Search

Multiple stringent confinement strategies should be used whenever possible

Research demonstrates that mutations that alter heterochromatin-induced gene silencing bring about changes in the chromosome inheritance in Drosophila melanogaster. Data show that mutation in the gene locus Suppressor of Position-Effect Variegation affects multiple aspects of chromosome structure and function.

Deletions in the Drosophila minichromosome Dp1187 were used to investigate the genetic interactions of trans-acting genes with the centromere. Mutations in several genes known to have a role in chromosome inheritance were shown to have dominant effects on the stability of minichromosomes with partially defective centromeres. Heterozygous mutations in the ncd and klp3A kinesin-like protein genes strongly reduced the transmission of minichromosomes missing portions of the genetically defined centromere, but had little effect on the transmission of minichromosomes with intact centromeres. Using this approach, ncd and klp3A were shown to require only the centromeric region of the chromosome for their roles in chromosome segregation. Increased gene dosage also affected minichromosome transmission and was used to demonstrate that the nod kinesin-like protein gene interacts genetically with the centromere, in addition to interacting with extracentromeric regions as demonstrated previously. The results presented in this study strongly suggest that dominant genetic interactions between mutations and centromere-defective minichromosomes could be used effectively to identify novel genes necessary for centromere function.

Hundreds of Drosophila melanogaster stocks are currently maintained at the Bloomington Drosophila Stock Center with mutations that have not been associated with sequence-defined genes. They have been preserved because they have interesting loss-of-function phenotypes. The experimental value of these mutations would be increased by tying them to specific genomic intervals so that geneticists can more easily associate them with annotated genes. Here, we report the mapping of 85 second chromosome complementation groups in the Bloomington collection to specific, small clusters of contiguous genes or individual genes in the sequenced genome. This information should prove valuable to Drosophila geneticists interested in processes associated with particular phenotypes and those searching for mutations affecting specific sequence-defined genes. [ABSTRACT FROM AUTHOR]

Balancer chromosomes are multiply inverted chromosomes that suppress meiotic crossing over and prevent the recovery of crossover products. Balancers are commonly used in Drosophila melanogaster to maintain deleterious alleles and in stock construction. They exist for all three major chromosomes, yet the molecular location of the breakpoints and the exact nature of many of the mutations carried by the second and third chromosome balancers has not been available. Here, we precisely locate eight of 10 of the breakpoints on the third chromosome balancer TM3, six of eight on TM6, and nine of 11 breakpoints on TM6B. We find that one of the inversion breakpoints on TM3 bisects the highly conserved tumor suppressor gene p53--a finding that may have important consequences for a wide range of studies in Drosophila. We also identify evidence of single and double crossovers between several TM3 and TM6B balancers and their normal-sequence homologs that have created genetic diversity among these chromosomes. Overall, this work demonstrates the practical importance of precisely identifying the position of inversion breakpoints of balancer chromosomes and characterizing the mutant alleles carried by them. [ABSTRACT FROM AUTHOR]

In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element-based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5-7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task. [ABSTRACT FROM AUTHOR]

Gene drive systems promote the spread of genetic elements through populations by assuring they are inherited more often than Mendelian segregation would predict (see the figure). Natural examples of gene drive from Drosophila include sex-ratio meiotic drive, segregation distortion, and replicative transposition. Synthetic drive systems based on selective embryonic lethality or homing endonucleases have been described previously in Drosophila melanogaster (1–3), but they are difficult to build or are limited to transgenic populations. In contrast, RNAguided gene drives based on the CRISPR/Cas9 nuclease can, in principle, be constructed by any laboratory capable of making transgenic organisms (4). They have tremendous potential to address global problems in health, agriculture, and conservation, but their capacity to alter wild populations outside the laboratory demands caution (4–7). Just as researchers working with self-propagating pathogens must ensure that these agents do not escape to the outside world, scientists working in the laboratory with gene drive constructs are responsible for keeping them confined (4, 6, 7)., Accepted Manuscript

