Drosophila DAxud1: A New Element in Transcriptional Pausing Complex Stabilization

BackgroundA rapid transcriptional response under an acute stimulus is common in all cellular systems and is an adaptation that allows tolerance to environmental changes. A gene group that has been studied because of its fast response and cytoprotective effects are the hsp genes (encodingHeat Shock Proteins(HSPs), conserved chaperones).. Under normal conditions, the mRNA and protein levels of the main hsp genes are low but they increase rapidly upon heat shock (HS). This is achieved due to the presence of an RNA Polymerase II pausing complex located +30-50 bp from TSS. This complex maintains a partially synthesized RNA strand of said length, poised to resume synthesis, and undergoes subsequent transcriptional inactivation to restore transcript levels after environmental stabilization.MethodsThe Gal4/UAS system was used to modify dAxud1 expression in a tissue specific manner. A DAxud1-GFP fusion was expressed in salivary glands to perform polytene chromosome immunofluorescence and chromatin immunoprecipitation. DAxud1 genome occupancy data was achieved expressing Dam-DAxud1 in imaginal wing discs using Gal4/UAS (TaDa-seq).ResultsUsing TaDa-seq, we demonstrate that DAxud1 protein is present mainly near the TSS of significant occupied genes, most frequently in the first intron. This results also revealed DAxud1 is present in hsp genes, mainly in promoter zone. Following these results, we found that, under dAxud knockdown, larvae and adults flies have a diminished thermotolerance, despite showing an increase in hsp transcripts in larval tissues. We performed polytene chromosome immunofluorescence for DAxud1-GFP, revealing extensive, but dynamic localization on chromatin in hsp70 loci. This was confirmed with chromatin immunoprecipitation. We also found that DAxud1 overexpression leads to an enrichment of RNA Polymerase II at the 5’ end of the hsp70 gene, with a decrease in its transcripts. Importantly, we show interaction of DAxud1 with NELF-B, a component of the transcriptional pausing complex, and knockdown of both genes individually has similar effects on hsp70 transcription.ConclusionDAxud1 protein is a component of chromatin, that relocates under stress conditions such as heat shock, playing a role in maintaining RNA Polymerase II stalled at the 5’ of hsp70, possibly through a pausing mechanism based on its interaction with NELF-B.

An analysis on the divergence of Chironomid spp. based on the study of 18S rRNA and polytene chromosome organization in the species revealing the role of environment on speciation

Background Nine species of Chironomus evolved throughout the world were measured for their divergence with regard to their DNA sequences concerning 18S rRNA since it is conserved for a specific species. With the advancement of the field of molecular evolution, cytogenetics requires further correlation between molecular architecture and morphological features of a species to compare amongst others to decipher their role in speciation. Therefore, divergence of DNA sequences of the Chironomus were compared with differences in the polytene chromosome features of most of the species under this investigation to evaluate underlying correlation among them, if any, to finally establish a novel method of molecular classification broadly applicable in cytogenetics studies. Results When Chironomus javanus Kieffer was considered as a reference organism, an in silico pair-wise alignment of sequences for the 18S rRNA gene regions of the other eight different species of the same genus exhibited nucleotide sequence homology ranging from 67 to 98%. This divergence of the species under consideration might be due to environmental impact causing alteration of nitrogenous bases probably due to mismatch pairing in DNA replication. This may be suggested as a cause of evolution of species in nature. A concomitant study on the polytene chromosome band patterns of majority of these species belonging to this series also indicated a divergence ranging from 10% to 30%. Conclusions Sequence analysis based on 18S rRNA of nine species of Chironomus under this investigation shows a similarity in the polytene chromosome organization in most of the Chironomid species of the series. Hence, molecular divergence in the species is consistent with cytological difference among Chironomid species. Therefore, molecular data based on 18S rRNA and cytological characters based on the polytene chromosome features of the Chironomid species may be useful for their taxonomical recognition. Moreover, variations concerning two aspects of this study may be correlated to their environmental distinctions.

Architecture of promoters of house-keeping genes in polytene chromosome interbands of drosophila melanogaster

This is the first study to investigate the molecular-genetic organization of polytene chromosome interbands located on both molecular and cytological maps of Drosophila genome. The majority of the studied interbands contained one gene with a single transcription initiation site; the remaining interbands contained one gene with several alternative promoters, two or more unidirectional genes, and “head-to-head” arranged genes. In addition, intricately arranged interbands containing three or more genes in both unidirectional and bidirectional orientation were found. Insulator proteins, ORC, P-insertions, DNase I hypersensitive sites, and other open chromatin structures were situated in the promoter region of the genes located in the interbands. This area is critical for the formation of the interband, an open chromatin region in which gene transcription and replication are combined.

Gene dunce localization in the polytene chromosome of Drosophila melanogaster long span batch of adjacent chromosomal structures

The molecular and chromosomal localization of the dunce gene was studied. This gene (167.3 kb) consists almost entirely of introns, in which a cluster of seven short tissue-specific genes is located. On the basis of the results of FISH analysis of the gene fragments, we established that the dunce gene is located within nine chromosomal structures (four bands and five interbands), which contradicts the common idea that genes are located in only one structure (band or interband) or at the boundary of these structures. Our results are quite unexpected and original and greatly expand the current understanding of the genetic organization of interphase chromosomes.

A Simple Key for Identifying the Sibling Species of the Malaria Vector Anopheles gambiae (Giles) Complex by Polytene Chromosome Cytogenetics

It has been established that Anopheles gambiae complex sibling species are the major Plasmodium malaria vectors in Africa; however, not all the sibling species transmit the infection. Easier molecular methods, PCR-based assays, have been developed to distinguish the several members of the A. gambiae complex. However, malaria vector research in less developed countries, particularly sub-Saharan Africa, is being hampered by the lack of PCR facilities in laboratories and the cost of carrying out the assay within lack of funding. Hence, the present study was designed to develop a simple identification key, based on an affordable method of polytene chromosome cytotaxonomy, for identifying the major P. falciparum vectors. The Identification Key was successfully used to identify two members of the A. gambiae complex, A. gambiae sensu stricto and A. arabiensis, which are the most potent malaria vectors in Africa; even so, it could not be used to establish the infective and the refractory strains.