CYTOGENETICS COMPILED TOPIC NOTES

TOPIC 1: ORIGIN AND IMPORTANCE OF CYTOGENETICS

1.1: Cytogenetics and Its Branches

What is Cytogenetics?

Cytogenetics is the study of chromosomes and the related disease states cause by abnormal chromosome
number and/or structure. The cell is the basic structural and functional unit of all organisms. The
chromosomes that cell contain are the containers of the hereditary factors which we call as the “gene”.
Chromosomes in cells that are abnormal in number and size have an abnormal effect to what the organism
will eventually become.

Cytogenetics if dissected is from two branches of science:

Cytology- study of cells

Genetics- study of heredity

Therefore, CYTOGENETICS tries to understand how cells produce what organism will eventually look like
including the genetic disorders it has. In modern times, cytogenetics or genetics itself has reached the
molecular level. This resulted to biochemical and molecular genetics.

What are the fields of Genetics?

1. Transmission Genetics- A scientist working in the field of transmission genetics examines the
relationship between the transmission of genes from parent to offspring and the outcome of the
offspring’s traits.
a. Example: How can two brown-eyed parents produce a blue-eyed child?
b. The following questions are under transmission genetics:
i. How are chromosomes transmitted during cell division and gamete formation?
ii. What are the common patterns of inheritance for genes?
2. Molecular Genetics- The goal of molecular genetics is to understand how the genetic material works
at the molecular level. It is understanding the molecular features of DNA and how these features
underlie the expression of genes.
a. The following questions are under Molecular genetics:
i. What is the composition and conformation of chromosomes?
ii. How is the genetic material copied?
iii. How is gene expression regulated so it occurs under the appropriate conditions, in
the appropriate cell type, and the correct stage of development?
iv. What is the molecular nature of mutation?
3. Population Genetics- this field helps us understand how process such as natural selection have
resulted in the prevalence of individuals that carry particular alleles. Population geneticists are
particularly interested in genetic variation and how that variation is related to an organism’s
environment. In this field, the frequencies of alleles are of central importance.
a. The following are questions under Population Genetics:
i. Why are two or more different alleles of a gene maintained in a population?
ii. What factors alter the prevalence of alleles within a population?
iii. What are the contributions of genetics and environment in the outcome of a trait?
iv. How do genetics and environment influence quantitative traits, such as size and
weight?

*Natural Selection- the process whereby organism better adapted to their environment tend to survive
and produce more offspring. The theory of its action was first fully expounded by Charles Darwin and is
now believed to be the main process that brings about evolution. (Definition from Oxford Dictionary)

1.2: The History of Cytogenetics

Historical Development

By the middle of the 19th century, the universality of cell division as the central phenomenon in the
reproduction of organisms was established, and Virchow expressed it in the famous aphorism “Omnis
cellula e cellula”. From this time on, the study of cells and of heredity and evolution converged, as was
stated by Wilson: “Heredity appears as a consequence of genetic continuity of the cells by division”

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Notable Persons and Discoveries:

• Van Beneden, Flemming, Strasburger, Boveri and others- observed germ cells which
supported the theory of the continuity of the germ plasm proposed by Weisman in 1883.
o GERM THEORY states
o that the transference of hereditary factors from one generation to the next takes place
through the continuity of what he called ‘germ plasm’, located on the sex elements
(Sperm and Egg), and not through somatic cells.
• Discovery of Fertilization in Animals- foreseen by O. Hertwig but observed directly by H. Fol
(1879)
• Discovery of Fertilization in Plants- observed by Strasburger.; both discovery led to the theory
that the cell nucleus is the bearer of the physical basis of heredity
• Roux- postulated that chromatin, the substance of the nucleus that constitutes the chromosome,
must have a linear organization
• Weismann- stated that hereditary units are disposed along the chromosomes in an orderly
manner
• Gregor Mendel- discovered the Fundamental Laws of Heredity in 1865, but at that time
cytologic changes produced in the sex cells were not sufficiently known to permit an
interpretation of the independent segregation of hereditary characters. For this and other
reasons, little attention was paid to Mendel’s work until the botanists Correns, Tschermack
and De Vries in 1901 independently rediscovered Mendel’s Law

1865/ 1866 Gregor Mendel published his investigations into inheritance of pea plants
1890 Theodor Boveri suggested that chromosomes are involved with inheritance
1900 Walter Sutton observed chromosomes in grasshopper cells
1900/1901 Mendel's work was rediscovered by three scientists: Hugo De Vries, Erich von
Tschermack, and Carl Correns
1902 Archibald Garrod discovered that some diseases must be inherited

1903 Sutton and Boveri, working independently, suggested that each egg of sperm cell
contains only one of each chromosome pair
1905 Edmund Beecher Wilson and Nettie Stevens, working independently, proposed that
certain chromosomes determine sex. They show that a single Y chromosome
determines maleness, and two copies of the X chromosome determine femaleness
1906 Bateson gave the term ‘genetics’
1909 Wilhelm Johannsen used the term 'gene' to describe the carrier of heredity,
'genotype' to describe an organism's genetic make-up, and 'phenotype' to describe
an organism's outward appearance
1910 Thomas Hunt Morgan proved that genes are carried on chromosomes. He also
showed that some characteristics are carried on the sex chromosome
1911/1913 Alfred Henry Sturtevant mapped the genes o the fruit fly’s sex chromosome
1912 Sir William Henry Bragg and his son discover that X-rays can be used to study the
molecular structure of simple crystals, such as salt
1926 Morgan published the ‘Theory of the Gene’
1928 Frederick Griffith discovered 'transformation' in bacteria
1944 Oswald Avery, Colin MacLeod, and Maclyn Mccatty used bacteria to show that DNA is
the hereditary material
1949 Erwin Chargaff finds that the amounts of adenine and thymine in DNA are about the
same, as area the amounts of guanine and cytosine
1953 James Watson and Francis Crick proposed that the DNA molecule is a double-
stranded helix
1963-1966 Marshall Nirenberg and Heinrich Matthaei work out the genetic code
1977 DNA from virus is sequenced for the first time by Frederick Sanger, Walter Gilbert
and Allan Maxam, working independently
1983 Kary Mullis discovered the Polymerase Chain Reaction (PCR), enabling lengths of
DNA to be multiplied
1987 Rebecca Cann, Mark Stoneking, and Allan Wilson analyze mitochondrial DNA in
different human races. They declared that humans have a common ancestor who
lived 200,000 years ago

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 1989 The first Human gene is sequences by Francis Collins and Lap-chee Tsui. It is the
gene that cause cystic fibrosis
1990 The Human Genome Project is launched
1993 Cystic fibrosis became the first genetic disease to be treated using gene therapy
1995 The genome of H.influenzae is sequenced. This is the first complete genome of an
organism
2000 First draft sequences of human genome are released at the same time by the Human
Genome Project and Celera genomics
2003 The Human Genome Project is successfully completed on 14th of April

Reference: Camara, J.S and Oclay, A. (2012). Cytogenetics: Principles and Application. Dagupan City:
Space Browser Publishing p.4-5

TOPIC 2: CYTOLOGICAL BASIS OF HEREDITY

2.1: Origin and Importance of Cytogenetics

The body of all living organisms (bacteria, blue green algae, plants and animals) except viruses has
cellular organizations and may contain one or many cells. The organisms with only one cell in their body
are called unicellular organisms (e.g bacteria, blue green algae, some algae, Protozoa). The organisms
having many cells in their body are called multicellular organism (e.g most plants and animals).

Any cellular organisms may contain only one type of cell from the following types of cells:
A. Prokaryotic cells; B. Eukaryotic Cells
*The term prokaryotic and Eukaryotic were suggested by Hans Ris in the 1960’s.

PROKARYOTIC CELLS:

From Gr. pro=primitive or before; karyon=nucleus; they are small, simple and most primitive.
They are probably the first to come into existence about 3.5 billion years ago; essentially a one-
envelop system organized in depth. It consists of central nuclear components (viz., DNA molecule,
RNA Molecule and nuclear proteins) surround by cytoplasmic ground substance; the cytoplasm of
a prokaryotic cell lacks in well defined cytoplasmic organelles; nuclear envelope; nucleoli,
cytoskeleton, centrioles and basal bodies.

Ex. Bacteria

EUKARYOTIC CELLS:

From Gr., eu=well; karyon= nucleus; they have evolved from the prokaryotic cells and the first
eukaryotic (nucleated) cells may have arisen 1.4 billion years ago (Vidal, 1983); essentially two
envelope systems and they are much larger than prokaryotic cells. Secondary membranes envelop
the nucleus and other internal organelles;

The eukaryotic cells are true cells which occur in the plants and animals.

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Reference: Verma, P.S and Agarwal, V.K (2005). Cell Biology, Genetics, Molecular Biology, Evolution and
Ecology. Ram Nagar, New Delhi: S. Chand & Company LTD. p.42-43; 54

2.2: CELL STRUCTURE AND ITS FUNCTION

The cell is the basic unit of organizations or structure of all living matter. Within a selective and retentive
semipermeable membrane, it contains a complete set of different kinds of units necessary to permit its own
growth and reproduction from simple nutrients.

All forms of life, except viruses, consist of cells; Consists of two distinct areas which in living cells, are in
constant motion: Cytoplasm and Nucleus

Cell Structure

Structure & Organelles Function

Cytoplasm -major portion of the protoplasmic substance
contained in the cell membrane
- several organelles (functionally important for the
survival of the cell and their presence or size may
vary between different organisms and different
tissues) are found esp. in eukaryotes

Endoplasmic Reticulum -an organelle where lipid production and some

protein translation occurs
-Rough ER has attached ribosomes
-Smooth ER has no attached ribosomes; functions
in lipid metabolism (both catabolism and
anabolism; they synthesize a variety of
phospholipids, cholesterol and steroids);
glycogenolysis and drug detoxfication
Golgi Apparatus -a cup-shaped organelle which is located near the
nucleus in many cell;
-consists of a set of cisternae (i.e, closed fluid-
filled flattened membranous sacs or vesicles)
- Function:
1. packaging of secretory materials that are to be
discharged from cell
2. The processing of proteins
3. The synthesis of certain polysaccharides and
glycolipid
4. The sorting of proteins destined for various
location
5. Proliferation of membranous element for the
plasma membrane
6. Formation of acrosome of the spermatozoa

Ribosome – small particles which may be free floating in the

cytoplasm or attached to the ER
- play an important role in the synthesis of protein
-May exist either in the free state in the cytosol or
attached to RER

Mitochondria -structures where most of the cellular energy is

produced in the form of ATP
Chloroplast -plastids in plant cell which contain chlorophyll
and serve as the photosynthetic factory of the
plants

* both mitochondria and chloroplasts are found in

plants, algae and some protozoans
* they also posses their own genomic DNA which
are circular and are not complexed with proteins
unlike the nuclear genome

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Centrosome –unlike organelles, not bound by membranes to
separate it from the surrounding cytosol
- found in most animals and lower plant cells
-consists of two cylindrical structures called
centrioles

* The centrosome serves as the organizing

unit for microtubules

Microtubule -hollow tubes of dynamic protein polymers

composed of subunits that contain 1 mol of α
tubulin and 1 mol of β tubulin;
-extend and retract to provide shape and
structure to eukaryotic cell
-they form the network that internal components
move along to their proper destination within the
cell

Spindle Fibers -attach to chromosomes during the early stages

of mitosis and meiosis are also composed of
microtubules

*Thus, centrosome is essential for the correct

formation of spindle fibers and the proper
movement of eukaryotic chromosomes during
mitosis and meiosis.
*In some organisms, such as fungi, the spindle
pole body serves the function of the centrosome.

