John Bedbrook

High level expression of introduced chimaeric genes in regenerated transformed plants.

Promoter DNA sequences from a petunia chlorophyll a/b binding protein gene were fused to octopine synthase DNA sequences and the resulting chimaeric genes were introduced into petunia and tobacco cells. Populations of transformed regenerated petunia plants containing the chimaeric genes were examined so that the expression of any particular construction could be compared between independent transformants. Substantial variation was observed between transformants in the level of chimaeric gene expression. In general, transcriptional fusions in which a linker sequence interrupted the 5′‐untranslated region gave rise to less chimaeric mRNA accumulation than a translational fusion. In the most actively expressing transformants the amount of mRNA from the introduced chimaeric genes was half that of the endogenous wild‐type gene. Transcription initiated at the same place in the chimaeric and endogenous genes. Construction of the translational cab/ocs fusion caused three amino acid changes in the octopine synthase protein and functional octopine synthase enzyme was absent from plants in which mRNA for the chimaeric gene was abundantly expressed.

The molecular basis of sulfonylurea herbicide resistance in tobacco

The enzyme acetolactate synthase (ALS) is the target enzyme for the sulfonylurea and imidazolinone herbicides. We describe the isolation and characterization of the ALS genes from two herbicide‐resistant mutants, C3 and S4‐Hra, of Nicotiana tabacum. There are two distinct ALS genes in tobacco which are 0.7% divergent at the amino acid sequence level. The C3 mutant has a single Pro–Gln replacement at amino acid 196 in one ALS gene. This gene is termed the class I gene and is equivalent to the SuRA locus. The S4‐Hra mutant has two amino acid changes in the other ALS gene. This gene is termed the class II gene or the SuRB locus. The S4‐Hra mutant includes a Pro–Ala substitution at amino acid 196 and a Trp–Leu substitution at amino acid 573. Gene reintroduction experiments have confirmed that these amino acid substitutions are responsible for the herbicide resistance phenotypes. Transgenic plants carrying these genes are highly resistant to sulfonylurea herbicide applications.

Differential expression of the eight genes of the petunia ribulose bisphosphate carboxylase small subunit multi-gene family.

Of the eight nuclear genes in the plant multi‐gene family which encodes the small subunit (rbcS) of Petunia (Mitchell) ribulose bisphosphate carboxylase, one rbcS gene accounts for 47% of the total rbcS gene expression in petunia leaf tissue. Expression of each of five other rbcS genes is detected at levels between 2 and 23% of the total rbcS expression in leaf tissue, while expression of the remaining two rbcS genes is not detected. There is considerable variation (500‐fold) in the levels of total rbcS mRNA in six organs of petunia (leaves, sepals, petals, stems, roots and stigmas/anthers). One gene, SSU301, showed the highest levels of steady‐state mRNA in each of the organs examined. We discuss the differences in the steady‐state mRNA levels of the individual rbcS genes in relation to their gene structure, nucleotide sequence and genomic linkage.

Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens.

Analysis of clones isolated from a cosmid DNA library indicates that the Serratia marcescens chromosome contains at least two genes, chiA and chiB, which encode distinct secreted forms of the enzyme chitinase. These genes have been characterized by inspection of chitinase activity and secreted proteins in Escherichia coli strains containing subclones of these cosmids. The two chitinase genes show no detectable homology to each other. DNA sequence analysis of one of the genes predicts an amino acid sequence with an N‐terminal signal peptide typical of genes encoding secreted bacterial proteins. This gene was mutagenized by cloning a neomycin phosphotransferase gene within its coding region, and the insertion mutation was recombined into the parental S. marcescens strain. The resulting chiA mutant transconjugant showed reduced chitinase production, reduced inhibition of fungal spore germination and reduced biological control of a fungal plant pathogen.

Relative strengths of the 35S califlower mosaic virus, 1', 2', and nopaline synthase promoters in transformed tobacco sugarbeet and oilseed rape callus tissue

SummaryThe 35S promoter of cauliflower mosaic virus and promoters from the nopaline synthase, 1′ and 2′ genes of Agrobacterium tumefaciens T-DNA were fused to the bacterial octopine synthase and chitinase gene coding regions. These chimaeric gene constructions were introduced into tobacco, sugarbeet and oilseed rape cells and their relative levels of expression measured by primer extension analysis of RNA isolated from pooled populations of stably transformed calli. In tobacco callus, the 35S promoter provided the highest levels of gene expression, followed by the 2′, 1′ and nopaline synthase promoters. While the ranking of these promoters is conserved in sugarbeet and oilseed rape callus, there is between-species variation in the relative strength of these promoters. In all three species, transcription initiation is conserved for each of the chimaeric gene constructions. Additional constructions in which the 5′ untranslated leader of a petunia chlorophyll a/b binding protein gene is substituted for DNA downstream of the 35S transcription start site demonstrates that heterologous 5′ leader sequences can be utilized to augment steady-state levels of reporter gene expression

