Biomedical subjects

E R Lozovskaya

Publications and source records attributed to E R Lozovskaya.

At least 19 recordsLinked to original sources

PubMed2000

Self-inflicted wounds, template-directed gap repair and a recombination hotspot. Effects of the mariner transposase.

Aberrant repair products of mariner transposition occur at a frequency of approximately 1/500 per target element per generation. Among 100 such mutations in the nonautonomous element peach, most had aberrations in the 5' end of peach (40 alleles), in the 3' end of peach (11 alleles), or a deletion of peach with or without deletion of flanking genomic DNA (29 alleles). Most mariner mutations can be explained by exonuclease "nibble" and host-mediated repair of the double-stranded gap created by the transposase, in contrast to analogous mutations in the P element. In mariner, mutations in the 5' inverted repeat are smaller and more frequent than those in the 3' inverted repeat, but secondary mutations in target elements with a 5' lesion usually had 3' lesions resembling those normally found at the 5' end. We suggest that the mariner transposase distinguishes between the 5' and 3' ends of the element, and that the 5' end is relatively more protected after strand scission. We also find: (1) that homolog-dependent gap repair is a frequent accompaniment to mariner excision, estimated as 30% of all excision events; and (2) that mariner is a hotspot of recombination in Drosophila females, but only in the presence of functional transposase.

Animals↗

PubMed1999

Genome size as a mutation-selection-drift process.

A novel method for estimating neutral rates and patterns of DNA evolution in Drosophila takes advantage of the propensity of non-LTR retrotransposable elements to create nonfunctional, transpositionally inactive copies as a product of transposition. For many LINE elements, most copies present in a genome at any one time are nonfunctional "dead-on-arrival" (DOA) copies. Because these are off-shoots of active, transpositionally competent "master" lineages, in a gene tree of a LINE element from multiple samples from related species, the DOA lineages are expected to map to the terminal branches and the active lineages to the internal branches, the primary exceptions being when the sample includes DOA copies that are allelic or orthologous. Analysis of nucleotide substitutions and other changes along the terminal branches therefore allows estimation of the fixation process in the DOA copies, which are unconstrained with respect to protein coding; and under selective neutrality, the fixation process estimates the underlying mutational pattern. We have studied the retroelement Helena in Drosophila. An unexpectedly high rate of DNA loss was observed, yielding a half-life of unconstrained DNA sequences approximately 60-fold faster in Drosophila than in mammals. The high rate of DNA loss suggests a straightforward explanation of the seeming paradox that Drosophila has many fewer pseudogenes than found in mammalian species. Differential rates of deletion in different taxa might also contribute to the celebrated C-value paradox of why some closely related organisms can have very different DNA contents. New data presented here rule out the possibility that the transposition process itself is highly mutagenic, hence the observed linear relation between number of deletions and number of nucleotide substitutions is most easily explained by the hypothesis that both types of changes accumulate in unconstrained sequences over time.

Animals↗

PubMed1998

Drosophila virilis has atypical kinds and arrangements of histone repeats.

Genomic and P1 clone DNAs of Drosophila virilis were analyzed to determine the structure and organization of histone genes in this species. The species contains unique and variable repeat types, in comparison with the related species Drosophila melanogaster, with quartet repeats lacking the H1 gene and multi-length variant quintet repeats containing the H1 gene. Unexpectedly, the H1-containing repeats are highly polymorphic in length, and thus not in a strict tandem arrangement, while the H1-less repeats are very uniform and tandemly reiterated. Despite such differences, the relative positions and transcriptional polarities of the histone gene subtypes of one subcloned quintet are similar to the major histone repeat type of D. melanogaster. For the first time, the histone H1 gene has been shown to be associated with other histone gene subtypes and is present at both chromosomal loci. DNA sequence variants of the H1 gene have been mapped to individual P1 clones and found to be in a partitioned organization. The P1 cloning system has proved useful in completely retrieving a complex repetitive locus in vitro and in examining the structure and organization of the histone genes of D. virilis.

Animals↗

PubMed1998

Factors contributing to the hybrid dysgenesis syndrome in Drosophila virilis.

