Functional interactions between the gene tetanic and the Shaker gene complex of Drosophila

Different phenotypes associated with the tetanic (tta) mutation such as appendage contraction, maternal effect and low viability and fertility are enhanced by one extra dose of the Shaker gene complex (ShC). The tta mutation is lethal with two extra doses of ShC. In addition, tta embryos have a defective nervous system. In this paper, I analyse the interaction between tta and ShC to gain insight into their relationship. Aneuploid analysis suggests that the lethality is due to an interaction of the tta mutation with the maternal effect (ME) region of this gene complex. Mutations in the ME region of ShC partially suppress this interaction. Trans-heterozygous combinations of MEI[l(1)305] and MEIII [l(1)459] mutations causes dominant lethality in a tta background. Trans-heterozygous combinations of an MEII [l(1)1359] mutation with the cited MEI and MEIII mutations are lethal in a tta background. Double mutant combinations and gene dosage experiments, suggest that tta also interacts with the viable (V) region of ShC. These specific genetic interactions indicate that tta and the ME and V regions of ShC are functionally related. These results, together with the previous electrophysiological, molecular and biochemical studies on these mutants suggest an interaction at the protein level. Thus, in the case of the V region, the tta gene product may modulate the activity of the K⁺ channels encoded in this region. Furthermore, the extreme dosage sensitivity of the interaction between tta and ShC suggests a stoichiometric requirement for the different gene products involved, which might be physically associated and form heteromultimers.     

Functional relationships between genes of the Shaker gene complex of Drosophila

Different mutations belonging to the HLI and HLII complementation groups of the haplolethal (HL) region of the Shaker complex (ShC) are described. The HLI complementation group includes viable (hdp), recessive lethals [l(1)1614], semidominant lethals [l(1)8384] and dominant lethals [l(1)5051,l(1)9916, l(1)13193], lack-of-function alleles that affect nervous system, cuticle and muscle development. The HLI complementation group encodes troponin I. HLII lack-of-function mutations [l(1)174 and l(l)4058] affect nervous system development. The semidominant lethal HLI mutation 1(1)8384 shows differential complementation with other mutations in the ME and HL regions of ShC. Thus, heterozygous combinations of l(1)8384 with ME mutations l(1)162 and l(1)387 are poorly viable. The same phenomenon is observed for heterozygotes of l(1)8384 with HL mutations l(1)1199, l(1)2288 and l(1)3014. These specific interactions indicate the existence of functional relationships among the genetic elements of ShC. The implications for the understanding of the functional organization of ShC are discussed.     

Functional relationships between genes of the Shaker gene complex of Drosophila

Different mutations belonging to the HLI and HLII complementation groups of the haplolethal (HL) region of the Shaker complex (ShC) are described. The HLI complementation group includes viable (hdp), recessive lethals [l(1)1614], semidominant lethals [l(1)8384] and dominant lethals [l(1)5051,l(1)9916, l(1)13193], lack-of-function alleles that affect nervous system, cuticle and muscle development. The HLI complementation group encodes troponin I. HLII lack-of-function mutations [l(1)174 and l(l)4058] affect nervous system development. The semidominant lethal HLI mutation 1(1)8384 shows differential complementation with other mutations in the ME and HL regions of ShC. Thus, heterozygous combinations of l(1)8384 with ME mutations l(1)162 and l(1)387 are poorly viable. The same phenomenon is observed for heterozygotes of l(1)8384 with HL mutations l(1)1199, l(1)2288 and l(1)3014. These specific interactions indicate the existence of functional relationships among the genetic elements of ShC. The implications for the understanding of the functional organization of ShC are discussed.     

Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus microplus associated with resistance to flumethrin but not to cypermethrin

A mutation in the domain II S4–5 linker region of the para-sodium channel gene has been associated previously with synthetic pyrethroid (SP) resistance in the cattle tick (Rhipicephalus microplus) in Australia. This is a C → A mutation at nucleotide position 190, which results in a leucine to isoleucine amino acid substitution (L64I). In a survey of 15 cattle tick populations with known SP resistance status, sourced from Queensland and New South Wales in Australia, there was a strong relationship (r = 0.98) between the proportion of ticks carrying the L64I homozygous resistant genotype and the survival percentage after exposure to a discriminating concentration of cypermethrin in the bioassay, as expected. However, among populations resistant only to flumethrin, the L64I homozygous genotype was not found. The sequence obtained for a 167 bp region including domain II S4–5 linker in flumethrin-resistant ticks identified a G → T non-synonymous mutation at nucleotide position 214 that results in a glycine to valine substitution (G72V). The frequency of the G72V homozygous genotype in each population was found to be moderately related to the survival percentage at the discriminating concentration of flumethrin in the larval packet test (r = 0.74). However, a much stronger relationship between genotype and resistance to flumethrin was observed when the heterozygotes of L64I and G72V were added to the G72V homozygotes (r = 0.93). These results suggest that there is an interaction between the two mutations in the same gene, such that flumethrin resistance might be conferred by either two copies of the G72V mutation or by being a L64I and G72V heterozygote.     

Synergistic interaction between particular X-chromosome deletions andSex-lethal causes female lethality inDrosophila melanogaster

We studied the effect on female viability oftrans-heterozygous combinations of X-chromosome deficiencies andSxl ^f1, a null allele ofSex-lethal. Twentyfive deficiencies, which together covered 80% of the X chromosome, were tested. Seven of thesetrans-hcterozygous combinations caused significant levels of female lethality. Two of the seven interacting deficiencies include the previously known sex determination genessans fills andsisterless-a. Four of the remaining uncover X-chromosomal regions that were not hitherto known to contain sex determination genes. These newly identified regions are defined by deficienciesDf(1)RA2 (7D10; 8A4-5),DJ(1)KA14 (7F1-2; 8C6),Df(1)C52 (8E; 9C-D) andDf(1)NI9 (17A1; 18A2). These four deficiencies were characterized further to determine whether it was the maternal or zygotic dosage that was primarily responsible for the observed lethality of female embryos,daughterless andextra macrochaetae, two known regulators ofSxl, influence the interaction of these deficiencies withSxl.     

Maternal and Zygotic Interactions between the Abnormal Oocyte Mutation and the Scute Inversion in DROSOPHILA MELANOGASTER

The authors have studied the interaction between the abnormal oocyte mutation and an inversion of the X chromosome, In( 1)sc⁴, which has a proximal breakpoint in or near the heterochromatic region (ABO) that maternally interacts with the abo product. It has been demonstrated that the presence of X chromosomes carrying this inversion, besides a marked increase in the severity of the maternal effect of the abo mutation, produces a zygotic effect resulting in the lethality of the progeny of stocks homozygous for abo and sc⁴. These results indicate that the sc⁴ inversion carries an abnormal region indispensable for the development of abo zygotes from sc⁴;abo mothers.     

MouseV k gene classification by nucleic acid sequence similarity

Analyses of immunoglobulin (Ig) variable (V) region gene usage in the immune response, estimates ofV gene germline complexity, and other nucleic acid hybridization-based studies depend on the extent to which such genes are related (i. e., sequence similarity) and their organization in gene families. While mouseIgh heavy chainV region (V _H ) gene families are relatively well-established, a corresponding systematic classification ofIgk light chainV region (V _k ) genes has not been reported. The present analysis, in the course of which we reviewed the known extent of theV _k germline gene repertoire andV _k gene usage in a variety of responses to foreign and self antigens, provides a classification of mouseV _k genes in gene families composed of members with >80% overall nucleic acid sequence similarity. This classification differed in several aspects from that ofV _H genes: only someV _k gene families were as clearly separated (by >25% sequence dissimilarity) as typicalV _H gene families; mostV _k gene families were closely related and, in several instances, members from different families were very similar (>80%) over large sequence portions; frequently, classification by nucleic acid sequence similarity diverged from existing classifications based on amino-terminal protein sequence similarity. Our data have implications forV _k gene analyses by nucleic acid hybridization and describe potentially important differences in sequence organization betweenV _H andV _k genes.     

patufet, the gene encoding the Drosophila melanogaster homologue of selenophosphate synthetase, is involved in imaginal disc morphogenesis

