Relations between Factors Controlling Crossing over and Mutability in Males of DROSOPHILA ANANASSAE

Several stocks, selected because they carried previously identified factors governing either crossing over in males or mutability, were examined to determine whether the effects of these factors are restricted to one or the other process. Neither of two dominant enhancers of male crossing over had detectable effects on Minute mutation frequencies among progenies of assayed F₁ males. Genetically equivalent F₁ males monitored for crossing over showed no unequivocal effect of either of three mutators (two dominant and one extrachromosomal) or of a suppressor of mutability. However, one combination of a dominant crossover enhancer with a dominant mutator showed synergistic increases in both crossover and Minute frequencies, and the possibility exists that a single extrachromosomally transmitted element suppresses both male crossing over and mutability. This suppressor element (or elements) had been previously diagnosed in the pc stock which, in this study, was discovered to have also a dominant enhancer of male crossing over and a dominant mutator occupying separable loci in the third chromosome. The pc enhancer of male crossing over differs from the dominant enhancer in another stock with respect to the regional distribution of crossovers, and the pc mutator is distinguished from another 3-linked mutator by its preferential induction of mutations at the Delta locus.     

Autosomal recessive lethal mutations in two mutator stocks of Drosophila ananassae.

The frequency of recessive lethals in the 2nd chromosome was examined in two mutator stocks of Drosophila ananassae, ca and ca; px. They are characterized respectively by possessing an extrachromosomal clastogenic mutator in males, and by the retrotransposon "tom", which induces Om mutability only in females. The frequencies of recessive lethal mutations in the 2nd chromosome among progenies from males and females of the ca; px stock are 0.35 and 0.34 percent, respectively. Similarity of these frequencies indicates that tom does not induce recessive lethals in females. In contrast to the ca; px stock, the frequency of recessive lethals in males of the ca mutator stock was estimated to be 1.54 percent for the 2nd chromosome. No visible mutants except Minutes were recovered. Some recessive lethals derived from ca stock males were associated with chromosomal rearrangements. Being consistent with its high rate of Minute mutation it was demonstrated that the ca clastogenic mutator also induced recessive lethals.     

Nucleocytoplasmic Relations in a Mutator-Suppressor System of DROSOPHILA ANANASSAE

A partially characterized mutator-suppressor system, previously identified in the ca; stw stock of Drosophila ananassae, was shown to exist in the ca ancestral stock; it consists of a clastogenic mutator of sperm chromosomes and a supressor that functions in the oöcyte soon after fertilization. Transmission of these components was monitored by Minute mutation frequencies produced by the progeny of recurrently backcrossed hybrid females derived from reciprocal outcrosses of the ca stock. In this way, the mutator was shown to be an extrachromosomally transmitted element whose propagation depends upon nuclear genes. Suppressivity was found to be determined by nuclear genes, some of which are expressed only after a delay of several generations. Neither the mutator nor its suppressor appear to be infectious. Measurement of dominant lethal frequencies showed that the suppressor is completely effective in repair of premutational lesions induced by the mutator. The properties of this mutator-suppressor system were compared with those of hybrid dysgenesis in Drosophila melanogaster.     

Mutators in SACCHAROMYCES CEREVISIAE: MUT1–1, MUT1–2 and MUT2–1

The purpose of this study was to characterize two mutator stocks of yeast which were induced and selected on the basis of high spontaneous reversion rates of the suppressible "ochre" nonsense allele lys1-1. In the mutator stock VA-3, a single mutation, designated mut1-1, is responsible for the increase in the reversion rate of the ochre alleles lys1-1 and arg4-17. In stock VA-105, there are two separate mutator mutations. Tetrad analysis data showed these two loci are loosely linked. Based on complementation data, one of these mutations is at the same locus as mut1-1 and designated mut1-2. The second mutator of stock VA-105 was designated mut2-1. All three mutators are recessive. Both mut1-1 and mut1-2 give a high mutation rate for ochre nonsense suppressor (SUP) loci, but not for the ochre nonsense alleles. On the contrary, the mutation rates of the ochre alleles are greatly reduced. With the mutant mut2-1 there were mutations at both the lys1-1 site and its suppressors; mut2-1 is as effective as mut1-2 but not as effective as mut1-1 in inducing reversions of a missense mutant, his1-7. Neither mut1-1, mut1-2 nor mut2-1 were effective in inducing reversions of a putative frameshift mutation, hom3-10, or in inducing forward mutations to canavanine resistance.     

Isolation and characterization of mutants with deletions in dnaQ, the gene for the editing subunit of DNA polymerase III in Salmonella typhimurium.

dnaQ (mutD) encodes the editing exonuclease subunit (epsilon) of DNA polymerase III. Previously described mutations in dnaQ include dominant and recessive mutator alleles as well as leaky temperature-sensitive alleles. We describe the properties of strains bearing null mutations (deletion-substitution alleles) of this gene. Null mutants exhibited a growth defect as well as elevated spontaneous mutation. As a consequence of the poor growth of dnaQ mutants and their high mutation rate, these strains were replaced within single colonies by derivatives carrying an extragenic suppressor mutation that compensated the growth defect but apparently not the mutator effect. Sixteen independently derived suppressors mapped in the vicinity of dnaE, the gene for the polymerization subunit (alpha) of DNA polymerase III, and one suppressor that was sequenced encoded an altered alpha polypeptide. Partially purified DNA polymerase III containing this altered alpha subunit was active in polymerization assays. In addition to their dependence on a suppressor mutation affecting alpha, dnaQ mutants strictly required DNA polymerase I for viability. We argue from these data that in the absence of epsilon, DNA replication falters unless secondary mechanisms, including genetically coded alteration in the intrinsic replication capacity of alpha and increased use of DNA polymerase I, come into play. Thus, epsilon plays a role in DNA replication distinct from its known role in controlling spontaneous mutation frequency.     

Cytotype regulation by telomeric P elements in Drosophila melanogaster: evidence for involvement of an RNA interference gene

P elements inserted at the left telomere of the X chromosome evoke the P cytotype, a maternally inherited condition that regulates the P-element family in the Drosophila germline. This regulation is completely disrupted in stocks heterozygous for mutations in aubergine, a gene whose protein product is involved in RNA interference. However, cytotype is not disrupted in stocks heterozygous for mutations in two other RNAi genes, piwi and homeless (spindle-E), or in a stock heterozygous for a mutation in the chromatin protein gene Enhancer of zeste. aubergine mutations exert their effects in the female germline, where the P cytotype is normally established and through which it is maintained. These effects are transmitted maternally to offspring of both sexes independently of the mutations themselves. Lines derived from mutant aubergine stocks reestablish the P cytotype quickly, unlike lines derived from stocks heterozygous for a mutation in Suppressor of variegation 205, the gene that encodes the telomere-capping protein HP1. Cytotype regulation by telomeric P elements may be tied to a system that uses RNAi to regulate the activities of telomeric retrotransposons in Drosophila.     

Independent Production of Co2 Sensitivity by Nuclear Gene DLY and a Transmissible Agent from Delayed-Recovery DROSOPHILA MELANOGASTER

Flies of stocks designated delayed-recovery by McCrady and Sulerud (1964) remain temporarily paralyzed after exposure to carbon dioxide. This condition is similar to CO2-sensitivity, which occurs in flies infected with the maternally transmitted sigma viruus, but is due, at least in part, to the third chromosome mutant gene Dly. Because earlier work indicated that extracts of delayed-recovery flies could occasionally transmit CO2 sensitivity when injected into resistant recipients, we have tested the possibility that some delayed-recovery stocks contain a sigma-like transmissible virus, in addition to the Dly gene. We found that TDR-orange, a stock derived from the original delayed-recovery line, and temperature-cured populations of the same stock, both contain some agent that is transmissible by injection. TDR-BC3f, a stock derived by backcrossing through the male line to eliminate maternally transmitted factors, does not appear to contain such an infectious agent, but remains sensitive to CO2. These observations lead us to the conclusion that the originally described delayed-recovery stocks harbor an infectious extrachromosomal agent, in addition to possessing the Dly gene, and each is capable of producing a sensitivity to carbon dioxide.     

Omnipotent Suppressors Effective in psi Strains of SACCHAROMYCES CEREVISIAE: Recessiveness and Dominance

We have characterized recessive and dominant omnipotent suppressor mutations obtained by conversion of the leu2-1 UAA mutation and the met8-UAG mutation in a ψ⁺ strain of Saccharomyces cerevisiae. The suppressors that act recessively upon these markers fell into two complementation groups; the sup47 and sup36 suppressors show linkage to the tyr1 locus and the aro1 locus, respectively. Of the suppressors acting dominantly upon both markers, those linked to the tyr1 locus are alleles of the SUP46 ribosomal mutation. The sup47 suppressors differ from the SUP46 suppressors not only in their suppressor activities in heterozygous diploids but also in their map positions relative to the tyr1 locus and their effects on the S11 ribosomal protein. The remaining dominant suppressors are not alleles of sup36 as judged by linkage analysis. The recessive suppressors and the dominant suppressors also differ in their effects on cell growth.     

Mutations in the gene 5 DNA polymerase of bacteriophage T7 suppress the dominant lethal phenotype of gene 2.5 ssDNA binding protein lacking the C-terminal phenylalanine

Gene 2.5 of bacteriophage T7 encodes a ssDNA binding protein (gp2.5) essential for DNA replication. The C-terminal phenylalanine of gp2.5 is critical for function and mutations in that position are dominant lethal. In order to identify gp2.5 interactions we designed a screen for suppressors of gp2.5 lacking the C-terminal phenylalanine. Screening for suppressors of dominant lethal mutations of essential genes is challenging as the phenotype prevents propagation. We select for phage encoding a dominant lethal version of gene 2.5, whose viability is recovered via second-site suppressor mutation(s). Functional gp2.5 is expressed in trans for propagation of the unviable phage and allows suppression to occur via natural selection. The isolated intragenic suppressors support the critical role of the C-terminal phenylalanine. Extragenic suppressor mutations occur in several genes encoding enzymes of DNA metabolism. We have focused on the suppressor mutations in gene 5 encoding the T7 DNA polymerase (gp5) as the gp5/gp2.5 interaction is well documented. The suppressor mutations in gene 5 are necessary and sufficient to suppress the lethal phenotype of gp2.5 lacking the C-terminal phenylalanine. The affected residues map in proximity to aromatic residues and to residues in contact with DNA in the crystal structure of T7 DNA polymerase-thioredoxin.     

Identification and Characterization of Genes That Interact with Lin-12 in Caenorhabditis Elegans

We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-17, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup-17 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup-17 and lag-2, suggest that both genes act at approximately the same time as lin-12 in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1.     

Mutations Affecting the Meiotic and Mitotic Divisions of the Early Caenorhabditis Elegans Embryo

We describe interactions between maternal-effect lethal mutations in four genes of Caenorhabditis elegans whose products appear to be involved in the meiotic and mitotic divisions of the one-cell embryo. Mitosis is disrupted by two dominant temperature-sensitive gain-of-function maternal-effect lethal mutations, mei-1(ct46) and mel-26(ct61), and by recessive loss-of-function maternal-effect lethal mutations of zyg-9. The phenotypic defects resulting from these mutations are similar. Doubly mutant combinations show a strong enhancement of the maternal-effect lethality under semipermissive conditions, suggesting that the mutant gene products interact. We isolated 15 dominant suppressors of the gain-of-function mutation mei-1(ct46). Thirteen of these suppressors are apparently intragenic, but 11 of them suppress in trans as well as cis. Two extragenic suppressors define a new gene, mei-2. The suppressor mutations in these two genes also result in recessive maternal-effect lethality, but with meiotic rather than mitotic defects. Surprisingly, most of these suppressors are also able to suppress mel-26(ct61) in addition to mei-1(ct46). The products of the four genes mei-1, mei-2, zyg-9 and mel-26 could be responsible for some of the specialized features that distinguish the meiotic from the mitotic divisions in the one-cell embryo.     

Extragenic suppressors of loss-of-function mutations in the aspergillus FlbA regulator of G-protein signaling domain protein.

We showed previously that two genes, fl bA and fadA, have a major role in determining the balance between growth, sporulation, and mycotoxin (sterigmatocystin; ST) production by the filamentous fungus Aspergillus nidulans. fadA encodes the alpha subunit for a heterotrimeric G-protein, and continuous activation of FadA blocks sporulation and ST production while stimulating growth. fl bA encodes an A. nidulans regulator of G-protein signaling (RGS) domain protein that antagonizes FadA-mediated signaling to allow development. To better understand FlbA function and other aspects of FadA-mediated growth control, we have isolated and characterized mutations in four previously undefined genes designated as sfaA, sfaC, sfaD, and sfaE (suppressors of flbA), and a new allele of fadA (fadAR205H), all of which suppress a fl bA loss-of-function mutation ( fl bA98). These suppressors overcome fl bA losses of function in both sporulation and ST biosynthesis. fadAR205H, sfaC67, sfaD82, and sfaE83 mutations are dominant to wild type whereas sfaA1 is semidominant. sfaA1 also differs from other suppressor mutations in that it cannot suppress a fl bA deletion mutation (and is therefore allele specific) whereas all the dominant suppressors can bypass complete loss of fl bA. Only sfaE83 suppressed dominant activating mutations in fadA, indicating that sfaE may have a unique role in fadA- fl bA interactions. Finally, none of these suppressor mutations bypassed fl uG loss-of-function mutations in development-specific activation.     

Inhibition of Growth by Amber Suppressors in Yeast

Strains of the yeast Saccharomyces cerevisiae that contain highly efficient amber (UAG) suppressors grow poorly on nutrient medium, while normal or nearly normal growth rates are observed when these strains lose the supressors or when the suppressors are mutated to lower efficiencies. The different growth rates account for the accumulation of mutants with lowered efficiencies in cultures of strains with highly efficient amber suppressors. Genetic analyses indicate that one of the mutations with a lowered efficiency of suppression is caused by an intragenic mutation of the amber supressor. The inhibition of growth caused by excessive suppression is expected to be exacerbated when appropriate suppressors are combined together in haploid cells if two suppressors act with a greater efficiency than a single suppressor. Such retardation of growth is observed with combinations of two UAA (ochre) suppressors (Gilmore 1967) and with combinations of two UAG suppressors when the efficiencies of each of the suppressors are within a critical range. In contrast, combinations of a UAA suppressor and a UAG suppressor do not affect growth rate. Apparently while either excessive UAA or excessive UAG suppression is deleterious to yeast, a moderate level of simultaneous UAA and UAG suppression is not.     

Genetic and molecular analysis of eight tRNA(Trp) amber suppressors in Caenorhabditis elegans

Over 100 revertants of five different amber mutants were analyzed by Southern blot hybridization using synthetic oligomers as probes to detect a single base change at the anticodon, CCA to CTA (amber), of tRNA(Trp) genes of Caenohrabditis elegans. Of the 12 members of the tRNA(Trp) gene family, a total of eight were converted to amber suppressor alleles. All eight encode identical tRNAs; three of these are new tRNA(Trp) suppressors, sup-21, sup-33 and sup-34. Previous results had suggested that individual suppressor tRNA genes were expressed differentially in a cell-type- or developmental stage-specific manner. To extend these observations to the new genes and to test the specificity of expression against additional genes, cross suppression tests of these eight amber suppressors were carried out against amber mutations in several different genes including genes likely to be expressed in the same cell-type: three nervous system-affecting genes, two muscle structure-affecting genes and two genes presumed to be expressed in hypodermis. Seven out of eight suppressors could be distinguished one from another by the spectrum of their suppression efficiencies. These results also provide further evidence of cell-type-specific patterns of expression in the nervous system, muscle and hypodermis. The suppression pattern of the suppressor against the two muscle-affecting genes, unc-15 and unc-52, suggested that either the suppressors are expressed in a developmental stage-specific manner or that the unc-52 products are expressed in cell-types other than muscle, possibly hypodermis.     

Suppression and Function of X-Linked Lethal and Sterile Mutations in CAENORHABDITIS ELEGANS

We have expanded our collection of recessive lethal and sterile mutants in the region of the X chromosome balanced by mnDp1(X;V), about 15% of the X linkage map, to a total of 54 mutants. The mutations have been mapped with respect to 20 overlapping deficiencies and five X duplications, and they have been assigned to 24 genes by complementation testing. Nine mutants are hermaphrodite-sterile: one of these is a sperm-defect mutant, two have abnormal gonadogeneses and six, in five genes, are maternally influenced mutants, producing inviable zygote progeny. One of the gonadogenesis mutants and two of the maternally influenced mutants are male fertile. All but one of the maternally influenced mutants give cross progeny when mated with wild-type males. Forty-three mutants were tested for suppression by homozygous sup-5(e1464), which is believed to be specific for null alleles. Ten mutants that were judged by independent criteria not to be null mutants are not suppressed. Nine of the other 33 mutants, in nine genes, are suppressed, five in both heterozygous and homozygous suppressor stocks and four only in homozygous suppressor stocks.     

Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene.

