearby conditions
➔ Motile cilia- found in respiratory tract, uterine tubes, ventricles of the brain,
efferent ductules of testes; sweeps substances in the same direction
➔ Axoneme- core of cilia that is the structural basis for ciliary movement; Has 9
+ 2 structure of microtubules
● Flagella- Whiplike structure with axoneme identical to cilium; movement is more
undulating
● Centrioles- Each basal body of a cilium or flagellum is a single centriole oriented
perpendicular to plasma membrane
 ❖ Membrane Transport
➢ Passive transport- require no ATP (ex: Filtration, diffusion, osmosis)
➢ Active transport- consumes ATP (ex: Active transport and vesicular transport)
➢ Carrier-mediated mechanisms- use a membrane protein to transport substances
from one side of the membrane to the other
➢ Filtration- process in which particles are driven through a selectively
permeable membrane by hydrostatic pressure
➢ Simple diffusion- the net movement of particles from area of high
concentration to area of low concentration
➢ Osmosis- flow of water from one side of a selectively permeable membrane to
the other; reverse osmosis the the pressure applied to one side that overrides
pressure
➢ Aquaporins- channel proteins in plasma membrane specialized for passage of
water
➢ Channel Mediated Transport- Proteins form aqueous pores allowing specific
solutes to pass across the membrane that allow much faster transport than
carrier proteins

*osmolarity- number of osmoles of solute per liter of solution

*osmolality- number of osmoles of solute per kilogram of water
*tomicity- ability of a solution to affect fluid volume and pressure in a cell (ex: hypotonic,
isotonic, hypertonic)

➢ Coupled Transport- Some solutes “go along for the ride” with a carrier protein
or an ionophore
➢ Carrier-mediated transport
➔ Facilitated diffusion- carrier-mediated transport of solute through a
membrane down its concentration gradient; no ATP. The carrier
changes confirmation, then releases solute on other side of membrane
➔ Active transport- carrier-mediated transport of solute through a
membrane up (against) its concentration gradient; needs ATP (3
mechanisms: coupled carriers, ATP-driven pump and light-driven
pump) ex: sodium-potassium pump
➔ Secondary active transport- Steep concentration gradient maintained
between one side of the membrane and the other (water behind a dam)
➢ Vesicular transport- processes that move large particles, fluid droplets, or
numerous molecules at once through the membrane in vesicles—bubble like
enclosures of membrane
➔ Endocytosis- vesicular processes that bring material into the cell
1. Phagocytosis- aka cell eating; macrophages
2. Pinocytosis- cell drinking; taking in droplets of ECF containing
molecules useful in cell
 3. Receptor-mediated endocytosis- particles bind to specific
receptors on plasma membranes; Clathrin-coated vesicle. It's
more selective
➔ Exocytosis- discharging material from the cell
 CYTOGENETICS
Chromosomes and Cell Division

❖ Chromosomes- threadlike gene carrying structure found in the nucleus.

❖ Chromatin- complex of DNA and proteins that make up a eukaryotic chromosome
❖ Chromatid- usually paired and parallel strands of a duplicated chromosome joined by
a single centromere
❖ Telomere- protective structure at each end of eukaryotic chromosome

❖ Terminologies
➢ Gene- discrete unit of hereditary information consisting of a specific
nucleotide sequence in DNA (or RNA in viruses)
➢ Aneuploidy- abnormal number of chromosomes, and is a type of chromosome
abnormality
➢ Allele- alternative versions of a gene that produce distinguishable phenotypic
effects
➢ Homologous chromosome- 2 chromosomes composing a pair, having the same
length, centromere position and staining pattern
➢ Sex Chromosome- are the chromosome that determine the sex of organism
➢ Autosome- a chromosome that is not directly involved in determining sex, as
opposed to a sex chromosome
➢ Somatic Number- basic number of chromosomes in the somatic cells on an
individual or a species (2n)
➢ Polyploid- multiple copies of a chromosome

*Chromosomes 1-22: called autosomes

❖ Homologous pairs (1903)- closely matched in size and shape. The two members of
each pair of a homologous pair of chromosomes carry alleles for the same genes and,
therefore, affect the same traits (Chromosomal Theory of Inheritance)
❖ Sex chromosomes- determine the gender of the organism.

❖ Mapping Genes
➢ Locus- location of a gene on a chromosome.

