The Effects of Temperature Shock on Transcription and Replication in Rhynchosciara americana (Diptera: Sciaridae)

The chromosomal response to temperature shock in Rhynchosciara americana is similar to that observed in other Diptera. After a 33 degrees C/90 min or a 36 degrees C/30 min shock the reaction for RNA polymerase II (RpII) is enhanced at five loci. The most prominent of these was identified by in situ hybridization as the site of the hsp70 gene. At 33 degrees C, an accumulation of heat shock factor (HSF) and an increase in the level of RpII was observed at some heat shock loci after 5 min and reached a maximum after 15 min at most loci. The pattern of accumulation of HSF and RpII at individual heat shock loci was similar and their increases were generally coordinated among the loci. RpII gradually decreased at sites active prior to shock, the rate of decrease varying with the site. The B2 DNA puff retained RpII for a significant length of time while the histone locus still contained RpII after a shock of 90 min. With a 36 degrees C/30 min shock, the size of the heat shock puffs and the intensities of HSF and RpII peaked at 1-4 h post stress. The level of HSF declined rapidly after 1 h while the level of RpII remained high for an additional 4 h. The reaction of the DNA puffs to heat shock varied. Usually they did not regress completely and retained traces of RpII. BrdU incorporation continued at both amplifying and non-amplifying bands after shock but on average it appeared depressed for about 24 h post stress.     

Local enrichment with homopolymeric (dA/dT) DNA in genomes of some lower dipterans and Drosophila melanogaster

An investigation into the chromosomal localization of homopolymeric dA/dT was carried out with species of the genera Rhynchosciara, Chironomus, Drosophila and several other taxa. In situ hybridisation probing mitotic and polytene chromosomes with RNA homopolymers was performed, followed by immunological detection of the DNA/RNA hybrid. Use of this method allowed us to assess specific regions of some dipteran genomes, where the signal was generally, but not always, located in heterochromatic regions. Human and Drosophila chromosome regions known to contain dA/dT runs of up to 153 bp were devoid of consistent labelling. The stability of the rA/dT hybrid formed in situ was in agreement with the T(m) for long rA/dT hybrid complexes, suggesting that the method used in this work is able to identify unusually long homopolymeric dA/dT tracts.     

The chromosomes of Rhynchosciara baschanti (Diptera: Sciaridae): molecular cytogenetic comparisons with taxa in the americana-like group.

Polytene chromosome analysis is presented for Rhynchosciara baschanti, a species belonging to the americana-like group of Rhynchosciara. R. baschanti chromosomes show morphological differences in centromeric and telomeric regions compared to two other members within the group, R. americana and R. hollaenderi. In addition, fixed band and autosomal inversion differences were noted. Physical mapping data showed synteny among the taxa under study for DNA puffs and single-copy or histone gene probes, whereas rDNA and poly-(r)A probes showed different diagnostic patterns. The activity of developmentally active genes and the pattern of thymidine incorporation into DNA puff sites of R. baschanti are consistent with those found in the two previously studied species, except for lower levels of expression at some of these sites. These results suggest that differential duplication of specific DNA sequences, in particular repetitive and homopolymeric DNA, has played a role in the chromosomal evolution of these Rhynchosciara species. Inversions and band dimorphisms have also occurred, but the processes leading to their maintenance and fixation appear to have been slow, since these three species are in general chromosomally monomorphic.     

Structural constraints in expansion segments from a midge 26S rDNA

DNA sequences representing approximately 40% of the large-subunit rRNA gene from the lower dipteran Chironomus thummi were analyzed. Once aligned with their Drosophila counterparts, sequence and base content comparisons were carried out. Sequence identity was found to be high overall, except for six regions that displayed a local bias in nucleotide composition toward AT. These regions were identified as expansion segments D3, D4, D5, D6, D7a, and D12. Besides base sequence divergence, differences in length were observed between the respective variable domains of the two species, particularly for D7a. Prediction of secondary structure showed that the folding of the Chironomus expansion segments analyzed is in agreement with the general patterns proposed for eukaryotic LSU rRNA. The comparison with Drosophila revealed also that the Chironomus secondary structures of the variable domains are supported by multiple compensatory substitutions or even compensatory insertions. Chironomus D7a displayed an unusual structural feature with respect to the insect D7a models that have been inferred up to now. The structural constraint observed in the expansion segments of Diptera so distantly related as midges and Drosophila suggests that these regions contribute to some functional role. Concerning the D7a of insects so far analyzed, there can be, in addition to a conserved secondary structure, a nucleotide composition constraint that might be important for the process giving rise to the alpha and beta halves of the 26S rRNA. Correspondence to: E. Gorab