Muzzammil Hussain

Student ID; 109727113

Human molecular genetics-II

1). Please explain differentiation events in early human

development, including morula, blastocyst, inner cell mass,
trophoblast, epiblast, hypoblast, etc.

There are several differentiation events in early human development in which

cells become specialized during development. In these events as the animal
embryos develop, their cells become progressively more specialized
(differentiated), and their potency becomes more restricted.

The mammalian zygote and its immediate cleavage descendants which are
usually up to the 8-16-cell stage are totipotent. Each such cell or blastomere can
give rise to all possible cells of the organism. However, following this, the cells
become more restricted in their ability to give rise to different cell types. Only a
small minority of the cells in the very early mammalian embryo give rise to the
proper organism. Mostly, other cells are devoted to making four kinds of
extraembryonic membranes. As well as protecting the embryo and later the
fetus, the extra embryonic membranes provide it with nutrition, respiration, and
excretion. The Morula stage is the 16-cell stage in mammals, in which the
embryo appears as a solid ball of cells, but it is possible to discriminate between
cells on the outside of the cluster and those in the interior. Fluid begins to be
secreted by cells so that in the subsequent blastocyst stages, the ball of cells is
hollow with fluid occupying much of the interior. A clear distinction can now
be seen between two separate cell layers: an outer layer of cells (trophoblast)
and a small group of internal cells (the inner cell mass). The outer cells
(trophoblast) will ultimately give rise to one of the extraembryonic membranes,
the chorion, which later combines with maternal tissue to form the placenta.
The Inner cell mass will give rise to the embryo proper plus the other
extraembryonic membranes. Even before mammalian embryos implant in the
uterine wall, the inner cell mass begins to differentiate into two layers, the
Epiblast and the Hypoblast. The epiblast gives rise to some extraembryonic
tissue as well as all the cells of the later stage embryo and fetus, but the
hypoblast is exclusively devoted to making extraembryonic tissues. The cells of
the inner cell mass have traditionally been considered to be pluripotent: they
can give rise to all of the cells of the embryo, but unlike totipotent cells they
cannot give rise to extraembryonic structures derived from the trophoblast. At
any time up until the late blastocyst, the potency of the embryonic cells is
demonstrated by the ability of the embryo to form twins. The embryo proper is
formed from the embryonic epiblast. At an early stage, germ-line cells are set
aside. Some of the embryonic epiblast cells are induced by signals from
neighbouring extraembryonic cells to become primordial germ cells. At a later
stage, gastrulation occurs. Here, the embryo undergoes radical changes, and the
non-germ-line cells are organized into three fundamental layers of cells.

The three germ layers, which are ectoderm, mesoderm, and endoderm, and they
will give rise to all the somatic tissues. The constituent cells of the three germ
layers are multipotent, and their differentiation potential is restricted. The
ectoderm cells of the embryo give rise to epidermis, neural tissue, and neural
crest, but they cannot normally give rise to kidney cells (mesoderm-derived) or
liver cells (endoderm derived). Cells from each of the three germ layers undergo
a series of sequential differentiation steps. Eventually, unipotent progenitor cells
give rise to terminally differentiated cells with specialized functions.
Morula is an early-stage embryo consisting of 16 cells which are called
Blastomeres in a solid ball contained within the zona pellucida.

Blastocyst is a structure formed in the early development. It possesses an inner

cell mass (ICM) which subsequently forms the embryo. The outer layer of the
blastocyst consists of cells collectively called the trophoblast. This layer
surrounds the inner cell mass and a fluid-filled cavity known as the blastocoel.

Trophoblast gives rise to the chorion and amnion that surround the embryo.
The placenta derives from the embryonic chorion and the underlying uterine
tissue of the mother.

Inner cell mass (ICM) is also known as the embryoblast or pluriblast is the
mass of cells inside the primordial embryo that will eventually give rise to the
definitive structures of the fetus.

Epiblast (also known as the primitive ectoderm) is one of two distinct layers
arising from the inner cell mass in the mammalian blastocyst. It derives the
embryo proper through its differentiation into the three primary germ layers,
ectoderm, mesoderm and endoderm, during gastrulation. The amnionic
ectoderm and extraembryonic mesoderm also originate from the epiblast.

Hypoblast is one of two distinct layers arising from the inner cell mass in the
mammalian blastocyst. The hypoblast gives rise to the yolk sac, which in turn
gives rise to the chorion.

