scholarly journals Comparison of targeted-gene replacement frequencies in Drosophila melanogaster at the forked and white loci.

1996 ◽  
Vol 16 (7) ◽  
pp. 3535-3544 ◽  
Author(s):  
D H Lankenau ◽  
V G Corces ◽  
W R Engels
Keyword(s):  
Drosophila Melanogaster ◽  
Gene Conversion ◽  
Gene Targeting ◽  
Strand Break ◽  
Gene Replacement ◽  
P Element ◽  
White Gene ◽  
Success Rates ◽  
Conversion Frequency

P element-induced gene conversion has been previously used to modify the white gene of Drosophila melanogaster in a directed fashion. The applicability of this approach of gene targeting in Drosophila melanogaster, however, has not been analyzed quantitatively for other genes. We took advantage of the P element-induced forked allele, f(hd), which was used as a target, and we constructed a vector containing a modified forked fragment for converting f(hd). Conversion frequencies were analyzed for this locus as well as for an alternative white allele, w(eh812). Combination of both P element-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 element-induced gap repair can be applied with similar success rates at the forked locus and in the white gene. The average conversion frequency at forked was 0.29%, and that at white was 0.17%. These frequencies indicate that in vivo gene targeting in Drosophila melanogaster should be applicable for other genes in this species at manageable rates. We also confirmed the homolog dependence of reversions at the forked locus, indicating that P elements transpose via a cut-and-paste mechanism. In a different experiment, we attempted conversion with a modified forked allele containing the su(Hw) binding site. Despite an increased sample size, there were no conversion events with this template. One interpretation (under investigation) is that the binding of the su(Hw) product prevents double-strand break repair.

The Effect of Heterologous Insertions on Gene Conversion in Mitotically Dividing Cells in Drosophila melanogaster

Genetics ◽  
2002 ◽  
Vol 161 (1) ◽  
pp. 249-258
Author(s):  
Angela M Coveny ◽  
Tammy Dray ◽  
Gregory B Gloor
Keyword(s):  
Drosophila Melanogaster ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
P Element ◽  
Double Strand Break Repair ◽  
Break Site ◽  
In Trans ◽  
Break Repair ◽  
In Cis

Abstract We examined the influence that heterologous sequences of different sizes have on the frequency of double-strand-break repair by gene conversion in Drosophila melanogaster. We induced a double-strand break on one X chromosome in female flies by P-element excision. These flies contained heterologous insertions of various sizes located 238 bp from the break site in cis or in trans to the break, or both. We observed a significant decrease in double-strand-break repair with large heterologous insertions located either in cis or in trans to the break. Reestablishing the homology by including the same heterologous sequence in cis and in trans to the double-strand break restored the frequency of gene conversion to wild-type levels. In one instance, an allelic nonhomologous insertion completely abolished repair by homologous recombination. The results show that the repair of a double-strand break by gene conversion requires chromosome pairing in the local region of the double-strand break.

scholarly journals The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length

2021 ◽  
Author(s):  
Dipti Vinayak Vernekar ◽  
Giordano Reginato ◽  
Céline Adam ◽  
Lepakshi Ranjha ◽  
Florent Dingli ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Meiotic Recombination ◽  
Strand Break ◽  
Dna Helicase ◽  
Clamp Loader ◽  
Repair Synthesis ◽  
Dna Repair Synthesis ◽  
Gene Conversions

Abstract Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLβ heterodimer, to meiotic recombination intermediates. The molecular actors inhibited by this complex are elusive. The Pif1 DNA helicase is known to stimulate DNA polymerase delta (Pol δ) -mediated DNA synthesis from D-loops, allowing long synthesis required for break-induced replication. We show that Pif1 is also recruited genome wide to meiotic DNA double-strand break (DSB) sites. We further show that Pif1, through its interaction with PCNA, is required for the long gene conversions observed in the absence of MutLβ recruitment to recombination sites. In vivo, Mer3 interacts with the PCNA clamp loader RFC, and in vitro, Mer3-MutLβ ensemble inhibits Pif1-stimulated D-loop extension by Pol δ and RFC-PCNA. Mechanistically, our results suggest that Mer3-MutLβ may compete with Pif1 for binding to RFC-PCNA. Taken together, our data show that Pif1’s activity that promotes meiotic DNA repair synthesis is restrained by the Mer3-MutLβ ensemble which in turn prevents long gene conversion tracts and possibly associated mutagenesis.

Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

scholarly journals Local transposition of P elements in Drosophila melanogaster and recombination between duplicated elements using a site-specific recombinase.

