D protein of miniF plasmid acts as a repressor of transcription and as a site-specific resolvase.

Two activities of the D protein of the miniF plasmid have been found. Divergent promoters in ori-1 ("primary" replicative origin) of miniF are both repressed in cells which produce D protein. The mobilization of plasmids containing the ori-1 region by the F conjugation system is also repressed by D protein. In the former case D appears to act as a transcriptional repressor, whereas in the latter case D protein acts by resolving cointegrates of F and the mobilized plasmid. D protein resolves dimers whose monomer units contain the rfsF sequence needed for recA-independent, site-specific recombination of F. The nucleotide sequence of the D gene was determined. The D gene region contains two oppositely-oriented open reading frames which have the same reading phase and substantially overlap. Transposon insertion mutants were used to show that the gene for D protein occupies the top-strand (left-to-right) open reading frame.

Plasmid-determined bleomycin resistance in Staphylococcus aureus.

A 1580-bp fragment of Staphylococcus aureus plasmid pUB110 encoding resistance to the DNA synthesis inhibitor bleomycin has been cloned and sequenced. A DNA sequence containing an open reading frame of 405 bp was subcloned into several expression vectors and bleomycin resistance was expressed at high level in Escherichia coli under the control of lambda PL promoter. On induction, a ca. 14,000-Da protein was detected by gel electrophoresis. The bleomycin resistance determinant of the gram-positive plasmid pUB110 was compared to that of the enterobacterial transposon Tn5; limited regions of close relatedness could be identified.

Optimizing the expression in E. coli of a synthetic gene encoding somatomedin-C (IGF-I).

Double-stranded DNA encoding the human hormone somatomedin-C (SMC) has been synthesized. This synthetic gene has been inserted into a plasmid bearing the strong leftward promoter (PL) of bacteriophage lambda and expressed in E. coli. Codons for the N-terminal region of SMC which maximized the hormone's synthesis were chosen in an SMC-lac z fusion assay. The amounts of SMC accumulated in E. coli were influenced by mutations at two chromosomal loci, lon and htpR.

Cloning and nucleotide sequence of the Salmonella typhimurium pepM gene.

The pepM gene coding for a methionine-specific aminopeptidase was cloned from Salmonella typhimurium and its nucleotide sequence determined. The gene encoded a 264 amino acid protein that was homologous to a similar protein from Escherichia coli. The sequence of an overproducer mutant allele, pepM100, contained a single base change in the likely--35 region of the pepM promoter that increased its homology to the consensus promoter sequence. A region downstream from the pepM coding sequence contained extensive inverted repeats and was homologous to sequences found elsewhere in both Salmonella and other bacterial species.

Alternative RNA processing events in human calcitonin/calcitonin gene-related peptide gene expression.

Two mRNAs generated as a consequence of alternative RNA processing events in expression of the human calcitonin gene encode the protein precursors of either calcitonin or calcitonin gene-related peptide (CGRP). Both calcitonin and CGRP RNAs and their encoded peptide products are expressed in the human pituitary and in medullary thyroid tumors. On the basis of sequence comparison, it is suggested that both the calcitonin and CGRP exons arose from a common primordial sequence, suggesting that duplication and rearrangement events are responsible for the generation of this complex transcription unit.

Molecular cloning and expression of human tumor necrosis factor and comparison with mouse tumor necrosis factor.

U-937 cells, a monocytic line derived from a human histiocytic lymphoma, were induced for human tumor necrosis factor (TNF) secretion into the medium and were used for the preparation of TNF mRNA. Biological activity of the latter was quantified in a Xenopus laevis oocyte injection system. TNF mRNA was enriched by gradient centrifugation and this size-fractionated mRNA was used for synthesis of cDNA and inserted into the unique PstI site of pAT153. A recombinant plasmid containing human TNF cDNA was selected by colony hybridization using an internal fragment of a mouse TNF cDNA clone [Fransen, L., Mueller, R., Marmenout, A., Tavernier, J., Van der Heyden, J., Kawashima, E., Chollet, A., Tizard, R., Van Heuverswyn, H., Van Vliet, A., Ruysschaert, M. R. & Fiers, W. (1985) Nucleic Acids Res. 13, 4417-4429] as a probe. The sequence of this human TNF cDNA is in agreement with the one published by Pennica et al. [Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg, P. H., Derynck, R., Palladino, M. A., Kohr, W. J., Aggarwal, B. B. & Goeddel, D. V. (1984) Nature (Lond.) 312, 724-729]. The 157-amino-acid-long mature sequence is about 80% homologous to mouse TNF and its hydrophilicity plot is also very similar, in spite of the apparent species specificity of TNF. In contrast to mouse TNF, it contains no potential N-glycosylation site. When compared to other cytokines, like IFN-beta, IFN-gamma, or IL-2, there is a remarkably high preference for G X C pairs in the third-letter positions. Expression of the TNF cDNA in monkey COS cells or in Escherichia coli gives rise to a protein having similar biological and serological properties as natural human TNF. A human genomic clone was also identified and sequenced; it was found to be in good agreement with the one recently published by Shirai et al. [Shirai, T., Yamaguchi, H., Ito, H., Todd, C. W. & Wallace, R. B. (1985) Nature (Lond.) 313, 803-806], except for some differences in the introns and 5'-untranslated region.