Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress

The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants.Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.     

A map of barley chromosome 2 using isozymic and morphological markers

The F₂ progeny of a cross between a chromosome 2 multiple marker stock and an adapted cultivar of barley were analyzed for four morphological markers and electrophoretic patterns of eight leaf isozymes. TheIdh-2 locus was linked to thePer-5 locus (27.96±5.07 cM) and to thee locus (10.26±3.13 cM). Also, thePer-5 ande loci were located on the short arm of chromosome 2. In additionIdh-2 was also located on barley chromosome 2 and was linked to thev locus (13.18±3.56 cM), which is located on the long arm of chromosome 2. Two other marker genes,li andwst,,B, were linked (26.50±5.24 cM) on chromosome 2 but segregate independently of the other loci evaluated. This project was supported by funds from the U.S.-Spain Joint Committee for Scientific and Technological Cooperation.     

Heterogeneity of adrenocortical ferredoxin

Bovine adrenocortical ferredoxin (adreno-ferredoxin) was purified from adrenocortical mitochondria by an improved method that included hydrophobic chromatography on Toyopearl gels. The purified ferredoxin was electrophoretically homogeneous. It was further separated into five fractions by hydrophobic chromatography on a TSK-gel phenyl-5PW column with a high-pressure liquid chromatography system. The properties of the three main fractions were examined. The fractions had identical absorption spectra and almost the same activity in an NADPH-cytochrome c reducing system. Their amino-terminal sequences all corresponded to the reported sequence, but the carboxyl-terminal residues were glycine or serine, not alanine as reported. These results indicate that these adreno-ferredoxins had additional amino acid residues at the carboxyl end. It seems that adreno-ferredoxin extracted from mitochondria undergoes proteolytic attack during purification to become heterogeneous.     

Disulfides of the lutropin receptor

Affinity cross-linking of the lutropin receptor with 125I-human choriogonadotropin (hCG) on porcine granulosa cells produced four distinct homone-receptor complexes under reducing conditions. They contain 18-, 24-, 28-, and 34-kDa components (Ji, I., Bock, J. H., and Ji, T. H. (1985) J. Biol. Chem. 260, 12815-12821). Photoaffinity labeling and cross-linking produced 136-, 102-, and 74-kDa hCG-receptor complexes under reducing conditions and the 136-kDa complex under nonreducing conditions. In addition, the unreduced 102-kDa complex was seen in photoaffinity labeling but not in cross-linking. When the unreduced 136-kDa complex was reduced, the 102- and 74-kDa complexes were generated, indicating release of the 34- and the 28-kDa components in two steps. When the unreduced 102-kDa complex was reduced, the 74-kDa complex was produced, indicating the release of a 28-kDa component. The 74-kDa complex could not be reduced but was cleaved by alkaline treatment to produce the hCG alpha beta dimer. The results indicate that the 24-kDa component is released from the 74-kDa complex, since the apparent mass of the hCG alpha beta dimer on gels is 50 kDa. The 24-kDa component appears to be the initial site for photoaffinity labeling or cross-linking and to be disulfide linked to the 28-kDa component which is in turn disulfide linked to the 34-kDa component. These intercomponent disulfides exist in some receptors but not all. Formation of the disulfide-linked 136-kDa band required the presence of a sulfhydryl-blocking agent, N-ethylmaleimide. In particular, the 34-kDa component was vulnerable to reduction. There was no significant evidence of disulfides between the hormone and any of the receptor components.     

Role of endogenous complement in monoclonal IgM antibody-dependent leukemia suppression in vivo: participation of C3b

The mechanism by which McAb of the IgM isotype causes prolonged survival of leukemic rats was investigated. The participation of endogenous C in the suppression of IgM-sensitized leukemia cells was demonstrated by the observations that a) suppression was abrogated in CVF-treated rats, and b) the CVF effect was partially reversed if C3b was provided on the surface of IgM-sensitized leukemia cells.     

Composition of cross-linked 125I-follitropin-receptor complexes

Both of the alpha and beta subunits of intact human follitropin (FSH) were radioiodinated with 125I-sodium iodide and chloramine-T and could be resolved on sodium dodecyl sulfate-polyacrylamide gels. Radioiodinated FSH was affinity-cross-linked with a cleavable (nondisulfide) homobifunctional reagent to its membrane receptor on the porcine granulosa cell surface as well as to a Triton X-100-solubilized form of the receptor. Cross-linked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65, 83, and 117 kDa, in addition to the hormone bands. The hormone alpha beta dimer band corresponded to 43 kDa. Formation of the three bands requires the 125I-hormone to bind specifically to the receptor with subsequent cross-linking. Binding was prevented by an excess of the native hormone but not by other hormones. A monofunctional analog of the cross-linking reagent failed to produce the three bands. Reagent concentration-dependent cross-linking revealed that their formation was sequential; smaller complexes formed first and then larger ones. When gels of cross-linked complexes were treated to cleave covalent cross-links and then electrophoresed in a second dimension, 18-, 22-, and 34-kDa components were released, in addition to the alpha and beta subunits of the hormone.     

Genetic engineering of coryneform bacteria

The coryneforms are a diverse group of bacteria which includes animal and plant pathogens as well as non-pathogenic bacteria. Although they are of significant economic and health importance, their genetics is poorly understood. The development of genetic engineering techniques for coryneforms and initial gene cloning studies are discussed.     

Effects of short-term treatment with calcium on the parathyroid gland of the rat,under particular consideration of the alteration of storage granules

Summary Short-term effects of CaCl₂-treatment on parathyroid cells of the rat, especially on their storage granules, were studied at the ultrastructural level. After an injection of 4% CaCl₂, serum calcium levels (SCL) rapidly increased from 9.1 mg/dl (controls) to a maximum of 14.9 mg/dl at 20 min. At 5 min after the injection, the number of type-I storage granules (large core) [NSG-I] and that of type-II storage granules (small core) [NSG-II] remained unchanged, in spite of elevated SCL (12.4 mg/dl). As soon as SCL rose to 13.2 mg/dl at 7.5 min, NSG-I gradually decreased to a minimum at 30 min; in contrast, NSG-II gradually increased to a maximum at 30 min. Vacuolar bodies also increased together with the augmentation of type-II storage granules. The average diameter of the core of the storage granules decreased significantly after the injection. Protein A-gold method for immunocytochemistry showed that the cores of these granules contain parathormone. Acid-phosphatase activity was occasionally found in storage granules of both types, especially in those of type II. It is concluded (i) that type-I storage granules may be transformed into vacuolar bodies via type-II granules as a result of hydrolysis, and (ii) that these processes may be accelerated during hypercalcemia.     

Pilot-scale production of l-phenylalanine from d-glucose

Effects of inoculum size and total sugar content on both l-phenylalanine productivity and titre have been investigated using a tyrosine auxotrophic regulatory mutant of Escherichia coli. Fermentations were carried out in a 500 litre pilot fermenter with intermittent feeding of d-glucose plus phosphate. It was found that the productivity was not greatly affected by inoculum size. However, the l-phenylalanine titre was significantly affected by total sugar content. Relatively high productivities of up to 0.35–0.40 g l-phenylalanine l^?1 h^?1 have been achieved at l-phenylalanine titres of 14–15 g l^?1.