Cytoplasmic Dynein Function Is Essential in Drosophila Melanogaster

The microtubule motor cytoplasmic dynein has been implicated in a variety of intracellular transport processes. We previously identified and characterized the Drosophila gene Dhc64C, which encodes a cytoplasmic dynein heavy chain. To investigate the function of the cytoplasmic dynein motor, we initiated a mutational analysis of the Dhc64C dynein gene. A small deletion that removes the chromosomal region containing the heavy chain gene was used to isolate EMS-induced lethal mutations that define at least eight essential genes in the region. Germline transformation with a Dhc64C transgene rescued 16 mutant alleles in the single complementation group that identifies the dynein heavy chain gene. All 16 alleles were hemizygous lethal, which demonstrates that the cytoplasmic dynein heavy chain gene Dhc64C is essential for Drosophila development. Furthermore, our failure to recover somatic clones of cells homozygous for a Dhc64C mutation indicates that cytoplasmic dynein function is required for cell viability in several Drosophila tissues. The intragenic complementation of dynein alleles reveals multiple mutant phenotypes including male and/or female sterility, bristle defects, and defects in eye development.     

Distinct cell-autonomous functions of RETINOBLASTOMA-RELATED in Arabidopsis stem cells revealed by the Brother of Brainbow clonal analysis system

Mutations that cause lethality in the gametophyte phase pose a major challenge for studying postfertilization gene function. When both male and female haploid cells require a functional gene copy, null alleles cause developmental arrest before the formation of the zygote, precluding further investigation. The Arabidopsis thaliana Rb homolog RETINOBLASTOMA-RELATED (RBR) has an important function in the stem cell niche, but its requirement in both male and female gametophytes has prevented full loss-of-function studies. To circumvent this obstacle, we designed a clonal deletion system named BOB (Brother of Brainbow) in which null mutant sectors marked by double fluorescence are generated in a fully complemented wild-type background. In this system, both copies of a complementing RBR transgene are eliminated by tissue-specific and inducible CRE expression, and homozygous mutant clones can be distinguished visually. Since mutant sectors can be produced in a homozygous, rather than a heterozygous, background, this system facilitates clonal deletion analysis not only for gametophytic lethal alleles but also for any type of mutation. Using the BOB system, we show that RBR has unique cell-autonomous functions in different cell types within the root stem cell niche.     

Male-Specific Lethal Mutations of DROSOPHILA MELANOGASTER

A total of 7,416 ethyl methanesulfonate (EMS)-treated second chromosomes and 6,212 EMS-treated third chromosomes were screened for sex-specific lethals. Four new recessive male-specific lethal mutations were recovered. When in homozygous condition, each of these mutations kills males during the late larval or early pupal stages, but has no detectable effect in females. One mutant, mle^ts, is a temperature sensitive allele of maleless, mle (Fukunaga, Tanaka and Oishi 1975), while the other three mutants identify two new loci: male-specific lethal-1 (msl-1) (two alleles) at map position 2-53.3 and male-specific lethal-2 (msl-2) at 2-9.0.——The male-specific lethality associated with these mutants is not related to the sex per se of the mutant flies, since sex-transforming genes fail to interact with these mutations. Moreover, the presence or absence of a Y chromosome in males or females has no influence on the male-specific lethal action of these mutations. Finally, no single region of the X chromosome, when present as a duplication, is sufficient to rescue males from the lethal effects of msl-1 or msl-2. These results suggest that the number of complete X chromosomes determines whether a fly homozygous for a male-specific lethal mutation lives or dies.     

The Drosophila splicing regulator sex-lethal directly inhibits translation of male-specific-lethal 2 mRNA.

Male-specific expression of the protein male-specific-lethal 2 (MSL-2) controls dosage compensation in Drosophila. msl-2 gene expression is inhibited in females by Sex-lethal (SXL), an RNA binding protein known to regulate pre-mRNA splicing. An intron present at the 5' untranslated region (UTR) of msl-2 mRNA contains putative SXL binding sites and is retained in female flies. Here we show that SXL plays a dual role in the inhibition of msl-2 expression. Cotransfection of Drosophila Schneider cells with an SXL expression vector and a reporter containing the 5' UTR of msl-2 mRNA resulted in retention of the 5' UTR intron and efficient accumulation of the unspliced mRNA in the cytoplasm, where its translation was blocked by SXL, but not by the intron per se. Both splicing and translation inhibition by SXL were recapitulated in vitro and found to be dependent upon SXL binding to high-affinity sites within the intron, showing that SXL directly regulates these events. Our data reveal a coordinated mechanism for the regulation of msl-2 expression by the same regulatory factor: SXL enforces intron retention in the nucleus and subsequent translation inhibition in the cytoplasm.     

