Developmental consequences of mutations in the 84B alpha-tubulin gene of Drosophila melanogaster

Developmental effects of six mutations in the gene encoding the majority of alpha-tubulin in all tissues at all stages of Drosophila melanogaster development have been examined. All six alleles produce at least partially stable alpha 84B protein. In genetic assays, two of these alleles approximate the null condition. The other four alleles appear to form a graded series of hypomorphs. The two most severe alleles produce a semidominant maternal-effect polyphasic lethality, plus a predominantly larval recessive zygotic lethality. Clonal analysis of one of these alleles suggests it is a cell lethal. Worsening of the lethal phenotype (negative complementation) occurs in most interallelic heterozygotes involving these two mutations. As hemizygotes, the other four alleles are predominantly larval/pupal lethals. Partial complementation is achieved by most interallelic heterozygotes involving these four alleles. Phenotypic defects associated with the six tubulin mutation include disrupted embryos, pseudopupae, pharate adults with defects in various cuticular pattern elements, pharate adults with retarded head development, adults with leg tremors and extremely short life spans, and viable but sterile adults with bristle defects.     

Genetic rescue of lethal genotypes in the mouse

Deletions of gene sequences in chromosome 7 of the mouse are known to interfere with biochemical and cellular development differentiation with lethal effects in homozygotes. The presence of the corresponding wild-type alleles in Cattanach's translocation (chromosomes 7 to X) is able to “rescue” potentially lethal females if they are made heterozygous for the translocation-carrying X chromosome. This holds true for those chromosome 7 deletions with perinatally lethal effects, whereas “rescue” is not readily accomplished with the deletions that cause early embryonic lethality. Females homozygous for the relevant deletion sequences and heterozygous for the translocation-carrying X chromosome are mosaics of two cell types: those in which the wild-type alleles included in the translocated piece complement the depleted sequences, resulting in a normal cellular phenotype, and those with the ordinary X chromosome expressing the lethal phenotype. The developmental interactions between the two cell types and their role in the mechanisms responsible for survival of females homozygous for lethal deletions are discussed. The failure of “rescue” of embryonic lethals reflects as yet unknown temporal and functional aspects of X-inactivation early embryogenesis.     

Identification of Genomic Regions Required for DNA Replication during Drosophila Embryogenesis

A collection of Drosophila deficiency stocks was examined by bromodeoxyuridine (BrdU) labeling of embryos to analyze the DNA replication patterns in late embryogenesis. This permitted us to screen 34% of the genome for genes that when absent in homozygous deficiencies affect the cell cycle or DNA replication. We found three genomic intervals that when deleted result in cessation of DNA replication in the embryo, 39D2-3;E2-F1, 51E and 75C5-7;F1. Embryos deleted for the 75C5-7;F1 region stop DNA replication at the time in embryogenesis when a G(1) phase is added to the mitotic cell cycle and the larval tissues begin to become polytene. Thus, this interval may contain a gene controlling these cell cycle transitions. DNA replication arrests earlier in embryos homozygous for deletions for the other two regions. Analysis of the effects of deletions in the 39D2-3;E2-F1 region on DNA replication showed that the block to DNA replication correlates with deletion of the histone genes. We were able to identify a single, lethal complementation group in 51E, l(2)51Ec, that is responsible for the cessation of replication observed in this interval. Deficiencies that removed one of the Drosophila cdc2 genes and the cyclin A gene had no effect on replication during embryogenesis. Additionally, our analysis identified a gene, pimples, that is required for the proper completion of mitosis in the post-blastoderm divisions of the embryo.     

The albino deletion complex and early postimplantation survival in the mouse

The albino deletion complex in the mouse represents 37 overlapping chromosomal deficiencies that have been arranged into at least twelve complementation groups. Many of the deletions cover regions of chromosome 7 that contain genes necessary for early embryonic development. The work reported here concentrates on two of these deletions (c6H, c11DSD), both of which were known to be lethal around the time of gastrulation when homozygous. A detailed embryological analysis has revealed distinct differences in the lethal phenotype associated with the c6H and c11DSD deletions. c6H homozygous embryos are grossly abnormal at day 7.5 of gestation, whereas c11DSD homozygous embryos appear abnormal at day 8.5 of gestation. There is no development of the extraembryonic ectoderm in c6H homozygotes, whereas extensive development of this tissue type occurs in c11DSD homozygotes. The visceral endoderm is abnormally shaped and the parietal endoderm appears to be overproduced in c6H homozygotes; these structures are not affected in c11DSD homozygotes. The embryonic ectoderm is runted in both types of embryo and it is not possible to obtain homozygous embryo-derived stem-cell lines for either deletion. Mesoderm formation occurs in the c11DSD but not in the c6H homozygotes. The c11DSD deletion chromosome complements the c6H chromosome in that the lethal phenotype of the compound heterozygote is similar to that of the c11DSD homozygote. These results suggest that a gene(s) necessary for normal development of the extraembryonic ectoderm is present in the c11DSD but deficient in the c6H deletion chromosome.     

