Oncovirus-induced permanent genetic instability in Drosophila melanogaster

Mutant alleles of a system of genetic instability induced by oncoviral DNAs were shown to demonstrate an unstable manifestation 500 generations after their emergence. A cytogenetic analysis of oncovirus-induced unstable lines has revealed numerous chromosome rearrangements. For the Lobe alleles of this system, a specific chromosome rearrangement, Df(2L) = 35C-36B, was found on the left arm of chromosome 2. We used recessive lethal mutations involving DNA rearrangements in a successful construction of cross systems for "explosive" instability.     

Gene instability induced by mobile genetic elements in Drosophila melanogaster]

The genetic instability of Drosophila melanogaster genes induced by the mobile genetic elements is reviewed. The main attention is paid to genetic instability depended on types of crossing. Data on the possibility of genetic instability induction by the chemical and physical (X-rays, heat-shock) agents and their complex effect are cited. It was shown that a number of agents which cause mutagenic effect realize their action by involving of mobile genetic elements.     

Hsp90 modulates CAG repeat instability in human cells

The Hsp90 molecular chaperone has been implicated as a contributor to evolution in several organisms by revealing cryptic variation that can yield dramatic phenotypes when the chaperone is diverted from its normal functions by environmental stress. In addition, as a cancer drug target, Hsp90 inhibition has been documented to sensitize cells to DNA-damaging agents, suggesting a function for Hsp90 in DNA repair. Here we explore the potential role of Hsp90 in modulating the stability of nucleotide repeats, which in a number of species, including humans, exert subtle and quantitative consequences for protein function, morphological and behavioral traits, and disease. We report that impairment of Hsp90 in human cells induces contractions of CAG repeat tracks by tenfold. Inhibition of the recombinase Rad51, a downstream target of Hsp90, induces a comparable increase in repeat instability, suggesting that Hsp90-enabled homologous recombination normally functions to stabilize CAG repeat tracts. By contrast, Hsp90 inhibition does not increase the rate of gene-inactivating point mutations. The capacity of Hsp90 to modulate repeat-tract lengths suggests that the chaperone, in addition to exposing cryptic variation, might facilitate the expression of new phenotypes through induction of novel genetic variation.     

CAG*CTG repeat instability in cultured human astrocytes

Cells of the central nervous system (CNS) are prone to the devastating consequences of trinucleotide repeat (TNR) expansion. Some CNS cells, including astrocytes, show substantial TNR instability in affected individuals. Since astrocyte enrichment occurs in brain regions sensitive to neurodegeneration and somatic TNR instability, immortalized SVG-A astrocytes were used as an ex vivo model to mimic TNR mutagenesis. Cultured astrocytes produced frequent (up to 2%) CAG·CTG contractions in a sequence-specific fashion, and an apparent threshold for instability was observed between 25 and 33 repeats. These results suggest that cultured astrocytes recapitulate key features of TNR mutagenesis. Furthermore, contractions were influenced by DNA replication through the repeat, suggesting that instability can arise by replication-based mechanisms in these cells. This is a crucial mechanistic point, since astrocytes in the CNS retain proliferative capacity throughout life and could be vulnerable to replication-mediated TNR instability. The presence of interruptions led to smaller but more frequent contractions, compared to a pure repeat, and the interruptions were sometimes deleted to form a perfect tract. In summary, we suggest that CAG·CTG repeat instability in cultured astrocytes is dynamic and replication-driven, suggesting that TNR mutagenesis may be influenced by the proliferative capacity of key CNS cells.     

Autonomous transposition of gypsy mobile elements and genetic instability in Drosophila melanogaster

Summary The laboratory imitator strain (MS) of Drosophila melanogaster is characterized by an elevated frequency of spontaneous mutation (10^–3–10^–4). Mutations occur in both sexes at premeiotic stages of germ cell development. The increased mutability is a characteristic feature of MS itself, since it appears in the absence of outcrossing. Most of the mutations arising in this strain are unstable: reversions to wild type, high frequency mutation to new mutant states and replicating instability were observed. We have investigated the localization of the transposable genetic elements mdg1, 412, mdg3, gypsy (mdg4), copia and P in the X chromosomes of the MS and in the mutant lines y, ct, sbt derived from it by in situ hybridization. The P element was not found in any of these strains. The distributions of mdg1, 412, mdg3 and copia were identical in the X chromosomes of the MS and its derivatives. However, the sites of hybridization with gypsy differ in the various lines tested. In the polytene chromosomes of MS animals significant variation in location and number of copies of the gypsy element was demonstrated between different larvae; copy numbers as high as 30–40 were observed. These results suggest autonomous transposition of gypsy in the MS genome while several other mobile elements remain stable.     

