Cloning of the Human Monocarboxylate Transporter MCT3 Gene: Localization to Chromosome 22q12.3–q13.2

Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). The retinal pigment epithelium (RPE) expresses a unique member of this family, MCT3. A portion of the human MCT3 gene was cloned by polymerase chain reaction using primers designed from rat RPE MCT3 cDNA sequence. The human genomic sequence was used to design primers to clone human MCT3 cDNA and to identify a bacterial artificial chromosome clone containing the human MCT3 gene. The human MCT3 cDNA contained a 1512-nucleotide open reading frame with a deduced amino sequence 85% identical to rat MCT3. Comparison of the cDNA and genomic sequences revealed that the MCT3 gene was composed of five exons distributed over 5 kb of DNA. The exon–intron borders were conserved between the human and the chicken MCT3 genes. Using radiation hybrid mapping, the MCT3 gene was mapped to chromosome 22 between markers WI11639 and SGC30687. A search of chromosome 22 in the Sanger Centre database confirmed the location of the human MCT3 gene at 22q12.3–q13.2.     

Cloning of the human monocarboxylate transporter MCT3 gene: localization to chromosome 22q12.3-q13.2.

Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). The retinal pigment epithelium (RPE) expresses a unique member of this family, MCT3. A portion of the human MCT3 gene was cloned by polymerase chain reaction using primers designed from rat RPE MCT3 cDNA sequence. The human genomic sequence was used to design primers to clone human MCT3 cDNA and to identify a bacterial artificial chromosome clone containing the human MCT3 gene. The human MCT3 cDNA contained a 1512-nucleotide open reading frame with a deduced amino sequence 85% identical to rat MCT3. Comparison of the cDNA and genomic sequences revealed that the MCT3 gene was composed of five exons distributed over 5 kb of DNA. The exon-intron borders were conserved between the human and the chicken MCT3 genes. Using radiation hybrid mapping, the MCT3 gene was mapped to chromosome 22 between markers WI11639 and SGC30687. A search of chromosome 22 in the Sanger Centre database confirmed the location of the human MCT3 gene at 22q12.3-q13.2.     

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     

Sequence and genomic structure of the human adult skeletal muscle sodium channel alpha subunit gene on 17q.

The amino acid sequence of the sodium channel alpha subunit from adult human skeletal muscle has been deduced by cross-species PCR-mediated cloning and sequencing of the cDNA. The protein consists of 1836 amino acid residues. The amino acid sequence shows 93% identity to the alpha subunit from rat adult skeletal muscle and 70% identity to the alpha subunit from other mammalian tissues. A 500 kb YAC clone containing the complete coding sequence and two overlapping lambda clones covering 68% of the cDNA were used to estimate the gene size at 35 kb. The YAC clone proved crucial for gene structure studies as the high conservation between ion channel genes made hybridization studies with total genomic DNA difficult. Our results provide valuable information for the study of periodic paralysis and paramyotonia congenita, two inherited neurological disorders which are caused by point mutations within this gene.     

Organization of the mouse microfibril-associated glycoprotein-2 (MAGP-2) gene

A 1.4-kb EST clone encoding mouse microfibril-associated glycoprotein-2 (MAGP-2), identified by its similarity with the reported human cDNA, was used to screen a mouse 129 genomic bacterial artificial chromosome (BAC) library. The mouse gene contains 10 exons spanning 16 kb, located on the distal region of Chromosome (Chr) 6. The exons range in size from 24 to 963 bp, with the ATG located in exon 2. The tenth and largest exon contains 817 bp of 3′ untranslated sequence, including a B2 repetitive element. Northern analysis demonstrates abundant expression of MAGP-2 mRNA in skeletal muscle, lung, and heart. Sequence analysis of additional cDNA clones suggests that the two mRNA forms of MAGP-2 in the mouse arise from alternative polyadenylation site usage. The promoter does not contain an obvious TATA box, and the sequence surrounding the start site does not conform to the consensus for an initiator promoter element. Additionally, the mouse promoter contains 22 copies of a CT dinucleotide repeat sequence located ∼155 bp 5′ to exon 1. Received: 27 August 1999 / Accepted: 2 November 1999     

Molecular cloning of cDNA for the catalytic subunit of rat liver type 2A protein phosphatase, and detection of high levels of expression of the gene in normal and cancer cells

A cloned cDNA encoding a catalytic subunit of type 2A protein phosphatase from a rat liver cDNA library was obtained by use of a synthetic oligonucleotide corresponding to the tryptic peptide sequence of the purified enzyme. There was only a single amino acid difference between the deduced amino acid sequence of the clone obtained and those of the catalytic subunits, 2A alpha, of the rabbit skeletal muscle, porcine kidney and human liver enzymes, suggesting that this clone was a rat 2A alpha cDNA. On Northern blot analysis using a cDNA fragment as a probe, three mRNA species were detected in rat liver: a major mRNA of 2.0 kb and a minor one of 2.7 kb under high stringency conditions, and also a 1.1 kb mRNA under low stringency conditions. The 2A alpha gene was found to be highly expressed in various tissues of rat, especially the brain. High levels of expression of the gene were also detected in mouse NIH3T3 cells and their transformants, and in human cancer cell lines as well as a human immortalized cell line.     

