Disruption of the pelota gene causes early embryonic lethality and defects in cell cycle progression

Mutations in either the Drosophila melanogaster pelota or pelo gene or the Saccharomyces cerevisiae homologous gene, DOM34, cause defects of spermatogenesis and oogenesis in Drosophila, and delay of growth and failure of sporulation in yeast. These phenotypes suggest that pelota is required for normal progression of the mitotic and meiotic cell cycle. To determine the role of the pelota in mouse development and progression of cell cycle, we have established a targeted disruption of the mouse PELO: Heterozygous animals are variable and fertile. Genotyping of the progeny of heterozygous intercrosses shows the absence of Pelo(-/-) pups and suggests an embryo-lethal phenotype. Histological analyses reveal that the homozygous Pelo deficient embryos fail to develop past day 7.5 of embryogenesis (E7.5). The failure of mitotic active inner cell mass of the Pelo(-/-) blastocysts to expand in growth after 4 days in culture and the survival of mitotic inactive trophoplast indicate that the lethality of Pelo-null embryos is due to defects in cell proliferation. Analysis of the cellular DNA content reveals the significant increase of aneuploid cells in Pelo(-/-) embryos at E7.5. Therefore, the percent increase of aneuploid cells at E7.5 may be directly responsible for the arrested development and suggests that Pelo is required for the maintenance of genomic stability.     

Heat-shock proteins, Hsp84 and Hsp86, of mice and men: two related genes encode formerly identified tumour-specific transplantation antigens

Mouse cDNA clones have been isolated with the help of Drosophila melanogaster 82-kDa heat-shock protein (Hsp82)-coding sequences as hybridization probe. Sequencing of the overlapping mouse clones reveals a long open reading frame (ORF) that encodes a polypeptide of 83.3 kDa which shows about 80% similarity to the respective Drosophila Hsp82 amino acid sequence. The N-terminal half of this cDNA cross-hybridizes to a different class of mouse cDNA clones indicating a related gene. Northern blot hybridization experiments reveal a 2.6-kb poly(A)+RNA when probed with the hsp84 clone and a 2.85-kb signal with the hsp84-related cDNA. The amino acid sequences deduced from the contiguous ORF of the hsp84 and the hsp84-related cDNA coincide with the N-terminal sequence of formerly identified 84-kDa and 86-kDa tumour-specific transplantation antigens (Ullrich et al., 1986). In addition, the amino acid composition of the putative 84-kDa mouse Hsp described here is very similar to that of the 84-kDa tumour antigen described by Ullrich et al. (1986). Both observations corroborate the assumption that these Hsps are identical to the described 84-kDa and 86-kDa tumour-specific transplantation antigens. Using these mouse hsp gene clones as hybridization probes we also isolated the corresponding pair of human cDNA clones. Comparison of the respective sequences reveals a strong evolutionary constraint on these two genes in mouse and man.     

Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.     

Identification, genomic organization, and mRNA expression of LACTB, encoding a serine beta-lactamase-like protein with an amino-terminal transmembrane domain.

Database searching with bacterial serine beta-lactamases identified mouse expressed sequence tags (ESTs) with significant similarity scores.The cloned mouse cDNA encodes a novel 551-amino-acid protein, LACTB, with a predicted amino-terminal transmembrane domain but no signal peptide. It contains an active site motif related to C-class beta-lactamases. Homologues were detected in sequence data from human, rat, cow, rabbit, pig, toad, zebrafish, and Caenorhabditis elegans, but not in Saccharomyces cerevisiae or Drosophila melanogaster. The genes were mapped to human chromosome 15q22.1 and mouse chromosome 9. Sequencing of a 14.7-kb fragment of mouse genomic DNA defined six exons. A virtual human cDNA and a 549-residue protein, predicted from unfinished genomic sequence, showed the same intron/exon structure. Northern blot analysis showed expression of the 2.3-kb mRNA predominantly in mouse liver and human skeletal muscle. This is the first reported vertebrate example of this microbial peptidase family.     

Drosophila melanogaster transferrin. Cloning, deduced protein sequence, expression during the life cycle, gene localization and up-regulation on bacterial infection.