2). Choose three animal models to explain their major advantages

and disadvantages.

Drosophila melanogaster (fruit fly) belongs to invertebrates. The major

advantage is easy to breed (GT = 12-14 days); It has sophisticated genetics and
large numbers of mutants available. The major disadvantage is body plan of
Drosophila is different from that of vertebrates and it cannot be stored frozen.
Danio rerio (zebrafish) belongs to fish. The major advantage is relatively easy
to breed (GT = 3 months) and maintains large populations, it has good genetics.
The major disadvantage of zebrafish is small embryo size makes manipulation
difficult.

Mus musculus (mouse) belongs to Mammals. The major advantage is relatively

easy to breed (GT = 2 months), It has sophisticated genetics and many different
strains and mutants. The major disadvantage of the mouse is small embryo size
makes manipulation difficult implantation of the embryo makes it difficult to
access.

3). a. List three morphogenetic processes in development,

including the examples. b. List three processes resulting from
altered cell adhesion, including the examples

a. Morphogenetic processes in development

Process Example

Gain of cell-cell adhesion Condensation of cells of the cartilage

mesenchyme in vertebrate limb bud
Cell-matrix interaction Migration of neural crest cells and
germ cells
Cell fusion Formation of trophoblast and
myotubes in mammals.

b. Processes resulting from Altered cell adhesion

Process Example
 Ingression The movement of a cell from the
surface of an embryo into its interior.
Egression The movement of a cell from the
interior of an embryo to the external
surface.
Intercalation The opposite of delamination in
which the cells from multiple cell
layers merge into a single epithelial
sheet.

4). a. What is the function of acrosome vesicle in sperm? b.

Explain the detailed structure & function of the primitive streak.
How to form the trilaminar embryo?

a. The acrosomal vesicle (or acrosome) at the anterior end contains digestive
enzymes. Fertilization begins with the attachment of sperm to the zona
pellucida followed by the release of enzymes from the acrosomal vesicle,
causing local digestion of the zona pellucida. The head of the sperm then fuses
with the plasma membrane of the oocyte and the sperm nucleus passes into the
cytoplasm. Within the oocyte, the haploid sets of sperm and egg chromosomes
are initially separated from each other and constitute, respectively, the male and
female pronuclei. They subsequently fuse to form a diploid nucleus. The
fertilized oocyte is known as the zygote.

b. In mammals, birds, and reptiles, the major structure characterizing

gastrulation is a linear one, the primitive streak. This transient structure
appears, at about day 15 in human development, as a faint groove along the
longitudinal midline of the now oval-shaped bilaminar germ disk. Over the
course of the next day, the primitive streak develops a groove (primitive
groove), which deepens and elongates to occupy close to half the length of the
embryo. By day 16, a deep depression (the primitive pit), surrounded by a
slight mound of epiblast (the primitive node), is evident at the end of the
groove, near the centre of the germ disk.

The process of gastrulation is extremely dynamic, involving very rapid cell

movements. At day 14-15 in human development, the epiblast cells near the
primitive streak begin to proliferate, flatten, and lose their connections with one
another. These flattened cells develop pseudopodia that allow them to migrate
downward through the primitive streak into the space between the epiblast and
the hypoblast. Some of the ingressing epiblast cells invade the hypoblast and
displace its cells, leading eventually to the complete replacement of the
hypoblast by a new layer of epiblast-derived cells, the definitive endoderm.
Starting on day 16, some of the migrating epiblast cells diverge into the space
between the epiblast and the newly formed definitive endoderm to form a third
layer, the intraembryonic mesoderm. When the intraembryonic mesoderm and
definitive endoderm have formed, the residual epiblast is now described as the
ectoderm, and the new three-layered structure is referred to as the trilaminar
germ disk. The ingressing mesoderm cells migrate in different directions, some
laterally and others toward the anterior, and others are deposited on the midline
The cells that migrate through the primitive pit in the center of the primitive
node and come to rest on the midline form two structures.

• The prechordal (also called prochordal) plate is a compact mass of mesoderm

to the anterior of the primitive pit. The prechordal plate will induce important
cranial midline structures such as the brain.