Zea mays chloroplast ribosomal RNA genes are part of a 22,000 base pair inverted repeat

Zea mays chloroplast rDNA exists in two identical units. Each unit contains one sequence for the 16, 23 and 5S rRNAs in the order given. The 16 and 23S sequences in each unit are separated by a 2100 base pair (bp) spacer. The DNA sequence for 5S RNA is closely linked to that for the 23S RNA. Within the above unit, the three RNAs are transcribed from a single DNA strand. The two rDNA units on the circular chloroplast DNA molecule are separated from each other by 18,500 bp in one direction and by 106,100 bp in the other direction. The two rDNA units have an inverted orientation with respect to each other. Each rDNA unit is part of a 22,000 bp sequence which is repeated with inverted orientation.

Novel cis-acting elements in petunia Cab gene promoters

SummaryIn order to identify specific cis-acting elements which regulate the expression of the divergent Cab22R and Cab22L genes of Petunia, we conducted systematic mutational studies of the 1 kb intergenic promoter region. Sequence analysis revealed three GATA box sequence repeats positioned between the TATA and CAAT box elements. These GATA elements are conserved in corresponding promoter regions of all LHCII Type I Cab genes in Petunia and other dicotyledonous plants we have examined. Sitespecific mutations in the CAAT box and the GATA box elements of the Cab22R promoter resulted in 8-fold and 5-fold reductions in Cab22R transcript levels respectively. A deletion of 52 bp, adjacent and upstream from the CAAT box (-92 to- 145) in the Cab22R promoter reduced transcript levels 20-fold. This deletion contains a region of 13 pb which is conserved between many Petunia Cab genes. These results indicate that the quantitative expression of the Cab22 promoters is regulated by multiple cis-acting elements including CAAT and GATA box elements as well as sequences located between -92 and -145. The deletion of the region between -92 and -145 is partially compensted by homologous sequences present in the adjacent divergent promoter Cab22L.

Recombination between bacterial plasmids leading to the formation of plasmid multimers

We report here the formation of plasmid multimers in E. coli. Multimers are covalently closed, circular molecules composed of tandem repeats of the monomer plasmid. Multimer formation occurs at high frequency in rec+, recB-C-, and recF- hosts. Multimer formation is not detected in recA- hosts and occurs at reduced frequency in recB-C-F- hosts. We conclude that multimer formation is mediated by a single reciprocal recombination event occurring at homologous regions on the two plasmids.

Inhibition of gene expression in transformed plants by antisense RNA

We report the successful suppression of nopaline synthase (EC 1.5.1.19) enzymatic activity in the leaves of tobacco plants via the overproduction of RNAs complementary to the nopaline synthase (nos) mRNA. Several different regions of the nos gene were fused, in antisense orientation, to the promoter from a strongly expressed petunia chlorophyll a/b-binding protein gene. These constructions were directly introduced into a tobacco line which contained a single copy of the wild-type nos gene and transgenic plants were regenerated. The degree of nopaline synthase suppression in the leaves of the double transformants ranged up to 85% and was dependent on the particular region of the nos gene present in the antisense RNA. The most effective nos antisense sequences were derived from the 3′ half of the nos gene transcript. In addition, we report a new sensitive method for the detection and quantitation of nopaline synthase activity in crude plant extracts.

Optimizing the expression of chimeric genes in plant cells

SummaryThe Serratia marcescens chiA gene encodes a secreted chitinase activity which contributes to the fungal growth inhibition exhibited by this bacterium. The coding region from the chiA gene was fused to the promoter and 3′ polyadenylation region of the Agrobacterium nopaline synthase gene. Site-directed mutagenesis of specific nucleotides surrounding the initiating AUG of the coding sequence of this chimeric gene resulted in up to an eight-fold increase in the amount of chitinase protein detected in transformed plant tissue. Analysis of the chiA mRNA indicated that these nucleotides also affected mRNA levels. At least 50% of the chitinase protein produced in transformed tobacco cells was the same molecular weight as the S. marcescen secreted protein.