The Streptomyces plasmid SCP2*: its functional analysis and development into useful cloning vectors

Detailed restriction maps of the plasmid SCP2* and its deletion derivative pSCP103 were constructed. DNA fragments carrying hygromycin (Hyg), thiostrepton (Thio) or viomycin-resistance (VioR) determinants were inserted into pSCP103, and various segments were deleted from the resulting plasmids. Changes in plasmid phenotypes associated with these insertions and deletions allowed the localisation and characterisation of plasmid replication, stability, transfer and fertility functions. Several useful cloning vectors were constructed. They are able to maintain large (greater than 30 kb) DNA inserts, with stable inheritance at a low copy number (1-2 per chromosome) and without structural rearrangements, in Streptomyces hosts. The vectors have a broad host range in the genus Streptomyces. One of them (pIJ903) is a shuttle vector for Streptomyces and Escherichia coli.     

An analysis of tobacco mosaic virus replicative structures synthesized in vitro

Summary The RNA structures synthesized in vitro by a crude enzyme complex from tobacco mosaic virus (TMV)-infected leaves have been analyzed; the major viral-specific products were similar to TMV-replicative form (RF) and-replicative intermediate (RI) in electrophoretic behavior and ribonuclease sensitivity. Synthesis of these RF-like and RI-like structures neither required nor responded to added viral RNA, but did require all four ribonucleotide triphosphates. Enriched radiolabeled RF-like and RI-like RNA fractions were isolated from non-denaturing agarose gels by electroelution and hybridized to a collection of TMV sequences cloned into bacteriophage M13. Enriched RF-RNA hybridized to sequences of both plus and minus polarity, while enriched RI-RNA hybridized only to inserts of minus polarity, indicating only plus strand synthesis in this fraction. Most of the label incorporated into the plus strand of the enriched RF-RNA was found near the 3-end of this strand, while most of the label incorporated into enriched RI-RNA was found several hundred bases from the 5-end of the plus strand.Paper presented at the first International Congress of Plant Molecular Biology (Savannah, GA, 1985).     

Experimental Histomoniasis in the Guinea Fowl,Numida meleagris*

Guinea fowl were inoculated rectally with Histomonas meleagridis to produce histomoniasis. The birds were infected readily by this unnatural route. Severe cecal involvement was frequent and long-lasting but liver lesions and death were rare. Turkeys given the same inoculum had high levels of liver involvement and mortality. Guinea fowl responded much less severely to infection with H. meleagridis when infected by a natural route (ingestion of a vector) rather than by rectal inoculation. Thus, naturally acquired infections with H. meleagridis appear to be of small consequence in the guinea fowl.     

Secretion of the sweet-tasting plant protein thaumatin byStreptomyces lividans

Summary To produce and direct the export inStreptomyces lividans of the sweet plant protein thaumatin, thaumatin II cDNA was fused in the correct reading frame to the -galactosidase leader peptide, under the control of the -galactosidase promoter and ribosome binding site. The export of the recombinant thaumatin may allow the correct formation of the thaumatin disulfide bonds. The recombinant thaumatin was purified from the medium on an S-Sepharose column and detected with western blots by sheep -thaumatin antibodies. The recombinant thaumatin was the same size as authentic thaumatin and changed position on an acrylamide gel in response to reduction by 2-mercaptoethanol in the same manner.     

Functional Expression of the Alkane-Inducible Monooxygenase System of the Yeast: Candida tropicalis IN Saccharomyces cerevisiae

The genes for the alkane-inducible monooxygenase system of the yeast Candida tropicalis, namely a cytochrome P450_alk (P450_alk) and a NADPH cytochrome P450 oxidoreductase (NCPR) gene, were transferred in Saccharomyces cerevisiae. The P450_alk gene was expressed in this host with the help of the yeast alcohol dehydrogenase I (ADHI) promoter and terminator, whereas the NCPR gene could be expressed with its own structural elements. The presence of P450_alk in S. cerevisiae microsomal fractions resulted in a new acquired lauric acid terminal hydroxylation activity. Moreover, the same activity, coupled with the appearance of 12-hydroxylauric acid derivatives, could be obtained by the addition of lauric acid to intact cells expressing P450_alk. The coordinate expression of the P450_alk and NCPR genes in S. cerevisiae elevated the turnover rate of the P450_alk monooxygenase activity about 2-fold.     

