[Variability of continuous insect cell lines and their identification].

Continuous insect cell lines make a special object of research in biology. Insect cells in the established lines differ in the number of attributes from both normal differentiated, and embryonic cells. The period of genome destabilization necessarily precedes cell line immortalization. Genome destabilization is manifested by changes in genome size, cell karyotype, amplification of some retrotransposone families, and induction of their expression. The existence of significant genetic variability in one line puts a problem of searching for invariant attributes providing culture identification and defining the limits of normal polymorphism of cells in the culture. Using the vast collection of insect continuous cell lines stored at the N. I. Vavilov Institute of General Genetics RAS, nine lines were identified by RFLP method of mitochondrial DNA. Variability of DNA-polymorphisms, cellular karyology, morphology, immunological and biochemical attribute in the culture is discussed.

Diversity of mitochondrial DNA lineages in South Siberia.

To investigate the origin and evolution of aboriginal populations of South Siberia, a comprehensive mitochondrial DNA (mtDNA) analysis (HVR1 sequencing combined with RFLP typing) of 480 individuals, representing seven Altaic-speaking populations (Altaians, Khakassians, Buryats, Sojots, Tuvinians, Todjins and Tofalars), was performed. Additionally, HVR2 sequence information was obtained for 110 Altaians, providing, in particular, some novel details of the East Asian mtDNA phylogeny. The total sample revealed 81% East Asian (M*, M7, M8, M9, M10, C, D, G, Z, A, B, F, N9a, Y) and 17% West Eurasian (H, U, J, T, I, N1a, X) matrilineal genetic contribution, but with regional differences within South Siberia. The highest influx of West Eurasian mtDNAs was observed in populations from the East Sayan and Altai regions (from 12.5% to 34.5%), whereas in populations from the Baikal region this contribution was markedly lower (less than 10%). The considerable substructure within South Siberian haplogroups B, F, and G, together with the high degree of haplogroup C and D diversity revealed there, allows us to conclude that South Siberians carry the genetic imprint of early-colonization phase of Eurasia. Statistical analyses revealed that South Siberian populations contain high levels of mtDNA diversity and high heterogeneity of mtDNA sequences among populations (Fst = 5.05%) that might be due to geography but not due to language and anthropological features.

[Influence of the genotype on the polytene chromosome puffing and bioelectrical properties of cellular nuclei of Drosophila melanogaster treated with ecdysone in vivo and in vitro experiments]. / Vliianie genotipa na pufing politennykh khromosom i bioélektricheskie svoistva kletochnykh iader Drosophila melanogaster v sviazi s deistviem ékdizona v opytakh in vivo i in vitro.

Activity of polytene chromosome puffs at the 0-hour prepupae stage and bioelectric properties of cellular nuclei of salivary glands were investigated under the influence of the hormone 20-OH-ecdysone in vitro in highly inbred selected lines LA (low activity) and HA (high activity) and their F1 hybrids LA x HA and Ha x LA. The inadaptive line LA differs from the line HA by the smaller size of the puffs. The hybrids exceed the best parental line by the size of the majority of the investigated puffs. In the course of investigation of F1 hybrids Ha x LA chromosomes the phenomenon of heteropuffing has been revealed at the asinapsis sites. In vitro impact of ecdysone increases the electrokinetic potential of cellular nuclei; in hybrids this effect expressed more strongly than in parental lines. The data obtained indicate genetic differences in puffing activity in Drosophila polytene chromosomes as a result of inbreeding, destabilizing selection and heterosis effect, and they also confirm the correlation of bioelectric properties of the nuclear genome with regulation of genetic activity of a cellular nucleus.

[Molecular genetic differentiation of ethnic populations in Southern and Eastern Siberia based on mitochondrial DNA polymorphism]. / Molekuliarno-geneticheskaia differentsiatsiia étnicheskikh grupp Iuzhnoi i Vostochnoi Sibiri po dannym o polimorfizme mitokhondrial'noi DNK.