Nucleus – the dark staining body within the cytoplasm

- contains the primary genome of the cell which is
organized as linear, double-stranded DNA that is
complexed w/ protein(nucleoprotein)
- primary director of cellular activity and
inheritance
- surrounded by a double membrane that
appears in active contact w/ ER and the cell
membrane.
- nuclear content consists of a dark network or
chromatin (during cell division becomes distinct
bodies or chromosomes)

Nucleoli/Nucleolus -1 or more spherical bodies may be found

attached to specific chromosome regions
-the site where ribosomes are manufactured;
where ribosomal DNA transcribes most of rRNA
molecules
Nuclear Envelope -comprises two nuclear membranes- inner
nuclear membrane which is lined by nuclear
lamina and an outer nuclear membrane which
is continuous with RER
-the nuclear envelope is interrupted by structures
called pores or nucleopores which contains
pore complexes that regulates the exchange
between nucleus and cytoplasm.

COMPARISON OF PROKAYORTIC AND EUKARYOTIC CELLS

Prokaryotes Eukaryotes

Typical organisms bacteria, archaea protists, fungi, plants, animals

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 ~ 10-100 µm (sperm cells, apart
Typical size ~ 1-10 µm
from the tail, are smaller)

real nucleus with double

Type of nucleus nucleoid region; no real nucleus
membrane

linear molecules (chromosomes)

DNA circular (usually)
with histone proteins

RNA-synthesis inside the nucleus

RNA-/protein-synthesis coupled in cytoplasm
protein synthesis in cytoplasm

Ribosomes 50S+30S 60S+40S

highly structured by
Cytoplasmatic structure very few structures endomembranes and a
cytoskeleton

flagella and cilia containing

Cell movement flagella made of flagellin microtubules; lamellipodia and
filopodia containing actin

one to several thousand (though

Mitochondria none
some lack mitochondria)

Chloroplasts none in algae and plants

single cells, colonies, higher

Organization usually single cells multicellular organisms with
specialized cells

Mitosis (fission or budding)

Cell division Binary fission (simple division)
Meiosis

DNA Content 1 × 106 to 5 × 106 1.5 × 107 to 5 × 109

Reference: Verma, P.S and Agarwal, V.K (2005). Cell Biology, Genetics, Molecular Biology, Evolution and
Ecology. Ram Nagar, New Delhi: S. Chand & Company LTD. p.42-43; 54

Powerpoint Prepared by Mr. Von Carlo P. Dela Tore, M.S.C

2.3: THE CHROMOSOME

Chromosomes are complex structures located in the cell nucleus. They are composed of DNA, histone and
non-histone proteins, RNA, and polysaccharides. They are basically the “packages” that contain the DNA.
Normally chromosomes cannot be seen with a light microscope but during cell division, they become
condensed enough to be easily analyzed at 1000x. To collect cells with their chromosomes in this condensed
state they are exposed to a mitotic inhibitor which blocks formation of the spindle and arrests cell division
at the metaphase stage.

Discovered by Karl von Nageli (1842) after staining techniques were developed that made them visible

Heinrich Wilhelm Waldeyer (1888)- coined the term chromosome (means “colored body”)

Genes are located on the chromosomes which exist as chromatin network in the non dividing cells

Chromatin

- linear eukaryotic chromosomes are composed of a complex double-stranded DNA and protein; Exist in
two forms:

a. Euchromatin – found in a loosely packed state; involved in gene duplication, gene transcription
and phenogenesis or phenotypic expression of gene through some type of protein synthesis

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 b. Heterochromatin- highly condensed and readily visible organization; exist both in the region of
centromere and in sex chromatin and is late replicating one

What does chromosomes do?

The unique structure of chromosomes keeps DNA tightly wrapped around spool-like protein called
“histones”. Without such packaging, DNA molecules would be too long to fit inside cells. For example, if all
of the DNA is a single human cell were unwound from their histones and placed end-to-end, they would
stretch 6 feet; they are a key part of cell division that ensures DNA is accurately copied and distributed in
the vast majority of cell divisions.

The chromosome structure

Chromosomes

• Under the microscope, chromosome appear as thin, thread-like structures. They all have
a short arm designated as “P” meaning petite and a long arm designated as “Q”, they are
separated by a primary constriction called the “centromere”

• The centromere is the location of spindle attachment and is an integral part of the chromosome;
essential for the normal movement and segregation of chromosomes during cell division

• Chromosomes maintain constant size and shape at specific stages of the cell cycle

• Condensed chromosomes may be as short as 0.25 μ (in fungi

and birds) or as long as 30 μ in Trillium sp.

• Kinetochore - the proteinaceous structure on the surface of

the centromere to which the microtubules attach

Each DNA Molecule That Forms a Linear Chromosome Must

Contain a Centromere, Two Telomeres, and Replication
Origins

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Figure 4-22. The three DNA sequences required to produce a Eukaryotic chromosome that can be replicated
and then segregated at mitosis. Each chromosome has multiple origins of replication, one centromere, and
two telomeres. Shown here is the sequence of events a typical chromosome follows during the cell cycle.
The DNA replicates in interphase beginning at the origins of replication and proceeding bidirectionally from
the origins across the chromosome. In M phase, the centromere attaches the duplicated chromosomes to
the mitotic spindle so that one copy is distributed to each daughter cell during mitosis. The centromere
also helps to hold the duplicated chromosomes together until they are ready to be moved apart. The
telomeres form special caps at each chromosome end.

-Secondary Constriction

- further constriction may be observed in some chromosomes; this may include pinching off of a
small chromosomal section called the satellite
-these secondary constrictions are often associated with regions where the nucleolus is formed or
attached

-Nucleolus-forming regions

- the organization of the nucleolus is the function of a specific point on a particular chromosome.
When a nucleolus is visible, it can be seen to be attached to this nucleolus-organizing region. The
chromosome where this region is located is known as the nucleolus organizer.

Chromomeres and knobs

-When a mitotic chromosome is stretched out it would be observed to consist of a string of

characteristic particles of unequal sizes at unequal distances apart. The smaller “beads on the string” are
called chromomeres (visible dark bands); the larger ones are called knobs.

TYPES OF CHROMOSOME

Human metaphase chromosomes come in three basic shapes and can be categorized according to the
length of the short and long arms and also the centromere location:

1. Metacentric- these chromosomes have short and long arms of equal length with centromere in the
middle.
2. Sub-metacentric- these chromosomes have short and long arms of unequal length with the
centromere more towards one side
3. Acrocentric- These chromosomes have a centromere very near to one end and have very small
short arms. They frequently have a secondary constrictions on the short arms that connect very
small pieces of DNA, called “Stalks” and “Satellites”, to the centromere. The stalks contain aenes
which code for ribosomal RNA.

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Chromosome Complement

- a particular species possesses a constant number of chromosomes.

Cells may be either:

1. Diploid – contain two sets of chromosomes

- found in most cells of eukaryotic organisms

- 2n=46 (humans)

*humans have 23 homologous chromosome pairs

Homologous chromosomes or homologs -members of the same chromosome pair

2. Haploid – contain one set of chromosomes

- include reproductive cells or gametes

- eggs and sperm are haploid cells that contain that contain one member of each
homologous pair (n=23)

Species 2n

Human being (Homo sapiens) 46

Garden pea (Pisum sativum) 14

Fruit fly (Drosophila melanogaster) 8

House mouse (Mus musculus) 40

Roundworm (Ascaris sp.) 2

Pigeon (Columba livia) 80

Boa constrictor (Constrictor constrictor) 36

Cricket (Gryllus domesticus) 22

Lily (Lily longiflorum) 24

Indian fern (Ophioglossum reticulatum) 1260

1. Homomorphic chromosome pairs

- homologous pairs are made up of identical partners

2. Heteromorphic chromosome pair

- have unequal size and composition (eg. Sex chromosomes)

a. homogametic sex (females in mammals)

- because all their gametes contain an X chromosome

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 b. heterogametic sex (males in mammals)

- because their gametes contain either an X or Y.

Reference: Verma, P.S and Agarwal, V.K (2005). Cell Biology, Genetics, Molecular Biology, Evolution and
Ecology. Ram Nagar, New Delhi: S. Chand & Company LTD. p.42-43; 54

Camara, J.S and Oclay, A. (2012). Cytogenetics: Principles and Application. Dagupan City: Space Browser
Publishing p.4-5

Powerpoint Prepared by Mr. Von Carlo P. Dela Tore, M.S.C

2.4: CELL CYCLE

Cell cycle refers to the regular and repetitive physical and chemical process taking place within the cell. A
cell cycle is a cycle which means there is no fixed starting point. The first major phase of the cell is the
interphase. Then, the second major phase- the M Phase. Cell cycle is simply cell reproduction.

A. Interphase

1. G1 phase

2. S-phase

3. G2 phase

B. Mitosis phase

Interphase

-cells spends most of their lives in interphase. In this phase of the cell cycle, cells are not actively
dividing.; chromosomes are uncondensed throughout interphase. During G1, cells undergo a period of
rapid growth, and the chromosomes are unduplicated. During the S phase, cells begin to prepare for division
during interphase by duplicating its chromosomes. At the end of S Phase, all the chromosomes are therefore
duplicated chromosomes. During G2, the cell again grows and complete the preparation for division (mitosis
or M Phase).

1. G1 (First Gap Phase)

• First stage of interphase
• The protein synthesis and RNA synthesis within the cell resumes that was interrupted
during the process of mitosis
• Growth and young cell maturation occurs, which accomplish the physiological function
• The phase during which the cell cycle starts with synthesis of RNA and protein required by
the young cells for their growth and maturity. G1 phase is usually termed as the prior to
DNA Synthesis phase.
2. S Phase
• The second stage of Interphase
• DNA synthesis takes place; S stands for synthesis

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 • Soon after the G1 phase, DNA checking and subsequent repair occurs during the variable
pause phase before the transition of the cell cycle to the S phase
• The S phase of the interphase deals with the semi-conservative synthesis of DNA occurs.
• Replication of cellular DNA begins with the S phase, which when gets duplicated with the
cell containing nearly double the amount of chromosomes, the cells from the S phase move
into the G2 Phase
3. G2 Phase (Second Gap Phase)
• The third stage of interphase
• There is an increase in the synthesis of the RNA and the protein, which is followed by
another round of proof reading and subsequent repair among the newly synthesized DNA
sequences before the cell cycle transits to the mitotic cycle
• The mitotic spindle formed from the cytokinetic fibers start forming and the cell ensures
the number of chromosomes and the organelles present, which further leads the cell cycle
from the interphase to the mitotic phase.

Typical Cell Cycle in a cell culture

INITIATION OF MITOSIS

• Mitosis Promoting Factor

o protein complex which initiates the mitotic phase of the cell cycle
o made of two proteins:
1. Cyclin B - one that oscillates in quantity during the cell cycle

2. CDC2- one whose quantity is constant

- encoded by the cdc2 gene
- a kinase (an enzyme that transfers a phosphate group on to a protein by
phosphorylation)

*CDC2 kinase is only functional when it is combined with cyclin which is referred as
cyclin-dependent kinase (CDK)
*Once mitosis has been initiated, anaphase-promoting complex (APC) or
cyclosome:
1. degrades the Cyclin B protein of MPF
2. permits the separation of the sister chromatids at the start of anaphase

CELL CYCLE CHECKPOINTS

Checkpoints refers some points in the cell cycle which allow the cell to make sure that various events
have been properly completed before the next phase begins

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 1. G1/S Checkpoint-
a. If the G1/S checkpoint detects damage, the p53 protein targets the cell for regulated
death (apoptosis)

2. Spindle Attachment Checkpoint

a. ensures that spindle fibers are attached to every kinetochore before the sister
chromatids attempt to separate
b. involves several proteins including mitotic arrest-deficient protein 2 (MAD2)
(binds to kinetochore)

MITOSIS

Mitosis only occurs among somatic cells or body cells.

It is a form of eukaryotic cell division that produces
two daughter cells with the same genetic component
as the parent cell. (- 2n = 2n)

In some single-celled organisms mitosis forms the

basis of asexual reproduction. In diploid multicellular
organisms sexual reproduction involves the fusion of
two haploid gametes to produce a diploid zygote.
Mitotic divisions of the zygote and daughter cells are
then responsible for the subsequent growth and
development of the organism. In the adult organism,
mitosis plays a role in cell replacement, wound
healing and tumor formation.

