CAENORHABDITIS ELEGANS Deficiency Mapping

Six schemes were used to identify 80 independent recessive lethal deficiencies of linkage group (LG) II following X-ray treatment of the nematode Caenorhabditis elegans. Complementation tests between the deficiencies and ethyl methanesulfonate-induced recessive visible, lethal and sterile mutations and between different deficiencies were used to characterize the extents of the deficiencies. Deficiency endpoints thus helped to order 36 sites within a region representing about half of the loci on LG II and extending over about 5 map units. New mutations occurring in this region can be assigned to particular segments of the map by complementation tests against a small number of deficiencies; this facilitates the assignment of single-site mutations to particular genes, as we illustrate. Five sperm-defective and five oocyte-defective LG II sterile mutants were identified and mapped. Certain deficiency-by-deficiency complementation tests allowed us to suggest that the phenotypes of null mutations at two loci represented by visible alleles are wild type and that null mutations at a third locus confer a visible phenotype. A segment of LG II that is about 12 map units long and largely devoid of identified loci seems to be greatly favored for crossing over.     

Spindle-E is essential for gametogenesis in the silkworm,Bombyx mori

As a defense mechanism against transposable elements, the PIWI-interacting RNA (piRNA) pathway maintains genomic integrity and ensures proper gametogenesis in gonads. Numerous factors are orchestrated to ensure normal operation of the piRNA pathway. Spindle-E (Spn-E) gene was one of the first genes shown to participate in the piRNA pathway. In this study, we performed functional analysis of Spn-E in the model lepidopteran insect, Bombyx mori. Unlike the germline-specific expression pattern observed in Drosophila and mouse, BmSpn-E was ubiquitously expressed in all tissues tested, and it was highly expressed in gonads. Immunofluorescent staining showed that BmSpn-E was localized in both germ cells and somatic cells in ovary and was expressed in spermatocytes in testis. We used a binary transgenic CRISPR/Cas9 system to construct BmSpn-E mutants. Loss of BmSpn-E expression caused derepression of transposons in gonads. We also found that mutant gonads were much smaller than wild-type gonads and that the number of germ cells was considerably lower in mutant gonads. Quantitative real-time PCR analysis and TUNEL staining revealed that apoptosis was greatly enhanced in mutant gonads. Further, we found that the BmSpn-E mutation impacted gonadal development and gametogenesis at the early larval stage. In summary, our data provided the first evidence that BmSpn-E plays vital roles in gonadal development and gametogenesis in B. mori.     

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.

     

Genetic analysis of sterile mutants in the dpy-5 unc-13 (I) genomic region of Caenorhabditis elegans

Essential genes were identified in the 1.5-map unit dpy-5 unc-13 region of chromosome I in the Caenorhabditis elegans genome by rescuing lethal mutations using the duplication sDp2. In this paper, we report the mapping and complementation testing of lethal mutations, 45 of which identify 18 new, essential genes. This analysis brings the number of essential genes defined by the sDp2 rescue of lethal mutants to 97; 64 of these map between dpy-5 and unc-13. 61% of these essential genes are identified by more than one allele. Positioning of the mutations was done using the breakpoints of six duplications. The mutant phenotypes of 14 loci essential for fertility were characterized by Nomarski microscopy and DAPI staining. None of the mutants were rescued by wild-type male sperm. The cytological data showed that four genes produced mutants with defects in gonadogenesis, let-395, let-603, let-605 and let-610. Mutations in seven genes, let-355, let-367, let-384, let-513, let-544, let-545 and let-606, affected germ cell proliferation or gametogenesis. Mutants for the remaining three genes, let-370, let-599 and let-604, produced eggs that failed to develop or hatch, thereby acting as maternal effect lethals. We observed a nonrandom distribution of arrest phenotypes with regard to map position. Received: 8 May 1996 / Accepted : 27 January 1997     

