A stationary-phase gene in Saccharomyces cerevisiae is a member of a novel, highly conserved gene family.

The regulation of cellular growth and proliferation in response to environmental cues is critical for development and the maintenance of viability in all organisms. In unicellular organisms, such as the budding yeast Saccharomyces cerevisiae, growth and proliferation are regulated by nutrient availability. We have described changes in the pattern of protein synthesis during the growth of S. cerevisiae cells to stationary phase (E. K. Fuge, E. L. Braun, and M. Werner-Washburne, J. Bacteriol. 176:5802-5813, 1994) and noted a protein, which we designated Snz1p (p35), that shows increased synthesis after entry into stationary phase. We report here the identification of the SNZ1 gene, which encodes this protein. We detected increased SNZ1 mRNA accumulation almost 2 days after glucose exhaustion, significantly later than that of mRNAs encoded by other postexponential genes. SNZ1-related sequences were detected in phylogenetically diverse organisms by sequence comparisons and low-stringency hybridization. Multiple SNZ1-related sequences were detected in some organisms, including S. cerevisiae. Snz1p was found to be among the most evolutionarily conserved proteins currently identified, indicating that we have identified a novel, highly conserved protein involved in growth arrest in S. cerevisiae. The broad phylogenetic distribution, the regulation of the SNZ1 mRNA and protein in S. cerevisiae, and identification of a Snz protein modified during sporulation in the gram-positive bacterium Bacillus subtilis support the hypothesis that Snz proteins are part of an ancient response that occurs during nutrient limitation and growth arrest.     

Isolation, characterization, and mapping of the mouse and human WDR8 genes, members of a novel WD-repeat gene family

The Trp-Asp (WD) motif has been shown to exist in a number of proteins. Genes containing repeats of the WD motif compose a large gene family associated with a variety of cellular functions and can be divided into a number of functional subfamilies. By means of the differential display method using ttw, a mouse model for the early stage of ectopic ossification, we have identified a novel mouse gene, Wdr8 (WD repeat domain 8), which contains two WD repeats, together with its human orthologue. The human and mouse WDR8 genes encode 460 and 462 amino acids, respectively, with 89% identity, and are expressed in almost all tissues, including bone and cartilage, and in bone-forming cells, including osteoblasts and chondrocytes. Wdr8 expression in cartilage was differentially displayed by stimuli for ectopic ossification in ttw and was observed strongly only at a transition period from hypertrophic to mineralizing stages in ATDC5, a chondrogenic cell line that exhibits endochondral ossification, suggesting a potential role for Wdr8 in the process of ossification. The WDR8 protein is highly conserved among a variety of species, but is distinctly different from other WD-repeat proteins, indicating that it represents a novel subfamily of the WD-repeat gene family.     

Isolation and mapping of a human lung-specific gene, TSA1902, encoding a novel chitinase family member.

Using differential display technique, we have isolated a novel human gene expressed specifically in the lung. Two forms of the gene, designated TSA1902, were transcribed by alternate mRNA splicing. The transcribed mRNAs, termed TSA1902-L and TSA1902-S, putatively encode proteins of 368 and 315 amino acids, respectively, which show high similarity to human chitotriosidase protein. The N-terminal region of TSA1902-L protein contains the conserved active site residues (DXXDXDXE) of the catalytic center of various chitinases which are essential for chitinase activity. The deduced protein sequence of TSA1902-S, however, does not possess this active site, with the N-terminal 54 amino acids present in TSA1902-L protein having been deleted. Both proteins lacked the secretory sequence of N-termini and, judging from the hydropathy profile, may be soluble proteins in the cytoplasm. Chromosomal mapping by radiation hybrid analysis localized this gene to the chromosome 1p13.1-p21.3.     

