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S A Wasserman

Publications and source records attributed to S A Wasserman.

At least 37 records · Page 2Linked to original sources
PubMed1996

A gradient of cactus protein degradation establishes dorsoventral polarity in the Drosophila embryo.

Dorsoventral polarity in the Drosophila embryo is established by a signaling pathway active on the ventral and ventrolateral surfaces of the embryo. Signal transduction via the protein kinase Pelle frees the Rel-related protein Dorsal from its cytoplasmic inhibitor Cactus, allowing Dorsal to translocate into ventral and ventrolateral nuclei and direct gene expression. Here, we show by immunochemical analyses that Pelle-mediated signaling induces the spatially graded degradation of Cactus. Using a tissue culture system which reconstitutes Pelle-dependent Cactus degradation, we show that a motif in Cactus resembling the sites of signal-dependent phosphorylation in the vertebrate homologs IkappaB-alpha and IkappaB-beta is essential for Pelle-induced Cactus degradation. Substitution of four serines within this motif with nonphosphorylatable alanine residues generated a mutant Cactus that still functions as a Dorsal inhibitor but is resistant to induced degradation. Injection of RNA encoding this altered form of Cactus has a dominant negative effect on establishment of dorsoventral polarity in the embryo. We conclude that dorsoventral signaling results in a Cactus concentration gradient and propose that signal-dependent phosphorylation directs the spatially regulated proteolysis of Cactus protein.

Amino Acid Sequence↗
PubMed1996

Meiotic cell cycle requirement for a fly homologue of human Deleted in Azoospermia.

Infertility resulting from a severe defect in sperm production affects 2% of men worldwide. Of these men with azoospermia, the absence of sperm in semen, one in eight carry de novo deletions for a specific region of the Y chromosome. A candidate gene for the Y-chromosome azoospermia factor (AZF) has been identified and named Deleted in Azoospermia (DAZ). Here we describe the cloning and characterization of the Drosophila gene boule, which is a homologue of DAZ. The two genes encode closely related proteins that contain a predicted RNA-binding motif, and both loci are expressed exclusively in the testis. Loss of boule function results in azoospermia; meiotic divisions are blocked, although limited spermatid differentiation occurs. Histological examination of boule testes with cell-cycle markers indicates that the primary defect is at the meiotic G2/M transition. These results support the hypothesis that DAZ is the human AZF, and indicate that Boule and DAZ have an essential meiotic function in fly and human spermatogenesis.

Amino Acid Sequence↗
PubMed1995

The pelota locus encodes a protein required for meiotic cell division: an analysis of G2/M arrest in Drosophila spermatogenesis.

During Drosophila spermatogenesis, germ cells undergo four rounds of mitosis, an extended premeiotic G2 phase and two meiotic divisions. In males homozygous for mutations in pelota, the germline mitotic divisions are normal, but the cell cycle arrests prior to the first meiotic division; pelota males are therefore sterile. Chromosomes begin to condense in these mutants, but other meiotic processes, including nuclear envelope breakdown and spindle formation, do not occur. The arrest phenotype closely resembles that of mutations in the Drosophila cdc25 homolog twine. Although meiosis is blocked in pelota and twine homozygotes, spermatid differentiation continues. pelota is also required for patterning in the eye and mitotic divisions in the ovary. We have cloned the pelota locus and show it encodes a 44 x 10(3) M(r) protein with yeast, plant, worm and human homologs.

Amino Acid Sequence↗
PubMed1995

Interaction of the pelle kinase with the membrane-associated protein tube is required for transduction of the dorsoventral signal in Drosophila embryos.

Within the Drosophila embryo, tube and the protein kinase pelle transduce an intracellular signal generated by the transmembrane receptor Toll. This signal directs import of the rel-related protein dorsal into ventral and ventrolateral nuclei, thereby establishing dorsoventral polarity. We show by immunolocalization that tube protein associates with the plasma membrane during interphase. We also find that tube sequences required for signaling interact with pelle in a yeast two-hybrid assay. We demonstrate that fusion of the pelle catalytic domain to the transmembrane receptor torso is sufficient to induce ventral fates; this activity is independent of Toll or tube. Lastly, we find that fusion of the tube protein to torso also induces ventral fates, but only in the presence of functional pelle. We propose a model wherein tube activates pelle by recruiting it to the plasma membrane, thereby propagating the axis-determining signal.

