Sedat lab website at http://www.msg.ucsf.edu/sedat/polytene_chrom.html

Polytene Chromosomes

Polytene chromosomes are giant chromosomes common to many dipteran (two-winged) flies. They begin as normal chromosomes, but through repeated rounds of DNA replication without any cell division (called endoreplication), they become large, banded chromosomes (see figure). For unknown reasons, the centromeric regions of the chromosomes do not endoreplicate very well. As a result, the centromeres of all the chromosomes bundle together in a mass called the chromocenter.Polytene chromosomes are usually found in the larvae, where it is believed these many-replicated chromosomes allow for much faster larval growth than if the cells remained diploid. Simply because each cell now has many copies of each gene, it can transcribe at a much higher rate than with only two copies in diploid cells.The polytene chromosomes at the right are from the salivary glands of the fruit fly Drosophila melanogaster. the bands on each chromosome are like a road map, unique to each chromosome and well defined enough to allow high resolution mapping of each chromosome. The Drosophila Genome Project uses polyene chromosomes as a framework for the map.

1

Size of interphase chromosomes in normal somatic cells

Alberts et al. (2002) Molecular Biology of the Cell at http://www.ncbi.nlm.nih.gov/books/NBK26931/

interphase

metaphase

early prophase

late anaphase late telophase

10 µm

2

Alberts et al. (2000) http://www.ncbi.nlm.nih.gov/books/NBK28337/

Polytene (multi-stranded) chromosomes are large, structured interphase chromosomes

3

The banding pattern is reproducible and provides a physical map

Wehner & Gehring (2007) Zoologie, 24. Aufl. 4

Salivary glands undergo partial endoreplication (replication without division) and are partially polyploid (1024n)

centromer

Edgar and Orr-Weaver (2001) Endoreplication Cell Cycles- More for Less5

Endoreplication can serve mass production of protein

Images: http://www.killi.co.uk/drosophila1.php; Biyasheva et al. (2001) Glue Secretion in the Drosophila Salivary Gland; http://www.hereke.com/images/cocoons.jpg; http://www.blogspan.org/blogs/permalinks/10-2006/mate-attracting-chemicals-in-silk-worms.html

Green glue (sgs3Nterm::gfp fusion) before and after secretion

Glue: Drosophila pupae adhering to fly bottle wall

silk protein: Silk moth adults, larvae, cocoons

6

Actively transcribed regions appear as "puffs"

Wehner & Gehring (2007) Zoologie, 24. Aufl. 7

Polytene chromosomes allow to detect localization of protein (antibody staining) and mRNA (in situ hybridization) at high resolution

Schiemann et al. (2010) Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila

8

Large structural mutations are visible in polytene chromosomes: here, a heterozygous inversion is forced into a loop to keep the sequence alignment of the two homologous chromosomes

Wehner & Gehring (2007) Zoologie, 24. Aufl. 9

Protocol: Aceto-Orcein staining of larval salivary glands I1. put one drop of 45% acetic acid into a glass dish

2. put one drop of aceto-orcein onto a coverslip that lies on the black plate (for contrast)

3. choose large wandering L3 larvae from the wall

4. pre-cool for a minute in a drop of water on ice-cooled metal plate to immobilize and anaesthesize

5. dissect in the drop of 45% acetic acid: grab larva by mouthhooks and in the middle and tear carefully - tear out mouthhooks and glands (see picture), discs and brain may also be attached

6. transfer salivary glands (see picture) into one drop of aceto-orcein staining, leave for 10-15 minutes

Images: Biyasheva et al. (2001) Glue Secretion in the Drosophila Salivary Gland ; http://www.tritechresearch.com/dros.html ; http://www.muhlenberg.edu/depts/biology/courses/bio240/polytene.html

10

Protocol: Aceto-Orcein staining of larval salivary glands II

7. add two drops of lacto-acetic acid to the orcein drop

8. carefully adhere the liquid and coverslip to a slide and turn around

9. push out excess liquid onto the coverslip with the eraser end of a pencil, watch that the glands stay inside. take off excess liquid (keep coverslip dry if possible), optionally cover with a kleenex, and tap onto the coverslip with the eraser end of a pencil for 30 seconds to spread chromosomes

10. blot coverslip down onto onto a using your thumb and kleenex to take off excess liquid, press firmly onto the coverslip

11. seal rims with nailpolish. nailpolish will spread onto wet parts of coverslip, avoid if possible.

11

Protocol: Aceto-Orcein staining of larval salivary glands III

12. Use phase contrast objectives 10x to 40x; match objective to phase stop on the condenser (below the specimen). There is almost no contrast with bright field, BF. Look for chromosomes with 10x objective, then use the 40x to see bands.

If you have difficulty finding chromosomes, focus first onto the level of the slide at the edge of the coverslip (something you easily find), then move stage to where the squashed salivary glands are. Blurry filaments are out of focus chromosomes. One can recognize individual chromosome arms by their ends, length and banding pattern.

Image: Wehner & Gehring (2007) Zoologie, 24. Aufl. 12