Human and mouse mitochondrial orthologs of bacterial ClpX

We have determined the cDNA sequence and exon/intron structure of the human CLPX gene encoding a human ortholog of the E. coli ClpX chaperone and protease subunit. The CLPX gene comprises 14 exons and encodes a 633-amino acid-long precursor polypeptide. The polypeptide contains an N-terminal putative mitochondrial transit peptide, and expression of a full-length ClpX cDNA tagged at its C-terminus (Myc-His) shows that the polypeptide is transported into mitochondria. FISH analysis localized the CLPX gene to human Chromosome (Chr) 15q22.1-22.32. This localization was refined by radiation hybrid mapping placing the CLPX gene 4.6 cR distal to D15S159. Murine ClpX cDNA was sequenced, and the mouse Clpx locus was mapped to a position between 31 and 42 cM offset from the centromere on mouse Chr 9. Experimental observations indicate the presence of a pseudogene in the mouse genome and sequence variability between mouse ClpX cDNAs from different strains. Alignment of the human and mouse ClpX amino acid sequences with ClpX sequences from other organisms shows that they display the typical modular organization of domains with one AAA⁺ domain common to a large group of ATPases and several other domains conserved in ClpX orthologs linked by non-conserved sequences. Notably, a C-4 zinc finger type motif is recognized in human and mouse ClpX. This motif of so far unknown function is present only in a subset of the known ClpX sequences. Received: 5 April 2000 / Accepted: 14 June 2000     

Characterization and chromosomal mapping of a mouse ortholog of the late-infantile ceroid-lipofuscinosis gene CLN2

Late-infantile ceroid-lipofuscinosis (CLN2) is an autosomal recessively inherited, neurodegenerative disease in humans. The CLN2 locus has been mapped to Chromosome (Chr) 11p15, and its sequence and genomic organization have recently been reported. In the present study, the cDNA sequence, exon/intron organization, and chromosomal localization of a mouse ortholog of the CLN2 gene are described. The mouse cDNA contains an open reading frame that predicts a protein product of 562 amino acids. The mouse and human coding regions are 86% and 88% identical at the nucleic acid and amino acid levels, respectively. One less codon appears in the mouse cDNA when compared with the human ortholog. The mouse gene (Cln2) spans more than 6 kb and consists of 13 exons separated by introns ranging in size from 111 to 1259 bp. Length polymorphism in an (AC)_n microsatellite in intron 3 of the mouse Cln2 gene was used to perform segregation analysis with The Jackson Laboratory DNA Panel Mapping Resource. On the basis of this analysis, the Cln2 gene was localized to a region of mouse Chr 7 that corresponds to human Chr 11p15. Characterization of the mouse Cln2 gene will facilitate generation of a mouse model for late-infantile ceroid-lipofuscinosis by gene targeting and identification of functionally important regions of the Cln2 protein. Received: 25 May 1999 / Accepted: 22 July 1999     

Genomic structure and comparison of mouse tissue-specific alkaline phosphatase genes

A full-length human placental alkaline phosphatase (AP) cDNA was used to identify and clone related genes from mouse genomic libraries. We report the cloning, sequence, and structural comparison of the mouse embryonic and intestinal AP genes and a putative AP pseudogene. All three mouse genes are composed of 11 exons interrupted by 10 small introns (70-261 bp) with an organization analogous to that of the three human tissue-specific AP genes. Introns interrupt the coding sequences at identical positions in all three mouse and human tissue-specific AP genes. The deduced amino acid sequence of the isozymes predicts proproteins of 529, 559, and 466 amino acids for embryonic AP, intestinal AP, and pseudo-AP, respectively. A repetitive sequence inserted in exon XI of the mouse intestinal AP gene codes for a unique stretch of 41 amino acids, 20 of which are threonines. This insertion has disrupted a region recognized as being responsible for phosphatidylinositol anchorage of human placental AP to the cytoplasmic membrane. Phylogenetic analysis indicates that the three mouse AP isozymes form a distinct group separate from the human tissue-specific AP isozymes, suggesting the taxon-specific evolution of the AP genes as opposed to independent evolution of AP genes expressed in specific tissues.     

Cloning, Structure, and Chromosome Localization of the Mouse Glutaryl-CoA Dehydrogenase Gene

Glutaryl-CoA dehydrogenase (GCDH) is a nuclear-encoded, mitochondrial matrix enzyme. In humans, deficiency of GCDH leads to glutaric acidemia type I, an inherited disorder of amino acid metabolism characterized by a progressive neurodegenerative disease. In this report we describe the cloning and structure of the mouse GCDH (Gcdh) gene and cDNA and its chromosomal localization. The mouse Gcdh cDNA is 1.75 kb long and contains an open reading frame of 438 amino acids. The amino acid sequences of mouse, human, and pig GCDH are highly conserved. The mouse Gcdh gene contains 11 exons and spans 7 kb of genomic DNA. Gcdh was mapped by backcross analysis to mouse chromosome 8 within a region that is homologous to a region of human chromosome 19, where the human gene was previously mapped.     

