A Drosophila heat shock response represents an exception rather than a rule amongst Diptera species.

Heat shock protein 70 (Hsp70) is the major player that underlies adaptive response to hyperthermia in all organisms studied to date. We investigated patterns of Hsp70 expression in larvae of dipteran species collected from natural populations of species belonging to four families from different evolutionary lineages of the order Diptera: Stratiomyidae, Tabanidae, Chironomidae and Ceratopogonidae. All investigated species showed a Hsp70 expression pattern that was different from the pattern in Drosophila. In contrast to Drosophila, all of the species in the families studied were characterized by high constitutive levels of Hsp70, which was more stable than that in Drosophila. When Stratiomyidae Hsp70 proteins were expressed in Drosophila cells, they became as short-lived as the endogenous Hsp70. Interestingly, three species of Ceratopogonidae and a cold-water species of Chironomidae exhibited high constitutive levels of Hsp70 mRNA and high basal levels of Hsp70. Furthermore, two species of Tabanidae were characterized by significant constitutive levels of Hsp70 and highly stable Hsp70 mRNA. In most cases, heat-resistant species were characterized by a higher basal level of Hsp70 than more thermosensitive species. These data suggest that different trends were realized during the evolution of the molecular mechanisms underlying the regulation of the responses of Hsp70 genes to temperature fluctuations in the studied families.

[Genetic polymorphism of flax Linum usitatissimum based on use of molecular cytogenetic markers].

Using a set of approaches based on the use of molecular cytogenetic markers (DAPI/C-banding, estimation of the total area of DAPI-positive regions in prophase nuclei, FISH with 26S and 5S rDNA probes) and the microsatellite (SSR-PCR) assay, we studied genomic polymorphism in 15 flax (Linum usitatissimum L.) varieties from different geographic regions belonging to three directions of selection (oil, fiber, and intermediate flaxes) and in the k-37 x Viking hybrid. All individual chromosomes have been identified in the karyotypes of these varieties on the basis of the patterns of differential DAPI/C-banding and the distribution of 26S and 5S rDNA, and idiograms of the chromosomes have been generated. Unlike the oil flax varieties, the chromosomes in the karyotypes of the fiber flax varieties have, as a rule, pericentromeric and telomeric DAPI-positive bands of smaller size, but contain larger intercalary regions. Two chromosomal rearrangements (chromosome 3 inversions) were discovered in the variety Luna and in the k-37 x Viking hybrid. In both these forms, no colocalization of 26S rDNA and 5S rDNA on the satellite chromosome was detected. The SSR assay with the use of 20 polymorphic pairs of primers revealed 22 polymorphic loci. Based on the SSR data, we analyzed genetic similarity of the flax forms studied and constructed a genetic similarity dendrogram. The genotypes studied here form three clusters. The oil varieties comprise an independent cluster. The genetically related fiber flax varieties Vita and Luna, as well as the landrace Lipinska XIII belonging to the intermediate type, proved to be closer to the oil varieties than the remaining fiber flax varieties. The results of the molecular chromosomal analysis in the fiber and oil flaxes confirm their very close genetic similarity. In spite of this, the combined use of the chromosomal and molecular markers has opened up unique possibilities for describing the genotypes of flax varieties and creating their genetic passports.

[The role of chromosomal regions anchored to the nuclear envelope in the functional organization of chromosomes].

The functional characteristics of the DNA fragments responsible for chromosome attachment to the nuclear envelope during the interphase (neDNAs) have been studied. The neDNAs flanking the transgene have been found to promote a steadily high rate of its expression, irrespective of the site of its insertion into the host chromosomes. At the same time, neDNAs themselves have no transcription regulatory functions.

[Structural and functional analysis of the representatives of a new class of retroelements in Drosophila species].

Various copies of mobile element Penelope previously described in Drosophila virilis have been investigated in different strains of D. virilis and transgenic strains of D. melanogaster transformed by P-based constructs containing full-size copy of Penelope. It has been demonstrated that most of Penelope copies in both species carry large terminal inverted repeats (TIR) and contain deletions of various size at their 5'end of ORF. Junctions between TIR and ORF usually carry microhomology of various length enabling to postulate a hypothesis explaining the molecular mechanism underlying the origin of such complex structures. Penelope copies usually carry 34 bp repeat located in direct orientation at both end of ORF and target site duplications of various length.