• The notochordal process is a hollow tube that sprouts from the primitive pit
and grows in length as cells proliferating in the region of the primitive node add
on to its proximal end. The notochordal process and adjacent mesoderm induce
the overlying embryonic ectoderm to form the neural plate, the precursor of the
central nervous system. At the same time as the notochordal process extends,
the primitive streak regresses. By day 20 of human development, the
notochordal process is completely formed, but it then transforms from a hollow
tube to a solid rod, the notochord. The notochord will later induce the
formation of components of the nervous system.

5). a. Explain the principal derivatives of the three germ layers,

especially for the differentiation of various lateral mesoderm. b.
Use EGF developmental pathway to explain evolutionary
conservation.

a. The three germ layers formed during gastrulation will eventually form all
tissues of the embryo. The connective tissue of the head and the cartilage of the
skull and of structures derived from branchial arches are a mixture of
ectodermal and mesodermal tissue, as shown. Although the notochord persists
in adults in some primitive vertebrates, in mammals and other higher vertebrates
it becomes ossified in regions of forming vertebrae and contributes to the center
of the intervertebral disks. Some of the embryonic mesoderm cells goon to form
extraembryonic mesoderm.
 principal derivatives of the three germ layers of the early embryo give

rise to the many different tissues of the mature organism.

The ingressing mesoderm cells that migrate laterally condense into rodlike and
sheetlike structures on either side of the notochord. There are three main
structures

• The paraxial mesoderm, a pair of cylindrical condensations lying

immediately adjacent to and flanking the notochord, first develops into a series
of whorl-like structures known as somitomeres, which form along the antero-
posterior axis through the third and fourth weeks of human development. The
first seven cranial somitomeres will eventually go on to form the striated
muscles of the face, jaw, and throat, but the other somitomeres develop further
into discrete blocks of segmental mesoderm known as somites. Cervical,
thoracic, lumbar, and sacral somites will establish the segmental organization of
the body by giving rise to most of the axial skeleton (including the vertebral
column), the voluntary muscles, and part of the dermis of the skin.

• The intermediate mesoderm, a pair of less pronounced cylindrical

condensations, just lateral to the paraxial mesoderm, later develops into the
urinary system, parts of the genital system, and kidneys.

• The remainder of the lateral mesoderm forms a flattened sheet, known as the
lateral plate mesoderm. Starting on day 17 of human development, each of the
lateral plates splits horizontally into two layers separated by a space that will
become the body cavity, the coelom. The dorsal layer is known as the somatic
(or parietal) mesoderm or somatopleure. It becomes applied to the inner surface
of the ectoderm and will give rise to the inner lining of the body wall. The
ventral layer, adjacent to the endoderm, is the splanchnic (or visceral)
mesoderm or splanchnopleure, and it will give rise to the linings of the visceral
organs.

b. In mammals, epidermal growth factor (EGF) binds to its receptor, EGFR,

to initiate a Ras-Raf-MAP kinase cascade and promote the proliferation of
epidermal cells. Equivalent pathways exist in D. melanogaster and C. elegans,
but the pathways perform different functions. In Drosophila, the equivalent
pathway is used to promote differentiation of one of the eight photoreceptor cell
types during eye development, and in C. elegans it is used to stimulate the
division and differentiation of vulval cells. One of the best examples of
evolutionary conservation involves the homeotic genes, which seem to be
present in all animals and to have very similar functions. The fundamental role
of these genes in pattern formation is demonstrated by the ability of orthologous
genes from very different species to substitute for each other. Complete rescue
of the mutant phenotype has been achieved for some Drosophila mutants by
introducing the orthologous human gene. For example, the apterous mutant has
no wings, but addition of the normal allele of this Drosophila mutation to the
mutant embryo, or the human counterpart LHX2, results in a normal phenotype.
However, there are also significant differences between species, even those that
are closely related. There are very great differences between the vertebrate
embryos before gastrulation, reflecting different strategies for nutrient
acquisition, and the process of gastrulation is rather different between human
and mouse embryos, with a flat bilaminar germ disk in humans and a cup-
shaped one in mouse embryos. Sex determination mechanisms are also very
diverse. Not all mammals use the human model of XY sex determination, and
many reptiles dispense with the use of heteromorphic sex chromosomes
altogether by relying on the temperature of the environment to specify the sex
of the embryo.

6). Use fruit fly to explain polytene chromosomes, spatially and

temporally restricted expression of transgenes & the P element.