Mitosis, although a continuous process, is conventionally divided into five stages: prophase, prometaphase,
metaphase, anaphase and telophase.

1. Prophase
a. -Chromatin in the nucleus begins to condense and becomes visible in the light
microscope as chromosomes
b. The nucleolus
disappears
c. Centrioles begin moving
to opposite ends of the cell
and fibers extend from the
centromeres; some fibers
cross the cell to form the
mitotic spindle
d. The number of
microtubules that attach to
each kinetochore differs in
different species; 1
microtubule attaches per
kinetochore in yeast 4-
7/kinetochore in cells of rat
fetus 70 to 150 attach in the
plant Haemanthus katherinae
2. Prometaphase
a. The nuclear membrane dissolves, marking the beginning of prometaphase
b. Proteins attach to the centromeres creating the kinetochores
c. Microtubules attach at the kinetochores and the chromosomes begin moving
3. Metaphase

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 a. Spindle fibers align the chromosomes along the middle of the cell nucleus; line is
referred to as the metaphase plate; this organization helps to ensure that in the next
phase, when the chromosomes are separated each new nucleus will receive one copy
of each chromosome
4. Anaphase
a. The paired chromosomes separate at the kinetochores and move to opposite side of
the cell. Motion results from a combination of kinetochore movement along the spindle
microtubules and through the physical interaction of polar microtubules
5. Telophase
a. Chromatids arrive at opposite poles of cell, and new membranes form around the
daughter nuclei
b. The chromosomes disperse and are no longer visible under the light microscope
c. The spindle fibers disperse, and cytokinesis or the partitioning of the cell may also
begin during this stage

CYTOKINESIS

In animal cells, cytokinesis results when a fiber ring composed of protein called actin around the center of
the cell contracts pinching the cell into two daughter cells, each with one nucleus. In plant cells, the rigid
wall requires that a cell plate be synthesized between the two daughter cells.

This separation of the genetic material in a mitotic nuclear division (or karyokinesis) is followed by a
separation of the cell cytoplasm in a cellular division (or cytokinesis) to produce two daughter cells.

SIGNIFICANCE OF MITOSIS

Mitosis is important for the maintenance of the chromosomal set; each cell formed receives chromosomes
that are alike in composition and equal in number to the chromosomes of the parent cell.

*With this stability ensured, single-celled organisms could thrive and multicellular organisms could evolve.

PROPHASE

ME

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METAPHASE TELOPHASE

ANAPHASE

MEIOSIS

The main goal of mitosis is to preserve the chromosomes of the parent cells to the daughter cells in order
to attain genetic continuity. This means that the information of one kind of skin cell is the same in all skin
cells. This kind of cell division happens in all cells except to sex cells.

Sex cells (gametes), which are haploid cells, undergo a special type of cell division which is properly
called “meiosis”. The goal of meiosis is to reduce the number of chromosomes in half, so that when
fertilization occurs, the number of chromosomes will be reestablished.

PHASES OF MEIOSIS

Two successive nuclear divisions occur, Meiosis I (Reduction) and Meiosis II (Division). Meiosis produces
4 haploid cells. Mitosis produces 2 diploid cells. The old name for meiosis was reduction/division. Meiosis
I reduces the ploidy level from 2n to n (reduction) whole Meiosis II divides the remaining set of
chromosomes in a mitosis-like process (division).

MEIOSIS I

1. Prophase I
a. Has a unique event- the pairing (by an as yet undiscovered mechanism) of homologous
chromosomes.
b. Synapsis – is the process of linking of the replicated homologous chromosomes. The
resulting chromosome is termed tetrad, being composed of two chromatids from each
chromosome, forming a thick (4-strand) structure
c. Crossing over- may occur at this point; during crossing-over chromatids break and may be
reattached to a different homologous chromosomes; crossing over between homologous
chromosomes produce chromosomes with new association of genes and alleles
d. Prophase I is further subdivided:

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 i. Leptotene- means thin thread; chromosomes first become visible as long, thread-
like structures; initial phase of condensation of the chromosomes
ii. Zygotene- means paired thread; marked by lateral pairing, or synapsis, of
homologous chromosomes, beginning at the chromosome tips
iii. Pachytene- means thick thread; condensation of the chromosome continues;
throughout this period, the chromosomes continue to shorten and thicken;
genetic exchange or crossing over happens here; but crossing over is not
apparent until the transition to diplotene
iv. Diplotene- means double thread; the synapsed chromosomes begin to separate,
and the diplotene chromosomes begin to double; each cross connection called
chiasma, is formed by a breakage and rejoining between nonsister chromatids
v. Diakenesis- final period; means moving apart; the homologous chromosomes
seem to repel each other and the segments not connected by chiasmata move
apart; near the end of Diakenesis, the formation of a spindle is initiated and the
nuclear envelope breaks down.
2. METAPHASE I
a. Tetrads line-up along the equator of the spindle
b. Spindle fibers attach to the centromere region of each homologous chromosome pair;
other metaphase events as in mitosis
3. ANAPHASE I
a. when tetrads separate, and are drawn to opposite poles by the spindle fibers. The
centromeres in Anaphase I remain intact
4. TELOPHASE I
a. Similar to telophase of mitosis, except that only one set of (replicated) chromosomes is in
each “cell”
b. Depending on species, new nuclear envelopes may or may not form; some animal cells
may have division of the centrioles during this phase

*At the beginning of Telophase I, each half of the cell has a complete haploid set of chromosomes, but
each chromosome is still composed of two sister chromatids
*Cytokinesis usually occurs simultaneously with Telophase I forming two hapoloid daughter cells
*No chromosome replication occurs between the end of meiosis I and the beginning of meiosis II, as the
chromosomes are already replicated

MEISOSIS II

1. Prophase II
a. A spindle apparatus forms
b. In late prophase II, chromosomes, each still composed of two chromatids, move toward
the opposite plate
2. Metaphase II
a. Chromosomes are positioned in the metaphase plate as in mitosis
b. Because of crossing over in meiosis I, the two sister chromatids of each chromosomes
are “not” genetically identical
c. The kinetochores of sister chromatids are attached to microtubules extending from
opposite poles
3. Anaphase II
a. The centromeres of each chromosome finally separate, and the sister chromatids come
apart
b. The sister chromatids of each chromosome now move as two individual chromosome
toward opposite poles
4. Telophase and Cytokinesis
a. Nuclei form, the chromosome begin decondensing, and cytokinesis occurs
b. Meiotic division of one parent cell produces four daughter cells, each with a haploid set
of (unreplicated) chromosome
c. Each of the four daughter cells is genetically distinct from the other daughter cells and
from the parent cell

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TOPIC 3: Inheritance; Mendelian Principle

3.1: Mendelian Genetics

Gregor Mendel

• The pioneer of classical geneticist

• Austrian Monk, born in what is now Czech Republic in 1822
• Son of peasant farmer, studied Theology and was ordained priest Order St. Augustine.
• Went to the university of Vienna, where he studied botany and learned the Scientific Method
• Worked with pure lines of peas for eight years
• Prior to Mendel, heredity was regarded as a "blending" process and the offspring were essentially
a "dilution" of the different parental characteristics
• Mendel was the first biologist to use Mathematics – to explain his results quantitatively.
• Mendel predicted
o The concept of genes
o That genes occur in pairs
o That one gene of each pair is present in the gametes

Mendel’s Pea Experiment

1. Choosing true-breeding pea plants

Mendel chose a pea plants that is true breeding for trait like round seeds. He chose a pea plant
that is also true breeding for the contrasting trail like a wrinkled seed. (True breeding means
all of the offspring look like the parents, i.e all round or all wrinkled)

2. Cross pollinating the parent plants.

He cross-pollinated the true-breeding round-seed pea plant to the true-breeding wrinkled

seeded pea plant. These pea plants can to be known as the ‘parental generation’

3. The result of cross-pollination of P is F1.

The parents (parental generation, or simple ‘P’) are allowed to self-fertilize. These generation
produced progenies, or offspring. The offspring of the cross is called first filial generation, or
simple F1. In the case round vs. wrinkled, it was observed that the F1 is round seeded and
not-wrinkled-seeded. In short, the round seeded trait is expressed; the wrinkled-seeded trait
is masked.

4. Allowing self-fertilization of F1 producing F2

Mendel allowed the first filial generation to self-fertilize. The result of the self-fertilization is a
new generation of progenies called second filial-generation or simple F2. In the case of the
round-seeded F1, some of its progenies are usually round-seeded and a few are wrinkled-
seeded. In short, the round-seeded trait still appeared; the wrinkled-seeded trait reappeared.

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MENDEL’s POSTULATE

Mendel, after his experiments, arrived to some generalizations which are known as postulates. Mendel did
not actually state them as postulates, nor did he propose a theory of inheritance.

1. Traits are controlled by heritable factors

2. Factors are passed from parent to offspring in reproductive cells
3. Each individual contains pairs of factors in every cell except reproductive cells
4. Paired factors segregate during the formation of reproductive cells so that each reproductive cell
gets one of the factors of a pair.
5. There is an equal chance that a reproductive cell will get one or the other factor of a pair.
6. Each factors from one parent has an equal chance of combining either with the identical factor or
with the other factor from the other parent during fertilization.
7. Sometimes one factor dominates the other factor; in such cases, the dominant factor controls the
feature of the plant
8. When two or more traits are under consideration, the factors for each trait assort independently to
the reproductive cells.

MENDEL’S LAW OF INHERITANCE/ MENDEL’S PRINCIPLE

1. Principle of Dominance
a. IF two contrasting traits (round vs. wrinkled) code for the same character (Seed shape),
one trait s dominant while the other is recessive. The dominant trait is always expressed
in F1; while the recessive trait is masked only to reappear in the F2 generation. The round
and wrinkled are called alleles, the alternative versions of a gene.
2. Law of Segregation
a. The two alleles for a heritable character separate or segregate during gamete formation,
and end up in different gametes. Thus, an egg or a sperm gets only one of the two alleles
that are present in the body cells of the organism
3. Law of Independent Assortment
a. Each pair of alleles segregate independently of other pairs of alleles during gamete
formation. This follows at least tracing the inheritance of two heritable characters; Genes
do not influence each other with regard to the sorting of alleles into gametes; every
possible combination of alleles for every gene is equally likely to occur; Genes get shuffled
– these many combinations are one of the advantages of sexual reproduction
b. Example in dihybrid crosses

3.2: Monohybrid and Dihybrid cross

MONOHYBRID CROSS

Is a genetic mix between two individuals who have a homozygous genotypes or genotypes that have
completely dominant or completely recessive alleles, which result in opposite phenotypes of certain genetic
trait.

PUNNETT SQUARE

• A useful tool to do genetic crosses

• For a monohybrid cross, you need a square divided by four that looks
like a window pane
• We use the Punnett square to predict the genotypes and phenotypes
of the offspring.

Steps in Using the PUNNETT SQUARE

STEPS:

1. determine the genotypes of the parent organisms

2. write down your "cross" (mating)

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 3. draw a p-square
4. Parent genotypes:

TT and t t

Cross TT  tt

5. "split" the letters of the genotype for each parent & put them "outside" the p-square
6. determine the possible genotypes of the offspring by filling in the p-square
7. summarize results (genotypes & phenotypes of offspring)

T- Dominant allele Tall Plant

t- recessive allele Short Plant

Parents

TT- Homozygous Tall plant

tt- heterozygous short plant

TTxtt

T T
Genotype:
t Tt Tt 100% Heterozygous Tall Plants

Phenotype:

100% Tall Plants

Tt Tt
t

Another Example:

Heterozygous Cross

Tt x TT

T T

T TT TT

t Tt Tt

Genotype:

50% Homozygous Tall Plants (TT, TT)

50% Heterozygous Tall Plants (Tt, Tt)

Phenotype

100% Tall Plants (T or the dominant allele is expressed)

DIHYBRID CROSS

• is a cross between two different lines/genes that differ in two observed traits

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 • a cross that shows the possible offspring for two traits
• Studying the inheritance of two characters simultaneously

How are Dihybrid Crosses Possible?

