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.

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.

A chimaeric hygromycin resistance gene as a selectable marker in plant cells.

We show here that plant cells are sensitive to the antibiotic hygromycin-B(4). We also show that a chimaeric gene consisting of the nopaline synthase (nos) gene regulatory elements and the E. coli derived hygromycin phosphotransferase (hpt) gene, when transferred to plants' cells, confers resistance to hygromycin B. The chimaeric nos-hpt gene enables efficient selection of DNA transfer to plant cells when used in conjunction with Ti plasmid-derived binary vectors in cocultivation experiments.

Reconstitution of plant nitrate reductase by Escherichia coli extracts and the molecular cloning of the chlA gene of Escherichia coli K12.

Extracts from Escherichia coli, wild type and chlB, chlC, chlD, chlE, and chlG, but not chlA mutants, were able to reconstitute the nitrate reductase activity in Nicotiana tabacum cnx68 and Hyoscyamus muticus MA-2 mutant extracts. Because cnx68 and MA-2 lack the molybdenum cofactor required for nitrate reductase activity, these results indicate that the functional chlA gene is essential to produce the molybdenum cofactor in E. coli. A clone containing a gene capable of complementing the chlA mutation SA493 was obtained on a large cosmid pJT1. A 1.9 kb BclI fragment subcloned from pJT1 in the vector plasmid pBR322 was shown to complement the chlA SA493 mutant when inserted in either of the two possible orientations. This suggested that the chlA gene was contained intact, including its own promoter, on the 1.9 kb BclI fragment.

Cloning and characterization of ribosomal RNA genes from wheat and barley.

Wheat and barley DNA enriched for ribosomal RNA genes was isolated from actinomycin D-CsCl gradients and used to clone the ribosomal repeating units in the plasmid pAC184. All five chimeric plasmids isolated which contained wheat rDNA and eleven of the thirteen which had barley rDNA were stable and included full length ribosomal repeating units. Physical maps of all length variants cloned have been constructed using the restriction endonucleases Eco Rl, Bam Hl, Bgl II, Hind III and Sal I. Length variation in the repeat units was attributed to differences in the spacer regions. Comparison of Hae III and Hpa II digestion of cereal rDNAs and the cloned repeats suggests that most methylated cytosines in natural rDNA are in -CpG-. Incomplete methylation occurs at specific Bam Hl sites in barley DNA. Detectable quantities of ribosomal spacer sequences are not present at any genomic locations other than those of the ribosomal RNA gene repeats.

Directive segregation in the basis of colE1 plasmid incompatibility.

Incompatibility between colE1 plasmids in Escherichia coli can be explained by competition for a limited number of replication sites. These sites ensure directive segregation of plasmids to daughter cells on cell division. The number of sites can be more than one only if a linear segregation mechanism is postulated.

Location of the single gene for the large subunit of ribulosebisphosphate carboxylase on the maize chloroplast chromosome.

The structural gene for the large subunit of ribulosebisphosphate carboxylase (E.C. 4.1.1.39) in Zea mays is shown to be contained within a 2500 base pair sequence of chloroplast DNA. One copy of this DNA sequence is present in each circular maize chloroplast DNA molecule. It maps approximately 30,000 base pairs from the 5' end of the closest of two sets of rRNA genes and approximately 71,000 base pairs from the other set of rRNA genes.

Maize plastid gene expressed during photoregulated development.

RNAs larger than about 6 S prepared from etioplasts of dark-grown maize seedlings, and from plastids at later stages of light-induced development, were labeled in vitro and hybridized to separated fragments of maize chloroplast DNA digested with endonucleases. The major nonribosomal RNA present in developing plastids, but virtually undetectable in etioplasts, hybridizes to chloroplast DNA Bam fragment 8 and has been mapped on the maize plastid chromosome. Other aliquots of RNA from plastids were translated in a rabbit reticulocyte-derived system. Developing plastids, and mature chloroplasts, but not etioplasts, contain mRNA for an approximately 34,500 dalton polypeptide. The simultaneous appearance, during light-induced maize plastid development, of RNA which hybridizes to Bam 8 and is translated into a 34,500 dalton protein indicates that photoregulated expression of a single gene is being observed.

Maize chloroplast DNA fragment encoding the large subunit of ribulosebisphosphate carboxylase.

In vitro linked transcription-translation of chloroplast DNA has been used to show that the large subunit of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] is encoded by Zea mays chloroplast DNA. A BamHI-generated chloroplast DNA sequence cloned in Escherichia coli is shown to direct the in vitro synthesis of this protein identified as large subunit by its size, serological properties, and limited proteolytic digestion products.

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.

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.

Endonuclease recognition sites mapped on Zea mays chloroplast DNA.

The closed-circular DNA molecules of 85 x 10(6) daltons from Zea mays chloroplasts were isolated, digested with the restriction endonucleases Sal I, Bam I, and EcoRI, and the resulting fragments sized by agarose gel electrophoresis. A map of maize chloroplast DNA showing the relative location of all the Sal I recognition sequences and many of the Bam I and EcoRI recognition sites was determined. A DNA sequence representing approximately 15% of the Zea mays chloroplast genome is repeated. The two copies of this sequence are in an inverted orientation with respect to one another and are separated by a nonhomologous sequence representing approximately 10% of the genome length. The inverted repeats contain the genes for chloroplast ribosomal RNAs.

Location, rate and asymmetry of ds-RNA synthesis during replication of TYMV in Chinese cabbage.

The ds-RNA induced by TYMV infection in the nuclei of infected Chinese cabbage leaf cells became labelled with [32P]phosphate most rapidly before production of virus could be detected. In contrast, the ds-RNA associated with the chloroplasts became labelled most rapidly during the period of maximum virus production.