The Mutation Masculinizer (Man) Defines a Sex-Determining Gene with Maternal and Zygotic Functions in Musca Domestica L

In Musca domestica, the primary signal for sex determination is the dominant factor M, which is assumed to regulate a postulated female-determining gene F. Presence of M prevents expression of F so that male development ensues. In the absence of M, F can become active, which dictates the female pathway. The existence of F is inferred from F(D), a dominant factor that is epistatic to M. We describe a new mutation masculinizer, which has all the properties expected for a null or strongly hypomorphic allele of F: (1) it maps to the same chromosomal location as F(D), (2) homozygous man/man animals develop as males, (3) homozygous man/man clones generated in man/+ female larvae differentiate male structures, (4) man has a sex-determining maternal effect. About a third of the morphological males synthesize yolk proteins, which indicates that they are intersexual in internal structures. The maternal effect of man is complete in offspring that derive from homozygous man/man pole cells transplanted into female hosts. In this case, all man/+ progeny become fertile males that do not produce yolk proteins. A sex-determining maternal effect has previously been demonstrated for F(D). Like F, maternal man(+) is needed for zygotic man(+) to become active, providing further evidence that man is a loss-of-function allele of F.     

Wobble inosine tRNA modification is essential to cell cycle progression in G(1)/S and G(2)/M transitions in fission yeast

Inosine (I) at position 34 (wobble position) of tRNA is formed by the hydrolytic deamination of a genomically encoded adenosine (A). The enzyme catalyzing this reaction, termed tRNA A:34 deaminase, is the heterodimeric Tad2p/ADAT2.Tad3p/ADAT3 complex in eukaryotes. In budding yeast, deletion of each subunit is lethal, indicating that the wobble inosine tRNA modification is essential for viability; however, most of its physiological roles remain unknown. To identify novel cell cycle mutants in fission yeast, we isolated the tad3-1 mutant that is allelic to the tad3(+) gene encoding a homolog of budding yeast Tad3p. Interestingly, the tad3-1 mutant cells principally exhibited cell cycle-specific phenotype, namely temperature-sensitive and irreversible cell cycle arrest both in G(1) and G(2). Further analyses revealed that in the tad3-1 mutant cells, the S257N mutation that occurred in the catalytically inactive Tad3 subunit affected its association with catalytically active Tad2 subunit, leading to an impairment in the A to I conversion at position 34 of tRNA. In tad3-1 mutant cells, the overexpression of the tad3(+) gene completely suppressed the decreased tRNA inosine content. Notably, the overexpression of the tad2(+) gene partially suppressed the temperature-sensitive phenotype and the decreased tRNA inosine content, indicating that the tad3-1 mutant phenotype is because of the insufficient I(34) formation of tRNA. These results suggest that the wobble inosine tRNA modification is essential for cell cycle progression in the G(1)/S and G(2)/M transitions in fission yeast.     

Flavodoxin mutants of Escherichia coli K-12

The flavodoxins are flavin mononucleotide-containing electron transferases. Flavodoxin I has been presumed to be the only flavodoxin of Escherichia coli, and its gene, fldA, is known to belong to the soxRS (superoxide response) oxidative stress regulon. An insertion mutation of fldA was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (fldA(+)) allele could carry it. A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene, fldB. Unlike the fldA mutant, an fldB insertion mutant is a viable prototroph in the presence or absence of oxygen. A high-copy-number fldB(+) plasmid did not complement the fldA mutation. Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I. An fldB-lacZ fusion was not induced by H(2)O(2) and is therefore not a member of the oxyR regulon. However, it displayed a soxS-dependent induction by paraquat (methyl viologen), and the fldB gene is preceded by two overlapping regions that resemble known soxS binding sites. The fldB insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a soxS-lacZ fusion. Therefore, fldB is a new member of the soxRS (superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds. However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown.     

