Human gastric intrinsic factor: characterization of cDNA and genomic clones and localization to human chromosome 11

A human gastric intrinsic factor (IF) cDNA clone was isolated using a rat cDNA clone as a probe. Comparison of the predicted amino acid sequence revealed 80% identity of human IF with rat IF. These cDNA clones were used to isolate and map two overlapping clones encoding the human IF gene. The first exon of the cloned region (exon 2) contains 30 bp of the 5' untranslated region, the signal peptide, and the first 8 amino acids of the mature protein. Exons 3-10 encode the remainder of the coding and 3' noncoding regions. Southern analysis of genomic DNA indicated the presence of a single human IF gene and also revealed the presence of strong hybridizing sequences in genomic DNA from monkey, rat, mouse, cow, and human, suggesting that the IF gene is well conserved. The IF gene was localized to human chromosome 11 by concurrent cytogenetic and cDNA probe analysis of a panel of human X mouse somatic cell hybrids. Southern analysis of genomic DNA from patients with congenital pernicious anemia (lacking intrinsic factor) revealed normal restriction fragment patterns, suggesting that a sizable gene deletion was not responsible for the deficiency.     

Molecular cloning and functional expression of two splice forms of human N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase.

We have isolated and sequenced human cDNA and mouse genomic DNA clones encoding N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase (phosphodiester alpha-GlcNAcase) which catalyzes the second step in the synthesis of the mannose 6-phosphate recognition signal on lysosomal enzymes. The gene is organized into 10 exons. The protein sequence encoded by the clones shows 80% identity between human and mouse phosphodiester alpha-GlcNAcase and no homology to other known proteins. It predicts a type I membrane-spanning glycoprotein of 514 amino acids containing a 24-amino acid signal sequence, a luminal domain of 422 residues with six potential N-linked glycosylation sites, a single 27-residue transmembrane region, and a 41-residue cytoplasmic tail that contains both a tyrosine-based and an NPF internalization motif. Human brain expressed sequence tags lack a 102-base pair region present in human liver cDNA that corresponds to exon 8 in the genomic DNA and probably arises via alternative splicing. COS cells transfected with the human cDNA expressed 50-100-fold increases in phosphodiester alpha-GlcNAcase activity proving that the cDNA encodes the subunits of the tetrameric enzyme. Transfection with cDNA lacking the 102-base pair region also gave active enzyme. The complete genomic sequence of human phosphodiester alpha-GlcNAcase was recently deposited in the data base. It showed that our cDNA clone was missing only the 5'-untranslated region and initiator methionine and revealed that the human genomic DNA has the same exon organization as the mouse gene.     

Gene structure of human and mouse methylenetetrahydrofolate reductase (MTHFR)

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine. A human cDNA for MTHFR, 2.2 kb in length, has been expressed and shown to result in a catalytically active enzyme of approximately 70 kDa. Fifteen mutations have been identified in the MTHFR gene: 14 rare mutations associated with severe enzymatic deficiency and 1 common variant associated with a milder deficiency. The common polymorphism has been implicated in three multifactorial diseases: occlusive vascular disease, neural tube defects, and colon cancer. The human gene has been mapped to chromosomal region 1p36.3 while the mouse gene has been localized to distal Chromosome (Chr) 4. Here we report the isolation and characterization of the human and mouse genes for MTHFR. A human genomic clone (17 kb) was found to contain the entire cDNA sequence of 2.2 kb; there were 11 exons ranging in size from 102 bp to 432 bp. Intron sizes ranged from 250 bp to 1.5 kb with one exception of 4.2 kb. The mouse genomic clones (19 kb) start 7 kb 5′ exon 1 and extend to the end of the coding sequence. The mouse amino acid sequence is approximately 90% identical to the corresponding human sequence. The exon sizes, locations of intronic boundaries, and intron sizes are also quite similar between the two species. The availability of human genomic clones has been useful in designing primers for exon amplification and mutation detection. The mouse genomic clones will be helpful in designing constructs for gene targeting and generation of mouse models for MTHFR deficiency. Received: 28 January 1998 / Accepted: 9 April 1998     