Animals↗
PubMed1994

Incarcerated hernia after laparoscopically assisted vaginal hysterectomy.

Herniation of small bowel through a fascial defect (Richter hernia) created at laparoscopy is a rare complication, but it occurred in a 54-year-old woman after a laparoscopically assisted vaginal hysterectomy. One must question whether or not increasing cannula sizes are associated with greater numbers of laparoscopic complications.

Female↗
PubMed1994

Dorsal, a Drosophila Rel-like protein, is phosphorylated upon activation of the transmembrane protein Toll.

The nuclear import of dorsal, a Drosophila Rel homolog, is directed by a spatially restricted extracellular ligand in blastoderm embryos. We have demonstrated both that dorsal is an embryonic phosphoprotein and that its phosphorylation state is regulated by an intracellular signaling pathway initiated by the transmembrane receptor Toll. Immunoblot analysis of cytoplasm from precisely staged embryos revealed that the phosphorylation state of dorsal is altered during the time period that Toll is activated. Moreover, mutations that constitutively activate Toll stimulated dorsal phosphorylation, while mutations that block Toll activation reduced the level of dorsal phosphorylation. We further demonstrated that signal-dependent dorsal phosphorylation is modulated by three intracellular proteins, pelle, tube, and cactus. Using double-mutant embryos, we then explored the nature of the kinase activity responsible for dorsal phosphorylation. We found that free dorsal is a substrate for a signal-independent kinase activity. In addition, our results imply that dorsal is a substrate for a Toll-dependent kinase. These results are consistent with the hypothesis that phosphorylation of Rel-related proteins may be required for the proper nuclear localization and transcriptional activity of these proteins.

Animals↗
PubMed1994

Diaphanous is required for cytokinesis in Drosophila and shares domains of similarity with the products of the limb deformity gene.

We show that the Drosophila gene diaphanous is required for cytokinesis. Males homozygous for the dia1 mutation are sterile due to a defect in cytokinesis in the germline. Females trans-heterozygous for dia1 and a deficiency are sterile and lay eggs with defective eggshells; failure of cytokinesis is observed in the follicle cell layer. Null alleles are lethal. Death occurs at the onset of pupation due to the absence of imaginal discs. Mitotic figures in larval neuroblasts were found to be polyploid, apparently due to a defect in cytokinesis. The predicted 123 x 10(3) M(r) protein contains two domains shared by the formin proteins, encoded by the limb deformity gene in the mouse. These formin homology domains, which we have termed FH1 and FH2, are also found in Bni1p, the product of a Saccharomyces cerevisiae gene required for normal cytokinesis in diploid yeast cells.

Amino Acid Sequence↗
PubMed1993

pelle encodes a protein kinase required to establish dorsoventral polarity in the Drosophila embryo.

The pelle gene is required for the nuclear import of dorsal protein that establishes dorsoventral polarity in Drosophila embryos. We report here the genetic mapping and molecular characterization of pelle. DNA sequence analysis revealed that pelle encodes a protein of 501 amino acids, the last 292 of which comprise a protein kinase catalytic domain. Microinjection of in vitro synthesized transcripts containing site-directed mutations indicates that the kinase catalytic domain is required for biological activity. This domain is most similar to that of the rat and mos protein kinases and is predicted to have a serine and threonine specificity. These results provide direct evidence for the role of phosphorylation in the in vivo regulation of a rel-like transcription factor.

Amino Acid Sequence↗
PubMed1993

Domain mapping of tube, a protein essential for dorsoventral patterning of the Drosophila embryo.

