Organization of the genetic

material of pro- and eukaryotic

cells and the structure of the
nucleus
• Genetics, 5th ed. chapter 2.2, 4.1-4.3, 8
• Essential Genetics, 5th ed. chapter 1.2,
3.1, 3.4, 3.5
• Molecular Biology of the Cell, 4th ed.
chapter 4, 12 p669-677
• Essential Cell Biology 3rd ed. chapter 5
 Organization of the prokaryotic and eukaryotic
cells

ø = 0,5 - 2 μm ø = 2 - 100 μm
Veu ≈ 1000 Vpro
 bacteriophage:
cca. 50 m DNA)

Human:
Cca 4 cm
DNA/chromosome
(linear)

Escherichia coli: 1-2-3 rule: 1 μm dsDNA =

Cca 1.4 mm DNA in one circular 2 MDa (2×106 Da) = 3 kbp
bacterial chromosome
Localisation of chromosomes 18 and 19
in an interphase nucleus.
Human karyogram
Paires of
autosomes:

#1 to #22

Pair of sex
chromosomes:

X and Y
Schematic drawing of the cell nucleus
 Electron micrograph of
the cell nucleus

• Euchromatin: less
dense, less compact,
genetically active
• Heterochromatin: more
dense, more compact,
transcriptionally
nucleolus inactive.
– Constitutive
euchromatin heterochromatin:
telomer,
centromer
nuclear envelope
– Facultative
nuclear heterochromatin:
lamina e.g. on inactive X
heterochromatin chromosome
 The functions of the nucleus

• Stores genes on chromosomes

• Organizes genes into chromosomes to allow cell
division
• Transports regulatory factors and gene products
via nuclear pores
• Produces messages (mRNAs) that code for proteins
• Produces ribosomal subunits in the nucleolus
Structurally, the nucleus is composed
of three main parts:

• 1) the nuclear envelope

+ the nuclear lamina
• 2) the nucleolus
• 3) the chromatin
1) The nuclear envelope
Electron micrograph of the nuclear pores

0.5 m
A nuclear pore complex has four structural
subunits:
a, lumenal subunit, which
contain transmembrane proteins
that ancor the complex to the
nuclear membrane.
b, columns subunit, which form
the bulk of the pore wall.
c, ring subunit, which form the
cytosolic and nuclear face of the
complex.
d, annular subunit, which
extends toward the center of the
pore.
The nuclear pores are the gateways,
that allows import and export
Nuclear import - export

Signal sequences (NLS)

Electron micrograph of the nuclear lamina, a network
of intermediate filaments
 The nuclear face of the inner membrane is
coated with the nuclear lamina.

cytoplasm

perinuclear space lamin B receptors outher and inner

membrane

nucleoplasm chromatine lamin B and A, C proteins.

 The changes of the nuclear envelope during the cell cycle

pore comlex
DNA
inner membrane
lamins outher membrane

FUSION OF THE COATED PHOSPHORYLATION

CHROMOSOMES OF LAMINS
nuclear
NUCLEUS IN INTERPHASE envelope
vesicle

chromosome
LATE
TELOPHASE chromatid
phosphorylated
lamins
PROPHASE

FUSION OF NUCLEAR
ENVELOPE VESICLES DEPHOSPHORYLATION
OF LAMINS
EARLY TELOPHASE
Mutations in the gene of lamin A result different syndromes

lipodystrophy

cardiomyopathy

muscular distrophy
2) The nucleolus
• nuclear organelle
• produces
ribosomes
(partially)
• associated with the
nucleolar organiser
region (NOR)
• A nucleus may
contain 1,2 or
several nucleoli.
NOR: rRNA genes
on p arm of
acrocentric
chromosomes
(13,14,15,21,22)
Function of
the
nucleolus
 pre rRNA
dense fibrillar
components

ribosome subunits granular

component

fibrillar
center

DNA loops with rRNA genes

In non-dividing cells; In dividing cells during In dividing cells, at
In dividing cells during the interphase the beginning of the
the interphase (after DNA replication): M (division) phase:
(before DNA replication):
Chromatin fiber: 1 dsDNA Chromatin fiber: 2 dsDNA Chromosome:
+ proteins + proteins 2 dsDNA + proteins
Chromatin: 46 dsDNA + Chromatin: 92 dsDNA + Chromatide: one of
proteins proteins the dsDNA + proteins
Packaging into
chromosomes results in a
104 x abbreviation
in length of the DNA.

beads-on-a-string form of
11 nm chromatin fiber
chromatin loops
(nucleosome fiber)
proteins of nuclear matrix
30nm chromatin fiber
(solenoid)
INTERPHASE

PROPHASE METAPHASE

entire
metaphase chromosome
 Chromatin isolated directly from an interphase nucleus appears in the
electron microscope as a thread 30 nm thick.

(A) 30 nm chromatin fiber

(B) 11 nm chromatin fiber

nucleosome core
particle
Consists of DNA
wound around a
core of proteins
formed from histones.
Figure 5-22 (part 1 of 2) Essential Cell Biology (© Garland Science 2010)
Figure 5-22 (part 2 of 2) Essential Cell Biology (© Garland Science 2010)
 The structure of the 30 nm fiber is variable
One model for the 30 nm fiber is a spiral or solenoid (helical coil) arrangement that
consists of 6 nucleosomes/turn with a fifth histone, typically one molecule of histone 1,
bound to the DNA on the inside of
the solenoid.

„zig-zag”
model
Condensins responsible for the generation
of coiled coil structure
The chromatin loop (or domain) is a unit of

• ~20-200kb
• Supercoiling
• DNA replication
– Region replicates early
when active
• tissue-specific gene
regulation and transcriptional
competence
RO: replication origin

SAR(s): binding sites for

DNA and topoisomerase II
known as scaffold-
attachement regions
 Nuclear Matrix
• It is the proteinaceous nuclear structure remaining after
nuclease, salt and detergent treatments of isolated nuclei
which maintains features of the overall nuclear architecture
including the nuclear lamina-pore complex, the nucleolus and
the nonchromatin fibrogranular matrix.
• It helps control the shape of chromosomes and regulate
heterochromatin and euchromatin
• Enzymes (DNA polymerases,
RNA polymerases, ect.) are Figure 07.07: The
nuclear matrix is a
linked to it, and the DNA moves. network of
filaments bound to
the nuclear
envelope and to
• The nuclear matrix proteins are DNA.