EMBRYONIC GENETIC LOAD IN THE HIGHBUSH BLUEBERRY,VACCINIUM CORYMBOSUM (ERICACEAE)

Hand-pollinations of 28 autotetraploid V. corymbosum accessions from a single population resulted in lower self- than outcross seed set. Fertility varied widely, ranging from clones that were effectively female sterile to individuals with high seed yields in both matings. Self- and outcross fertility were highly correlated. A genetic load model was invoked to explain these phenomena. Reduced self-fertility was attributed to homozygosity for sublethal mutations at loci controlling embryo development, or to loss of heterotic interactions at these loci. Near zero cross-fertility in some clones may be evidence of partially dominant mutational load. Estimates of the number of lethal equivalents per zygote carried by individuals in this population ranged from 2.2 to 20.4, with a mean of 9.6. Embryonic genetic load at the individual level was significantly correlated with heterozygosity at nine enzyme loci. Low pollen viability and reduced receptivity to pollen from any source were also noted in the low fertility genotypes. It is suggested that gametic, gametophytic, and embryonic development are symptomatic of the amount of genetic load carried by individuals.     

A screen for lethal mutations in the chromosomal region 59AB suggests that bellwether encodes the alpha subunit of the mitochondrial ATP synthase in Drosophila melanogaster

We report the isolation and complementation mapping of lethal mutations within the 59AB region on the second chromosome of Drosophila melanogaster. The newly induced lethal mutations in this region define four different complementation groups. Using existing and newly induced deficiencies, these loci can be assigned to three different chromosomal intervals. Moreover, complementation analysis with chromosomes carrying various P element insertions, in combination with a molecular characterization of the corresponding insertion sites, suggests that the previously described male sterile mutation bellwether is an allele of an essential gene that encodes the alpha subunit of the mitochondrial ATP synthase. Received: 25 April 1998 / Accepted: 27 May 1998     

The distribution of randomly recovered X-ray-induced sex-linked genetic effects in Drosophila melanogaster

Cytogenetic analysis of more than 1500 randomly recovered lethal X chromosomes derived from 2000 and 3000 r X-ray exposures of post-meiotic male germ cells has made possible a plot of the distribution in different regions of the X chromosome of: (1) gene mutations associated with cytologically normal chromosomes, (2) mutations associated with chromosomal rearrangement breakpoints, (3) deficiencies, and (4) rearrangement breakpoints whether or not they are associated with mutations. The distribution of point mutations, vital loci and rearrangement breakpoints in different regions of the X chromosome is not proportional to either the number of bands or the relative DNA content. Further, the density of vital loci (those capable of mutating to a lethal allele) is quite different in some regions as compared to others. For example, vital loci in the 3AB region, which has been thoroughly studied by Judd and others, are at least as numerous as bands; whereas, the 3CD region, equally long, has only two vital loci. Other regions densely populated with vital loci include 1B, 1F-2A, 10A, 11A, and 19EF; sparsely populated regions include 6EF and 10B-10E. It seems reasonable to conclude that the recovered X-ray-induced mutants available for analysis do not represent a random sample of those initially induced in the exposed male germ cells.     

Interacting Genes That Affect Microtubule Function in Drosophila Melanogaster: Two Classes of Mutation Revert the Failure to Complement between Hay(nc2) and Mutations in Tubulin Genes

The recessive male sterile mutation haync2 of Drosophila melanogaster fails to complement certain beta 2-tubulin and alpha-tubulin mutations, suggesting that the haywire product plays a role in microtubule function, perhaps as a structural component of microtubules. The genetic interaction appears to require the presence of the aberrant product encoded by haync2, which may act as a structural poison. Based on this observation, we have isolated ten new mutations that revert the failure to complement between haync2 and B2tn. The revertants tested behaved as intragenic mutations of hay in recombination tests, and fell into two phenotypic classes, suggesting two functional domains of the hay gene product. Some revertants were hemizygous viable and less severe than haync2 in their recessive phenotype. These mutations might revert the poison by restoring the aberrant product encoded by the haync2 allele to more wild-type function. Most of the revertants were recessive lethal mutations, indicating that the hay gene product is essential for viability. These more extreme mutations could revert the poison by destroying the ability of the aberrant haywirenc2 product to interact structurally with microtubules. Flies heterozygous for the original haync2 allele and an extreme revertant show defects in both the structure and the function of the male meiotic spindle.     