The fruit fly Drosophila melanogaster has been studied extensively for decades,
during which a vast amount of information has been built up about gene
function.

Polytene chromosomes which are present in the salivary gland cells in the
larval stages, these interphase chromosomes arise through repeated DNA
replication without separation into daughter nuclei. However, there is a
arrangement in which 1024 copies of a normally single DNA duplex are
arranged side by side. For which, these exceptional interphase chromosomes are
visible under the light microscope, and so chromosome breakpoints and
hybridized DNA clones can be mapped on chromosomes at high resolution.

Spatially and temporally restricted expression of transgenes is possible with

the GAL4-UAS system of conditional gene expression. Large-scale
mutagenesis screens are possible. In many cases, loss of function does not result
in a mutant phenotype, but transgene misexpression often gives clues to gene
function by producing dominant/dominant-negative phenotypes. One generation
screen for suppressors/ enhancers of dominant mutant phenotypes can identify
interacting genes. The yeast Flp-FRT recombinase system can be used to induce
mitotic clones and so form homozygous patches, permitting the observation of
phenotypes of lethal recessive mutations at late stages of development. Mitotic
recombination can also be used in a one-generation screen to score mutant
phenotypes in clones and recover lethal mutations that affect late development.

The P element is this Drosophila transposable element which permits several

types of experimental manipulation, including mutagenesis and transgenesis.
Unequal recombination between adjacent P-element inserts can also produce
precise deletions.

7). How comparative genomics can help to validate predicted

genes and identify novel genes?

Comparative genomics helps validate predicted genes and

Identifies novel genes

When the sequences of metazoan genomes were first reported, large numbers of
novel genes were predicted for which there was little or no supportive
experimental evidence. Often gene prediction relied rather heavily on the
identification of open reading frames (ORFs). However, apparent ORFs can
occur by chance within noncoding DNA. Confidence that an ORF is
functionally significant increases according to its length, but smaller ORFs are
more ambiguous. Gene predictions can also be made by using ESTs (expressed
sequence tags), but these could reflect occasional artifacts in making cDNA
libraries. A more robust way to validate a predicted gene is by supportive data
from clearly recognizable sequence counterparts in related species.
Comparative genomics has been used to great advantage in re-annotating
genomes. Early examples included genome sequencing of multiple different
Saccharomyces and Drosophila species to help annotate the previously studied
S. cerevisiae and D. melanogaster genomes. Many originally predicted genes in
the latter two genomes were discounted when equivalent ORFs or similar codon
substitution frequencies were not consistently found in orthologous sequences
in the other Saccharomyces or Drosophila species. Comparative genomics also
helps uncover novel genes by identifying sufficient sequence conservation. For
example. 1275 novel C. elegans genes were predicted only after comparison
with the nematode C. briggsae.

Comparative genomics has also been of great help in identifying novel genes
that produce functional non-coding RNA. Short RNA genes can easily be
overlooked in bioinformatics analyses of a single genome sequence. but
comparative genome analyses can identify signatures of purifying selection in
non-coding sequences as well as protein-coding DNA, and so they can focus
attention on functionally important noncoding DNA regions that have
subsequently been shown to be genes. Comparative genomics has also helped in
the prediction of conserved small RNA genes, notably miRNA genes.

8). a. What is exon shuffling? b. What are the functions of gene

duplication? Also explain orthologs, paralogs,
neofunctionalization & subfunctionalization of duplicated genes.

Exon shuffling

Protein domains are rarely restricted to one type of protein. For example, the
protein-binding kringle domains of lipoprotein(a) are widely found in blood-
clotting factors and fibrinolytic proteins, and the type II domains of fibronectin
are found in many cell surface receptors and extracellular matrix proteins.
Different exon shuffling mechanisms can give rise to the spreading of protein
domains to different proteins. Non-allelic recombination is one possibility, but
transposons are likely to have been important contributors. In particular,
retrotransposons offer the possibility of a copy-and-paste mechanism such that
domains are retained in the donor gene and copied into an acceptor gene.