Mendel Concluded that due to the Law of Independent Assortment, different traits are not linked and have
equal probability of showing up in offspring.

Example: Tall Purple plants, Tall White plant, short purple plants and short white plants.
Keep in mind, dominance will skew the ratios, but all of these outcomes are POSSIBLE!

Is the inheritance of one character affected by the inheritance of another?

THE LAW OF INDEPENDENT ASSORTMENT

 It appears that the inheritance of seed shape has no influence over the inheritance of seed colour

 The two characters are inherited INDEPENDENTLY

 The pairs of alleles that control these two characters assort themselves independently

Yellow (Y) is dominant to green (y).

Round (R) is dominant to wrinkled (r).

Steps to solving dihybrid crosses:

1) Make gametes for each parent.

2) Put gametes of one parent on top of square and the other parent on the side of square.

3) Fill in squares.
R r Y y
4) Determine ratio of offspring. R

r
Example: RrYy x RrYy Y

WRONG WAY OF DOING IT!

F1 -> F2 Dihybrid

• When the YyRr plant is mated, it makes 4 kinds of gamete. Each gamete gets 1 copy of each gene,
so 1/2 are Y and 1/2 are y. Independently, 1/2 are R and 1/2 are r. Thus, 1/4 of the gametes are
YR, 1/4 are Yr, 1/4 are yR, and 1/4 are yr. This happens for both male and female gametes.

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 • The gametes combine randomly. The combinations are shown in a 16 cell Punnett square.

Law of independent assortment:

• each pair of alleles segregates into gametes independently

• 4 sets of gametes are produced in equal probability

• YR, Yr, yR, yr

• Only true for genes on separate chromosomes! How come?

Mendel's second law.

• For unlinked genes, the alleles from each gene segregate into the gametes independently of one
another.

• Some genes are linked, which means that they don't segregate independently of each other and
thus don't give the 9:3:3:1 ratio of F2 offspring.

EXAMPLE:

Fur Color: Coat Texture:

B: Black R: Rough
b: White r: Smooth
In this example, we will cross a heterozygous individual with another heterozygous individual. Their
genotypes will be: BbRr x BbRr

1. First, you must find ALL possible gametes that can be made from each parent.
2. Remember, each gamete must have one B and one R.

3. Possible gametes:

a. BR
Next, arrange all possible gametes for one
b. Br parent along the top of your Punnett
Square, and all possible gametes for the
c. bR
other parent down the side of your Punnett
d. br Square…

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CMBS MLS114: CYTOGENETICS- TOPIC NOTES A.Y. 2020-2021 Page 21 of 46
3.3: Non-Mendelian Patterns of Inheritance

It is no doubt that Mendel was able to explain what, in many years, other people could not explain.
However, there are traits which do not conform to the discovered laws of Mendel.

1. Incomplete Dominance
a. In incomplete dominance, one allele of a pair is not fully dominant over its partner, so a
heterozygous phenotype somewhere in between the two homozygous phenotypes
emerges. An example is the case of snapdragons (starr et al., 160p)
b. When an organism is heterozygous for a trait, it will show a third phenotype; the third
phenotype is a blend of the other two
c. Example: cross between white and red flower the offspring becomes pink
2. Codominance/Multiple Alleles
a. A pair of non-identical alleles specific two phenotypes, which are both expressed at the
same time in heterozygotes.
b. Example is the AB blood type; Both A and B antigen is expressed
3. Pleiotropy
a. Most genes have multiple phenotypic effects, a property called pleiotropy (Gr.
pleion=more).
b. For example- pleiotropic alleles are responsible for the multiple symptoms associated with
certain heredity diseases in humans, such as cystic fibrosis and sickle-cell disease
4. Epistasis
a. (Gr. word meaning “stopping”) a gene at one locus alters the phenotypic expression of a
gene at a second locus
b. Example- in mice and many other mammals; black coat is dominant to brown. Let’s
designate B and b as the two alleles for this character. For a mouse to have a brown fur,
its genotype must be bb. But there is more to the story. A second gene determines whether
or not pigment will be deposited in the hair. The dominant allele symbolized as C (for
color), results in the deposition of either black or brown pigment, depending on the
genotype at the first locus. But if the mouse is homozygous recessive for the second locus
(cc, then the coat is white (albino), regardless of the genotype of the black/brown locus.
The gene for pigment deposition is sai to be epistatic to the gene that codes for black or
brown pigment (Reexe et al., 262p)
5. Polygenic Inheritance
a. Mendel studied characters that could be classified on an either-or-basis, such as purple
versus white flower color. But many characters like skin color and height, vary in the
population along a continuum (in gradations). These are called quantitative variations
usually indicate polygenic inheritance, an additive effect of two or more genes on a single
phenotypic character (the converse of pleiotropy, where a single gene affects several
phenotypic characters)

Reference: Verma, P.S and Agarwal, V.K (2005). Cell Biology, Genetics, Molecular Biology, Evolution and
Ecology. Ram Nagar, New Delhi: S. Chand & Company LTD. p.42-43; 54

Camara, J.S and Oclay, A. (2012). Cytogenetics: Principles and Application. Dagupan City: Space Browser
Publishing p.4-5

Powerpoint Prepared by Mr. Von Carlo P. Dela Tore, M.S.C

TOPIC 4: DNA REPLICATION AND DNA SYNTHESIS

DNA (Deoxyribonucleic Acid) and RNA (Ribonucleic Acid), the principal genetic materials of living organisms,
are chemically called nucleic acids and are complex molecules largen than most proteins and contains
Carbon, Oxygen, Hydrogen, Nitrogen and Phosphorous.

4.1: The DNA and RNA

DNA (Deoxyribonucleic Acid)

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The 3-Dimensional structure of DNA was discovered in 1953 by Watson and Crick in Cambridge, using the
experimental data of Wilkins and Franklin in London, for which they won a Nobel prize. Ms. Franklin however
died before the award and the Nobel Prize is never awarded posthumously.

The main features of the structure are:

1. DNA is a double-stranded, so there are two polynucleotide stands alongside each other.
2. The strands are antiparallel, i.e they run in opposite directions thus 5’ to 3’ is parallel to 3’ to 5’
3. The two strands are wound round each other to form a double helix (not a spiral)
4. The two strands are joined together by hydrogen bonds
5. The bases therefore form base pairs, which are like rungs of ladder.
6. The base pairs are specific. A only binds to T (and T to A); and C only binds to G (G with C)
7. These are called complementary base pairs (or sometimes Watson-Crick base pairs). (A-T and G-
C)
8. This means that whatever the sequence of bases along one strand, the sequence of bases on the
other strand must be complementary to it.

RNA (Ribonucleic Acid)

Like DNA, RNA is polymeric nucleic acid of four monomeric robotids or ribonucleotids.

Each ribonucleotide contains a pentose sugar (D-ribose); a molecule of phosphate group and nitrogen base.
The nitrogen bases of RNA are two purines; The four ribonucleotides also occur freely in nucleoplasm but
in the form of triphosphate of ribonucleotisides such as ATP and uridine triphosophate (UTP)

RNA molecule may be either single stranded or double stranded but not helical like DNA molecules

3 Different Types of RNA

1. mRNA- Messenger RNA- this carries information from the nucleus to the ribosomes which are
sites for protein synthesis. The coding sequence of the mRNA determines the amino acid sequence
in the protein. The mRNA is a straight molecule extends from 5’ to 3’ end. It is transcribed from a
DNA template. On the mRNA nucleotides are arranged into codons consisting of 3 bases each.
Each such codon specifies an amino acid.
2. tRNA- transfer RNA- this RNA type is a small chain of about 80 nucleotides; tRNA transfers
specific amino acid molecules to a growing polypeptide chain. The tRNA has a clover lead model
with 5 arms each with a specific function. The tRNA also has an anticodon region that can base
pair with the codon region on the mRNA.
3. rRNA- Ribosomal RNA- synthesized in the nucleolus. In the cytoplasm, ribosomal RNA and
protein combine together to form a nucleoprotein called a ribosomes. The ribosome and mRNA
bind to carry out protein synthesis

DNA Replication

The Enzymes used in DNA Replication

Enzymes are proteins which are created in order to complete a certain task within the body, and in most
cases catalyze chemical reactions. In this case, enzymes carry out the process of DNA Replication.

1. DNA Helicase- Unzips strand of DNA by breaking hydrogen bonds between base pairs of
nucleotide. In short, the DNA is cut down in the middle into two strands
2. Single site bonding proteins- clasps onto both DNA strands to prevent the re-zipping of DNA
3. DNA Polymerase- adds nucleotides to DNA Strands. Also serves as the corrector for mismatched
nucleotides.
4. DNA Ligase- Binds Okazaki fragments together within the lagging strand, with forms one
complete DNA strand

Lagging vs. Leading Strand

DNA has two sides that are coiled up. One side has a 5’ end, and the complementary side has a 3’ end.
This means that on one side, the sequence begins with a Phosphorus, and the other side begins with a
sugar. Because both strands are built from the 5’ end to the 3’ end, both strands must be built in a different
way. The leading strand is the strand that is continuously built throughout DNA Replication. The lagging

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strand must be built differently because both strands built from the 5’ to 3’ direction. The lagging strand is
built piece, and those pieces are later connected into one full DNA strand with the help of DNA Ligase.

Leading Strand runs 3’ to 5’ (left -> right)

Lagging Strand runs 5’ to 3’

The Process of DNA Replication from Beginning to End

1. DNA Replication begins with one strand of DNA

2. DNA helicase then runs throughout the DNA strand and breaks it apart into two halves
3. Single sit bonding proteins hold back what has been unzipped to ensure that the sides do not
rebind.
4. After, DNA Polymerase attaches the primer (where DNA attaches to), and begins to lengthen the
new strands of DNA by adding nucleotide sequences to it
5. While this is occurring, on the lagging strand (5’ to 3’) the process is happening in the same fashion,
but the new DNA on this strand is being built in small segments called the “Okazaki Fragments”
which will then be bind by the DNA Ligase into one complete strand of new DNA
6. DNA Replication has now been completed.

4.2: From Gene to Protein (Transcription and Translation)

The Flow of Genetic Information Overview

• The information content of DNA is in the form of specific sequences of nucleotides

• The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins

• Proteins are the links between genotype and phenotype

• Gene expression, the process by which DNA directs protein synthesis, includes two stages:
transcription and translation

How was the fundamental relationship between genes and proteins discovered?

1.Evidence from the study of Metabolic defects

• In 1909, British physician Archibald Garrod first suggested that genes dictate phenotypes through
enzymes that catalyze specific chemical reactions

• He thought symptoms of an inherited disease reflect an inability to synthesize a certain enzyme

• Linking genes to enzymes required understanding that cells synthesize and degrade molecules in
a series of steps, a metabolic pathway

2. Nutritional Mutants in Neurospora: Scientific Inquiry

• George Beadle and Edward Tatum exposed bread mold to X-rays, creating mutants that were
unable to survive on minimal medium as a result of inability to synthesize certain molecules

• Using crosses, they identified three classes of arginine-deficient mutants, each lacking a different
enzyme necessary for synthesizing arginine

• They developed a one gene–one enzyme hypothesis, which states that each gene dictates
production of a specific enzyme

3. The Products of Gene Expression: A Developing Story

• Some proteins aren’t enzymes, so researchers later revised the hypothesis: one gene–one
protein

• Many proteins are composed of several polypeptides, each of which has its own gene

• Therefore, Beadle and Tatum’s hypothesis is now restated as the one gene–one polypeptide
hypothesis

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BASIC PRINCIPLES OF TRANSCRIPTION AND TRANSLATION

• RNA is the intermediate between genes and the proteins for which they code

• Transcription is the synthesis of RNA under the direction of DNA

• Transcription produces messenger RNA (mRNA)

• Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA

• Ribosomes are the sites of translation

• In prokaryotes, mRNA produced by transcription is immediately translated without more processing

• In a eukaryotic cell, the nuclear envelope separates transcription from translation

• Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA

• A primary transcript is the initial RNA transcript from any gene

• The central dogma is the concept that cells are governed by a cellular chain of command: DNA →
RNA → protein

The Genetic Code

• There are 20 amino acids, but there are only four nucleotide bases in DNA

• How many bases correspond to an amino acid?