A germline-specific gap junction protein required for survival of differentiating early germ cells

Germ cells require intimate associations and signals from the surrounding somatic cells throughout gametogenesis. The zero population growth (zpg) locus of Drosophila encodes a germline-specific gap junction protein, Innexin 4, that is required for survival of differentiating early germ cells during gametogenesis in both sexes. Animals with a null mutation in zpg are viable but sterile and have tiny gonads. Adult zpg-null gonads contain small numbers of early germ cells, resembling stem cells or early spermatogonia or oogonia, but lack later stages of germ cell differentiation. In the male, Zpg protein localizes to the surface of spermatogonia, primarily on the sides adjacent to the somatic cyst cells. In the female, Zpg protein localizes to germ cell surfaces, both those adjacent to surrounding somatic cells and those adjacent to other germ cells. We propose that Zpg-containing gap junctional hemichannels in the germ cell plasma membrane may connect with hemichannels made of other innexin isoforms on adjacent somatic cells. Gap junctional intercellular communication via these channels may mediate passage of crucial small molecules or signals between germline and somatic support cells required for survival and differentiation of early germ cells in both sexes.     

X-Linked Female-Sterile Loci in DROSOPHILA MELANOGASTER

We have examined the number of X-linked loci specifically required only during oogenesis. Complementation analyses among female-sterile (fs) mutations obtained in two mutagenesis screens--GANS' and MOHLER's--indicate that any fs locus represented by two or more mutant alleles in GANS' collection are usually present in MOHLER's collection. However, when a locus is represented by a single allele in one collection, it is generally not present in the other collection. We propose that this discrepancy is due to the fact that most "fs loci" represented by less than two mutant alleles are, in fact, vital (zygotic lethal) genes, and that the fs alleles are hypomorphic mutations of such genes. In support of this hypothesis we have identified lethal alleles at 12 of these "fs loci." The present analysis has possibly identified all maternal-effect lethal loci detectable by mutations on the X chromosome and has allowed us to reevaluate the number of "ovary-specific fs" loci in the Drosophila genome. Finally, germline clone analysis of a large number of fs mutations was performed in order to estimate the relative contribution of germline and somatic cell derivatives to oogenesis and to embryonic development. All the maternal-effect lethal loci tested are germline-dependent.     

Hypomorphic lethal mutations and their implications for the interpretation of lethal complementation studies in Drosophila

In a small region of the X chromosome of Drosophila melanogaster, we have found that a third of the mutations that appear to act as lethals in segmental haploids are viable in homozygous mutant individuals. These viable mutations fall into four complementation groups. The most reasonable explanation of these mutations is that they are a subset of functionally hypomorphic alleles of essential genes: hypomorphic mutations with activity levels above a threshold required for survival, but below twice that level, should behave in this manner. We refer to these mutations as "haplo-specific lethal mutations." In studies of autosomal lethals, haplo-specific lethal mutations can be included in lethal complementation tests without being identified as such. Accidental inclusion of disguised haplo-specific lethals in autosomal complementation tests will generate spurious examples of interallelic complementation.     