A hybrid dysgenesis syndrome in Drosophila virilis is associated with the mobilization of at least four unrelated transposable elements designated Helena, Paris, Penelope and Ulysses. We carried out 42 crosses between eight strains differing in transposable element copy number in order to assess their contributions to hybrid dysgenesis. Linear regression and stepwise regression analysis was performed to estimate the correlation between the difference in euchromatic transposable element number between the parental flies of different strains involved in the crosses and the percentage, in the progeny of these crosses, of males with atrophic gonads. Male gonadal atrophy is a typical manifestation of the D. virilis hybrid dysgenesis syndrome. About half the variability in the level of male gonadal atrophy can be attributed to Penelope and Paris/Helena. Other factors also seem to play a significant role in hybrid dysgenesis in D. virilis, including maternally transmitted host factors and/or uncontrolled environmental variation. In the course of this work a novel transposable element named Telemac was found. Telemac is also mobilized in hybrid dysgenesis but does not appear to play a major causative role.

Animals↗

PubMed1997

A framework physical map of Drosophila virilis based on P1 clones: applications in genome evolution.

The analysis of patterns of genome evolution may help to evaluate the evolutionary forces that shape the composition and organization of the genome. Comparisons between the physical maps of divergent species can be used to identify conserved blocks of closely linked genes whose synteny is possibly under selective constraint. We have used in situ hybridization to determine the genomic position of 732 randomly selected clones from a bacteriophage P1 library of Drosophila virilis. The resulting map includes at least one clone in each of 69% of the subdivisions into which the D. virilis polytene chromosomes are divided. A subset of these clones was used to carry out a comparative physical analysis of chromosome 2 from D. virilis and from Drosophila montana. A number of discrepancies with the classical scenario of chromosome evolution were noted. The D. virilis P1 clones were also used to determine the physical relations between ten genes that are located in the X chromosome of Drosophila melanogaster between the markers crn (2F1) and omb (4C5-6). In this region, which is approximately 2 Mb in length, there have been at least six breakpoints since the divergence of the species, and six of the genes are found at widely scattered locations in the D. virilis X chromosome. However, a block of four functionally unrelated genes, including white, roughest, Notch, and dunce, seems to be conserved between the two species.

Animals↗

PubMed1997

What restricts the activity of mariner-like transposable elements.

A number of mechanisms have recently been described that might be important in restricting the level of activity of mariner-like transposable elements (MLEs) in natural populations. These mechanisms include overproduction inhibition, in which increasing the dose of transposase decreases net activity. Another mechanism is mediated by certain missense mutations, in which a mutant transposase protein impairs the activity of the wild-type transposase in heterozygous mutant/nonmutant genotypes. A further mechanism is the potential for transposase titration by defective elements that retain transposase binding activity. The issue of regulation is not only of theoretical importance in understanding the molecular and evolutionary genetics of MLEs, but also of practical significance in learning how best to use MLEs in the germline transformation of insect pests and disease vectors.

Animals↗

PubMed1997

Discordant rates of chromosome evolution in the Drosophila virilis species group.

In Drosophila, the availability of polytene chromosome maps and of sets of probes covering most regions of the chromosomes allows a direct comparison of the organization of the genome in different species. In this work, we report the localization, in Drosophila virilis, D. montana, and D. novamexicana, of > 100 bacteriophage PI clones containing approximately 65 kilobase inserts of genomic DNA from D. virilis. Each clone hybridizes with a single euchromatic site in either chromosome 1 or chromosome 3 in D. virilis. From these data, it is possible to estimate the minimum number of inversions required to transform the map positions of the probes in one species into the map positions of the same probes in a related species. The data indicate that, in the D. virilis species group, the X chromosome has up to four times the number of inversions as are observed in chromosome 3. The first photographic polytene chromosome maps for D. montana and D. novamexicana are also presented.

Animals↗

PubMed1997

Modern thoughts on an ancyent marinere: function, evolution, regulation.

