SMC1: an essential yeast gene encoding a putative head-rod-tail protein is required for nuclear division and defines a new ubiquitous protein family

The smc1-1 mutant was identified initially as a mutant of Saccharomyces cerevisiae that had an elevated rate of minichromosome nondisjunction. We have cloned the wild-type SMC1 gene. The sequence of the SMC1 gene predicts that its product (Smc1p) is a 141-kD protein, and antibodies against Smc1 protein detect a protein with mobility of 165 kD. Analysis of the primary and putative secondary structure of Smc1p suggests that it contains two central coiled-coil regions flanked by an amino- terminal nucleoside triphosphate (NTP)-binding head and a conserved carboxy-terminal tail. These analyses also indicate that Smc1p is an evolutionary conserved protein and is a member of a new family of proteins ubiquitous among prokaryotes and eukaryotes. The SMC1 gene is essential for viability. Several phenotypic characteristics of the mutant alleles of smc1 gene indicate that its product is involved in some aspects of nuclear metabolism, most likely in chromosome segregation. The smc1-1 and smc1-2 mutants have a dramatic increase in mitotic loss of a chromosome fragment and chromosome III, respectively, but have no increase in mitotic recombination. Depletion of SMC1 function in the ts mutant, smc1-2, causes a dramatic mitosis-related lethality. Smc1p-depleted cells have a defect in nuclear division as evidenced by the absence of anaphase cells. This phenotype of the smc1- 2 mutant is not RAD9 dependent. Based upon the facts that Smc1p is a member of a ubiquitous family, and it is essential for yeast nuclear division, we propose that Smc1p and Smc1p-like proteins function in a fundamental aspect of prokaryotic and eukaryotic cell division.     

The Drosophila DSP1 gene encoding an HMG 1-like protein: genomic organization,evolutionary conservation and expression

The gene that encodes the dorsal switch protein (DSP1) has been isolated from a Drosophila melanogaster cosmid library. It is organized into seven exons and six introns. The relative position of the introns within the region coding for the high mobility group (HMG) domains are identical to those of vertebrate HMG 1/2 genes. The close similarity between DSP1 and HMG 1/2 genes strongly suggests that these genes derived from a common ancestral gene. DSP1 encodes, at least, two distinct mRNAs that differ in the length of their 5′-untranslated region and coding sequence. Detailed sequence analysis shows that alternative splicing of precursor mRNA gives rise to the two isoform mRNAs found in Drosophila cells.     

Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1.

We have isolated a novel liver-specific organic anion transporter, LST-1, that is expressed exclusively in the human, rat, and mouse liver. LST-1 is a new gene family located between the organic anion transporter family and prostaglandin transporter. LST-1 transports taurocholate (Km = 13.6 microM) in a sodium-independent manner. LST-1 also shows broad substrate specificity. It transports conjugated steroids (dehydroepiandrosterone sulfate, estradiol-17beta-glucuronide, and estrone-3-sulfate), eicosanoids (prostaglandin E2, thromboxane B2, leukotriene C4, leukotriene E4), and thyroid hormones (thyroxine, Km = 3.0 microM and triiodothyronine, Km = 2.7 microM), reflecting hepatic multispecificity. LST-1 is probably the most important transporter in human liver for clearance of bile acids and organic anions because hepatic levels of another organic anion transporter, OATP, is very low. This is also the first report of the human molecule that transports thyroid hormones.     

Expression of MUL, a gene encoding a novel RBCC family ring-finger protein, in human and mouse embryogenesis

In mammals, the SWI/SNF complex is involved in chromatin remodelling in a wide range of cellular events for which regulatory factors require access to DNA. In the present study, we analyzed in early postimplantation mouse embryos the expression pattern of BRM (SNF2alpha) and BRG1 (SNF2beta), which are both ATPase subunits of this complex. Contrarily to the previous studies conducted in adult mice, showing the ubiquitous and overlapping expressions of BRM and BRG1, we show that BRM expression is restricted to mesodermal tissues involved in early vasculogenesis and heart morphogenesis.     