– the most abundant proteins, highly conserved in mammals

– species, cell type and tissue specific, developmental specific, cell
growth and proliferation specific, human cancer specific
 aaaaaaaaaaaaa
aaaaaaaaaaaaa
aaaaaaaaaaaaa
aaaaaaaaaaaaa
aaaaaaaaaaaaa
aaaaaaaaaaaaa aaaaaaaaaa

aaaaaaaaaaaaa
aaaaaaaaaaa
DNA observed in 20-200 kb loops
attached to a nuclear matrix in interphase
or to a chromosome scaffold in metaphase chromosomes.
(Major components of the nuclear matrix are lamins,
topoisomerase II and components of centromeres and
telomeres. Scaffold may be different and less
complex than matrix.)
Cell cycle and cell division
 Cell division is in very close relationship with
several important biological processes:

• Growth
• Development
• Reproduction
• Regeneration
• Inheritance
• Cancer
• The events that take place inside the cell from
one division to the next are collectively called the
CELL CYCLE
• Main steps of the cell
cycle:
– Reproductive Signal: to
initiate cell division
– The replication of the DNA
within the nucleus
– The packaging and
segregation of the replicated
DNA into two new nuclei
(chromosomes, nuclear
division)
– The division of the cytoplasm
(cytokinesis)
 1h

5h

Restriction point
(START)

8h
10 h

The cell cycle consists of interphase and M-phase

1 ds DNA replicating
2 ds DNAs
DNA

1 chromosome =
2 ds DNAs =
2 sister chromatides
 Condensation of the chromatin into
chromosomes
Condensins
coat the DNA
and make it
more
compact.
The sister
chromatids
are held
together by
cohesin.
During mitosis,
the cohesin
is removed,
except at the
centromere.
Chromosome movement during cell
division
 Microtubule-organizing center (MTOC)

• Centrosome is the
microtubule
organizing centre of
the animal cell
(MTOC).
• Centrosome-matrix
can be found
around the 2
centriolum.
• The centrosome is
replicated during
the interphase.
• During mitosis the
centrosomes are
moving toward the
opposite poles of
the cell.
 • A) Three classes of
microtubules of the
mitotic spindle.
• (B) Phase contrast
(polar) micrograph of an
isolated mitotic
spindle in metaphase.
The kinetochor
• Electron micrograph of
an anaphase chromatid
with microtubules
attached to its
kinetochor.
• Each kinetochor forms a
plaque on the surface of
the centromere.
• Most kinetochors have a
trilamilar structure.
• The number of
microtubules bound to a
metaphase kinetochor
varies from 1 to 40.
 Review of Mitosis
Prophase –mitotic spindle starts to form;
chromosomes condense

Prometaphase – nuclear envelope disappears;

mitotic spindle forms;
chromosomes attach to spindle

Metaphase – chromosomes aligned

along equator

Anaphase – chromatids separate;

chromosomes migrate to poles

Telophase – chromosomes decondense;

nuclear envelope forms

+ Citokinezis – cleavage furrow with

contractile ring splits cell in two
 Changes of chromosome number and DNA
content during the cell cycle

1 DNA molecule = 1 chromosome

2 DNA=
1 chrom.
The sexual
reproductive cycle

• Meiosis is specialized cell

division used for sexual
reproduction.
The genetic information
in the chromosomes
is shuffled, and the cells,
called gametes, typically get
one-half of the original DNA
complement.
 Meiosis
• The First Meiotic Division: Reduction
(number of chromosomes is reduced)
• The Second Meiotic Division: Equation
Reproduction: Asexual and Sexual

• In multicellular organisms, somatic cells each

contain two sets of chromosomes: DIPLOID
CELL.
• In each recognizable pair of chromosomes,
one comes from each of the two parents.
• The members of the pair are called
homologous chromosomes and have
corresponding but generally not identical
genetic information.
Human karyogram
Paires of
autosomes:

#1 to #22

Pair of sex
chromosomes:

X and Y
 Meiosis and sexual reproduction
AIM:
To keep, to stabilize the
diploid set of chromosomes
from generation to
generation.

To increase genetic
variability in the population.

Haploid cells contain just one homolog of each pair. The

number of chromosomes in a single set is denoted by n.
When haploid gametes fuse in fertilization, they create the
zygote, which is 2n, or diploid.
Comparison of mitosis and meiosis
MITOSIS MEIOSIS

Daughter cells Genetically identical Not identical, unique

Ploidy of daughter Diploid or haploid Haploid
cells
Accomplishments
1. Reproduction of 1. Reduction of
parental cell chromosome
2. Equal distribution number
of DNA to daughter 2. Independent
cells assortment
3. Recombination by
crossing over
 Mitosis & Meiosis

(individually)
• Events through the
first cell division of
meiosis:
– prophase
– prometaphase
– metaphase
– anaphase
– telophase

– cytokinesis
• The five stages of
meiotic prophase I.
– leptotene
– zygotene
– pachytene
– diplotene
– diakinesis
Stages of the meiotic prophase I.
Leptotene
Stages of the meiotic prophase I
Zygotene
Stages of the meiotic prophase I
Pachytene
• Electron micrograph of
a synaptonemal
complex from a meiotic
cell in pachytene
Stages of the meiotic prophase I
Diplotene
 Lampbrush chromosomes in an amphibian oocyte

2
3

Homologous replicated chromosomes pair to form this highly

extended, four stranded structure. This stage may persist
for months or years.
Stages of the meiotic prophase I
Diakinesis
• Paired homologous
chromosomes in meiotic
division I.
• A single crossover event has
occured earlier in prophase to
create one chiasma.

Bivalents with 3 chiasmata

Prometaphase I: nuclear envelope and
nucleoli disappear.
• Spindle forms; kinetochores of both
chromatids of a chromosome attach to
the same half-spindle.
• Which member of each homologous pair
goes to which pole is random.

Metaphase I: chromosomes are at the

equatorial plate; homologous pairs held
together by chiasmata.
Anaphase I: homologous chromosomes separate;
each chromosome consists of two chromatids.
• The individual chromosomes are pulled to the
poles, with one homolog of a pair going to one
pole and the other homolog going to the opposite
pole.

Telophase I: occurs in some organisms.

