Mitosis –Meiosis-Karyotyping

DR.DEEPAK N KHEDEKARASST.PROFESSOR

DEPT.OF ANATOMYLTMMC &GH,MUMBAI.

2014

We are all born with a unique genetic blueprint, which lays out the basic characteristics of our personality as well as our physical health and appearance... And yet, we all know that life experiences do change us…………………………………………….Anonymous

Cell cycle…

• Consist of interphase-mitosis-interphase-

mitosis

• Interphase consist of…

G1 or preduplication phase in which most of

the body cells exist and function.

S or synthesis phase - DNA duplication

occurs(tetraploid material in diploid no.)

G2-premitotic phase

Cell cycle…

Mitosis and Meiosis…

• Mitosis:

-division of somatic (body) cells

• Meiosis

-division of gametocytes (sex cells)

What is the purpose of mitosis?

• Cell division

• Products genetically identical products

• Growth of organ….growth of organism

• Mitosis results in the distribution of identical

copies of the parent cells genome to the

daughter cells.

Stages of Mitosis…(ipmat)

• Interphase

• Prophase

• Metaphase

• Anaphase

• Telophase

Interphase…

• Interesting things starts happening…

1. Cell preparing to divide

2. Genetic material doubles

Prophase…

Chromosome pair up

1. Chromosomes thicken and shorten…

-become visible

-2 chromatids joined by centromere

2. Centrioles move to the opposite sides of the

nucleus

3. Nucleolus disappears

4. Nuclear membrane disintegrate

Prophase…

Metaphase…

Chromosomes meet in the middle

1. Chromosomes arrange at equator of cell

2. Become attached to spindle fibres by

centromeres

Metaphase…

Anaphase…

Spindle fibres contract pulling chromatids

• Chromosomes get pulled apart one towards

each pole of the cell.

• Chromosomes become grouped at each of the

cell.

• Form. of 2 grps with diploid no. chrmosomes

• Cytoplasmic division also starts

Anaphase…

Anaphase…

Telophase…

Now there are two groups forming 2 cells.

1. Chromosomes uncoil

2. Spindle fibres disintegrate

3. Centrioles replicate

4. Nuclear membrane reappears.

5. Cell division completed (cytokinesis)

Telophase…

Meiosis…• Occur only in gonads -gametes.ie spermatocyte

and oocyte

• Consist of 2 divisions.

• 4 daughter cells produced in c/o male gamete only 1 daughter cell in c/o female

• Each daughter cell has half the chromosomes of the parent

Meiosis…

to reduce the chromosome number in the

gamete to 23

ensure that every gamete is genetically unique.

S

T

A

G

E

S

Meiosis 1…

• Prophase 1

Leptotene

Zygotene

Pachytene

Diplotene

• Metaphase 1

• Anaphase 1

Prophase 1…(lzpd)

• Leptotene -chromosome become visible, beaded

in appearance,chromomeres

• Zygotene - (synapsis,conjugation)

chromosome come together

Point to point contact of homologous pair

• Pachytene-

chr.become shorter,thicker forms chromatids

Bivalent pair consist of 4 chromatids.

Crossing over occurs

Meiosis…

A homologous pair of parental chromosomes (e.g. chromosome 7)

In meiosis I each chromosome duplicates producing two sister chromatids

Crossing-over(Recombination)

Gene re-assortment by crossing-over

meiosis II

Meiosis…

• Diplotene-homologous pair move apart

• Diakinesis-end of prophase, nucleoli

disappear, nuclear membrane dissolution,

• movememnts of chromosome to equator.

• Dictyotene-reticular arrangement of

chromosome

Meiosis 2…

Normal monosomic gametes

Normal meiosisReduction division

MEIOSIS I

MEIOSIS II

Results of crossing-over not shown

Replicate DNA

MEIOSIS I

MEIOSIS II

Results of crossing-over not shown

Replicate DNA

Nondisjunction

during meiosis I

Non-disjunction

Disomic gametes Nullisomic gametes

MEIOSIS I

MEIOSIS II

Results of crossing-over not shown

Replicate DNA Nondisjunction during

meiosis II

Non-disjunction

Disomic Nullisomic Monosomic Monosomic gametes

Normal disomy

Mitosis

Non-disjunction

Normal disomy Trisomy Monosomy (lethal to cell)

Somatic mosaicism (eg trisomy 21) as a result of mitotic non-

disjunction

MeioticNon-disjunction

(Trisomy 21: 75% meiosis 1)

Trisomy Monosomy (lethal)

The effects of non-disjunction in meiosis.

The non-disjunction involves only the single pair of chromosomes (meiosis I) or the single chromosome (meiosis II) shown; all the other chromosomes (not shown) disjoin and segregate normally.

Karyotyping andChromosomal banding

Karyotype…

The karyotype is the complete chromosomal

set of the nucleus of the cell.

• 1: To understand the structure of the

chromosomes

• 2: to understand the various banding

techniques and their application

Karyotype…

• A diagrammatic representation of all of the pairs

of chromosomes arranged in order of size is

called an ideogram.

