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Biomedical subjects

F Cannon

Publications and source records attributed to F Cannon.

15 recordsLinked to original sources
PubMed1994

Alfalfa yield response to inoculation with recombinant strains of Rhizobium meliloti with an extra copy of dctABD and/or modified nifA expression.

The construction of rhizobial strains which increase plant biomass under controlled conditions has been previously reported. However, there is no evidence that these newly constructed strains increase legume yield under agricultural conditions. This work tested the hypothesis that carefully manipulating expression of additional copies of nifA and dctABD in strains of Rhizobium meliloti would increase alfalfa yield in the field. The rationale for this hypothesis is based on the positive regulatory role that nifA plays in the expression of the nif regulon and the fact that a supply of dicarboxylic acids from the plant is required as a carbon and energy source for nitrogen fixation by the Rhizobium bacteroids in the nodule. These recombinant strains, as well as the wild-type strains from which they were derived, are ideal tools to examine the effects of modifying or increasing the expression of these genes on alfalfa biomass. The experimental design comprised seven recombinant strains, two wild-type strains, and an uninoculated control. Each treatment was replicated eight times and was conducted at four field sites in Wisconsin. Recombinant strain RMBPC-2, which has an additional copy of both nifA and dctABD, increased alfalfa biomass by 12.9% compared with the yield with the wild-type strain RMBPC and 17.9% over that in the uninoculated control plot at the site where soil nitrogen and organic matter content was lowest. These increases were statistically significant at the 5% confidence interval for each of the three harvests made during the growing season. Strain RMBPC-2 did increase alfalfa biomass at the Hancock site; however, no other significant increases or decreases in alfalfa biomass were observed with the seven other recombinant strains at that site. At three sites where this experiment was conducted, either native rhizobial populations or soil nitrogen concentrations were high. At these sites, none of the recombinant strains affected yield. We conclude that RMBPC -2 can increase alfalfa yields under field conditions of nitrogen limitation, low endogenous rhizobial competitors, and sufficient moisture.

Base Sequence↗
PubMed1990

Organ-specific modulation of gene expression in transgenic plants using antisense RNA.

We have shown leaf-specific inhibition GUS gene expression in transgenic Nicotiana plants using an antisense RNA with a 41-base homology spanning the translation start codon of the gene. GUS was expressed from the nominally constitutive 35S promoter and the antisense RNA was expressed from the light-regulated ca/b promoter of Arabidopsis thaliana. A range of GUS inhibition from 0 to 100% was obtained by screening a small population of transgenic plants and the specific levels of inhibition observed were stably inherited in two generations. An antiGUS 'gene' dosage effect was observed in plants which were homozygous for antiGUS. RNA detection results suggest that duplex formation with the 41 base pair antiGUS RNA destabilized the GUS mRNA and that an excess of antisense RNA was not required. Our results demonstrate the potential of antisense RNA as a strategy for obtaining plant mutants, especially 'down mutations' in essential genes where only a short 5' sequence of the mRNA is required. They also suggest that the 'position effect' on gene expression could be used in conjunction with an antisense RNA strategy to provide a versatile approach for crop improvement.

Base Sequence↗
PubMed1988

Nucleotide sequence and mutagenesis of the nifA gene from Azotobacter vinelandii.

The nucleotide sequence of the nifA gene from Azotobacter vinelandii was determined. This gene encodes an Mr = 58,100 polypeptide that shares significant sequence identity when compared to nifA-encoded products from other organisms. Interspecies comparisons of nifA-encoded products reveal that they all have a consensus ATP binding site and a consensus DNA binding site in highly conserved regions of the respective polypeptides. The nifA gene immediately precedes the nifB-nifQ gene region but is unlinked to the major nif gene cluster from A. vinelandii. A potential regulatory gene precedes and is apparently cotranscribed with nifA. Mutant strains that have a deletion or a deletion plus an insertion within nifA are incapable of diazotrophic growth and they fail to accumulate nitrogenase structural gene products.

Amino Acid Sequence↗
PubMed1987

Comparative organization of nitrogen fixation-specific genes from Azotobacter vinelandii and Klebsiella pneumoniae: DNA sequence of the nifUSV genes.

In the facultative anaerobe Klebsiella pneumoniae 17 nitrogen fixation-specific genes (nif genes) have been identified. Homologs to 12 of these genes have now been isolated from the aerobic diazotroph Azotobacter vinelandii. Comparative studies have indicated that these diverse microorganisms share striking similarities in the genetic organization of their nif genes and in the primary structure of their individual nif gene products. In this study the complete nucleotide sequence of the nifUSV gene clusters from both K. pneumoniae and A. vinelandii were determined. These genes are identically organized on their respective genomes, and the individual genes and their products exhibit a high degree of interspecies sequence homology.

