The Organization of Pericentromeric Heterochromatin in Polytene Chromosome 3 of the Drosophila melanogaster Line with the Rif11; SuURES Su(var)3-906 Mutations Suppressing Underreplication

Although heterochromatin makes up 40% of the Drosophila melanogaster genome, its organization remains little explored, especially in polytene chromosomes, as it is virtually not represented in them due to underreplication. Two all-new approaches were used in this work: (i) with the use of a newly synthesized Drosophila line that carries three mutations, Rif11, SuURESand Su(var)3-906, suppressing the underreplication of heterochromatic regions, we obtained their fullest representation in polytene chromosomes and described their structure; (ii) 20 DNA fragments with known positions on the physical map as well as molecular genetic features of the genome (gene density, histone marks, heterochromatin proteins, origin recognition complex proteins, replication timing sites and satellite DNAs) were mapped in the newly polytenized heterochromatin using FISH and bioinformatics data. The borders of the heterochromatic regions and variations in their positions on arm 3L have been determined for the first time. The newly polytenized heterochromatic material exhibits two main types of morphology: a banding pattern (locations of genes and short satellites) and reticular chromatin (locations of large blocks of satellite DNA). The locations of the banding and reticular polytene heterochromatin was determined on the physical map.

A method to estimate the frequency of chromosomal rearrangements induced by CRISPR/Cas9 multiplexing in Drosophila

Using CRISPR/Cas9 to simultaneously induce mutations in two or more target genes, commonly referred to as multiplexing, may result in chromosomal rearrangements such as inversions or translocations. While this may be undesirable in some contexts, the ability to recover chromosomal rearrangements targeted to specific sites in the genome is potentially a powerful tool. Before developing such tools, however, it is first important to measure the frequency with which chromosome rearrangements are induced by CRISPR/Cas9 multiplexing. To do this, we have developed a self-selecting screening system using a Drosophila line that carries an autosomal pericentric inversion in what is known as the autosynaptic form. All progeny of normal females crossed to males of this autosynaptic stock are lethal due to excessive aneuploidy. If an inversion is induced within the female germline, and if it is analogous to the inversion in the male autosynaptic line, then it is possible to recover progeny in which aneuploidy is reduced and viability is restored. Using this self-selection method, we screened 130 females and recovered one new autosynaptic element. Salivary gland polytene chromosome analysis, PCR, and sequencing confirmed the recovery of a breakpoint induced precisely between the two sgRNA target sites. Overall, we demonstrate that CRISPR/Cas9 multiplexing can induce chromosomal rearrangements in Drosophila. Also, in using this particular system, the recovery of chromosomal rearrangements was not a high frequency event.

THE INFLUENCE OF HYPERCAPNIA FOR RESISTANCE TO STRESS AND SPONTANEOUS LOCOMOTOR ACTIVITY OF DROSOPHILA MELANOGASTER DIFFERENT LINES

The effect of hypercapnia for resistance to fruit flies hyperthermic stress, life expectancy at alimentary and water deprivation and spontaneous locomotor activity. Canton-S and Oregon-R test Drosophila lines were divided into low and high are resistant to the action of carbon dioxide and contained in hypercapnic gas medium (5 % CO2) for seven generations. Under the influence of hypercapnia increased resistance to fruit flies hyperthermic stress. Life expectancy line Drosophila Canton-S high are resistant to the action of CO2 alimentary conditions of water deprivation increased 5 % and time of extinction of a half of individuals to 19 % compared with the control. In Drosophila line Oregon-R low and high are resistant to the action of CO2. the average life expectancy had a tendency to decrease i7-8 % compared with the control. It is shown that the spontaneous motor activity of both drosophila lines highly resistant to CO2 was higher compared to the low resistant. The number of flies with a positive phototaxis after adaptation to hypercapnia reduced in Drosophila all the experimental groups.

Transcriptional activation of the minimal human Proα1(I) collagen promoter: obligatory requirement for Sp1

A construct containing human Proα1(I) collagen gene promoter/enhancer-driven chloramphenicol acetyltransferase (CAT), pCOL-KT, failed to be expressed significantly in Sp1-deficient Schneider Drosophila line 2 (SL2) cells. However, CAT expression was induced 200-fold in SL2 cells co-transfected with pCOL-KT and pPACSp1, an Sp1-expression vector driven by the Drosophila actin 5C promoter. Elimination of the four potential Sp1-binding sites from pCOL-KT (pCOL-KTΔI), by removal of the first intron, did not abrogate Sp1-mediated induction of CAT. Even more significantly, a minimal Proα1(I) collagen promoter (-100 to +117 bp), containing a TATA box (-28 to -25 bp) and one putative Sp1-binding site (-87 to -82 bp), elicited strong Sp1-induced transactivation. Furthermore, mutation of the Sp1 motif in the minimal Proα1(I) collagen promoter-CAT construct abolished Sp1-induced expression of the reporter gene. Purified Sp1 protein bound specifically to DNA fragments of the Proα1(I) minimal promoter encompassing the putative Sp1-binding site; Sp1 binding could be competed out by a double-stranded oligonucleotide containing the wild-type Sp1 sequence, while an oligonucleotide containing a mutated Sp1 site failed to compete. Based on these results, we postulate that Sp1 plays an obligatory role in the transcriptional activation of the human Proα1(I) collagen gene. Additionally, we propose that a bona fide Sp1 motif, located most proximal to the TATA box, is necessary and sufficient for Sp1-mediated activation of the minimal Proα1(I) collagen promoter.