Developmental Genetics of the 2e-F Region of the Drosophila X Chromosome: A Region Rich in "Developmentally Important" Genes

We have analyzed the 2E1-3A1 area of the X chromosome with special attention to loci related to embryogenesis. Published maps indicate that this chromosomal segment contains ten bands. Our genetic analysis has identified 11 complementation groups: one recessive visible (prune), two female steriles and eight lethals. One of the female sterile loci is fs(1)k10 for which homozygous females produce both egg chambers and embryos with a dorsalized morphology. The second female sterile is the paternally rescuable fs(1)pecanex in which unrescued embryos have a hypertrophic nervous system. Of the eight lethal complementation groups two are recessive embryonic lethals: hemizygous giant (gt) embryos possess segmental defects, and hemizygous crooked neck (crn) embryos exhibit a twisted phenotype. Analysis of these mutations in the female germ line indicates that gt does not show a maternal effect, whereas normal activity of crn is required for germ cell viability. Analysis of the maternal effect in germ line clones of the remaining six recessive lethal complementation groups indicates that four are required for germ cell viability and one produces ambiguous results for survival of the germ cells. The remaining, l(1)pole hole, is a recessive early pupal lethal in which embryos derived from germ line clones and lacking wild-type gene activity exhibit the "torso" or "pole hole" phenotype.     

The gene fl(2)d is required for various Sxl-controlled processes in Drosophila females

Summary In Drosophila melanogaster, the gene Sex-lethal (Sxl) controls the processes of sex determination, dosage compensation, oogenesis and sexual behaviour. The control of Sxl is by alternative splicing of its primary RNA. We have identified a gene, female-lethal-2-d (fl(2)d), which is needed for the female-specific splicing of Sxl RNA and which also has a vital function independent of Sxl. Here we analyse other aspects of the gene fl(2)d. Specifically, we have analysed the effect of the temperature-sensitive mutation fl(2)d ¹ on the viability of adult flies homozygous for this mutation. We have found that the viability of the mutant females is reduced, while that of the mutant males is not affected. In addition, the capacity of the mutant females to be inseminated is considerably reduced, whilst all the mutant males are able to inseminate females. These effects on females are suppressed by Sxl ^M1. However, the fat body cells of fl(2)d ¹ homozygous females are able to synthesize yolk proteins at the restrictive temperature. We have also carried out, in males, a clonal analysis of fl(2)d ², a mutation lethal in both sexes. We have found that the clones are fully viable. We conclude that the gene fl(2)d seems to be necessary during the adult life of females for the processes that require Sxl ⁺ activity. Moreover, the Sxl-independent vital function of fl(2)d seems to be required in both sexes only during larval development. Offprint requests to: L. Sánchez     

Behaviour of cells mutant for an EGF receptor homologue of Drosophila in genetic mosaics

The Drosophila homologue of the epidermal growth factor receptor (DEGFr or DER, also called torpedo or top) has many mutant alleles that cause either embryonic lethality (both early and late), pupal lethality or female sterility, possibly corresponding to degrees of hypomorphism. We have studied the clonal behaviour of some lethal alleles in genetic mosaics in the imaginal development of thorax, head and tergite epidermis. These alleles cause reduced cell viability to different degrees (measured in frequency and size of clones), smaller cell sizes, abnormal patterning of sensory-organ differentiation and lack of differentiation of macro-chaetae and veins. These effects are cell-autonomous but also cause abnormal differentiation in wild-type cells surrounding the clones. In addition, we have studied the phenotypes of double mutant combinations of viable top alleles with wing-pattern mutants, some related to other Drosophila proto-oncogenes, to reveal gene interactions in the role(s) of DER in cell proliferation and differentiation. We discuss how those complex cell-behaviour phenotypes and genetic interactions are related to the molecular nature of the DER.     

Viability of Female Germ-Line Cells Homozygous for Zygotic Lethals in DROSOPHILA MELANOGASTER

We have analyzed the viability of different types of X chromosomes in homozygous clones of female germ cells. The chromosomes carried viable mutations, single-cistron zygotic-lethal and semi-lethal mutations, or small (about six chromosome band) deletions. Homozygous germ-line clones were produced by recombination in females heterozygous for an X-linked, dominant, agametic female sterile.

