Molecular characterization of two homologs of the Caenorhabditis elegans cadmium-responsive gene cdr-1: cdr-4 and cdr-6

A novel cadmium-inducible gene, cdr-1, was previously identified and characterized in the nematode Caenorhabditis elegans and found to mediate resistance to cadmium toxicity. Subsequently, six homologs of cdr-1 were identified in C. elegans. Here, we describe two homologs: cdr-4, which is metal inducible, and cdr-6, which is noninducible. Both cdr-4 and cdr-6 mRNAs contain open reading frames of 831 nt and encode predicted 32-kDa integral membrane proteins, which are similar to CDR-1. cdr-4 expression is induced by arsenic, cadmium, mercury, and zinc exposure as well as by hypotonic stress. In contrast, cdr-6 is constitutively expressed at a high level in C. elegans, and expression is not affected by these stressors. Both cdr-4 and cdr-6 are transcribed in postembryonic pharyngeal and intestinal cells in C. elegans. In addition, cdr-4 is transcribed in developing embryos. Like CDR-1, CDR-4 is targeted to intestinal cell lysosomes in vivo. Inhibition of CDR-4 and/or CDR-6 expression does not render C. elegans more susceptible to cadmium toxicity; however, there is a significant decrease in their lifespan in the absence of metal. Although nematodes in which CDR-4 and/or CDR-6 expression is knocked down accumulate fluid in the pseudocoelomic space, exposure to hypertonic conditions did not significantly affect growth or reproduction in these nematodes. These results suggest that CDR expression is required for optimal viability but does not function in osmoregulation.     

Changes in Nuclear Receptor and Vitellogenin Gene Expression in Response to Steroids and Heavy Metal in Caenorhabditis elegans

To gain basic understanding of the reproductive and developmental effects of endocrine disrupting chemicals in invertebrates, we have used C. elegans as an animal model. The completion of the C. elegans genome sequence brings to bear microarray analysis as a tool for these studies. We previously showed that the C. elegans genome was responsive to vertebrate steroid hormones, and changes in gene expression of traditional biomarkers used in environmental studies were detected; i.e., vitellogenin (vtg), cytochrome P450 (cyp450), glutathione-S-transferase (gst) and heat shock proteins (hsp). The data were interpreted to suggest that exogenous lipophilic compounds can be metabolized via cytochrome P450 proteins, and that the resulting metabolites can bind to members of the Nuclear Receptor (NR) class of proteins and regulate gene expression. In the present study, using DNA microarrays, we examined the pattern of gene expression after progesterone (10(-5), 10(-7) M), estradiol (10(-5) M), cholesterol (10(-9) M) and cadmium (0.1, 1 and 10 μM) exposure, with special attention to the members of NRs. Of approximately 284 NRs in C. elegans, expression of 25 NR genes (representing 9% of the total NRs in C. elegans) was altered after exposure to steroids. Of note, each steroid activated or inhibited different subsets of NR genes, and only estradiol regulated NR genes implicated in neurogenesis. These results suggest that NRs respond to a variety of exogenous steroids, which regulate important metabolic and developmental pathways. The response of the C. elegans genome to cholesterol and cadmium was analyzed in more detail. Cholesterol is a probable precursor to signaling molecules that may interact with NRs and we focused on expression of genes related to lipid metabolism (cyp450), transport and storage (i.e., vitellogenin). Worms exposed to cadmium respond principally by activating the expression of genes encoding stress-responsive proteins, such as mtl-2 and cdr-1, and no significant changes in expression of NRs or vtg genes were observed. The possible implications of these results with regard to the evolution of steroid receptors, endocrine disruption and the role of vitellogenin as a lipid transporter are discussed.     

Characterization of ATDRG1, a member of a new class of GTP-binding proteins in plants

We report the initial characterization of an Arabidopsis thaliana cDNA (atdrg1), a member of a new class of GTP-binding proteins (G-proteins) in plants. The predicted ATDRG1 protein contains all five structural motifs characteristic of the G-protein superfamily. Apart from these motifs, the amino acid sequence differs substantially from all known G-proteins except for a recently discovered new family named developmentally regulated G-proteins (DRGs). Sequences closely related to atdrg1 are found in species as distant as human (80% amino acid conservation), Drosophila (74%), yeast (77%) and Caenorhabditis elegans (77%). The remarkable evolutionary conservation of these proteins suggests an important, but as yet unclear role. Phylogenetic analysis of the available homologous sequences strongly suggests a diphyletic origin of the eukaryotic DRG proteins. Northern analysis shows high levels of atdrg1 mRNA in all Arabidopsis tissues studied, and homologues of atdrg1 are present throughout the plant kingdom. In situ hybridization reveals that atdrg1 is highly expressed in actively growing tissues and reproductive organs. Southern analysis indicates the presence of either one or two copies of atdrg1 in the Arabidopsis genome. Immunolocalization studies show that the protein is present in cytoplasmic vesicles found mainly in actively growing tissues suggesting a putative role for ATDRG1 in either the regulation of vesicle transport or the regulation of enzymes involved in storage protein processing.     