• Nuclear envelope reaggregates; followed by an
interphase called interkinesis.
• In other organisms, meiosis II begins
immediately.
 Meiosis: A Pair of Nuclear
Divisions
• The second meiotic division separates the
chromatids.
• Meiosis II is similar to mitosis but one difference
is that DNA does not replicate before meiosis II.
• The number of chromosomes in the resulting
cells is therefore half that found in diploid mitotic
cells.
• In meiosis II, sister chromatids are not identical
(because of crossing over in meiosis I)
• There is no crossing-over in meiosis II.
• Two major sources of
genetic variability
• (A) independent
assortment
• (B) recombination by
crossing over
Independent assortment
The result of independent
assortment:

Humans have 23 pairs of homologous

chromosomes;

223 different gametes can be produced in a

human by the independent assortment
(it is more than 8 million)

246 different zygotes can be formed due to the

random fusion of the gametes
70,368,744,000,000 kind
 n = DNA content
(here not sets of
chromosomes)

46 chromosomes,
46 DNA

46 chromosomes,
92 DNA

46 chromosomes, 23 chromosomes,
46 DNA 46 DNA

23 chromosomes, 23 chromosomes,
23 DNA 23 DNA
• Stages of oogenesis I.
• The stages of oogenesis II.
• In mammals primary oocytes are
formed between 3 and 8 months
of gestation in the embryo and
remain arrested in meiotic division
I, until the female becomes
sexually mature.
• A small number periodically
matures under the influence of
hormones, completing meiotic
division I, to become secondary
oocytes.
• Oocyte maturation is arrested at
metaphase of meiosis II, when the
egg is released from the ovary
(ovulation).
• The secondary oocyte completes
meiosis II only after fertilization.
• Stages of
spermatogenesis I.
• Stages of
spermatogenesis II.
• New cells enter meiosis
continually from the time
of puberty.
• Each cell that begins
meiosis gives rise to 4
mature gametes.
• Mature sperm forms by an
elaborate process of cell
differentiation.
• About twice as many cell
divisions occur in the
production of a sperm as
in the production of an
egg (56 or 27 divisions
occur from the zygote to
the mature sperm or egg,
respectively.
 Polytene chromosomes
from salivary gland of
Drosophila

This giant chromosomes

consist up to a thousand
copies of a chromosome bound
together in register.
Activitiy of genes can
efficiently be studied on
polytene chromosomes.
Chromosome analysis

Sampling:
• Amniocentesis (week 15)
• Chorionic villus sampling (week 10 – 12)
• Bone marrow biopsy (stem cells)
• Venipuncture (blood drawn -
lymphocytes)
• In some cases, an abnormality may occur
as the cells are growing in the lab dish.
Karyotype tests should be repeated to
confirm that an abnormal chromosome
problem is actually in the body of the
patient.
week 10 - 12
Chorionic villus sampling
1. specific mitogenic agent is used to make the cells to divide (to start
the cell cycle)
e.g. phytohaemagglutinine for blast transformation (create
lymphoblasts from lymphocytes)
2. block the cell cycle in metaphase
e.g. colchicin prevents the polymerization of microtubules (so
the forming of the mitotic spindle)
Fluorescence in situ hybridization
(FISH)
• Sequences specific for one
chromosome, chromosome
arm, or microdisected
chromosome band are
converted to probes labeled
with a fluorescent dye.
Probe sets for other
chromosomes can be made
similarly, each with a
different fluorescent dye, or
specific mixtures of dyes.

• Hybridization, under FISH conditions, with one chromosome's paint probe results
in specific labeling of that chromosome.
• Simultaneous hybridization with all probe sets results in a chromosome spread in
which each of the chromosomes in a human haploid chromosome set appears a
different color when viewed with a fluorescence microscope.
• The technique is known as chromosome painting and is used to detect
chromosome translocations, breakages and other anomalies.
Denver nomenclature
Human ideogram, G-bands

Paris nomenclature
reverse banding
Synonym for R-banding
stain. A reverse Giemsa
chromosome banding
method that produces
bands complementary to
G-bands; induced by
treatment with high
temperature, low pH, or
acridine orange staining;
often used together with
G-banding on human
karyotype to determine
whether there are
deletions.
Male cat, Q-
bands

Female cat, T-
bands

Human female, Q-
bands
 Unordered karyogram Ordered karyogram
(metaphase spread)
comparison

summarization of several ordered

karyograms of different cells / tissues

Karyotype: 46, XX
Diagnosis: normal female

human ideogram
Technical terms in cytogenetic
investigation
Hungarian usage English-American usage

karyogram metaphase spread

ordered karyogram karyotype
Karyogram analysis Karyotype analysis
Result: karyotype Description of karyotype ↓
↓
46 XX, 46XY
idiogram ideogram
characteristic for a species
(human, mouse, etc.)
46,XX
46,XY
 Chromosomal abnormalities
• Numerical abnormalities:
caused by nondisjunction
– meiotic failures during
gametogenesis:
homologous chromosomes
fail to separate during
anaphase I, or sister
chromatids fail to separate
during anaphase II
– mitotic failures of somatic
cells causing mosaicism
• Structural abnormalities:
results of improper
recombination or DNA
duplication or chromatid
segregation
– deletion
– insertion
– translocation
– inversion
– duplication
– isochromosome
• Euploid = contains all
chromosomes (complete
sets), normally:
• diploid somatic cells (2n)
• haploid gametes (germ
cells - n)

(euploid)
 Meiotic failures – disorders in
chromosome number
• Nondisjunction occurs when homologous
chromosomes fail to separate during anaphase I,
or sister chromatids fail to separate during
anaphase II.
• The result is a condition called aneuploidy.

• euploid = contains all chromosomes (complete

sets)
• aneuploid = not euploid, suppose normally a diploid
– 2n -1 = monosomic
– 2n + 1 = trisomic
– 2n - 2 = nullisomic
Mitotic nondisjunction and mosaicism
 Mitotic chromosome delay and mosaicism
diploid monosomic
cell line cell line

Bilateral gynandromorph
 Chimeras
Chimare: an animal which has
two or more different
populations of genetically
distinct cells that originated in
different zygotes

The chimeric animal shown here is a baby

"geep", made by combining a goat and
sheep embryo. Notice the chimerism
evident in the skin - big patches of skin on
front and rear legs are covered with wool,
representing the sheep contribution of the
animal, while a majority of the remainder of
the body is covered with hair, being derived
from goat cells.
Variance in the number of chromosome sets
• Polyploids have extra whole sets of chromosomes, and
this abnormality in itself does not prevent mitosis.