Drosophila melanogaster transferrin cDNA was cloned from an ovarian cDNA library by using a PCR fragment amplified by two primers designed from other dipteran transferrin sequences. The clone (2035 bp) encodes a protein of 641 amino acids containing a signal peptide of 29 amino acids. Like other insect transferrins, Drosophila transferrin appears to have a functional iron-binding site only in the N-terminal lobe. The C-terminal lobe lacks iron-binding residues found in other transferrins, and has large deletions which make it much smaller than functional C-terminal lobes in other transferrins. In-situ hybridization using a digoxigenin labeled transferrin cDNA probe revealed that the gene is located at position 17B1-2 on the X chromosome. Northern blot analysis showed that transferrin mRNA was present in the larval, pupal and adult stages, but was not detectable in the embryo. Iron supplementation of the diet resulted in lower levels of transferrin mRNA. When adult flies were inoculated with bacteria (Escherichia coli), transferrin mRNA synthesis was markedly increased relative to controls.     

Coding sequence, genomic organization, chromosomal localization, and expression pattern of the signalosome component Cops2: the mouse homologue of Drosophila alien

The Drosophila alien gene is highly homologous to the human thyroid receptor interacting protein, TRIP15/COPS2, which is a component of the recently identified signalosome protein complex. We identified the mouse homologue of Drosophila alien through homology searches of the EST database. We found that the mouse cDNA encodes a predicted 443-amino-acid protein, which migrates at approximately 50 kDa. The gene for the mouse alien homologue, named Cops2, includes 12 coding exons spanning approximately 30 kb of genomic DNA on the central portion of mouse chromosome 2. Mouse Cops2 is widely expressed in embryonic, fetal, and adult tissues beginning as early as E7.5. Mouse Cops2 cDNA hybridizes to two mRNA bands in all tissues at approximately 2.3 and approximately 4 kb, with an additional approximately 1.9-kb band in liver. Immunostaining of native and epitope tagged proteins localized the mouse Cops2 protein in both the cytoplasm and the nucleus, with larger amounts in the nucleus in some cells.     

cDNA cloning of human oxysterol-binding protein and localization of the gene to human chromosome 11 and mouse chromosome 19

Cellular cholesterol metabolism is regulated primarily through sterol-mediated feedback suppression of the activity of the low-density lipoprotein receptor and several enzymes of the cholesterol biosynthetic pathway. We previously described the cloning of a rabbit cDNA for the oxysterol-binding protein (OSBP), a cytosolic protein of 809 amino acids that may participate in these regulatory events. We now use the rabbit OSBP cDNA to clone the human OSBP cDNA and 5' genomic region. Comparison of the human and rabbit OSBP sequences revealed a remarkably high degree of conservation. The cDNA sequence in the coding region showed 94% identity between the two species, and the predicted amino acid sequence showed 98% identity. The human cDNA was used to determine the chromosomal localization of the OSBP gene by Southern blot hybridization to panels of somatic cell hybrid clones containing subsets of human or mouse chromosomes and by RFLP analysis of recombinant inbred mouse strains. The OSBP locus mapped to the long arm of human chromosome 11 and the proximal end of mouse chromosome 19. Along with previously mapped genes including Ly-1 and CD20, OSBP defines a new conserved syntenic group on the long arm of chromosome 11 in the human and the proximal end of chromosome 19 in the mouse.     

Exon-intron organization and chromosomal localization of the mouse monoglyceride lipase gene

Monoglyceride lipase (MGL) functions together with hormone-sensitive lipase to hydrolyze intracellular triglyceride stores of adipocytes and other cells to fatty acids and glycerol. In addition, MGL presumably complements lipoprotein lipase in completing the hydrolysis of monoglycerides resulting from degradation of lipoprotein triglycerides. Cosmid clones containing the mouse MGL gene were isolated from a genomic library using the coding region of the mouse MGL cDNA as probe. Characterization of the clones obtained revealed that the mouse gene contains the coding sequence for MGL on seven exons, including a large terminal exon of approximately 2.6 kb containing the stop codon and the complete 3' untranslated region. Two different 5' leader sequences, diverging 21 bp upstream of the predicted translation initiation codon, were isolated from a mouse adipocyte cDNA library. Western blot analysis of different mouse tissues revealed protein size heterogeneities. The amino acid sequence derived from human MGL cDNA clones showed 84% identity with mouse MGL. The mouse MGL gene was mapped to chromosome 6 in a region with known homology to human chromosome 3q21.     

