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1.
Genetics ; 209(1): 173-193, 2018 05.
Article in English | MEDLINE | ID: mdl-29531012

ABSTRACT

Covalent attachment of ubiquitin to substrate proteins changes their function or marks them for proteolysis, and the specificity of ubiquitin attachment is mediated by the numerous E3 ligases encoded by animals. Mind Bomb is an essential E3 ligase during Notch pathway signaling in insects and vertebrates. While Caenorhabditis elegans encodes a Mind Bomb homolog (mib-1), it has never been recovered in the extensive Notch suppressor/enhancer screens that have identified numerous pathway components. Here, we show that C. elegans mib-1 null mutants have a spermatogenesis-defective phenotype that results in a heterogeneous mixture of arrested spermatocytes, defective spermatids, and motility-impaired spermatozoa. mib-1 mutants also have chromosome segregation defects during meiosis, molecular null mutants are intrinsically temperature-sensitive, and many mib-1 spermatids contain large amounts of tubulin. These phenotypic features are similar to the endogenous RNA intereference (RNAi) mutants, but mib-1 mutants do not affect RNAi. MIB-1 protein is expressed throughout the germ line with peak expression in spermatocytes followed by segregation into the residual body during spermatid formation. C. elegans mib-1 expression, while upregulated during spermatogenesis, also occurs somatically, including in vulva precursor cells. Here, we show that mib-1 mutants suppress both lin-12 and glp-1 (C. elegans Notch) gain-of-function mutants, restoring anchor cell formation and a functional vulva to the former and partly restoring oocyte production to the latter. However, suppressed hermaphrodites are only observed when grown at 25°, and they are self-sterile. This probably explains why mib-1 was not previously recovered as a Notch pathway component in suppressor/enhancer selection experiments.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Glucagon-Like Peptide 1/metabolism , Receptors, Notch/metabolism , Spermatogenesis/genetics , Ubiquitin-Protein Ligases/genetics , Alleles , Animals , Caenorhabditis elegans Proteins/genetics , Gene Expression Regulation, Developmental , Loss of Function Mutation , Mutation , Phenotype , Signal Transduction , Ubiquitin-Protein Ligases/metabolism
2.
Genetics ; 191(2): 477-91, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22446317

ABSTRACT

Secretory vesicles are used during spermatogenesis to deliver proteins to the cell surface. In Caenorhabditis elegans, secretory membranous organelles (MO) fuse with the plasma membrane to transform spermatids into fertilization-competent spermatozoa. We show that, like the acrosomal vesicle of mammalian sperm, MOs undergo acidification during development. Treatment of spermatids with the V-ATPase inhibitor bafilomycin blocks both MO acidification and formation of functional spermatozoa. There are several spermatogenesis-defective mutants that cause defects in MO morphogenesis, including spe-5. We determined that spe-5, which is on chromosome I, encodes one of two V-ATPase B paralogous subunits. The spe-5 null mutant is viable but sterile because it forms arrested, multi-nucleate spermatocytes. Immunofluorescence with a SPE-5-specific monoclonal antibody shows that SPE-5 expression begins in spermatocytes and is found in all subsequent stages of spermatogenesis. Most SPE-5 is discarded into the residual body during spermatid budding, but a small amount remains in budded spermatids where it localizes to MOs as a discrete dot. The other V-ATPase B subunit is encoded by vha-12, which is located on the X chromosome. Usually, spe-5 mutants are self-sterile in a wild-type vha-12 background. However, an extrachromosomal transgene containing wild-type vha-12 driven by its own promoter allows spe-5 mutant hermaphrodites to produce progeny, indicating that VHA-12 can at least partially substitute for SPE-5. Others have shown that the X chromosome is transcriptionally silent in the male germline, so expression of the autosomally located spe-5 gene ensures that a V-ATPase B subunit is present during spermatogenesis.