Ploidy Levels
• let n = number of chromosomes in basic set
– n = monoploid
– 2n = diploid
– 3n = triploid
– 4n = tetraploid
– 6n = hexaploid

• Although mitosis usually is unimpaired, meiosis is

problematic, especially for odd numbers of sets, as in
triploidy
Karyogram of an aborted embryo

69, XXX

Triploidy, viable only in mosaic form

Down-syndrome
47,XX +21
Incidence of Down's syndrome with increasing maternal age
Incidenceat chorionic villus
Maternal Age At Birth
sampling
20 ~1 in 750 1 in 1500
30 ~1 in 450 1 in 900
35 1 in 240 1 in 400
40 1 in 110 1 in 100
45 1 in 13 1 in 30
 Phenotypic map of trisomy 21

• Amyloid precursor protein – Alzheimer disease

• Purine synthetic enzymes – increased level of purines
• Oncogene ETS-2 – increased risk of leukemia
• Alpha-A crystalline – increased frequency of cataract
The epicantal fold
Pätau-syndrome
47, XX +13
Edwards-syndrome
47, XY +18
 X-inactivation (lyonization)
• It is a process by which one of the two copies of the X chromosome
present in female mammals is inactivated. The inactive X chromosome
is silenced by its being packaged in such a way that it has a
transcriptionally inactive structure called heterochromatin (Barr body).
As nearly all female mammals have two X chromosomes, X-inactivation
prevents them from having twice as many X chromosome gene
products as males, who only possess a single copy of the X
chromosome (dosage compensation). The choice of which X
chromosome will be inactivated is random during the embrionic
development in placental mammals such as humans, but once an X
chromosome is inactivated it will remain inactive throughout the lifetime
of the cell and its descendants in the organism (Mary Lyon).
• The X-inactive specific transcript (Xist) gene encodes a large non-
coding RNA that is responsible for mediating the specific silencing of the
X chromosome from which it is transcribed. The inactive X chromosome
is coated by Xist RNA, whereas the Xa is not. The Xist gene is the only
gene which is expressed from the Xi but not from the Xa. X
chromosomes which lack the Xist gene cannot be inactivated. Artificially
placing and expressing the Xist gene on another chromosome leads to
silencing of that chromosome.
One of the X chromosomes is
inactivated in females
X chromatin (Barr-body) in cells of
normal females
X inactivation results in dosage
compensation
Functional mosaicism caused by
random X-inactivation
Rainbow C. C., the clone of
Rainbow
Turner-syndrome
45,X
• isochromosome: arms
become separated not
sister chromatides
– long arm isochromosome
(e.g.: iXq, trisomy of the
long arm, monosomy of the
short arm)
– short arm isochromosome
(e.g.: iXp, trisomy of the
short arm, monosomy of
the long arm)
Turner-syndrome
46,X iXq
Klinefelter-syndrome
47,XXY
FISH images of metaphase chromosomes and interphase nuclei obtained
from peripheral blood sample of the patient.
Metaphase chromosomes and interphase cells hybridized with CEP X/CEP Y
probe.
Two red (chromosome X) and one green (chromosome Y) signals in the
metaphase showing 47,XXY chromosome constitution
testosterone level is low
Calico cat

female (XX)

sterile male (XXY)

48,XXXY More severe clinical
presentation than
47,XXY. Usually are
mentally retarded
49,XXXXY Moderate to severe
mental retardation;
marked hypogonadism;
skeletal abnormalities;
congenital heart disease
48,XXYY Taller but much like
47,XXY; phenotypic
overlap with XYY
47, XXX

Triple X condition
Double Y condition
When chromosomal sex do not match with gonads
symptoms symptoms
similar to similar to
Klinefelter- Turner-
syndrome syndrome
 When chromosomal sex do not match with genitalia
• real hermaphroditism (ovotesticular intersex)
• female and male gonads, more often oogenesis, than spermiogenesis,
usually they are sterile, but pregnancy also has occurred
• they can be 46,XX/46,XY chimeras, even temporally, but it can caused
even by autosomal disorder
• pseudohermaphroditism (ovarian [→ovaries and penis, ovarian
masculinization = virilization] or testicular [→ testis and vagina, testicular
feminization] intersex = gonad dysgenesis)
• testicular feminization = Morris-syndrome = complete androgen
insensitivy syndrome (CAIS)
• 46,XY, female, testis in the abdomen, testosterone level is high, no
uterus, feminization of external genitals, X-linked recessive, androgen
receptor mutation
• congenital adrenal hyperplasia
• 46,XX, male, ovarian development normal, excessive production of
androgens cause the masculanization of the external genitals,
autosomal recessive, enzymatic defect in cortisol biosythesis
 Mutations of larger areas, gene and
chromosome mutations
Results of improper recombination or DNA duplication:
• deletion
• insertion
• translocation
• inversion
• duplication
 Mutations of larger areas, gene and
chromosome mutations
Results of improper recombination or DNA duplication:
• deletion (fracture and loss)
– terminal or interstitial
• insertion (incorporation, unidirectional interstitial
translocation)
• translocation (change the place)
– terminal or interstitial (last one equals with insertion)
– unidirectional or reciprocal (e.g. for the last one: Robertsonian-
translocation)
– balanced or unbalanced
• inversion (change the orientation)
– paracentric (centromer is not involved) or pericentric (centromer
is involved)
• duplication (doubling)
– tandem (AB AB) or reversal (AB BA), on the same or on an other
chromosome
 Disorders in chromosome structure
Results of improper recombination or DNA duplication:
• deletion (fracture and loss)
– terminal or interstitial
• insertion (incorporation, unidirectional interstitial
translocation)
• translocation (change the place)
– terminal or interstitial (last one equals with insertion)
– unidirectional or reciprocal (e.g. for the last one: Robertsonian-
translocation = centromeric fusion)
– balanced or unbalanced
• inversion (change the orientation)
– paracentric (centromer is not involved) or pericentric (centromer
is involved)
• duplication (doubling)
– tandem (AB AB) or reversal (AB BA), on the same or on an other
chromosome
balanced carrier
45, XX -13,-14,+t(13;14)
balanced carrier
45, XY -14,-21,+t(14;21)
unbalanced carrier
46, XX -14,+t(14;21)
 14 21

balanced carrier t(14; 21)

(45, XX, -14, -21, +t(14;21)

A C B

unbalanced carrier
(46, XX, -14, +t(14;21)
 14 21

balanced carrier t(14; 21)

(45, XX, -14, -21, +t(14;21)

A C B

unbalanced carrier
(46, XX, -14, +t(14;21)
balanced carrier
She can have only baby
45, XX -21,-21+t(21;21) with Down-syndrome
unbalanced carrier
46, XX -21,+t(21;21)
TRANSLOCATION

Philadelphia chromosome is designated Ph (or Ph') chromosome and the

translocation is termed t(9;22)(q34.1;q11.2).
CML is diagnosed in the Cytogenetics lab by the
presence of the Philadelphia chromosome. This
is an acquired translocation involving
chromosomes 9 and 22

