8/20/2019 Drosophila tissue and organ development: Polytene chromosomes, endoreplication and puffing

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Genes involved in tissue and organ development

Polytene chromosomes, endoreduplication and puffing

In dividing diploid cells the DNA synthetic phase (S phase) is regularly followed by mitosis (M phase).
The alternation of G1, S, G2, M and G1 phases is called the cell cycle. In certain circumstances, the M
phase does not follow the S phase, and repeated cycles of DNA synthesis results in polyploidy. The
process of recurrent duplication cycle without consequent mitosis is called endoreduplication. Instances of
polyploid chromosomes in Drosophila include ovary nurse cells, follicle cells surrounding oocytes,
abdominal histoblasts (see Escargot), fat body cells, gut cells, and cells of the late prepupal salivary gland.
During the process of polyploidization, chromosomes become multistranded. In Drosophila the degree of
polyteny is estimated to be approximately 1024 in salivary gland chromosomes, i.e., about 512 in each
homolog.

Polyploid chromosomes exhibit a banded structure that is reproducible from individual to individual. In
Drosophila there are thousands of recognizable bands. In situ hybridization of cloned complementary
DNA of identified genes to banded polyploid chromosomes allows the localization of genes to individual
chromosome bands. Chromosomal rearrangements are easily documented by comparing the order of
bands between individuals, lines or even species. The degree of rearrangement observed between species
is indicative of their evolutionary distance. Drosophila melanogaster has four chromosomes, three
autosomes and a pair of sex chromosomes.

The reference system proposed by Bridges divides the limbs of salivary gland chromosomes into 102
sections called "divisions" designated by number from 1 to 102. Each of the five main limbs (X, 2L, 2R,
3L, and 3R) contains 20 divisions; the short chromosome 4 contains only two divisions. The divisions are
started with a prominent band and divided further into 6 subdivisions, each designated with capital letters
from A to F. Each subdivision starts with a sharp band. Thus each individual band of salivary gland
chromosomes can be identified by giving the division number, subdivision, and the number of the band
starting from the beginning of the subdivision. Bridges presents the following minimum numbers of bands
for the salivary gland chromosomes of Drosophila melanogaster: 537 bands for the X chromosome, 1032
bands for the second chromosome, 1047 bands for the third chromosome, and 34 bands for the fourth
chromosome, totalling a minimum of 2650 bands for the whole genome. In this initial count doublets were
listed as single bands; more recent interpretations give the total number of bands as 3286 (Sorsa, 1988).

Below:
Polytene chromosomes of Drosophila melanogaster as sketched by Calvin B. Bridges in 1935.
Select thumbnail for larger image.

Chromosomes X and 4

Chromosomes 2L and 2R

Chromosomes 3L and 3R

Bridges, C.B. (1935). Salivary chromosome maps with a key to the banding
of the chromosomes of Drosophila melanogaster. J. Hered. 26: 60-64.

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8/20/2019 Drosophila tissue and organ development: Polytene chromosomes, endoreplication and puffing

In late prepupal salivary gland chromosomes, not all DNA in each of the chromosomes is polyploid.
Approximately a third of the Drosophila genome is represented by heterochromatin, and heterochromatic
regions are underrepresented in polytene chromosomes as these regions do not undergo endoreduplication.
For example, the rolled locus is found in a heterochromatic region of chromosome 2 that is considered to
remain condensed (and for the most part transcriptionally inactive) throughout all or most of the cell cycle.
rolled lies in what is considered to be alpha heterochromatin, a chromosome region that makes up the
chromocenter of polytene salivary gland chromosomes. The chromocenter is not thought to be
polytenized, that is, it is not thought to undergo repeated rounds of DNA replication resulting in multiple
copies of active genes. The chromocenter is thought to be made up of DNA and protein in a dense, tightly
knit structure that is transcriptionally inactive. Such heterochomatic regions, which make up 30% of the
Drosophila genome, have a much lower density of genes as compared to euchromatin. rolled gene activity
is unusual in that it requires the surrounding heterochromatin for gene function. rolled gene activity is
severly impaired by bringing rolled close to any euchromatic position; however, these position effects can
be reversed by chromosomal rearrangements that bring the rolled gene closer to any block of autosomal or
X chromosome heterochromatin (Eberl, 1993).

Puffing is the term that describes structural changes in polytene chromosomes. If one observes polytene
chromosomes during the late prepupal stage, different bands appear to be puffed up. For 40 years, this has
been understood to be the result of gene activity. Puffs then, afford a view of the temporal sequence of
gene activation. A temporal pattern to puffing in the salivary glands of late prepupal flies is inducible by
ecdysone injection and is therefore under control of the Ecdysone receptor. A small number of genes react
by puffing within minutes of exposure to ecdysone, and a much larger number (>100) react within hours.
It is hypothesized that the time sequence of puffing represents a genetic hierarchy of gene activation. Early
puffs are independent of protein synthesis while late puffs require prior protein synthesis (Ashburner,
1990). For more information on the hierarchy of gene activation during metamorphosis, see Ecdysone
receptor, FTZ-F1 and Broad.

In recent years, transcription factors and chromosomal proteins have been localized to various bands.
Binding of these proteins is thought to have functional significance and to reflect the activity of these
proteins in gene regulation. For more information on the binding of various proteins and RNA species to
bands, see HP1/Su(var)205, Polycomb, Male sex lethal 2, and suppressor of Hairy wing.

An example of binding of specific proteins to polytene chromosomes is found in a study of the protein
CHD1 (chromo-ATPase/helicase-DNA-binding domain). Proteins related to CHD1 via the helicase
domain have been shown to exist in large multiprotein complexes. For example SNF2/SWI2/Brm proteins
(see Brahma and ISWI) are thought to participate in ATP-dependent remodeling of chromatin. Antibodies
to CHD1 localize this protein to extended chromatin (interbands) and regions associated with high
transcriptional activity (puffs) on polytene chromosomes from salivary glands. These observations support
the idea that CHD1 functions to alter chromatin structure in a way that facilitates gene expression (Stokes,
1996).

Polyploidization by endoreduplication requires regulation of the cell cycle. What makes one region of the
chromosome become polyploid while another remains underreplicated. Information about the roles of cell
cycle genes in the regulation of polyploidization can be found in Cyclin E, Escargot, and Origin
recognition complex 2.

References

Ashburner, M. (1990). Puffs, genes, and hormones revisited. Cell 61:1-3

Eberl, D. F., Duyf, B. J. and Hilliker, A. J. (1993). The role of heterochromatin in the expression of a
heterochromatic gene, the rolled locus of Drosophila melanogaster. Genetics 134: 277-92

Sorsa, V. (1988). Chromosome maps of Drosophila. Vols. I and II. Boca Raton, Florida: CRC Press

Stokes, D., G., Tartof, K. D. and Perry, R. P. (1996). CHD1 is concentrated in interbands and puffed
regions of Drosophila polytene chromsomes. Proc. Natl. Acad. Sci. 93: 7137-42.

Genes involved in tissue and organ development

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