Isolation of a gene encoding a chaperonin-like protein by complementation of yeast amino acid transport mutants with human cDNA.
A human cDNA library in lambda-yes plasmid was used to transform a strain of Saccharomyces cerevisiae with defects in histidine biosynthesis (his4-401) and histidine permease (hip1-614) and with the general amino acid permease (GAP) repressed by excess ammonium. We investigated three plasmids complementing the transport defect on a medium with a low concentration of histidine. Inserts in these plasmids hybridized with human genomic but not yeast genomic DNA, indicating their human origin. mRNA corresponding to the human DNA insert was produced by each yeast transformant. Complementation of the histidine transport defect was confirmed by direct measurement of histidine uptake, which was increased 15- to 65-fold in the transformants as compared with the parental strain. Competitive inhibition studies, measurement of citrulline uptake, and lack of complementation in gap1- strains indicated that the human cDNA genes code for proteins that prevent GAP repression by ammonium. The amino acid sequence encoded by one of the cDNA clones is related to T-complex proteins, which suggests a "chaperonin"-like function. We suggest that the human chaperonin-like protein stabilizes the NPR1 gene product and prevents inactivation of GAP.
Department of Pediatric, University of Rochester Medical School, NY 14642.
G B
GB
Segel
T R
TR
Boal
T S
TS
Cardillo
F G
FG
Murant
M A
MA
Lichtman
F
F
Sherman
D000595:Amino Acid Sequence; D000641:Ammonia / Q000494:pharmacology; D001483:Base Sequence; D001692:Biological Transport; D015139:Blotting, Southern; D018833:Chaperonins; D002956:Citrulline / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005796:Genes; D005816:Genetic Complementation Test; D006639:Histidine / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment
D011506:Proteins; D012333:RNA, Messenger; D002956:Citrulline; D006639:Histidine; D000641:Ammonia; D004247:DNA; D018833:Chaperonins