A full‐length cDNA clone of 4.3 kb encoding the human ATP‐citrate lyase enzyme has been isolated by screening a human cDNA library with the recently isolated rat ATP‐citrate lyase cDNA clone [Elshourbagy et al. (1990) J. Biol. Chem. 265, 1430]. Nucleic‐acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1105 amino acids in length with a calculated molecular mass of 121419 Da. Comparison of the human and rat ATP‐citrate lyase cDNA sequences reveals 96.3% amino acid identity throughout the entire sequence. Further sequence analysis identified the His765 catalytic phosphorylation site, the ATP‐binding site, as well as the CoA binding site. The human ATP‐citrate lyase cDNA clone was subcloned into a mammalian expression vector for expression in African green monkey kidney cells (COS) and Chinese hamster ovary cells (CHO) cells. Transfected COS cells expressed detectable levels of an enzymatically active recombinant ATP‐citrate lyase enzyme. Stable, amplified expression of ATP‐citrate lyase in CHO cells was achieved by using coamplification with dihydrofolate reductase. Resistant cells expressed high levels of enzymatically active ATP‐citrate lyase (3 pg/cell/d). Site‐specific mutagenesis of His765→Ala diminishes the catalytic activity of the expressed ATP‐citrate lyase protein. Since catalysis of ATP‐citrate lyase is postulated to involve the formation of phosphohistidine, these results are consistent with the pattern of earlier observations of the significance of the histidine residue in catalysis of the human ATP‐citrate lyase.

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The murine submandibular gland (SMG) produces a novel class of highly acidic salivary proteins encoded by one or more highly abundant mRNA transcripts. In inbred mice, these transcripts are encoded by members of a multigene family comprising approximately 8-12 homologues. Most, and probably all, of these homologues are clustered at a new locus near belted (bt) on chromosome 15, which we designate Spt (salivary protein). Although physically closely linked, Spt genes differ in their patterns of expression both in strains of mice and in their tissues. One gene, Spt-1, is expressed at high levels in the SMG of all inbred strains examined. This gene is also expressed at significant levels in the lacrimal gland. A second gene, Spt-2, appears to be present as a single copy in some strains and as two copies in others. This gene is expressed at high levels only in the SMG of those strains carrying two copies, and Spt-2 mRNA is not detectable in the SMG of strains carrying only one copy. In contrast to Spt-1, the Spt-2 gene is not expressed at detectable levels in the lacrimal gland.

Synthesis of heat shock proteins (hsps) is a universal response of cells to heat shock. Recent evidence suggests that hsps are involved in the repair of damage caused by heat or other protein-denaturing insults. We have determined kinetics of induction for the 70 kD heat shock protein (hsp 70) and hsp 70 mRNA in adult salamanders, Eyrycea bislineata. We demonstrate that the extent of expression of the hsp protein and mRNA is proportional to the heat dose but differs with respect to tissue. Hsp 70 synthesis was shown to be controlled primarily at the level of mRNA transcription. Hsp 70 mRNA accumulation is prolonged in the adult animal when compared to cultured cells from other vertebrate ectotherms given similar heat shocks. Additionally, we show that the hottest of four animals captured in the field on a hot summer day had synthesized hsp 70 mRNA in its tissues. These findings are discussed relative to the thermal biology of vertebrate ectotherms.Exposure of cultured cells to near-lethal temperatures induces the heat shock response. Although other changes in cellular metabolism occur, the heat shock response consists principally of the induction of the heat shock proteins (hsps) in affected cells. The heat shock response appears to be universal in eukaryotes, regardless of their thermoregulatory strategy.

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