Exon shuffling between genes can be mediated by transposable elements.

b. Gene duplication can permit increased gene dosage but its major value is
to permit functional complexity

Human genes have been duplicated by various duplication mechanisms. Gene

families are a major general feature of metazoan genomes, and one gene in a
hundred is estimated to be duplicated and fixed in the population every million
years. When a gene duplicates within a genome. the two resulting gene copies
are initially identical. In rare cases, the duplicated genes may be maintained to
produce identical, or essentially identical, gene products because increased
amounts of gene products are advantageous in some way. The large numbers of
genes that make essentially identical copies of different ribosomal RNA and
histone proteins come into this category.

In response to an altered environment. other genes may undergo duplication to

provide a selective advantage through increased gene dosage, as illustrated in
the recent adaptation of human populations to starch diets. Starch is digested
using the enzyme salivary amylase. The chimpanzee has a single salivary
amylase gene, but in humans a series of evolutionarily very recent tandem gene
duplications has produced multiple copies of such genes at the AMYl gene
cluster at Ip21. AMYl copy number varies significantly between haplotypes,
and AMYl copy number positively correlates with the expression of salivary
amylase. Human populations that historically consume a high-starch diet have
significantly more AMYl copies than populations that traditionally consume a
low-starch diet; an increased AMYl copy number seems to be an adaptive
response to increased starch in the diet.

Gene duplication to provide increased gene dosage is comparatively rare. More

frequently, after gene duplication, the sequences of the two genes diverge quite
extensively. One of the duplicated gene copies may become a pseudogene.
Alternatively, two divergent functional gene copies are retained. The divergent
gene copies may acquire different properties and often they come to be
expressed in different ways that are functionally advantageous. Gene
duplication is thus thought to be a major motor that drives increasing functional
complexity. The two homologous gene copies are described as paralogs (as
opposed to orthologs, which are present in different species).
Orthologs and paralogs

Retrogenes offer an immediate possibility for functional divergence: because

the gene copy is made at the cDNA level. it lacks the promoter and both
neighbouring and intronic regulatory elements of the parent gene. Continued
expression of a retrogene is therefore dependent on regulatory sequences in the
neighbourhood of its integration site that is usually rather different from the
sequences that regulate the parent gene. As a result, the parent gene and
retrogene copy can be expressed in different ways, often in different cell types.
After being exposed to a different cellular (and molecular) environment, the
retrogene can come to acquire different functions.

Genes duplicated at the genomic DNA level can also acquire different
functions. One possibility is that one of the duplicate genes acquires a
distinctive new function (neofunctionalization). Pure neofunctionalization is
rare, however. Usually, both genes undergo expression changes over
evolutionary time. Sometimes the duplicated genes acquire complementary
mutations in different Cis-regulatory regions so that they diverge in expression
while between them initially maintaining the expression domains of the
ancestral gene. As a result, different subsets of the functions of the ancestral
gene can be partitioned between the duplicated daughter genes
(subfunctionalization). Studies of whole-genome duplication indicate that
many duplicated genes can be retained over long time scales.

Functional divergence of duplicated genes. (A) Neofunctionalization. (B) Subfunctionalization

Homologs, genes that have significant sequence identity suggesting a close

evolutionary relationship. can be one of two types.

Paralogs are closely related genes present in a single genome as a result of gene
duplication. Paralogs are often identical in sequence immediately after gene
duplication.

Orthologs are genes present in the genomes of different species that are directly
related through descent from a common ancestor.

The Neofunctionalization term represents one of the duplicated genes that

retain the function of the ancestral gene. The other paralog undergoes mutations
that result in its having altered characteristics, leading it to acquire a new
function.

The Subfunctionalization term represents the duplicated gene copies

undergoing complementary deleterious mutations, often at the level of
regulatory elements. In the example, the ancestral gene is imagined to be
regulated by two sets of upstream control elements so that it is expressed in two
different cell types, or different tissue types, or at different developmental
stages. Mutations inactivate one set of control elements in one paralog and the
other set in the second paralog so that the expression patterns and functions of
the ancestral gene are partitioned between the two daughter genes.

9). Explain the four ways of activating (proto)-oncogenes, using

examples.

Oncogene activation Involves a gain of function. This can be quantitative (an

increase in the production of an unaltered product) or qualitative (the production
of a subtly modified product as a result of a mutation, or a novel product from a
chimeric gene created by a chromosomal rearrangement). These changes are
dominant and normally affect only a single allele of the gene.

Activation by amplification

Many cancer cells contain multiple copies of a structurally normal oncogene.

