Mobile Genetic Element gypsy(MDG4) in Drosophila melanogasterStrains: Structural Characteristics and Regulation of Transposition

Distribution of two structural functional variants of the gypsy(MDG4) mobile genetic element was examined in 44 strains of Drosophila melenogaster. The results obtained suggest that less transpositionally active gypsyvariant is more ancient component of the Drosophilagenome. Using Southern blotting, five strains characterized by increased copy number of gypsywith significant prevalence of the active variant over the less active one were selected for further analysis. Genetic analysis of these strains led to the suggestion that some of them carry factors that mobilize gypsyindependently from the cellular flamencogene known to be responsible for transposition of this element. Other strains probably contained a suppressor of the flam ^–mutant allele causing active transpositions of the gypsy. Thus, the material for studying poorly examined relationships between the retrovirus and the host cell genome was obtained.     

Interlineage distribution and characteristics of the structure of two subfamilies of Drosophila melanogaster MDG4 (gypsy) retrotransposon

The distribution of two variants of MDG4 (gypsy) was analyzed in several Drosophila melanogaster strains. Southern blot hybridization revealed the inactive variant of MDG4 in all strains examined and active MDG4 only in some of them. Most of the strains harboring the active MDG4 variant were recently isolated from natural populations. It is of interest that the active MDG4 prevailed over the inactive one only in strains carrying the mutant flamenco gene. Several lines were analyzed in more detail. The number of MDG4 sites on salivary-gland polytene chromosomes was established via in situ hybridization, and MDG4 was tested for transposition using the ovoD test.     

The Distribution in Different Strains and Characteristic Features of Two Subfamilies of Drosophila melanogaster Retrotransposon MDG4 (gypsy)

The distribution of two variants of MDG4 (gypsy) was analyzed in severalDrosophila melanogasterstrains. Southern blot hybridization revealed the inactive variant of MDG4 in all strains examined and active MDG4 only in some of them. Most of the strains harboring the active MDG4 variant were recently isolated from natural populations. It is of interest that the active MDG4 prevailed over the inactive one only in strains carrying the mutantflamenco gene. Several lines were analyzed in more detail. The number of MDG4 sites on salivary-gland polytene chromosomes was established via in situ hybridization, and MDG4 was tested for transposition using the ovo^D test.     

Retrotransposon gtwin: structural analysis and distribution in Drosophila melanogaster strains

A search for noncanonical variants of the gypsy retrotransposon (MDG4) in the genome of the Drosophila melanogaster strain G32 led to the cloning of four copies of the poorly studied 7411-bp gtwin element. Sequence analysis showed that gtwin belongs to a family of endogeneous retroviruses, which are widespread in the Drosophila genome and have recently been termed insect erantiviruses. The gtwin retrotransposon is evolutionarily closest to MDG4, as evident from a good alignment of their nucleotide sequences including ORF1 (the pol gene) and ORF3 (the env gene), as well as the amino acid sequences of their protein products. These regions showed more than 75% homology. The distribution of gtwin was studied in several strains of the genus Drosophila. While strain G32 contained more than 20 copies of the element, ten other D. melanogaster strains carried gtwin in two to six copies per genome. The gtwin element was not detected in D. hydei or D. virilis. Comparison of the cloned gtwin sequences with the gtwin sequence available from the D. melanogaster genome database showed that the two variants of the mobile element differ by the presence or absence of a stop codon in the central region of ORF3. Its absence from the gtwin copies cloned from the strain G32 may indicate an association between the functional state of ORF3 and amplification of the element.     

Comparative analysis of the localization and mobility of retrotransposons in sibling species Drosophila simulans and Drosophila melanogaster]