Structures of mouse Rep-8 cDNA and genomic clones

A mouse homologue of the human Rep-8 gene was cloned by PCR methods using degenerate oligonucleotide primers corresponding to highly conserved regions between human and mouse genes, and by the Marathon–Ready cDNA amplification method. The full-length mouse Rep-8 contains 1422 nucleotides and codes for a protein of 277 amino acids with a calculated mol. wt. of 31,519. The overall amino acid sequence homology between mouse and human Rep-8 proteins was 73%, and the overall nucleic acid sequence similarity was 65%. The predicted amino acid sequence of mouse Rep-8 has leucine zipper-like motifs in the N-terminal region, similar to the human Rep-8 protein. Rep-8 exists as a single-copy gene and is expressed in both the early and late embryonic stages of mouse development, suggesting that the Rep-8 gene product has an important role in embryogenesis. The genomic structure of the mouse Rep-8 gene was characterized extensively so that a gene targeting strategy could be used to develop an understanding of the biological function(s) of this interesting gene and its product.     

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.     

Isolation, sequence analysis and characterization of cDNA clones coding for the C chain of mouse C1q. Sequence similarity of complement subcomponent C1q, collagen type VIII and type X and precerebellin.

A mouse macrophage lambda gt11 cDNA library was screened using a genomic DNA clone coding for the C-chain gene of human C1q. Approximately 600,000 recombinant phage plaques were hybridized with peroxidase-labeled human C-chain probe and detected by enhanced chemiluminescence. Five positive clones were obtained. The size of the full-length cDNA is 1019 bp. The sequence identity of the nucleotide sequence with human C1q C chain is 79%, the identity of the deduced amino acid sequences is 73%. The mouse C1q C chain exhibits the same structural features as the human C chain, e.g. conservation of the cysteine residues. Like the mouse A chain, the mouse C chain has an RGD sequence that may be recognized by receptors of the integrin family. No RGD sequences have been found in any of the human C1q chains. The size of the C-chain mRNA (1.2 kb) and its tissue distribution (macrophages being the cell type with the highest mRNA concentration) are identical to the mRNA of the mouse A and B chains. Alignment of human and mouse C1q A, B and C chains exhibits two blocks of highly conserved residues within the C-terminal globular regions. Three other proteins, collagen type VIII and type X and precerebellin share this similarity with C1q, indicating the structural and probably functional importance of these regions within the non-collagenous domains of the molecules.     

Mouse cathepsin F: cDNA cloning, genomic organization and chromosomal assignment of the gene

A murine cysteine protease of the papain family was identified by dbEST-database search. A 1.87kb full-length cDNA encoding a predicted polypeptide of 462 amino acids was sequenced. Since the encoded polypeptide shows more than 80% sequence identity with human cathepsin F, it is most likely that this cDNA represents the murine homologue of cathepsin F, and it was therefore named accordingly. Murine cathepsin F exhibits a domain structure typical for papain-like cysteine proteases, a 20 amino acid N-terminal hydrophobic signal sequence followed by an extraordinarily long propeptide of 228 amino acids and the domain of the mature protease comprising 214 amino acids. The mature region contains all features characteristic of a papain-like cysteine protease, including the highly conserved cysteine, histidine and asparagine residues of the 'catalytic triad'. Genomic clones covering the murine cathepsin F gene were isolated. The mouse cathepsin F gene consists of 14 exons and 13 introns and spans 5.8kb. Murine cathepsin F was mapped to chromosome 19, a region with synteny homology to a region of human chromosome 11 to which human cathepsin F has been mapped previously. Northern blot analysis of RNA from multiple tissues revealed a ubiquitous expression of cathepsin F in mouse and man.     

Isolation and characterization of non-muscle tropomyosin cDNAs of human and mouse origin

Several cDNA clones of human and mouse non-muscle tropomyosin have been isolated. All the human clones possess a common 23 bp sequence immediate 5' of the initiation codon. However, in the further upstream regions, the nucleotide sequences diverge. Two of the mouse cDNA clones pPSI-8 and pPSI-14 have identical nucleotide sequence in the coding region sequenced. However, 5' of the initiation codon these clones have only 40 identical nucleotides and further upstream the nucleotide sequences diverge. Analysis of the genomic DNAs of mouse cells indicated the possibility of a common gene giving rise to both the tropomyosin cDNAs differing in their 5' ends.     