NABP1, a novel RORgamma-regulated gene encoding a single-stranded nucleic-acid-binding protein

RORgamma2 (retinoid-related orphan receptor gamma2) plays a critical role in the regulation of thymopoiesis. Microarray analysis was performed in order to uncover differences in gene expression between thymocytes of wild-type and RORgamma-/- mice. This analysis identified a novel gene encoding a 22 kDa protein, referred to as NABP1 (nucleic-acid-binding protein 1). This subsequently led to the identification of an additional protein, closely related to NABP1, designated NABP2. Both proteins contain an OB (oligonucleotide/oligosaccharide binding) motif at their N-terminus. This motif is highly conserved between the two proteins. NABP1 is highly expressed in the thymus of wild-type mice and is greatly suppressed in RORgamma-/- mice. During thymopoiesis, NABP1 mRNA expression is restricted to CD4+CD8+ thymocytes, an expression pattern similar to that observed for RORgamma2. These observations appear to suggest that NABP1 expression is regulated either directly or indirectly by RORgamma2. Confocal microscopic analysis showed that the NABP1 protein localizes to the nucleus. Analysis of nuclear proteins by size-exclusion chromatography indicated that NABP1 is part of a high molecular-mass protein complex. Since the OB-fold is frequently involved in the recognition of nucleic acids, the interaction of NABP1 with various nucleic acids was examined. Our results demonstrate that NABP1 binds single-stranded nucleic acids, but not double-stranded DNA, suggesting that it functions as a single-stranded nucleic acid binding protein.     

Cloning and characterization of human CAGLP gene encoding a novel EF-hand protein.

The EF-hand proteins, containing conserved Ca2+ binding motifs, play important roles in many biological processes. Through data mining, a novel human gene, CAGLP (calglandulin-like protein) was predicted and subsequently isolated from human skeleton muscle. The open reading frame of CAGLP is 543 bp in length, coding a putative Ca2+ binding protein with four EF-hand motifs. The deduced amino acid sequence of CAGLP displays high similarity with Bothrops insularis snake protein calglandulin (80%). The results of PCR amplification using cDNA from 17 human tissues indicated that human CAGLP is expressed in prostate, thymus, heart, skeleton muscle, bone marrow and ovary. Functional CAGLP::EGFP (enhanced green fluorescent protein) fusion protein revealed that CAGLP accumulated through-out Hela cells. Western blot using anti-EGFP antibodies indicated that the CAGLP protein has a molecular weight of about 19 kD. A phylogenetic tree showed that CAGLP and calglandulin may be orthologous proteins representing a distinct group in the EF-hand proteins.     

CLAN, a novel human CED-4-like gene

Proteins governing cell death form the basis of many normal processes and contribute to the pathogenesis of many diseases when dysregulated. Here we report the cloning of a novel human CED-4-like gene, CLAN, and several of its alternatively spliced isoforms. These caspase-associated recruitment domain (CARD)-containing proteins are expressed at varying degrees in normal human tissues and may contribute to a number of intracellular processes including apoptosis, cytokine processing, and NF-kappa B activation. The CARD of the CLAN proteins binds a number of other CARD-containing proteins including caspase-1, BCL10, NOD2, and NAC. Once their physiologic functions are uncovered, CLAN proteins may prove to be valuable therapeutic targets.     

Neuron navigator: a human gene family with homology to unc-53, a cell guidance gene from Caenorhabditis elegans

We have cloned the gene neuron navigator-1 (NAV1), a human homolog of unc-53, a gene involved in axon guidance in Caenorhabditis elegans. Duplications during evolution gave rise to three human homologs located on chromosomes 1q32.1, 11p15.1, and 12q21.1. NAV1 and NAV2 are expressed in the developing brain. NAV1, NAV2, and NAV3 expression is detected in adult heart, kidney, and brain, respectively. NAV1 encodes a protein lacking, in the aminoterminal part, a CH domain present in the other NAV genes. The first exon of NAV1 arose through an ancient internal duplication of sequences that also gave rise to exon 8 of NAV3 and exon 7 of NAV2. A detailed study of the NAV environment on the different chromosomes reveals incomplete micro-syntheny between the three regions. Through analysis of the phylogenetic relationships for three different gene families in the NAV environment, we reconstructed part of the events that formed these regions.     

