Cytogenetic and molecular aspects of position effect variegation in Drosophila melanogaster

A new genetic model system for studying position effect variegation in Drosophila melanogaster was found. It allows the analysis of genetic inactivation and changes in chromosome morphology in the same cells. In T(1;2)dor ^var7 strains the 2B5 early ecdysone puff, and the ecs locus which maps in this puff are translocated into the vicinity of centromeric heterochromatin. The ecs locus plays a key role in the system of ecdysone puffs: genetic damage to this locus results in loss of sensitivity of cells to the hormone and, as a consequence, ecdysone-induced puffs do not develop. In the T(1;2)dor ^var7 chromosome the ecs and at least five adjoining loci are inactivated in a variegated fashion. In the salivary gland cells of T(1;2)dor ^var7/ ecs^lt435 0 h prepupae which do not show the ecdysone puffs, the morphology of the 2B region was analysed. In all cases where the ecs locus was inactivated, a dense block of chromatin reminiscent of a solid band was found in the 2B region instead of the four bands 2B1–2, 3–4, 5 and 6. Sometimes compaction of the chromatin reached the 2A1–2 or even 1E1–4 bands. Formation of the compact block of chromatin coincided with late replication in this region. In situ hybridization of polytene chromosomes with a DNA clone from the ecs locus showed that when the dense chromatin block was present, no DNA was accessible for hybridization in 2B5. Hybridization of DNA of another clone located in the region of the translocation breakpoint (2B7–8) was found only in polytene chromosomes of larvae grown at 25° C, and never in those grown at 18° C, independently of the morphology of the 2B5 puff. The possibility that in the case of block formation both late replication and, as a consequence, underreplication of chromosome DNA take place, is discussed.Dedicated to Professor W. Beermann on the occasion of his 65th birthday     

Concerted transpositions of mobile genetic elements coupled with fitness changes in Drosophila melanogaster

In an inbred low-activity (LA) strain of Drosophila melanogaster with a low level of fitness and a complex of inadaptive characters, in situ hybridization reveals an invariant pattern of distribution of three copia-like elements (mdg-1, mdg-3, and copia). Rare, spontaneous, multiple transpositions of mobile elements in the LA strain were shown to be coupled with a drastic increase of fitness. A changed pattern of various types of mobile elements was also observed on selecting the LA strain for higher fitness. High-fitness strains show transpositions of mobile elements to definite chromosomal sites ("hot spots"). Concerted changes in the location of three different mobile elements were found to be coupled with an increase of fitness. The mdg-1 distribution patterns were also examined in two low-fitness strains independently selected from the high-fitness ones. Fitness decrease was accompanied by mdg-1 excision from the hot spots of their location usually detected in the high-fitness strains. The results suggest the existence of a system of adaptive transpositions of mobile elements that takes part in fitness control.      

DNA underreplication in intercalary heterochromatin regions in polytene chromosomes of Drosophila melanogaster correlates with the formation of partial chromosomal aberrations and ectopic pairing

We studied the influence of the Suppressor of Underreplication (SuUR) gene expression on the intercalary heterochromatin (IH) regions of Drosophila melanogaster polytene chromosomes. We observed a strong positive correlation between increased SuUR expression, underreplication extent, amount of DNA truncation, and formation of ectopic contacts in IH regions. SuUR overexpression from heat shock-driven transgene results in the formation of partial chromosomal aberrations whose breakpoints map exclusively to the regions of intercalary and pericentric heterochromatin. It is important to note that all these effects are seen only if SuUR overexpression is induced during early stages of chromosome polytenization. Therefore, we developed the idea that ectopic pairing results from the joining of free DNA ends, which are formed as a consequence of underreplication.     

Comparative genomic and phylogenetic analysis of short-chain dehydrogenases/reductases with dual retinol/sterol substrate specificity

Human short-chain dehydrogenases/reductases with dual retinol/sterol substrate specificity (RODH-like enzymes) are thought to contribute to the oxidation of retinol for retinoic acid biosynthesis and to the metabolism of androgenic and neuroactive 3alpha-hydroxysteroids. Here, we investigated the phylogeny and orthology of these proteins to understand better their origins and physiological roles. Phylogenetic and genomic analysis showed that two proteins (11-cis-RDH and RDHL) are highly conserved, and their orthologs can be identified in the lower taxa, such as amphibians and fish. Two other proteins (RODH-4 and 3alpha-HSD) are significantly less conserved. Orthologs for 3alpha-HSD are present in all mammals analyzed, whereas orthologs for RODH-4 can be identified in some mammalian species but not in others due to species-specific gene duplications. Understanding the evolution and divergence of RODH-like enzymes in various vertebrate species should facilitate further investigation of their in vivo functions using animal models.     

