A Study of Rdna Magnification Phenomenon in a Repair-Recombination Deficient Mutant of DROSOPHILA MELANOGASTER

The rDNA magnification process consists of a rapid and inheritable rDNA increase occurring in bobbed males: in a few generations the bb loci acquire the wild-type rDNA value and reach a bb⁺ phenotype.—We have analyzed the rDNA magnification process in the repair-recombination-deficient mutant mei9^a, both at the phenotypical and rDNA content levels. In mei9^a bb double mutants the recovery of bb⁺ phenotype is strongly disturbed and the rDNA redundancy value fails to reach the wild-type level. The strong effect of this meiotic mutation on rDNA magnification suggests a close relationship between this phenomenon and the repair-recombination processes.     

The conditions required for the induction of petite yeast mutants by fluorinated pyrimidines

Summary Newly magnified bobbed loci, combined with bb⁺ or bb^o loci, are in certain cases unstable. This may lead either to a reversion to the original bobbed mutation, or to lethal bobbed mutations. We name this instability modification. Modification occurs very early during the first divisions following fecondation of eggs, in embryos heterozygous for a magnified bobbed locus and a bb⁺ or bb^o locus. The phenomenon of modification is consistent with the model proposed by Ritossa (1972) to account for the phenomenom of magnification.     

rDNA magnification in D. melanogaster: state of rDNA copies following the first step.

Summary D. melanogaster males of bb/O genetic constitution undergoing rDNA magnification were mated singly to XXbb ⁺/O females, yielding bb/O male progeny, and to X_NO-w sn bb ⁺ fameles, yielding bb/X_NO- females. The male and female offspring were scored for the bb ⁺ phenotype.Results show that there is a higher percentage of bb ⁺ flies in the bb/O male progeny than in bb/X_NO- females progeny, in single crosses as well as in the combined data. rRNA/DNA hybridization experiments agree with this observation, by showing that the rDNA content in the progeny of premagnified flies was higher in the sons than in the daughters.These data indicate that the increase of ribosomal RNA genes is not due to a stable event such as an unequal mitotic sister exchange, whereas they do not contrast with the extracopy model.     

Regulation of rRNA gene number in Drosophila melanogaster: new aspects resulting from the use of free duplications

Summary We have isolated a bobbed (bb) mutant on the free duplication Dp(1; f)122bb ⁺ and we have measured the rDNA content of the bb ⁺ and the bb loci in genetic combinations in which none of the phenomena involved in the change of the rDNA redundancy occurs. We have also measured the rDNA content of the two bb loci carried by the free duplications in two different genetic combinations: (1) and females in which there are two attached X chromosomes completely deleted for the nucleolus organizer (NO) regions and there-fore the only rDNA is contributed by the free duplication; (2) X/Dp122bb ⁺ and X/Dp122bb males, in which there are two bb loci, one on the X chromosome and the other on the X free duplication.The bb ⁺ and the bb duplications produced an overall increase of the rDNA content in the two genetic conditions tested.These results are not in favour of both a cis and trans effect of the regulator locus (cr ⁺ locus) hypothesised as being involved in the disproportionate replication of rRNA genes.     

Autonomy of the wingless mutation in Drosophila melanogaster

Summary T(Y;2) translocations were used to cytologically localise the wingless locus of Drosophila melanogaster. We found that an existing T(Y;2), which is an insertion of a segment of 2L into the Y chromosome, has wg ⁺ within this insert. This Y chromosome was used to generate an attached XY chromosome containing wg ⁺. The mutation claret-nondisjunctional (ca ^nd) was used to induce the loss of this XY chromosome and thus generate gynandromorphs with wg ¹/wg ¹ male tissue and wg ⁺/wg ¹/wg ¹ female tissue. Analysis of these gynanders demonstrated that a genotypically wingless mutant hemithorax is usually also phenotypically mutant in these half body mosaics; thus wg ¹ is discautonomous. This observation is of interest as it is known that wg is not cell autonomous.     

Different behaviour of Xbb+ and Xbb chromosomes in D. melanogaster with only one nucleolus organizer.