The Unc-45 Gene of Caenorhabditis Elegans Is an Essential Muscle-Affecting Gene with Maternal Expression

We have isolated three novel alleles of the unc-45 locus in C. elegans, that are recessive lethals. Two of these alleles, when homozygous, result in a nearly total loss of muscle contraction with a concomitant arrest of development and a displacement of muscle cells. The third allele is similar, but showed maternal rescue by a wild-type allele. All previously identified unc-45 alleles were temperature sensitive and, although they produced paralysis of adult animals, all were homozygous viable. Prior genetic studies with these temperature sensitive alleles had suggested that at least one function of the unc-45 gene product was to interact with the major myosin heavy chain isoform, MHC B, of body wall muscles. Our observations of the lethal alleles suggest that the unc-45 product normally interacts with additional muscle components in both the body wall and pharyngeal muscles. In particular, we suggest that the unc-45 product might interact with all four myosin heavy chains: MHC B; MHC A; and the pharyngeal isoforms, MHC C and MHC D. Maternal rescue of the lethality of the third allele shows that the unc-45 gene product is present in the oocytes, although it may not be necessary until late in development when myofilaments begin to assemble.     

Construction and characterization of deletions with defined end points in Drosophila using P elements in trans

We used P-element transposase-mediated "male recombination" between two P elements in trans to create genetic deletions that removed a number of loci, including the gene encoding the neuropeptide crustacean cardioactive peptide (CCAP). Two classes of recombinant chromosomes were produced. Approximately one-quarter were viable when homozygous or hemizygous, whereas the remaining lines caused homozygous and hemizygous lethality. Preliminary analyses using PCR and CCAP immunohistochemistry suggested that, whereas the DNA of the viable lines was largely intact, most lethal lines contained chromosomal deletions that were roughly bounded by the insertion sites of the two P elements used. Southern blot analyses of select lethal lines showed that the DNA flanking the deletion was indeed grossly intact whereas the intervening DNA could not be detected. Sequencing across the deletion in three of these lethal lines identified a single line bearing intact genomic DNA on either side of the deletion separated by 30 bp of P-element DNA. The method described here suggests a simple procedure for creating deletions with defined end points. Importantly, it can use preexisting P-element insertion strains and does not rely on the use of transposable elements that are engineered to cause specific DNA rearrangements.     

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 Passover Locus in Drosophila Melanogaster: Complex Complementation and Different Effects on the Giant Fiber Neural Pathway

Drosophila melanogaster bearing the Passover mutation fail to jump in response to a light-off stimulus. Pas also disrupts some of the synapses between the neurons of the giant fiber system which mediate this escape behavior. We have mapped Pas to the 19E subdivision of the polytene X chromosome. Our genetic analyses reveal that deletions of either of two nonoverlapping regions fail to fully complement Pas. Heterozygotes of Pas with chromosomal deletions in the vicinity of polytene band 19E3 exhibit the full set of neuronal defects shown by Pas homozygotes. Alleles of the R-9-29 complementation group, which maps to band 19E3, exhibit a complex pattern of complementation with Pas. Heterozygotes combining the lethal R-9-29 alleles with Pas are all viable, some complement the neuronal defects of Pas, but most exhibit these defects. The viable shaking-B2 mutation also fails to complement Pas, the R-9-29 alleles or the 19E3 deficiencies. The R-9-29 locus may contain two functional domains, one required for viability the other for normal neuronal phenotype, trans-Heterozygotes bearing mutant alleles or a deficiency of the first region (19E3) together with deficiencies of the second region (19E5-6) also exhibit some of the neuronal defects shown by the Passover mutant. Deficiencies which delete the entire 19E3 to 19E6 interval do not produce this phenotype when heterozygous with a normal X chromosome. Thus normal function requires a cis-interaction between the two regions. These findings raise the possibility that the gene mutated by Pas is split or separated from a cis-activator by at least one other gene.     

Genetic Characterization of Small Ovaries, a Gene Required in the Soma for the Development of the Drosophila Ovary and the Female Germline

The small ovary gene (sov) is required for the development of the Drosophila ovary. Six EMS-induced recessive alleles have been identified. Hypomorphic alleles are female sterile and have no effect on male fertility, whereas more severe mutations result in lethality. The female-sterile alleles produce a range of mutant phenotypes that affect the differentiation of both somatic and germline tissues. These mutations generally produce small ovaries that contain few egg cysts and disorganized ovarioles, and in the most extreme case no ovarian tissue is present. The mutant egg cysts that develop have aberrant morphology, including abnormal numbers of nurse cells and patches of necrotic cells. We demonstrate that sov gene expression is not required in the germline for the development of functional egg cysts. This indicates that the sov function is somatic dependent. We present evidence using loss-of-function and constitutive forms of the somatic sex regulatory genes that sov activity is essential for the development of the somatic ovary regardless of the chromosomal sex of the fly. In addition, the genetic mapping of the sov locus is presented, including the characterization of two lethal sov alleles and complementation mapping with existing rearrangements.     