Cloning and Mapping of the cDNA for Human Sarcosine Dehydrogenase, a Flavoenzyme Defective in Patients with Sarcosinemia

Sarcosine dehydrogenase is a liver mitochondrial matrix flavoenzyme that is defective in patients with sarcosinemia, a rare autosomal metabolic defect characterized by elevated levels of sarcosine in blood and urine. Some patients also exhibit mental retardation and growth failure. A full-length cDNA for human sarcosine dehydrogenase was isolated from an adult liver cDNA library. The first 22 residues in the deduced amino acid sequence exhibit features expected for a mitochondrial targeting sequence. The predicted mass of the mature human liver sarcosine dehydrogenase (99,505 Da) is in good agreement with that observed for rat liver sarcosine dehydrogenase ( approximately 100,000 Da). Human sarcosine dehydrogenase exhibits 89% identity with rat liver sarcosine dehydrogenase and strong homology ( approximately 35% identity) with rat liver dimethylglycine dehydrogenase, a sarcosine dehydrogenase-related protein from Rhodobacter capsulatus, and the regulatory subunit from bovine pyruvate dehydrogenase phosphatase. The human sarcosine dehydrogenase gene is at least 75.3 kb long and located on chromosome 9q34. The adult human liver clone is assembled from 21 exons (1-6, 7a, 8a, 9-21). Two smaller cDNA clones, isolated from adult liver and infant brain libraries, were assembled from the same sarcosine dehydrogenase gene by the use of alternate polyadenylation and splice sites. This is the first report of the genomic structure of the sarcosine dehydrogenase gene in any species. The observed chromosomal location is consistent with genetic studies with a mouse model for sarcosinemia that map the mouse gene to a region of mouse chromosome 2 syntenic with human 9q33-q34. The availability of the SDH gene sequence will enable characterization of the genotypes of sarcosinemia patients with different phenotypes.     

Chromosomal localization and genomic organization of the human Linker for Activation of T cells (LAT) gene

We have mapped the LAT gene by radiation hybrid mapping and fluorescence in situ hybridization to chromosome 16p11.2. The complete cDNA sequence of LAT was generated using assembled sequences of cDNA fragments already available. BLAST analysis using the cDNA sequence led to the identification of BAC clone CTB-134H23 (GenBank Accession No. AC112166). The genomic structure of the human LAT gene consists of 11 exons, encompassing 5.7 kb. Alternative splicing variants were identified.     

Cloning, genomic sequence, and chromosome mapping of Scyb11, the murine homologue of SCYB11 (alias betaR1/H174/SCYB9B/I-TAC/IP-9/CXCL11)

A T-cell attracting CXC chemokine phylogenetically related to MIG and SCYB10 was recently characterized and termed SCYB11 (alias betaR1/H174/SCYB9B/I-TAC/IP-9/CXCL11). Here, we cloned the cDNA of the murine homologue of this protein, Scyb11, from interferon-gamma/lipopolysaccharide-stimulated RAW264.7 mouse macrophage-like cells. The nucleotide sequence of Scyb11 shares 63% identity with its human counterpart. It encodes a 100 amino acid immature protein of 11,265 Da which contains a putative signal peptide of 21 amino acids. The molecular mass of the mature protein was calculated to be 9,113 Da. Sequence identity of the murine and human SCYB11 proteins is 68%. Phylogenetic tree analysis of mouse CXC chemokines places SCYB11 together with the murine homologues of MIG and SCYB10 (Crg-2/muIP-10) on an individual branch. A genomic sequence was obtained by genome walking and subcloning DNA fragments from a BAC clone containing Scyb11. Like human SCYB11, Scyb11 contains 4 exons with intron/exon boundaries at positions comparable to the human gene. Whereas introns 2 and 3 are of similar length in the murine and human genes, intron 1 of Scyb11 contains 1,260 bp more than intron 1 of the human gene. Intron 1 of Scyb11 is also characterized by a 201-bp stretch with repetitive sequences of high cryptic simplicity. Using a BAC clone containing Scyb11, this gene could be mapped to chromosome 5 at position 5E3. Since human SCYB11 is localized on 4q21.2, this result confirms the mouse/human homology of the two chromosome regions.