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W. Engels |
We have cloned a mutS homolog from Drosophila melanogaster called spellchecker1 (spel1) and have constructed spel1 mutant flies. MutS proteins promote the correction of DNA mismatches and serve important roles in DNA replication, recombination, and repair. The spel1 gene belongs to a subfamily of mutS first characterized by the MSH2 gene of yeast and which also includes hMSH2, one of the two major hereditary nonpolyposis colon cancer loci of humans. Like msh2 mutants in other species, we find that flies lacking the spel1 gene suffer a highly increased rate of instability in long runs of dinucleotide repeats when analyzed after 10-12 fly generations. Using a new assay, we have also discovered that mutations in spel1 decrease the stability of a dinucleotide repeat when it is copied into the site of a double-strand break during gene conversion. Contrary to the case in mammalian cells, spel1 deficiency does not affect tolerance of flies to a methylating agent nor does it affect resistance to gamma-irradiation.
Several eukaryotic homologs of the Escherichia coli RecQ DNA helicase have been found. These include the human BLM gene, whose mutation results in Bloom syndrome, and the human WRN gene, whose mutation leads to Werner syndrome resembling premature aging. We cloned a Drosophila melanogaster homolog of the RECQ helicase family, Dmblm (Drosophila melanogaster Bloom), which encodes a putative 1487-amino-acid protein. Phylogenetic and dot plot analyses for the RECQ family, including 10 eukaryotic and 3 prokaryotic genes, indicate Dmblm is most closely related to the Homo sapiens BLM gene, suggesting functional similarity. Also, we found that Dmblm cDNA partially rescued the sensitivity to methyl methanesulfonate of Saccharomyces cerevisiae sgs1 mutant, demonstrating the presence of a functional similarity between Dmblm and SGS1. Our analyses identify four possible subfamilies in the RECQ family: (1) the BLM subgroup (H. sapiens Bloom, D. melanogaster Dmblm, and Caenorhabditis elegans T04A11.6); (2) the yeast RECQ subgroup (S. cerevisiae SGS1 and Schizosaccharomyces pombe rqh1/rad12); (3) the RECQL/Q1 subgroup (H. sapiens RECQL/Q1 and C. elegans K02F3.1); and (4) the WRN subgroup (H. sapiens Werner and C. elegans F18C5.2). This result may indicate that metazoans hold at least three RECQ genes, each of which may have a different function, and that multiple RECQ genes diverged with the generation of multicellular organisms. We propose that invertebrates such as nematodes and insects are useful as model systems of human genetic diseases.
The complete structure of the cysteine proteinase (CP1) gene reveals two large 5' introns as well as a small third intron. Deletion studies have shown that null mutations for the locus are female sterile with partial male sterility as well as wing and pigmentation effects. Null alleles can be produced by either deletions to the left or deletions to the right of a P element insertion in the long second intron of the gene. A nearby phenylalanyl tRNA synthetase gene (Pts) was also identified.
Somewhere in Latin America a single P element copy found its way into the genome of Drosophila melanogaster from another insect species. Once there, these transposable elements made use of their new hosts’ DNA repair mechanism to increase their copy number while transposing to new genomic positions. Within the span of a few decades they spread worldwide to encompass nearly the entire species. The only populations to escape this invasion were the stocks that were maintained in the laboratories of early Drosophila geneticists and were thus reproductively isolated from the rest of the species. This remarkable scenario was followed by a P element invasion of another kind. Within the last 10 years these elements have become ubiquitous tools for Drosophila geneticists of all stripes and have changed the way Drosophila research is conducted.
The Drosophila genome has many families of transposable elements, some of which have been studied in detail, and others are known only superficially (Berg and Howe 1989). Particular attention has been given to the P family (reviewed by Engels 1989), which has been the subject of intensive research for nearly two decades . There are two reasons for this special interest. First, the population biology and recent evolutionary history of P elements suggests a remarkable scenario of horizontal transfer from another species into D. melanogaster followed by rapid spread through the global population. The other reason is the wide array of technical applications that have made P elements an indispensable tool for manipulating the Drosophila genome.
P element induced gene conversion has been previously used to modify the white gene of Drosophila in a directed fashion. The applicability of this approach of gene targeting in Drosophila, however, has not been analyzed quantitatively for other genes. We took advantage of the P-induced forked allele, f hd , which was used as a target , and we constructed a vector containing a modified forked fragment for converting fhd. Conversion frequencies were analyzed for this locus as well as for an alternative white allele, weh812 . Combination of both P induced mutant genes allowed the simultaneous analysis of conversion frequencies under identical genetic, developmental and environmental conditions. This paper demonstrates that gene conversion through P-induced gap repair can be applied with similar success-rates at the forked locus as at the white gene. The average conversion frequencies at forked were 0.29% and at white 0.16%. These frequencies indicate that in vivo gene targeting in Drosophila should be applicable for other genes in this species at managable rates. A different experiment revealed evidence that may indicate that a protein (Su(Hw)), which imparts control on chromatin-condensation, may interfere with the gap repair process.