All the zygotic-viable mutants are also viable in germ cells. Of 16 deletions tested (uncovering a total of 93 bands) only 2 (of 4 and 5 bands) are germ-cell viable. Mutations in 15 lethal complementation groups in the zeste-white region were tested. When known, the most extreme alleles at each locus were tested. Only in five loci (33%) were the mutants viable in the germ line. Similar studies of the same deletions and point-mutant lethals in epidermal cells show that 42% of the bands and 77% of the lethal alleles are viable. Thus, germ-line cells have more stringent cell-autonomous genetic requirements than do epidermal cells.

The eggs recovered from clones of three of the germ-cell viable zw mutations gave embryos arrested early in embryogenesis, although genotypically identical embryos derived from heterozygous oogonia die as larvae or even hatch as adult escapers. For two genes, homozygosis of the mutations tested also caused embryonic arrest of heterozygous female embryos, and in one case, the eggs did not develop at all. Germ-line clones of one quite leaky mutation gave eggs that were indistinguishable from normal. The abundance of genes whose products are required for oogenesis, whose products are required in the oocyte, and whose activity is required during zygotic development is discussed.

     

Cloning and characterisation of the Schizosaccharomyces pombe rad32 gene: a gene required for repair of double strand breaks and recombination.

A new Schizosaccharomyces pombe mutant (rad32) which is sensitive to gamma and UV irradiation is described. Pulsed field gel electrophoresis of DNA from irradiated cells indicates that the rad32 mutant, in comparison to wild type cells, has decreased ability to repair DNA double strand breaks. The mutant also undergoes decreased meiotic recombination and displays reduced stability of minichromosomes. The rad32 gene has been cloned by complementation of the UV sensitive phenotype. The gene, which is not essential for cell viability and is expressed at a moderate level in mitotically dividing cells, has significant homology to the meiotic recombination gene MRE11 of Saccharomyces cerevisiae. Epistasis analysis indicates that rad32 functions in a pathway which includes the rhp51 gene (the S.pombe homologue to S.cerevisiae RAD51) and that cells deleted for the rad32 gene in conjunction with either the rad3 deletion (a G2 checkpoint mutation) or the rad2 deletion (a chromosome stability and potential nucleotide excision repair mutation) are not viable.     

Genetic identification of a female partial-sterile mutant in soybean.

We report here the genetic identification of a female partial-sterile mutant derived from soybean mutant L67-3483. L67-3483, which originated from the cultivar Clark after X-ray irradiation, is male and female fertile. All F1 plants in reciprocal pollinations of L67-3483 with 'Clark', 'Minsoy', or 'BSR 101' were female partial sterile. Partial sterility is expressed in the heterozygous condition at a single locus and upon self-pollination this locus exhibits a 1:1 segregation pattern. This locus is located on the terminus of the soybean molecular linkage group D1b+W, between simple sequence repeat (SSR) markers Satt157 and Satt266, and is linked to each by 5.3 and 1.2 cM, respectively. This gene is transmitted through both female and male gametes and there was no segregation distortion of SSR markers linked to this gene. We concluded that this female partial-sterile gene is a new mutation class, and differs from the previously reported mutation classes in soybean, i.e., sporophytic mutation, gametophytic female-specific mutation, and general gametophytic mutation. Restriction of recombination around the mutant gene suggested that this gene is located near or within (a) small inversion(s) or adjacent to (a) chromosomal deletion(s).     

A novel gene, ecl1(+), extends the chronological lifespan in fission yeast.

We have identified a novel gene from Schizosaccharomyces pombe that we have named ecl1(+) (extender of the chronological lifespan). When ecl1(+) is provided on a high-copy number plasmid, it extends the viability of both the Deltasty1 MAP kinase mutant and the wild-type cells after entry into the stationary phase. ecl1(+) encodes an 80-amino acid polypeptide that had not been annotated in the current database. The ecl1(+)-mRNA increases transiently when the growth phase is changed from the log phase to the stationary phase. The Ecl1 protein is localized in the nucleus. Calorie restriction extends the chronological lifespan of wild-type and Deltaecl1 cells but not ecl1(+)-overproducing cells. The Deltapka1 mutant shows little, if any, additional extension of viability when Ecl1 is overproduced. The ste11(+) gene that is negatively controlled by Pka1 is up regulated when Ecl1 is overproduced. From these results we propose that the effect of Ecl1 overproduction may be mainly linked to and negatively affects the Pka1-dependent pathway.