The mariner/Tc1 superfamily of transposable elements is one of the most diverse and widespread Class II transposable elements. Within the larger assemblage, the mariner-like elements (MLEs) and the Tc1-like elements (TLEs) are distinct families differing characteristically in the composition of the "D,D(35)E" cation-binding domain. Based on levels of sequence similarity, the elements in each family can be subdivided further into several smaller subfamilies. MLEs and TLEs both have an extraordinarily wide host range. They are abundant in insect genomes and other invertebrates and are found even in some vertebrate species including, in the case of mariner, humans, in which one element on chromosome 17p has been implicated as a hotspot of recombination. In spite of the extraordinary evolutionary success of the elements, virtually nothing is known about their mode of regulation within genomes. There is abundant evidence that the elements are disseminated to naive host genomes by horizontal transmission, and there is a substantial base of evidence for inference about the subsequent population dynamics. Studies of engineered mariner elements and induced mutations in the transposase have identified two mechanisms that may be operative in mariner regulation. One mechanism is overproduction inhibition, in which excessive wild-type transposase reduces the rate of excision of a target element. A second mechanism is dominant-negative complementation, in which certain mutant transposase proteins antagonize the activity of the wild-type transposase. The latter process may help explain why the vast majority of MLEs in nature undergo "vertical inactivation" by multiple mutations and, eventually, stochastic loss. There is also evidence that mariner/Tc1 elements can be mobilized in hybrid dysgenesis; in particular, certain dysgenic crosses in Drosophila virilis result in mobilization of a TLE designated Paris as well as the mobilization of other unrelated transposable elements.

Animals↗

PubMed1997

Regulation of the transposable element mariner.

The mariner/Tcl superfamily of transposable elements is widely distributed in animal genomes and is especially prevalent in insects. Their wide distribution results from their ability to be disseminated among hosts by horizontal transmission and also by their ability to persist in genomes through multiple speciation events. Although a great deal is known about the molecular mechanisms of transposition and excision, very little is known about the mechanisms by which transposition is controlled within genomes. The issue of mariner/Tcl regulation is critical in view of the great interest in these elements as vectors for germline transformation of insect pests and vectors of human disease. Several potentially important regulatory mechanisms have been identified in studies of genetically engineered mariner elements. One mechanism is overproduction inhibition, in which excessive wild-type transposase reduces the rate of excision of a target element. A second mechanism is mediated by certain mutant transposase proteins, which antagonize the activity of the wild-type transposase. The latter process may help explain why the vast majority of MLEs in nature undergo 'vertical inactivation' by multiple mutations and, eventually, stochastic loss. Another potential mechanism of regulation may result from transposase titration by defective elements that retain their DNA binding sites and ability to transpose. There is also evidence that some mariner/Tcl elements can be mobilized in a type of hybrid dysgenesis.

Animals↗

PubMed1996

High intrinsic rate of DNA loss in Drosophila.

Pseudogenes are common in mammals but virtually absent in Drosophila. All putative Drosophila pseudogenes show patterns of molecular evolution that are inconsistent with the lack of functional constraints. The absence of bona fide pseudogenes is not only puzzling, it also hampers attempts to estimate rates and patterns of neutral DNA change. The estimation problem is especially acute in the case of deletions and insertions, which are likely to have large effects when they occur in functional genes and are therefore subject to strong purifying selection. We propose a solution to this problem by taking advantage of the propensity of retrotransposable elements without long terminal repeats (non-LTR) to create non-functional, 'dead-on-arrival' copies of themselves as a common by-product of their transpositional cycle. Phylogenetic analysis of a non-LTR element, Helena, demonstrates that copies lose DNA at an unusually high rate, suggesting that lack of pseudogenes in Drosophila is the product of rampant deletion of DNA in unconstrained regions. This finding has important implications for the study of genome evolution in general and the 'C-value paradox' in particular.

Animals↗

PubMed1996

Germline transformation of Drosophila virilis mediated by the transposable element hobo.

A laboratory strain of Drosophila virilis was genetically transformed with a hobo vector carrying the miniwhite cassette using a helper plasmid with an hsp70-driven hobo transposase-coding sequence. The rate of transformation was 0.5% per fertile GO animal. Three transgenic insertions were cloned and characterized and found to be authentic hobo insertions. These results, together with the known widespread distribution of hobo in diverse insect species, suggest that hobo and related transposable elements may be of considerable utility in the germline transformation of insects other than D. melanogaster.

Animals↗

PubMed1996

Molecular phylogeny and genome evolution in the Drosophila virilis species group: duplications of the alcohol dehydrogenase gene.