The tube protein plays an essential role in the signal transduction pathway that establishes dorsoventral polarity in the Drosophila melanogaster embryo. Characterization of each of four tube mutants revealed a substitution or insertion in the amino-terminal half of the protein. This portion of the tube protein is also evolutionarily conserved, as demonstrated by isolation and sequencing of the Drosophila virilis tube gene. Moreover, RNA microinjection assays and germline transformation experiments demonstrated that the amino-terminal domain alone provides substantial levels of gene function: constructs encoding only the amino-terminal domain restore dorsoventral polarity to embryos lacking any maternal tube function. In the carboxyterminal domain, sequence conservation is concentrated in the five octapeptide repeats. Although the repeat-containing domain by itself provides no rescue of the tube maternal effect phenotype, it is necessary for wild-type levels of tube activity. This domain is thus likely to play an ancillary role in axis formation.

Amino Acid Sequence↗
PubMed1993

Toward a molecular genetic analysis of spermatogenesis in Drosophila melanogaster: characterization of male-sterile mutants generated by single P element mutagenesis.

We describe 83 recessive autosomal male-sterile mutations, generated by single P element mutagenesis in Drosophila melanogaster. Each mutation has been localized to a lettered subdivision of the polytene map. Reversion analyses, as well as complementation tests using available chromosomal deficiencies, indicate that the insertions are responsible for the mutant phenotypes. These mutations represent 63 complementation groups, 58 of which are required for spermatogenesis. Phenotypes of the spermatogenesis mutants were analyzed by light microscopy. Mutations in 12 loci affect germline proliferation, spermatocyte growth, or meiosis. Mutations in 46 other loci disrupt differentiation and maturation of spermatids into motile sperm. This collection of male-sterile mutants provides the basis for a molecular genetic analysis of spermatogenesis.

Animals↗
PubMed1991

Supercoiled DNA-directed knotting by T4 topoisomerase.

The mechanism by which the type 2 topoisomerase from bacteriophage T4 mediates knotting of negatively supercoiled DNA was deduced from an analysis of product topology. The knotted products were nicked and then subjected to electrophoresis in order to separate species on the basis of the minimum number of crossings in the knotted form. Knots with defined numbers of crossings were purified and the configuration of these crossings determined in the electron microscope by the RecA coating method. The product knots were exclusively of the twist form, in which an interwound region is entrapped by a single interlock of two looped ends. The interwound region was of negative sign in greater than 98% of the molecules examined, whereas the single interlock was equally likely to be positive or negative. These results are interpreted in terms of a model for knot formation in which random strand passage mediated by the topoisomerase links bent or branched portions of a superhelix that has a specific interwound geometry. Superhelix interwinding and DNA contacts stabilized by excess enzyme molecules explain the very high frequency of knotting.

DNA Topoisomerases, Type I↗
PubMed1991

Genetic and molecular characterization of tube, a Drosophila gene maternally required for embryonic dorsoventral polarity.

Loss of maternal function of the tube gene disrupts a signaling pathway required for pattern formation in Drosophila, causing cells throughout the embryo to adopt the fate normally reserved for those at the dorsal surface. Here we demonstrate that tube mutations also have a zygotic effect on pupal morphology and that this phenotype is shared by mutations in Toll and pelle, two genes with apparent intracellular roles in determining dorsoventral polarity. We then describe the isolation of a functionally full-length tube cDNA identified in a phenotypic rescue assay. The tube mRNA is expressed maximally early in embryogenesis and again late in larval development, corresponding to required periods of tube activity as defined by distinct maternal and zygotic loss-of-function phenotypes in tube mutants. Sequence analysis of the cDNA indicates that the tube protein contains five copies of an eight-residue motif and shares no significant sequence similarity with known proteins. These results suggest that tube represents a class of protein active in signal transduction at two stages of development.

Amino Acid Sequence↗
PubMed1991

Ultrasound and genetic features of a term triploid pregnancy.