Acridine orange accumulation in acid organelles of normal and vacuolated frog skeletal muscle fibres

The spatial distribution of acid membrane organelles and their relationships with normal and vacuolated transverse tubules has been studied in living frog skeletal muscle fibres using confocal microscopy. Acridine orange (AO) was used to evaluate acid compartments, while a lipophilic styryl dye, RH 414, was employed to stain the membranes of the T-system. AO accumulated in numerous spherical granules located near the poles of nuclei and between myofibrils where they were arranged in short parallel rows, triplets or pairs. AO granules could be divided into three groups: green (monomeric AO), red (aggregated AO), and mixed green/red. As demonstrated by lambda-scanning, most granules were mixed. Double staining of muscle fibres with AO and RH 414 revealed almost all AO granules located near the transverse tubules. Vacuolation of the T-system was induced by glycerol loading and subsequent removal. The close juxtaposition of AO granules and the T-system was preserved in vacuolated fibres. The lumens of vacuoles did not accumulate AO. It is concluded that AO granules represent an accumulation of AO in lysosome-related organelles and fragmented Golgi apparatus and a possible functional role of the spatial distribution of such acidic compartments is discussed.     

Bioenergetic parameters of lamprey and frog liver mitochondria during metabolic depression and activity

The objective of this study is to elucidate the role of mitochondria in reversible metabolic depression of hepatocytes of the Baltic lamprey (Lampetra fluviatilis) taking place in the last year of its life cycle and to compare their main bioenergetic parameters with those of the frog (Rana temporaria) and the white outbred mouse (Mus musculus). Using isolated mitochondria as a model, we have revealed significant seasonal variations in the main bioenergetic parameters of the lamprey liver. These changes indicate that the metabolic depression is mediated by prolonged reversible alterations of mitochondrial functions, which manifest in low activity of the mitochondrial respiratory chain, low oxidative phosphorylation, low content of mitochondrial adenine nucleotides, high level of reduced mitochondrial pyridine nucleotides and leaky mitochondrial membranes observed in winter. The enhanced ion membrane permeability of winter lamprey liver mitochondria is found to be sensitive to EGTA and to cyclosporine A in combination with ADP and Mg²⁺ and is likely mediated opening the mitochondrial permeability transition pore in its low conductance state. The sharp activation of oxidation and phosphorylation in the lamprey liver mitochondria followed by spawning and death of the animal is observed in spring. The possible causes of the phenomenon and the differences obtained between lamprey, frog and mouse are under discussion.     

Biochemical characterization of human epidermal retinol dehydrogenase 2

The mRNA encoding a putative human enzyme named Epidermal Retinol Dehydrogenase 2 (RDH-E2) was found to be significantly elevated in psoriatic skin [Y. Matsuzaka, K. Okamoto, H. Tsuji, T. Mabuchi, A. Ozawa, G. Tamiya, H. Inoko, Identification of the hRDH-E2 gene, a novel member of the SDR family, and its increased expression in psoriatic lesion, Biochem. Biophys. Res. Commun. 297 (2002) 1171-1180]. This finding led the authors to propose that RDH-E2 may be involved in the pathogenesis of psoriasis through its potential role in retinoic acid biosynthesis and stimulation of keratinocyte proliferation. However, enzymatic activity for RDH-E2 has never been demonstrated. RDH-E2 is a member of the short-chain dehydrogenase/reductase (SDR) superfamily of proteins, and is most closely related to the group of SDRs comprised of both NAD(+)- and NADP(+)-dependent enzymes with activities toward retinoid and steroid substrates. In this study, we began the characterization of RDH-E2 protein in order to determine whether it might play a role in retinoic acid biosynthesis. The results of this study show that, similarly to other SDR-type retinol dehydrogenases, RDH-E2 appears to be associated with the membranes of endoplasmic reticulum. Furthermore, RDH-E2 expressed in Sf9 insect cells as a fusion to the C-terminal His(6)-tag and purified using Ni(2+)-affinity chromatography recognizes all-trans-retinol and all-trans-retinaldehyde as substrates and exhibits a strong preference for NAD(+)/NADH as cofactors. Specific activity of RDH-E2 toward all-trans-retinoids is much lower than that of other retinoid-active SDRs, such as human RoDH4 or RDH10. The preference for NAD(+) suggests that RDH-E2 is likely to function in the oxidative direction in vivo, further supporting its potential role in the oxidation of retinol to retinaldehyde for retinoic acid biosynthesis in human keratinocytes.     

The Effects of Gasomediators on the Са2+-Dependent Potassium Permeability of the Erythrocyte Membrane

Biophysics - We investigated the effects of the H2S and CO gasomediators on Ca2+-dependent potassium channels and an anion exchanger, which participate in the induction of the hyperpolarization...