Isolation and characterization of a mouse betaine-homocysteine S-methyltransferase gene and pseudogene.
Betaine-homocysteine S-methyltransferase (BHMT) is one of the enzymes involved in the branch point metabolism of homocysteine. Elevated levels of plasma homocysteine may be a risk factor for the development of vascular disease; however, whether BHMT has a significant role in the regulation of plasma levels of homocysteine remains to be determined. As a prelude to creating a mouse strain deficient in BHMT activity, we screened a lambda library containing mouse SvJ 129 genomic DNA for the mouse BHMT gene using random probes made from the human cDNA. One genomic isolate was completely sequenced and found to encode an intronless BHMT pseudogene (mBHMT-ps). mBHMT-ps was then used as a template for the generation of random probes that were used to screen a BAC library containing mouse 129 Sv/Ev genomic DNA. In order to discriminate between pseudogenes and the authentic BHMT gene, a secondary PCR-based screen was employed which used primers designed from the pseudogene sequence that would predictably amplify across introns. Using this strategy, we isolated six mouse genomic clones that tested positive for the presence of all seven introns characteristic of the human gene, and the BHMT gene of one clone was completely sequenced. Like the human BHMT gene, the mouse gene spans 21kb and is encoded by eight exons interrupted by seven introns. The structure of the mouse BHMT gene is described herein as well as the 5'-flanking region of the gene adjacent to exon 1, which we demonstrate is capable of conferring basal promoter activity in Chinese Hamster Ovary cells.
Neece
D J
DJ
Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, 61801, USA.
Griffiths
M A
MA
Garrow
T A
TA
D000818:Animals; D001483:Base Sequence; D050940:Betaine-Homocysteine S-Methyltransferase; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005091:Exons; D005796:Genes / Q000235:genetics; D006801:Humans; D007438:Introns; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008780:Methyltransferases / Q000235:genetics*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011544:Pseudogenes; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D011993:Recombinant Fusion Proteins; D004247:DNA; D008156:Luciferases; D008780:Methyltransferases; C494548:BHMT protein, human; D050940:Betaine-Homocysteine S-Methyltransferase; C494549:Bhmt protein, mouse