Breast cancers often amplify ERBB2 (= HERZ) and sometimes MYC; a related
gene, MYCN, is usually amplified in late-stage neuroblastomas and
rhabdomyosarcomas. Hundreds of extra copies may be present. They can exist
as small paired chromatin bodies separated from the chromosomes (double
minutes) or as insertions within the normal chromosomes (homogeneously
staining regions). The genetic events producing these may be quite complex
because they usually contain sequences derived from several different
chromosomes. Similar gene amplifications are seen in cells exposed to strong
artificial selective regimes-for example amplified dihydrofolate reductase genes
in cells selected for resistance to methotrexate. In all cases, the result is a great
increase in the quantity of the gene product. Amplification of a specific
oncogene in tumor cells can be studied by fluorescence in situ hybridization
(FISH) or by staining with the appropriate antibody. Alternatively, a genome-
wide search for amplified sequences can be made by using comparative
genomic hybridization. or by analyzing the intensity of signals on a SNP chip .
The same techniques also reveal any loss of material, which may point to tumor
suppressor genes.

Activation by point mutation

The three RAS family genes-HRAS, KRAS, and NRAS-encode small proteins
that mediate signalling by receptor tyrosine kinases on the cell surface. Binding
of ligand to the receptor triggers the binding of GTP to the Ras protein, and
GTP-Ras transmits the signal onward in the cell. Ras proteins have GTPase
activity, which rapidly converts GTP-Ras to the inactive GDP-Ras and switches
the signal off. Specific point mutations in RAS genes are frequently found in
cells from a variety of tumors including colon, lung, breast, and bladder cancers.
These almost invariably encode substitutions of amino acids 12, 13, or 61.
These all have the effect of decreasing the GTPase activity of the protein so that
the GTP-Ras is inactivated more slowly, leading to an excessive cellular
response to the signal from the receptor.

Another oncogene that is frequently activated by point mutations is BRAF.

This encodes a tyrosine kinase that relays the signal from activated Ras proteins
to the ERK kinase, eventually stimulating gene transcription. Two-thirds of
malignant melanomas, and a large number of other tumors, have an amino acid
substitution in the kinase domain of BRAF that permanently activates it. A
single mutation, p.V599E, accounts for 80% of all BRAF mutations in
malignant melanoma. BRAF is also sometimes activated by gene fusion.

Activation by a translocation that creates a novel chimeric gene

The best-known example of activation by a translocation that creates a novel

chimeric gene is the Philadelphia (Ph ') chromosome, a small acrocentric
chromosome seen in 90% of patients with chronic myeloid leukemia. The Ph'
chromosome is one product of a balanced reciprocal 9;22 translocation. The
breakpoint on chromosome 9 is within an intron of the ABL1 oncogene. The
translocation joins the 3' part of the ABU genomic sequence onto the 5' part of
the BCR (breakpoint cluster region) gene on chromosome 22, creating a novel
fusion gene. This chimeric gene is expressed to produce a tyrosine kinase
related to the ABU product but with abnormal transforming properties.

Many other tumor-specific recurrent rearrangements that produce chimeric

oncogenes have now been recognized. This mechanism is seen in 15-25% of
leukemias, lymphomas, and sarcomas, but has been reported in only 1 % or less
of the common solid epithelial tumors. The apparent scarcity of gene fusions in
epithelial tumors may, however, be deceptive, and be due to technical
difficulties. A review by Mitelman and colleagues gives a valuable discussion
and many examples. All known examples are cataloged in the Mitelman
database of chromosomal aberrations in cancer. At present, 358 different
fusions have been recognized, involving 337 different genes. Many genes are
promiscuous-that is, they are found in fusions with various different partners.
The MLL gene at llq23 is the most extreme example, having been noted in
fusions with more than 40 different partners. Fusions can be produced not only
by translocations but also by inversions or, occasionally, by deletions that
remove sequences that normally separate two genes.