A P element insertion flanked by 13 RFLP marker sites was used to examine male recombination and gene conversion at an autosomal site. The great majority of crossovers on chromosome arm 2R occurred within the 4-kb region containing the P element and RFLP sites. Of the 128 recombinants analyzed, approximately two-thirds carried duplications or deletions flanking the P element. These rearrangements are described in more detail in the accompanying report. In a parallel experiment, we examined 91 gene conversion tracts resulting from excision of the same autosomal P element. We found the average tract length was 1463 bp, which is essentially the same as found previously at the white locus. The distribution of conversion tract endpoints was indistinguishable from the distribution of crossover points among the non rearranged male recombinants. Most recombination events can be explained by the "hybrid element insertion" model, but, for those lacking a duplication or deletion, a second step involving double strand gap repair must be postulated to explain the distribution of crossover points.
We studied P element-induced recombination in germline mitotic cells by examining the structure of the recombinant chromosomes. We found that most recombinants retain a mobile P element at the site of the recombination, usually with either a deletion or a duplication immediately adjacent to the P end at which the crossover occurred. The sizes of these deletions and duplications ranged from a few base pairs to well over 100 kb. These structures fit the "hybrid element insertion" (HEI) model of male recombination in which the two P element copies on sister chromatids combine to form a "hybrid element" whose termini insert into a nearby position on the homolog. The data suggest that P-induced recombination can be used as an efficient means of generating flanking deletions in the vicinity of existing P elements. These deletions are easily screened using distant flanking markers, and they can be chosen to extend in a given direction depending on which reciprocal recombinant type is selected. Furthermore, the retention of a mobile P element allows one to extend the deletion or generate additional variability at the site by subsequent rounds of recombination.
P element-induced chromosome breakage on the X chromosome of Drosophila melanogaster was repaired six times more frequently when a homologous template was located anywhere on the X rather than on an autosome. Cis-trans comparisons confirmed that recombinational repair was more frequent when the interacting sequences were physically connected. These results suggest that the search for homology between the broken ends and a matching template sequence occurs preferentially in the cis configuration. This cis advantage operates over more than 15 megabases of DNA.
P element-induced gap repair was used to copy nonhomologous DNA into the Drosophila white locus. We found that nearly 8000 base pairs of nonhomologous sequence could be copied in from an ectopic template at essentially the same rate as a single base substitution at the same location. An in vitro-constructed deletion was also copied into white at high frequencies. This procedure can be applied to the study of gene expression in Drosophila, especially for genes too large to be manipulated in other ways. We also observed several types of more complex events in which the copied template sequences were rearranged such that the breakpoints occurred at direct duplications. Most of these can be explained by a model of double strand break repair in which each terminus of the break invades a template independently and serves as a primer for DNA synthesis from it, yielding two overlapping single-stranded sequences. These single strands then pair, and synthesis is completed by each using the other as template. This synthesis-dependent strand annealing (SDSA) model is discussed as a possible general mechanism in complex organisms.
We describe here a family of P elements that we refer to as Type I repressors. These elements are identified by their repressor functions and their lack of any deletion within the first two-thirds of the cannonical P sequence. Elements belonging to this repressor class were isolated from P strains and were made in vitro. We found that Type I repressor elements could strongly repress both a cytotype-dependent allele and P element mobility in somatic and germline tissues. These effects were very dependent on genomic position. Moreover, we observed that an element's ability to repress in one assay positively correlated with its ability to repress in either of the other two assays. The Type I family of repressor elements includes both autonomous P elements and those lacking exon 3 of the P element. Fine structure deletion mapping showed that the minimal 3´ boundary of a functional Type I element lies between nucleotide position 1950 and 1956. None of 12 elements examined with more extreme deletions extending into exon 2 made repressor. We conclude that the Type I repressors form a structurally distinct group that does not include more extensively deleted repressors such as the KP element described previously.
We studied the process by which whd, a P element insertion allele of the Drosophila white locus, is replaced by its homolog in the presence of transposase. These events are interpreted as the result of double-strand gap repair following excision of the P transposon in whd. As templates for this repair we used a series of alleles derived from whd through P element mobility. One group of them, referred to collectively as whd-F, carried fragments of the P element that had lost some of the sequences needed in cis for mobility. The other group, whd-D, had lost all of the P insert and carried a deletion of some of the flanking DNA from white. The average replacement frequency was 43% for whd-F alleles and 7% for the whd-D alleles. Some of the former were converted at frequencies exceeding 50%. Our data suggest that the high conversion frequencies for the whd-F templates can be attributed at least in part to an elevated efficiency of repair of unexpanded gaps, possibly due to the closer match between whd-F sequences and the unexpanded gap endpoints. In addition, we found that the gene substitutions were almost exclusively in the direction of whd being replaced by the whd-F or whd-D allele rather than the reverse. The template alleles were usually unaltered in the process. This asymmetry implies that the conversion process is unidirectional and that the P fragments are not good substrates for P element transposase. Our results help elucidate a highly efficient double-strand gap repair mechanism in Drosophila that can also be used for gene replacement procedures involving insertions and deletions. They also help explain the rapid spread of P elements in populations.
