Polytene Chromosome Map

Isolated Chinese hamster metaphase chromosomes were resuspended in 4 M ammonium acetate and spread on a surface of distilled water or 0.15 to 0.5 M ammonium acetate. The DNA was released in the form of a regular series of rosettes connected by interrossette DNA. The mean length of the rosette DNA was 14 μm, similar to the mean length of 10 μm for chromomere DNA of Drosophila polytene chromosomes. The mean interrosette DNA was 4.2 μm. SDS gel electrophoresis of the chromosomal nonhistone proteins showed them to be very similar to nuclear nonhistone proteins except for the presence of more actin and tubulin. Nuclear matrix proteins were present in the chromosomes and may play a role in forming the rosettes. Evidence that the rosette pattern is artifactual versus the possibility that it represents a real organizational substructure of the chromosomes is reviewed.

An hsc70 homologue gene (Rahsc70) of the diptera Rhynchosciara americana was isolated and characterized. We were able to determine the mRNA sequence from an EST of salivary gland cDNA library, and a Rahsc70 cDNA cassette was used as a probe to isolate the genomic region from a genomic library. The mRNA expression of this gene parallels the 2B puff expansion, suggesting its involvement in protein processing, since this larval period corresponds to a high synthetic activity period. During heat shock stress conditions, hsc70 expression decreased. In situ hybridization of polytene chromosomes showed that the Rahsc70 gene is located near the C3 DNA puff. The cellular localization of Hsc70 protein showed this protein in the cytoplasm and in the nucleus.

Drosophila melanogaster protein kinase CK2 (DmCK2) is a Ser/Thr protein kinase composed of catalytic a and regulatory ß subunits associated as an a2a2 heterotetramer. Using the two-hybrid system, we have screened a Drosophila embryo cDNA library in order to identify proteins that interact with DmCK2a. One of these cDNAs encodes a novel previously undescribed zinc-finger protein, which we call ZFP47. ZFP47 interacts with DmCK2a but not with DmCK2ß, indicating that this interaction is specific for the catalytic subunit of CK2. In situ hybridization to polytene chromosomes indicates that the corresponding gene is located at the 72A interval of chromosome III. Sequence analysis indicates that ZFP47 contains a consensus site for phosphorylation by CK2, 4 C2H2-type zinc-fingers, and a bipartite nuclear localization signal. Consistent with the prediction of a site for phosphorylation by CK2, we demonstrate that ZFP47 is phosphorylated by CK2 purified from Drosophila embryos. These studies ...

Non-LTR retrotransposons, also known as long interspersed nuclear elements (LINEs), are transposable elements that encode a reverse transcriptase and insert into genomic locations via RNA intermediates. The sequence analysis of a cDNA library constructed from mRNA of the salivary glands of R. americana showed the presence of putative class I elements. The cDNA clone with homology to a reverse transcriptase was the starting point for the present study. Genomic phage was isolated and sequenced and the molecular structure of the element was characterized as being a non-LTR retrotransposable element. Southern blot analysis indicated that this transposable element is represented by repeat sequences in the genome of R. americana. Chromosome tips were consistently positive when this element was used as probe in in-situ hybridization. Real-time RT-PCR showed that this retrotransposon is transcribed at different periods of larval development. Most interesting, the silencing of this retrotransposon in R. americana by RNA interference resulted in reduced transcript levels and in accelerated larval development.

Puffing activity patterns of the five large polytene chromosomes of Drosophila subobscura were studied during the late third-larval instar and through the prepupal period. A total of 166 loci active in some of the eleven stages studied were described. The distribution of these active loci per chromosome is the following: 25 on chromosome A, 33 on chromosome J, 31 on chromosome U, 34 on chromosome E and 43 on chromosome O. Seven principal patterns of puffing activity were defined taking into account the different curves of the puffing histograms. Gene activities per chromosome as well as total were analysed. Three peaks of gene activity at the beginning, middle and ending of prepupation can be observed. U is the most active chromosome and A (the sex chromosome), and J the least active. Chromosomes E and O show a medium activity. A possible biological explanation for these results is discussed.

A technique for the short term organ culture of larval salivary glands of D. melanogaster is described. Cultured Puff Stage 1 glands respond to 20-OH ecdysone by initiating the cycle of puffing activity characteristic of late larval development and puparium formation. This puffing cycle involves the sequential activation of at least 125 puffs. Their response to ecdysone allows these puffs to be divided into 3 main classes: a) PS1 puffs that regress (e.g. 25AC); b) puffs activated very rapidly (within 5 min) (e.g. 23E, 74EF, 75B) and c) puffs activated only after longer periods (>4 h) (e.g. 62E, 78D, 22C, 63E and 82F). The detailed behaviour of representatives of each class is described. These data support Clever's distinction of ‘early’ and ‘late’ ecdysone responsive sites.

Chromatin insulators are required for proper temporal and spatial expression of genes in metazoans. Here, we have analyzed the distribution of insulator proteins on the 56F–58A region of chromosome 2R in Drosophila polytene chromosomes to assess the role of chromatin insulators in shaping genome architecture. Data show that the suppressor of Hairy-wing protein [Su(Hw)] is found in three structures differentially associated with insulator proteins: bands, interbands, and multi-gene domains of coexpressed genes. Results show that bands are generally formed by condensation of chromatin that belongs to genes containing one or more Su(Hw) binding sites, whereas, in interbands, Su(Hw) sites appear associated with open chromatin. In addition, clusters of coexpressed genes in this region form bands characterized by the lack of CP190 and BEAF-32 insulator proteins. This pattern correlates with the distribution of specific chromatin marks and is conserved in nurse cells, suggesting that this organization may not be limited to one cell type but represents the basic organization of interphasic chromosomes.

Widely regarded beliefs aboutDrosophila histone gene copy numbers and developmental requirements have been generalized from fairly limited data since studies on histone gene arrangements and copy numbers have been largely confined to a single species,D. melanogaster. Histone gene copy numbers and chromosomal locations were examined in three species:D. melanogaster, D. hydei andD. hawaiiensis. Quantitative whole genome blot analysis of DNA from diploid tissues revealed a tenfold variability in histone gene copy numbers for these three species. In situ hybridization to polytene chromosomes showed that the histone DNA (hDNA) chromosomal location is different in all three species. These observations lead us to propose a relationship between histone gene reiteration and chromosomal position.