Physical and Genetic Characterization of a 75-Kilobase Deletion Associated with A(l), a Recessive Lethal Allele at the Mouse Agouti Locus

The agouti locus (A) of the mouse determines the timing and type of pigment deposition in the growing hair bulb, and several alleles at this locus are lethal when homozygous. Apparent instances of intragenic recombination and complementation between different recessive lethal alleles have suggested that the locus has a complex structure. We have begun to investigate the molecular basis of agouti gene action and recessive lethality by using a series of genetically linked DNA probes and pulsed field gel electrophoresis to detect structural alterations in radiation-induced agouti mutations. Hybridization probes from the Src and Emv-15 loci do not reveal molecular alterations in DNA corresponding to the ae, ax, and al alleles, but a probe from the parotid secretory protein gene (Psp) detects a 75-kilobase (kb) deletion in DNA containing the non-agouti lethal allele (al). The deletion is defined by a 75-kb reduction in the size of BssHII, NotI, NruI and SacII high molecular weight restriction fragments detected with the Psp probe and is located between 25 kb and 575 kb from Psp coding sequences. Because the genetic distance between A and Emv-15 is much less than A and Psp, there may be a preferred site of recombination close to Psp, or suppression of recombination between A and Emv-15. The al deletion has allowed us to determine the genotype of mice heterozygous for different recessive lethal alleles. We find that three different recessive lethal complementation groups are present at the agouti locus, two of which are contained within the al deletion.     

Genetic characterization of the 44D-45B region of the Drosophila melanogaster genome based on an F2 lethal screen

We have performed an F₂ genetic screen to identify lethal mutations that map to the 44D-45B region of the Drosophila melanogaster genome. By screening 8500 mutagenized chromosomes for lethality over Df(2R)Np3, a deficiency which encompasses nearly 1% of the D. melanogaster euchromatic genome, we recovered 125 lines with lethal mutations that represent 38 complementation groups. The lethal mutations have been mapped to deficiencies that span the 44D-45B region, producing an approximate map position for each complementation group. Lethal mutations were analyzed to determine the phase of development at which lethality occurred. In addition, we have linked some of the complementation groups to P element-induced lethals that map to 44D-45B, thus possibly providing new alleles of a previously tagged gene. Some of the complementation groups represent potentially novel alleles of previously identified genes that map to the region. Several genes have been mapped by molecular means to the 44D-45B region, but do not have any reported mutant alleles. This screen may have uncovered mutant alleles of these genes. The results of complementation tests with previously identified genes in 44D-45B suggests that over half of the complementation groups identified in this screen may be novel. Received: 13 July 1999 / Accepted: 4 November 1999     

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.

     

The gene for the intermediate chain subunit of cytoplasmic dynein is essential in Drosophila

The microtubule motor cytoplasmic dynein powers a variety of intracellular transport events that are essential for cellular and developmental processes. A current hypothesis is that the accessory subunits of the dynein complex are important for the specialization of cytoplasmic dynein function. In a genetic approach to understanding the range of dynein functions and the contribution of the different subunits to dynein motor function and regulation, we have identified mutations in the gene for the cytoplasmic dynein intermediate chain, Dic19C. We used a functional Dic transgene in a genetic screen to recover X-linked lethal mutations that require this transgene for viability. Three Dic mutations were identified and characterized. All three Dic alleles result in larval lethality, demonstrating that the intermediate chain serves an essential function in Drosophila. Like a deficiency that removes Dic19C, the Dic mutations dominantly enhance the rough eye phenotype of Glued(1), a dominant mutation in the gene for the p150 subunit of the dynactin complex, a dynein activator. Additionally, we used complementation analysis to identify an existing mutation, shortwing (sw), as an allele of the dynein intermediate chain gene. Unlike the Dic alleles isolated de novo, shortwing is homozygous viable and exhibits recessive and temperature-sensitive defects in eye and wing development. These phenotypes are rescued by the wild-type Dic transgene, indicating that shortwing is a viable allele of the dynein intermediate chain gene and revealing a novel role for dynein function during wing development.     

Mast cell growth factor maps near the steel locus on mouse chromosome 10 and is deleted in a number of steel alleles.