Comparison of wild type and genetically engineered nuclear polyhedrosis viruses of Autographa californica for mortality, virus replication and polyhedra production in Trichoplusia ni larvae

Virus replication and polyhedra production of two polyhedron-positive recombinant nuclear polyhedrosis viruses of Autographa californica, AcJHE.KK and AcAaIT which encode juvenile hormone esterase and scorpion toxin, respectively, were compared with those of a plaque purified wild-type nuclear polyhedrosis virus, AcMNPV-C6, in Trichoplusia ni larvae. Though average times required to kill the T. ni larvae increased with the age of the larvae, killing time by either recombinant virus was significantly shorter than that by wild-type virus. Killing time was reduced ca. 30% for AcAaIT-infected larvae and 5 to 8% for AcJHE.KK-infected larvae as compared to that for AcMNPV-C6-infected larvae. The average weight of larvae infected with AcAaIT was significantly lower than that of larvae infected with AcJHE.KK and AcMNPV-C6. The mean numbers of polyhedra produced in each larva inoculated with AcAaIT and AcJHE.KK were ca. 20% and 60%, respectively, of those for AcMNPV-C6. Total virus titers in AcMNPV-C6-infected larvae were significantly higher than those in AcJHE.KK- and AcAaIT-infected larvae until 2 days post infection.     

Molecular analysis of two functional homologues of the S 3 allele of the Papaver rhoeas self-incompatibility gene isolated from different populations

The S ₃ allele of the S gene has been cloned from Papaver rhoeas cv. Shirley. The sequence predicts a hydrophilic protein of 14.0 kDa, showing 55.8% identity with the previously cloned S ₁ allele, preceded by an 18 amino acid signal sequence. Expression of the S ₃ coding region in Escherichia coli produced a form of the protein, denoted S₃e, which specifically inhibited S₃ pollen in an in vitro bioassay. The recombinant protein was ca. 0.8 kDa larger than the native stigmatic form, indicating post-translational modifications in planta, as was previously suggested for the S₁ protein. In contrast to other S proteins identified to date, S₃ protein does not appear to be glycosylated. Of particular significance is the finding that despite exhibiting a high degree of sequence polymorphism, secondary structure predictions indicate that the S₁ and S₃ proteins may adopt a virtually identical conformation. Sequence analysis also indicates that the P. rhoeas S alleles share some limited homology with the SLG and SRK genes from Brassica oleracea. Previously, cross-classification of different populations of P. rhoeas had revealed a number of functionally identical alleles. Probing of western blots of stigma proteins from plants derived from a wild Spanish population which contained an allele functionally identical to the Shirley S ₃ allele with antiserum raised to S₃e, revealed a protein (S ₃ s) which was indistinguishable in pI and M _r from that in the Shirley population. A cDNA encoding S ₃ s was isolated, nucleotide sequencing revealing a coding region with 99.4% homology with the Shirley-derived clone at the DNA level, and 100% homology at the amino acid level.     

The Biology of the Tospoviruses

The tospoviruses are a diverse, cosmopolitan and economically important genus of plant viruses. In the recent past, interest in the tospoviruses has been rekindled with the resurgence and expansion of tomato spotted wilt virus and the appearance of new tospoviruses, including impatiens necrotic spot. This renewed interest in the tospoviruses, accompanied with the many recent advances in plant virology techniques, particularly those utilising molecular biology, have resulted in a rapid growth of our understanding of these viruses. This paper provides a review of the tospoviruses, encompassing all the major aspects of their biology, including the recent changes in the classification of the genus and current knowledge on molecular biology, vector relations, control and diagnosis.     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

Augmentation of antitumor effect by combined administration with interleukin-2 and sizofiran,a single glucan,on murine EL-4 lymphoma

The antitumor effect of the combined administration with recombinant human interleukin-2 (rIL-2) and sizofiran (SPG), a single glucan of Shizophyllum commune Fries, was studied in vivo in C57BL/6 mice intraperitoneally inoculated with EL-4 lymphoma. The effect was evaluated by a) comparing the survival time of the mice, b) analysis of the intraperitoneal cell population in Giemsa-stained specimens, c) surface marker analysis of peritoneal exudative cells with flow cytometry, d) cytotoxic assay of cells against EL-4 and Yac-1 lymphoma, and e) elimination of some cell populations by monoclonal antibodies, to identify the antitumor-effector cells showing cytotoxic activity. The survival of mice given both rIL-2 and SPG was significantly longer than the control mice or those given SPG alone or rIL-2 alone. It was demonstrated that the administration of SPG and/or rIL-2 to the EL-4 lymphoma-bearing mice activated immune-response cells in the peritoneal cavity such as T lymphocytes, NK cells, or macrophages, which might be effective in reducing lymphoma cells. The combination of rIL-2 and SPG administration appears to activate the antitumor- immune response at the tumor site more effectively than when either agent was administered alone.