Summary We have examined the rDNA content of male and female adult flies having only one nucleolus organizer (NO), using X chromosomes carrying wild or partially deleted bobbed loci (Xbb ⁺/O, Xbb ⁺/X_NO-and Xbb/O, Xbb/X_NO ^-).The results show that in Xbb ⁺/O and Xbb ⁺/X_NO ^-flies, where only somatic gene compensation is supposed to occur, the rDNA increase, although less pronounced than previously reported, is directly proportional to the number of rRNA genes initially present in the nucleolus organizer. In Xbb/O and in Xbb/X_NO ^-flies the rDNA increase is relatively much higher than that observed in flies carrying bb ⁺ instead of bb. It is suggested that this may be due to rDNA premagnification and somatic gene compensation occurring simultaneously in the former flies.On leave of absence from International Institute of Genetics and Biophysics, Naples, Italy     

Male-Sterilizing Interactions between Duplications and Deficiencies for Proximal X-Chromosome Material in DROSOPHILA MELANOGASTER

The genetic limits of sixty-four deficiencies in the vicinity of the euchromatic-heterochromatic junction of the X chromosome were mapped with respect to a number of proximal recessive lethal mutations. They were also tested for male fertility in combination with three Y chromosomes carrying different amounts of proximal X-chromosome-derived material (B^SYy⁺, y⁺Ymal¹²⁶ and y⁺Ymal⁺). All deficiencies that did not include the locus of bb and a few that did were male-fertile in all male-viable Df(1)/Dp(1;Y) combinations. Nineteen bb deficiencies fell into six different classes by virtue of their male-fertility phenotypes when combined with the duplicated Y chromosomes. The six categories of deficiencies are consistent with a formalism that invokes three factors or regions at the base of the X, one distal and two proximal to bb, which bind a substance critical for precocious inactivation of the X chromosome in the primary spermatocyte. Free duplications carrying these regions or factors compete for the substance in such a way that, in the presence of such duplications, proximally deficient X chromosomes are unable to command sufficient substance for proper control of X-chromosome gene activity preparatory to spermatogenesis. We conclude that there is no single factor at the base of the X that is required for the fertility of males whose genotype is otherwise normal.     

Study of several factors producing a variation of the reversion frequency at the bobbed locus in Drosophila melanogaster (author''s transl)

Summary The proportion of magnified individuals in the progeny of bobbed males is dependant upon different factors: the bobbed allele used (Fig. 3), the age of the male (Table 2, 3) and the individual male tested (Table 1). From this, it can be thought that the first event leading to magnification happens before the separation of the germ line in the egg.A number of individuals carrying bobbed alleles more extreme than the parental one were observed (Table 4); these alleles were lethal in combination with Ybb^-. It cannot yet be acertained whether they are produced during the first step of magnification process itself or whether they originate later on.     

Preferential mutagenesis of lacZ integrated at unique sites in the Escherichia coli chromosome

To study the variation in spontaneous mutation frequencies in different chromosomal domains, a mini-Mu-kan-lacZ ⁻transposable element was constructed to insert the lacZ ⁻(Trp570 → Opal) allele into many different loci in the Escherichia coli chromosome. Papillation on MacConkey lactose plates was used to screen for mini-Mu insertion mutants with elevated levels of spontaneous mutagenesis of lacZop → LacZ⁺ candidates were then screened for normal mutation frequencies in other genes. Two different insertion mutants with this enhanced mutagenesis phenotype were isolated from 14 000 colonies, and named plm-1 (preferential lacZmutagenesis) and plm-2. The frequency of LacZ⁻→ LacZ⁺ mutations in these plm mutants was over 400-fold higher than that in isogenic strains containing mini-Mu-kan-lacZop insertions at other loci. Six Lac⁺ reversion (or suppression) mutations obtained from each of the two plm mutants were mapped by P1 transduction and all were found to be linked to the Kan^r gene in the mini-Mu-kan-lacZop, suggesting that a localized mutagenic event is responsible for the preferential mutagenesis. Furthermore, both the LacZ⁺→ LacZ⁻and Kan^r→ Kan^s mutant frequencies of these Lac⁺ revertants were in the range of 10⁻³ to 10⁻², indicating that this putative localized mutagenesis is neither allele nor gene specific. To identify the plm loci, the chromosomal regions flanking the mini-Mu insertion sites were cloned and sequenced. A computer-assisted database search of homologous sequences revealed that the plm-1 locus is identical to the mutS gene; the mini-Mu insertion most probably results in the production of a truncated MutS protein. We suggest that the enhanced lacZ mutation frequency in plm-1 may be associated with an active process involving the putative truncated MutS protein. The DNA sequence of the plm-2 locus matched a putative malate oxidoreductase gene located at 55.5 min of the E. coli chromosome. Received: 1 August 1996 / Accepted: 3 April 1997     