Molecular cloning and characterization of the human S100A14 gene encoding a novel member of the S100 family

S100 proteins form a growing subfamily of proteins related by Ca2+-binding motifs to the Efhand Ca2+-binding protein superfamily. By analyzing a human lung cancer cell line subtraction cDNA library, we have identified and characterized a new member of the human S100 family that we named S100A14 (GenBank acc. no. NM_020672). It encodes a mRNA present in several normal human tissues of epithelial origin, with the highest level of expression in colon. The full-length cDNA is 1067 nt in length, with a coding region predicting a protein of 104 amino acids that is 68% homologous to the S100A13 protein. The deduced amino acid sequence of the human S100A14 and its mouse homolog (identified as GenBank entry) contains two EF-hand Ca2+-binding domains, a myristoylation motif, a glycosylation site, and several potential protein kinase phosphorylation sites. We have mapped this gene to human chromosome 1q21, within a region where at least 15 other S100 genes are tightly clustered. A 3.2-kb genomic fragment containing the entire S100A14 was cloned and sequenced. The gene is split into four exons and three introns spanning a total of 2165 bp of genomic sequence. We examined the intracellular distribution of the epitope-tagged S100A14 protein in two human lung carcinoma cell lines and one immortalized monkey cell line. Pronounced staining was observed in the cytoplasm, suggesting an association with the plasma membrane and in the perinuclear area. We also provide evidence for heterogenic expression of S100A14 in tumors, demonstrating its overexpression in ovary, breast, and uterus tumors and underexpression in kidney, rectum, and colon tumors, a pattern suggesting distinct regulation with potentially important functions in malignant transformation.     

PWP2, a member of the WD-repeat family of proteins,is an essentialSaccharomyces cerevisiae gene involved in cell separation

WD-repeat proteins contain four to eight copies of a conserved motif that usually ends with a tryptophan-aspartate (WD) dipeptide. TheSaccharomyces cerevisiae PWP2 gene, identified by sequencing of chromosome III, is predicted to contain eight so-called WD-repeats, flanked by nonhomologous extensions. This gene is expressed as a 3.2-kb mRNA in all cell types and encodes a protein of 104 kDa. ThePWP2 gene is essential for growth because spores carrying thepwp2Δ1::HIS3 disruption germinate before arresting growth with one or two large buds. The growth defect ofpwp2Δ1::HIS3 cells was rescued by expression ofPWP2 or epitope-taggedHA-PWP2 using the galactose-inducibleGAL1 promoter. In the absence of galactose, depletion of Pwp2p resulted in multibudded cells with defects in bud site selection, cytokinesis, and hydrolysis of the septal junction between mother and daughter cells. In cell fractionation studies, HA-Pwp2p was localized in the particulate component of cell lysates, from which it would be solubulized by high salt and alkaline buffer but not by nonionic detergents or urea. Indirect immunofluorescence microscopy indicated that HA-Pwp2p was clustered at multiple points in the cytoplasm. These results suggest that Pwp2p exists in a proteinaceous complex, possibly associated with the cytoskeleton, where it functions in control of cell growth and separation.     

The TIS11 primary response gene is a member of a gene family that encodes proteins with a highly conserved sequence containing an unusual Cys-His repeat.

The TIS11 primary response gene is rapidly and transiently induced by both 12-O-tetradecanoylphorbol-13-acetate and growth factors. The predicted TIS11 protein contains a 6-amino-acid repeat, YKTELC. We cloned two additional cDNAs, TIS11b and TIS11d, that contain the YKTELC sequence. TIS11, TIS11b, and TIS11d proteins share a 67-amino-acid region of sequence similarity that includes the YKTELC repeat and two cysteine-histidine containing repeats. TIS11 gene family members are not coordinately expressed: (i) unlike TIS11, the TIS11b and TIS11d mRNAs are detectable in quiescent Swiss 3T3 cells and are not dramatically induced by 12-O-tetradecanoylphorbol-13-acetate; (ii) cycloheximide superinduction does not occur for TIS11b and TIS11d; and (iii) unlike TIS11, TIS11b expression is extinguished in PC12 pheochromocytoma cells.     