Genetics ◽  
1994 ◽  
Vol 137 (2) ◽  
pp. 551-563 ◽  
Author(s):  
K G Golic
Keyword(s):  
Drosophila Melanogaster ◽  
Sister Chromatid ◽  
P Element ◽  
White Gene ◽  
Direct Repeats ◽  
Site Specific ◽  
P Elements ◽  
Dicentric Chromosomes ◽  
A Site ◽  
Different Levels

Abstract The transposase source delta 2-3(99B) was used to mobilize a P element located at sites on chromosomes X, 2 and 3. The transposition event most frequently recovered was a chromosome with two copies of the P element at or near the original site of insertion. These were easily recognized because the P element carried a hypomorphic white gene with a dosage dependent phenotype; flies with two copies of the gene have darker eyes than flies with one copy. The P element also carried direct repeats of the recombination target (FRT) for the FLP site-specific recombinase. The synthesis of FLP in these flies caused excision of the FRT-flanked white gene. Because the two white copies excised independently, patches of eye tissue with different levels of pigmentation were produced. Thus, the presence of two copies of the FRT-flanked white gene could be verified. When the P elements lay in the same orientation, FLP-mediated recombination between the FRTs on separated elements produced deficiencies and duplications of the flanked region. When P elements were inverted, the predominant consequence of FLP-catalyzed recombination between the inverted elements was the formation of dicentric chromosomes and acentric fragments as a result of unequal sister chromatid exchange.

scholarly journals Maternal repression of the P element promoter in the germline of Drosophila melanogaster: a model for the P cytotype.

Genetics ◽  
1993 ◽  
Vol 135 (1) ◽  
pp. 149-160 ◽  
Author(s):  
B Lemaitre ◽  
S Ronsseray ◽  
D Coen
Keyword(s):  
Drosophila Melanogaster ◽  
Maternal Effect ◽  
Molecular Mechanisms ◽  
Maternal Inheritance ◽  
P Element ◽  
Fusion Genes ◽  
P Elements ◽  
Somatic Tissues ◽  
By Products

Abstract The transposition of P elements in Drosophila melanogaster is regulated by products encoded by the P elements themselves. The P cytotype, which represses transposition and associated phenomena, exhibits both a maternal effect and maternal inheritance. The genetic and molecular mechanisms of this regulation are complex and not yet fully understood. In a previous study, using P-lacZ fusion genes, we have shown that P element regulatory products were able to inhibit the activity of the P promoter in somatic tissues. However, the repression observed did not exhibit the maternal effect characteristic of the P cytotype. With a similar approach, we have assayed in vivo the effect of P element regulatory products in the germline. We show that the P cytotype is able to repress the P promoter in the germline as well as in the soma. Furthermore, this repression exhibits a maternal effect restricted to the germline. On the basis of these new observations, we propose a model for the mechanism of P cytotype repression and its maternal inheritance.

scholarly journals Homology Requirements for Targeting Heterologous Sequences During P-Induced Gap Repair in Drosophila melanogaster  1

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 689-699 ◽  
Author(s):  
Tammy Dray ◽  
Gregory B Gloor
Keyword(s):  
Drosophila Melanogaster ◽  
Point Mutations ◽  
Double Strand Breaks ◽  
Yellow Gene ◽  
P Element ◽  
White Gene ◽  
Base Pairs ◽  
Strand Breaks ◽  
Distal Point ◽  
White Locus

The effect of homology on gene targeting was studied in the context of P-element-induced double-strand breaks at the white locus of Drosophila melanogaster. Double-strand breaks were made by excision of P-whd, a P-element insertion in the white gene. A nested set of repair templates was generated that contained the 8 kilobase (kb) yellow gene embedded within varying amounts of white gene sequence. Repair with unlimited homology was also analyzed. Flies were scored phenotypically for conversion of the yellow gene to the white locus. Targeting of the yellow gene was abolished when all of the 3′ homology was removed. Increases in template homology up to 51 base pairs (bp) did not significantly promote targeting. Maximum conversion was observed with a construct containing 493 bp of homology, without a significant increase in frequency when homology extended to the tips of the chromosome. These results demonstrate that the homology requirements for targeting a large heterologous insertion are quite different than those for a point mutation. Furthermore, heterologous insertions strongly affect the homology requirements for the conversion of distal point mutations. Several aberrant conversion tracts, which arose from templates that contained reduced homology, also were examined and characterized.