Amber, ochre and opal suppressor tRNA genes have been generated by using oligonucleotide directed site-specific mutagenesis to change one or two nucleotides in a human serine tRNA gene. The amber and ochre suppressor (Su+) tRNA genes are efficiently expressed in CV-1 cells when introduced as part of a SV40 recombinant. The expressed amber and ochre Su+ tRNAs are functional as suppressors as demonstrated by readthrough of the amber codon which terminates the NS1 gene of an influenza virus or the ochre codon which terminates the hexon gene of adenovirus, respectively. Interestingly, several attempts to obtain the equivalent virus stock of an SV40 recombinant containing the opal suppressor tRNA gene yielded virus lacking the opal suppressor tRNA gene. This suggests that expression of an efficient opal suppressor derived from a human serine tRNA gene is highly detrimental to either cellular or viral processes.     

Identification of an amber nonsense mutation in the rosy516 gene by germline transformation of an amber suppressor tRNA gene.

Seven xanthine dehydrogenase and cross-reacting material negative Drosophila melanogaster rosy stocks were screened for amber and ochre nonsense mutations. Amber and ochre nonsense suppressors were created by site-directed mutagenesis starting from a wild-type tRNA(Tyr) gene. The suppressor tRNA genes were subcloned into a pUChsneo transformation vector providing heat-shock controlled neomycin resistance. The seven rosy stocks were germline transformed with amber and ochre tDNA(Tyr), and the G1 generation was screened for Geneticin resistance. Surviving rosy516 flies transformed with the amber suppressor showed an eye colour intermediate between the original ry516 stock and the wild-type, suggesting that ry516 is an amber nonsense mutant. This was confirmed by sequencing the relevant part of the ry516 gene; the analysis revealed a C-to-T transition in a CAG glutamine codon at nucleotide 1522 of the wild-type rosy gene.     

Impairment of cytotype regulation of P-element activity in Drosophila melanogaster by mutations in the Su(var)205 gene

Cytotype regulation of transposable P elements in the germ line of Drosophila melanogaster is associated with maternal transmission of P elements inserted at the left telomere of the X chromosome. This regulation is impaired in long-term stocks heterozygous for mutations in Suppressor of variegation 205 [Su(var)205], a gene implicated in the control of telomere length. Regulation by TP5, a structurally incomplete P element at the X telomere, is more profoundly impaired than regulation by TP6, a different incomplete P element inserted at the same site in a TAS repeat at the X telomere. Genetic analysis with the TP5 element indicates that its regulatory ability is not impaired in flies whose fathers came directly from a stock heterozygous for a Su(var)205 mutation, even when the flies themselves carry this mutation. However, it is impaired in flies whose grandfathers came from such a stock. Furthermore, this impairment occurs even when the Su(var)205 mutation is not present in the flies themselves or in their mothers. The impaired regulatory ability of TP5 persists for at least several generations after TP5 X chromosomes extracted from a long-term mutant Su(var)205 stock are made homozygous in the absence of the Su(var)205 mutation. Impairment of TP5-mediated regulation is therefore not directly dependent on the Su(var)205 mutation. However, it is characteristic of the six mutant Su(var)205 stocks that were tested and may be related to the elongated telomeres that develop in these stocks. Impairment of regulation by TP5 is also seen in a stock derived from Gaiano, a wild-type strain that has elongated telomeres due to a dominant mutation in the Telomere elongation (Tel) gene. Regulation by TP6 is not impaired in the Gaiano genetic background. The regulatory abilities of the TP5 and TP6 elements are therefore not equally susceptible to the effects of elongated telomeres in the mutant Su(var)205 and Gaiano stocks.     

Dominant Suppressors of a Muscle Mutant Define an Essential Gene of CAENORHABDITIS ELEGANS

The sup-11 I locus of C. elegans was defined by rare dominant suppressors of unc-93(e1500) III, a mutation that affects muscle structure. All ten of these dominant suppressors have a recessive "scrawny" phenotype. Two additional classes of sup-11 alleles were identified. One class, null alleles, was obtained by reversion of the dominant suppressor activity. These null alleles are recessive embryonic lethals, indicating that sup-11 is an essential gene. Members of the second class, rare semidominant revertants of the "scrawny" phenotype, are partial suppressors of unc-93(e1500). The genetic properties of the dominant suppressor mutations suggest that they are rare missense mutations that confer a novel activity to the sup-11 protein. We consider some of the ways that sup-11 alleles might suppress unc-93(e1500), including the possibilities that the altered sup-11 proteins restore function to a protein complex or are modified products of a gene that is a member of an unc-93 gene family.