Drosophila melanogaster Scramblases modulate synaptic transmission

Scramblases are a family of single-pass plasma membrane proteins, identified by their purported ability to scramble phospholipids across the two layers of plasma membrane isolated from platelets and red blood cells. However, their true in vivo role has yet to be elucidated. We report the generation and isolation of null mutants of two Scramblases identified in Drosophila melanogaster. We demonstrate that flies lacking either or both of these Scramblases are not compromised in vivo in processes requiring scrambling of phospholipids. Instead, we show that D. melanogaster lacking both Scramblases have more vesicles and display enhanced recruitment from a reserve pool of vesicles and increased neurotransmitter secretion at the larval neuromuscular synapses. These defects are corrected by the introduction of a genomic copy of the Scramb 1 gene. The lack of phenotypes related to failure of scrambling and the neurophysiological analysis lead us to propose that Scramblases play a modulatory role in the process of neurotransmission.     

A small genetic region that controls dihydroorotate dehydrogenase in Drosophila melanogaster

A locus is described that controls levels of mitochondrial dihydroorotate dehydrogenase (EC 1.3.3.1) in Drosophila melanogaster. The effects of alleles of the locus, Dhod, are manifest in preparations from whole organisms as well as in partially purified mitochondrial preparations; however, other mitochondrial functions do not appear to be appreciably affected by Dhod genotypes. The locus maps near p in the proximal portion of the right arm of chromosome 3. Flies trisomic for a chromosome segment including that region display elevated enzyme levels, implying that an enzyme structural gene is in that vicinity. Furthermore, Dhod alleles are semidominant in heterozygotes, suggesting that the dosage-sensitive element detected in the trisomics is actually the Dhod locus. These findings are discussed relative to the role of dihydroorotate dehydrogenase in the de novo pyrimidine biosynthetic pathway and relative to other pathway mutants that have been described in Drosophila.This work was supported by NSF Grants PCM 76-17214 to W. Cohen and PCM 78-14164 To J. Rawls, as well as NIH Research Career Development Award 1 KO4 AM00676 to J. Rawls.     

A misexpression screen identifies genes that can modulate RAS1 pathway signaling in Drosophila melanogaster

Differentiation of the R7 photoreceptor cell is dependent on the Sevenless receptor tyrosine kinase, which activates the RAS1/mitogen-activated protein kinase signaling cascade. Kinase suppressor of Ras (KSR) functions genetically downstream of RAS1 in this signal transduction cascade. Expression of dominant-negative KSR (KDN) in the developing eye blocks RAS pathway signaling, prevents R7 cell differentiation, and causes a rough eye phenotype. To identify genes that modulate RAS signaling, we screened for genes that alter RAS1/KSR signaling efficiency when misexpressed. In this screen, we recovered three known genes, Lk6, misshapen, and Akap200. We also identified seven previously undescribed genes; one encodes a novel rel domain member of the NFAT family, and six encode novel proteins. These genes may represent new components of the RAS pathway or components of other signaling pathways that can modulate signaling by RAS. We discuss the utility of gain-of-function screens in identifying new components of signaling pathways in Drosophila.     

Orientation dependence of trinucleotide CAG repeat instability in Saccharomyces cerevisiae.

To examine the chromosomal stability of repetitions of the trinucleotide CAG, we have cloned CAG repeat tracts onto the 3' end of the Saccharomyces cerevisiae ADE2 gene and placed the appended gene into the ARO2 locus of chromosome VII. Examination of chromosomal DNA from sibling colonies arising from clonal expansion of strains harboring repeat tracts showed that repeat tracts often change in length. Most changes in tract length are decreases, but rare increases also occur. Longer tracts are more unstable than smaller tracts. The most unstable tracts, of 80 to 90 repeats, undergo changes at rates as high as 3 x 10(-2) changes per cell per generation. To examine whether repeat orientation or adjacent sequences alter repeat stability, we constructed strains with repeat tracts in both orientations, either with or without sequences 5' to ADE2 harboring an autonomously replicating sequence (ARS; replication origin). When CAG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively stable regardless of the orientation of ADE2. When CTG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively unstable regardless of the orientation of ADE2. Removal of the ARS as well as other sequences adjacent to the 5' end of ADE2 alters the orientation dependence such that stability now depends on the orientation of ADE2 in the chromosome. These results suggest that the proximity of an ARS or another sequence has a profound effect on repeat stability.     

Structural instability of 297 element in Drosophila melanogaster

297 element Southern pattern modifications previously detected in mutation accumulation lines of Drosophila melanogaster were further investigated by in situ hybridisation, Southern blotting with different combinations of genomic digest-probe, and PCR. Only one out of the nine pattern modifications studied could be interpreted as an excision and was detectable by in situ hybridisation to polytene chromosomes. Results were consistent with most pattern modifications being small rearrangements within the body of the element. In agreement with the existence of spontaneous rearrangements of this kind is the observation that many genomic copies of element 297 are defective and these are not limited to heterochromatin. These findings have important implications for the models of transposable element (TE) number regulation as well as for the study of genome evolution. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of cellular factors that recognize UV-damaged DNA in Drosophila melanogaster.