Meiotic recombination is suppressed near the histone-defined border of euchromatin and heterochromatin on chromosome 2L of Drosophila melanogaster

Genome ◽  
2016 ◽  
Vol 59 (4) ◽  
pp. 289-294 ◽  
Author(s):  
Alistair B. Coulthard ◽  
Rhodri W. Taylor-Kamall ◽  
Graham Hallson ◽  
Anna Axentiev ◽  
Don A. Sinclair ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Histone Modifications ◽  
Meiotic Recombination ◽  
Pericentric Heterochromatin ◽  
P Element ◽  
White Gene ◽  
Chromosomal Proteins ◽  
Chromatin States ◽  
Genetic Property ◽  
Element Insertion

In Drosophila melanogaster, the borders between pericentric heterochromatin and euchromatin on the major chromosome arms have been defined in various ways, including chromatin-specific histone modifications, the binding patterns of heterochromatin-enriched chromosomal proteins, and various cytogenetic techniques. Elucidation of the genetic properties that independently define the different chromatin states associated with heterochromatin and euchromatin should help refine the boundary. Since meiotic recombination is present in euchromatin, but absent in heterochromatin, it constitutes a key genetic property that can be observed transitioning between chromatin states. Using P element insertion lines marked with a su(Hw) insulated mini-white gene, meiotic recombination was found to transition in a region consistent with the H3K9me2 transition observed in ovaries.

scholarly journals Delimiting regulatory sequences of the Drosophila melanogaster Ddc gene.

1986 ◽  
Vol 6 (12) ◽  
pp. 4548-4557 ◽  
Author(s):  
J Hirsh ◽  
B A Morgan ◽  
S B Scholnick
Keyword(s):  
Drosophila Melanogaster ◽  
Germ Line ◽  
Regulatory Elements ◽  
P Element ◽  
Developmental Expression ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Flanking Sequences

We delimited sequences necessary for in vivo expression of the Drosophila melanogaster dopa decarboxylase gene Ddc. The expression of in vitro-altered genes was assayed following germ line integration via P-element vectors. Sequences between -209 and -24 were necessary for normally regulated expression, although genes lacking these sequences could be expressed at 10 to 50% of wild-type levels at specific developmental times. These genes showed components of normal developmental expression, which suggests that they retain some regulatory elements. All Ddc genes lacking the normal immediate 5'-flanking sequences were grossly deficient in larval central nervous system expression. Thus, this upstream region must contain at least one element necessary for this expression. A mutated Ddc gene without a normal TATA boxlike sequence used the normal RNA start points, indicating that this sequences is not required for start point specificity.

scholarly journals Restriction-stimulated homologous recombination of plasmids by the RecE pathway of Escherichia coli.

Genetics ◽  
1992 ◽  
Vol 130 (1) ◽  
pp. 37-49 ◽  
Author(s):  
A Nussbaum ◽  
M Shalit ◽  
A Cohen
Keyword(s):  
Escherichia Coli ◽  
Strand Break ◽  
Dsb Repair ◽  
Conversion Frequency ◽  
Break Site ◽  
Recombination Pathway ◽  
Dna Ends ◽  
Kinetics Of ◽  
Recombination Assay

Abstract To test the double-strand break (DSB) repair model in recombination by the RecE pathway of Escherichia coli, we constructed chimeric phages that allow restriction-mediated release of linear plasmid substrates of the bioluminescence recombination assay in infected EcoRI+ cells. Kinetics of DSB repair and expression of recombination products were followed by Southern hybridization and by the bioluminescence recombination assay, respectively. Plasmid recombinants were analyzed with restriction endonucleases. Our results indicate that a DSB can induce more than one type of RecE-mediated recombination. A DSB within the homology induced intermolecular recombination that followed the rules of the DSB repair model: (1) Recombination was enhanced by in vivo restriction. (2) Repair of the break depended on homologous sequences on the resident plasmid. (3) Break-repair was frequently associated with conversion of alleles that were cis to the break. (4) Conversion frequency decreased as the distance from the break increased. (5) Some clones contained a mixture of plasmid recombinants as expected by replication of a heteroduplex in the primary recombinant. The rules of the DSB repair model were not followed when recombination was induced by a DSB outside the homology. Both the cut and the uncut substrates were recipients in conversion events. Recombination events were associated with deletions that spanned the break site, but these deletions did not reach the homology. We propose that a break outside the homology may stimulate a RecE-mediated recombination pathway that does not involve direct participation of DNA ends in the homologous pairing reaction.

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