The distribution of four retrotransposon families (MDG1, MDG3, MDG4 and copia) on polytene chromosomes of different (from 9 to 15) Drosophila simulans strains is studied. The mean number of MDG1 and copia euchromatic hybridization sites (3 sites for each element) is drastically decreased in D. simulans in comparison with D. melanogaster (24 and 18 sites respectively). The mean number of MDG3 sites of hybridization is 5 in D. simulans against 12 in D. melanogaster. As for MDG4 both species have on the average about 2-3 euchromatic sites. The majority of MDG1 and copia and about a half of MDG3 euchromatic copies are localized in restricted number of sites (hot spots) on D. simulans polytene chromosomes. In D. melanogaster these elements are scattered along the chromosomes though there are some hot spots too. It appears that euchromatic copies of MDG1 and copia are considerably less mobile in D. simulans in contrast to D. melanogaster. Some common hot spots of retrotransposon localization in D. simulans and D. melanogaster were earlier described as intercalary heterochromatin regions in D. melanogaster. The level of interstrain variability of MDG4 hybridization sites is comparable in both species. Comparative blot-analysis of adult and larval salivary gland DNA shows that MDG1 and copia are situated mainly in euchromatic regions of D. melanogaster chromosomes. In D. simulans genome they are located mainly in heterochromatic regions underreplicated in salivary gland polytene chromosomes. There are interspecies differences in the distribution of retrotransposons in beta-heterochromatic chromosome regions.     

Maintenance of the copy number of retrotransposon MDG3 in the Drosophila melanogaster genome]

The genomes of laboratory stocks and natural population of Drosophila melanogaster contain 8-12 copies of retrotransposon MDG3 detected by in situ hybridization. Construction of genotypes with decreased MDG3 copy number using X-chromosome and chromosome 3 free of MDG3 copies results in appearance of hybrid genomes carrying up to 7-10 copies, instead of 2-4 copies expected. New MDG3 copies are detected in different genome regions, including the 42B hot spot of their location. The chromosomes, where new clusters of MDG3 were observed, carry conserved "parental pattern" of MDG1 arrangement. The data obtained suggest the existence of genomic mechanism for maintenance of retrotransposon copy number on a definite level.     

Retrotransposon <Emphasis Type="Italic">gtwin</Emphasis>: Structural analysis and distribution in <Emphasis Type="Italic">Drosophila</Emphasis> strains

A search for noncanonical variants of the gypsy retrotransposon ( MDG4 ) in the genome of the Drosophila melanogaster strain G32 led to the cloning of four copies of the poorly studied 7411-bp gtwin element. Sequence analysis showed that gtwin belongs to a family of endogeneous retroviruses, which are widespread in the Drosophila genome and have recently been termed insect erantiviruses. The gtwin retrotransposon is evolutionarily closest to MDG4, as evident from a good alignment of their nucleotide sequences including ORF2 (the pol gene) and ORF3 (the env gene), as well as the amino acid sequences of their protein products. These regions showed more than 75% homology. The distribution of gtwin was studied in several strains of the genus Drosophila. While strain G32 contained more than 20 copies of the element, ten other D. melanogaster strains carried gtwin in two to six copies per genome. The gtwin element was not detected in D. Hydei or D. Virilis. Comparison of the cloned gtwin sequences with the gtwin sequence available from the D. melanogaster genome database showed that the two variants of the mobile element differ by the presence or absence of a stop codon in the central region of ORF3. Its absence from the gtwin copies cloned from the strain G32 may indicate an association between the functional state of ORF3 and amplification of the element.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 23–29.Original Russian Text Copyright © 2005 by Kotnova, Karpova, Feoktistova, Lyubomirskaya, Kim, Ilyin.     

Analysis of the primary structure of DNA sequences flanking MDG1

MDG is a very important component of the Drosophila genome. MDG have many sites of localisation in chromosomes and can change their localisation. Perhaps the process of MDG integration has some specificity. To study this problem we sequenced the flanking region of MDG1 DNA. The analysis of this sequences reveals the following features. 1. The 5'-flanking sequences contain 7 TATA-boxex, 5 of which form a cluster. 2. The 3'-flanking sequences contain TTTAAA block which is similar to TATA-box for alpha- and gamma-casein genes of mammals. 3. The flanking region are rich in repeated sequences, the longest of which TCCTCCT (R) and TTCTTC (R2) are on the 5'-flank and on the 3'-flank respectively, so that the whole structure is: 5'-R1NNR1-MDG1-R2NNR2-3', where N is some nucleotide. 5'-flanking sequences are AT-rich, while the 3'-flank contains 10 consecutive thymidines 4 nucleotides apart from MDG1. The MDG1 and MDG "17.6" share several common repeats in the flanking sequences, the longest of which TACTTACAT is 63 bases upstream MDG1 and 11 bases upstream MDG "17.6". This sequence differs strongly from the consensus enhancer sequence.     