Expression profile of mouse BWF1, a protein with a BEACH domain, WD40 domain and FYVE domain

We isolated a mouse cDNA encoding a protein that contains a BEACH domain, 5 WD40 repeats and a FYVE domain, which we designated as BWF1. The mRNA is approximately 10 kb in size and encodes a protein consisting of 3508 amino acids with a predicted molecular weight of 385 kDa. BWF1 has 45% homology with the Drosophila protein, blue cheese (BCHS). The BWF1 gene consists of 67 exons, which span 270 kb of genomic sequence, and has been mapped to mouse chromosome 5. Northern blot analysis revealed that it was strongly expressed in the liver, moderately in the kidney and testis, and weakly in the brain of adult mice. During the development of the mouse brain, BWF1 mRNA was abundant on embryonic day (E) 14-16; after birth, the level of BWF1 mRNA expression decreased markedly to reach the adult level at postnatal day 3. In situ hybridization analysis revealed that the expressed BWF1 mRNA was restricted to the marginal region both in E14 and E16 embryonic brain, but became diffuse after birth. Confocal microscopy studies of the epitope-tagged BWF1 protein showed that the protein was a cytoplasmic one.     

A Human Homologue (BICD1) of theDrosophila Bicaudal-DGene

We previously isolated a cDNA fragment homologous to theDrosophila Bicaudal-Dgene (Bic-D) using a hybridization selection procedure with cosmids derived from the short arm of human chromosome 12. A PCR-mediated cDNA cloning strategy was applied to obtain the coding sequence of the human homologue (BICD1) and to generate a partial mouse (Bicdh1) cDNA. TheDrosophila Bicaudal-Dgene encodes a coiled coil protein, characterized by five α-helix domains and a leucine zipper motif, that forms part of the cytoskeleton and mediates the correct sorting of mRNAs for oocyte- and axis-determining factors during oogenesis. Analysis of the predicted amino acid sequence of theBICD1cDNA clones indicates that the sequence similarity is essentially limited to the amphipatic helices and the leucine zipper, but the conserved order of these domains suggests a similar function of the protein in mammalians. A database search further indicates the existence of a second human homologue on chromosome arm 9q and aCaenorhabditis eleganshomologue. Northern blot analysis indicates that both the human and the murine homologues produce an mRNA species of 9.5 kb expressed in brain, heart, and skeletal muscle and during mouse embryonic development. The conserved structural characteristics of theBICD1protein and its expression in muscle and especially brain suggest thatBICD1is a component of a cytoskeleton-based mRNA sorting mechanism conserved during evolution.     

Identification of the candidate ALS2 gene at chromosome 2q33 as a human aldehyde oxidase gene

Denver, Tokyo, and Salt Lake City investigators recently published different complimentary deoxyribonucleic acid (cDNA) sequences for human liver xanthine dehydrogenase/xanthine oxidase (XD/XO). The gene encoding the Denver cDNA was subsequently linked to juvenile familial amyotrophic lateral sclerosis (JFALS) at chromosome 2q33 and has been proposed as the ALS2 locus. The present investigation was undertaken to elucidate the differences between the three cDNA sequences, and we provide evidence that the Denver cDNA encodes aldehyde oxidase (AO): first, the Denver cDNA sequence diverged significantly from the Tokyo and Salt Lake City cDNA sequences which were very similar; second, the deduced protein sequence from the Denver cDNA was very similar to the amino acid sequence of purified rabbit liver AO protein; third, the deduced Denver protein sequence was 76% identical to the encoded 101 amino acid long peptides from partial cDNAs for rabbit and rat AO and 81.7% identical to 300 amino acids from an incomplete cDNA encoding bovine AO; fourth, the Denver gene was expressed in liver, kidney, lung, pancreas, prostate, testes, and ovary while the Tokyo XD gene was expressed predominantly in liver and small intestine; fifth, the Denver gene was previously mapped to chromosome 2q33 which is syntenic to the mouse AO locus on chromosome 1. Our results have revealed dramatic similarities in protein and DNA sequence in the human molybdenum hydroxylases, have uncovered unanticipated complexity in the human molybdenum hydroxylase genes, and advance the potential for AO derived oxygen radicals in JFALS and other human diseases.