Structure, sequence and expression of the hepatitis delta (delta) viral genome.

Biochemical and electron microscopic data indicate that the human hepatitis delta viral agent contains a covalently closed circular and single-stranded RNA genome that has certain similarities with viroid-like agents from plants. The sequence of the viral genome (1,678 nucleotides) has been determined and an open reading frame within the complementary strand has been shown to encode an antigen that binds specifically to antisera from patients with chronic hepatitis delta viral infections.

Human Cu/Zn superoxide dismutase cDNA: isolation of clones synthesising high levels of active or inactive enzyme from an expression library.

The molecular cloning and nucleotide sequence of the cDNA for human Cu/Zn superoxide dismutase (SOD) is reported. The tacI promoter has been used to direct the synthesis in E. coli of this SOD which is soluble, stable, and of normal specific activity. The N-terminal methionine is removed from this protein. A construction with a ribosome binding site identical to that of the lacz gene 5' of the initiator methionine codon, resulted in low levels of SOD. An in vitro mutagenesis procedure was used to randomize the four nucleotides preceding the initiator methionine codon and the silent third positions of the codons specifying the second and third amino acids. Analysis of a sample of 500 clones showed that ca. 25 clones synthesised 5% or more of soluble cell protein as SOD. The nucleotide sequences of high level expressors showed a predominance of A and T residues in the variable positions 5' of the initiator methionine codon. An SOD mutant (ala4----gln) was discovered during the sequencing and shown to lack dismutation activity. Secondary structure predictions for the 5' regions of the mRNAs from high and low level expressors support the hypothesis that initiation of translation is much reduced if part of the region complementary to 16s rRNA is base paired in a stem structure.

Synthesis in yeast of a functional oxidation-resistant mutant of human alpha-antitrypsin.

Cumulative damage to lung tissue by leukocyte elastase is thought to be responsible for the development of pulmonary emphysema, an irreversible lung disease characterized by loss of lung elasticity. It is also thought to be involved in the rapidly developing and usually fatal adult respiratory distress syndrome. The primary defence against elastase damage is the anti-protease known as alpha 1-antitrypsin, a glycosylated serum protein of 394 amino acids. Oxidation of the methionine 358 residue located at the active centre of alpha 1-antitrypsin results in a dramatic decrease in inhibitory activity towards elastase which effectively inactivates the protective function. It has been suggested that this oxidation sensitivity has a regulatory function and allows tissue breakdown at sites of inflammation by inactivation of alpha 1-antitrypsin by oxygen radicals released by phagocytes. In the above diseases, however, the oxidative inactivation of alpha 1-antitrypsin is probably of major importance in allowing lung damage by elastase. An oxidation-resistant alpha 1-antitrypsin required for emphysemics and provide treatment for acute inflammatory respiratory conditions. To further the possibility of therapy for the above conditions, we describe here the synthesis in yeast of active, non-glycosylated, human alpha 1-antitrypsin. Site-directed mutagenesis has been used to construct an active, oxidation-resistant derivative containing a single methionine to valine substitution at the active centre. This demonstrates the potential of engineered modifications to protein molecules designed to improve their physiological function.

The sequence of human serum albumin cDNA and its expression in E. coli.

A recombinant plasmid has been constructed which contains the mature protein coding region of the human serum albumin (HSA) gene. Bacteria containing this plasmid synthesize HSA protein under control of the E. coli trp promoter-operator. The DNA sequence and predicted protein sequence of HSA were determined from the cDNA plasmid and are compared to existing data obtained from direct protein sequencing. The DNA sequence predicts a mature protein of 585 amino acids preceded by a 24 amino acid "prepro" peptide.

Similar sequences from the 5' end of cowpea mosaic virus RNAs.

Similar, large (N approximately 40) oligonucleotides are released from each of the two cowpea mosaic virus genomic RNAs by digestion with ribonuclease T1. Each large oligonucleotide was radiolabeled at the 3' end, and the nucleotide sequence was determined by base-specific partial degradation and analysis of the fragments. Specific deoxyoligonucleotides that are complementary to a portion of each large oligonucleotide were primers in reverse transcription reactions with intact virion RNA as template. Sequences to the 5' side of the large oligonucleotide sequences, and extending almost to the 5' end of each RNA, were read from analyses of transcripts that had been terminated by the incorporation of 2',3'-dideoxynucleoside-5'-phosphate residues. We report sequences of 87 and 79 nucleotide residues, respectively, from the two virus RNAs. The two sequences show many similarities and can be folded into a similar pattern to accommodate base pairing.

Oxalate and spin-labeled oxalate as probes of the anion binding site of human transferrin. Metal to anion distance.

The spin-labeled anion N-[4-(2,2,6,6-tetramethylpiperidin-1-oxyl)] oxamate has been synthesized and characterized. In the presence of this compound, a specific iron-transferrin-anion complex is formed, as evidenced by the development of a characteristic red color. No EPR signal was observed for the nitroxyl radical in the protein complex, presumably due to broadening of the signal by the paramagnetic metal ion. Failure to observe a signal implies that the metal to nitroxyl distance is less than or equal to 6 A. This suggests that the anion is directly attached to the metal ion in the protein. The pH dependence of iron dissociation from iron-transferrin-oxalate is also reported. This complex is more stable at low pH than iron-transferrin-carbonate.