• Every organism has a standard karyotype, which

provides a frame of reference for the analysis of

mutations.

Procedure..

• It involves metaphase chromosomes derived

from somatic cells by their culture.

• They are obtained for analysis and

photomicrographed.

• Individual chromosomes are photographed.

• Photographs are arranged in orders according

standard classification.

• Ideogram or karyotype

Karyotype…

• Karyotypic analysis is the study of all of the

visible traits of chromosomes in a typical cell.

Ex 1: distinct species of Drosophila that are

very similar in appearance are distinguished

easily by karyotypic analysis, which reveals

numerous chromosomal inversions and

translocations.

Chromosome structure…

• Composed of chromatin, a combination of

nuclear DNA and proteins

• For karyotyping, cells are captured in

metaphase

metaphase stage in mitosis at which

chromosomes are aligned along the cell

equator

Chromosome preparation…

Peripheral blood culture involves-

1.Collection of blood sample

2.Planting

3.Incubation

4.Harvesting

5.Staining

Planting...

Collected blood transferred to culture vials

• Vials contain-

culture medium

Serum for nourishment of cultured cells.

Phytoheamagglutinin mitotic agent promote

rate

Antibiotics to prevent unwanted growth.

• Incubation at 37 c for 3 days.

• Harvesting- at 70 hrs

• Addition of colchicine to arrest cells in

mitosis.

Staining or Chromosomal Banding…

• Chromosomes are stained with a dye

• comprised of alternating light and dark stripes,

or bands, that appear along its length after

staining.

• Used to identify each chromosome

• To diagnose chromosomal aberrations

.

G-Banding Technique

• Giemsa is the most commonly used

• Staining a metaphase chromosome with a

Giemsa stain is referred to as G-banding.

• G-banding techniques require pretreating

• Chromosomes are pretreated with either salt

or a proteolytic (protein-digesting) enzyme.

G-Banding Technique …

• “GTG banding” refers to the process in which G-banding is preceded by treating with trypsin

• G-banding preferentially stains the regions of DNA that are rich in adenine and thymine.

• The regions of the chromosome that are rich in guanine and cytosine have little affinity for the dye and remain light.

G-Banding Technique …

• Standard G-band staining techniques allow

between 400 and 600 bands to be seen on

metaphase chromosomes.

• With high-resolution G-banding techniques, as

many as two thousand different bands have

been catalogued on the twenty-four human

chromosomes.

G-banded metaphase spread : N female

G-banded karyotype from a normal female.

Q-Banding Technique…

• Quinacrine mustard, an alkylating agent,

was the first chemical to be used for

chromosome banding

• They observed alternating bright and dull

bands under fluorescent microscope

Q-Banding Technique…

• Quinacrine-bright bands were composed

primarily of DNA that was rich in the bases

adenine and thymine

• Quinacrine-dull bands were composed of DNA

that was rich in the bases guanine and

cytosine

Q-Banding…

R-Banding Technique…

• R-banding is the reverse pattern of G bands

• G-positive bands are light with R-banding method,

and vice versa

• R-banding involves pretreating cells with a hot salt

solution that denatures DNA that is rich in adenine

and thymine

• The chromosomes are then stained with Giemsa.

• R-banding is helpful for analyzing the structure of

chromosome ends, since these areas usually stain

light with G-banding.

C-Banding Technique…

• C-banding stains areas centromeric region and

area of secondary consriction

• This area of heterochromatin, which is tightly

packed and repetitive DNA.

C banding…

NOR-Staining…

• NOR-staining, where NOR is an abbreviation for

“nucleolar organizing region,”

• silver staining method ,identifies genes for

ribosomal RNA that active in a previous cell

cycle

NOR-Staining…

Fluorescent in situ Hybridization (FISH)

• Revolutionised concept of chromosome analysis,

is a molecular cytogenetic technique.

• To analyze chromosome resolution at the DNA or gene level

• FISH can be performed on dividing (metaphase) and non-dividing (interphase) cells to identify numerical and structural abnormalities resulting from genetic disorders.

FISH …• Utilizes Probes; there are 3 catogories…

1.Repetitive sequences that bind to the centromereof a chromosome

2. DNA segments, representative of the entire chromosome, that will bind to and cover the entire length of a particular chromosome

3.DNA segments from specific genes or regions on a chromosome that have been previously mapped or identified.

FISH …

• Probes are “Tagged” with fluorecentnucleotides.

• This is done by attaching nucleotides to small molecules such as biotin digoxygenin, or dinitrophenyl, to which fluorescent antibodies can later be bound

• The cells are then viewed with a fluorescence microscope.

• The fluorescent signals represent the probe(s) that is bound to the chromosomes

Chromosome Painting

Clinical applications…

• Clinical diagnosis

• Gene mapping

• Role in cancer-detection of Ph.chromosome

(CML)

• Recurrent abortions

• Prenatal Diagnosis.

References:

• Bhatnagar Kothari Book

• Human Genetics:S.D.Gangane

• Genetics by Richard Robinson, The Macmillan

Science Library, Volume 1; Page 125-129.