Proliferation in imaginal discs requires cell growth and is linked to patterning processes controlled by secreted cell-signalling molecules. To identify new genes involved in the control of cell proliferation we have screened a collection of P-lacW insertion mutants that result in lethality in the larval/pupal stages, and characterized a novel gene, patufet (ptuf). Inactivation of ptuf by a P element insertion in the 5′ untranslated region leads to aberrant imaginal disc morphology characterized by a reduction in mass of discs and disorganisation of disc cells where no folding or patterning can be detected. Moreover, apoptotic cells can be observed in these small and abnormal mutant discs. To examine the role of ptuf we have studied its clonal behaviour in genetic mosaics generated by mitotic recombination. The mutation causes reduced cell viability, smaller cell size and stops vein differentiation. Non-autonomous effects, such as abnormal differentiation of wild-type cells surrounding the clones, are also observed. We have cloned the ptuf gene of Drosophila melanogaster and found that it encodes a selenophosphate synthetase, which is the first identified in insects. Mutant flies transformed with the full-length cDNA show complete reversion of lethality and disc phenotype. Northern blot analysis and in situ hybridization indicate that the ptuf gene is expressed in imaginal discs as well as at different stages of development. The synthesis of selenoproteins by the selenophosphate synthetase, the role of selenoproteins in the maintenance of the oxidant/antioxidant balance of the cell and its possible implications in imaginal disc morphogenesis are discussed.     

Rapid detection of CAA/CAG repeat polymorphism in the AIB1 gene using DHPLC

Denaturing high-performance liquid chromatography (DHPLC) has been used for rapid and accurate DNA mutation analysis; to extend the DNA fragment lengths analysis. Recently, polymorphism in polyglutamine-coding region of Amplified In Breast cancer gene 1 (AIB1) was analyzed as an independent genetic risk factor influencing breast cancer onset in carriers of mutation in breast cancer predisposing gene 1 (BRCA1). We have implemented efficient, cost-effective and rapid method for analysis of the AIB1 polyglutamine repeat polymorphism based on DHPLC analysis (WAVE system) of unlabeled PCR products. This strategy can be useful for genotyping of other trinucleotide repeat polymorphisms using DHPLC in medium/high throughput settings.     

Comparative Genomics of Community-Acquired ST59 Methicillin-Resistant Staphylococcus aureus in Taiwan: Novel Mobile Resistance Structures with IS1216V

Methicillin-resistant Staphylococcus aureus (MRSA) with ST59/SCCmecV and Panton-Valentine leukocidin gene is a major community-acquired MRSA (CA-MRSA) lineage in Taiwan and has been multidrug-resistant since its initial isolation. In this study, we studied the acquisition mechanism of multidrug resistance in an ST59 CA-MRSA strain (PM1) by comparative genomics. PM1’s non-β-lactam resistance was encoded by two unique genetic traits. One was a 21,832-bp composite mobile element structure (MES_PM1), which was flanked by direct repeats of enterococcal IS1216V and was inserted into the chromosomal sasK gene; the target sequence (att) was 8 bp long and was duplicated at both ends of MES_PM1. MES_PM1 consisted of two regions: the 5′-end side 12.4-kb region carrying Tn551 (with ermB) and Tn5405-like (with aph[3′]-IIIa and aadE), similar to an Enterococcus faecalis plasmid, and the 3′-end side 6,587-bp region (MES_cat) that carries cat and is flanked by inverted repeats of IS1216V. MES_cat possessed att duplication at both ends and additional two copies of IS1216V inside. MES_PM1 represents the first enterococcal IS1216V-mediated composite transposon emerged in MRSA. IS1216V-mediated deletion likely occurred in IS1216V-rich MES_PM1, resulting in distinct resistance patterns in PM1-derivative strains. Another structure was a 6,025-bp tet-carrying element (MES_tet) on a 25,961-bp novel mosaic penicillinase plasmid (pPM1); MES_tet was flanked by direct repeats of IS431, but with no target sequence repeats. Moreover, the PM1 genome was deficient in a copy of the restriction and modification genes (hsdM and hsdS), which might have contributed to the acquisition of enterococcal multidrug resistance.     