A P4‐ATPase subunit of the Cdc50 family plays a role in iron acquisition and virulence in Cryptococcus neoformans

The pathogenic fungus Cryptococcus neoformans delivers virulence factors such as capsule polysaccharide to the cell surface to cause disease in vertebrate hosts. In this study, we screened for mutants sensitive to the secretion inhibitor brefeldin A to identify secretory pathway components that contribute to virulence. We identified an ortholog of the cell division control protein 50 (Cdc50) family of the noncatalytic subunit of type IV P‐type ATPases (flippases) that establish phospholipid asymmetry in membranes and function in vesicle‐mediated trafficking. We found that a cdc50 mutant in C. neoformans was defective for survival in macrophages, attenuated for virulence in mice and impaired in iron acquisition. The mutant also showed increased sensitivity to drugs associated with phospholipid metabolism (cinnamycin and miltefosine), the antifungal drug fluconazole and curcumin, an iron chelator that accumulates in the endoplasmic reticulum. Cdc50 is expected to function with catalytic subunits of flippases, and we previously documented the involvement of the flippase aminophospholipid translocases (Apt1) in virulence factor delivery. A comparison of phenotypes with mutants defective in genes encoding candidate flippases (designated APT1, APT2, APT3, and APT4) revealed similarities primarily between cdc50 and apt1 suggesting a potential functional interaction. Overall, these results highlight the importance of membrane composition and homeostasis for the ability of C. neoformans to cause disease.     

Molecular Cloning and Chromosomal Localization of the MouseGpr37Gene Encoding an Orphan G-Protein-Coupled Peptide Receptor Expressed in Brain and Testis

We report the cloning of the mouse ortholog of the humanGPR37gene, which encodes an orphan G-protein-coupled receptor highly expressed in brain tissues and homologous to neuropeptide-specific receptors ([20],Genomics 45:68–77;[45],Biochem. Biophys. Res. Commun. 233:559–567). The genomic organization of theGPR37gene is conserved in both mouse and human species with a single intron interrupting the receptor-coding sequence within the presumed third transmembrane domain. Comparative genetic mapping of theGPR37gene showed that it maps to a conserved chromosomal segment on proximal mouse chromosome 6 and human chromosome 7q31. The mouseGpr37gene contains an open reading frame coding for a 600-amino-acid protein 83% identical to the humanGPR37gene product. The predicted mouse GPR37 protein contains seven putative hydrophobic transmembrane domains, as well as a long (249 amino acid residues), arginine- and proline-rich amino-terminal extracellular domain, which is also a distinctive feature of the human GPR37 receptor. Northern blot analysis of mouse tissues withGpr37-specific probes revealed a main 3.8-kb mRNA and a much less abundant 8-kb mRNA, both expressed in the brain. A 3-kb mRNA is also expressed in the testis. Both the mouse and the humanGPR37genes may belong to a class of highly conserved mammalian genes encoding a novel type of G-protein-coupled receptor predominantly expressed in the brain.     

The murine gene encoding the highly conserved Sm B protein contains a nonfunctional alternative 3' splice site.

The cDNA and partial genomic nucleotide (nt) sequences were derived for the mouse Sm B polypeptide and compared to the cDNA and genomic sequences encoding human Sm B. The deduced amino acid (aa) sequences from the mouse and human genes are identical with the exception of a single conserved aa substitution, accounting for the ability of anti-Sm antibodies to recognize the Sm polypeptides from a broad range of species. The genomic sequence of mouse B gene is similar to the human B genomic locus that extends from exon 6 to exon 7. These loci include conservation of both 3' alternative splice sites and putative branch points required to process B and B' mRNAs in human cells. However, the nt sequence downstream from the putative distal 3' splice junction and single nt flanking the 3' splice site consensus sequence, differ between mouse and human B. This results in a murine mRNA with a different predicted secondary structure around the distal 3' splice site when compared to humans. Thus, secondary structural constraints in the mRNA or changes in the exon sequence might prevent recognition of this alternative splice site to form B' mRNA in murine tissues.     

Cloning and expression of a plant homologue of the small subunit of the Golgi-associated clathrin assembly protein AP19 from Camptotheca acuminata

Clathrin-coated vesicles (CCVs) are involved in selective protein transport in eukaryotes. AP-1 and AP-2 are protein complexes found in the CCVs of the Golgi apparatus and the plasma membrane respectively. AP19 is the smallest polypeptide chain components of AP-1. We have identified a cDNA clone (CAP19) encoding a putative homologue for the assembly protein AP19 from the Chinese medicinal tree, Camptotheca acuminata. The deduced polypeptide contains 161 amino acids and has a predicted M _r of 18 820. DNA blot analysis suggests that the AP19s of C. acuminata are encoded by a small gene family. CAP19 was expressed ubiquitously throughout the plant suggesting that it may be involved in general Golgi-mediated secretion.     

