Developmental regulation of a cyclin-dependent kinase inhibitor controls postembryonic cell cycle progression in Caenorhabditis elegans

C. elegans cki-1 encodes a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, and functions to link postembryonic developmental programs to cell cycle progression. The expression pattern of cki-1::GFP suggests that cki-1 is developmentally regulated in blast cells coincident with G1, and in differentiating cells. Ectopic expression of CKI-1 can prematurely arrest cells in G1, while reducing cki-1 activity by RNA-mediated interference (RNAi) causes extra larval cell divisions, suggesting a role for cki-1 in the developmental control of G1/S. cki-1 activity is required for the suspension of cell cycling that occurs in dauer larvae and starved L1 larvae in response to environmental signals. In vulva precursor cells (VPCs), a pathway of heterochronic genes acts via cki-1 to maintain VPCs in G1 during the L2 stage.     

Molecular cloning and expression of human and mouse tyrosylprotein sulfotransferase-2 and a tyrosylprotein sulfotransferase homologue in Caenorhabditis elegans

Tyrosine O-sulfation, a common post-translational modification in eukaryotes, is mediated by Golgi enzymes that catalyze the transfer of the sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate to tyrosine residues in polypeptides. We recently isolated cDNAs encoding human and mouse tyrosylprotein sulfotransferase-1 (Ouyang, Y. B., Lane, W. S., and Moore, K. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2896-2901). Here we report the isolation of cDNAs encoding a second tyrosylprotein sulfotransferase (TPST), designated TPST-2. The human and mouse TPST-2 cDNAs predict type II transmembrane proteins of 377 and 376 amino acid residues, respectively. The cDNAs encode functional N-glycosylated enzymes when expressed in mammalian cells. In addition, preliminary analysis indicates that TPST-1 and TPST-2 have distinct specificities toward peptide substrates. The human TPST-2 gene is on chromosome 22q12.1, and the mouse gene is in the central region of chromosome 5. We have also identified a cDNA that encodes a TPST in the nematode Caenorhabditis elegans that maps to the right arm of chromosome III. Thus, we have identified two new members of a class of membrane-bound sulfotransferases that catalyze tyrosine O-sulfation. These enzymes may catalyze tyrosine O-sulfation of a variety of protein substrates involved in diverse physiologic functions.     

Mammalian homologue of the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth is a protein kinase C zeta-interacting protein

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C zeta (PKCzeta) as a bait, we have cloned a gene coding for a novel PKCzeta-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKCzeta and weakly with that of PKCepsilon. In the COS-7 cells coexpressing FEZ1 and PKCzeta, FEZ1 was present mainly in the plasma membrane, associating with PKCzeta and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCzeta. When the constitutively active mutant of PKCzeta was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCzeta activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCzeta stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCzeta.     

Identification of a novel, tissue-specific calpain htra-3; a human homologue of the Caenorhabditis elegans sex determination gene

We cloned a novel human gene encoding a tissue-specific calpain, termed htra-3, which is highly homologous to the tra-3 sex determination gene of Caenorhabditis elegans. The predicted htra-3 polypeptide had similarity to the calpain large subunits in domain organization throughout domains I to III, but the sequences of domain IV lacked calcium-binding motifs. Northern blot analysis revealed high expression in the colon, small intestine and testis. Radiation hybrid mapping localized the htra-3 gene to chromosome 11q14 (2.53cR apart from WI-3895). Western blot analysis demonstrated that the approximately 73-kDa htra-3 protein was transiently expressed in COS-7 cells. These observations, together with the genetic information in C.elegans, suggest a unique function for htra-3 protein.     