[Comparative genome analysis in pea Pisum sativum L. varieties and lines with chromosomal and molecular markers].

C banding, Ag-NOR staining, FISH with pTa71 (45S rDNA) and pTa794 (5S rDNA), and RAPD-PCR analysis were used to study the genome and chromosome polymorphism in four varieties (Frisson, Sparkle, Rondo, and Finale) and two genetic lines (Sprint-2 and SGE) of pea Pisum sativum L. A comparison of the C-banding patterns did not reveal any polymorphism within the varieties. The most significant between-variety differences were observed for the size of C bands on satellite chromosomes 4 and 7. All grain pea varieties (Frisson, Sparkle, and Rondo) had a large C band in the satellite of chromosome 4 and a medium C band in the region adjacent to the satellite thread on chromosome 7. C bands were almost of the same size in the genetic lines and vegetable variety Finale. In all accessions, 45S rDNA mapped to the secondary constriction regions of chromosomes 1, 3, and 5. The signal from chromosome 5 in the lines was more intense than in the varieties. Ag-NOR staining showed that the transcriptional activity of the 45S rRNA genes on chromosome 7 was higher than on chromosome 4 in all accessions. No more than four Ag-NOR-positive nucleoli were observed in interphase nuclei. Statistical analysis of the total area of Ag-NOR-stained nucleoli did not detect any significant difference between the accessions examined. RAPD-PCR analysis revealed high between-variety and low within-variety genomic polymorphism. Chromosomal and molecular markers proved to be promising for genome identification in pea varieties and lines.

The expression of esterase S gene of Drosophila virilis in Drosophila melanogaster.

Drosophila melanogaster was transformed with the esterase S gene from Drosophila virilis. This gene is strongly activated in ejaculatory bulbs of mature males of Drosophila virilis. The closely related gene from Drosophila melanogaster is activated in ejaculatory ducts. The tissue- and stage-specific expression of incomplete genomic copy of the esterase S gene integrated into the Drosophila melanogaster genome is the same as in Drosophila virilis. These data show that tissue and stage specificity is determined by relatively small 5' regulatory region of the esterase S gene. The comparison between deduced amino-acid sequences of the esterase S of Drosophila virilis and esterase 6 of Drosophila melanogaster was performed. These sequences revealed 50% homology.

[5S genes of the loach: determination of the primary structure of the transcription termination region and nontranscribed spacer]. / 5S geny v'iuna: opredelenie pervichnoi struktury oblasti terminatsii transkriptsii i netranskribiruemogo speisera.

The primary structure of a 5S gene spacer has been determined by sequencing three cloned 5S rDNA fragments of the loach genome. The region of the spacer adjacent to the 3'-termini of the gene structural part was shown to comprise an AT-rich sequence (24 bp long) including an oligo (T)6-9 block corresponding to the terminator of RNA polymerase III. The results supported our previous data about the 5S rRNA precursor synthesis during the transcription of cloned 5S rDNAs injected into oocyte nuclei. The comparative sequence analysis revealed a homology between the spacer region from -54 to -26 bp and the 5'-termini (1-18 bp) and the 3'-termini (108-117 bp) stretches of the gene coding region. In addition, both the coding part and nontranscribed spacer of 5S DNA include highly diversed repeats which are homologous to the 3'-termini sequence (111-+8 bp) of the gene. Apparently the nontranscribed spacer of loach 5S genes derived in evolution from numerous replicated 5S genes as a result of subsequent base elimination and substitution in the most of the 5S rRNA coding sequences.

[Organization of genes coding for 5S rRNA in the loach Misgurnus fossilis L]. / Organizatsiia genov, kodiruiushchikh 5S rRNK v'iuna Misgurnus fossilis L.

The organization of 5S rRNA genes in the loach Misgurnus fossilis L. was studied. 5S rDNA was cloned in Escherichia coli using the HindIII-fragments of loach genome DNA fused into the plasmid pBR322 restricted at the same site. The recombinant clones were tested by colony hybridization. The presence of the 5S rDNA structural sequences in cloned fragments was determined by the Southern procedure of hybridization with 5S [32P]rRNA of the loach and transcription in the oocyte test system. It was found that the size of 5S rDNA repetitive units corresponds to 240-250 bp and 450-460 bp. By the CsCl centrifugation and restriction analysis it was shown that the 5S genes in the loach genome are arranged in clusters (5-30 repeats per cluster), the smaller repeat was found to contain one coding sequence while the larger repeat contains two coding sequences of 5S rDNA.