Drosophila virilis is a prominent reference species for comparison with Drosophila melanogaster in regard to patterns and mechanisms of molecular and genomic evolution. Sequences were determined for 11 Adh genes from 8 species of the D. virilis species group, including species from both the virilis phylad and the montana subphylad. The genome of D. virilis contains a 6-kb duplication that includes the entire Adh coding region. The pattern of sequence identity within the duplication strongly suggests a recent gene-conversion event bordered by 36-bp indels. As in other Drosophila, the amino-acid coding region of Adh is encoded by three exons interrupted by two short introns. The promoter region includes 16 blocks of sequence that are well conserved in D. virilis, Drosophila hydei, and D. melanogaster. The developmental profile of Adh transcription suggests a distal/proximal promoter switch analogous to that in D. melanogaster. Duplicate Adh genes were also found in Drosophila montana and Drosophila lacicola, which apparently originated independently of that in D. virilis. The Adh genes in all species of the D. virilis group have among the lowest codon bias of any Adh genes so far reported in the genus Drosophila. Taking the low codon bias into account, we estimate the time of divergence between the virilis and montana clades as 9.0 +/- 0.7 Mya and the approximate time of divergence of D. virilis from other members of the virilis phylad as 2.6 +/- 0.4 Mya. The region of the D. virilis genome containing Adh, as well as the chromosome as a whole, gives evidence of extensive rearrangements relative to the genome of D. melanogaster.

Alcohol Dehydrogenase↗

PubMed1995

Diverse transposable elements are mobilized in hybrid dysgenesis in Drosophila virilis.

We describe a system of hybrid dysgenesis in Drosophila virilis in which at least four unrelated transposable elements are all mobilized following a dysgenic cross. The data are largely consistent with the superposition of at least three different systems of hybrid dysgenesis, each repressing a different transposable element, which break down following the hybrid cross, possibly because they share a common pathway in the host. The data are also consistent with a mechanism in which mobilization of a single element triggers that of others, perhaps through chromosome breakage. The mobilization of multiple, unrelated elements in hybrid dysgenesis is reminiscent of McClintock's evidence [McClintock, B. (1955) Brookhaven Symp. Biol. 8, 58-74] for simultaneous mobilization of different transposable elements in maize.

Amino Acid Sequence↗

PubMed1995

P1 clones from Drosophila melanogaster as markers to study the chromosomal evolution of Muller's A element in two species of the obscura group of Drosophila.

Thirty P1 clones from the X chromosome (Muller's A element) of Drosophila melanogaster were cross-hybridized in situ to Drosophila subobscura and Drosophila pseudoobscura polytene chromosomes. An additional recombinant phage lambda Dsuby was also used as a marker. Twenty-three (77%) of the P1 clones gave positive hybridization on D. pseudoobscura chromosomes but only 16 (53%) did so with those of D. subobscura. Eight P1 clones gave more than one hybridization signal on D. pseudoobscura and/or D. subobscura chromosomes. All P1 clones and lambda Dsuby hybridized on Muller's A element (X chromosome) of D. subobscura. In contrast, only 18 P1 clones and lambda Dsuby hybridized on Muller's A element (XL chromosomal arm) of D. pseudoobscura; 4 additional P1 clones hybridized on Muller's D element (XR chromosomal arm) of this species and the remaining P1 clone gave one hybridization signal on each arm of the X chromosome. This latter clone may contain one breakpoint of a pericentric inversion that may account for the interchange of genetic material between Muller's A and D elements in D. pseudoobscura. In contrast to the rare interchange of genetic material between chromosomal elements, profound differences in the order and spacing of markers were detected between D. melanogaster, D. pseudoobscura and D. subobscura. In fact, the number of chromosomal segments delimited by identical markers and conserved between pairwise comparisons is small. Therefore, extensive reorganization within Muller's A element has been produced during the divergence of the three species. Rough estimates of the number of cytologically detectable inversions contributing to differentiation of Muller's A element were obtained. The most reliable of these estimates is that obtained from the D. pseudoobscura and D. melanogaster comparison since a greater number of markers have been mapped in both species. Tentatively, one inversion breakpoint about every 200 kb has been produced and fixed during the divergence of D. pseudoobscura and D. melanogaster.

Animals↗

PubMed1995

Genomic regulation of transposable elements in Drosophila.