A gene encoding a novel isoform of the PR-1 protein family from tomato is induced upon viroid infection

A Lycopersicon esculentum cDNA clone encoding an acidic-type pathogenesis-related protein (PR-lal) was isolated, sequenced and characterized. It contains an open reading frame of 175 amino acids and the mature protein, after cleavage of the 21 amino acid signals peptide, has a pl of 5.24. The protein shows highest homology (75% identity) with the basic pathogenesis-related prb-lb protein from tobacco. The PR-lal gene shows constitutive expression in roots from tomato plants. It is expressed in leaves and stems upon viroid infection, and appears to be induced by ethylene. Comparative studies of this gene and a related basic isoform of PR-1 indicate that the expression of these two members of the PR-1 gene family in tomato may be differentially regulated upon viroid infection.The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X71592.     

Isolation and characterization of a cDNA encoding a novel member of the human regenerating protein family: Reg IV

Human Reg and Reg-related genes constitute a multi-gene family belonging to the calcium (C-type) dependent lectin superfamily. Regenerating gene family members are expressed in the proximal gastrointestinal (GI) tract and ectopically at other sites in the setting of tissue injury. By high-throughput sequence analysis of a large inflammatory bowel disease library, two cDNAs have been isolated which encode a novel member of this multigene family. Based on primary sequence homology, tissue expression profiles, and shared exon-intron junction genomic organization, we assign this gene to the regenerating gene family. Specific protein structural differences suggest that the current three regenerating gene subtypes should be expanded to four. We demonstrate that Reg IV has a highly restricted tissue expression pattern, with prominent expression in the gastrointestinal tract. Reg IV mRNA expression is significantly up-regulated by mucosal injury from active Crohn's disease or ulcerative colitis.     

phoR1, a gene encoding a new histidine protein kinase Myxococcus xanthus

A soil bacterium able to undergo multicellular development and a coordinated gliding in swarms, requires an accurate regulatory network of phosphorelay proteins. Inorganic phosphate is a limiting nutrient in soil and its importance in regulation is critical. As a step towards studying phosphate regulation and its influence in the developmental process in this bacterium, we screened a Myxococcus xanthus library for clones with phosphatase activity, and found four different ones. The deduced sequence of one of the cloned inserts is similar to that of the classic transmembrane histidine protein kinase of the sensor family of the two-component signal transduction systems with a high sequence similarity to the sensor kinase in the Pho regulon of Bacillus subtilis PhoR. This gene has been named phoR1 and its deduced amino acid sequence consists of 455 residues with a predicted molecular mass of 48.5 kDa. The M. xanthus PhoR1 deduced sequence contains all the characteristic histidine protein kinase motifs in the same order and with the same spacing. A hydropathy profile indicates two membrane-spanning segments located at the extreme N-terminus, according to the putative sensor role of this domain. A gene-disrupted mutant is unable to produce normal mature fruiting bodies and produces fewer spores. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cloning and characterization of a Saccharomyces cerevisiae gene encoding a new member of the ubiquitin-conjugating protein family

Ubiquitin-conjugating enzymes (E2s), which participate in the post-translational conjugation of ubiquitin to proteins, are encoded by a multigene family in the yeast Saccharomyces cerevisiae. E2s function in a variety of cellular activities including intracellular proteolysis, DNA repair, sporulation, and cell cycle traverse. Here, we report the cloning and characterization of a new member of the yeast UBC gene family, UBC8. UBC8 encodes a 206-amino acid protein containing a highly acidic carboxyl terminus. The primary structure of the protein is similar to that of all other known E2s, with the highest homology being to the E2 (23 kDa) of wheat germ. Haploid strains in which the UBC8 gene is disrupted are viable, and the disruption does not produce any obvious phenotype. The UBC8 protein, produced in Escherichia coli, forms thiol ester adducts with ubiquitin and, apparently, diubiquitin, but does not transfer ubiquitin to histones.