Superunstable systems in Drosophila melanogaster: analysis of mutations in the ocelliless locus

Six super-unstable mutations were obtained after crossing the M-cytotype strain of Drosophila melanogaster, where transpositions of the Stalker mobile element occur, with the pi 2 strain. One of these mutations appeared at the ocelliless locus. Detailed genetic analysis of this mutation and its numerous super-unstable derivatives was performed. Eleven allelic classes with different expression of the oc phenotype were isolated. The main features of super-instability at the ocelliless locus have been described and found to be common for all super-unstable systems obtained in our experiments. The role of the P element in the induction of super-instability is discussed.     

Participation of mobile element hobo in transposition events in the long-term instability system of Drosophila melanogaster]

The lines with an active hobo elements as well as those without any hobo fragments were hybridized with the y2sc1waG line. This resulted in the appearance of a number of mutations at the white, miniature, and some other loci. The authors analysed, in which way the hobo transposable elements take part in mutagenesis in these crosses. Most of the white mutants obtained were analysed and transpositions of hobo and Stalker elements were demonstrated. Both independent and simultaneous transpositions were found. It was shown by means of the Southern blot analysis that additional hobo or Stalker insertion into or close to the parental unknown waG insertion resulted in mutant white phenotype's shift toward both extreme and partial reversion. Possible participation in mutagenesis of other mobile elements is also under debate.     

Non-random distribution of spontaneous and high temperature-induced recessive lethal mutations in the X-chromosome of Drosophila]

Frequency and localization of spontaneous and induced by high temperature (37 degrees C) recessive lethal mutations in X-chromosome of females belonging to the 1(1) ts 403 strain defective in synthesis of heat-shock proteins (HSP) were studied. No differences in frequencies of both spontaneous and induced lethals between 1(1) ts 403 and control strain were found, thus implying that the disturbances in HSP synthesis have no effect on this process in oocytes of Drosophila melanogaster females. Surprisingly, distribution of spontaneous and induced lethals along the X-chromosome of 1(1) ts 403 strain appeared to be non-random: they primarily are located in its distal portion (1-44 cM of genetic map or in I-II sections of the Bridges cytogenetic map). This correlates with non-random distribution of mobile elements in the X-chromosome of D. melanogaster (Leibovich, 1990).     

A Hybrid Dysgenesis Syndrome in Drosophila Virilis

A new example of ``hybrid dysgenesis' has been demonstrated in the F(1) progeny of crosses between two different strains of Drosophila virilis. The dysgenic traits were observed only in hybrids obtained when wild-type females (of the Batumi strain 9 from Georgia, USSR) were crossed to males from a marker strain (the long-established laboratory strain, strain 160, carrying recessive markers on all its autosomes). The phenomena observed include high frequencies of male and female sterility, male recombination, chromosomal nondisjunction, transmission ratio distortion and the appearance of numerous visible mutations at different loci in the progeny of dysgenic crosses. The sterility demonstrated in the present study is similar to that of P-M dysgenesis in Drosophila melanogaster and apparently results from underdevelopment of the gonads in both sexes, this phenomenon being sensitive to developmental temperature. However, in contrast to the P-M and I-R dysgenic systems in D. melanogaster, in D. virilis the highest level of sterility (95-98%) occurs at 23-25°. Several of the mutations isolated from the progeny of dysgenic crosses (e.g., singed) proved to be unstable and reverted to wild type. We hypothesize that a mobile element (``Ulysses') which we have recently isolated from a dysgenically induced white eye mutation may be responsible for the phenomena observed.     