The relationship between first and second chromosome segregation ratios from Drosophila melanogaster males bearing Segregation Distorter

Summary Drosophila melanogaster males heterozygous for the second chromosome locus Segregation Distorter preferentially transmit this chromosome to their progeny due to a dysfunctioning of SD ⁺-bearing sperm. SD males with a normal sex chromosome constitution produce more females than males among SD ⁺ progeny. This report shows that this unequal recovery of sexes is enhanced from XY/Y; SD/SD ⁺ males and enhanced still further from XY/O; SD/SD ⁺ males. It is argued that the probability that a SD ⁺-bearing sperm will dysfunction is related to its sex chromsome complement, with the relative probabilities of dysfunction ranked O> Y> X> XY. It is shown that a modified probit analysis accounts for the relationship between sex ratio and second chromosome segregation frequency for all paternal genotypes. Finally, SD/SD ⁺ males show no increase in sex chromosome nondisjunction with respect to a control.R. E. Denell was supported by U.S.P.H.S. Training Grant No. GM00337 and by a U.S.P.H.S. Postdoctoral Fellowship; George L. Gabor Miklos was supported by A.E.C. Contract No. AT (04-3)-34 PA150.     

Interaction of alleles of therelA, relC andspoT genes inEscherichia coli: Analysis of the interconversion of GTP, ppGpp and pppGpp

Summary Mutants in thespoT gene have been isolated as stringent second site revertants of therelC mutation. These show varying degrees of the characteristics associated with thespoT1 gene,viz relative amount and absolute levels of both pppGpp and ppGpp and the decay rate of the latter. The entry of³H-guanosine into GTP and ppGpp pools inspoT ⁺ andspoT1 cells either growing exponentially or during amino acid starvation was determined, and the rate of ppGpp synthesis and its decay constant calculated. During exponential growth the ppGpp pool is 2-fold higher, its decay constant 10-fold lower, and its synthesis rate 5-fold lower inspoT ^- than inspoT ⁺ cells; during amino acid starvation the ppGpp pool is 2-fold higher, its decay constant 20-fold lower, and its synthesis rate 10-fold lower inspoT than inspoT ⁺ cells. In one of the “intermediate”spoT mutants the rate of entry of³H-guanosine into GTP, ppGpp and pppGpp was measured during amino acid starvation. The data form the basis of a model for the interconversion of the guanosine nucleotides in which the flow is:GDP→GTP→pppGpp→ppGpp→Y. Calculations of the rates of synthesis and conversion of pppGpp and ppGpp under various conditions in variousspoT ⁺ andspoT ^- strains indicate that the ppGpp concentration indirectly controls the rate of pppGpp synthesis. ThespoT1 allele was introduced into various relaxed mutants. It was shown that many phenomena associated with the relaxed response ofrelC and “intermediate”relA mutants were phenotypically suppressed when thespoT1 allele was introduced into these mutants. These double mutants exhibit ppGpp accumulation, rate of RNA accumulation, rate of β-galactosidase synthesis, and heat lability of β-galactosidase synthesized during amino acid starvation similar to the stringent wild-type. It is concluded that the relaxed response is due directly to the lack of ppGpp and that the stringest response is due directly to ppGpp.     