Karyotype showing the Philadelphia

chromosome.
46,XX,t(9;22)(q34.1;q11.2)
DELETION
Cri-du-chat-syndrome
46, XX 5p-
• ring chromosome: terminal deletion
of the ends of the chromosome and
the attachment of the two ends
IMPRINTING
 Possible effects of deletion

necdin
Inversion
Kariotyping
 Unordered karyogram Ordered karyogram
(metaphase spread)
comparison

summarization of several ordered

karyograms of different cells / tissues

Karyotype: 46, XX
Diagnosis: normal female

human ideogram
 Chromosomal abnormalities
• Numerical abnormalities:
caused by nondisjunction
– meiotic failures during
gametogenesis:
homologous chromosomes
fail to separate during
anaphase I, or sister
chromatids fail to separate
during anaphase II
– mitotic failures of somatic
cells causing mosaicism
• Structural abnormalities:
results of improper
recombination or DNA
duplication or chromatid
segregation
– deletion
– insertion
– translocation
– inversion
– duplication
– isochromosome
 Numerical chromosomal abnormalities
• Euploid: only complete
chromosomal set(s)
– Normal (human):
• haploid (n) gametes
• diploid (2n) somatic cells
– Abnormal (human: none of
them is viable):
• diploid (2n) gametes
• triploid (3n), tetraploid
(4n), pentaploid (5n) stb –
so poliploid somatic cells
(or gametes)
• Aneuploid: not only complete
chromosomal sets
– Monosomy: 2n-1
Viable: only monosomy of X
• Turner syndrome: 45,X
– Trisomy: 2n+1
Viable (can be born alive):
• Down syndrome : 47,X_,+21 (_: X
vagy Y)
• Pätau syndrome : 47,X_,+13
• Edwards syndrome : 47,X_,+18
• Klinefelter syndrome : 47,XXY
• Triplo-X condition: 47,XXX
• Double-Y condition: 47,XYY
– Tetrasomy (2n+2), pentasomy
(2n+3): viable only in case of sex
chromosomes (e.g.: 48,XXXY)
46,XX
46,XY
 Karyogram of an aborted embryo

Triploidy, not viable 69,XXX

Down-syndrome
47,XX+21
Pätau-syndrome
47,XX+13
Edwards-syndrome
47,XY+18
Turner-syndrome
45,X
Klinefelter-syndrome
47,XXY
47,XXX
 Mutations of larger areas, gene and
chromosome mutations
Results of improper recombination or DNA duplication:
• deletion (fracture and loss)
– terminal or interstitial
• insertion (incorporation, unidirectional interstitial
translocation)
• translocation (change the place)
– terminal or interstitial (last one equals with insertion)
– unidirectional or reciprocal (e.g. for the last one: Robertsonian-
translocation)
– balanced or unbalanced
• inversion (change the orientation)
– paracentric (centromer is not involved) or pericentric (centromer
is involved)
• duplication (doubling)
– tandem (AB AB) or reversal (AB BA), on the same or on an other
chromosome
Cri-du-chat-syndrome
46, XX 5p-
46,XY,t(3;21)
 CML is diagnosed in the Cytogenetics lab by the presence of the Philadelphia
chromosome. This is an acquired translocation involving chromosomes 9 and 22

Karyotype showing the Philadelphia chromosome.

46,XX,t(9;22)(q34.1;q11.2)
balanced carrier of Robertsonian translocation
45, XX -13,-14,+t(13;14)
balanced carrier of Robertsonian translocation
45, XY -14,-21,+t(14;21)
unbalanced carrier of Robertsonian translocation
46, XX -14,+t(14;21)
balanced carrier of Robertsonian translocation
She can have baby only
45, XX -21,-21+t(21;21) with Down syndroma
unbalanced carrier of Robertsonian translocation
46, XX -21,+t(21;21)
 KEY

• 1: deletion; 2: duplication; • 47,XXY

3: paracentric inversion; • 49,XXXXY
4: pericentric inversion • 45,XX,-13,-14,+t(13;14)
• 46,X,delXq or 46,X,Xq- • 47,XXX
• 45,X • 46,XY,-14,+t(14;21)
• 46,X,i(Xq) • 45,XY,-14,-21,+t(14;21)
• 48,XYY,+21 • 46,XX,5p-
• 46,XY-21,+t(21;21) • 45,XY,-7
• 47,XX,+21 • 47,XY,+15
• 47,XXY • 46,XX
• 47,XXX • 46,XY
• 47,XX,+13 • 69,XXY
• a, 45,X • 69,XXX
b, 46,X,i(Xq)
• 46,XY,t(16;22)
• 47,XY,+18
• 47,XY,+9
• 45,XY,-14,-21,+t(14;21)
• 47,XXY
• 46,XY,-14,+t(14;21)
• 47,XX,+8
• 47,XY,i(Xq)
• 46,XY,t(4;11)
• 46,XY,t(9;22)
• 47,XY,t(2;5),+7
The genome is the complete DNA sequence
of a gamete, individual, population
or species.
Gene: A hereditery unit.
A sequence/segment of chromosomal DNA
that codes for a functional product:
protein (polypeptide ) or RNA
(tRNA, rRNA, snRNA, snoRNA, microRNA,
etc)
Prokaryotic genome, prokaryotic gene.
• Eukaryotic genome, eukaryotic gene.
Transcription
Characteristics of the
prokaryotic genome and genes

• The genome is a single, circular,

covalently closed DNA molecule
(but there are exceptions)
• There are no histones in bacteria
• They may contain other genetic
elements like plasmids or phages
• The genome is small ( a few millions bp)
the gene density is high and almost 100% is coding.
• There are very few repeating sequences
In prokaryotes genes involved in the same function
are organized into operons (regulatory units)and
their activity is regulated together. A single
promoter controls all of these genes.
 The operon and its transcription

Operon:
Regulatory gene
Regulatory sequences
Structural genes
 Transcription in prokaryotes

The
transcription
and
translation
are coupled.

5’ untranslated 3’ untranslated
LEADER sequence coding region mRNS TRAILER seq

The transcriptional start site usually preceeds

the translational start codon (ATG) forming a LEADER
sequence. The SHINE-DALGARNO sequence (the ribosomal
binding site) is part of the leader sequence.
 Transcription in prokaryotes

A gén/ek genes
structure kódoló

mRNA

5’ untranslated 3’ untranslated
LEADER sequence coding region TRAILER seq

Most of the mRNA found in bacteria are policistronic

mRNA.
 Transcription and translation
in prokaryotes

The number of protein coding genes (cistrons)

is equal to the number of synthesized
proteins. The primary transcript is not
processed.

