Rapid mapping of genomic P1 clones: the mouse L-isoaspartyl/D-aspartyl methyltransferase gene.
We report the mapping of the gene for the murine protein-L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1. 77) from a 129 mouse strain. This gene encodes an enzyme present in all tissues that can catalyze the first step of a repair reaction in which age-damaged proteins containing abnormal l-isoaspartyl (or d-aspartyl) residues can be converted to forms containing normal l-aspartyl residues. We first mapped the restriction sites from a genomic P1 clone using a rapid method generally applicable to all bacteriophage P1 clones containing large DNA inserts. We show that a single pulsed-field electrophoresis blot can be used to map an entire 89-kb P1 clone insert for eight restriction endonucleases with an error of no more than 2% of the length of the fragment, or 1 kb at the middle of the insert. In this method, we combine complete restriction endonuclease digestion at rare sites within the P1 vector with partial restriction endonuclease digestion within the insert. After size separation by pulsed-field gel electrophoresis and blotting, the fragments are detected by Southern hybridization with probes to the vector. This method is potentially useful for restriction mapping other large DNA clones such as artificial chromosomes. We then determined the positions of the exons of the methyltransferase gene by restriction mapping of long PCR fragments. The previously unidentified exon 8, which encodes the -DEL C-terminus of the more acidic isozyme II, was sequenced and mapped 5. 3 kb from the end of exon 7.
MacLaren
D C
DC
Department of Chemistry and Biochemistry, University of California, Los Angeles, California, 90024-1569, USA.
Clarke
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S
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D017931:DNA Primers; D005091:Exons; D015723:Gene Library; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000096:biosynthesis / Q000235:genetics*; D015183:Restriction Mapping
D017931:DNA Primers; D015345:Oligonucleotide Probes; D004247:DNA; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase