The chalcone synthase multigene family of Petunia hybrida (V30): differential,light-regulated expression during flower development and UV light induction

We have analysed the expression of the 8–10 members of the gene family encoding the flavonoid biosynthetic enzyme chalcone synthase (CHS) from Petunia hybrida. During normal plant development only two members of the gene family (CHS-A and CHS-J) are expressed. Their expression is restricted to floral tissues mainly. About 90% of the total CHS mRNA pool is transcribed from CHS-A, wheares CHS-J delivers about 10% in flower corolla, tube and anthers. Expression of CHS-A and CHS-J during flower development is coordinated and (red) light-dependent. In young seedlings and cell suspension cultures expression of CHS-A and CHS-J can be induced with UV light. In addition to CHS-A and CHS-J, expression of another two CHS genes (CHS-B and CHS-G) is induced in young seedlings by UV light, albeit at a low level. In contrast to CHS genes from Leguminoseae, Petunia CHS genes are not inducible by phytopathogen-derived elicitors. Expression of CHS-A and CHS-J is reduced to a similar extent in a regulatory CHS mutant, Petunia hybrida Red Star, suggesting that both genes are regulated by the same trans-acting factors. Comparison of the promoter sequences of CHS-A and CHS-J reveals some striking homologies, which might represent cis-acting regulatory sequences.     

Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme

The enzymology of methanol utilization in thermotolerant methylotrophic Bacillus strains was investigated. In all strains an immunologically related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting conditions this enzyme constituted a high percentage of total soluble protein. The methanol dehydrogenase from this organism was purified to homogeneity and characterized. In cell-free extracts the enzyme displayed biphasic kinetics towards methanol, with apparent K _m values of 3.8 and 166 mM. Carbon assimilation was by way of the fructose-1,6-bisphosphate aldolase cleavage and transketolase/transaldolase rearrangement variant of the RuMP cycle of formaldehyde fixation. The key enzymes of the RuMP cycle, hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), were present at very high levels of activity. Failure of whole cells to oxidize formate, and the absence of formaldehyde-and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS. A comparison of the levels of methanol dehydrogenase and HPS in cells of Bacillus sp. C1 grown on methanol and glucose suggested that the synthesis of these enzymes is not under coordinate control.Abbreviations RuMP ribulose monophosphate - HPS hexulose-6-phosphate synthase - HPI hexulose-6-phosphate isomerase - MDH methanol dehydrogenase - ADH acohol dehydrogenase - PQQ pyrroloquinoline, quinone - DTT dithiothreitol - NBT nitrobluetetrazolium - PMS phenazine methosulphate - DCPIP dichlorophenol indophenol     

Regulation of reduced nitrogen assimilation in Rhodobacter capsulatus E1F1

The phototrophic bacterium Rhodobacter capsulatus E1F1 assimilates ammonia and other forms of reduced nitrogen either through the GS/GOGAT pathway or by the concerted action of l-alanine dehydrogenase and aminotransferases. These routes are light-independent and very responsive to the carbon and nitrogen sources used for cell growth. GS was most active in cells grown on nitrate or l-glutamate as nitrogen sources, whereas it was heavily adenylylated and siginificantly repressed by ammonium, glycine, l-alanine, l-aspartate, l-asparagine and l-glutamine, under which conditions specific aminotransferases were induced. GOGAT activity was kept at constitutive levels in cells grown on l-amino acids as nitrogen sources except on l-glutamine where it was significantly induced during the early phase of growth. In vitro, GOGAT activity was strongly inhibited by l-tyrosine and NADPH. In cells using l-asparagine or l-aspartate as nitrogen source, a concerted induction of l-aspartate aminotransferase and l-asparaginase was observed. Enzyme level enhancements in response to nitrogen source variation involved de novo protein synthesis and strongly correlated with the cell growth phase.Abbreviations ADH l-alanine dehydrogenase - AOAT l-alanine:2-oxoglutarate aminotransferase - Asnase l-asparaginase - GOAT Glycine: oxaloacetate aminotransferase - GOGAT Glutamate synthase - GOT l-aspartate: 2-oxoglutarate aminotransferase - GS Glutamine synthetase - HPLC High-Pressure Liquid Chromatography - MOPS 2-(N-morpholino)propanesulfonic acid - MSX l-methionine-d,l-sulfoximine     

Regulation of branched-chain amino acid biosynthesis in Streptomyces fradiae,a producer of tylosin

Synthesis of threonine dehydratase in Streptomyces fradiae was positively influenced by valine and negatively by isoleucine. However, these two amino acids had no effect on the activity of this enzyme. Synthesis of threonine dehydratase in -aminobutyrate resistant mutants of S. fradiae was pronouncedly less sensitive to the positive effect of valine and this change in regulation led to valine overproduction. Synthesis of acetohydroxy acid synthase is regulated in a similar manner to that of threonine dehydratase, however a lower level of expression was detected in -aminobutyrate resistant mutants. And again, no effect of branched-chain amino acids on acetohydroxy acid synthase activity was observed. It follows that in S. fradiae synthesis of threonine dehydratase is the main regulatory mechanism governing production and the mutual ratio of synthesized valine and isoleucine.Abbreviations -AB -aminobutyrate - AHAS acetohydroxy acid synthase - -KB -ketobutyrate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - TD threonine dehydratase - Trans. B. transaminase of branched-chain amino acids - VDH valine dehydrogenase     

Immunological studies of ferredoxin-nitrite reductases and ferredoxin-glutamate synthases from photosynthetic organisms