Cloning and tissue expression of the mouse ortholog of AIM1, a βγ-crystallin superfamily member

We report the isolation of the murine ortholog of AIM1, a human gene whose expression is associated with the reversal of tumorigenicity in an experimental model of melanoma. Mouse and human AIM1 are more than 90% identical in amino acid sequence in the βγ-crystallin repeats and the C-terminal domain, and more than 75% identical in the extended N-terminal domain. Consistent with the isolated cDNA representing the authentic AIM1 ortholog, linkage analysis localized mouse Aim1 to proximal mouse Chromosome (Chr) 10 in a conserved linkage group with genes localized to human Chr band 6q21. Searches of EST databases identified a second AIM1-like gene in both mouse and human, suggesting the existence of a gene family. Northern analysis demonstrates Aim1 is expressed most abundantly in adult skin, lung, heart, liver, and kidney and is temporally regulated during embryogenesis. Aim1 is expressed highly in the shaft region of the hair follicles and the presumptive ectoderm, but not at detectable levels in melanocytes or melanocyte precursor cells. Received: 18 February 1998 / Accepted: 8 May 1998     

Characterization of centrin genes from Ochromonas danica (Chrysophyceae) and Scytosiphon lomentaria (Phaeophyceae)

Centrin, the EF‐hand Ca²⁺‐binding protein is localized at the basal apparatus of flagella and in centrioles in many eukaryotic cells. In the present study, centrin genes of the heterokont algae have been clarified for the first time. We isolated and analyzed cDNA and genomic DNA of centrin genes from the crysophycean alga Ochromonas danica Prings (UTEX LB1298) and the brown alga Scytosiphon lomentaria (Lyngbye) Link. The centrin gene of Ochromonas contained an open reading frame of 163 amino acids. The deduced protein, named Odcen, exhibited 85%, 78% and 59% homology to Chlamydomonas, human and Arabidopsis centrin, respectively. The centrin genes of Scytosiphon contained an open reading frame of 164 amino acids. The deduced protein, named Slcen, exhibited 84%, 77% and 59% homology to Chlamydomonas, human and Arabidopsis centrin, respectively. Both Odcen and Slcen possessed N‐terminal extensions before the conserved amino acid among various centrins, four EF‐hand domains and an aromatic amino acid at the C‐terminus. Southern blot hybridization suggested that the centrin gene occurs as a single copy gene in both Ochromonas and Scytosiphon genomes. Comparison of the sequence of the cDNA and the genomic DNA revealed that the Odcen gene was split into three fragments by introns and Slcen gene consisted of five fragments. The junctions of all introns of both genes conformed to the GT–AG rule. The introns of Slcen gene were considerably long and, as a result, the Slcen gene was approximately seven times longer than Odcen gene.     

Isolation and characterization of the gene encoding a manganese peroxidase from <Emphasis Type="Italic">Lentinula edodes</Emphasis>

A genomic DNA sequence and cDNA encoding a putative manganese peroxidase were isolated from the white-rot basidiomycete Lentinula edodes. The gene, called lemnp1, consists of a 1985-bp open reading frame interrupted by 16 introns and was flanked by an upstream region having putative CAAT, TATA, and heat shock elements and by a downstream region having polyadenylation signals. The lemnp1 gene encodes a protein of 364 amino acids that shows high sequence homology to manganese peroxidases of other basidiomycetes. The deduced N-terminal amino acid sequence is different from the L. edodes manganese peroxidase reported previously.     

PLAC1, an Xq26 Gene with Placenta-Specific Expression

A novel human X-linked gene shows placenta-specific expression and has been named PLAC1. The gene maps 65 kb telomeric to HPRT at Xq26 and has been completely sequenced at the cDNA and genomic levels. The mouse orthologue Plac1 maps to the syntenically equivalent region of the mouse X chromosome. In situ hybridization studies with the antisense mRNA during mouse embryogenesis detect Plac1 expression from 7.5 dpc (days postcoitum) to 14.5 dpc in ectoplacental cone, giant cells, and labyrinthine trophoblasts. The putative human and murine PLAC1 proteins are 60% identical and 77% homologous. Both include a signal peptide and a peptide sequence also found in an interaction domain of the ZP3 (zona pellucida 3) protein. These results make PLAC1 a marker for placental development, with a possible role in the establishment of the mother–fetus interface.     

The solution structure of the forkhead box‐O DNA binding domain of Brugia malayi DAF‐16a

Brugia malayi is a parasitic nematode that causes lymphatic filariasis in humans. Here the solution structure of the forkhead DNA binding domain of Brugia malayi DAF‐16a, a putative ortholog of Caenorhabditis elegans DAF‐16, is reported. It is believed to be the first structure of a forkhead or winged helix domain from an invertebrate. C. elegans DAF‐16 is involved in the insulin/IGF‐I signaling pathway and helps control metabolism, longevity, and development. Conservation of sequence and structure with human FOXO proteins suggests that B. malayi DAF‐16a is a member of the FOXO family of forkhead proteins. Proteins 2014; 82:3490–3496. © 2014 Wiley Periodicals, Inc.