Genetic factors affecting allelic recombination at the histidine-3 locus ofNeurospora crassa

Summary In addition to the generec-4, other genetic factors affect the frequency of allelic recombination in thehis-3 locus. One dominant factor, designated asrec-6 ⁺, in association withrec-4 ⁺ causes greater reduction in prototrophic frequency than obtained withrec-4 ⁺ alone. The action ofrec 6 ⁺ in crosses recessive homozygous forrec-4 is not established at the present. The effect ofrec-6 ⁺ is recognised only with onehis-3 allele but not with another. Interaction ofrec-4 ⁺ orrec-4 with other genetic factors can give approximately ten fold variation in the prototrophic frequencies obtained with a pair of alleles. It is suggested that the control of the rate of mutations during meiosis might be one of the roles of the recombination genes.     

Role of surface exclusion genes in lethal zygosis in Escherichia coli K12 mating

Summary We find that diaminopimelic acid in the recipient membrane is released into the medium during bacterial matings, indicating that membrane damage was inflicted on the recipient by the donor, probably for forming a channel for DNA transfer. When the damage is extensive, as in matings with an excess of Hfr bacteria, the F^- bacteria are killed (lethal zygosis). The transfer of a large amount of DNA in Hfr matings appears to enhance the killing. In analogous F⁺xF^- (Nal^r) matings, on the other hand, killing of F^- bacteria does not occur unless F plasmid transfer is inhibited by a substance like nalidixic acid. The F^- bacteria are killed, suggesting that F plasmids contain genes that express immunity to lethal zygosis in the recipient. For example, bacteria containing surface exclusion-deficient mutants of F plasmids, such as traS ^- and traT ^-, induce lethal zygosis in F^- bacteria and are susceptible to it. Various tra ^- polar mutants that abolish surface exclusion are also susceptible to lethal zygosis when mated with Hfr bacteria. Kinetic experiments indicate that in F⁺ (wild type) x F^- matings, immunity to lethal zygosis is expressed in the F^- recipient within 1/4 division time, whereas a complete expression of surface exclusion requires more than 1 division time. Thus, a complete change in all receptor sites seems to be required for the expression of surface exclusion.     

Juvenile hormone involvement in Drosophila melanogaster male reproduction as suggested by the Methoprene-tolerant mutant phenotype

Juvenile hormone (JH) involvement in male reproduction is poorly understood. In Drosophila melanogaster adults, JH deficiency has been shown to result in lowered protein synthesis in male accessory glands. To probe additional roles, we have examined males homozygous for a null allele of Methoprene-tolerant (Met). This gene is involved in the action of JH, possibly at the JH receptor level, and Met²⁷ null mutants reflect a diminution of JH action. Met²⁷ males were found to have reduced protein accumulation in male accessory glands and to court and mate wild-type females much less avidly than do either Met⁺ or Met²⁷; Met⁺ transgenic males. Exposure of Met²⁷ males to methoprene partially rescued the courtship deficiency. However, sperm transfer as reflected by fertility of Met²⁷ fathers was found to be similar to that of Met⁺. Taken together with previous work examining the JH-deficient mutant apterous, these results corroborate JH involvement in protein synthesis in the male accessory glands and suggest a role for JH in promoting male mating behavior in these flies.     

Mutual regulation of magnified bobbed loci in Drosophila melanogaster

Summary rDNA magnification in D. melanogaster increases the redundancy of that locus to a value higher than the wild type. A magnified locus (bb ^m) can lose the express copies according to rDNA content of partner sexual chromosome. This paper is a study of behaviour of two bb ^m loci put together to show their interaction.     

Effect of tra mutations on F factor-specified immunity to lethal zygosis

Summary Hfr, F⁺, and F-prime cells are, unlike F^– cells, insensitive to an excess of Hfr donor cells, indicating that there is an F factor mediated immunity to lethal zygosis (Ilz). Results with Flac episomes carrying traJ, traS or various polar mutations in the tra region indicate that this immunity is independent of surface exclusion, of traJ control, and of all known genes within the tra operon. However, analysis of a series of strains with deletions in the F factor, extending from the right into the tra region, suggests that a gene for immunity to lethal zygosis is located within the tra region. We therefore conclude that Ilz is genetically complex, and present a hypothesis to account for these results.     