❖ Regulation of Cell Cycle- if unregulated, it can result to cancer

➢ Metastasis- cell breaks free from original cancerous mass and resides in new
area in the body
➢ Checkpoints- between G1 and S also between G2 and M

❖ History
➢ Archibald Garrod (1909)- first to suggest that genes dictate phenotypes
through proteins produced
 ➢ Beadle & Ephrussi (1930s)- Studied mutations affecting eye color in
Drosophila. They also treated Neurospora with x-rays

*Crossing over- Exchange of genetic material between chromatids of homologous

chromosomes which happens meiosis (prophase I); it disrupts linkage groups

❖ Chemical Nature of the Gene

➢ 1860s- Frederich Meisner studied fundamental constituents of life; he
Discovered unknown substances containing carbon, nitrogen, oxygen, and
phosphorus. He named it nucelin but was changed nucleic acid by his students
after discovering it’s acidic

❖ Karyotyping- karyon = kernel, seed or nucleus; Used for the complete set of
chromosomes in a species or an individual. It describes the number of chromosomes
and what they look like under a light microscope
➢ Amniocentesis- obtaining amniotic fluid which has cells from the fetus
➢ Chorionic villi sampling- removing cells from the chorion with fetal tissue
➢ Chromosomes at metaphase under a microscope:
➔ Light bands- Replicate early in S phase Less condensed chromatin
Transcriptionally active Gene and GC rich
➔ Centromere-
➔ Telomere- and protein cap Ensures replication to tip Tether to nuclear
membrane
➔ Dark (G) bands- Replicate late Contain condensed chromatin AT rich
➢ Chromosomes are arranged into seven groups based on size and centromere
location.
➢ Centromeres can be found in:
➔ Median/metacentric (middle)
➔ Acrocentric- near to one end
➔ Submedian/ submetacentric- in between the first two
➔ Telocentric- have the centromere at one end
➢ Groups:
1. Group A- chromosomes 1-3 are largest with median centromere
2. Group B- chromosomes 4-5 are large with submedian centromere
3. Group C- chromosomes 6-12 are medium sized with submedian
centromere; similar to chromosome X
4. Group D- chromosomes 13-15 are medium sized with acrocentric
centromere
5. Group E- chromosomes 16-18 are short with median or submedian
centromere
6. Group F- chromosomes 19-20 are short with median centromere
7. Group G- chromosomes 21-22 are very short with acrocentric
centromere; similar to chromosome Y
 ❖ Chromosome Anomalies- Cause their effects by altering the amounts of products of
the genes involved
➢ Classifications:
➔ Numerical- usually due to de novo error in meiosis
● Aneuploidy
a. Monosomy
b. trisomy
● Polyploidy
a. triploidy
➔ Structural- may be due to de novo error in meiosis or inherited
● Translocation- Robertsonian (centric fusion)
● Deletion
● Duplication
● Inversion
➔ Different cell lines- occurs post-zygotically
● Mosaicism

MOST FREQUENT NUMERICAL ANOMALIES IN LIVEBORN

Autosomes Sex chromosomes All Chromosomes

Down syndrome (trisomy Turner syndrome 45,X Triploidy (69 chromosomes)

21: 47,XX,+21)

Edwards syndrome (trisomy Klinefelter syndrome

18: 47,XX,+18) 47,XXY

Patau syndrome (trisomy 13:

47,XX+13)

❖ Anaphase I- homologous chromosomes separate from the metaphase plate

➢ Nondisjunction- If chromosomes do not properly separate

❖ Summary of Chromosome Anomalies

➢ Down Syndrome- caused by trisomy of chromosome 21
➢ Patau Syndrome- caused by trisomy of chromosome 13
➢ Edwards Syndrome- caused by trisomy of chromosome 18
➢ Klinefelter’s Syndrome- most common male chromosomal disease, otherwise
known as 47, XXY is caused by an extra X chromosome
➢ Turner’s Syndrome- from a single X chromosome (45, X or 45, X0)
➢ Cri du Chat Syndrome- from a truncated short arm on chromosome 5. The
name comes from the babies' distinctive cry, caused by abnormal formation of
the larynx.
➢ 1p36 Deletion Syndrome- from the loss of part of the short arm of
chromosome 1
➢ Angelman Syndrome- 50% of cases have a segment of the long arm of
chromosome 15 missing; a deletion of the maternal genes, Ex. Imprinting
Disorder
➢ Prader-Willi Syndrome- 50% of cases have a segment of the long arm of
chromosome 15 missing; a deletion of the paternal genes
➢ Triple X Syndrome- form of chromosomal variation characterized by the
presence of an extra X chromosome in each cell of a human female
➢ XYY Syndrome- an aneuploidy (abnormal number) of the sex chromosomes
in which a human male receives an extra Y-chromosome, giving a total of 47
chromosomes instead of the more usual 46. 1 in 1000 in males
➢ 22q11.2 DELETION SYNDROME ( DIGEORGE SYNDROME)- caused by
the deletion of a small piece of chromosome 22 at a location designated
22q11.2—signifying its location on the long arm of one of the pair of
chromosomes 22, on region 1, band 1, sub-band 2
➢ Wolf Hirschhorn Syndrome- partial deletion of chromosomal material of the
short arm of chromosome 4 (del(4p16.3)).
➢ Acute Promyelocytic Leukemia- Translocation between chromosomes 15 and
17
 CYTOGENETICS
Mutations