We used P transposable element mobilization to study the repair of double-strand DNA breaks in Drosophila melanogaster premeiotic germ cells. The distribution of conversion tracts was found to be largely unaffected by changes in the length of sequence homology between the broken ends and the template, suggesting that only a short match is required. However, the frequency of repair was highly sensitive to single-base mismatches within the homologous region, ranging from 19% reversion when there were no mismatches to 5% when 15 mismatches were present over a 3455 base-pair span.
The P family of transposable genetic elements is thought to be a recent addition to the Drosophila melanogaster genome. New evidence suggests that the elements came from another Drosophila species, possibly carried by parasitic mites. The transposition mechanism of P elements involves DNA gap repair which may have facilitated their rapid spread through D. melanogaster worldwide. These results provide new insight into the process of a transposon’s invasion into a new species and the potential risk of extinction such an invasion might entail.
Transposable elements of the P family in Drosophila are thought to transpose by a cut-and-paste process that leaves a double-strand gap. The repair of such gaps resulted in the transfer of up to several kilobase pairs of information from a homologous template sequence to the site of P element excision by a process similar to gene conversion. The template was an in vitro-modified sequence which was tested at a variety of genomic positions. Characterization of 123 conversion tracts provided a detailed description of their length and distribution. Most events were continuous conversion tracts that overlapped the P insertion site without concomitant conversion of the template. The average conversion tract was 1379 base pairs, and the distribution of tract lengths fit a simple model of gap enlargement. The conversion events occurred at sufficiently high frequencies to form the basis of an efficient means of directed gene replacement.
P element transposons in Drosophila melanogaster are capable of mobilizing incomplete P elements elsewhere in the genome, and of inducing recombination. This recombination is usually only of the order of 1% or less. We show that two P elements, located at exactly homologous sites, induce levels of recombination of 20% or higher. The recombination appears to be exact, as determined by the lack of phenotypic effects in recombinant products and the lack of size changes detectable by Southern hybridization. Female recombination is increased, but to a lesser extent than male recombination. Somatic recombination levels are also elevated. Alternative explanations for the high recombination levels are given in terms of the consequences of repair of an excision site and in terms of recombination as part of the replicative transposition process.
We examine the third phase of Wright’s shifting balance theory of evolution, the exportation by migration of favorable gene combinations from a fitter subgroup to the rest of the population. The equations are deterministic and studied numerically. Most of the models studied involve 2-9 loci in which all intermediates between two extreme genotypes are equally unfit. If the favored combination consists of dominant alleles it is usually fixed even if the migration rate is two orders of magnitude less than the selection coefficient, and if the combination is recessive, one order. Although Wright thought of migration as being essentially one-way, two-way migration does not significantly alter the results. We conclude that, whatever weaknesses the Wright theory may have, they are not in Phase III.
P transposable elements in Drosophila melanogaster can undergo precise loss at a rate exceeding 13% per generation. The process is similar to gene conversion in its requirement for a homolog that is wild type at the insertion site, and in its reduced frequency when pairing between the homologs is inhibited. However, it differs from classical gene conversion by its high frequency, its requirement for P transposase, its unidirectionality, and its occurrence in somatic and pre-meiotic cells. The results suggest a model of P element transposition in which jumps occur by a "cut-and-paste" mechanism, but are followed by double-strand gap repair to restore the P element at the donor site. The results also suggest a technique for site-directed mutagenesis in Drosophila.
The P element insertion Delta2-3(99B) has previously been shown to activate incomplete P elements elsewhere in the genome. We show that this element, in conjunction with a second incomplete P element, P[CaSpeR], also induces recombination in the male germ-line. In the absence of the P[CaSpeR] element, Delta2-3(99B) induces recombination at a much lower level. The recombination is induced preferentially in the region of the P[CaSpeR] element. Recombinant chromosomes contain the P[CaSpeR] element in more than 50% of cases, and alternative models of transposon replication and preferential chromosome breakage are put forward to explain this finding. As is the case with male recombination induced by P-M dysgenic crosses, recombination appears to be pre-meiotic in a high proportion of cases. The Delta2-3(99B) element is known to act in somatic cells. Correspondingly, we show that the Delta2-3(99B) - P[CaSpeR] combination elevates the incidence of somatic recombination.
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