The development of a molecular cloning system in higher plants.
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Biomedical subjects
Publications and source records attributed to R B Meagher.
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The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide.
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A procedure for the large-scale enzymatic synthesis of beta-carboxymuconate is described. When used as a growth substrate, beta-carboxymuconate selected for mutant strains of Pseudomonas putida that were permeable to polycarboxylic acid intermediates of the beta-ketoadipate pathway. One mutant organism, strain PRS2110, was investigated in detail. It differed from the parental strain in that it possessed a beta-carboxymuconate uptake system that was formed when the compound was supplied exogenously to the cells. The uptake system was not induced by beta-carboxymuconate supplied endogenously during growth with p-hydroxybenzoate. These observations suggested that beta-carboxymuconate was contained within a physical compartment of enzymes during growth with p-hydroxybenzoate. Support for this hypothesis came from the demonstration that enzymes of the beta-ketoadipate pathway were held together by weak chemical interactions during the chromatography of crude extracts of benzoategrown P. putida on diethylaminoethyl-cellulose columns.
We present the sequence of the nuclear-encoded ribosomal small-subunit RNA from soybean. The soybean 18S rRNA sequence of 1807 nucleotides (nt) is contained in a gene family of approximately 800 closely related members per haploid genome. This sequence is compared with the ribosomal small-subunit RNAs of maize (1805 nt), yeast (1789 nt), Xenopus (1825 nt), rat (1869 nt), and Escherichia coli (1541 nt). Significant sequence homology is observed among the eukaryotic small-subunit rRNAs examined, and some sequence homology is observed between eukaryotic and prokaryotic small-subunit rRNAs. Conserved regions are found to be interspersed among highly diverged sequences. The significance of these comparisons is evaluated using computer simulation of a random sequence model. A tentative model of the secondary structure of soybean 18S rRNA is presented and discussed in the context of the functions of the various conserved regions within the sequence. On the basis of this model, the short base-paired sequences defining the four structural and functional domains of all 18S rRNAs are seen to be well conserved. The potential roles of other conserved soybean 18S rRNA sequences in protein synthesis are discussed.
The Mitchell variety of Petunia hybrida possesses a superfamily of actin genes which contains between 100 and 200 members that can be divided into at least six highly divergent subfamilies. The segregation of restriction fragment length polymorphisms among 96 plants from two backcrosses between the Violet 23 and Red 51 Petunia varieties and the Violet 23 x Red 51 hybrid was examined using gene-specific probes from six Petunia actin gene subfamilies. These data were compared with the genotypes of each plant at 11 marker loci which are distributed among the seven chromosomes of Petunia and which determine flower, pollen, and isozyme phenotypes. From these analyses, members of these six actin gene subfamilies were mapped to five locations on five Petunia chromosomes: the PAc9, PAc1, PAc4, and PAc2 subfamilies are on chromosomes I, II, III, and VII respectively; the PAc3 and PAc7 subfamilies are tightly linked on chromosome IV. All members of the PAc4 subfamily cosegregated as a cluster of genes. These data are discussed regarding gene amplification in plants.