Cloning and analysis of a constitutive heat shock (cognate) protein 70 gene inducible by L-glutamine.
An intronless gene encoding a protein of 652 amino acid residues with an M(r) of 71,266, showing between 79% and 59% identity in nucleotide sequence with heat shock protein 70 (HSP 70) genes of Bremia lactucae (a parasitic Oomycete of lettuce) and a wide range of organisms that include humans, was isolated from the nonparasitic Oomycete Achlya klebsiana. While the gene appears to be constitutively expressed, L-glutamine augmented its expression particularly under conditions of nutritional stress. L-Glutamine enhanced the transcription of a 2.4-kilobase poly(A)+ RNA simultaneously in the same way as it elevated the cellular level of the HSP 70-like protein. A polyclonal antibody (affinity-purified) raised in rabbit against the purified monomeric (M(r) 120,000) form of an NAD-specific glutamate dehydrogenase (Yang, B., and LéJohn, H.B. (1994) J. Biol. Chem. 269, 4506-4512) immunoprecipitated the HSP 70-like protein, and it was used to study the kinetics of induction of this stress-related protein and the effect of proteinase inhibitors on its metabolism. By using as probes four partial length cDNA clones, nine overlapping DNA fragments of the organism's genome carrying the HSP 70-like protein gene were isolated from a genomic library. The nucleotide sequence of the gene, including its boundaries, was determined by using these genomic clones. The 5'-untranslated boundary of the gene displayed the classical nucleotide arrangement of heat shock elements as well as CCAAT and TATA box motifs. Within the coding region are the typical conserved amino acid heat shock protein signatures 1 and 2 at the predicted locations. By primer extension and S1 nuclease protection mapping system, we estimated that the gene is probably transcribed into a message of 2.2 kilobases.
LéJohn
H B
HB
Department of Microbiology, University of Manitoba, Winnipeg, Canada.
Cameron
L E
LE
Yang
B
B
MacBeath
G
G
Barker
D S
DS
Williams
S A
SA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D005800:Genes, Fungal; D005947:Glucose / Q000494:pharmacology; D005969:Glutamate Dehydrogenase / Q000235:genetics*; D005973:Glutamine / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism; D009868:Oomycetes / Q000235:genetics*; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012995:Solubility
D017931:DNA Primers; D018076:DNA, Complementary; D004271:DNA, Fungal; D006360:Heat-Shock Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D005973:Glutamine; D005969:Glutamate Dehydrogenase; D005947:Glucose; D009584:Nitrogen