A wat1 mutant of fission yeast is defective in cell morphology

The organization of the actin cytoskeleton plays an integral role in cell morphogenesis of all eukaryotes. We have isolated a temperature-sensitive mutant in Schizosaccharomyces pombe, wat1-1, in which acting patches are delocalized, resulting in an elliptically shaped cell phenotype. Molecular cloning and DNA sequencing of wat1 ⁺ showed that the gene encodes a 314 residue protein containing WD-40 repeats. Cells lacking wat1 ⁺ are slow growing but viable at 25° C and temperature-sensitive for growth above 33° C. At restrictive temperature, wat1-d strains are phenotypically indistinguishable from wat1-1. When combined with a deletion for the wat1 ⁺ gene, cdc mutants failed to elongate at restrictive temperature and exhibited alterations in actin patch localization. This analysis suggests that wat1 ⁺ is required directly or indirectly for polarized cell growth in S. pombe. Wat1p and a functional, epitope-tagged, version of Wat1p can be overproduced without inducing alterations in cell morphology. Received: 18 September 1996 / Accepted: 22 October 1996     

Genetic instability in Drosophila melanogaster

Summary An unstable long tandem duplication which includes the white locus twice, marked with w ^sp in the left and w ^17G in the right locus, when kept in males has been found to produce red-eyed sons which have lost the long duplication and with it the w ^sp and w ^17G mutants. Such exceptions were produced also when w ^17G had been exchanged for w ^a.Stocks originating from these exceptions are unstable, producing: 1) zeste males, also unstable, 2) w ^- deletions, stable, 3) transpositions of the white locus to sites in other chromosomes.The instability is interpreted as the effect of an IS element, within or adjacent to the white locus, which is supposed to retain a duplication of the proximal zeste interacting part of this locus. According to the orientation of the IS element the duplicated part can be active or inactive, giving a zeste or red eye phenotype.The frequency of exceptional offspring after X-ray treatment of the red and zeste unstable stocks have been compared to stable stocks with corresponding genotypes.     

Complementation studies with the repair-deficient uvrD3, uvrE156, and recL152 mutations in Escherichia coli

Summary Previous work showed that the mutations uvrD3, uvrE156, and recL152 were closely linked and increased UV-sensitivity. They were phenotypically distinguishable in that only the uvrD3 mutation significantly decreases host cell reactivation of UV-irradiated phage (Hcr^-) and repair of methylmethane sulfonate (MMS)-induced damage, and only the uvrE156 mutation increased mutation rates (Mut^-). MMS-resistant revertants of a uvrD3 mutant were still UV-sensitive and fell into two phenotypic classes, Hcr^- Mut⁺ (non-mutator) and Hcr⁺ Mut^-. In this work complementation tests were done by examining UV-and MMS-sensitivity and host cell reactivation in heterogenotes containing combinations of uvrD3, uvrE156, recL152, and the MMS-resistant mutations derived from uvrD3. The mutations could not complement each other in the repair of UV-damage, the one trait all had in common, indicating that they were in one gene. For the most part, the different mutations were able to complement each other in respect to traits in which one was deficient and the other had wild type activity.     

Cloning of caf1 +, caf2 + and caf4? + from Schizosaccharomyces pombe : their involvement in multidrug resistance, UV and pH sensitivity

We previously identified four nuclear genes (caf1 ⁺ to caf4 ⁺) in Schizosaccharomyces pombe, mutations in which can confer caffeine resistance. Here we report the cloning and sequencing of caf1 ⁺, caf2 ⁺ and caf4 ⁺. All three genes are allelic to genes (hba1 ⁺ , crm1 ⁺ and trr1 ⁺ , respectively) involved in multidrug resistance mechanisms or in stress response systems. In agreement with this the caffeine-resistant mutants caf1(hba1)-21, caf2(crm1)-3 and caf4(trr1)-83 are also resistant to brefeldin. Disruption of caf1(hba1) ⁺ and caf4(trr1) ⁺ makes cells sensitive to high pH. The overlapping ranges of pleiotropic effects and the genetic interaction detected between caf1(hba1) ⁺ and caf2(crm1) ⁺ suggest that the three genes function in interlinked systems. Received: 9 March 1998 / Accepted: 16 September 1998