Using a gel electrophoresis DNA band-shift assay, we have identified 2 DNA-binding protein complexes in wild-type Drosophila embryonic cells which have high affinity for UV-irradiated, double-stranded DNA. Screening of Drosophila mutants deficient in DNA repair led to the identification of 5 mutants which lacked either one of the 2 protein complexes. Four excision repair-deficient mutants (mus-201, phr, mus-308 and mus-205) lacked one protein complex (Factor 2). The other protein complex (Factor 1) was not detectable in the post-replication repair-deficient mutant mus-104. These findings might suggest the possible involvement of these gene products in lesion recognition and repair of UV-induced photoproducts in DNA.     

A SCA7 CAG/CTG repeat expansion is stable in Drosophila melanogaster despite modulation of genomic context and gene dosage

CAG and CTG repeat expansions are the cause of at least a dozen inherited neurological disorders. In these so-called "dynamic mutation" diseases, the expanded repeats display dramatic genetic instability, changing in size when transmitted through the germline and within somatic tissues. As the molecular basis of the repeat instability process remains poorly understood, modeling of repeat instability in model organisms has provided some insights into potentially involved factors, implicating especially replication and repair pathways. Studies in mice have also shown that the genomic context of the repeat sequence is required for CAG/CTG repeat instability in the case of spinocerebellar ataxia type 7 (SCA7), one of the most unstable of all CAG/CTG repeat disease loci. While most studies of repeat instability have taken a candidate gene approach, unbiased screens for factors involved in trinucleotide repeat instability have been lacking. We therefore attempted to use Drosophila melanogaster to model expanded CAG repeat instability by creating transgenic flies carrying trinucleotide repeat expansions, deriving flies with SCA7 CAG90 repeats in cDNA and genomic context. We found that SCA7 CAG90 repeats are stable in Drosophila, regardless of context. To screen for genes whose reduced function might destabilize expanded CAG repeat tracts in Drosophila, we crossed the SCA7 CAG90 repeat flies with various deficiency stocks, including lines lacking genes encoding the orthologues of flap endonuclease-1, PCNA, and MutS. In all cases, perfect repeat stability was preserved, suggesting that Drosophila may not be a suitable system for determining the molecular basis of SCA7 CAG repeat instability.     

Enteric pathogens modulate metabolic homeostasis in the Drosophila melanogaster host

On quotidian basis, living beings work out an armistice with their microbial flora and a scuffle with invading pathogens to maintain a normal state of health. Although producing virulence factors and escaping the host's immune machinery are the paramount tools used by pathogens in their “arm race” against the host; here, we provide insight into another facet of pathogenic embitterment by presenting evidence of the ability of enteric pathogens to exhibit pathogenicity through modulating metabolic homeostasis in Drosophila melanogaster. We report that Escherichia coli and Shigella sonnei orally infected flies exhibit lipid droplet deprivation from the fat body, irregular accumulation of lipid droplets in the midgut, and significant elevation of systemic glucose and triglyceride levels. Our findings indicate that these detected metabolic alterations in infected flies could be attributed to differential regulation of peptide hormones known to be crucial for lipid metabolism and insulin signaling. Gaining a proper understanding of infection-induced alterations succours in curbing the pathogenesis of enteric diseases and sets the stage for promising therapeutic approaches to quarry infection-induced metabolic disorders.     

Retrotransposon MDG4 and its role in genetic instability of a mutator strain of Drosophila melanogaster

This article summarizes the results of a ten-year study of genetic instability of a mutator strain of Drosophila melanogaster caused by transposition of the gypsy retrotransposon. The results of other authors working with an analogous system are analyzed. Possible mechanisms are suggested for the interaction of gypsy with the cell gene flamenco that participates in transposition control of this mobile element.     

A novel GC-rich dispersed repeat sequence in Drosophila melanogaster.

A novel family of dispersed repeat sequences from Drosophila melanogaster is described. Sequence analysis of two members of this family show them to contain greater than 75% GC bases. These are comprised of multiple repeats of GGX triplets interspersed occasionally with CGPy and TTPy. Southern blotting shows that these repeats are not transposable elements. Twenty four homologous recombinants have been localised by in situ hybridization to seven sites in the Drosophila genome. Polyadenylated RNAs homologous to this repeat family are expressed in a complex pattern which is developmentally regulated. We suggest that this family encodes a set of glycine-rich domains in Drosophila proteins.