In Drosophila melanogaster, P element transposition has been a productive means of insertional mutagenesis. Thousands of genes have been tagged with natural and engineered P element constructs. Nevertheless, chromosomes carrying P element insertions tend to have high levels of background mutations from P elements inserting and excising during transposition. Consequently, the phenotypes seen when P element-bearing chromosomes are homozygous are often not attributable to the P insertions themselves. In this study, 178 strains in the Bloomington Drosophila Stock Center collection with P insertions on the second chromosome were complementation tested against molecularly defined chromosomal deletions and previously characterized single-gene mutations to determine if recessive lethality or sterility is associated with the P insertions rather than background mutations. This information should prove valuable to geneticists using these strains for experimental studies of gene function. [ABSTRACT FROM AUTHOR]

Balancers are rearranged chromosomes used in Drosophila melanogaster to maintain deleterious mutations in stable populations, preserve sets of linked genetic elements and construct complex experimental stocks. Here, we assess the phenotypes associated with breakpoint-induced mutations on commonly used third chromosome balancers and show remarkably few deleterious effects. We demonstrate that a breakpoint in p53 causes loss of radiation-induced apoptosis and a breakpoint in Fucosyltransferase A causes loss of fucosylation in nervous and intestinal tissue--the latter study providing new markers for intestinal cell identity and challenging previous conclusions about the regulation of fucosylation. We also describe thousands of potentially harmful mutations shared among X or third chromosome balancers, or unique to specific balancers, including an Ankyrin 2 mutation present on most TM3 balancers, and reiterate the risks of using balancers as experimental controls. We used long-read sequencing to confirm or refine the positions of two inversions with breakpoints lying in repetitive sequences and provide evidence that one of the inversions, In(2L)Cy, arose by ectopic recombination between foldback transposon insertions and the other, In(3R)C, cleanly separates subtelomeric and telomeric sequences and moves the subtelomeric sequences to an internal chromosome position. In addition, our characterization of In(3R)C shows that balancers may be polymorphic for terminal deletions. Finally, we present evidence that extremely distal mutations on balancers can add to the stability of stocks whose purpose is to maintain homologous chromosomes carrying mutations in distal genes. Overall, these studies add to our understanding of the structure, diversity and effectiveness of balancer chromosomes. [ABSTRACT FROM AUTHOR]

As part of an ongoing effort to generate comprehensive resources for the experimental analysis of fourth chromosome genes in Drosophila melanogaster, the Fourth Chromosome Resource Project has used CRISPR mutagenesis with single guide RNAs to isolate mutations in 62 of the 80 fourth chromosome, protein-coding genes. These mutations were induced on a fourth chromosome bearing a basal FRT insertion to facilitate experimental approaches involving FLP recombinase-induced mitotic recombination. To permit straightforward comparisons among mutant stocks, most of the mutations were generated on isogenic fourth chromosomes, which were then crossed into a common genetic background. Of the 119 mutations, 84 are frameshift mutations likely to be null alleles, 29 are small, in-frame deletions and 6 have yet to be characterized molecularly. The mutations were tested for recessive lethal, female sterile and visible phenotypes. Stable stocks for most of the mutations have been submitted to repositories in the United States and Japan for public distribution., (© The Author(s) 2025. Published by Oxford University Press on behalf of The Genetics Society of America.)

In Drosophila melanogaster , hormone-secreting enteroendocrine cells are important for communication from the midgut to other tissues. Many lexA, GAL4, and split-GAL4 drivers that direct gene expression in enteroendocrine cells also confer expression in hormone-secreting cells of the central nervous system. This study examines the midgut expression of selected lexA, GAL4, and split-GAL4 transgenes carrying enhancer fragments previously associated with panneuronal gene expression to assess the experimental usefulness of these drivers for distinguishing the endocrine influences of CNS versus midgut cells on physiological processes., (Copyright: © 2022 by the authors.)