Heterochromatin, Position Effect, and Genetic Silencing

Genomes of higher eukaryotes consist of two types of chromatin: euchromatin and heterochromatin. Heterochromatin is densely packed material typically localized in telomeric and pericentric chromosome regions. Euchromatin transferred by chromosome rearrangements in the vicinity of heterochromatin is inactivated and acquires morphological properties of heterochromatin in the case of position effect variegation. One of the X chromosomes in mammal females and all paternal chromosome set in coccides become heterochromatic. The heterochromatic elements of the genome exhibit similar structural properties: genetic inactivation, compaction, late DNA replication at the S stage, and underrepresentation in somatic cells. The genetic inactivation and heterochromatin assembly are underlain by a specific genetic mechanism, silencing, which includes DNA methylation and posttranslational histone modification provided by the complex of nonhistone proteins. The state of silencing is inherited in cell generations. The same molecular mechanisms of silencing shared by all types of heterochromatic regions, be it unique or highly repetitive sequences, suggest the similar organization of these regions. No type of heterochromatin is a permanent structure as they all are formed at the strictly definite stages of early embryogenesis. Based on the bulk of evidence accumulated today, heterochromatin can be regarded as a morphological manifestation of genetic silencing.     

Acridine orange distribution and fluorescence spectra in myoblasts and single muscle fibers

Acridine orange (AO) fluorescence spectra in nuclei and cytoplasm of living myoblasts L6J1 and frog single muscle fibers have been studied using spectral scanning system of Leica TCS SL confocal microscope. AO fluorescence spectra in salt solutions dependent on free AO concentrations or in complex with DNA have also been obtained. Myoblast nuclei fluoresced in the green spectral region with maximum at about 530 nm; nucleoli had the brightest fluorescence. The fluorescence of nuclear chromatin was not uniform. Similar fluorescence of nuclei and nucleoli was observed in frog single muscle fibers. Uniform, weak, green fluorescence was observed in the myoblast cytoplasm. In the sarcoplasm of muscle fibers, AO green fluorescence was seen in A discs. In the cytoplasm of myoblasts and muscle fibers stained with AO, different red, yellow, and green fluorescent granules, which were acidic organelles, were visualized. The comparison of AO fluorescence spectra in living cells with AO fluorescence spectra in buffer solutions with different AO concentrations and AO in complex with DNA enables the estimation of the AO concentration in acidic granules. It is important for the evaluation of these cellular organelles functions in intracellular transport, adaptation, and apoptosis, as well as in a number of pathological processes.     

Evidence that proteosome inhibitors and chemical chaperones can rescue the activity of retinol dehydrogenase 12 mutant T49M

Retinol dehydrogenase 12 (RDH12) is a microsomal enzyme that catalyzes the reduction of all-trans-retinaldehyde to all-trans-retinol when expressed in cells. Mutations in RDH12 cause severe retinal degeneration; however, some of the disease-associated RDH12 mutants retain significant catalytic activity. Our previous study (Lee et al., 2010 [9]) demonstrated that the catalytically active T49M and I51N variants of RDH12 undergo accelerated degradation through the ubiquitin-proteosome system, which results in reduced levels of these proteins in the cells. Here, we investigated whether the stabilization of T49M or I51N RDH12 protein levels through the inhibition of proteosome activity or improved folding could rescue their retinaldehyde reductase activity. For the T49M variant, the inhibition of proteosome activity resulted in an increased level of T49M protein in the microsomal fraction. The higher level of the T49M variant in microsomes correlated with the higher microsomal retinaldehyde reductase activity. T49M-expressing living cells treated with the inhibitors of proteosome activity or with dimethyl sulfoxide exhibited an increase in the conversion of retinaldehyde to retinol, consistent with the recovery of functional RDH12 protein. On the other hand, accumulation of the I51N variant in the microsomes did not result in higher retinaldehyde reductase activity of the microsomes or cells. These results provide a proof of concept that, at least in the case of the T49M variant, the prevention of accelerated degradation could lead to restoration of its function in the cells. This finding justifies further search for more efficient and clinically relevant compounds for stabilizing the T49M variant activity.     

Informational Content of Polytene Chromosome Bands and Puffs

Abstract

Three widely documented mechanisms of chloride transport across plasma membranes are anion-coupled antiport, sodium-coupled symport, and an electrochemical coupling process. No direct genetic evidence has yet been provided for primary active chloride transport despite numerous reports of cellular Cl-stimulated adenosine triphos-phate (ATP)ases coexisting in the same tissue with uphill chloride transport that could not be accounted for by the three common chloride transport processes. Ch-stimulated ATPases are a common property of practically all biological cells, with the major location being of mitochondrial origin. It also appears that plasma membranes are sites of Cl–stimulated ATPase activity. Recent studies of Cl'-stimulated ATPase activity and chloride transport in the same membrane system, including liposomes, suggest a mediation by the ATPase in net movement of chloride up its electrochemical gradient across plasma membranes. Further studies, especially from a molecular biological perspective, are required to confirm a direct transport role to plasma membrane-localized Ch-stimulated ATPases.     

