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We have characterized the vaccinia virus 11‐kd late promoter through 5′ and 3′ deletions and site‐directed mutagenesis. The promoter function appears to be contained within an approximately 30‐bp fragment, which after translocation is able to direct RNA synthesis late in infection at a reduced level. We demonstrate that a TAAAT sequence in the proximal part of the promoter is essential for its function. This cis‐acting element is highly conserved within vaccinia virus late promoters and overlaps the site of transcription initiation. Deletions or mutations within this conserved element completely inactivate the promoter. The evidence indicates that the TAAAT motif functions as a TATA box. The region immediately upstream of the TAAAT motif determines the promoter strength.

A 240‐bp DNA fragment encoding a peptide, designated ENV(80), homologous to a conserved part of the gp41 transmembrane glycoprotein of human immunodeficiency virus (HIV) was chemically synthesized and inserted into different plasmid expression vectors. Escherichia coli transformants containing these plasmid constructs produced upon induction high amounts of either an ENV(80) peptide of relative molecular mass (Mr) of 10,000 or the same ENV(80) peptide N‐terminally fused to E. coli chloramphenicol acetyltransferase (CAT) or to mouse dihydrofolate reductase (DHFR) having Mr of 36,000 and 31,000 respectively. All polypeptides containing the ENV(80) sequences were strongly reactive with antibodies present in sera from AIDS virus‐infected individuals, but not with control sera. The strategy of gene assembly allowed the expression of ENV(80) subfragments fused to DHFR. The serodiagnosis of 15 positive sera by Western blot analysis using these bacterially synthesized ENV(80) subfragments revealed the presence of several immunoreactive epitopes on the 80‐amino acid polypeptide which were recognized differently by the various patients.

We describe a general purification method for recombinant proteins based upon the selective interaction between a poly-histidine peptide, which is fused to the protein of interest, and a novel metal chelate adsorbent. The principle of the technique is illustrated with mouse dihydrofolate reductase. DNA elements coding for adjacent histidines were fused to the mouse dihydrofolate reductase gene. Subsequent expression in E. coli resulted in the production of hybrid proteins that could be purified by immobilized metal ion affinity chromatography, followed by removal of the histidine affinity peptide with carboxypeptidase A.

The highest structural diversity of GABAA-receptor subunits is observed among members of the α-subunit class. Using subunit-specific antisera, the receptors containing the α2-subunit were characterized. Western blots revealed an apparent molecular size of 52 kDa for the α2-subunit. Immunohistochemically, the α2-subunit was most preponderant in areas which lack the α1-subunit, e.g. striatum and olfactory bulb granule cell layer, suggesting that these two subunits represent largely distinct receptor subtypes. Pharmacologically, the receptor population which was immunoprecipitated by the α2-subunit-specific antisera displayed a drug binding profile characterized by a low affinity for CL 218872, βCCM and zolpidem. This is in striking contrast to the high affinities of these ligands displayed by receptors immunoprecipitated by the α1-subunit-specific antiserum. Thus, the α-and the α2-subunit characterize two GABAA-receptor populations which greatly differ in brain distribution and pharmacological profile.

Synthetic 32P-labeled oligonucleotides have been used to identify the prostatic proline-rich polypeptide (PRP) mRNA which has partially been characterized. The 14-mer d(G-G-T-T-C-T-G-C-A-T-A-A-T-G) complementary to the coding sequence for His-Tyr-Ala-Glu-Pro, a sequence element occurring in all 38-residue PRP variants, hybridizes specifically with a 12.5-kilobase mRNA which is clearly androgen-controlled. This oligonucleotide was used as an efficient primer for the construction of a PRP-specific lambda gt10 cDNA library. The nucleotide sequence of the inserts from several recombinant clones has been determined. This structural analysis revealed a PRP mRNA encoding a large precursor containing a number of tandemly repeated units. Each repeat codes for a sequence of 100 amino acids in which the highly conserved PRP sequence is embedded. From this polyprotein the large number of PRP variants must be generated by a post-translational processing mechanism which is still unknown. The high degree of conservation of both nucleotide and amino acid sequence in the entire unit also indicates that the PRP gene(s) likely evolved by multiplication of a 300-base pair ancestral DNA sequence. This has resulted in a noninterrupted repetitive DNA coding segment which is detected at the genomic level.

We investigated the stability of fusion proteins composed of the signal peptide of the heat-labile enterotoxin of Escherichia coli and three polypeptides: the bacterial cytoplasmic chloramphenicol acetyltransferase, the mouse dihydrofolate reductase, and human immune interferon. We demonstrate that these proteins are rapidly degraded as a result of being targeted to the secretion apparatus of E. coli, with the extent of degradation varying among the three fusion proteins. Four lines of experimental evidence are presented in support of this suggestion. First, the chimeric polypeptides containing a functional signal peptide were detected in low amounts in vivo. When a mutation was introduced in the signal peptide, resulting in lack of recognition by the secretion apparatus, the chimeric proteins accumulated at high levels in the cytoplasm of the cell. Second, both the wild-type and mutant polypeptides accumulated in a purified and reconstituted in vitro translation system from E. coli and were equally susceptible to digestion by an exogenous protease. Third, the chimeric polypeptides lacking the signal peptide accumulated in a stable form in vivo. Fourth, the precursors of the proteins containing a functional signal peptide accumulated in a secA ts mutant at the restrictive temperature when secretion was blocked, suggesting that degradation is tightly linked to the secretion apparatus.