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KARYOTYPE


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Karyotype:

A karyotype is the number and appearance ofchromosomes in the nucleus of a eukaryotic cell.

The term is also used for the complete set of

chromosomes in a species or an individual organism

Karyotypes describe the number of chromosomes,

and what they look like under a light microscope

Attention is paid to their length, the position of the

centromeres, banding pattern, any differencesbetween the sex chromosomes, and any other

physical characteristics


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Idiogram:

An ideogram or ideograph (from Gk. idea "idea"+ grafo "to write") is a graphic symbol that

represents an idea or concept When the chromosomes are depicted (by rearranging a

microphotograph) in a standard format - is known as a

karyogram or ideogram/idiogram - in pairs, ordered by

size and position of centromere for chromosomes ofthe same size


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Historical:

Prior to 1950: sections of testis (from dead humans)

1952: T. C. Hsu employed skin and spleen from 4month old fetus..good chromosome spreads

It was found after this article had been sent to pressthat the well spread metaphases and mitoticanaphases were the result of an accident. Instead of

being washed in isotonic saline, the cultures had

been washed in hypotonic Tyrode solution before

fixation

The diploid human chromosome number was reported

as 48!!!


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1956:

Tjio and Levan - used connective tissue cells fromlungs of legally aborted human embryo

Cultured in bovine amniotic fluid

Added colchicine 12hrs. prior to making chromosome

preparations

Employed Hsus hypotonic treatment to get spreads

We do not wish to generalize our present findings into

a statement that the chromosome number of man is2n=46, but it is hard to avoid the conclusion that this

would be the most natural explanation of our

observations


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Two further developments:

Ford & Harnden obtained cells from human bone

marrow and skin biopsies..showed the possibility ofmaking chromosome preparations from live adults!

Hungerford Human WBC can be stimulated to cometo mitosis by using Phytohaemagglutinin


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Karyotypes are used to study chromosomalaberrations

Used to determine other macroscopically visible

aspects of an individual's genotype, such as sex

To see the chromosomes and determine their size and

internal pattern, they are chemically labelled with a

dye ("stained")

The pattern of individual chromosomes is calledchromosome banding


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Karyotyping can be done using a sample of blood, bone

marrow, amniotic fluid, or placental tissue.

Possible due to cell culture techniques

Bone marrow dividing mitotic cells

Amniotic fluid contains fetal nucleated cells

Chorionic villi mixture of maternal and fetal cells

Human WBC


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Cells separated out from blood/amniotic fluid by

centrifugation

Stimulated to divide by chemical/hormonal treatment

Grown in special culture media

Cells treated with colchicine/colcemid

Hypotonic solution

Air dry preparation

Staining spread cells


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Cell culture:

100ml McCoys 5A medium

12.5ml fetal calf serum

2ml L-glutamine

2.5ml reconstituted PHA

2ml penicillin/streptomycin/gentamycin sulfate

To 9.5ml of this mixture

0.4ml of whole blood/Heparinized whole blood

Incubated at 37C

10 g/ml Colcemid G incubate for 1-1.5 hrs prior toharvesting cells

Total incubation time 70-72 hrs.

Cells quickly harvested for air dry preparations


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Metaphase spreads are drawn using camera lucida or

photographed

Homologous chromosomes can be recognized by size,

shape (caliper) and/or banding pattern (G, C, R, Q)

The photographed chromosomes are cut out,

matched with its partner and arranged from largestto smallest on a chart

Human idiogram consists of 7 groups:

A: 1-3 E: 16-18

B: 4, 5 F: 19, 20

C: 610, 11, 12, X G: 21, 22, Y

D: 13-15


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Normal karyotypes for women contain two Xchromosomes and are denoted 46,XX;

Men have both an X and a Y chromosome

denoted 46,XY.

However, some individuals have other karyotypes

with added or missing sex chromosomes

47,XYY, 47,XXY, 47,XXX and 45,X.

The karyotype 45,Y does not occur, as an embryo

without an X chromosome cannot survive


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GTL-Banding:

GTL-Banding /G-Banding - Giemsa/Trypsin/Leishman

banding.

Devised by Dr. Giemsa uses trypsin to partially digest the

histones the chromosome relax, letting the Leishmanstain bind to the exposed DNA

Preferentially stains the regions of DNA - rich in AT

The resulting bands are specific to each chromosome andthe order and size of the band enables us to distinguish

and compare homologous pairs of chromosomes


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G-Banding


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G-Banded Karyotype


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1971: at a conference in Paris, scientists got together to draw up a system of

numbering of the bands and sub bands.

The result is the Paris Conference ideogram.. In making the assignments,however, they did make one mistake.

Chromosome 22 has more DNA than chromosome 21 and thus the numbers

should have been reversed. Since an extra chromosome 21 was already

associated with Down syndrome, it was decided not to change the numbering


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Q-banding: Quinacrine gives bands that fluoresce on exposure to UV

light. The patterns can be correlated with G-bands.

Disadvantage: Intensity of fluorescence fades rapidly. Photographs

must be made within a few minutes of staining.


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R-banding:

It is the reverse pattern of G bands so that G-

positive bands are light with R-banding methods,

and vice versa. 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


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Helpful for analyzing the structure of chromosome ends,

since these areas usually stain light with G-banding


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C-banding: stains areas of heterochromatin, which

is tightly packed and repetitive DNA


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NOR-staining: refers to a silver staining method.

Identifies genes for ribosomal RNA that were active in

a previous cell cycle


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6 characteristics of karyotypes are usually observed and compared:

1. Differences in absolute sizes of chromosomes. Chromosomes can vary in

absolute size by as much as twenty-fold between genera of the same family -

Probably reflects different amounts of DNA duplication.

2. Differences in the position of centromeres - brought about by

inversions/translocations

3. Differences in relative size of chromosomes - can only be caused by segmental

interchange of unequal lengths.4. Differences in basic number of chromosomes - may occur due to successive

unequal translocations which finally remove all the essential genetic material

from a chromosome, permitting its loss without penalty to the organism

(dislocation hypothesis)

Humans have one pair fewer chromosomes than the great apes, but thegenes have been mostly translocated (added) to other chromosomes

5. Differences in number and position of satellites

6. Differences in degree and distribution ofheterochromatic regions


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Implications:

Chromosomal types and number

Numerical variations Aneuploids/Ploids Structural variations

Genetic mosaics

Phylogenetic relationships between species

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