Using the data on mitochondrial DNA (mtDNA) polymorphism, genetic structures of the ethnic groups inhabiting South and East Siberia, including Altaians, Buryats, Tuvinians, Todjins, Tofalars, Yakuts, and Evenks were described. Mitochondrial gene pools of the populations examined were characterized by different ratios between Mongoloid (M*, C, D, E/G, G, A, B, and F) and Caucasoid (H, HV, I, J, K, T, U, and X) mtDNA lineages. All the populations studied carried a marked Mongoloid component, maximum frequency of which was observed in Evenks (92.4%) and Buryats (90.1%). Maximum frequencies of Caucasoid mtDNA lineages were detected in Tofalars (20.7%) and Yakuts (14.5%). Statistically significant interpopulation differences regarding the frequencies of mtDNA haplogroups were observed between all populations examined, excluding the pairs of Evenks-Yakuts, Evenks-Tuvinians, and Tuvinians-Todjins. Differentiation of the ethnic groups inhabiting South and East Siberia, as well as Central and Middle Asia, is discussed based on genetic, linguistic, and anthropological data.

[Combined effect of heat shock and ecdysone on the transcription of Drosophila melanogaster polytene chromosomes]. / Kombinirovannoe deistvie teplovogo shoka i ékdizona na transkriptsiipolitennykh khromosom Drosophila melanogaster.

The combined action of ecdysone and temperature elevation (heat shock) on D. melanogaster polytene chromosome transcription has been investigated. It was shown that heat shock while blocking the transcription in ecdysone--induced puffs is not effective in decreasing their size. Thus we are able to observe chromatin decondensation without transcription. The indirect immunofluorescence using an antiserum directed against DNA/RNA hybrids revealed an intense fluorescence in the polytene chromosomes of heat shocked larvae both in the actively transcribed heat shock puffs and in ecdysone stimulated "primary puffs" with blocked transcription. Ecdysone introduced into the media during heat shock is unable to induce any puffs. On the other hand the hormone introduced into the media after temperature elevation (30' of heat shock + 1 hour of ecdysone stimulation) induces some of the "primary puffs". A new approach for cloning the genes inducible by ecdysone and other inducible loci as well has been developed using the data obtained.

Cytogenetic analysis of the 2B3-4-2B11 region of the X chromosome of Drosophila melanogaster. II. Changes in 20-OH ecdysone puffing caused by genetic defects of puff 2B5.

Larvae homozygous or hemizygous for the l(1)t435 mutation located within the early ecdysteroid puff 2B5, or carrying a deletion of the 2B5 band, die at the end of the third larval instar. In the salivary gland chromosomes of these larvae only intermoult puffs are detected. If these salivary glands are incubated in vitro with 20-OH ecdysone for 6 h the intermoult puff 68 C remains large, some early puffs (74EF and 75B) are induced to 30-40% of their normal size, other early (63F) and all late puffs (62E, 78D, 82F and 63E) are not induced at all. Puff 2B5 reaches its normal size but does not regress after 6 h incubation with 20-OH ecdysone, as it does in normal stocks. The data obtained in this study show the existence of a locus (or loci) in the band (puff) 2B5 which is necessary for the normal response of the salivary gland chromosomes to the hormone 20-OH ecdysone.

[The effect of alpha- and beta-ecdysone on differentiation of imaginal disks of Drosophila melanogaster cultivated in vitro]. / Vliianie alpha- i beta-ekdyzona na differentsirovku imaginal'nykh diskov Drosophila melanogaster, kul'tiviruemykh in vitro