Clinically, the specific gene fusions present in a patient's leukemic cells have an
important bearing on the management and prognosis. They can be identified by
a targeted FISH assay. For example, to check for the 9;22 translocation,
differently colored FISH probes for the BCR and ABU genes can be hybridized
to an interphase cell. If the translocation is present, there will be one BCR
signal, one ABL signal, and two fusion signals from the reciprocal products of
the translocation.
Activation by translocation into a transcriptionally active chromatin region

Burkitt lymphoma is especially common in malarial regions of Central Africa

and Papua New Guinea. Mosquitoes and Epstein-Barr virus are believed to have
some role in the etiology, but activation of the MYC oncogene is a central
event. A characteristic chromosomal translocation, t(8;14)(q24;q32), is seen in
75-85% of patients. The remainder have t(2;8)(pI2;q24) or t(8;22)(q24;qll).
Each of these translocations juxtaposes the MYC oncogene (normally located at
8q24) close to an immunoglobulin (IG) locus. This may be IGH at 14q32, IGK
at 2p12, or IGL at 22qll. Unlike the translocations shown in, these
translocations do not create novel chimeric genes. Instead. they bring the
oncogene under the influence of regulatory elements that normally ensure high
expression of the immunoglobulin genes in antibody-producing B cells. In the
8; 14 translocation, the MYC and IGH genes are in opposite transcriptional
orientations, head to head. Often, depending on the precise breakpoint, exon 1
of the MYC gene (which is noncoding) is not included in the translocated
material. Deprived of its normal upstream controls and placed in an active
chromatin domain, MYC is expressed at an inappropriately high level. Between
25% and 65% of all B-cell malignancies involve the activation of one or another
oncogene by an immunoglobulin enhancer, and many T-cell malignancies
involve a similar activation by an enhancer at a T-cell receptor locus.
Predictably, these rearrangements are characteristic of leukemias and
lymphomas, but not solid tumors.

10). a. How Rb controls cell cycle? b. What are the important

functions of p53? What are the relationships between Mdm2,
p14-ARF, p21-WAF1/CIP1 & p53?

a. pRb: a key regulator of progression through G1 phase

The RB1 gene was identified through its role in retinoblastoma, but it is widely
expressed and helps control the cycling of all cells. The gene product, pRb, is a
110 kD nuclear protein. Some cells contain two related proteins, pi 07 and p130,
giving some redundancy in the Rb pathway. The lack of this redundancy in
certain cells probably explains why a loss of RB1 function results in very
specific types of tumor.

pRb binds and inactivates the cellular transcription factor E2F, the function of
which is required for cell cycle progression. At 2-4 hours before a cell enters the
S phase, complexes of D cyclins and Cdk4 or Cdk6 phosphorylate pRb. This
inactivates it, allowing E2F to become free. In cells with loss-of-function
mutations in RB1, E2F is inappropriately activated. Several viral oncoproteins
(adenovirus E1A, SV40-T antigen, and human papillomavirus E7 protein)
achieve the same result by binding and sequestering or degrading pRb, thus
favouring cell cycle progression.

b. p53: the guardian of the genome

The pS3 transcription factor, encoded by the TP53 gene, has been called the
guardian of the genome because of its central role in preventing inappropriate
cell cycling. Tumor cells with absent or non-functional pS3 may continue to
replicate damaged DNA and do not undergo apoptosis.

Normally, pS3 levels in a cell are low. The Mdm2 protein ubiquitylates pS3,
which targets it for degradation. MDM2 is itself a transcriptional target of pS3,
so there is a negative feedback loop that keeps pS3 concentrations low. MDM2
is an oncogene that is amplified in many sarcomas. Signals from a whole range
of cellular stress sensors, including sensors of DNA damage, leading to
phosphorylation of pS3. Phosphorylated pS3 is no longer a substrate for Mdm2,
and hence the level of pS3 in the cell rises. This increases pS3-dependent
transcription of genes such as that encoding p21 WAF LlCIP1, an inhibitor
ofCdk2 and hence of cell cycling, and of genes such as PUMA, EAX, and
NOXA that control apoptosis.

p53 has two relatives, p63 and p73, with functions partly overlapping those of
p53. However, TP53 is the major target of mutations in cancer. Loss or
mutation of TP53 is probably the commonest single genetic change in cancer.
TP53 maps to 17p 13, and this is one of the commonest regions of loss of
heterozygosity in a wide range of tumours. Tumours that have not lost TP53
very often have mutated versions of it. To complete the picture of TP53 as a
tumour suppressor gene, constitutional mutations in TP53 are found in families
with the dominantly inherited Li-Fraumeni syndrome (OMIM 151623).
Affected family members suffer multiple primary tumours, typically including
soft tissue sarcomas, osteosarcomas, tumors of the breast, brain, and adrenal
cortex, and leukemia.