Human and mouse homologues of the Drosophila melanogaster tweety (tty) gene: a novel gene family encoding predicted transmembrane proteins

We have cloned cDNA for TTYH1, a human homologue of the Drosophila melanogaster tweety (tty) gene. The 450-residue predicted protein shows 27% amino acid sequence identity (51% similarity) to the Drosophila protein, which contains an additional C-terminal repetitive region. A second Drosophila homologue exhibits 42% identity (65% similarity) to the tty protein. Mouse (Ttyh1), macaque, and Caenorhabditis elegans homologues were also identified, and the complete coding sequence for the mouse gene was determined. The mouse protein is 91% identical to the human protein. Hydrophobicity analysis of the tty-related proteins indicates that they represent a new family of membrane proteins with five potential membrane-spanning regions. The yeast FTR1 and FTH1 iron transporter proteins and the mammalian neurotensin receptors 1 and 2 have a similar hydrophobicity profile, although there is no detectable sequence homology to the tty-related proteins. This suggests that the tweety-related proteins could be involved in transport of iron or other divalent cations or alternatively that they may be membrane-bound receptors. TTYH1 was mapped to chromosome 19q13.4 by FISH and by radiation hybrid mapping using the Stanford G3 panel.     

eat-5 and unc-7 represent a multigene family in Caenorhabditis elegans involved in cell-cell coupling

The Drosophila melanogaster genes Passover and l(1)ogre and the Caenorhabditis elegans gene unc-7 define a gene family whose function is not known. We have isolated and characterized the C. elegans gene eat-5, which is required for synchronized pharyngeal muscle contractions, and find that it is a new member of this family. Simultaneous electrical and video recordings reveal that in eat-5 mutants, action potentials of muscles in the anterior and posterior pharynx are unsynchronized. Injection of carboxyfluorescein into muscles of the posterior pharynx demonstrates that all pharyngeal muscles are dye-coupled in wild-type animals; in eat-5 mutants, however, muscles of the anterior pharynx are no longer dye-coupled to posterior pharyngeal muscles. We show that a gene fusion of eat-5 to the green fluorescent protein is expressed in pharyngeal muscles. unc-7 and eat-5 are two of at least sixteen members of this family in C. elegans as determined by database searches and PCR-based screens. The amino acid sequences of five of these members in C. elegans have been deduced from cDNA sequences. Polypeptides of the family are predicted to have four transmembrane domains with cytoplasmic amino and carboxyl termini. We have constructed fusions of one of these polypeptides with beta-galactosidase and with green fluorescent protein. The fusion proteins appear to be localized in a punctate pattern at or near plasma membranes. We speculate that this gene family is required for the formation of gap junctions.     

Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans.

A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology.     

Isolation and characterization of two evolutionarily conserved murine kinases (Nek6 and nek7) related to the fungal mitotic regulator, NIMA

Entrance and exit from mitosis in Aspergillus nidulans require activation and proteolysis, respectively, of the NIMA (never in mitosis, gene A) serine/threonine kinase. Four different NIMA-related kinases were reported in mammals (Nek1-4), but none of them has been shown to perform mitotic functions related to those demonstrated for NIMA. We describe here the isolation of two novel murine protein kinase genes, designated nek6 and nek7, which are highly similar to each other (87% amino acid identity in the predicted kinase domain). Interestingly, Nek6 and Nek7 are also highly similar to the F19H6.1 protein kinase of Caenorhabditis elegans (76 and 73% amino acid identity in the kinase domain, respectively), and phylogenetic analysis suggests that these three proteins constitute a novel subfamily within the NIMA family of serine/threonine kinases. In contrast to the other documented NIMA-related kinases, Nek6/7 and F19H6.1 harbor their catalytic domain in the C-terminus of the protein. Immunofluorescence suggests that Nek6 and Nek7 are cytoplasmic. Linkage analysis, using the murine BXD recombinant inbred strain panel, localized nek6 to chromosome 2 at 28 cM. Using a mouse/hamster radiation hybrid panel, we assigned the nek7 gene to chromosome 1 at approximately 73 cM.     

Molecular analysis of neisserial Opa protein interactions with the CEA family of receptors: identification of determinants contributing to the differential specificities of binding

The carcinoembryonic antigen (CEA) gene family members, CEACAM1, CEACAM3, CEACAM5 and CEACAM6, are bound by the Opa outer membrane proteins of pathogenic Neisseria spp., whereas CEACAM8 is not. In this study, we demonstrate that the closely related CEACAM4 and CEACAM7, which are also members of the CEA family, are not Opa receptors. We exploited the high conservation between CEACAM6 and CEACAM8 to generate an extensive set of chimeric receptors in order to delineate the sequences necessary for Opa binding. Using a transfection-based infection system, we showed that binding of Opa₅₂ involves residues 27–42, which are predicted to form β-strand C and short loops adjacent to it, and residues lying between amino acids 60 and 108 in the amino-terminal domain. The replacement of residues 27–29 in CEACAM6 with the CEACAM1 or CEACAM5 sequences generated recombinant CEACAM6 receptors that are bound by CEACAM1/CEACAM5-specific Opa variants. Together, our data demonstrate that Opa proteins bind to residues exposed on the GFCC' face of the N-terminal domain of CEACAM receptors, and identify an amino acid triplet sequence that is responsible for the differential binding of Opa proteins to CEACAM1, CEACAM5 and CEACAM6.