Cloning and restriction fragment length polymorphism analysis of a cDNA for swine PIT-1, a gene controlling growth hormone expression*

A partial swine cDNA which encodes the functional domain of PIT-1 was isolated by the polymerse chain reaction (PCR). The swine PIT-1 cDNA clone is 95% identical at the protein level to the rat Pit-1 gene. Thus, Pit-l's known function in control of rat growth hormone and prolactin expression is likely to be conserved in swine. This swine cDNA clone was used to investigate genetic variability at PIT-1 in several American and Chinese breeds. Polymorphic BamIII fragments were found in pure-bred Meishan animals (n= 13), but only monomorphic fragments in five American breeds (n= 36).     

Alterations in the hepatic enzymes following experimental lead poisoning

An investigation on the influence of lead toxicity on some of the hepatic enzymes was studied in rats both after a shorter interval of 15 d and after longer intervals of 60 and 90 d. Three different doses of lead as 5, 10, and 50 mg/kg body wt were administered orally on every alternate day. Whereas significant inhibition of succinic dehydrogenase was seen following lead poisoning, the activity acid and alkaline phosphatase increased with lead intoxication. The histoarchitecture of the liver was grossly intact. Liver accumulated less lead compared to kidney at 60 and 90 d.     

Purification and characterization of recombinant human testis angiotensin-converting enzyme expressed in Chinese hamster ovary cells.

Enzymatically active human testis angiotensin-converting enzyme (ACE) was expressed in Chinese hamster ovary (CHO) cells stably transfected with each of three vectors: p omega-ACE contains a full-length testis ACE cDNA under the control of a retroviral promoter; and pLEN-ACEVII and pLEN-ACE6/5, in which full-length and membrane anchor-minus testis ACE cDNAs, respectively, are under the control of the human metallothionein IIA promoter and SV40 enhancer. In every case, active recombinant human testis ACE (hTACE) was secreted in a soluble form into the culture media, up to 2.4 mg/liter in the media of metal-induced, high-producing clones transfected with one of the pLEN vectors. In addition, membrane-bound recombinant enzyme was recovered from detergent extracts of cell pellets of CHO cells transfected with either p omega-ACE or pLEN-ACE-VII. Recombinant converting enzyme was purified to homogeneity by single-step affinity chromatography of conditioned media and detergent-extracted cell pellets in 85 and 70% overall yield, respectively. Purified hTACE from all sources comigrated with the native testis isozyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) approximately 100 kDa. The native and recombinant proteins cross-reacted equally with anti-human kidney ACE antiserum on Western blotting. The catalytic activity of recombinant angiotensin-converting enzyme, in terms of angiotensin I and 2-furanacryloyl-Phe-Gly-Gly hydrolysis, chloride activation, and lisinopril inhibition, was essentially identical to that of the native enzyme. The facile recovery in high yield of fully active hTACE from the media of stably transfected CHO cells provides a suitable system for investigating structure-function relationships in this enzyme.     

Multiple xylanases of Cellulomona fimi are encoded by distinct genes

Cellulomonas fimi genomic DNA encoding xylanase activity has been cloned and expressed in Escherichia coli. As judged by DNA hybridization and restriction analysis, twelve xylanase-positive clones carried a minimum of four different xylanase (xyn) genes. The encoded enzymes were devoid of cellulase activity but three of the four bound to Avicel.     

Synthesis and expression of genes encoding tuna, pigeon, and horse cytochromes c in the yeast Saccharomyces cerevisiae.

Genes encoding tuna, pigeon, and horse cytochromes c were constructed with synthetic oligodeoxyribonucleotides having preferred codons and portions of the iso-1-cytochrome c-encoding gene from the yeast Saccharomyces cerevisiae. The genes were ligated into an expression vector, which contains the normal 5'- and 3'-untranslated regions of the yeast iso-1-cytochrome c gene, and were integrated in single copy into the chromosome. Yeast strains were also constructed with multiple integrated copies of the pigeon gene. The heterologous and normal mRNA levels of the single-copy strains were equivalent. Although the N-terminal methionines were completely cleaved in the heterospecific proteins, the levels of trimethylation of Lys72 and acetylation of N-terminal glycines ranged from 39-78% and 10-70%, respectively. Horse cytochrome c was produced at a nearly normal level, whereas the pigeon and tuna cytochromes c were produced at approx. 40% of the normal levels. The levels of the cytochromes c and growth of the mutant yeast strains indicated that the heterospecific cytochromes c had approx. 50% specific activity in vivo.     

The role of the epidermal growth factor-1 and hydrophobic stack domains of human factor IX in binding to endothelial cells.

To determine the function and specificity in factor IX of the first epidermal growth factor (EGF)-like domain and the eight-amino acid hydrophobic stack encoded by exon C (residues 39-46), these domains were replaced by the corresponding polypeptide regions of factor X and chimeric proteins were produced in human embryo kidney cells. Both chimeras were activated by factor XIa at a rate similar to plasma factor IX and exhibited calcium-dependent fluorescence quenching similar to plasma factor IX. Both chimeras competed equally for binding to the endothelial cell receptor. Our findings make it unlikely that the first EGF-like domain or the hydrophobic stack of factor IX are responsible for the specific binding of factor IX to its endothelial cell receptor.     

Efficient expression of a Trypanosoma cruzi antigen in Escherichia coli and Staphylococcus aureus and its rapid purification

A Trypanosoma cruzi antigen gene was closed into a fusion vector based on the IgG binding domain of Staphylococcus aureus protein A. This vector transformed into Escherichia coli or Staphylococcus aureus and produced about 12 mg fusion protein/l culture. In E. coli, the product remained intracellular while in S. aureus it was excreted into the growth medium. The hybrid protein was purified by IgG Sepharose affinity chromatography. The presence of a cleavage site for enterokinase between protein A and the T. cruzi antigen in the fusion protein allowed the efficient release of the unfused antigen by enzymatic treatment. Further affinity chromatography through IgG Sepharose resulted in the production of the T. cruzi antigen free of protein A.The authors are with the Department of Molecular Genetics, BioSidus S.A., Constitución 4234, 1254, Buenos Aires, Argentina.