Positioning of protein-coding genes on the soybean chloroplast genome

Seven major plastid protein encoding genes were positioned on the soybean chloroplast DNA by heterologous hybridization. These include the genes for the alpha, beta and epsilon subunits of the CF₁ component of ATP synthase (atpA, atpB and atpE respectively), for subunit III of the CF₀ component of ATP synthase (atpH), for the cytochrome f (cytF), for the ‘32 Kd’ thylakoid protein (psbA), and for the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL), all of which map in the large single copy region. The atpB, atpE and rbcL genes are located in the region adjacent to one of the segments of the inverted repeat. The genetic organization of the soybean chloroplast DNA is compared to that of other plastid genomes.     

Expression ofKlebsiella pneumoniae nif genes inProteus mirabilis

Self-transmissible plasmids carryinghis andnif genes fromKlebsiella pneumoniae have been introduced into threehis mutants ofProteus mirabilis: strains 5006-1, WR19 and WR20. Expression ofhis by the transconjugants was unequivocal, if slightly temperature-sensitive, but none was Nif⁺ when tested for acetylene reduction in anaerobic glucose medium using inocula from rich or glucose-minimal aerobic agar cultures. Succinate or pyruvate in place of glucose, low glucose, lower temperature or elevated Na₂MoO₄ did not allownif expression and no nitrogenase MoFe-protein peptide was detected immunologically after exposure to conditions in which diazotrophic enterobacteria, normal or genetically constructed, derepressnif.One strain,P. mirabilis WR19, carrying thehis nif Km^r plasmid pMF250 was examined in detail. Thenif activator genenifA was introduced on the plasmid pCK1. Such derivatives remained Nif^- when tested, after aerobic growth on rich agar media, with normal or low glucose, with succinate or with elevated Mo. However, pre-conditioning by aerobic growth on glucose-minimal agar led to subsequent anaerobic expression ofnif in glucose medium from pMF250 in WR19 carrying pCK1. NH ₄ ⁺ or proline could serve as N-source in the glucose-minimal agar. Maximum activity was about 5% of that ofK. pneumoniae in our assay conditions. Material cross-reacting with anti-serum to the nitrogenase MoFe protein was formed. Nitrogenase activity was not switched off by NH ₄ ⁺ .P. mirabilis WR19 (pCK1) showed NH ₄ ⁺ -constitutive temperature-sensitive kanamycin resistance (anif-related phenotype of this plasmid) in aerobic glucose minimal medium. Expression ofnif inP. mirabilis WR19 (pCK1, pMF250) was NH ₄ ⁺ -repressible despite the constitutivenifA character of pCK1 and introduction of thentrA ⁺ plasmid pMM17 did not alter this phenotype. However, pCK1 did not give rise to NH ₄ ⁺ -constitutive diazotrophy in the wild-typeK. pneumoniae M5al. A construct of WR19 carrying pMF250 and constitutiventrC plasmid (pMD45) remained Nif^- even after pre-growth on glucose-minimal media.We conclude (a) thatP. mirabilis forms a gene product functionally equivalent to that ofntrA inK. pneumoniae, (b) that it forms no functional equivalent of thentrC product in our growth conditions. The need for pre-conditioning on aerobic glucose media remains perplexing.Non-common abbreviation NFDM Nitrogen-free-Davis-Mingioli medium     

Conserved plasmid/chromosome sequences in fast- and slow-growing rhizobia that nodulate the same plant