[Intragenomic polymorphism of the primary structure of 5S rRNA gene variants of the loach (Misgurnus fossilis L.). Determination of the transcriptional activity]. / Vnutrigenomnyi polimorfizm pervichnoi struktury variantov genov 5S rRNK v'iuna (Misgurnus fossilis L.). Opredelenie transkriptsionnoi aktivnosti.

The primary structure of 12 cloned repeats of loach oocyte 5S rRNA genes was determined. The heterogeneity of nucleotide sequences was revealed in the coding regions and spacer of the genes. The results of the study on in vivo transcriptional activity of the cloned 5S rRNA gene variants are consistent with the localisation of site specific base substitutions in the coding part affecting the transcription. We have compared the nucleotide sequences of loach 5S rRNA gene variants and of Xenopus laevis, X. borealis and Bombyx mori 5S genes which can be actively transcribed in X. laevis oocyte nuclei. As a result we could propose a consensus nucleotide sequence in the internal control region (from 45-th up to 100-th nucleotide) of the eukaryotic 5S rRNA gene. This sequence comprises a RNA-polymerase III promotor and stretches interacting with transcriptional factors. We have considered the base substitutions in the nucleotide sequences of 5S gene variants exerted on the experimental model of loach 5S rRNA secondary structure. All base substitutions in actively transcribed genes do not influence the general double-stranded structures of the transcripts. However in 5S RNA transcripts from genes with low transcriptional activity base substitutions affecting the box c RNA-polymerase III promoter destroy hairpin II interacting with ribosomal proteins. We have concluded that two factors can restrict the divergency of 5S rRNA genes: (1) conservation of the nucleotide sequence in the gene internal control region, and (2) conservation of the general double stranded structures in 5S rRNA transcripts.

[Isolation of Drosophila melanogaster strain y+ snw sc wa transformed by a series of P-element mediated vectors using microinjections into early embryos]. / Poluchenie osobei Drosophila melanogaster linii y+ snw sc wa, transformirovannykh seriei vektorov na osnove P-élementa s pomoshch'iu mikroin''ektsii v rannie émbriony.

P-element-mediated transformation of Drosophila melanogaster was performed and the effectivity of transformation determined using the y+snwsc wa stock. Some new D. melanogaster stocks with sne and sn+ phenotypes were obtained as well as the stocks containing bacterial "neo" gene integrated into the genome.

[Detection of a new tissue-specific enhancer in the ct6 region of the regulatory region of the drosophila cut locus]. / Obnaruzhenie novykh tkanespetsificheskikh énkhanserov v raione ct6 reguliatornoi oblasti lokusa cut drozofily.

The cut locus of Drosophila is an interesting example of a complex eukaryotic locus responsible for the development of many tissues and organs. Most of this locus is regulatory. The entire locus was cloned by Tchurikov et al. in 1986 and Blochlinger et al. in 1988. The wing ctn enhancer located 80 kb upstream of the promoter was earlier found in a 2.7 kb EcoRI-BamHI DNA fragment. The locus region 65-80 kb remote from the promoter was assumed to control the development of wings and vibrissae. We have found a new enhancer region in the ct6 region of the locus, which was in a 5 kb BamHI-EcoRI DNA fragment adjacent to the ctn enhancer. This region is responsible for the expression of the reporter lacZ gene in many tissues and organs at all stages of Drosophila development (at least in the intestine, Malpighian tubules, thoracic and abdominal sensory organs, thoracic ganglia and in ring glands). Thus, the region located 75 kb upstream of the promoter has some properties of the locus control region (LCR).

[Generation of Kruppel phenocopies by injecting into Drosophila embryos RNA complementary to mRNA in parallel orientation]. / Poluchenie fenokopii Kruppel pri in''ektsiiakh v émbriony drozofily RNK, komplementarnoi mRNK v parallel'nom napravlenii.