Ribosome binding sites /SHINE-DALGARNO sequences

proteins
 The promoter

At the 5’ of each operon lies a (100-1000 base pairs

long) DNA sequence at which transcription initiated.
The RNA polymerase holoenzyme containing sigma-70 protein
binds on this region.
A typical promoter has 3 components, consisting of:
- the -10 (Pribnow box) and
- the -35 consensus sequences
- the transcriptional startpoint (+1)
 Transcription termination
• ρ (rho)–factor dependent or rho independent
Operons: The Basic Concept – Gene expression
in bacteria is controlled by the operon model
• A cluster of functionally related genes can be under
coordinated control by a single on-off “switch”
• The regulatory “switch” is a segment of DNA called an
operator usually positioned close to the the promoter
• An operon is the entire stretch of DNA that includes
the operator, the promoter, and the genes that they
control
• The operon can be switched off by a protein repressor
• The repressor prevents gene transcription by binding
to the operator and blocking RNA polymerase
• The repressor is the product of a separate regulatory
gene
• The repressor can be in an active or inactive form,
depending on the presence of other molecules
• A corepressor is a molecule that cooperates with a
repressor protein to switch an operon off
The lac Operon of E. coli
Inducible and Repressible Operons: Two
Types of Negative Gene Regulation

• An inducible operon is one that is usually off; a

molecule called an inducer inactivates the repressor
and turns on transcription
• The lac operon is an inducible operon and contains
genes that code for enzymes used in the hydrolysis
and metabolism of lactose: catabolic operon
• By itself, the lac repressor is active and switches
the lac operon off
• A molecule called an inducer inactivates the
repressor to turn the lac operon on
• A repressible operon is one that is usually on;
binding of a holorepressor to the operator shuts off
transcription
• The trp operon is a repressible operon
 Regulatory Promoter
gene
Operator

DNA lacI lacZ

No
RNA
made
3
mRNA RNA
5 polymerase

Active
Protein repressor

(a) Lactose absent, repressor active, operon off

lac operon

DNA lacI lacZ lacY lacA

RNA
polymerase
3
mRNA
mRNA
5 5

Protein -Galactosidase Permease Transacetylase

Allolactose Inactive
(inducer) repressor

(b) Lactose present, repressor inactive, operon on

 Positive Gene Regulation
• Catabolic operons are also subject to positive control
through a stimulatory protein, such as catabolite
activator protein (CAP), an activator of transcription
• When glucose (a preferred food source of E. coli) is
scarce, CAP is activated by binding with cyclic AMP
• Activated CAP attaches to the promoter of the lac operon
and increases the affinity of RNA polymerase, thus
accelerating transcription
• The lac operon is an inducible operon and contains genes
that code for enzymes used in the hydrolysis and
metabolism of lactose
• By itself, the lac repressor is active and switches the
lac operon off
• A molecule called an inducer inactivates the repressor to
turn the lac operon on
• Catabolite repression: glucose inhibits cAMP biosynthesis
and thus inhibits the lac operon
Fig. 18-5
Promoter
Operator
DNA lacI lacZ
CAP-binding site RNA
polymerase
Active binds and
cAMP CAP transcribes

Inactive Inactive lac

CAP repressor
Allolactose
(a) Lactose present, glucose scarce (cAMP level
high): abundant lac mRNA synthesized
Promoter Operator
DNA lacI lacZ
CAP-binding site RNA
polymerase less
likely to bind
Inactive
CAP Inactive lac
repressor

(b) Lactose present, glucose present (cAMP level

low): little lac mRNA synthesized
 E. coli can synthesize the amino acid
tryptophan

• By default the anabolic trp operon is on and

the genes for tryptophan synthesis are
transcribed

• When tryptophan is present, it binds to the trp

repressor protein, which turns the operon off

• The repressor is active only in the presence of

its corepressor tryptophan; thus the trp operon
is turned off (repressed) if tryptophan levels
are high
Fig. 18-2
Precursor
Natural
selection
Feedback has favored
inhibition bacteria
trpE gene
that produce
Enzyme 1 Regulation only the
of gene products
trpD gene needed by
expression
that cell

Enzyme 2 trpC gene A cell can

regulate the
production
trpB gene
of enzymes
by feedback
Enzyme 3 inhibition
or by gene
trpA gene
regulation

Tryptophan

(a) Regulation of enzyme (b) Regulation of enzyme

activity production
Fig. 18-3
trp operon

Promoter Promoter
Genes of operon
DNA trpR trpE trpD trpC trpB trpA
Regulatory Operator
gene Start codon Stop codon
3
mRNA RNA mRNA 5’
5 polymerase
E D C B A
Protein Inactive Polypeptide subunits that make up
repressor enzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on

DNA
No RNA made

mRNA

Protein Active
repressor
Tryptophan
(corepressor)
(b) Tryptophan present, repressor active, operon off
 The eukaryotic genome and structure
of the eukaryotic genes
• The genetic information is found in the nucleus and
organelles (mitochondria in animals and mitochondria
and chloroplasts in plants).
• The haploid human nuclear genome is 3.3 billion bp
and organized into chromosomes that contain linear
DNA molecules.
• The mitochondrial genome is small (16 kb), and
contains a few dozens of genes
• The gene density in the nuclear genome is low, only
2% is coding.
• The gene density in the mitochondria is high
• The ratio of repeated sequences is high (50%).
• The genes are not continuous, they are interrupted
with non-coding sequences called introns.
• The genes are not arranged into operons.
• There is no coupled transcription and translation.
• The regulation of transcription is more
sophisticated.
• The number of synthesized proteins is much higher
than the number of genes.
 Genes are located throughout the genome, but
tend to cluster in some regions and on some
chromosomes. Some of the genes are orginized
into families of related genes. Some regions are
gene poor, „gene deserts” can be found on certain
chromosomes.
Eukaryotic transcriptional control:
major considerations
1. Chromatin and its impact on transcription

2. Multiple regulatory sequences: proximal

and distant

3. Interplay among multiple general

transcription factors;
activators/repressors;
mediator/coactivators

4. Co-transcriptional RNA processing

5. Regulation of transcriptional regulators

 The genome is topologically
organized in 3D space
Chromosmal Teritories is a major feature of the
chromosome architecture. Each chromosome resides
in a separate territory in the nucleus. It
appears that during each cell division the CTs
are re-shuffled randomly. Homologous chromosomes
are not in proximity.
Each chromosome is comprised of many distinct chromatin
domains, referred to variably as topological domains or
topologically associating domains (TADs). TADs are a reflection
of looping events or “loop domains” in the genome.
These chromatin domains are stable for many cell divisions,
invariant across diverse cell types, and evolutionarily
conserved in related species.