The human homologue for the Caenorhabditis elegans cul-4 gene is amplified and overexpressed in primary breast cancers

Amplification is a key mechanism whereby a cancer cell increases the message level of genes that confer a selective advantage when they are overexpressed. In breast cancer, there are many chromosome regions present in multiple copies relative to overall DNA copy number (amplicons), and their target genes are unknown. Using differential display, we have cloned and sequenced the full coding region of a candidate amplicon target gene located on chromosome 13. This candidate is the human homologue of the Caenorhabditis elegans cul-4 gene, cul-4A, a member of the novel cullin gene family, which is involved in cell cycle control of C. elegans. cul-4A was amplified and overexpressed in 3 of 14 breast cancer cell lines analyzed, and it was overexpressed in 8 additional cell lines in which it was not amplified. The latter observation, indicating that its overexpression can occur by mechanisms other than gene amplification, suggests that cul-4A plays a key role in carcinogenesis. Moreover, cul-4A was found to be amplified in 17 of 105 (16%) cases of untreated primary breast cancers, and 14 of 30 cases analyzed (47%) were shown by RNA in situ hybridization to overexpress cul-4A. These results suggest that up-regulation of cul-4A may play an important role in tumor progression.     

Formation of the vulva in Caenorhabditis elegans: a paradigm for organogenesis

The genes involved in the inductive interactions that specify cell fates in the vulva of Caenorhabditis elegans are known in some detail. However, little is known about the morphogenesis of this organ. Using a combination of cell biological and anatomical approaches, we have determined a complete morphogenetic pathway of cellular events that lead to the formation of the vulva. These events include reproducible cell divisions, migrations, remodeling of adherens junctions, cell fusions and muscle attachments. In the course of these events, an epithelial channel comprising a stack of 7 toroidal cells is formed that connects the internal epithelium of the uterus with the external body epithelium, forming the vulva. Vulval muscles attach to the epithelial channel and the whole structure everts during the final molt. The mature vulva has rotational, two-fold symmetry. Using laser microsurgery, we found that the two halves of the vulva develop autonomously.     

Gas chromatographic assay of volatile anesthetics: some problems and a solution

A modification of the gas chromatographic procedure of Fink and Morikawa (ANESTHESIOLOGY 32:451-455, 1970) for measuring inhalation anesthetics is presented. Emphasis is placed on separating vehicular gas from anesthetic vapor by using appropriate column packing material and assay conditions.     

Cloning, expression and mapping of a novel RING-finger gene (RNF5), a human homologue of a putative zinc-finger gene from Caenorhabditis elegans

The RING-finger is a unique zinc-chelating domain involved in mediating protein-protein interactions. The extensive sequence homology within the RING-finger domain allowed us to clone a novel member of the RING-finger family of genes. This cDNA clone, designated RNF5 (Ring-finger protein 5), contained an open reading frame of 540 nucleotides. Its predicted amino acid sequence revealed significant homology to a hypothetical protein encoded by Caenorhabditis elegans cosmid C16C10.7. The expression of RNF5 was detected in a variety of human tissues. The RNF5 gene was mapped by fluorescence in situ hybridization to chromosome 6p21.31. Radiation hybrid mapping further assigned RNF5 to a region proximal to the major histocompatibility complex (MHC) on chromosome 6. RNF5 is the third RING-finger gene identified in the region proximal to MHC raising the possibility that the RING-finger family of genes may exist as a cluster in this region.     

Mapping a telomere using the translocation eT1(III;V) in Caenorhabditis elegans

TCR engagement activates phospholipase C gamma 1 (PLC gamma 1) via a tyrosine phosphorylation-dependent mechanism. PLC gamma 1 contains a pair of Src homology 2 (SH2) domains whose function is that of promoting protein interactions by binding phosphorylated tyrosine and adjacent amino acids. The role of the PLC gamma 1 SH2 domains in PLC gamma 1 phosphorylation was explored by mutational analysis of an epitope-tagged protein transiently expressed in Jurkat T cells. Mutation of the amino-terminal SH2 domain (SH2(N) domain) resulted in defective tyrosine phosphorylation of PLC gamma 1 in response to TCR/CD3 perturbation. In addition, the PLC gamma 1 SH2(N) domain mutant failed to associate with Grb2 and a 36- to 38-kDa phosphoprotein (p36-38), which has previously been recognized to interact with PLC gamma 1, Grb2, and other molecules involved in TCR signal transduction. Conversely, mutation of the carboxyl-terminal SH2 domain (SH2(C) domain) did not affect TCR-induced tyrosine phosphorylation of PLC gamma 1. Furthermore, binding of p36-38 to PLC gamma 1 was not abrogated by mutations of the SH2(C) domain. In contrast to TCR/CD3 ligation, treatment of cells with pervanadate induced tyrosine phosphorylation of either PLC gamma 1 SH2(N) or SH2(C) domain mutants to a level comparable with that of the wild-type protein, indicating that pervanadate treatment induces an alternate mechanism of PLC gamma 1 phosphorylation. These data indicate that the SH2(N) domain is required for TCR-induced PLC gamma 1 phosphorylation, presumably by participating in the formation of a complex that promotes the association of PLC gamma 1 with a tyrosine kinase.     