The Genetics of the DORSAL-BICAUDAL-D Region of DROSOPHILA MELANOGASTER

The chromosomal region 36C on 2L contains two maternal-effect loci, dorsal (dl) and Bicaudal-D (Bic-D), which are involved in establishing polarity of the Drosophila embryo along the dorsal-ventral and anterior-posterior axes, respectively. To analyze the region genetically, we isolated X-ray-induced dorsal alleles, which we recognized by virtue of the haplo-insufficient temperature-sensitive dorsal-dominant phenotype in progeny of single females heterozygous for a mutagenized chromosome. From the 20,000 chromosomes tested, we isolated three deficiencies, two inversions with breakpoint in dl and one apparent dl point mutant. One of the deficiencies, Df(2L)H20 (36A6,7; 36F1,2) was used to screen for EMS-induced lethal- and maternal-effect mutants mapping in the vicinity of dl and Bic-D. We isolated 44 lethal mutations defining 11 complementation groups. We also recovered as maternal-effect mutations four dl alleles, as well as six alleles of quail and one allele of kelch, two previously identified maternal-effect genes. Through complementation tests with various viable mutants and deficiencies in the region, a total of 18 loci were identified in an interval of about 30 cytologically visible bands. The region was subdivided into seven subregions by deficiency breakpoints. One lethal complementation group as well as the two maternal loci, Bic-D and quail, are located in the same deficiency interval as is dl.     

The multi sex combs gene of Drosophila melanogaster is required for proliferation of the germline

We present a genetic analysis showing that the Drosophila melanogaster gene multi sex combs (mxc; Santamaria and Randsholt 1995) is needed for proliferation of the germline. Fertility is the feature most easily affected by weak hypomorphic mutations of this very pleiotropic locus. Pole cell formation and early steps of gonadogenesis conform to the wild-type in embryos devoid of zygotic mxc ⁺ product. mxc mutant gonad phenotypes and homozygous mxc germline clones suggest a role for mxc ⁺ in control of germ cell proliferation during the larval stages. mxc ⁺ requirement is germ cell autonomous and specific in females, whilst in males mxc ⁺ product is also needed in somatic cells of the gonads. Although mxc can be classified among the Polycomb group (Pc-G) of genes, negative trans-regulators of the ANT-C and BX-C gene complexes, germline requirement for mxc appears independent of a need for other Pc-C gene products, and mxc gonad phenotypes are different from those induced by mutations in BX-C genes. We discuss the possible functions of the mxc ⁺ product which helps to maintain homeotic genes repressed and prevents premature larval haemocyte differentiation and neoplasic overgrowth, but promotes growth and differentiation of male and female gonads.F.D. and O.S. should be considered as equal first authors     

Anent the Genomics of Spermatogenesis in Drosophila melanogaster

An appreciable fraction of the Drosophila melanogaster genome is dedicated to male fertility. One approach to characterizing this subset of the genome is through the study of male-sterile mutations. We studied the relation between vital and male-fertility genes in three large autosomal regions that were saturated for lethal and male-sterile mutations. The majority of male-sterile mutations affect genes that are exclusively expressed in males. These genes are required only for male fertility, and several mutant alleles of each such gene were encountered. A few male-sterile mutations were alleles of vital genes that are expressed in both males and females. About one-fifth of the genes in Drosophila melanogaster show male-specific expression in adults. Although some earlier studies found a paucity of genes on the X chromosome showing male-biased expression, we did not find any significant differences between the X chromosome and the autosomes either in the relative frequencies of mutations to male sterility or in the frequencies of genes with male-specific expression in adults. Our results suggest that as much as 25% of the Drosophila genome may be dedicated to male fertility.     

Lethal bithorax complex mutations of Drosophila melanogaster show no germ line maternal effects

Three Ultrabithorax (Ubx) alleles and three different deficiencies of the bithorax complex (BX-C) of Drosophila melanogaster have been tested for maternal effects in the germ line. The dominant female sterile technique was used. The Ubx alleles and a deletion of the abdominal region of the BX-C are homozygous viable in germ line clones and show no maternal effects. Two deletions which lack the proximal portion of the BX-C are lethal in the female germ line indicating either that these deficiencies lack genes apart from the BX-C that are necessary for fertility or that there are BX-C genes that are essential for normal maternal germ line function. The significance of the bias in the isolation of only zygotic mutations at the BX-C are discussed with respect to these results.