The effect of alpha- and beta-ecdysone was studied upon differentiation of eye and leg imaginal discs of Drosophila cultivated together with neural ganglia in the medium C-39. In the medium without hormone, eye and leg imaginal discs from larvae of the early 3rd instar are not capable of differentiation. In these conditions, eye imaginal discs from larvae of the late 3rd instar become pigmented in 5 weeks and leg imaginal discs form sometimes segmented limbs on the 4th week of cultivation. In the medium with alpha- and beta-ecdysone, eye and leg imaginal discs from larvae of the early 3rd instar reveal only initial stages of differentiation. The effect of beta-ecdysone concentration was studied upon differentiation of leg and eye imaginal discs from larvae of the late 3rd instar. On decrease of beta-ecdysone concentration from 0.05 down to 0.005 mug/ml, initial stages of differentiation of leg imaginal discs proceed at a slower rate but, then, more complete differentiation with the formation of chitin and neuromuscular tissue is observed. In eye imaginal discs alpha-ecdysone at a concentration of 0.5 mug/ml and beta-ecdysone at a concentration of 0.005-0.5 mug/ml induce the pigment accumulation already at the 1st week of cultivation and beta-ecdysone at a concentration of 5 mug/ml fully inhibits the formation and accumulation of pigment.

[Chromosome variability in established cultures of Drosophila melanogaster embryonal cells]. / Khromosomnaia izmenchivost'v peresevaemykh kul'turakh émbrional'nykh kletok Drosophila melanogaster

Diploid cell lines with female karyotype for 7 years conserved the normal chromosome set. Tripoid and tetraploid chromosome sets also remained unchanged after two years of cultivation in both sublines. The karyotypes characterized by supernumerary X-chromosomes or deficient for one or more X-chromosomes are predominant among the aneuploids in diploid and euploid lines. The cloning technique allows to isolate the hyperploid variants only. The following karotypic changes were observed during prolonged cultivation: a loss of a single chromosome from the 4th pair; the X-autosome (third pair) translocation; the enlargement of a heterochromatic pricentromeric segment of the X-chromosome; the centric fusion of the X-chromosomes. The formation of 2 telocentric fragments as a result of the centromeric breakage of the autosome of the second pair is a typical chromosome aberration observed in diploid, triploid and tetraploid cell lines.

[Natural resistance of Drosophila melanogaster cells to 6-mercaptopurine and 8-azaguanine]. / O prirodnoi rezistentnosti kletok Drosophila melanogaster k 6-merkaptopurinu i 8-azaguaninu

The growth of cells and the activity of some enzymes in diploid embryonic cells of Drosophila and Mosquito which cultivated for a long time in the presence of 6-mercaptopurine and 8-azaguanine were studied. It is shown on the culture of Drosophila cells that a high level of the resistance to 6-mercaptopurine and 8-azaguanine correlates with the absence of hypoxanthine-guanine phyosphorybosyltransferase (HGPRT) in them. HGPRT activity was not also revealed in extracts obtained from flies of different lines, which draws to the conclusion on the natural resistance of Drosophila cells to hypoxanthine and quanine analogues. Similar resistance was found out in Mosquito cell lines Mos 20 A. The second enzyme of such a type, adenine phosphoribosyltransferase, is present in the cells of all investigated lines. This fact explaines the retention of the sensitivity to adenine analogues in these cells.

Influence of ecdysterone on the growth of cells and synthesis of macromolecules in established cell lines of Drosophila melanogaster.

Ecdysterone (beta-ecdysone) and 2-deoxy-alpha-ecdysone specifically inhibit the growth of established embryonic cells of D. melanogaster. A preparation of alpha-ecdysone is 100 times less active than ecdysterone. The action of ecdysterone is eliminated when it is removed within 24 h, but after 48 h the inhibiting influence of the hormone becomes irreversible. The diploid line, triploid and tetraploid sublines are sensitive to ecdysterone. The diploid subline with a spontaneously arisen translocation of an X-chromosome to an autosome of the third pair is substantially more resistant to ecdysterone. At 24 h after the addition of ecdysterone, there is a 2-5-fold suppression of the synthesis of total RNA; DNA synthesis is lowered to the same degree only after 48 h. The gross synthesis of proteins, measured according to the incorporation of C 14-lysine, was not suppressed even after 48 h.