gon-2, a gene required for gonadogenesis in Caenorhabditis elegans

The gonad of the Caenorhabditis elegans hermaphrodite is generated by the postembryonic divisions of two somatic precursors, Z1 and Z4, and two germline precursors, Z2 and Z3. These cells begin division midway through the first larval stage. By the end of the fourth larval stage, Z1 and Z4 produce 143 descendants, while Z2 and Z3 give rise to approximately 1000 descendants. The divisions of Z2 and Z3 are dependent on signals produced by Z1 and Z4, but not vice versa. We have characterized the properties of five loss-of-function alleles of a newly described gene, which we call gon-2. In gon-2 mutants, gonadogenesis is severely impaired; in some animals, none of the gonad progenitors undergo any postembryonic divisions. Mutations in gon-2 have a partial maternal effect: either maternal or zygotic expression is sufficient to prevent the severe gonadogenesis defects. By cell lineage analysis, we found that the priman, defect in gon-2 mutants is a delay (sometimes a complete block) in the onset and continuation of gonadal divisions. The results of upshift experiments using a temperature-sensitive allele suggest that zygotic expression of gon-2 begins early in embryogenesis, before the birth of Z1 and Z4. The results of downshift experiments suggest that Z1 and Z4 can generate the full complement of gonadal tissues even when gon-2 function is inhibited until the end of the second larval stage. Thus, gon-2 activity is probably not required for the specification of gonadal cell fates, but appears to be generally required for gonadal cell divisions.     

Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast

Autophagy is a bulk protein degradation process that is induced by starvation. The control mechanism for induction of autophagy is not well understood. We found that Tor, a phosphatidylinositol kinase homologue, is involved in the control of autophagy in the yeast, Saccharomyces cerevisiae. When rapamycin, an inhibitor of Tor function, is added, autophagy is induced even in cells growing in nutrient-rich medium. A temperature-sensitive tor mutant also leads to induction of autophagy at a nonpermissive temperature. These results indicate that Tor negatively regulates the induction of autophagy. Tor is the first molecule that is identified as a pivotal player in the starvation-signaling pathway of autophagy. Furthermore, we found that a high concentration of cAMP is inhibitory for induction of autophagy. APG gene products are involved in autophagy induced by starvation. Autophagy was not induced in apg mutants in the presence of rapamycin, indicating that the site of action of Tor is upstream of those of Apg proteins. In nutrient-rich medium, Apg proteins are involved also in the transport of aminopeptidase I from the cytosol to the vacuole. Tor may act to switch Apg function between autophagy and transport of aminopeptidase I.     

Homologs of the yeast longevity gene LAG1 in Caenorhabditis elegans and human

LAG1 is a longevity gene, the first such gene to be identified and cloned from the yeast Saccharomyces cerevisiae. A close homolog of this gene, which we call LAC1, has been found in the yeast genome. We have cloned the human homolog of LAG1 with the ultimate goal of examining its possible function in human aging. In the process, we have also cloned a homolog from the nematode worm Caenorhabditis elegans. Both of these homologs, LAG1Hs and LAG1Ce-1, functionally complemented the lethality of a lag1delta lac1delta double deletion, despite low overall sequence similarity to the yeast proteins. The proteins shared a short sequence, the Lag1 motif, and a similar transmembrane domain profile. Another, more distant human homolog, TRAM, which lacks this motif, did not complement. LAG1Hs also restored the life span of the double deletion, demonstrating that it functions in establishing the longevity phenotype in yeast. LAG1Hs mapped to 19p12, and it was expressed in only three tissues: brain, skeletal muscle, and testis. This gene possesses a trinucleotide (CTG) repeat within exon 1. This and its expression profile raise the possibility that it may be involved in neurodegenerative disease. This possibility suggests at least one way in which LAG1Hs might be involved in human aging.     

