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 kle bsiana. While the gene appears to be constitutively expressed, L-gluta mine augmented its expression particularly under conditions of nutriti onal stress. L-Glutamine enhanced the transcription of a 2.4-kilobas p oly(A)(+) RNA simultaneously in the same way as it elevated the cellul ar level of the HSP 70-like protein. A polyclonal antibody (affinity-p urified) raised in rabbit against the purified monomeric (M(r) 120,000 ) form of an NAD-specific glutamate dehydrogenase (Yang, B., and LeJoh n, H. B. (1994) J. Biol. Chem. 269, 4506-4512) immunoprecipitated the HSP 70-like protein, and it was used to study the kinetics of inductio n of this stress related protein and the effect of proteinase inhibito rs on its metabolism. By using as probes four partial length cDNA clon es, nine overlapping DNA fragments of the organism's genome carrying t he HSP 70-like protein gene were isolated from a genomic library. The nucleotide sequence of the gene, including its boundaries, was determi ned by using these genomic clones. The 5'-untranslated boundary of the gene displayed the classical nucleotide arrangement of heat shock ele ments as well as CCAAT and TATA box motifs. Within the coding region a re the typical conserved amino acid heat shock protein signatures 1 an d 2 at the predicted locations. By primer extension and S1 nuclease pr otection mapping system, we estimated that the gene is probably transc ribed into a message of 2.2 kilobases.