❖ Types of Mutations
➢ Substitution- one nucleotide is substituted for another; can sometimes
be harmless
➢ Insertion- DNA is inserted into a gene, either one nucleotide or many
by viruses and transposable elements
➢ Deletion- DNA bases are removed.
➢ Duplication- an entire gene is duplicated
➢ Transposition- DNA is moved to a new place in the genome because of
errors in meiosis or transposable elements
➢ Frame shift- renders a gene meaningless caused by small insertions and
deletions; Proteins built incorrectly

❖ Mutation- change in genotype other than by recombination. They’re the only

source of new alleles. 3 types:
1. Point mutation- Change in a single DNA Nucleotide (1 in 100,000
gametes or 0.25 point mutations/gamete)
2. Chromosome mutation- Rearrangements (including losses and gains)
of large pieces of DNA (ex: inversion)
3. Change in chromosome number- ex: polyploidy
*CCR-d32 allele- codes for a 32 base pair deletion that makes the protein nonfunctional;
homozygous individuals (it is effectively codominant) are essentially resistant to HIV-HIV
cannot infect their cells

Genetic Engineering

❖ Gene- fundamental, physical and functional unit of heredity responsible for the
physical and inheritable characteristics of an organism.

❖ Chemical Structure of Genes

➢ Prior 1950s- “gene” was used to stand for the unit by which some genetic
characteristics passed to generation
➢ 1953- Englishc chemist Francis Crick & American biologist James Watson
created the DNA structure
➢ DNA- very long chains/units made up of a combination of simple sugar and
phosphate group with a nitrogenous base attached to it
❖ Gene Splicing- process in which fragments of DNA from one or more different
microorganism are combined to form rDNA (recombinant DNA) and are made to
function within the cell of a host organism
Techniques:
➔ Gene transfer
➔ Gene therapy- Correcting defective gene that are responsible for disease
development
➢ Plasmid- circular form of DNA often used as a vector in genetic engineering
➢ Vector- an organism/ chemical that is used to transport a gene to the host cell
➢ Enzymes Used:
● Endonucleases- cut DNA molecule at some given location
● Exonucleases- removes one nitrogen base unit at a time
● Ligases- join two DNA segments together

➢ Paul Berg (1926-)- “father of genetic engineering”; developed a method for

joining the DNA from two different organisms, a monkey virus known as
SV40 and a virus called lambda phage
➢ Stanley Cohen (1922) and Herbert Boyer (1936)- Francisco, discovered an
enzyme that greatly increased the efficiency of the Berg procedure

❖ Gene Therapy
➢ 4 approaches:
1. normal gene inserted to compensate for the defective gene
2. abnormal gene replaced with a normal one
3. abnormal gene repaired through selective reverse mutation
4. change the regulation of gene pairs
➢ First gene therapy was performed on September 14th 1990 when Ashanti
DeSilva was treated for SCID. Doctors removed her white blood cells,
inserted the missing gene into the WBC and then put them back into her
bloodstream

❖ Genetic engineering- manipulation/alteration of structure of a gene to create a desired

characteristic in an organism; coined by Jack Williamson
➢ 1973- Herbert Boyer and Stanley Cohen created the first transgenic organism
by inserting antibiotic resistance genes into the plasmid of an E.coli bacterium
➢ 1986- first trials of genetically engineered plants occurred in France and USA
wherein tobacco plants were engineered to be resistant to herbicides

❖ Transgenic plants
➢ Flavr Savr tomato was a tomato engineered to have a longer shelf life
➢ 1995- Bt Potato was approved safe by the Environmental Protection Agency
➢ Bt-Cotton is a genetically modified cotton which is resistant to pests
➢ Golden Rice genetically modified to contain beta-carotene (a source of
Vitamin A).
❖ Transgenic Animals
➢ Glo fish
➢ Dolion
➢ Dolly- was born 5 July 1996 to three mothers (one provided the egg, another
the DNA and a third carried the cloned embryo to term).
➢ Zorse- originally done in England and Africa to try to produce a domestic
horse like animal that was resistant to diseases spread by a fly in Africa

❖ Basic Steps of Genetic Modification

1. Isolating a gene to be inserted
2. Inserting the gene in a Vector
3. Inserting Vector into the host
4. Multiplication of host cells by cloning
5. Extraction of desired product

❖ Benefits:
➢ Vaccination- involves injecting weak live, killed or inactivated forms of
viruses or their toxins into the person being immunized
➢ Gene therapy- genetic engineering of humans by replacing defective human
genes with functional copies

❖ Gene Transfer
➢ Insulin- by “Genentech”, first genetic engineering company, founded by
Robert Swanson and Herbert Boyer

❖ Cloning
Types:
1. Gene cloning- describe a collection of DNA fragments derived from the
genome of an organism and cloned randomly into suitable cloning vectors
(plasmids, phages).
2. Reproductive cloning- the production of a genetic duplicate of an existing
organism
3. Therapeutic cloning-creates embryonic Stem Cells