Codons: Triplets of Bases

• The flow of information from gene to protein is based on a triplet code: a series of
nonoverlapping, three-nucleotide words
• These triplets are the smallest units of uniform length that can code for all the amino acids
• Example: AGT at a particular position on a DNA strand results in the placement of the amino
acid serine at the corresponding position of the polypeptide to be produced
• During transcription, one of the two DNA strands called the template strand provides a
template for ordering the sequence of nucleotides in an RNA transcript
• During translation, the mRNA base triplets, called codons, are read in the 5 to 3 direction
• Each codon specifies the amino acid to be placed at the corresponding position along a
polypeptide
• Codons along an mRNA molecule are read by translation machinery in the 5 to 3 direction
• Each codon specifies the addition of one of 20 amino acids

CRACKING THE CODE

• All 64 codons were deciphered by the

mid-1960s

• Of the 64 triplets, 61 code for amino

acids; 3 triplets are “stop” signals to end
translation

• The genetic code is redundant but not

ambiguous; no codon specifies more than
one amino acid

• Codons must be read in the correct

reading frame (correct groupings) in
order for the specified polypeptide to be
produced

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Evolution of the Genetic Code

• The genetic code is nearly universal, shared by the simplest bacteria to the most complex
animals
• Genes can be transcribed and translated after being transplanted from one species to another

TRANSCRIPTION

• Transcription, the first stage of gene expression, can be examined in more detail

• RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and hooks
together the RNA nucleotides

• RNA synthesis follows the same base-pairing rules as DNA, except uracil substitutes for thymine

• The DNA sequence where RNA polymerase attaches is called the promoter; in bacteria, the
sequence signaling the end of transcription is called the terminator

• The stretch of DNA that is transcribed is called a transcription unit

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Synthesis of RNA Transcript

The three stages of transcription:

– Initiation

– Elongation

– Termination

RNA Polymerase Binding and Initiation of Transcription

• Promoters signal the initiation of RNA synthesis

• Transcription factors mediate the binding of RNA polymerase and the initiation of transcription

• The completed assembly of transcription factors and RNA polymerase II bound to a promoter is
called a transcription initiation complex

• A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes

Termination of Transcription

• The mechanisms of termination are different in bacteria and eukaryotes

• In bacteria, the polymerase stops transcription at the end of the terminator

• In eukaryotes, the polymerase continues transcription after the pre-mRNA is cleaved from the
growing RNA chain; the polymerase eventually falls off the DNA

Eukaryotic cells modify RNA after transcription

• Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched
to the cytoplasm

• During RNA processing, both ends of the primary transcript are usually altered

• Also, usually some interior parts of the molecule are cut out, and the other parts spliced together

Alteration of mRNA Ends

• Each end of a pre-mRNA molecule is modified in a particular way:

– The 5 end receives a modified nucleotide 5 cap

– The 3 end gets a poly-A tail

• These modifications share several functions:

– They seem to facilitate the export of mRNA

– They protect mRNA from hydrolytic enzymes

– They help ribosomes attach to the 5 end

Split Genes and RNA Splicing

• Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that
lie between coding regions

• These noncoding regions are called intervening sequences, or introns

• The other regions are called exons because they are eventually expressed, usually translated into
amino acid sequences

• RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding
sequence

RIBOZYMES

• Ribozymes are catalytic RNA molecules that function as enzymes and can splice RNA

• The discovery of ribozymes rendered obsolete the belief that all biological catalysts were proteins

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TRANSLATION

• The translation of mRNA to protein can

be examined in more detail

• A cell translates an mRNA message into

protein with the help of transfer RNA
(tRNA)

• Molecules of tRNA are not identical:

• Each carries a specific amino acid

on one end

• Each has an anticodon on the

other end; the anticodon base-
pairs with a complementary
codon on mRNA

Accurate translation requires two steps:

– First: a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-
tRNA synthetase

– Second: a correct match between the tRNA anticodon and an mRNA codon

• Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to
more than one codon

RIBOSOMES

Ribosomes facilitate specific

coupling of tRNA anticodons with
mRNA codons in protein synthesis

The two ribosomal subunits (large

and small) are made of proteins
and ribosomal RNA (rRNA)

A ribosome has three binding sites

for tRNA:

1. The P site holds the tRNA that

carries the growing polypeptide
chain
2. The A site holds the tRNA that
carries the next amino acid to be
added to the chain
3. The E site is the exit site, where
discharged tRNAs leave the
ribosome

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The three stages of translation:

– Initiation

– Elongation

– Termination

Ribosome Association and Initiation of Translation

• The initiation stage of translation brings together mRNA, a tRNA with the first amino acid, and the
two ribosomal subunits

• First, a small ribosomal subunit binds with mRNA and a special initiator tRNA

• Then the small subunit moves along the mRNA until it reaches the start codon (AUG)

• Proteins called initiation factors bring in the large subunit that completes the translation initiation
complex

Elongation of Polypeptide Chain

• During the elongation stage, amino acids are added one by one to the preceding amino acid

• Each addition involves proteins called elongation factors and occurs in three steps: codon
recognition, peptide bond formation, and translocation

Termination of Translation

• Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome

• The A site accepts a protein called a release factor

• The release factor causes the addition of a water molecule instead of an amino acid

• This reaction releases the polypeptide, and the translation assembly then comes apart

Completing and Targeting the Functional Protein

• Often translation is not enough to make a functional protein

• Polypeptide chains are modified after translation

• Completed proteins are targeted to specific sites in the cell

4.3: Mutations

• Mutations are changes in the genetic material of a cell or virus

• Point mutations are chemical changes in just one base pair of a gene

• The change of a single nucleotide in a DNA template strand can lead to the production of an
abnormal protein

Types of Point Mutations

Point mutations within a gene can be divided into two general categories

– Base-pair substitutions

– Base-pair insertions or deletions

*If you have at least access to the internet please download the powerpoint to see pictures*

1.Base- pair Substitutions

• A base-pair substitution replaces one nucleotide and its partner with another pair of nucleotides

• Silent mutations have no effect on the amino acid produced by a codon because of redundancy in
the genetic code

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 • Missense mutations still code for an amino acid, but not necessarily the right amino acid

• Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a
nonfunctional protein

2.Base-pair Insertion and Deletion

• Insertions and deletions are additions or losses of nucleotide pairs in a gene

• These mutations have a disastrous effect on the resulting protein more often than substitutions do

• Insertion or deletion of nucleotides may alter the reading frame, producing a frameshift
mutation

MUTAGENS

• Spontaneous mutations can occur during DNA replication, recombination, or repair

• Mutagens are physical or chemical agents that can cause mutations

ADDITIONAL NOTES & REVIEW:

• Archaea are prokaryotes, but share many features of gene expression with eukaryotes

• Bacteria and eukarya differ in their RNA polymerases, termination of transcription and ribosomes;
archaea tend to resemble eukarya in these respects

• Bacteria can simultaneously transcribe and translate the same gene

• In eukarya, transcription and translation are separated by the nuclear envelope

• In archaea, transcription and translation are likely coupled

WHAT IS GENE?

• The idea of the gene itself is a unifying concept of life

• We have considered a gene as:

– A discrete unit of inheritance

– A region of specific nucleotide sequence in a chromosome

– A DNA sequence that codes for a specific polypeptide chain

In summary, a gene can be defined as a region of DNA that can be expressed to produce a final functional
product, either a polypeptide or an RNA molecule

Reference: Powerpoint Lecture from Biology by Neil Campbell and Jane Reece

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TOPIC 5: STEM CELLS

5.1: Stem Cells and its Characteristics

Stem Cells

-A cell that has the ability to continuously divide and differentiate (develop) into various other kind(s) of
cells/tissues

-every cell in body can be traced back to a fertilized egg that came into existence from the union of egg
and sperm. But the body is made up of over 200 different types of cells. Not just one. All of these cell types
come from a pool of stem cells in the early embryo.

-During early development, as well as later in life, various types of stem cells give rise to the specialized or
differentiated cells that carry out the specific functions of the body, such as skin, blood, muscles and nerve
cell.

Stem Cell Characteristics

➢ ‘Blank cells’ (unspecialized)

➢ Capable of dividing and renewing themselves for long periods of time (proliferation and renewal)

➢ Have the potential to give rise to specialized cell types (differentiation)

TYPES OF STEM CELLS

1. Embryonic Stem Cells-

a. come from a five to six-day-old embryo. They have the ability to form virtually any type of
cell found in the human body
b. are derived from the part of a human embryo or fetus that will ultimately produce eggs or
sperm (gametes).
2. Adult Stem Cells-
a. are undifferentiated cells found among specialized or differentiated cells in a tissue or
organ after birth. Based on current research they appear to have a more restricted ability
to produce different cell types and to self-renew.

STEM CELL TYPE

Stem cell type Description Examples

Totipotent Each cell can develop into a new Cells from early (1-3 days)
individual embryos

Pluripotent Cells can form any (over 200) cell types Some cells of blastocyst (5 to
14 days)

Multipotent Cells differentiated, but can form a Fetal tissue, cord blood, and
number of other tissues adult stem cells

5.2: Application of Stem Cells

➢ Disease

• Diabetes, Spinal cord injury, Parkinson’s disease, heart disease

➢ Genetic based Disease

• Cystic fibrosis, Huntington’s

HOW THEY CAN COULD TREAT CERTAIN TYPES OF DISEASE?

1. Tissue Repair
a. Regenerate spinal cord, heart tissue, or any other major tissues in the body
2. Heart Disease

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 a. Adult bone marrow stem cells injected into the heart arteries are believed to improve
cardiac function in victims of heart failure or heart attack
3. Leukemia and cancer
a. Studies show that leukemia patients treated with stem cells emerge free of disease
b. Injections of stem cells have also reduced pancreatic cancers in some patients
4. Rheumatoid Arthritis
a. Adult stem cells may be helpful in jumpstarting repair of eroded cartilage
5. Type I Diabetes
a. Pancreatic cells do not produce insulin
b. Basic research focused on understanding how embryonic stem cells might be trained to
become pancreatic islet cells needed to secrete insulin

Unknowns in Stem Cell/Cloning Research

• It is uncertain that human embryonic stem cells in vitro can give rise to all the different cell types
of the adult body.
• It is unknown if stem cells cultured in vitro (apart from the embryo) will function as the cells do
when they are part of the developing embryo

Challenges to Stem Cell/Cloning Research

• Stem cells need to be differentiated to the appropriate cell type(s) before they can be used
clinically.

• Recently, abnormalities in chromosome number and structure were found in three human ESC
lines.

• Stem cell development or proliferation must be controlled once placed into patients.

• Possibility of rejection of stem cell transplants as foreign tissues is very high.

• Contamination by viruses, bacteria, fungi, and Mycoplasma possible.

• The use of mouse “feeder” cells to grow ESC could result in problems due to xenotransplantation
(complicating FDA requirements for clinical use).

• Ethics

Reference: Understanding Stem Cells: An overview of the science and issues from the national
academies retrieved on July 11, 2020 from
https://www.nap.edu/resource/11278/Understanding_Stem_cells.pdf
Powerpoint Prepared by Mr. Von Carlo P. Dela Tore, M.S.C

TOPIC 6: VARIATION IN CHROMOSOME STRUCTURE

6.1: Variation in Genetic Structure

New Chromosomes
Normal human somatic cells have 46 chromosomes: 22 pairs, or homologs, of autosomes
(Chromosome 1-22) and two sex chromosomes. This is called the diploid number. Females carry
two X-chromosomes (46, XX) while males have an X and Y (46, XY). Germ cells (egg and sperm)
have 23 chromosomes: one copy of each autosome plus a single six chromosomes. This is referred
to as the haploid number. One chromosome from each autosomal pair plus on sex chromosomes
is inherited from each parent. Mothers can contribute only an X chromosome to their children while
fathers can contribute either an X or a Y.