Three proteins involved in Caenorhabditis elegans vulval invagination are similar to components of a glycosylation pathway

We have molecularly analyzed three genes, sqv-3, sqv-7, and sqv-8, that are required for wild-type vulval invagination in Caenorhabditis elegans. The predicted SQV-8 protein is similar in sequence to two mammalian beta(1,3)-glucuronyltransferases, one of which adds glucuronic acid to protein-linked galactose-beta(1, 4)-N-acetylglucosamine. SQV-3 is similar to a family of glycosyltransferases that includes vertebrate beta(1, 4)-galactosyltransferases, which create galactose-beta(1, 4)-N-acetylglucosamine linkages. One model is therefore that SQV-8 uses a SQV-3 product as a substrate. SQV-7 is similar to members of a family of nucleotide-sugar transporters. The sqv genes therefore are likely to encode components of a conserved glycosylation pathway that assembles a C. elegans carbohydrate moiety, the absence of which perturbs vulval invagination.     

An activating mutation in a Caenorhabditis elegans Gs protein induces neural degeneration

Heterotrimeric guanine nucleotide-binding proteins (G proteins) act as signal-transducing molecules that connect serpentine-transmembrane receptors to a variety of intracellular effectors. We characterized a Caenorhabditis elegans G(s) gene, gsa-1, which encodes a G(s) alpha-subunit (G alpha(s)) that is expressed throughout the nervous system and in muscle cells. gsa-1 is an essential gene; a loss-of-function mutation in gsa-1 results in lethality at the first stage of larval development. Partial (mosaic) loss of G alpha(s) expression or overexpression of the protein results in reciprocal defects in movement and egg-laying, suggesting a role for G alpha(s) in the regulation of these behaviors. Expression of a constitutively active form of G alpha(s) from an inducible promotor results in hypercontraction of body-wall muscle cells and vacuolization and degeneration of neurons within hours of induction. Neurons that are susceptible to the degeneration induced by activated G alpha(s) are predominantly motoneurons located within the ventral nerve cord. Phenotypic analysis shows that the induced neural degeneration is not the result of programmed cell death but is probably caused by the activation of ion channels. A genetic suppressor of activated G alpha(s) was isolated that identifies a putative downstream target of G(s) signaling.     

Mutations causing neurodegeneration in Caenorhabditis elegans drastically alter the pH sensitivity and inactivation of the mammalian H+-gated Na+ channel MDEG1

The mammalian degenerin MDEG1 belongs to the nematode degenerin/epithelial Na+ channel superfamily. It is constitutively activated by the same mutations that cause gain-of-function of the Caenorhabditis elegans degenerins and neurodegeneration. ASIC and DRASIC, which were recently cloned, are structural homologues of MDEG1 and behave as H+-gated cation channels. MDEG1 is also a H+-activated Na+ channel, but it differs from ASIC in its lower pH sensitivity and slower kinetics. In addition to the generation of a constitutive current, mutations in MDEG1 also alter the properties of the H+-gated current. Replacement of Gly-430 in MDEG1 by bulkier amino acids, such as Val, Phe, or Thr, drastically increases the H+ sensitivity of the channel (half-maximal pH (pHm) approximately 4.4 for MDEG1, pHm approximately 6.7 for the different mutants). Furthermore, these replacements completely suppress the inactivation observed with the wild-type channel and increase the sensitivity of the H+-gated channel to blockade by amiloride by a factor of 10 without modification of its conductance and ionic selectivity. These results as well as those obtained with other mutants clearly indicate that the region surrounding Gly-430, situated just before the second transmembrane segment, is essential for pH sensitivity and gating.     