Molecular cloning of the full-length cDNA encoding mouse neutral ceramidase. A novel but highly conserved gene family of neutral/alkaline ceramidases

We report here the molecular cloning, sequencing, and expression of the gene encoding the mouse neutral ceramidase, which has been proposed to function in sphingolipid signaling. A full-length cDNA encoding the neutral ceramidase was cloned from a cDNA library of mouse liver using the partial amino acid sequences of the purified mouse liver ceramidase. The open reading frame of 2,268 nucleotides encoded a polypeptide of 756 amino acids having nine putative N-glycosylation sites. Northern blot analysis revealed that the mRNA of the ceramidase was expressed widely in mouse tissues, with especially strong signals found in the liver and kidney. The ceramidase activity of lysates of CHOP cells increased more than 900-fold when the cells were transformed with a plasmid containing the cDNA encoding ceramidase. We also cloned the ceramidase homologue from the cDNA library of mouse brain and found that the sequence of the open reading frame, but not the 5'-noncoding region, was identical to that of the liver. Interestingly, phylogenetic analysis of various ceramidases clearly indicated that neutral/alkaline ceramidases form a novel but highly conserved gene family that is evolutionarily different from lysosomal acid ceramidases.     

The identification of a novel human homologue of the SH3 binding glutamic acid-rich (SH3BGR) gene establishes a new family of highly conserved small proteins related to Thioredoxin Superfamily

The SH3 binding glutamic acid-rich (SH3BGR) gene was cloned in an effort to identify genes located to human chromosome 21, within the congenital heart disease region, and expressed in the developing heart. After the identification of SH3BGR, two human homologous genes, SH3BGRL and SH3BGRL3, were identified and mapped to chromosome Xq13.3 and 1p34.3-35, respectively. SH3BGRL and SH3BGRL3 code for small proteins similar to the N-terminal region of the SH3BGR protein. SH3BGRL3 protein shows a significant similarity to Glutaredoxin 1 of Escherichia coli, and all the three proteins are predicted to belong to Thioredoxin-like protein Superfamily. Here we describe the identification and characterization of an additional human homologue of SH3BGR, named SH3BGRL2. The SH3BGRL2 gene maps to chromosome 6q13-15 and its messenger RNA has a large 3' untranslated region containing several AUUUA repeats. SH3BGRL2 codes for a protein of 107 amino acids, which, like SH3BGRL and SH3BGRL3 proteins, is highly homologous to the N-terminal region of the SH3BGR protein and appears to be related to Glutaredoxins and to PKC-interacting cousin of thioredoxin homology domain. We propose that the identification of SH3BGRL2 establishes a novel family of human genes, coding for highly conserved small proteins belonging to Thioredoxin-like protein Superfamily.     

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.     

Molecular characterization of a novel gene family encoding ACT domain repeat proteins in Arabidopsis

In bacteria, the regulatory ACT domains serve as amino acid-binding sites in some feedback-regulated amino acid metabolic enzymes. We have identified a novel type of ACT domain-containing protein family in Arabidopsis whose members contain ACT domain repeats (the "ACR" protein family). There are at least eight ACR genes located on each of the five chromosomes in the Arabidopsis genome. Gene structure comparisons indicate that the ACR gene family may have arisen by gene duplications. Northern-blot analysis indicates that each member of the ACR gene family has a distinct expression pattern in various organs from 6-week-old Arabidopsis. Moreover, analyses of an ACR3 promoter-beta-glucuronidase (GUS) fusion in transgenic Arabidopsis revealed that the GUS activity formed a gradient in the developing leaves and sepals, whereas low or no GUS activity was detected in the basal regions. In 2-week-old Arabidopsis seedlings grown in tissue culture, the expression of the ACR gene family is differentially regulated by plant hormones, salt stress, cold stress, and light/dark treatment. The steady-state levels of ACR8 mRNA are dramatically increased by treatment with abscisic acid or salt. Levels of ACR3 and ACR4 mRNA are increased by treatment with benzyladenine. The amino acid sequences of Arabidopsis ACR proteins are most similar in the ACT domains to the bacterial sensor protein GlnD. The ACR proteins may function as novel regulatory or sensor proteins in plants.