Genetic Variation
Genetic variation refers to differences between members of the same species or those of different
species
• Allelic variations are due to mutations in particular genes
• Chromosomal aberrations are substantial changes in chromosome structure
▪ These typically affect more than one gene
▪ They are also called chromosomal mutations

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I.ALTERATION IN CHROMOSOME STRUCTURE
There are two primary ways in which the structure of chromosomes can be altered:
1.The total amount of genetic information in the chromosome change:
Decrease: Deficiencies/Deletion
Increase: Duplication and Insertion

2. Genetic material may remain the same in number, but is re arranged

a. Inversions
b. Translocations

Structural abnormalities involve changes in the structure of one or more chromosomes.

TOTAL AMOUNT OF GENETIC INFORMATION

1.DELETION
– involve loss of material from a single chromosome. The effects are typically severe since there
is a loss of genetic material
- deletion may be spontaneous of may be induced
-Mis-division of the centromere
-Radiation, UV, Chemicals, viruses may increase breakage
-Deletions do not revert because the DNA is gone (degraded)
-the effect of a deletion depends on what was deleted

TYPES OF DELETION
1.Terminal Deletion- It involved a single break and the terminal part of the chromosome is lost.

2.Interstitial Deletion- Deletion that does not involve the terminal parts of a chromosome

• A deletion in one allele of a homozygous wildtype organism may give a normal phenotype
• While the same deletion in the wild-type allele of a heterozygote would produce a mutant phenotype.
• Deletion of the centromere results in an acentric chromosome that is lost, usually with serious or lethal
consequences.
• No known living human has an entire autosome deleted from the genome.
• Example of human disorders caused by large chromosomal deletions:
o Cri-du-chat (“cry of the cat”) syndrome (OMIM 123450), resulting from deletion of part of the
short arm of chromosome 5
• Facial Dysmorphisms
• Including microcephaly, round face, hypertelorism, epicanthal folds, low-set ears, and micrognathia.

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• Severe psychomotor and mental retardation
• Other health problems associated with CdC:
• Poor-suck, hypotonia, respiratory and heart defects, growth retardation, and cleft palate and/or lip.
• CdC patients are generally very sociable, but may exhibit maladaptive behaviors such as
inattentiveness, hyperactivity, temper-tantrums, and self injury.

2.DUPLICATION
- Duplications result from doubling of chromosomal segments, and
occur in a range of sizes and locations

TYPES OF DUPLICATION

• Tandem duplications are adjacent to each other.

• Reverse tandem duplications result in genes arranged in the opposite
order of the original.
• Tandem duplication at the end of a chromosome is a terminal
tandem duplication

• An example is the Drosophila eye shape allele, Bar, that reduces the number of eye facets, giving the
eye a slit-like rather than oval appearance

• Another example is the Charcot–Marie–Tooth Disease

o The foot of a person with Charcot–Marie–Tooth disease:

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 o The lack of muscle, a high arch, and claw toes are signs of this genetic disease.
o caused by duplication of the gene encoding peripheral myelin protein 22 (PMP22) on
chromosome 17.

TOTAL AMOUNT OF GENETIC INFORMATION IS THE SAME BUT REARRANGED

1. INVERSIONS
-occur when there are two breaks within a single chromosome and the broken segment flips 180
degrees (inverts) and reattaches to form a chromosome that is structurally out-of-sequence.
-There is usually no risk for problems to an individual if the inversion is of familial origin (has been
inherited from a parent)
-There is a slightly increased risk if it is a de novo (new) mutation due possible to an interruption
of a key gene sequence; although an inversion carrier ay be completely normal, they are slightly
increased risk for producing chromosomally unbalanced embryo. This is because an inverted
chromosome has difficulty pairing with its normal homolog during meiosis, which can result if
gametes containing unbalanced derivative chromosomes if an unequal cross-over event occurs.

TWO TYPES OF INVERSION

How do inversions behave genetically?

• Crossing-over within the inversion loop of a paracentric inversion connects homologous centromeres in
a dicentric bridge while also producing an acentric fragment—a fragment without a centromere.
• Then, as the chromosomes separate in anaphase I, the centromeres remain linked by the bridge, which
orients the centromeres so that the noncrossover chromatids lie farthest apart.
• The acentric fragment cannot align itself or move and is, consequently, lost.
• Tension eventually breaks the bridge, forming two chromosomes with terminal deletions
• The gametes containing such deleted chromosomes may be inviable but, even if viable, the zygotes
that they eventually form are inviable.
• Hence, a crossover event, which normally generates the recombinant class of meiotic products, instead
produces lethal products.
• The overall result is a lower recombinant frequency. In fact, for genes within the inversion, the RF is
zero. For genes flanking the inversion, the RF is reduced in proportion to the relative size of the
inversion.
• Inversions affect recombination in another way too.
• Inversion heterozygotes often have mechanical pairing problems in the region of the inversion
• these pairing problems reduce the frequency of crossing-over and hence the recombinant frequency in
the region.

The net genetic effect of a pericentric inversion is the same as that of a paracentric one…
• In a pericentric inversion, because the centromeres are contained within the inverted region, the
chromosomes that have crossed over disjoin in the normal fashion, without the creation of a bridge.
• However, the crossover produces chromatids that contain a duplication and a deficiency for different
parts of the chromosome

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 • In this case, if a nucleus carrying a crossover chromosome is fertilized, the zygote dies because of
its genetic imbalance.
• Again, the result is the selective recovery of noncrossover chromosomes in viable progeny.

NOTE:
Two mechanisms reduce the number of recombinant products among the progeny
of inversion heterozygotes: elimination of the products of crossovers in the inversion loop and inhibition
of pairing in the region of the inversion.

What about homozygous inversions?

 In such cases the homologous inverted chromosomes pair and cross over normally, there are no bridges,
and the meiotic products are viable.

CONSEQUENCIES OF CHROMOSOME INVERSION IN HUMANS:

➢ lowered fertility due to production of unbalanced gametes

2.TRANSLOCATION
-Translocation involve exchange of material between two or more chromosomes. If a translocation is
reciprocal (balanced) the risk for problems to an individual is similar to that with inversions: usually none
if familial and slightly increased if de novo. Problems arise with translocations when gametes from a
balanced parent are formed which do not contain both translocation products. When such a gamete
combines with a normal gamete from the other parent result is an unbalanced embryo which is partially
monosomic for one chromosome and partially trisomic for the other.

There are two main types of Translocation:

1. Reciprocal (Balanced) Translocation
2.Robertsonian (unbalanced) Translocation

*Both types are capable of causing disease in human

ROBERTSONIAN TRANSLOCATIONS
-the transfer of genetic material occurs in only one direction
-are associated with phenotypic abnormalities or even lethality
-Example: Familial Down Syndrome
-In this condition, the majority of chromosome 21 is attached to chromosome 14
-The individual would have three copies of genes found on a large segment of chromosome 21;
Therefore, they exhibit the characteristic of Down Syndrome
-This translocation occurs as follows:
• Breaks occur at the extreme ends of the short arms of two non-homologous acrocentric
chromosomes

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 • The larger fragments fuse at their centromeic regions to form a single chromosome
• The small acrocentric fragments are subsequently lost
• This type of translocation is the most common type of chromosomal rearrangement in
humans
-Robertsonian Translocations are confined to chromosomes 13, 14, 15 21 (the acrocentric chromosomes)

CHROMOSOMAL MUTATIONS AND HUMAN TUMORS

• Most human malignant tumors have chromosomal mutations
• Most common are translocations
• There is much variation in chromosome abnormalities, however, and they include simple
rearrangements to complex changes in chromosome structure and number
• Many tumor types show a variety of mutations
• Some, however, are associated with specific chromosomal abnormalities

 Follicular lymphoma is a type of non-Hodgkin lymphoma.

 It develops when the body makes abnormal B-lymphocytes – the lymphoma cells. (B-lymphocytes are
white blood cells that fight infection).
 The lymphoma cells build up in lymph nodes. The most common symptom is a painless swelling in the
neck, armpit or groin.

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Burkitt’s Lymphoma vs. Small Noncleaved Non-Burkitt’s

Burkitt’s Lymphoma
• High grade tumor
• Uniform appearance of abnormal cells
• t(8;14); t(8;22) or t(8;2)
• Endemic in equatorial Africa
Small noncleaved non-Burkitt’s
• High-grade tumor
• Variability in size and shape of abnormal cells
• t(8;14); t(8:22) or t(8;2)

ISOCHROMES
• a structure where a chromosome has lost one of its arms, and the replacement arm is an exact mirror
image of the remaining arm
• Example: Pallister-Killian mosaic syndrome
o a developmental disorder that affects many parts of the body.
o characterized by extremely weak muscle tone (hypotonia) in infancy and early childhood,
intellectual disability, distinctive facial features, sparse hair, areas of unusual skin coloring
(pigmentation), and other birth defects.

II. ALTERATION IN CHROMOSOME NUMBER

Numerical abnormalities involve the loss and/or gain of a whole chromosome or chromosomes and can
include both autosome and sex chromosomes.
Examples include: Down Syndrome, Edward’s Syndrome and the likes

Each species has a characteristic number of chromosomes in the nuclei of its gametes and
somatic cells. The gametic chromosome number constitutes a basic set of chromosomes called genome. A
gamete cell contains single genome and is called haploid. When haploid gametes of both sexes (male and
female) unite in the process of fertilization a diploid zygote with two genomes is formed. The diploid zygote
undergoes embryological development and forms an adult animal which upon attaining sexual maturity
produces haploid gametes. And this alternation of generation continues between haploidic and diploidic
generation in most species. However, sometimes irregularities occur in nuclear division or “accidents” ( as
from radiations) may befall interphase chromosomes so that cells or entire organisms with aberrant
genomes may be formed. Such chromosomal aberrations may include whole genomes and entire single
chromosomes. Changes in number of whole chromosomes is called heteroploidy (see Burns and Bottino,
1989). Heteroploidy may involve entire sets of chromosomes (euploidy), or loss or addition of single whole
chromosomes (aneuploidy). Each may produce phenotypic changes, modifications of phenotypic ratio, or
alteration of linkage groups. Many are of some evolutionary significance.

EUPLOIDY
The term euploidy (Gr., eu = even or true; ploid = unit) designates genomes containing chromosomes that
are multiples of some basic number (x). The euploids are those organisms which contain balanced set or
sets of chromosomes in any number. The number of chromosomes in a basic set is called the monoploid
number, x. Those euploid types whose number of sets is greater than two are called polyploid. Thus, 1x is
monoploid, 2x is diploid; and the polyploid types are 3x (triploid), 4x (tetraploid), 5x (pentaploid), 6x

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 (hexaploid) and so on. The haploid (n) refers strictly to the number of chromosomes in gametes. In most
animals and many plants the haploid number and monoploid number are the same. Hence n or x (or 2n or
2x) can be used interchangeably. However, in case of polyploids the usage of n may create confusion. For
example, in modern wheat x and n are different. Wheat has 42 chromosomes, but careful study reveals
that in hexaploid there are six rather similar but not identical sets of seven chromosomes. So, 6x = 42, and
x = 7. However, gametes of wheat contain 21 chromosomes, hence, 2n = 42 and n = 21 (see Suzuki et
al., 1986). A triploid hybrid of wheat, from which a hexaploid Triticum spelta has been obtained due to
colchicine treatment, contains 2n = 3x = 21. Such haploids, since are obtained from the polyploids (i.e.,
cross of tetraploid emmer wheat and diploid goat grass), they are called polyhaploids, just to differentiate
them from the normal monoploids. The lower organisms such as bacteria and viruses are called haploids
because they have a single set of genetic elements. However, since they do not form gametes comparable
to those of higher organisms, the term monoploid would seem to be more appropriate (see Stansfield,
1986).