Isolation, pharmacology and gene organization of KPSFVRFamide: a neuropeptide from Caenorhabditis elegans

To date, 53 peptides with C-terminal RFamides have been identified by the genome sequencing project in the nematode, Caenorhabditis elegans. In this study the FMRFamide-related peptide (FaRP) KPSFVRFamide (879.90 Da [MH]+) was structurally characterized from extracts of the nematode, Caenorhabditis elegans. Two copies of KPSFVRFamide are encoded by a gene designated flp-9. RT-PCR identified a single cDNA product which was confirmed as flp-9 by sequence determination. Flp-9 cDNA was isolated from larval stages of C. elegans but was not detected in adult worms, indicating that its expression is may be developmentally regulated. KPSFVRFamide displays sequence homology to the nematode peptide, KPNFIRFamide (PF4). The physiological effects of KPSFVRFamide, PF4 and the chimeras, KPNFVRFamide and KPSFIRFamide, were measured on body wall muscle and the vagina vera of the parasitic nematode, Ascaris suum. KPNFVRFamide and KPNFIRFamide had Cl--dependent inhibitory activity on innervated and denervated muscle-preparations, whereas KPSFVRFamide and KPSFIRFamide did not elicit a detectable physiological effect. Although all 4 peptides had inhibitory effects on the vagina vera, KPSFVRFamide and KPSFIRFamide (threshold, >/=0.1 microM) were less potent than KPNFVRFamide and KPNFIRFamide (threshold, >/=10 nM).     

Genetic and molecular analysis of fox-1, a numerator element involved in Caenorhabditis elegans primary sex determination

fox-1 was previously identified as a candidate numerator element based on its overexpression phenotype. FOX-1 is an RRM-type RNA-binding protein, which can bind RNAs in vitro. Western analysis detects FOX-1 throughout development. fox-1::lacZ comes on ubiquitously early during embryogenesis. Postembryonically, fox-1::lacZ is expressed sex specifically in a subset of cells in the head and tail. We describe a Tc1-derived deletion allele [fox-1(Delta)] that removes the RRM domain. fox-1(Delta) confers no phenotype in XXs, but can rescue XO-specific lethality and feminization caused by duplications of the left end of the X. fox-1(Delta) synergizes with putative numerators, resulting in abnormal XX development. Genetic analysis indicated that fox-1(Delta) leads to a slight increase in xol-1 activity, while fox-1(gf) leads to partial loss of xol-1 activity, and xol-1 is epistatic to fox-1. RNase protection experiments revealed increased levels of the 2.2-kb xol-1 message in fox-1(Delta) animals, and reduced levels in fox-1(gf) animals. Additionally, fox-1(Delta) impairs male mating efficiency, which, we propose, represents another function of fox-1, independent of xol-1 and its role in sex determination.     

HOP-1, a Caenorhabditis elegans presenilin, appears to be functionally redundant with SEL-12 presenilin and to facilitate LIN-12 and GLP-1 signaling

Mutant presenilins have been found to cause Alzheimer disease. Here, we describe the identification and characterization of HOP-1, a Caenorhabditis elegans presenilin that displays much more lower sequence identity with human presenilins than does the other C. elegans presenilin, SEL-12. Despite considerable divergence, HOP-1 appears to be a bona fide presenilin, because HOP-1 can rescue the egg-laying defect caused by mutations in sel-12 when hop-1 is expressed under the control of sel-12 regulatory sequences. HOP-1 also has the essential topological characteristics of the other presenilins. Reducing hop-1 activity in a sel-12 mutant background causes synthetic lethality and terminal phenotypes associated with reducing the function of the C. elegans lin-12 and glp-1 genes. These observations suggest that hop-1 is functionally redundant with sel-12 and underscore the intimate connection between presenilin activity and LIN-12/Notch activity inferred from genetic studies in C. elegans and mammals.