An additional copy of the adenylate cyclase-encoding gene relieves developmental defects produced by a mutation in a vegetative incompatibility-controlling gene in Podospora anserina

To identify cellular functions involved in vegetative incompatibility in filamentous fungi, we have initiated the cloning of Podospora anserina (Pa) mod genes. These genes interfere with the lethal reaction triggered by interaction between incompatible het genes. A gene (Pa AC) has been cloned by complementation of developmental defects caused by a mutation in the mod-D gene. This gene encodes a protein of 2145 amino acids (aa) that exhibits strong similarities with many adenylate cyclases (AC). About 65% aa identity has been found between the sequence of the polypeptide encoded by this Pa AC gene and the AC of Neurospora crassa. The organization of peptidic domains in the polypeptide encoded by Pa AC is closely related to that of Saccharomyces cerevisiae CYRI. Restriction-fragment-length polymorphism (RFLP) and genetic analysis have shown that Pa AC and mod-D are distinct genes     

Evidence that toxin resistance in poison birds and frogs is not rooted in sodium channel mutations and may rely on “toxin sponge” proteins

Many poisonous organisms carry small-molecule toxins that alter voltage-gated sodium channel (Na_V) function. Among these, batrachotoxin (BTX) from Pitohui poison birds and Phyllobates poison frogs stands out because of its lethality and unusual effects on Na_V function. How these toxin-bearing organisms avoid autointoxication remains poorly understood. In poison frogs, a Na_V DIVS6 pore-forming helix N-to-T mutation has been proposed as the BTX resistance mechanism. Here, we show that this variant is absent from Pitohui and poison frog Na_Vs, incurs a strong cost compromising channel function, and fails to produce BTX-resistant channels in poison frog Na_Vs. We also show that captivity-raised poison frogs are resistant to two Na_V-directed toxins, BTX and saxitoxin (STX), even though they bear Na_Vs sensitive to both. Moreover, we demonstrate that the amphibian STX “toxin sponge” protein saxiphilin is able to protect and rescue Na_Vs from block by STX. Taken together, our data contradict the hypothesis that BTX autoresistance is rooted in the DIVS6 N→T mutation, challenge the idea that ion channel mutations are a primary driver of toxin resistance, and suggest the possibility that toxin sequestration mechanisms may be key for protecting poisonous species from the action of small-molecule toxins.     

A Chromosomal Translocation Causing Overproduction of Iso-2-Cytochrome c in Yeast

The CYC7–1 mutation in the yeast Saccharomyces cerevisiae causes the production of approximately 30 times the normal amount of iso-2-cytochrome c. Genetic analysis established that the CYC7–1 mutation is a reciprocal translocation involving the left arm of chromosome V and the right arm of chromosome XVI. The chromosome V arm was broken adjacent to the gene CYC7, which determines the primary structure of iso-2-cytochrome c, and this fragment containing the CYC7 gene was joined to the segment of chromosome XVI. It appears as though the elevation of iso-2-cytochrome c is caused by an abnormal controlling region adjacent to the structural region of the CYC7 gene.     

Genetic Analysis of the Achaete-Scute System of DROSOPHILA MELANOGASTER

Several mutations in the achaete-scute region of Drosophila have been analyzed phenotypically and cytologically. One group of them corresponds to point mutations, another to rearrangements with one breakpoint in this region. Trans heterozygotes of the different point mutations or of the different rearrangements show poor complementation or fail to complement; therefore, they could be interpreted as mutations affecting the same gene product. However, left-right inversion recombinants and duplication-deficiency combinations between rearrangements with different cytological breakpoints uncover a complex organization of the achaete-scute region. This region seems to contain several independent achaete and scute functions, as well as a lethal function, arranged as a tandem reverse repeat at both sides of a lethal locus. Since all of the mutants show the same phenotype qualitatively, though different quantitatively, we suggest that these functions are of a reiterative nature. The achaete-scute wild-type condition may well be dependent on a multimeric gene product made of several evolutionary related monomers.