lab3 polytene chromosome.ppt

5/19/2018 lab3 polytene chromosome.ppt

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Lab No ( 3 )

The Polytene

hromosomes


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Polytene Chromosomes

Polytene chromosomes are giant chromosomescommon to many dipteran (two-winged) flies.

They begin as normal chromosomes, but throughrepeated rounds of DNA replication without any celldivision (called endoreplication), they become large,banded chromosomes.

For unknown reasons, the centromeric regions of thechromosomes do not endoreplicate very well. As aresult, the centromeres of all the chromosomes bundletogether in a mass called the chromocenter.


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The salivary gland cells in the larval stages ofDrosophilacontain large, multistranded polytenechromosomes.

This laboratory procedure outlines themechanism by which salivary glands areremoved and prepared so that the polytenechromosomes may be observed.

Drosophila melanogasteris easier to dissect andremove the salivary glands from the larvae ofthis species.


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Polytene

Chromosomes

from SalivaryGlands


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Student Handout

Materials

For each s tudent group:

compound microscope

2 teasing needlesinsect pins

microscope slides

cover glasses

stock supply of Drosophila melanogasterlarvaedropping bottle of aceto-orcein stain

dropping bottle of 45% acetic acid solution


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Procedures

Remov ing the sal ivary glands:

1- Remove a large larva from the stock of D.melanogaster. Larger larvae are easier todissect. However, select an active larva and onethat has not started to pupate.

2- Using the compound microscope, dissect thelarva by placing one teasing needle on the

posterior aspect of the larva and the otherneedle at the anterior end, near the black mouthparts.

3- Carefully pull outward with the anterior needle.


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Procedure for removing Drosophila salivary glands

( cont )

4- There are two transparent salivary glands

located anteriorly in the larva.

5- Discard all of the larva except for the

salivary glands.


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Stain ing and Observ ing

1- Place 2 drops of aceto-orcein stain on thesalivary glands, and let it stand for 10 minutes.

2- Place a cover slip over the glands, and usingyour thumb and a paper towel, push down onthe slide. The pressure applied will squash theglands, rupture the nuclear membrane, and freethe chromosomes.

3- Using a compound microscope, observe theslide under low and high magnification.

4- Make the slide permanent by brushing along theedges of the cover slip with clear nail polish


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The Puffing

Puffing is the term that describes structural

changes in polytene chromosomes. If one

observes polytene chromosomes during

the late prepupal stage, different bandsappear to be puffed up. For 40 years, this

has been understood to be the result of

gene activity .


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Definition of polytene chromosome

A giant chromosomeproduced by an endomitotic processin which, following synapsisof the two homologues,

multiple rounds of replicationproduce chromatids thatremain synapsed together in a haploidnumber of

chromosomes. Polytene chromosomes have been studiedmostly in Drosophilasalivary glands, in which

chromosomes undergo 10 cycles of replicationwithoutseparation of the daughter chromosomes. This leads to1024 identical strands of chromatinaligned side by side.

These chromosomes are easy to see with a lightmicroscope because of their large size and precisealignment. The chromosomes are seen as distinct

alternating dark and light bands which has given rise to apolytene chromosomemap. The dark bands correspond to

more condensed regions of the chromatin, and the light(interband) regions are less dense regions.
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lab3 polytene chromosome.ppt

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