These chromatin domains have been considered the basic units of

chromosome folding, important secondary structure in chromosome
organization.
TADs create autonomous gene regulatory domains by constraining
the chromatin interactions among loci within each TAD to the
same domain. Consequences are:
-the “co-regulation” of genes within TADs,
-the blocking of the “spread” of activity between neighboring
TADs.
Genes within the same TADs share coordinated gene expression
profiles across different cell types and tissues. This effect
is not absolute, the spatial organization of chromatin
domains can change in response to regulation.

The transcriptional repressor CTCF and cohesin are known to be

associated with TAD formation.
 Different levels of the regulation
of gene expression in eukaryotes

• Transcriptional
regulation
• Epigenetic mechanism
of transcriptional
regulation
• RNA processing and
decay
• RNA interference
• Translational
control and
- post-translational
control
• DNA rearrangement
 Transcription
This process copies the template DNA strand to a
complementary RNA molecule. Catalyzed by RNA-
polymerase enzyme.

1. RNA-polimerase I: transcribes the rRNA genes

(except the 5S rRNA).

2. RNA polymerase II: transcribes the protein

coding genes

3. RNS-polymerase III: transcribes the tRNA

genes and the 5S rRNA.
Transcriptional Regulation of Gene
Expression
Eukaryotes regulate transcription by:
- Trans-acting regulatory proteins: transcription
factors (TF), these are mostly but not exclusively
DNA-binding proteins: transcriptional activator,
co-activator or repressor proteins
-Cis-acting regulatory DNA sequences: promoter,
enhancers, silencers, insulators, barriers and
locus control regions. These are binding sites for
enzymes and regulatory proteins.
General structural of a human gene
Gene is a segment
of a DNA
containing the
code for a
functional
macromolecule
(RNA or protein)
and the proximal
regulatory
sequences
necessary for its
expression.
 Cis-acting regulatory elements:
promoter
• Core promoter: binding site for RNA-
polymerase + TF complexes
– TATA box,
– CpG islands (housekeeping genes)
• Promoter proximal regulatory regions
(Upstream Promoter Elements): CAT box, GC
rich box, …
 Cis-acting regulatory elements:
enhancers; silencers, LCR
• Cis-acting DNA sequences that bind multiple transcription
factors to regulate gene expression.
• Enhancers enable genes to be transcribed only when proper
transcriptional activators are present. Can be either upstream
or downstream of the promoter and can act at a distance
(~1000’s of base pairs) from the gene, the orientation is
unimportant.
• Some genes are also subjected to regulation by transcriptional
silencers, that are binding sites for transcription factors
that, once recruited to the site prevent transcription of the
silenced genes
• LCR (locus control region): DNA sequences in eukaryotic cells
that control the expression of a family of genes.
 Trans-acting regulators:
transcription factors
• Transcription factors: mostly DNA-binding
proteins, bind to specific sequences on the DNA;
generally have two or more domains: DNA-binding
and protein-binding activator or repressor
domains
• Activators: proteins that, when bound to specific
DNA sequences near or far away from the genes,
activate transcription.
• Repressors: proteins that, when bound to specific
DNA sequences near or far away from the genes,
repress transcription.
Some regulatory proteins can function both as an
activator and a repressor.
Some TF-s bind to other proteins and not to DNA
directly.
Structural features of a human gene

„Cap” site terminátion

signal

Exon: sequence coding for amino acid sequence

of protein
Intron: intervening sequence, non-coding
sequence
 Constitutively expressed genes lack
CAT and TATA boxes
Promoters of house-keeping
genes often contains C and Transcriptionally active TF-s
G rich sequences, called
CpG islands.(CGI).
The CpG rich sequence
elements serve as binding
sites for specific TF-s.
CGI-s are targets of DNA
methylation which is
usually associated with
repression of gene
expression. Repressed by methylation
 Nucleotide sequence of the complete human -globin gene.
The sequence of the 5' to 3' strand of the gene is shown.
The distal and proximal regulatory sequences
and the assembly of transcription factors
• Transcription complex is an aggregate of protein
factors that combines with the promoter to initiate
transcription
• The basal transcription factors are proteins in the
complex that are used in the transcription of many
different genes (TBP: TATA-box-binding protein; TAFs:
TBP-associated proteins)
 The TATA-
binding protein
bends the DNA
in the promoter

• Transcriptional activation
occurs by a mechanism called
recruitment — the interaction
of TFs with the promoter and
with other proteins.
 Multiple enhancers and activators
can regulate an eukaryotic promoter
Sequence specific DNA-protein and protein-protein
interactions are important to activate the RNA
polymerase: combinatorial and cooperative
interactions. Loop formation in the chromatin
explains the interaction between distant sequences.

EM picture
The spatial organization of the eukaryotic
genome is linked to the functional
compartmentalization of the cell nucleus.
The eukaryotic genome has an extremely complex spatial
organization.

The physical distances between regulatory elements of

the genome, such as enhancers, promoters, insulators,
and CpG-islands, do not necessarily reflect genomic
distances.

Some remote regulatory elements appear to interact

physically with target promoters in the 3D nuclear
space due to loop formations.

These spatial contacts are thought to play a crucial

role in the regulation of transcription.
 Transcription factories: discrete sites
where transcription occurs in the nucleus.
Transcription occurs at a number of specialized, discrete
sites: in transcription factories composed of ~4–30 RNA
polymerase molecules and are associated with many other
molecules involved in transcriptional activation and mRNA
processing.
Transcription factories contribute to both the formation of
chromatin loops and the clustering of active and co-regulated
genes.
Separate factories can exist for RNA polymerase I and III;
the nucleolus is the prototype for transcription factories.

Green label:
localization of
RNA polymerase II
and III in the
nucleus
 Transcription factories in the nucleus
A promoter is only likely to initiate transcription if
tethered to a factory containing appropriate factors.

Motifs like enhancers, silencers, insulators, barriers,

and boundaries would work by tethering target promoters
close to, or distant from, suitable factories.
 Control of eukaryotic transcription initiation:
Ordered binding and function of activators and co-
activators result in cooperative formation of a
stable activated initiation complex

Sequence-specific
transcriptional activators;
+ Chromatin remodelers; and
Co-activators
Histone modifiers

Several activators cooperatively

interact with a single mediator
complex
The human β-globin cluster.
Five functional genes:5′-ε-Gγ-Aγ-δ-β-3′ genes.
Transcribed in that sequence during the human
development.