Structural studies of cDNA and the gene for the beta-subunit of cGMP phosphodiesterase from human retina]

cDNA clones encoding the beta-subunit of the photoreceptor cGMP phosphodiesterase-(PDE) were isolated from a human retinal library. The encoded polypeptide has 854 amino acid residues with calculated molecular mass of 98416 Da. Alignment of the deduced amino acid sequence with the previously analysed alpha-, beta- and alpha'-subunits of the bovine and mouse PDEs demonstrates highly significant similarities. We have also isolated, from a genomic library, two overlapping recombinant lambda phage clones containing 26 kb of the human PDE beta-subunit gene. The cloning of the human gene and the knowledge of its genomic organization will allow the rapid assessment of the role of this gene in the causation of human retinopathies.     

cDNA sequence, interspecies comparison, and gene mapping analysis of argininosuccinate lyase

A cDNA clone of the argininosuccinate lyase gene (ASL) was isolated from an adult human liver library by probing with synthetic oligonucleotide probes. This clone and a yeast genomic DNA fragment containing the ASL gene were sequenced using the M13-dideoxynucleotide method. Comparison of the yeast and human clones at the nucleotide and putative amino acid sequence levels indicated identities of 50 and 54%, respectively. The most conserved region of the yeast gene was used to detect human clones in the liver cDNA library to test phylogenetic screening capabilities of conserved genes. ASL was mapped to human chromosome 7pter----q22 using human-mouse somatic cell hybrid DNA and further mapped by in situ hybridization to chromosome 7cen----q11.2 on human metaphase chromosomes. The probe also detected a sequence on chromosome 22. Somatic cell hybrid DNA digested with PvuII revealed a mouse polymorphism between Balb/c and C3H mice in the ASL gene.     

Isolation of the Mouse Homologue of BRCA1 and Genetic Mapping to Mouse Chromosome 11

The BRCA1 gene is in large part responsible for hereditary human breast and ovarian cancer. Here we report the isolation of the murineBrca1homologue cDNA clones. In addition, we identified genomic P1 clones that contain most, if not all, of the mouseBrca1locus. DNA sequence analysis revealed that the mouse and human coding regions are 75% identical at the nucleotide level while the predicted amino acid identity is only 58%. A DNA sequence variant in theBrca1locus was identified and used to map this gene on a (Mus m. musculusCzech II × C57BL/KsJ)F1 × C57BL/KsJ intersubspecific backcross to distal mouse chromosome 11. The mapping of this gene to a region highly syntenic with human chromosome 17, coupled with Southern and Northern analyses, confirms that we isolated the murineBrca1homologue rather than a related RING finger gene. The isolation of the mouseBrca1homologue will facilitate the creation of mouse models for germline BRCA1 defects.     

Molecular cloning and characterization of the genes encoding the two subunits of Drosophila melanogaster calcineurin.

Genomic clones containing the full coding sequences of the two subunits of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin, were isolated from a Drosophila melanogaster genomic library using highly conserved human cDNA probes. Three clones encoded a 19.3-kDa protein whose sequence is 88% identical to that of human calcineurin B, the Ca(2+)-binding regulatory subunit of calcineurin. The coding sequences of the Drosophila and human calcineurin B genes are 69% identical. Drosophila calcineurin B is the product of a single intron-less gene located at position 4F on the X chromosome. Drosophila genomic clones encoding a highly conserved region of calcineurin A, the catalytic subunit of calcineurin, were used to locate the calcineurin A gene at position 21 EF on the second chromosome of Drosophila and to isolate calcineurin A cDNA clones from a Drosophila embryonic cDNA library. The structure of the calcineurin A gene was determined by comparison of the genomic and cDNA sequences. Twelve exons, spread over a total of 6.6 kilobases, were found to encode a 64.6-kDa protein 73% identical to either human calcineurin A alpha or beta. At the nucleotide level Drosophila calcineurin A cDNA is 67 and 65% identical to human calcineurin A alpha and beta cDNAs, respectively. Major differences between human and Drosophila calcineurins A are restricted to the amino and carboxyl termini, including two stretches of repetitive sequences in the carboxyl-terminal third of the Drosophila molecule. Motifs characteristic of the putative catalytic centers of protein phosphatase-1 and -2A and calcineurin are almost perfectly conserved. The calmodulin-binding and auto-inhibitory domains, characteristic of all mammalian calcineurins A, are also conserved. A remarkable feature of the calcineurin A gene is the location of the intron/exon junctions at the boundaries of the functional domains and the apparent conservation of the intron/exon junctions from Drosophila to man.