RNA preparations synthesized in vitro were used to study the influence of RNA interference on the Kruppel gene activity in Drosophila embryos. RNA complementary in parallel orientation to the mRNA fragment proved to induce the development of Kruppel phenocopies. The data obtained indicate that mechanisms of specific regulation of gene activity exist in Drosophila cells, which are sensitive to the formation of both parallel and antiparallel RNA-RNA duplexes that include mRNA of the corresponding gene.

[Preparation and primary genetic analysis of Drosophila melanogaster transformants line w'lz(b)/XXywf, containing mini-white genes, integrated in the genome during P-element-dependent transformation]. / Poluchenie i pervichnyi geneticheskii analiz transformantov Drosophila melanogaster linii w'lz(b)/XXywf, soderzhashchikh gen mini-white, integrirovannyi v genom pri P-élementzavisimoi transformatsii.

Transformation of Drosophila melanogaster using P-element-based vectors yielded 129 sublines, which carried mini-white gene copies in the different genome regions. Dependence of mini-white gene expression on the location, gene dosage, and sex of the transformed individuals was analyzed. The mutation lzb was shown to suppress mini-white gene expression, the degree of suppression depending on the location and dosage of the mini-white gene.

[The unusual mobile element Penelope and its behavior in distant Drosophila species]. / Neobychnyi mobil'nyi élement Penelope i ego povedenie v otdalennykh vidakh Drozofily.

The retroelement Penelope isolated from Drosophila virilis has a very unusual structure and codes for reverse transcriptase and an endonuclease belonging to the UvrC type. As shown previously, Penelope is a key element in induction of the hybrid dysgenesis syndrome described in D. virilis, which also involves mobilization of several unrelated mobile element families. Here we report a successful introduction of Penelope into the D. melanogaster genome by P element-mediated transformation. In the new host genome, Penelope is actively transcribed producing major transcript which coincides with that detected in dysgenic hybrids of D. virilis. In situ hybridization on D. melanogaster polytene chromosomes and Southern blotting revealed multiple transpositions of Penelope in the transformed D. melanogaster strains. We determined the structure of six Penelope copies inserted into D. melanogaster chromosomes. Some transformed D. melanogaster strains showed dysgenesis effects similar to those observed in hybrids from D. virilis dysgenic crosses.

[Localization of DNA probes for human ribosomal genes on barley chromosomes]. / Lokalizatsiia na khromosomakh iachmenia DNK-prob ribosomnykh genov cheloveka.

To estimate the possibility of plant genome mapping using human genome probes, the probes fluorescent in situ hybridization (FISH) of human 18S-28S rDNA (clon 22F9 from the LA-13NCO1 library) was carried out on chromosomes of the spring barley Hordeum vulgare L. As a control, wheat rDNA probe (clon pTa71) was taken. Hybridization of the wheat DNA probe revealed two major labelling sites on mitotic barley chromosomes 5I (7H) and 6I (6H), as well as several minor sites. With the human DNA probe, signals were detected in the major sites of the ribosomal genes on chromosomes 5I (7H) and 6I (6H) only when the chromosome preparations were obtained using an optimized technique with obligatory pepsin treatment followed by hybridization. Thus, this study demonstrates that physical mapping of plant chromosomes with human DNA probes that are 60 to 75% homologous to the plant genes is possible. It suggests principal opportunity for the FISH mapping of plant genomes using probes from human genome libraries, obtained in the course of the total sequencing of the human genomes and corresponding to the coding regions of genes with known functions.

[Introduction of a single transpositionally-active copy of MDG4 into the genome of a stable line of Drosophila melanogaster causes genetic instability]. / Vvedenie edinichnoi transpozitsionno-aktivnoi kopii MDG4 v genom stabil'noi linii Drosophila melanogaster vyzvaet geneticheskuiu nestabil'nost'.

A previously described system of a Drosophila melanogaster mutative strain (MS), which originates from a stable strain (SS), is characterized by genetic instability caused by transposition of the retrotransposon gypsy. New unstable strains were obtained by microinjections of the gypsy transposable copy into SS embryos. In situ hybridization experiments revealed amplification and active transposition of gypsy in SS derivatives. At the same time, introduction of the gypsy transposable copy into another stable strain (208) did not lead to appearance of genetic instability. Genetic instability in the MS system is apparently induced by a combination of two factors: the presence of a gypsy transposable copy and mutation(s) in the gene(s) regulating its transpositions.