Subject(s)
Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Secretory Vesicles/metabolism , Spermatogenesis/genetics , Amino Acid Sequence , Animals , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Gene Expression , Male , Molecular Sequence Data , Mutation , Protein Transport , Sequence Alignment , Spermatozoa/metabolism , Testis/metabolism , Vacuolar Proton-Translocating ATPases/genetics , Vacuolar Proton-Translocating ATPases/metabolism
3.
Am J Hum Genet ; 76(4): 663-71, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15717285

ABSTRACT

Previously, our group reported a five-generation family in which a balanced t(13;17) translocation is associated with a spectrum of skeletal abnormalities, including Robin sequence, hypoplastic scapulae, and a missing pair of ribs. Using polymerase chain reaction (PCR) with chromosome-specific markers to analyze DBA from somatic cell hybrids containing the derivative translocation chromosomes, we narrowed the breakpoint on each chromosome. Subsequent sequencing of PCR products spanning the breakpoints identified the breaks precisely. The chromosome 17 breakpoint maps approximately 932 kb upstream of the sex-determining region Y (SRY)-related high-mobility group box gene (SOX) within a noncoding transcript represented by two IMAGE cDNA clones. A growing number of reports have implicated chromosome 17 breakpoints at a distance of up to 1 Mb from SOX9 in some cases of campomelic dysplasia (CD). Although this multigeneration family has a disorder that shares some features with CD, their phenotype is significantly milder than any reported cases of (nonmosaic) CD. Therefore, this case may represent an etiologically distinct skeletal dysplasia or may be an extremely mild familial example of CD, caused by the most proximal translocation breakpoint from SOX9 reported to date. In addition, we have refined the breakpoint in a acampomelic CD case described elsewhere and have found that it lies approximately 900 kb upstream of SOX9.


Subject(s)
Bone Diseases, Developmental/genetics , Chromosomes, Human, Pair 17 , High Mobility Group Proteins/genetics , Transcription Factors/genetics , Base Sequence , Chromosome Breakage , Chromosome Mapping , Humans , Male , Molecular Sequence Data , SOX9 Transcription Factor , Translocation, Genetic
4.
FASEB J ; 18(15): 1834-50, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15576487

ABSTRACT

ADP-ribosylation factor (Arf) and Arf-like (Arl) proteins are a family of highly conserved 21 kDa GTPases that emerged early in the evolution of eukaryotes. These proteins serve regulatory roles in vesicular traffic, lipid metabolism, microtubule dynamics, development, and likely other cellular processes. We found evidence for the presence of 6 Arf family members in the protist Giardia lamblia and 22 members in mammals. A phylogenetic analysis was performed to delineate the evolutionary relationships among Arf family members and to attempt to organize them by both their evolutionary origins and functions in cells and/or organisms. The approximately 100 protein sequences analyzed from animals, fungi, plants, and protists clustered into 11 groups, including Arfs, nine Arls, and Sar proteins. To begin functional analyses of the family in a metazoan model organism, we examined roles for all three C. elegans Arfs (Arf-1, Arf-3, and Arf-6) and three Arls (Arl-1, Arl-2, and Arl-3) by use of RNA-mediated interference (RNAi). Injection of double-stranded RNA (dsRNA) encoding Arf-1 or Arf-3 into N2 hermaphrodites produced embryonic lethality in their offspring and, later, sterility in the injected animals themselves. Injection of Arl-2 dsRNA resulted in a disorganized germline and sterility in early offspring, with later offspring exhibiting an early embryonic arrest. Thus, of the six Arf family members examined in C. elegans, at least three are required for embryogenesis. These data represent the first analysis of the role(s) of multiple members of this family in the development of a multicellular organism.


Subject(s)
ADP-Ribosylation Factors/classification , ADP-Ribosylation Factors/physiology , Caenorhabditis elegans/embryology , Caenorhabditis elegans/enzymology , Phylogeny , ADP-Ribosylation Factors/genetics , Animals , Caenorhabditis elegans/genetics , Eukaryotic Cells/enzymology , Genomics , Green Fluorescent Proteins/metabolism , Membrane Proteins/classification , RNA Interference
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