Many spontaneous, chemical-induced, and radiation-induced dominant white spotting (W) and steel (Sl) mutations have been identified in the mouse. W and Sl mutations have similar phenotypic effects including deficiencies in pigment cells, germ cells, and blood cells, Numerous studies have suggested that W acts within the affected cell while Sl instead exerts its effects in the extracellular environment. Recent findings demonstrating that W encodes the c-kit proto-oncogene, a tyrosine kinase membrane receptor, have suggested that Sl encodes a ligand for c-kit. In the accompanying article we report the identification and purification of mast cell growth factor (MGF), a c-kit ligand. Here we describe the cloning of sequences encoding MGF. Furthermore, we show that Mgf maps near Sl in the distal region of mouse chromosome 10 and is deleted in a number of Sl alleles. These findings strongly support the notion that Sl encodes the mast cell growth factor.     

Developmental genetic analysis of lethal alleles at the ewg locus and their effects on muscle development in Drosophila melanogaster

The recessive X-linked mutation erect wing (ewg), in Drosophila melanogaster, was characterized as a flightless behavioral mutant which specifically lacked the dorsal longitudinal flight muscles [1]. This mutation was mapped distal to the X chromosomal locus yellow, and further to the cytological segment 1 A 1 to 1 B2-3 [2]. Several lethal complementation groups have been mapped to this interval [3]. Our complementation tests show that ewg is allelic to one lethal complementation group in the region 1 A 1 to 1 B2-3. A further analysis of ewg and several lethal alleles isolated at this locus was undertaken in the present investigation. Most of the lethal alleles at this locus lead to a late embryonic or early larval lethal phase, indicating that the ewg⁺ gene product is necessary for the development of more than just the dorsal longitudinal flight muscles. Intragenic complementation was observed for some of the ewg lethal alleles. Genetic mosaics with ewg lethal alleles showed that mutant cell clones in cuticular structures are viable. Mosaic analysis is consistent with a mesodermal defect associated with the locus.     

Mutants Resistant to anti-Microtubule Herbicides Map to a Locus on the Uni Linkage Group in Chlamydomonas Reinhardtii

We have used genetic analysis to study the mode of action of two anti-microtubule herbicides, amiprophos-methyl (APM) and oryzalin (ORY). Over 200 resistant mutants were selected by growth on APM- or ORY-containing plates. The 21 independently isolated mutants examined in this study are 3- to 8-fold resistant to APM and are strongly cross-resistant to ORY and butamiphos, a close analog of APM. Two Mendelian genes, apm1 and apm2, are defined by linkage and complementation analysis. There are 20 alleles of apm1 and one temperature-sensitive lethal (33°) allele of apm2. Mapping by two-factor crosses places apm1 6.5 cM centromere proximal to uni1 and within 4 cM of pf7 on the uni linkage group, a genetically circular linkage group comprising genes which affect flagellar assembly or function; apm2 maps near the centromere of linkage group VIII. Allele-specific synthetic lethality is observed in crosses between apm2 and alleles of apm1. Also, self crosses of apm2 are zygotic lethal, whereas crosses of nine apm1 alleles inter se result in normal germination and tetrad viability. The mutants are recessive to their wild-type alleles but doubly heterozygous diploids (apm1 +/+ apm2) made with apm2 and any of 15 apm1 alleles display partial intergenic noncomplementation, expressed as intermediate resistance. Diploids homozygous for mutant alleles of apm1 are 4-6-fold resistant to APM and ORY; diploids homozygous for apm2 are ts(-) and 2-fold resistant to the herbicides. Doubly heterozygous diploids complement the ts(-) phenotype of apm2, but they are typically 1.5-2-fold resistant to APM and ORY. From the results described we suggest that the gene products of apm1 and apm2 may interact directly or function in the same structure or process.     

A Genetic Analysis of the Stoned Locus and Its Interaction with Dunce, Shibire and Suppressor of Stoned Variants of Drosophila Melanogaster

The genetic complementation patterns of both behavioral and lethal alleles at the stoned locus have been characterized. Mosaic analysis of a stoned lethal allele suggests that stoned functions either in the nervous system or in both the nervous system and musculature, but is not required for gross neural development. The behavioral alleles stn(ts) and stn(C), appear to be defective in a diametrically opposite sense, show interallelic complementation, and indicate distinct roles for the stoned gene product in the visual system and in motor coordination. A number of other neurological mutations have been investigated for their possible interaction with the viable stoned alleles. Mutations at two loci, dunce and shibire, act synergistically with the stn(ts) mutations to cause lethality, but fail to interact with stn(C). A third variant (Suppressor of stoned) has been identified which can suppress the debilitation associated with the stn(ts) mutations. These data, together with a previously identified interaction between the stn(ts) and tan mutants, indicate a central role for the stoned gene product in neuronal function, and suggests that the stoned gene product interacts, either directly or indirectly, with the neural cAMP second messenger system, with the synaptic membrane recycling pathway via dynamin, and with biogenic amine metabolism.