Locus controll region: located 5’ 3’

upstream (5’from the ε gene)
required for establishing the
permissive chromatin structure
for the expression of the genes.
LCR-binding proteins promote the
formation of open chromatin that
allows transcription. 5’ 3’

(permisive chromatin: Cfp1 regulated H3/H4Ac, H3K4me3 modification,

H3K36me2 depletion, nucleosome deficiency)
 Chromatin Remodelling
• Remember that chromatin structure is dynamic and
changes in chromatin structure adds another level
of regulation to eukaryotic gene expression.
• Chromatin Remodeling is an active process. There
are several chromatin remodeling complexes (CRC)
that use energy provided by hydrolysis of ATP to
restructure chromatin .
• Remodeling complexes are recruited to promoters by
sequence-specific activators.
• Remodeling complexes restructure chromatin and
enable it to be transcribed.
Chromatin Remodeling Is an
Active Process

• There are numerous ATP-

dependent chromatin
remodeling complexes.

Transcription activation
often involves nucleosome
displacement at the
promoter.
 Chromatin Remodeling Is an
Active Process
• Remodeling complexes can alter, slide, or
displace nucleosomes.
• Some remodeling complexes can exchange one
histone for another in a nucleosome.

Remodeling changes
nucleosome organization
Enzymatic chemical modification histone
lead to changes in chromatin structures
Promoter Activation Involves
Multiple Changes to Chromatin

• Remodeling complexes can

facilitate binding of
histone modifying enzymes
and vice versa.

• Different modifications
and complexes facilitate
transcription elongation.
 Processing of eukaryotic pre-mRNA
Poly-A signal
sequence
Enhancer Proximal
Termination
(distal control elements) control elements region
Exon Intron Exon Intron Exon
DNA
Upstream Downstream
Promoter Transcription

Primary RNA Exon Intron Exon Intron Exon

Cleaved 3’ end
transcript 5’ of primary
RNA processing transcript

Intron RNA Poly-A

signal
Coding segment
mRNA 3
Start Stop
5 Cap 5’ UTR codon codon 3’ UTR Poly-A
tail

For primary transcripts (pre-messenger RNA)

containing multiple exons and introns, splicing
occurs before the transcription of the gene is
completed: co-transcriptional splicing.
Post-transcriptional regulation: RNA
splicing and processing
• Modifications of the eukaryotic pre-mRNA:

• 5' „cap” formation

• 3' poly-(A) tail formation
• Removal of the introns: splicing

• Alternative splicing
Pre-mRNA modification is a
co-transcriptional process
Capping of the 5’ end of nascent RNA
transcripts with m7G

•The cap is a modified GTP (7-

methyl-GTP).

• Capping helps stabilize mRNA

and enhances translation,
splicing and export into the
cytoplasm.
Sometimes
methylated

Sometimes
methylated
The splicing reaction proceeds in two steps

Step 1. Cleavage at the 5’ splice

site and joining of the 5’ end of the
intron to the branch point A within
the intron, producing a lariat-like
intermediate.
Step 2. Cleavage at the 3’ splice
site and simultaneous ligation of the
exons, resulting in excision of the
intron as a lariat-like structure.

Two transesterification reactions:

(1) The 5’ P of the intron is
attacked by the 2’-OH of the branch
site Adenosine, causing cleavage of a
3’, 5’-phosphodiester bond and
formation of a 2, 5’-phosphodiester
bond (not hydrolysis followed by
ligation). (2) The newly formed 3’-OH
of exon 1 attacks the 5’ P of exon 2,
causing cleavage of a phosphodiester
bond and formation of a new bond.

Two transesterification reactions:

the number of phosphodiester bonds
remains unchanged in either
reaction.
Spliceosome-mediated splicing of pre-mRNA
•Five snRNPs (U1, U2, U4, U5 and U6 small nuclear
ribonucleoprotein particles) containing 5 snRNAs
(U1, U2, U4, U5 and U6 small nuclear RNAs, ranging
from 107 to 210 nucleotides) and their associated
proteins (6-10 per snRNP) assemble on the pre-mRNA
to form the spliceosome.

•There are a total of ~100 proteins in the

spliceosome, some of which are not associated with
snRNPs. These non-snRNP proteins may contribute to
the specificity of recognition of the splice sites
by snRNPs and some of them contain RNA helicase
activity to help the rearrangements of base pairing
in snRNAs during the splicing cycle.

•U4 masks the catalytic activity of U6 in the

U4/U6/U5 tri-snRNPs prior to the actual
transesterification reactions.

•Massive rearrangements of base-pairing interactions

among various snRNAs converts the spliceosome into a
catalytically active form, which releases the U1 and
then the U4 snRNPs and brings U2 and U6 together.

•RNA molecules play key roles in directing the

alignment of splice sites (e.g. U1 and U2 base
pairing with the pre-mRNA) and in carrying out the
catalysis (a U2/U6 catalytic center).
 Splicing signals in the gene

5’donor splice 3’acceptor splice

site site
Branch site
5’exon 5’- UACUAAC-3’ 3’ exon
---AGGUAAGU-----------A--------(Py)nNCAGG..

Intron ~ 20 – 50 nt

The reactions are guided by specific sequences

in the pre-mRNA at both the 5’ and 3’ ends of
the introns (at the exon-intron boundary). The
excision should be very precise, not to disrupt
the reading frame.
Addition of the poly-A tail at
the 3’ end of the pre-mRNA
catalized by the poli(A)-
polimerase (PAP) enzyme.

The poly-A signal sequence:

AATAAAxxxxxxxG
It is the transcriptional
termination signal, too.
 Alternative Splicing

• Alternative splicing is a form of gene regulation

which results in the generation of alternative
mRNAs from a single gene. Alternative splicing
(together with alternative promoters) is an
important source of human genetic complexity.

• Different splice patterns may occur in different

tissues resulting in tissue-specific gene
expression.

• The coding capacity of the human genome is

enlarged by alternative spliceing!
 Alternative Splicing Results in
Different mature mRNAs and Proteins

Alternative splicing pathways results in the production

of multiple related, but different mRNAs, each of which
can be translated into protein.
 Gene silencing
• RNA interference (RNAi) is a biological process in
which RNA molecules inhibit gene expression, typically
by causing the destruction of specific mRNA molecules.
• RNA interference has an important role in defending
cells against viruses and transposons, but also in
directing development as well as gene expression in
general.

micro RNA-s (miRNAs)

• A microRNA is a small non-coding RNA molecule (~26
nucleotides), which functions in transcriptional and
post-transcriptional regulation of gene expression.
• Encoded by nuclear genes, miRNAs function via base-
pairing with complementary sequences within mRNA
molecules, usually resulting in translational
repression or target degradation.
 Gene silencing by miRNA
miRNA precursor RNA, pri-miRNA
In this process a
specific miRNA
triggers
degradation of Pre-miRNA
the mRNA or
inhibition of the
translation of
the mRNA
containing 3’UTR
sequences
complementer to
the miRNA.