Identification of a novel insertion sequence element in Streptococcus agalactiae. bspeller@imib.rwth-aachen.de

Gain and loss of bacterial pathogenicity is often associated with mobile genetic elements. A novel insertion sequence (IS) element designated ISSa4 was identified in Streptococcus agalactiae (group B streptococci). The 963bp IS element is flanked by 25bp perfect inverted repeats and led to the duplication of a 9bp target sequence at the insertion site. ISSa4 contains one open reading frame coding for a putative transposase of 287 aa and exhibits closest similarities to insertion elements of the IS982 family which has previously not been identified in streptococci. Analysis of different S. agalactiae strains showed that the copy number of ISSa4 in S. agalactiae varies significantly between strains. The S. agalactiae strain with the highest copy number of ISSa4 was nonhemolytic and harbored one copy inserted in cylB, which encodes the membrane-spanning domain of the putative hemolysin transporter (Spellerberg et al., 1999. Identification of genetic determinants for the hemolytic activity of Streptococcus agalactiae by ISS1 transposition. J. Bacteriol. 181, 3212-3219). Determination of the distribution of ISSa4 in different S. agalactiae strains revealed that ISSa4 could be detected only in strains isolated after 1996, which might indicate a recent acquisition of this novel insertion element by S. agalactiae.     

Intragenic suppression: Stalker,a retrovirus-like transposable element,can compensate for a deficiency at the cut locus of Drosophila melanogaster

A number of mutations at the cut locus were induced by non-precise exision of a silent P-element insertion which resulted in deletions at the regulatory region of the locus. Unexpectedly, a reversion of one of these mutations was found, which appears as a result of insertion of Stalker (a retrovirus-like mobile element) near the 1.3 kb deletion. Thus an insertion of a retrovirus-like mobile element can suppress the deficiency at the regulatory region of a gene.     

Superunstable systems in Drosophila melanogaster. Analysis of mutations in signed and cut loci

The lines of the M'-cytotype characterized by a long-term instability (which was shown to be conditioned by transpositions of the new mobile element, Stalker) were hybridized with the P-line. This resulted in the appearance of a number of superunstable mutations at the yellow, white, singed, ocelliless and some other loci. The authors analyzed four independently obtained families of superunstable mutations at the singed locus. A wide spectrum of derivatives and high frequency of mutations were demonstrated, as well as the regularities of allelic transitions. Besides this, mutagenesis at the cut locus was observed in the chromosomes carrying sn mutations with frequency of 5.05 x 10(4). By means of the blot analysis it has been shown that most of ct mutations are intragenic deficiencies, ranging from 1.3 to 3 Kb, whose appearance is, conceivably, attributed to the inaccuracy of the insertion excision (the insertion is present but fails to alter the phenotype) at the cut locus of the chromosomes with the superunstable sn-alleles. In the lines with the sn- and ct-mutations the transpositions of the P-element and the Stalker were found, which indicates their involvement in mutagenesis. The authors discuss possible effects of inserting the complicated constructions, based on the combinations of P-element and the Stalker, on the induction of superinstability.     

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.     

The nature of spontaneous and induced mutagenesis in a genetically unstable mutator strain of Drosophila melanogaster]

The spontaneous and induced frequencies of visible mutations by N-nitroso-N-ethylurea in male cells of Drosophila melanogaster genetically unstable mutator strain have been investigated. The spontaneous and induced by N-nitroso-N-ethylurea genetic instability in mutator strain have similar manifestation, that evidently testifies the existence of general mechanisms of the appearance of unstable mutations, namely the transpositions of the mobile genetic elements.     

Transposable Element-Induced Response to Artificial Selection in DROSOPHILA MELANOGASTER