[Chromosomal localization of 5S and 45S ribosomal DNA in species of Linum L. section Linum (syn=Protolinum and Adenolinum)]. / Khromosomnaia lokalizatsii 5S i 45S ribosomnoi DNK u vidov roda Linum L. sektsii Linum (syn.=Protolinum i Adenolinum).

Fluorescence in situ hybridization (FISH) was for the first time used to study the chromosomal location of the 45S (18-2.5S-26S) and 5S ribosomal genes in the genomes of five flax species of the section Linum (syn. Protolinum and Adenolinum). In L. usitatissimum L. (2n = 30), L. angustifolium Huds. (2n = 30), and L. bienne Mill. (2n = 30), a major hybridization site of 45S rDNA was observed in the pericentric region of a large metacentric chromosome. A polymorphic minor locus of 45S rDNA was found on one of the small chromosomes. Sites of 5S rDNA colocalized with those of 45S rDNA, but direct correlation between signal intensities from the 45S and 5S rDNA sites was observed only in some cases. Other 5S rDNA sites mapped to two chromosomes in these flax species. In L. grandiflorum Desf. (2n = 16) and L. austriacum L. (2n = 18), large regions of 45S and 5S rDNA were similarly located on a pair of homologous satellite-bearing chromosomes. An additional large polymorphic site of 45S and 5S rDNA was found in the proximal region of one arm of a small chromosome in the L. usitatissimum. L. angustifolium, and L. bienne karyotypes. The other arm of this chromosome contained a large 5S rDNA cluster. A similar location of the ribosomal genes in the pericentric region of the pair of satellite-bearing metacentrics confirmed the close relationships of the species examined. The difference in chromosomal location of the ribosomal genes between flax species with 2n = 30 and those with 2n = 16 or 18 testified to their assignment to different sections. The use of ribosomal genes as chromosome markers was assumed to be of importance for comparative genomic studies in cultivated flax, a valuable crop species of Russia, and in its wild relatives.

[The relative content of oocyte and somatic 5S rRNA at different stages of embryonic development as an index of the replacement of maternal ribosomes by ribosomes of the embryo in the loach]. / Otnositel'noe soderzhanie ootsitnoi i somaticheskoi 5S-rRNK na raznykh stadiiakh émbrional'nogo razvitiia kak pokazatel' zameny materinskikh ribosom ribosomami zarodysha u v'iuna.

Relative content of the oocyte and somatic 5S rRNA in loach Misgurnus fossilis L. during development was determined electrophoretically. Embryos before hatching contain 70% and swimming larvae no less than 50% of the oocyte 5S rRNA. We assume that the relative content of 5S rRNA fractions reflects the proportion between ribosomes synthesized during oogenesis and those synthesized in embryos and larvae. We calculated using previous data (Timofeeva, Kafiani, 1964) the rates of maternal ribosome decay and ribosome synthesis in the embryo. During organogenesis these rates appear to be 1.17-1.09 x 10(6) and 1.7 x 10(6) molecules/sec per embryo, respectively.

[Transport RNA in early embryogenesis of fish. 3. Certain problems of regulation of RNA synthesis at the early stages of development of the loach (Misgurnus fossilis (1)]. / Transportnye RNK v rannem embriogeneze ryb. Soobshchenie 3. Nekotorye voprosy reguliatsii sinteza tRNK na rannykh stadiiakh razvitiia Bìuna Misgurnus fossilis (1)

tRNA synthesis in the early loach embryos of different ploidy and factors of the activation of synthesis and the maturation of tRNA molecules at the mid-blastula stage have been studied. tRNA synthesis is activated at the early- and mid-blastula and in the beginning of gastrulation. The normal activation of synthesis and maturation of tRNA molecules require the embryo to be maintained in the contact with the yolk at the earlier developmental stages. The methionine starvation may be one of the factors limiting the rate of tRNA maturation. The activity of tRNA synthesis during blastulation was shown to depend on gene dosage. At this stage the paternal and maternal tRNA genes are transcribed independently. In the beginning of gastrulation, the type of tRNA synthesis control markedly changes and the effect of gene dose compensation manifests itself, that is typical for the control of rRNA synthesis as well. The data obtained are discussed with respect to the state of protein synthesizing system at the moment of activation of specific protein syntheses with the onset of morphogenesis.