Aerobiosis↗
PubMed1985

A molecular genetic study of nif expression in Klebsiella pneumoniae at the level of transcription, translation and nitrogenase activity.

A comprehensive study of nif expression in Klebsiella pneumoniae at the level of transcription, translation and nitrogenase activity during derepression and repression by NH+4 and O2 revealed that (1) transcription and translation rates remained coupled under all conditions; (2) these rates reached a peak during derepression and then decreased to a low level; (3) the transcription profile of nifLA had two peaks; the first was at 1 h before and the second coincided with that of the other operons; and (4) the peaks of nif transcription coincided with a trough in the profile of stringent regulation of RNA synthesis. Our results provide strong evidence that nif-specific repression by NH+4 and O2 occurs exclusively by transcription inhibition and that repression by O2 is independent of transcriptional regulation of the nifLA operon. We have also found evidence which together with the results of previous work shows that O2 repression of nifA mediated transcription involves the nifL gene product.

Ammonia↗
PubMed1983

Cloning and characterisation of nifLA regulatory mutations from Klebsiella pneumoniae.

A total of nine regulatory mutations in the nifLA operon of Klebsiella pneumoniae were cloned in the high copy-number plasmid vector pACYC184. The regulatory phenotypes of the resultant clones were then correlated with their restriction maps and their ability to synthesise nifL and nifA polypeptides in vivo. One mutation, nifL2401, was identified as a 400 bp. deletion within the nifL gene. This mutation was non-polar and caused a Nif+ phenotype which showed escape from repression by oxygen and low levels of fixed nitrogen. Identification of this deletion allows the first definitive allocation of a mutation with this phenotype to the nifL gene and provides further evidence for the role of the nifL gene product in nif-specific repression.

Chromosome Mapping↗
PubMed1983

The nif promoters of Klebsiella pneumoniae have a characteristic primary structure.

We have determined the precise point of transcription initiation for five nif (nitrogen fixation) operons of Klebsiella pneumoniae and sequenced the promoters. Our results show that nifF is transcribed in the opposite direction to that previously proposed for nif genes, that nifM is transcribed from two promoters, and that there is a promoter preceding nifU but not nifX, and we present a revised map of nif transcripts. The nif promoters have a characteristic structure of 26 bp located between positions--1 and --26 upstream of the site of transcription initiation: CTGG..8bp...TTGCA...9bp...Py(+1). This structure, which has two regions of conserved sequence, shows no homology to the "consensus promoter" of enteric bacteria. Our results suggest that the--10 homology is equivalent to a "Pribnow box" for promoters expressed under nitrogen-starved conditions and that the--23 homology confers activator specificity on the nif promoters.

Base Sequence↗
PubMed1982

Cloning of the glnA, ntrB and ntrC genes of Klebsiella pneumoniae and studies of their role in regulation of the nitrogen fixation (nif) gene cluster.

The glnA, ntrB and ntrC genes of Klebsiella pneumoniae have been cloned, on a 12 kb HindIII fragment, into the plasmid pACYC184. In a coupled in vitro transcription/translation system the resultant plasmid, pGE100, directed synthesis of five polypeptides (molecular weights 73, 53, 51, 39, 36 kd) from the cloned fragment. A number of plasmids were derived from pGE100 and studied by complementation analysis and in vitro transcription/translation in order to locate particular genes and identify their products. On the basis of the results presented here, together with previous genetic and physical characterisation of the glnA gene and its product in other enteric bacteria, we propose that the 53 kd polypeptide is the glnA gene product (glutamine synthetase monomer). Two polypeptides (36 kd and 51 kd) were synthesised from a 3 kb region previously defined as glnR. In E. coli and S. typhimurium this region comprises two genes ntrB and ntrC with products of 36 kd and 54 kd respectively. This analogy supports the idea that the 36 kd and 51 kd polypeptides are the products of the K. pneumoniae ntrB and ntrC genes respectively. Comparison of these assignments with the physical map of the region indicates a gene order glnA, ntrB, ntrC. Assessment of the Nif phenotype of a glnA-ntrC deletion strain carrying various clones from pGE100 demonstrated that glnA is not required for expression of the nif regulon and that of the three genes cloned, ntrC alone is sufficient for nif expression.

Cloning, Molecular↗