The P family of transposable elements in Drosophila melanogaster transpose with exceptionally high frequency when males from P strains carrying multiple copies of these elements are crossed to females from M strains that lack P elements, but with substantially lower frequency in the reciprocal cross. Transposition is associated with enhanced mutation rates, caused by insertion and deletion of P elements, and chromosome rearrangements. If P element mutagenesis creates additional variation for quantitative traits, accelerated response to artificial selection of progeny of M female female X P male male strain crosses is expected, compared with that from progeny of P female female X M male male strain crosses.--Divergent artificial selection for number of bristles on the last abdominal tergite was carried out for 16 generations among the progeny of P-strain males (Harwich) and M-strain females (Canton-S) and also of M-strain males (Canton-S) and P-strain females (Harwich). Each cross was replicated four times. Average realized heritability of abdominal bristle score for the crosses in which P transposition was expected was 0.244 +/- 0.017, 1.5 times greater than average heritability estimated from crosses in which transposition was expected to be rare (0.163 +/- 0.010). Phenotypic variance of abdominal bristle score increased by a factor of four in lines selected from M female female X P male male crosses when compared with those selected from P female female X M male male hybrids. Not all quantitative genetic variation induced by P elements is additive. A substantial fraction of nonadditive genetic variation is implicated by chromosomal analysis, which demonstrates deleterious fitness effects of the mutations when homozygous.--Several putative "quantitative" mutations were identified from chromosomes extracted from the selected lines; these will form the basis for further investigation at the molecular level of the genes controlling quantitative inheritance.     

Genetic instability and transposition of mobile element mdg4 in a mutator strain of Drosophila melanogaster

Laboratory mutator strain of Drosophila melanogaster is characterized by increased (up to 10(-3)-10(-4) frequency of spontaneous mutability. Mutations appear in premeiotic stages of gametes development. The majority of mutations were unstable (high frequencies of reversions, appearance of new mutations at the same and other loci, replicating instability). Localization of mobile elements mdg1, mdg2, mdg3, mdg4, copia and P element in X chromosomes of mutator individuals and its mutations y, ct, sbt was studied by hybridization in situ. In all strains P element was absent. The distribution of mdg1, mdg2, mdg3 and copia was identical in mutator strains and its derivatives, but distribution of mdg4 was different. The essential heterogeneity in localization of mdg4 and increased (up to 30-40) copy number in the mutator strain individuals was observed. The ability of single element mdg4 to autonomous transpositions was thus shown.     

Transposition of an intron in yeast mitochondria requires a protein encoded by that intron

The optional 1143 bp intron in the yeast mitochondrial 21S rRNA gene (omega +) is nearly quantitatively inserted in genetic crosses into 21S rRNA alleles that lack it (omega -). The intron contains an open reading frame that can encode a protein of 235 amino acids, but no function has been ascribed to this sequence. We previously found an in vivo double-strand break in omega - DNA at or close to the intron insertion site only in zygotes of omega + X omega - crosses that appears with the same kinetics as intron insertion. We now show that mutations in the intron open reading frame that would alter the translation product simultaneously inhibit nonreciprocal omega recombination and the in vivo double-strand break in omega - DNA. These results provide evidence that the open reading frame encodes a protein required for intron transposition and support the role of the double-strand break in the process.     

The double-strand-break repair model for recombination

We have isolated and characterized several members of the P transposable element family from a Drosophila melanogaster P strain. Large 2.9 kb elements are present as multiple highly conserved copies together with smaller (0.5-1.6 kb), heterogeneous elements. The complete DNA sequences of the 2.9 kb element and four small elements (previously isolated from hybrid-dysgenesis-induced mutations of the white locus) have been determined. Each small element appears to have arisen from the 2.9 kb element by a different internal deletion. P elements have 31 bp perfect inverse terminal repeats and upon insertion duplicate an 8 bp sequence found only once at the site of insertion. Three of the insertions into the white locus occurred at the same nucleotide, indicating a high degree of local site specificity for insertion. The basis of this specificity has been investigated by DNA sequence analysis of the sites where 18 P elements are found. A revertant of one of the white locus mutants has been found to result from precise excision of the P element, restoring the wild-type DNA sequence.     

