The normal phenotype of Pmm1-deficient mice suggests that Pmm1 is not essential for normal mouse development

Phosphomannomutases (PMMs) are crucial for the glycosylation of glycoproteins. In humans, two highly conserved PMMs exist: PMM1 and PMM2. In vitro both enzymes are able to convert mannose-6-phosphate (mannose-6-P) into mannose-1-P, the key starting compound for glycan biosynthesis. However, only mutations causing a deficiency in PMM2 cause hypoglycosylation, leading to the most frequent type of the congenital disorders of glycosylation (CDG): CDG-Ia. PMM1 is as yet not associated with any disease, and its physiological role has remained unclear. We generated a mouse deficient in Pmm1 activity and documented the expression pattern of murine Pmm1 to unravel its biological role. The expression pattern suggested an involvement of Pmm1 in (neural) development and endocrine regulation. Surprisingly, Pmm1 knockout mice were viable, developed normally, and did not reveal any obvious phenotypic alteration up to adulthood. The macroscopic and microscopic anatomy of all major organs, as well as animal behavior, appeared to be normal. Likewise, lectin histochemistry did not demonstrate an altered glycosylation pattern in tissues. It is especially striking that Pmm1, despite an almost complete overlap of its expression with Pmm2, e.g., in the developing brain, is apparently unable to compensate for deficient Pmm2 activity in CDG-Ia patients. Together, these data point to a (developmental) function independent of mannose-1-P synthesis, whereby the normal knockout phenotype, despite the stringent conservation in phylogeny, could be explained by a critical function under as-yet-unidentified challenge conditions.     

Tissue Distribution, Genomic Structure, and Chromosome Mapping of Mouse and Human Eukaryotic Initiation Factor 4E-Binding Proteins 1 and 2

Two related eukaryotic initiation factor-4E binding proteins (4E-BP1 and 4E-BP2) were recently characterized for their capacity to bind specifically to eIF4E and inhibit its function. Here, we determined the cDNA sequence, tissue distribution, genomic structure, and chromosome localization of murine and human 4E-BP1 and 4E-BP2. Mouse 4E-BP1 and 4E-BP2 consist of 117 and 120 amino acids and exhibit 91.5 and 95.0% identity, respectively, to their human homologues. 4E-BP1 mRNA is expressed in most tissues, but is most abundant in adipose tissue, pancreas, and skeletal muscle, while 4E-BP2 mRNA is ubiquitously expressed. The structures of the mouse 4E-BP1 and 4E-BP2 were determined. The 4E-BP1 gene consists of three exons and spans ∼16 kb. In addition, two 4E-BP1 pseudogenes exist in the mouse genome. The 4E-BP2 gene spans approximately 20 kb and exhibits an identical genomic organization to that of 4E-BP1, with the protein coding portion of the gene divided into three exons. There are no pseudogenes for 4E-BP2. The chromosomal locations of 4E-BP1 and 4E-BP2 were determined in both mice and humans by fluorescencein situhybridization analysis. Mouse 4E-BP1 and 4E-BP2 map to chromosomes 8 (A4-B1) and 10 (B4-B5), respectively, and human 4E-BP1 and 4E-BP2 localize to chromosomes 8p12 and 10q21–q22, respectively.     

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.     

Evolution of aspartyl proteases by gene duplication: the mouse renin gene is organized in two homologous clusters of four exons.

Overlapping recombinant clones that appear to encompass the entire renin gene, named Ren 1, have been isolated from a library of BALB/c mouse genomic DNA fragments. Based on restriction endonuclease mapping and DNA sequence analysis, Ren 1 spans 9.6 kb and contains nine exons interrupted by eight intervening sequences of highly variable size. The first exon, encoding the signal peptide of preprorenin, is separated from the eight following exons by a 3-kb intron. These eight exons are organized into two clusters of four separated by a 2-kb intron. DNA stretches encoding the aspartyl residues, which are part of the active site of renin, are located at homologous positions in both clusters. Our results show that aspartyl protease genes have arisen by duplication and fusion of an ancestral gene containing five exons. The estimated date of the duplication event of the mouse renin genes Ren 1 and Ren 2 is discussed.     