Transposable elements are a major source of genetic change, including the creation of novel genes, the alteration of gene expression in development, and the genesis of major genomic rearrangements. They are ubiquitous among contemporary organisms and probably as old as life itself. The long coexistence of transposable elements in the genome would be expected to be accompanied by host-element coevolution. Indeed, the important role of host factors in the regulation of transposable elements has been illuminated by recent studies of several systems in Drosophila. These include host factors that regulate the P element, a host mutation that renders the genome permissive for gypsy mobilization and infection, and newly induced mutations that affect the expression of transposon insertion mutations. The finding of a type of hybrid dysgenesis in D. virilis, in which multiple unrelated transposable elements are mobilized simultaneously, may also be relevant to host-factor regulation of transposition.

Animals↗

PubMed1994

Genome structure and evolution in Drosophila: applications of the framework P1 map.

Physical maps showing the relative locations of cloned DNA fragments in the genome are important resources for research in molecular genetics, genome analysis, and evolutionary biology. In addition to affording a common frame of reference for organizing diverse types of genetic data, physical maps also provide ready access to clones containing DNA sequences from any defined region of the genome. In this paper, we present a physical map of the genome of Drosophila melanogaster based on in situ hybridization with 2461 DNA fragments, averaging approximately 80 kilobase pairs each, cloned in bacteriophage P1. The map is a framework map in the sense that most putative overlaps between clones have not yet been demonstrated at the molecular level. Nevertheless, the framework map includes approximately 85% of all genes in the euchromatic genome. A continuous physical map composed of sets of overlapping P1 clones (contigs), which together span most of the euchromatic genome, is currently being assembled by screening a library of 9216 P1 clones with single-copy genetic markers as well as with the ends of the P1 clones already assigned positions in the framework map. Because most P1 clones from D. melanogaster hybridize in situ with chromosomes from related species, the framework map also makes it possible to determine the genome maps of D. pseudoobscura and other species in the subgenus Sophophora. Likewise, a P1 framework map of D. virilis affords potential access to genome organization and evolution in the subgenus Drosophila.

Animals↗

PubMed1994

Genome evolution: between the nucleosome and the chromosome.

Intermediate between DNA sequences and broad patterns of karyotypic change there is a major gap in understanding genome structure and evolution. The gap is at the megabase level between genes and chromosomes. New methods for analyzing large DNA fragments cloned in yeast or bacterial vectors provide experimental access to genome evolution at the megabase level by enabling the assembly of megabase-size contiguous regions. Genome evolution at the megabase level can also be studied using high-resolution genetic maps. Rates and patterns of genome evolution in mammals (mouse versus humans) and Drosophila (D. virilis versus D. melanogaster) are compared and contrasted. Opportunities for research in genome evolution using the new technologies are enumerated and discussed.

Animals↗

PubMed1993

A combined molecular and cytogenetic approach to genome evolution in Drosophila using large-fragment DNA cloning.

Methods of genome analysis, including the cloning and manipulation of large fragments of DNA, have opened new strategies for uniting molecular evolutionary genetics with chromosome evolution. We have begun the development of a physical map of the genome of Drosophila virilis based on large DNA fragments cloned in bacteriophage P1. A library of 10,080 P1 clones with average insert sizes of 65.8 kb, containing approximately 3.7 copies of the haploid genome of D. virilis, has been constructed and characterized. Approximately 75% of the clones have inserts exceeding 50 kb, and approximately 25% have inserts exceeding 80 kb. A sample of 186 randomly selected clones was mapped by in situ hybridization with the salivary gland chromosomes. A method for identifying D. virilis clones containing homologs of D. melanogaster genes has also been developed using hybridization with specific probes obtained from D. melanogaster by means of the polymerase chain reaction. This method proved successful for nine of ten genes and resulted in the recovery of 14 clones. The hybridization patterns of a sample of P1 clones containing repetitive DNA were also determined. A significant fraction of these clones hybridizes to multiple euchromatic sites but not to the chromocenter, which is a pattern of hybridization that is very rare among clones derived from D. melanogaster. The materials and methods described will make it possible to carry out a direct study of molecular evolution at the level of chromosome structure and organization as well as at the level of individual genes.

Animals↗