Heterochromatic DNA repeats in Drosophila and unusual gene silencing in yeast cells

The 1.25-kb heterochromatic Stellate repeats of Drosophila melanogaster are capable of stably persisting in transgenic constructs and silencing the white reporter gene (mosaic position effect variegation). This system reveals an unusual form of silencing, which is insensitive to known modifiers of position effect variegation. The unusual form of silencing was studied with yeast Saccharomyces cerevisiae, a simple eukaryotic model. To be transferred into yeast cells, the D. melanogaster Stellate repeats were cloned in the pYAC4 centromeric vector (CEN4, URA3, TRP1, HIS3). The HIS3 and/or URA3 genes could be inactive in plasmids consisting of pYAC4 and the Stellate insert in yeast cells. Deletion of D. melanogaster DNA from the plasmid was found to activate the URA3 and HIS3 genes. It was assumed that the genes were repressed rather than damaged in the presence of the Stellate repeats and that a new form of gene silencing was revealed in S. cerevisiae.     

Small RNAs and cancerogenesis

Disturbances of microRNA generation and functioning as inhibitors of gene expression at the translational level are considered as specific and diagnostic features of cancer. This review also highlights the role of short interfering RNA (siRNA) in modified epigenomic chromatin structure, which may cause cancer transformation. Future directions of cancer epigenomics are considered in the light of the involvement of siRNA in epigenomic modification of chromatin.     

The binuclear iron site of membrane-bound methane hydroxylase from <Emphasis Type="Italic">Methylococcus capsulatus</Emphasis> (Strain M)

The particulate membrane-bound methane hydroxylase (pMMOH) was isolated from methane-oxidizing cells of Methylococcus capsulatus (strain M). At SDS PAGE, pMMOH displays three bands: at 47 (α), 27 (β), and 25 kDa (γ). The ESR spectrum of pMMOH incubated with hydrogen peroxide (final concentration 20 mM) at 4°C exhibited, along with the copper signal of type II with g = 2.05, signals of cytochrome with g = 3.0 and of high-spin ferriheme with g = 6.00 After incubation at ?30°C, additional signals with g 8.5 and 13.5 were observed. These signals, which have not been recorded previously in pMMOH preparations, are due to an intermediate of the pMMOH active site, which arises in the reaction of hydrogen peroxide with pMMOH at ?30°C. It was established that this intermediate is a high-spin dimer [Fe(III)-Fe(IV)] with S = 9/2 and different degree of rhombic distortion of structure (it is responsible for both signals). Presumably, the signal with g = 8.5 also arises from the same dimer [Fe(III)-Fe(IV)], but with S = 7/2. The presence of the intermediate [Fe(III)-Fe(IV)] in pMMOH preparations suggests that the original state of the pMMOH active site is the dimer [Fe(III)-Fe(III)] which is located in the β-subunit and cannot be detected by ESR.     

Inactivation of reporter genes by cloned heterochromatic repeats of Drosophila melanogaster is accompanied by chromatin compaction

Cloned Stellate heterochromatic repeats caused unstable mosaic inactivation (position effect variegation; PEV) of the reporter gene mini-white. A number of known protein modifiers of the classical position effect induced by large heterochromatin blocks do not affect the expression of mini-white. This raises the question as to the specificity of chromatin compaction around the reporter gene. The inactivation of the mini-white gene has been found to be accompanied by a decrease in its methylation catalyzed by Escherichia coli dam-methyltransferase expressed in the genome of Drosophila. However, no changes in the nucleosome organization of mini-white have been found.     

The size and internal structure of a heterochromatic block determine its ability to induce position effect variegation in Drosophila melanogaster

In the In(1LR)pn2a rearrangement, the 1A-2E euchromatic segment is transposed to the vicinity of X heterochromatin (Xh), resulting in position effect variegation (PEV) of the genes in the 2BE region. Practically the whole X-linked heterochromatin is situated adjacent to variegated euchromatic genes. Secondary rearrangements showing weakening or reversion of PEV were obtained by irradiation of the In(1LR)pn2a. These rearrangements demonstrate a positive correlation between the strength of PEV of the wapl locus and the sizes of the adjacent heterochromatic blocks carrying the centromere. The smallest PEV-inducing fragment consists of a block corresponding to approximately 10% of Xh and containing the entire XR, the centromere, and a very proximal portion of XL heterochromatin. Heterochromatic blocks retaining the entire XR near the 2E region, but lacking the centromere, show no PEV. Reversion of PEV was also observed as a result of an internal rearrangement of the Xh blocks where the centromere is moved away from the eu-heterochromatin boundary but the amount of X heterochromatin remaining adjacent to 2E is unchanged. We propose a primary role of the X pericentromeric region in PEV induction and an enhancing effect of the other blocks, positively correlated with their size.     

Molecular evolution of two paralogous tandemly repeated heterochromatic gene clusters linked to the X and Y chromosomes of Drosophila melanogaster

Here we report the peculiarities of molecular evolution and divergence of paralogous heterochromatic clusters of the testis- expressed X-linked Stellate and Y-linked Su(Ste) tandem repeats. It was suggested that Stellate and Su(Ste) clusters affecting male fertility are the amplified derivatives of the unique euchromatic gene betaCK2tes encoding the putative testis-specific beta-subunit of protein kinase CK2. The putative Su(Ste)-like evolutionary intermediate was detected on the Y chromosome as an orphon outside of the Su(Ste) cluster. The orphon shows extensive homology to the Su(Ste) repeat, but contains several Stellate-like diagnostic nucleotide substitutions, as well as a 10-bp insertion and a 3' splice site of the first intron typical of the Stellate unit. The orphon looks like a pseudogene carrying a drastically damaged Su(Ste) open reading frame (ORF). The putative Su(Ste) ORF, as compared with the Stellate one, carries numerous synonymous substitutions leading to the major codon preference. We conclude that Su(Ste) ORFs evolved on the Y chromosome under the pressure of translational selection. Direct sequencing shows that the efficiency of concerted evolution between adjacent repeats is 5-10 times as high in the Stellate heterochromatic cluster on the X chromosome as that in the Y-linked Su(Ste) cluster, judging by the frequencies of nucleotide substitutions and single-nucleotide deletions.