Isolation of human purH gene expressed in the rodent transformant cells by subtractive enrichment of 3'-untranslated region of human transcript.
A subtraction procedure was developed for identification and isolation of a human gene transcribed in mouse transformant cells. The procedure was based on subtractive enrichment of the products that were amplified by the combination of reverse transcription and polymerase chain reaction from the 3'-untranslated region (3'-UTR) of human poly(A)+ RNA expressed in the mouse transformant cells. To assess the ability and usefulness of the procedure, we attempted to recover the human purH gene from a mouse transformant cell line, which was originally established by functional complementation using the human metaphase chromosome-mediated gene transfer technique from a mouse purH-negative mutant cell line. Using our procedure, a part of the human transcript in the transformant cells was successfully identified and isolated. The full-length cDNA was isolated using the 3'-UTR clone as a probe, and its biological activity was confirmed by introducing it into the mouse purH-negative mutant cells.
Yamauchi
M
M
Genome Research Group, National Institute of Radiological Sciences, Chiba, Japan.
Seki
N
N
Mita
K
K
Saito
T
T
Tsuji
S
S
Hongo
E
E
Morimyo
M
M
Shiomi
T
T
Koyama
H
H
Ayusawa
D
D
D000217:Acyltransferases / Q000235:genetics*; D000225:Adenine; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D005796:Genes; D005816:Genetic Complementation Test; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009709:Nucleotide Deaminases / Q000235:genetics*; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D012333:RNA, Messenger; D019877:Hydroxymethyl and Formyl Transferases; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D000217:Acyltransferases; D009709:Nucleotide Deaminases; C021436:IMP cyclohydrolase; D000225:Adenine