i. Monoploidy
• Monoploids have a single basic set of chromosomes, e.g., 7 in barley and 10 in corn.
Monoploidy is common in plants and rare in animals.
ii. Polyploidy
• Any organism with more than two genomes (2x) is called a polyploid. Many plant genera
include species whose chromosome numbers constitute a euploid series. For example, the
rose genus Rosa includes species with the somatic numbers 14, 21, 28, 35, 42 and 56.
These numbers are the multiples of 7. Therefore, this is a euploid series of the basic
monoploid number 7, which gives diploid, triploid, tetraploid, pentaploid, hexaploid and
octaploid species. Except diploids, rest of these belong to polyploid category. Ploidy levels
higher than tetraploid are not commonly encountered in natural populations, but our most
important crops and ornamental flowers are polyploids, e.g., wheat (Hexaploid 6x),
strawberries (octaploid, 8x), many commercial fruits and ornamental plants. Generally,
polyploidy is common in plants (more common in monocots) but rare in animals

ANEUPLOIDY
changes that involve parts of a chromosome set results in individuals, called aneuploids (Gr. aneu = uneven
; ploid = unit). Aneuploidy can be either due to the loss of one or more chromosomes (hypoploidy) or due
to addition of one or more chromosomes to the complete chromosome set (hyperploidy). Hypoploidy is
mainly due to the substraction (or loss) of a single chromosome, called monosomy (2n–1) or due to the
loss of one pair of chromosome called nullisomy (2n–2 ; two lost chromosomes are homologs). Likewise,
hyperploidy may involve addition of either a single chromosome, called trisomy (2n + 1) or a pair of
chromosomes, called tetrasomy (2n + 2). In the monoploid organisms, addition of single chromosome
produces disomy (n + 1). All of these aneuploids are probably produced by nondisjunction during mitosis
or meiosis.

i. Monosomy
• Diploid organisms which are missing one chromosome of a single pair are monosomic with the
genomic formula 2n –1. A monosomic individual forms gametes of two types, (n) and (n – 1).
The n –1 gametes do not survive in plants, but, in animals that may cause genetic imbalance,
which is manifested by high mortality or reduced fertility of resulted organism.
ii. Nullisomy
• An organism which has lost a chromosome pair is a nullosomic. The nullosomic organism has
the genomic formula (2n–2). A nullosomic diploid often does not survive, however, a nullosomic
polyploid (e.g., hexaploid wheat, 6x–2) may survive but exhibit reduced vigour and fertility.
iii. Trisomy
• Trisomics are those diploid organisms which have an extra chromosome (2n + 1). Since the
extra chromosome may belong to any one of different chromosomes of a haploid complement,
the number of possible trisomics will be equal to the haploid chromosome number. For
example, haploid chromosome number of barley is 7, consequently in it seven trisomics are
possible. Further, when the extra chromosome is identical to its homologs, such a trisomic is
called primary trisomic. There are also secondary and tertiary trisomics. While the secondary
trisomic means that the extra chromosome should be an isochromosome (i.e., both
chromosome arms genetically similar), a tertiary trisomic would mean that the extra
chromosome should be the product of translocation.

EXAMPLES:
• Down Syndrome (Trisomy 21)
o Down’s syndrome is named after the physician J.Langdon Down who first described this
genetic defect in 1866 and it was formally called mongolism or mongolian idiocy. It is
usually associated with a trisomic condition for one of the smallest human autosomes (i.e.,

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 chromosome 21). It is the most common chromosomal abnormality in live births (1/650
births).
o There are about 50 physical characteristics shown by DS infants soon after birth; include
mild or moderate mental retardation; eyes that slant up and out with internal epicanthal
folds; a tongue that is large, swollen and protruding ; small and under developed ears; a
single palmar crease; short stature; stubby fingers; an enlarged liver and spleen.
o Women over 45 years of age are about twenty times more likely to give birth to a child
with DS than women aged 20. Nondisjunction of chromosome pair 21 during oogenesis is
the main cause of occurrence of trisomy-21. This event is found to be affected either by
senescence of oocytes, virus infection, radiation damage, etc. (e.g., mothers who have
had infectious hepatitis prior to pregnancy may have three times more chances to give
birth to DS infants). Nondisjunction of chromosome pair 21 during spermatogenesis can
also produce child with DS, but paternal age does not seem to be associated with its
incidence
o Lastly, in about 2 to 5 per cent cases, the normal chromosome number is present (2n =
46), but the extra chromosome 21 is attached (translocated) to one of the larger
autosomes (usually chromosome 14).
• Edward’s Syndrome (Trisomy 18)
o First described in 1960 by John H. Edwards and his colleagues, trisomy18 is found to
contain an incidence of about 0.3 per 1000 births
o It is characterized by multiple malformations, primarily low-set ears; small receding lower
jaw; flexed and clenched fingers; cardiac malformations; and various deformaties of skull,
face and feet. Harelip and cleft palate often occurs. Death takes place around 3 to 4 months
of age. Trisomy-18 children show evidence of severe mental retardation, which is more
pronounced in females (the reason is still not clear).
o Like the Down’s syndrome, occurrence of Edward’s syndrome is too related with maternal
age (i.e., 35 to 45 year old mothers have more chance of giving birth to trisomy-18 infant).
• Patau’s Syndrome (Trisomy 13)
o his syndrome was described in 1960 by Klaus Patau and coworkers. Its incidence is about
0.2 per 1000 births
o Individuals with Patau syndrome appear to be markedly mentally retarded; have sloping
forehead, harelip and cleft palate. Polydactyly (both hands and feet) is almost always
present; the hands and feet are deformed. Cardiac and various internal defects (of kidney,
colon, small intestine) are common.
o Death usually occurs within hours or days, but the fetus may abort spontaneously. (Note
: Sex chromosomal variations will be described in Chapter 18 of Human Genetics).

List of Basic Syndromes

• Numeric Anomalies of Sex Chromosomes
o Turner Syndrome (45, X)
▪ 1:2000-2500
▪ Short stature- treated with hormonal therapy
▪ Gonadala dysgenesis, primary amenorrhoea
▪ Average intelligence, short webbed neck (pterygium colli); low posterior
hairline;broad/shield chest; palms and feet edema (newborns)
o Klinefelter’s Syndrome (47, XXY)
▪ 1:500-1000
▪ Tall stature; average intelligence; male psychosocial orientation; hypoplastic
testes, cryptochism; sterility- azoospermia; gynecomastia (enlargement of breast
in male)
o XYY Syndrome (Supermale; 47, XYY)
▪ Robust growth (proportional) especially to height
▪ Average intelligence; normal sexual development; normal fertility without risk of
chromosomal aberrations in offspring
▪ Controversy- affected psychosocial development
o XXX Syndrome (Superfemale; 47, XXX)
▪ 1:1000; no specific phenotype
▪ Average intelligence; normal sexual development; decreased fertility (spontaneous
abortions) without risk of chromosomal aberrations in offspring
▪ No increased occurrence of congenital disorder over to population risk
iv. Tetrasomy
• The diploid organisms having two extra chromosomes are known as tetrasomic. They have the
genomic formula 2n+2. All the 21 possible tetrasomics are available in wheat.

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*Numerical and structural abnormalities can be further divided into two main categories: constitutional,
those you are born with; and acquired, those that arise secondary changes to other diseases such as
cancer.

6.2:KARYOTYPING

Karyotyping is:
✓ simply a picture of a person's chromosomes.
✓ In order to get this picture, the chromosomes are isolated, stained, and examined under the
microscope.
✓ Most often, this is done using the chromosomes in the white blood cells.
✓ A picture of the chromosomes is taken through the microscope.

How are the chromosomes arranged in a karyotype?

➢ Since a karyotype is an organized profile of a person's chromosomes,
➢ Two chromosomes specify gender — XX for female and XY for male. The rest are arranged in
pairs, numbered 1 through 22, from largest to smallest.

What are examples of genetic abnormalities that can be seen on a karyotype?

➢ Karyotype analysis can reveal abnormalities, such as
✓ missing chromosomes,
✓ extra chromosomes,
✓ Deletions
✓ duplications
✓ and translocations
➢ These abnormalities can cause genetic disorders including Down syndrome, Turner
syndrome, Klinefelter syndrome, triple X syndrome, and Jacob syndrome.

References: Camara, J.S and Oclay, A. (2012). Cytogenetics: Principles and Application. Dagupan City: Space
Browser Publishing
Verma, P.S and Agarwal, V.K (2005). Cell Biology, Genetics, Molecular Biology, Evolution and Ecology. Ram
Nagar, New Delhi: S. Chand & Company LTD. p.42-43; 54
Powerpoint prepared by Mr. Von Carlo Dela Torre, M.S.C

TOPIC 7: HUMAN GENETIC DISORDERS

7.1:Genetic Disorder: Definition and Classification

Both environmental and genetic factors have roles in the development of any disease. A genetic disorder
is a disease caused by abnormalities in an individual's genetic material (genome). The four different types
of genetic disorders are:

• single-gene
• multifactorial
• chromosomal, and

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 • mitochondrial

1. Single-gene (also called Mendelian or monogenic) — This type is caused by changes

or mutations that occur in the DNA sequence of one gene. Genes code for proteins, the
molecules that carry out most of the work, perform most life functions, and even make up
the majority of cellular structures. When a gene is mutated so that its protein product can no
longer carry out its normal function, a disorder can result. There are more than 6,000 known
single-gene disorders, which occur in about I out of every 200 births. Some examples are
cystic fibrosis, sickle cell anemia, Marfan syndrome, Huntington's disease, and hereditary
hemochromatosis.
2. Multifactorial (also called complex or polygenic)- This type is caused by a combination
of environmental factors and mutations in multiple genes. For example, different genes that
influence breast cancer susceptibility have been found on chromosomes 6, 11, 13, 14, 15, 17,
and 22. Its more complicated nature makes it much more difficult to analyze than single-gene
or chromosomal disorders. Some of the most common chronic disorders are multifactorial.
Examples include heart disease, high blood pressure, Alzheimer's disease, arthritis, diabetes,
cancer, and obesity. Multifactorial inheritance also is associated with heritable traits such as
fingerprint patterns, height, eye color, and skin color.
3. Chromosomal - Chromosomes, distinct structures made up of DNA and protein, are located
in the nucleus of each cell. Because chromosomes are carriers of genetic material, such
abnormalities in chromosome structure as missing or extra copies or gross breaks and
rejoinings (translocations) can result in disease. Some types of major chromosomal
abnormalities can be detected by microscopic examination. Down syndrome or trisomy 21 is
a common disorder that occurs when a person has three copies of chromosome 21.
4. Mitochondrial - This relatively rare typeof genetic disorder is caused by mutations in the
non-chromosomal DNA of mitochondria. Mitochondria are small round or rod-like organelles
involved in cellular respiration and found in the cytoplasm of plant and animal cells. Each
mitochondrion may contain 5 to 10 circular pieces of DNA.

Why do we have genes that cause genetic disorder?

Many genes are named for the disorders to which they have been linked. This can be very
confusing. For example, the gene associated with hereditary hemochromatosis is called the
“hemochromatosis gene.” This name implies that the gene exists for the sole purpose of causing
disease, which of course is not the case. The normal function of a gene is to encode a protein,
not cause illness. Disease occurs when genes are unable to work properly. The hemochromatosis
gene actually codes for a membrane protein that works with other proteins to regulate iron
absorption in cells. Like other single-gene disorders, hemochromatosis occurs when a gene us
mutated in a ay that prevents it from encoding a normal, functional protein product.

7.2:Common Human Genetic Disorders

The following lists the common human genetic disorders in alphabetical order

• Achromatopsia (inability to see color)

• Adrenal Hypoplasia Congenita (reduction in adrenal gland function)
• Albinism/Hypopigmentation (no melanin pigment in eyes, skin and hair)
• Alzheimer’s (degenerative disease starting with memory loss)
• Amyblopia (poor or indistinct vision)
• Ataxia Telangiectasia (immunodeficiency disorder)
• Autism (brain development disorder)
• Batten Disease (Fatal, autosomal recessive neurodegenerative disorder)
• Best’s Disease (progressive vision loss)
• Cerebral Palsy (physical disability in human development)
• Color Blindness
• Cooley’s Anemia/Thalassemia (formation of abnormal hemoglobin molecules)
• Cystic Fibrosis (progressive disability due to multisystem failure)
• Cystinosis (autosomal recessive disorder of the renal tubules)
• Diabetes

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 • Down Syndrome (Impairment of cognitive ability, physical growth & facial appearance)
• Epidermolysis Bullosa (disorder of the autonomic nervous system)
• Familia Dystautonomia (disorder of the autonomic nervous system)
• G6PD (Glucose-6-phosphate Dehydrogenase) (Deficiency Anemia)
• Gaucher’s Disease (deficiency of the enzyme glucocerebrosidase)
• Glaucoma (disease of the optic nerve)
• Hemophilia/Bleeding Disorders (inefficient control over blood clotting or coagulation)
• Huntington’s Disease (abnormal body movements)
• Hurler Syndrome (abnormal body movements)
• Klinefelter Syndrome (small testicles and reduced fertility)
• Krabbe Disease (fatal degenerative disorder of nervous system)
• Leber Congenital Amaurosis (loss of vision)
• Leukodystrophies (progressive degeneration of the white matter of the brain)
• Muscular Dystrophy (progressive muscle weakness)
• Neimann-Pick Disease (disorder affecting lipid metabolism)
• Peutz-Jeghers Syndrome (bening hamartomatous polyps in gastrointestinal tract)
• Phenylketonuria (PKU) (deficiency in enzyme phenylalanine hydroxylase)
• Progeria (accelerated aging)
• Ptosis (dropping upper eyelid or breasts)
• Sickle cell Anemia (abnormal,rigid, sickle shape of red blood cells)
• Skeletal Dysplasias (Abnormal bone and cartilage development)
• Spina Bifida (incompletely formed spinal cord)
• Tay-Sachs Disease (usually affects nervous tissue of the brain)
• Werner Syndrome (premature aging)
• Williams Syndrome (“elfin” facial appearance, with low nasal bridge)

Reference: Camara, J.S and Oclay, A. (2012). Cytogenetics: Principles and Application. Dagupan City: Space
Browser Publishing

TOPIC 8: GENETIC ENGINEERING AND DNA TECHNOLOGY AND GENE THERAPY

8.1:Genetic Engineering and Recombinant DNA Technology

Recall about DNA Molecules

DNA is the keeper of the all the information needed to recreate an organism. All DNA is made up of a base
consisting of sugar, phosphate and one nitrogen base. There are four nitrogen bases, Adenine (A), Thymine
(T), guanine (G), and cytosine (C). The nitrogen bases are found in pair, with AT & T and G & C paired
together. The sequence of the nitrogen cases can be arranged in a in infinite ways, and their structure is
known as the famous “Double helix”. The sugar used in DNA is Deoxyribose. The four nitrogen bases are
the same for all organisms. The sequence and number of bases is what creates diversity.

DNA does not actually make the organism, it only makes proteins. The DNA is transcribed intro mRNA and
mRNA is translated into protein, and the protein then forms the organism. By changing the DNA sequence,
the way in which the protein is formed changes. This leads to either a different protein, or an inactive
protein.

WHAT IS A RECOMBINANT DNA?

Recombinant DNA is the general name for taking a piece of one DNA, and combining it with another strand
of DNA. Thus, the name recombinant.

It is also sometimes referred to as “chimera”. BY combining two or more different strands of DNA, scientists
are able to create a new strand of DNA. The most common recombinant process involves combining the
DNA of how different organisms.

THREE DIFFERENT METHODS RECOMBINANT DNA

• TRANSFORMATION
o First step in transformation is to select a piece of DNA to be inserted to a vector.

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 o Second step is to cut that piece of DNA with a restriction enzyme and then ligate the DNA
insert into the vector with DNA ligase. The insert contains a selectable marker which allows
for identification of recombinant molecules; an antibiotic marker is often used so a host
cell without a vector dies when exposed to a certain antibiotic, and the host with the vector
will live because it is resistant.
o The vector is inserted into a host cell, in a process called transformation. One example of
a possible host cell is E.coli. The host cells must be specially prepared to take up the foreign
DNA.
o Selectable markers can be for antibiotic resistance, color changes, or any other
characteristic which can distinguish transformed hosts from untransformed hosts. Different
vectors have different properties to make them suitable to different applications. Some
properties can include symmetrical cloning sites, size and high copy number.
• Non-Bacterial Transformation
o This is a process very similar to Transformation, which was described above.the only
difference between the two is that non-bacterial does not use bacteria such as E.coli for
host.
o In microinjection, the DNA is injected directly into the nucleus of the cell being transformed.
In biolistics, the host cells are bombarded with high velocity microprojectiles, such as
particles of gold or tungsten that have been coated with DNA.
• Phage Introduction
o Phage introduction is the process of transfection, which is equivalent to transformation
except a phase (a virus that infects a bacteria) is used instead of bacteria.
o In vitro packaging of a vector is used
o This use lambda or MI2 phages to produce plaques which contain recombinants
o The recombinants that are created can be identified by differences in the recombinants
and non-recombinants

How does rDNA Works?

Recombinant DNA works when the host cell expresses protein from the recombinant genes. A significant
amount of recombinant protein will not be produced by the host unless expression factors are added.
Protein expressions depend upon the gene being surrounded by a collection of signal which provide
instructions for the transcription and translation of the gene by the cell. These signals include the promoter,
the ribosome binding site, and the terminator. Expression vectors, in which the foreign DNA is inserted,
contains these signals. Signals are species specific. In the case of E.coli, these signals must be E.Coli signals
as E.coli is likely to understand the signals of human promoters and terminators.

Problems are encountered if the gene contains introns or contains signals which act as terminators to a
bacterial host. This result in premature termination and the recombinant protein may not be processed
correctly, be folded correctly, or may even be degraded.

Production of recombinant proteins in eukaryotic systems generally takes place in yeast and filamentous
fungi. The use of animal cells is difficult due to the fact that many need a solid support surface, unlike
bacteria, and have complex growth needs. However, some proteins are too complex to be produced in
bacterium, so eukaryotic cells must be used.

Why is rDNA important?

Recombinant DNA has been gaining in importance over the last few years, and recombinant DNA will only
become more important in the 21st century as genetic diseases become more prevalent and agricultural
area is reduced. Below are some of the areas where recombinant DNA will have an impact

• Better Crops (drought & heart resistance)

• Recombinant Vaccines (i.e. Hepatitis B)
• Prevention and cure of sickle anemia
• Prevention and cure of cystic fibrosis
• Production of clotting factors
• Production of insulin
• Production of recombinant pharmaceuticals
• Plants that produce their own insecticides
• Germ line and somatic gene therapy

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What is Genetic Engineering?

Genetic engineering is the process of identifying and isolating DNA from living or dead cell and introducing
it into another living cell. Before the genetic material is introduced, it may be altered in the laboratory.
When the genetic engineering process is successful, the new DNA becomes permanently incorporated into
the chromosomes of the new cell, and appear in the DNA of progeny cells as well. How can scientist do
genetic engineering? They use recombinant DNA technology.

Tools of Genetic Engineering

• Enzymes- such as exonuclease, endonucleases, restriction enzymes (=restriction endonuclease),

SI enzymes (to change cohesive ends of single stranded DNA fragments into blunt ends), DNA
ligases, alkaline phosphatases, reverse transcriptase, DNA polymerase
• Foreign DNA/Passenger DNA- it is a fragment

8.2:Gene Therapy

Gene therapy is a treatment that involves altering the genes inside your body’s cells to stop disease.

Genes contain your DNA- the code that controls much of your body’s form and function, from making you
grow taller to regulating your body systems. Genes that don’t work properly can cause disease. Gene
therapy replaces a faulty gene or adds a new gene in an attempt to cure disease or improve your body’s
ability to fight disease. Gene therapy holds promise for treating a wide range of diseases, including cancer,
cystic fibrosis, heart disease, diabetes, hemophilia and AIDS. Researchers are still studying how and when
to use gene therapy. Currently, in the United States, gene therapy is available only as part of a clinical trial.

Gene therapy is the insertion of genes into an individual’s cells and tissue to treat a disease, such as a
hereditary disease in which a deleterious mutant allele is replaced with a functional one. Although the
technology is still in its infancy, it has been used with some success. Scientific breakthroughs continue to
move gene therapy toward mainstream medicine.

Methods to replace or repair genes

There are a variety of different methods to replace or repair the genes targeted in gene therapy.

• A normal gene may be inserted into a nonspecific location within the genome to replace a
nonfunctional gene. This approach is most common
• An abnormal gene could be swapped for a normal gene through homologous recombination.
• The abnormal gene could be repaired through selective reverse mutation, which returns the gene
to its normal function.
• The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered
• Spindle transfer is used to replace the entire mitochondria that carry defective mitochondrial DNA

Gene therapy can be performed at two different levels :

• Patient therapy- In patient therapy, cells with healthy genes may be introduced in the affected
tissue, so that the healthy gene overcomes the defect without affecting the inheritance of the
patient.
o Patient therapy include the following steps:
▪ identification of a defective gene;
▪ isolation or synthesis of normal healthy gene;
▪ isolation of cells of the tissue, where the normal healthy gene has to function;
▪ introduction of healthy gene into the cell. During early 1990s, in the routine
exercise of patient therapy any gene is isolated and this isolated gene is either
directly injected into the cell or be carried by a virus (vector) to which it is linked
by recombinant DNA technique. After entering the cell, the gene may become a
part of nuclear DNA or remain free in cytoplasm like extra-chromosomal DNA.
However, in each case RNA is synthesized only at the rate of few copies per cell
in comparison to normal cells where thousands of copies are made.

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 • Embryo therapy- In embryo therapy the genetic constitution of embryo at the post-zygotic level
is altered so that the inheritance is altered.
o This technique involves the following steps:
▪ In vitro fertilization of the egg;
▪ Insertion of normal gene into embryo at post-zygotic level, either with viruses or
directly by microinjection; and
▪ Integration of inserted gene in host DNA, where it may or may not function.

Gene Therapy Issues

• Short-lived nature of gene therapy — Before gene therapy can become a permanent cure for any
condition, the therapeutic DNA introduced into target cells must remain functional and the cells
containing the therapeutic DNA must be long-lived and stable. Problems with integrating
therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene
therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of
gene therapy.
• Immune response — Anytime a foreign object is introduced into human tissues, the immune system
has evolved to attack the invader. The risk of stimulating the immune system in a way that reduces
gene therapy effectiveness is always a possibility. Furthermore, the immune system's enhanced
response to invaders that it has seen before makes it difficult for gene therapy to be repeated in
patients.
• Problems with viral vectors — Viruses, the carrier of choice in most gene therapy studies, present
a variety of potential problems to the patient —toxicity, immune and inflammatory responses, and
gene control and targeting issues. In addition, there is always the fear that the viral vector, once
inside the patient, may recover its ability to cause disease.
• Multigene disorders — Conditions or disorders that arise from mutations in a single gene are the
best candidates for gene therapy. Unfortunately, some of the most commonly occurring disorders,
such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused
by the combined effects of variations in many genes. Multigene or multifactorial disorders such as
these would be especially difficult to treat effectively using gene therapy.
• Chance of inducing a tumor (insertional mutagenesis) - If the DNA is integrated in the wrong place
in the genome for example in a tumor suppressor gene, it could induce a tumor. This has occurred
in clinical trials for X-Iinked severe combined immunodeficiency (X-SCID) patients, in which
hematopoietic stem cells were transduced With a corrective transgene using a retrovirus, and this
led to the development of T cell leukemia in 3 of 20 patients. Deaths have occurred due to gene
therapy, including that of Jesse Gelsinger.

References: Camara, J.S and Oclay, A. (2012). Cytogenetics: Principles and Application. Dagupan City: Space
Browser Publishing
Verma, P.S and Agarwal, V.K (2005). Cell Biology, Genetics, Molecular Biology, Evolution and Ecology. Ram
Nagar, New Delhi: S. Chand & Company LTD.

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