Characterization of mouse muc6 and evidence of conservation of the gel-forming mucin gene cluster between human and mouse

Using degenerate primers designed from conserved cysteine-rich domains of gel-forming mucins, we cloned two new mouse mucin cDNAs. Blast searching showed that they belong to the same new gene assigned to chromosome 7 band F5. This gene is clustered with the three secreted large gel-forming mucins Muc2, Muc5ac, and Muc5b in a region that exhibits synteny with human chromosome 11p15. Computer analysis and sequence alignments with mucin genes predict that the new gene is composed of 33 exons and spans 30 kb from the initiation ATG codon to the Stop codon. Sequence similarities, domain organization of the deduced peptide, and expression analysis allow us to conclude that this newly cloned mouse gene is Muc6, i.e., the mouse ortholog of human MUC6. Like those of their human homologs, the genomic order and arrangement of the four mucins within the cluster of mucin genes are conserved.     

Mapping and characterization of the mouse and human SS18 genes, two human SS18-like genes and a mouse Ss18 pseudogene.

We have previously isolated and characterized a mouse cDNA orthologous to the human synovial sarcoma associated SS18 (formerly named SSXT and SYT) cDNA. Here, we report the characterization of the genomic structure of the mouse Ss18 gene. Through in silico methods with sequence information contained in the public databases, we did the same for the human SS18 gene and two human SS18 homologous genes, SS18L1 and SS18L2. In addition, we identified a mouse Ss18 processed pseudogene and mapped it to chromosome 1, band A2-3. The mouse Ss18 gene, which is subject to extensive alternative splicing, is made up of 11 exons, spread out over approximately 45 kb of genomic sequence. The human SS18 gene is also composed of 11 exons with similar intron-exon boundaries, spreading out over about 70 kb of genomic sequence. One alternatively spliced exon, which is not included in the published SS18 cDNA, corresponds to a stretch of sequence which we previously identified in the mouse Ss18 cDNA. The human SS18L1 gene, which is also made up of 11 exons with similar intron-exon boundaries, was mapped to chromosome 20 band q13.3. The smaller SS18L2 gene, which is composed of three exons with similar boundaries as the first three exons of the other three genes, was mapped to chromosome 3 band p21. Through sequence and mutation analyses this gene could be excluded as a candidate gene for 3p21-associated renal cell cancer. In addition, we created a detailed BAC map around the human SS18 gene, placing it unequivocally between the CA-repeat marker AFMc014wf9 and the dihydrofolate reductase pseudogene DHFRP1. The next gene in this map, located distal to SS18, was found to be the TBP associated factor TAFII-105 (TAF2C2). Further analogies between the mouse Ss18 gene, the human SS18 gene and its two homologous genes were found in the putative promoter fragments. All four promoters resemble the promoters of housekeeping genes in that they are TATA-less and embedded in canonical CpG islands, thus explaining the high and widespread expression of the SS18 genes.     

Chromosomal localization of seven neuronal nicotinic acetylcholine receptor subunit genes in humans.

We have determined the chromosomal location of seven human neuronal nicotinic acetylcholine receptor subunit genes by genomic Southern analysis of hamster/human somatic cell hybrid DNAs. The beta 2 subunit gene was localized to human chromosome 1, the alpha 2 and beta 3 subunit genes were localized to human chromosome 8, the alpha 3, alpha 5, and beta 4 subunit genes were localized to human chromosome 15, and the alpha 4 subunit gene was localized to human chromosome 20. Mapping of the beta 2 subunit gene to chromosome 1 establishes a syntenic group with the amylase gene locus on human chromosome 1 and mouse chromosome 3, while mapping of the alpha 3 subunit gene to chromosome 15 confirms the existence of a syntenic group with the mannose phosphate isomerase gene locus on human chromosome 15 and mouse chromosome 9.