A 69,XXY triploid term infant is reported to demonstrate the prenatal sonographic findings of first trimester intrauterine growth retardation as an important early indicator of this diagnosis. Progressive lag in early sonographic dating for gestational age should raise suspicion of triploidy, which can lead to an early diagnosis, genetic counseling, and a realistic management plan.

Female↗
PubMed1990

Two novel annexins from Drosophila melanogaster. Cloning, characterization, and differential expression in development.

The annexins are a family of homologous Ca2(+)- and phospholipid-binding proteins that until now have only been found in vertebrates. cDNA clones encoding two novel annexins from Drosophila melanogaster were isolated and characterized. RNA blots indicate that the messages for the two Drosophila proteins are differentially expressed in development, with one message being expressed throughout development, while the other is only found in early embryos and adult flies. In situ hybridizations localize the two Drosophila genes to 93B and 19A-4,7. A similarly high degree of homology relates Drosophila annexins to different vertebrate annexins, indicating that the Drosophila annexins are not the invertebrate homologues of particular mammalian annexins but that they constitute novel members of the annexin gene family. In continuation with a recently established terminology, the Drosophila annexins will be named annexins IX and X. The biochemical properties of Drosophila annexin X were investigated using recombinant protein. Similar to vertebrate annexins, annexin X bound to liver membranes and liposomes containing phosphatidylserine in a calcium-dependent manner but not to liposomes containing phosphatidylcholine. In addition, annexin X partitioned into the detergent phase of Triton X-114 as a function of calcium. The conservation of the annexin family of Ca2(+)-binding proteins in invertebrates suggests that they have a basic function in cells which is not peculiar to vertebrate biology, and the availability of the Drosophila sequences will open avenues for mutational studies of these functions.

Amino Acid Sequence↗
PubMed1988

The helical repeat of double-stranded DNA varies as a function of catenation and supercoiling.

DNA in the cell is intertwined at several levels: one polynucleotide strand wraps helically around its complement and the double helix is in turn coiled in space. The higher-order intertwining most often takes the form of supercoiling of the helix axis, but can also be observed as the wrapping of one DNA duplex around another, as in catenation. We have investigated the relationship between intertwining at these three levels, the double helix, supercoiling, and catenation, using an approach that relies on comparative measurements of DNA linking numbers by gel electrophoresis. The method determines both the handedness of DNA catenanes and the change in helical repeat that accompanies catenation-induced supercoiling. For multiply-linked catenated rings of 3.5 kilobase pairs (kb), we conclude that the double helix unwinds by two-thirds of a turn for every right-handed supercoil involved in linking the two circles. Altering the geometry of the catenanes by linking rings of dissimilar size changes the effect of catenation on helical and superhelical parameters. Our experiments used intact DNA rings, but we note that linear DNA molecules, by virtue of their subdivision into closed loops or domains in vivo, can intertwine in the same ways.

DNA, Bacterial↗
PubMed1986

Biochemical topology: applications to DNA recombination and replication.

Processes of DNA rearrangement such as recombination or replication frequently have as products different subsets of the limitless number of distinguishable catenanes or knots. The use of gel electrophoresis and electron microscopy for analysis of these topological isomers has made it possible to deduce physical and geometric features of DNA structure and reaction mechanisms that are otherwise experimentally inaccessible. Quantitative as well as qualitative characterization is possible for any pathway in which the fate of a circular DNA can be followed. The history, theory, and techniques are reviewed and illustrative examples from recent studies are presented.

DNA↗
PubMed1985

Discovery of a predicted DNA knot substantiates a model for site-specific recombination.

The mechanism of site-specific genetic recombination mediated by Tn3 resolvase has been investigated by a topological approach. Extrapolation of a detailed model of synapsis and strand exchange predicts the formation of an additional DNA product with a specific knotted structure. Two-dimensional gel electrophoresis of DNA reacted in vitro revealed a product, about 0.1 percent of the total, with the appropriate mobility. A technique for determining DNA topology by electron microscopy was improved such that less than a nanogram of DNA was required. The structure of the knot was as predicted, providing strong evidence for the model and showing the power of the topological method.

Electrophoresis↗