Some behavioral features in Drosophila melanogaster lines carrying a flamenco gene mutation]

Olfactory sensitivity and locomotor activity was assayed in Drosophila melanogaster strains carrying a mutation of the flamenco gene, which controls transposition of the mobile genetic element 4 (MGE4) retrotransposon the gypsy mobile element. A change in olfactory sensitivity was detected. The reaction to the odor of acetic acid was inverted in flies of the mutator strain (MS), which carried the flam mutation and active MGE4 copies and were characterized by genetic instability. Flies of the genetically unstable strains displayed a lower locomotor activity. The behavioral changes in MS flies can be explained by the pleiotropic effect of the flam mutation or by insertion mutations which arise in behavior genes as a result of genome destabilization by MGE4.     

Heterochromatic regions in different Drosophila melanogaster stocks contain similar arrangements of moderate repeats with inserted copia-like elements (MDG1)

Seven out of twenty 30–50 kb genome fragments with an MDG1 copia-like element cloned in cosmids were found to carry homologous sequences which belong to a new family of non-mobile heterochromatic moderate repeats (the HMR family). These repeats along with the MDG1 copies inserted in them are under-replicated in polytene chromosomes. Such repeats may also be located in the intercalary heterochromatin site 12E of the X chromosome. Chromosomal heterochromatic regions are enriched with one of the two main genomic variants of MDG1, MDG1^het, identifiable by EcoRI restriction. From Southern DNA blot analysis the number of MDG1^het copies and their sites within the heterochromatin are invariant in all the stocks examined, while there is not a single MDG1 site along the polytene chromosomes shared by all the stocks in question.     

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.     

Transposon display supports transpositional activity of <Emphasis Type="Italic">P</Emphasis> elements in species of the <Emphasis Type="Italic">saltans</Emphasis> group of <Emphasis Type="Italic">Drosophila</Emphasis>

Mobilization of two P element subfamilies (canonical and O-type) from Drosophila sturtevanti and D. saltans was evaluated for copy number and transposition activity using the transposon display (TD) technique. Pairwise distances between strains regarding the insertion polymorphism profile were estimated. Amplification of the P element based on copy number estimates was highly variable among the strains (D. sturtevanti, canonical 20.11, O-type 9.00; D. saltans, canonical 16.4, O-type 12.60 insertions, on average). The larger values obtained by TD compared to our previous data by Southern blotting support the higher sensitivity of TD over Southern analysis for estimating transposable element copy numbers. The higher numbers of the canonical P element and the greater divergence in its distribution within the genome of D. sturtevanti (24.8%) compared to the O-type (16.7%), as well as the greater divergence in the distribution of the canonical P element, between the D. sturtevanti (24.8%) and the D. saltans (18.3%) strains, suggest that the canonical element occupies more sites within the D. sturtevanti genome, most probably due to recent transposition activity. These data corroborate the hypothesis that the O-type is the oldest subfamily of P elements in the saltans group and suggest that the canonical P element is or has been transpositionally active until more recently in D. sturtevanti.     

Screening and construction of Saccharomyces cerevisiae strains with improved multi-tolerance and bioethanol fermentation performance

In this study, a systemic analysis was initially performed to investigate the relationship between fermentation-related stress tolerances and ethanol yield. Based on the results obtained, two elite Saccharomyces cerevisiae strains, Z8 and Z15, with variant phenotypes were chosen to construct strains with improved multi-stress tolerance by genome shuffling in combination with optimized initial selection. After three rounds of genome shuffling, a shuffled strain, YZ1, which surpasses its parent strains in osmotic, heat, and acid tolerances, was obtained. Ethanol yields of YZ1 were 3.11%, 10.31%, and 10.55% higher than those of its parent strains under regular, increased heat, and high gravity fermentation conditions, respectively. YZ1 was applied to bioethanol production at an industrial scale. Results demonstrated that the variant phenotypes from available yeast strains could be used as parent stock for yeast breeding and that the genome shuffling approach is sufficiently powerful in combining suitable phenotypes in a single strain.