Apart from the ability to nodulate legumes, fast-and slow-growing rhizobia have few bacteriological traits in common. Given that there is only one pathway to nodulation, DNA sequences conserved in fast- and slow-growing organisms that nodulate the same host should be strongly enriched in infectivity genes. We tested this hypothesis with seven fast-growing and five slow-growing strains that produced responses varying from fully effective nodulation through various ineffective associations to non-nodulation on four different hosts (Lotus pedunculatus, Lupinus nanus, Macroptilium atropurpureum, and Vigna unguiculata). When restriction enzyme digested total DNA from 10 of the strains was separately hybridized with nick-translated plasmid DNA isolated from 4 fast-growing strains, variable but significant homologies were found with all 10 strains. Part of this homology was shown to be associated with the nifKDH genes for nitrogenase and part with putative nodulation genes carried on pC2, a cosmid clone containing a 37 kbp region of the large sym plasmid present in the fast-growing broad-host range Rhizobium sp. strain NGR234. Analysis of the extent of homology between the plasmids of 3 fastgrowing strains (NGR234, TAL 996 and UMKL 19) able to effectively nodulate Vigna unguiculata showed them to have homologous DNA fragments totalling 47 kbp. This core homology represents less than 12% of the total coding capacity of the sym plasmid present in each of these strains.Abbreviations Sym symbiotic sequences/plasmids - nod genes required for nodulation - nod putative nod genes - nif genes required for the synthesis of the enzyme nitrogenase     

Structural features are as important as sequence homologies inDrosophila heat shock gene upstream regions

Summary Pelham has shown that theDrosophila hsp 70 gene is not transcribed under heat shock conditions unless a given upstream region is present. Davidson et al. have recently compiled a list of sequences homologous to this region in otherDrosophila heat shock genes. They proposed that a set of unlinked genes, such as the heat shock genes, could be coordinately induced through an interaction in cis with a common regulatory molecule. That this interaction involves structural elements is suggested by the fact that these upstream regions share inverted repeats as well as areas of Z-DNA potential. Furthermore, using the Calladine-Dickerson rules for local helical parameters, we show that these regions share structural homology. This is significant because the presence of regions homologous to a derived consensus sequence does not necessarily imply structural similarity. Therefore, we suggest that these structural features are at least as important as the sequence homologies in enabling the heat shock response.     

Carbon monoxide sensitivity of cytochrome oxidase in wheat leaves in relation to dwarfing genes (Rht) in wheat cultivars

Summary Leaves of young seedlings of a number of tall cultivars of wheat, lacking the dwarfing Rht genes, readily responded to a brief 2 min exposure to CO, as assessed by in vivo aerobic assay of nitrate reductase. This test depends on the inhibition of cytochrome c oxidase by CO, which in turn renders cytosolic NADH available for the reduction of nitrate to nitrite in vivo. Semi-dwarf cultivars of wheat (Rht present) did not respond to CO in this way. Since CO forms a complex only with reduced cytochrome a₃, the results indicate differences in the redox state of cytochrome a₃, during in situ respiration of leaves from tall and semi-dwarf plants which are likely to be under genetic control.     

Mobilization of cloned luciferase genes into Vibrio harveyi luminescence mutants

A recombinant plasmid which carried a 5 kb fragment of Vibrio harveyi DNA containing the luxA and luxB genes was mobilized from Escherichia coli into luminescence-deficient mutants of V. harveyi. The cloned genes complemented a temperature sensitive luciferase mutation, but failed to complement lesions in two different aldehyde deficient mutants. Expression of the cloned genes was not subject to autoinduction in either E. coli or in V. harveyi.     

Comparison of the ribosomal RNA genes in four closely relatedCucurbitaceae

Cucurbitaceae are characterized by a high copy number for nuclear ribosomal RNA genes. We have investigated the genomic ribosomal DNA (rDNA) of four closely related species of this family with respect to structure, length heterogeneity, and evolution. InCucumis melo (melon) there are two main length variants of rDNA repeats with 10.7 and 10.55kb.Cucumis sativus (cucumber) shows at least three repeat types with 11.5, 10.5, and 10.2kb.Cucurbita pepo (zucchini) has two different repeat types with 10.0 and 9.3kb. There are also two different repeat types inCucurbita maxima (pumpkin) of about 11.2 and 10.5kb. Restriction enzyme mapping of the genomic rDNA of these four plants and of cloned repeats ofC. sativus shows further heterogeneities which are due to methylation or point mutations. By comparison of the restriction enzyme maps it was possible to trace some evolutionary events in the family ofCucurbitaceae. Some aspects of regulation and function of the middle repetitive rRNA genes (here between 2000 and 10000 copies) are discussed.