Insertional DNA and spontaneous mutation at the white locus in Drosophila simulans

A large body of data on molecular analyses of several multiallelic loci in Drosophila melanogaster has demonstrated a high incidence of mobile DNA element insertions among spontaneous mutations. In the sibling species D. simulans, the dispersed, middle repetitive, nomadic sequences are reduced to about one-seventh that of its sibling species (Dowsett and Young 1982). Does this reduced amount of middle repetitive DNA (or mobile DNA sequences) mean that in D. simulans the occurrence of insertion mutants will be rare compared with that of D. melanogaster? To test this possibility, we collected seven different spontaneous white mutants of D. simulans and studied their molecular gene structures. Five out of seven mutants had insertion sequences which varied in length from 0.4 kb to 16 kb. One bore a deletion spanning the w region and another showed no gross structural alteration. Thus the proportion of insertional mutations at the white locus in D. simulans is equivalent to that observed in D. melanogaster. Among the five insertional mutants, one, wmky, showed genetic instability; the other four were stable. wmky was found to mutate at a frequency of 2.1 x 10(-5) in meiotic cells and may also be unstable in somatic cells.     

Molecular genetic analysis of hobo mobile genetic element polymorphism in the genome of Drosophila melanogaster line subjected to long-term selection

The distribution of mobile genetic element hobo was examined in Drosophila melanogaster lines HA (high male mating activity) and LA (low male mating activity) before and after their isogenization using Southern blot hybridization. The probe containing a full-size hobo copy was shown to produce polymorphic multilocus hybridization with chromosomal DNA. The polymorphism was line-specific. A comparison of hybridization patterns in isogenic and original lines showed that isogenization in dysgenic crosses resulted in the appearance of additional hobo localization sites in LA but not in HA. The hobo destabilization in the LA genome correlated with genetic instability and the ability to induce H-E hybrid dysgenesis. The results obtained are discussed in relation to the possible role of hobo in inducing genetic variability in lines with low male mating activity, which may counteract deleterious consequences of inbreeding and selection in the negative direction.     

Mobile Element Insertions Causing Mutations in the Drosophila Suppressor of Sable Locus Occur in Dnase I Hypersensitive Subregions of 5''-Transcribed Nontranslated Sequences

The locations of 16 mobile element insertions causing mutations at the Drosophila suppressor of sable [su(s)] locus were determined by restriction mapping and DNA sequencing of the junction sites. The transposons causing the mutations are: P element (5 alleles), gypsy (3 alleles), 17.6, HMS Beagle, springer, Delta 88, prygun, Stalker, and a new mobile element which was named roamer (2 alleles). Four P element insertions occur in 5' nontranslated leader sequences, while the fifth P element and all 11 non-P elements inserted into the 2053 nucleotide, 5'-most intron that is spliced from the 5' nontranslated leader approximately 100 nucleotides upstream of the translation start. Fifteen of the 16 mobile elements inserted within a approximately 1900 nucleotide region that contains seven 100-200-nucleotide long DNase I-hypersensitive subregions that alternate with DNase I-resistant intervals of similar lengths. The locations of these 15 insertion sites correlate well with the roughly estimated locations of five of the DNase I-hypersensitive subregions. These findings suggest that the features of chromatin structure that accompany gene activation may also make the DNA susceptible to insertion of mobile elements.     

Characteristics of inserted mutations obtained in the P-M hybrid dysgenesis system in the 9F12-10A7 region of the Drosophila melanogaster X-chromosome

19 new mutations in the 9F12-10A7 region of Drosophila melanogaster X chromosome was obtained in the system of P-M hybrid dysgenesis. They appeared to be lethals, as judged from viability of homo- or hemizygous females. In situ hybridization of P DNA with polytene chromosomes revealed P-element insertion in the 10A1-2 band in the majority of the mutants. As a result of complementation analysis, all these mutations were localized at previously known loci: l(1)BP1, l(1)BP5, l(1)BP8, l(1)BP7. No insertion mutations were found at the vermilion locus. This can imply for non-random distribution of insertion mutations in the region studied. Further comparison of these mutations with previously EMS-induced ones revealed that insertion mutations are predominantly hypomorph lethals which do not influence the viability, morphology and fertility of homozygous males and females, but drastically reduce viability of hemizygous females.