Transpositions of mobile dispersed genes in Drosophila melanogaster and fitness of stocks

Summary In situ hybridization with polytene chromosomes was used to demonstrate the transposition of mobile dispersed genes (mdg)-1 and 3 following the selection of flies from low reproductive activity and vability (LA stock) for high reproductive activity, viability and fitness (LA⁺ and HA stocks).The inbred LA stock is continuously selected for low reproductive activity and viability and maintains at least for twentyfive generations a characteristic pattern of mdg-1 distribution in 14–15 sites. Inbred LA⁺ and HA stocks exhibit a changed pattern of mdg-1 locations and the number of sites reaches 21–25. Parallel and independent selection for higher viability may lead to similar characteristic changes in the localization of mdg-1.In several independent experiments we observed, within one generation, a spontaneous and saltatory growth of viability and fitness in the mass-bred LA stock. In these cases new mdg-1 and mdg-3 sites reproducibly appeared to within several bands, some of them characteristic of LA⁺ and HA stocks.We discuss the possible role of mdg in determining the quantitative characters of individuals and their fitness.     

Outcross-dependent transpositions of copia-like mobile genetic elements in chromosomes of an inbred Drosophila melanogaster stock

Summary Transpositions of copia-like mobile genetic elements (MDG1, MDG3 and copia) were studied in crosses of the inbred maladaptive LA line with other laboratory lines made in order to replace specific chromosome pairs in the LA line. Individuals with various hybrid genotypes displayed changed chromosomal patterns of mobile elements compared with the parent LA chromosomes. Variability of the chromosomal molecular structure in hybrids was observed when crossing over was suppressed in the process of hybrid genome constructions. Multiple transposition events were detected in hybrid genomes carrying the second chromosomal pair of the LA line, but not if it was replaced by the second chromosome of the Swedish-b stock. No transpositions were detected in control crosses that did not involve the LA line. Outcross-dependent MDG1 transposition hot spots in the LA second chromosome were found to coincide with previously established hot spots for spontaneous transpositions in the LA line coupled with a fitness increase. The data obtained demonstrate that crosses involving inversions suppressing crossing over cannot guarantee that the chromosomal molecular content will remain the same: it can change as a result of mobile element trans-positions.     

Cytogenetic and molecular aspects of position effect variegation in Drosophila

Variations in compaction of chromosomal material of the rearrangements Dp(1;f) 1337, Dp(1;f) R, Dp(1;1)pn2b, and T(1;4)w ^m258-21, which display an extended position effect, were characterized. Morphological changes found in these rearangements were assigned to two major types: (i) continuous compaction, in which bands and interbands located distal to the eu/heterochromatin junction fuse into one compacted block of chromatin. The extent of compaction is increased by enhancers of position effect (low temperature, removal of the Y or 2R chromosome heterochromatin). In extreme cases compaction extends over dozens of bands. (ii) Discontinuous compaction, in which at least two zones of compaction separated by morphologically normal zones can readily be identified. As a result, some regions located at a greater distance from heterochromatin may be compacted more frequently than others than map nearer to it. A few regions (1D, 2B1-12, 2D) were shown to be most frequently compacted in all rearrangements investigated. The 2B13-18, 2C1-2, 2E, and 2F regions exhibited the lowest frequencies of compaction. Compaction of the zone containing the 2B1-12 bands is always accompanied by inactivation of the ecs locus, which maps in the 2B3-5 puff. At the same time the 2C1-2 and 2E bands located nearer to the breakpoint can retain normal morphology and puffing in response to ecdysterone. The results are interpreted as morphological manifestations of the discontinuity of the spreading effect.by W. Beermann     

Long-term acclimation of barley photosynthetic apparatus to narrow-band red and blue light

Chloroplasts of barley plants grown under red light (RL, 660 nm) dramatically differed from the chloroplasts of plants raised under blue light (BL, 450 nm) or control plants (white light). The chloroplasts under RL had an extensive membrane system with high stacking degree and disordered irregular shaped stacks (shaggy-formed grana). After 5 h in darkness, dynamic rearrangements of chloroplast architecture in RL- and especially BL-grown plants were restricted compared with control plants. The light spectral quality affected the content and proportions of photosynthetic pigments. The leaves of RL-grown plants had the increased ratio of low-temperature fluorescence bands, F₇₄₁/F₆₈₃, corresponding to emission of PSI and PSII, respectively. This increase can be related to specific architecture of chloroplasts in RL-treated plants, providing close spacing between the two photosystems, which enhances energy transfer from PSII to PSI and facilitates the movement of LHCII toward PSI.