Polyclonal antiserum specific for ferredoxin-nitrite reductase (EC 1.7.7.1) from the green alga Chlamydomonas reinhardii recognized the nitrite reductase from other green algae, but did not cross-react with the corresponding enzyme from different cyanobacteria or higher plant leaves. An analogous situation was also found for ferredoxin-glutamate synthase (EC 1.4.7.1), using its specific antiserum. Besides, the antibodies raised against C. reinhardii ferredoxin-glutamate synthase were able to inactivate the ferredoxin-dependent activity of nitrite reductase from green algae.These results suggest that there exist similar domains in ferredoxin-nitrite reductases and ferredoxin-glutamate synthases from green algae. In addition, both types of enzymes share common antigenic determinants, probably located at the ferredoxin-binding domain. In spite of their physicochemical resemblances, no apparent antigenic correlation exists between the corresponding enzymes from green algae and those from higher plant leaves or cyanobacteria.Abbreviations Fd ferredoxin - GOGAT glutamate synthase - MV⁺ reduced methyl viologen (radical cation) - NiR nitrite reductase - PMSF phenylmethylsulphonyl fluoride - SDS sodium dodecyl sulfate     

In vitro study of transgenic tobacco expressing Arabidopsis wild type and mutant acetohydroxyacid synthase genes

Summary Genes coding for the enzyme acetohydroxyacid synthase, often referred to as acetolactate synthase (AHAS, ALS; EC 4.1.3.18), from wild type Arabidopsis thaliana and a sulfonylurea-resistant mutant line GH50 (csrl-1; Haughn et al. 1988) were introduced in Nicotiana tabacum. Both genes were expressed at high levels with the 35S promoter. The csrl-1 gene conferred high levels of resistance to chlorsulfuron whereas the wild type gene did not. As selectable markers, chimaeric AHAS genes yielded transgenic plants on chlorsulfuron but at much lower efficiencies than with a chimaeric neomycin phosphotransferase gene on kanamycin (Sanders et al. 1987). Shoot differentiation from leaf discs was delayed on chlorsulfuron by 4–6 weeks. This study indicated a role for mutant AHAS genes in the genetic manipulation of herbicide resistance in transgenic plants but as selectable markers for plant cells undergoing differentiation no advantage over other genes was perceived.     

CO2 fixation in halobacteria

Seven strains of extremely halophilic bacteria (Halobacterium spp., Halococcus spp., and Haloarcula sp.) fixed CO₂ under light and dark conditions. Light enhanced CO₂ fixation in Halobacterium halobium but inhibited it in Halobacterium volcanii and Haloarcula strain GN-1. Propionate stimulated ¹⁴CO₂ incorporation in some strains, but inhibited it in others. Semi-starvation in basal salts plus glycerol induced enhanced CO₂ fixation rates. ¹⁴CO₂ fixation in semi-starved cells was stimulated by NH ₄ ⁺ or pyruvate and inhibited by succinate and acetate in most strains. No possible reductant was found. In cell-free extracts of H. halobium, NH ₄ ⁺ but not propionate stimulated ¹⁴CO₂ fixation. No RuBP carboxylase activity was detected. The main ¹⁴C-labeled -keto acid detected after a 2-min incubation with ¹⁴CO₂ and pyruvate was pyruvate. Little or no -ketobutyrate was detected among the early products of propionate-stimulated CO₂ fixation. Glycine was the major amino acid synthesized during a 2-min incubation with NH ₄ ⁺ , propionate, and ¹⁴CO₂. Propionate-stimulated CO₂ fixation was sensitive to trimethoprim and insensitive to avidin. A novel pathway for non-reductive CO₂ fixation involving a glycine synthase reaction with CO₂, NH ₄ ⁺ , and a methyl carbon derived from the -carbon cleavage of propionate is tentatively proposed.Abbreviations used BBS buffered basal salts - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - DNPH 2,4-dinitrophenylhydrazine - DNP dinitrophenyl - TLC thin-layer chromatography - FH₄ tetrahydrofolate This work was supported by National Science Foundation grant PCM-8116330 and Petroleum Research Fund grant PRF 13704-AC2     

Lung burden of green nickel oxide aerosol and histopathological findings in rats after continuous inhalation

There are few inhalation studies of nickel carcinogenesis. In this study, Wistar male rats were exposed to green nickel oxide (NiO(G)) aerosols (mass median aerodynamic diameter, 0.6 μm) for 7 h/d, 5 d/wk for up to 12 mo. The average exposure concentration was controlled at 0.3 and 1.2 mg/m³ during the exposure. For histopathological examination and measurement of the nickel concentration in rat organs, the rats were sacrificed at 3, 6, and 12 mo of exposure and 8 mo clearance period following 12 mo of exposure. The nickel content in rat lungs that was observed up to 2.6 mg after 12 mo exposure, was proportional to the exposure concentration during the exposure. The clearance of the nickel from the lungs was very slow and the biological half time was determined 7.7 mo. Although the rats were exposed continuously to NiO(G), for 12 mo and kept for 8 mo clearance period, there were no malignant tumors in any of the exposed animals.     

Daucus carota cells contain a dihydrofolate reductase: thymidylate synthase bifunctional polypeptide

Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) activities from cell suspension cultures of Daucus carota were shown to copurify on (NH₄)₂SO₄ fractionation, DEAE Sephadex and methotrexate-Sepharose affinity chromatography and to share approximately the same M_r(183 kDa and 185 kDa respectively) as judged by gel filtration on Sephacryl S-200.The copurified protein migrated as a single band on polyacrylamide gel electrophoresis under denaturing conditions.Both activities could be eluted from the same position of the native gel.Moreover, methotrexate-resistant cell lines which overproduce DHFR revealed to have a parallel higher level of TS. It is therefore proposed and discussed that in carrot, similarly to protozoa, TS and DHFR are present on a single bifunctional polypeptide of 58 kDa.