A novel family of small cysteine-rich antimicrobial peptides from seed of Impatiens balsamina is derived from a single precursor protein.

Four closely related peptides were isolated from seed of Impatiens balsamina and were shown to be inhibitory to the growth of a range of fungi and bacteria, while not being cytotoxic to cultured human cells. The peptides, designated Ib-AMP1, Ib-AMP2, Ib-AMP3, and Ib-AMP4, are 20 amino acids long and are the smallest plant-derived antimicrobial peptides isolated to date. The Ib-AMPs (I. balsamina antimicrobial peptides) are highly basic and contain four cysteine residues which form two intramolecular disulfide bonds. Searches of protein data bases have failed to identify any proteins with significant homology to the peptides described here. Characterization of isolated cDNAs reveals that all four peptides are encoded within a single transcript. The predicted Ib-AMP precursor protein consists of a prepeptide followed by 6 mature peptide domains, each flanked by propeptide domains ranging from 16 to 35 amino acids in length. Such a primary structure with repeated alternating basic mature peptide domains and acidic propeptide domains has, to date, not been reported in plants.

A high efficiency technique for the generation of transgenic sugar beets from stomatal guard cells.

An optimized protocol has been developed for the efficient and rapid genetic modification of sugar beet (Beta vulgaris L.). A polyethylene glycol-mediated DNA transformation technique could be applied to protoplast populations enriched specifically for a single totipotent cell type derived from stomatal guard cells, to achieve high transformation frequencies. Bialaphos resistance, conferred by the pat gene, produced a highly efficient selection system. The majority of plants were obtained within 8 to 9 weeks and were appropriate for plant breeding purposes. All were resistant to glufosinate-ammonium-based herbicides. Detailed genomic characterization has verified transgene integration, and progeny analysis showed Mendelian inheritance.

Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment.

Zea mays transformants produced by particle bombardment of embryogenic suspension culture cells of the genotype A188 x B73 and selected on kanamycin or bialaphos were characterized with respect to transgene integration, expression, and inheritance. Selection on bialaphos, mediated by the bar or pat genes, was more efficient than selection on kanamycin, mediated by the nptII gene. Most transformants contained multicopy, single locus, transgene insertion events. A transgene expression cassette was more likely to be rearranged if expression of that gene was not selected for during callus growth. Not all plants regenerated from calli representing single transformation events expressed the transgenes, and a non-selectable gene (uidA) was expressed in fewer plants than was the selectable transgene. Mendelian inheritance of transgenes consistent with transgene insertion at a single locus was observed for approximately two thirds of the transformants assessed. Transgene expression was typically, but not always, predictable in progeny plants--transgene silencing, as well as poor transgene transmission to progeny was observed in some plant lines in which the parent plants had expressed the transgene.

A short N-proximal region of prochymosin inhibits the secretion of hybrid proteins from Escherichia coli.

A gene encoding bovine prochymosin (PC) was fused to the coding sequence (phoA) for the Escherichia coli alkaline phosphatase (AP) signal peptide and expressed in E. coli under the control of the phoA promoter. Upon induction, an AP-PC fusion protein was produced which was neither processed nor exported into the periplasm. We investigated this lack of secretion by constructing a series of gene fusions in which different regions of the PC gene were inserted between the coding regions of the AP leader and mature protein. Analysis of the cellular location of the proteins encoded by these fusions revealed that a region of PC (between amino acids 6 and 29) prevented processing and secretion of an AP-PC fusion when inserted near to the AP signal peptide. In contrast, when this 'blocking sequence' was inserted elsewhere in AP the hybrid proteins were efficiently processed and translocation was initiated.

Identification of nodX, a gene that allows Rhizobium leguminosarum biovar viciae strain TOM to nodulate Afghanistan peas.

Gene(s) conferring the ability of Rhizobium leguminosarum biovar viciae strain TOM to nodulate primitive peas (cultivar Afghanistan) had been located in a 2.0 kb region of its sym plasmid, pRL5JI. In this DNA, a single open reading frame of 1101 bp, corresponding to a gene, nodX was found. nodX is downstream of nodJ which is present in strain TOM and also in the sym plasmid of a typical strain of this biovar. nodX specifies a hydrophobic protein (of Mr 41,036) with no clear similarity to other proteins in data bases. Mutations in nodX abolished nodulation of Afghanistan peas but not nodulation of commercial peas. nodX-lacZ fusions were used to show that transcription of nodX was activated by root exudates from both commercial and Afghanistan peas and by defined flavonoids. Exudate from Afghanistan peas activated nod genes of typical strains of R. leguminosarum biovar viciae which fail to nodulate these peas; thus, their failure to nodulate these primitive peas is not due to a lack of activation of their nod genes by exudate from Afghanistan peas. A homologue of nodX exists in R. leguminosarum biovar trifolii (which nodulates clover) but not in typical strains of R. leguminosarum biovar viciae.

A family of related ATP-binding subunits coupled to many distinct biological processes in bacteria.

Many biological processes are coupled to ATP hydrolysis. We describe here a class of closely related ATP-binding proteins, from several bacterial species, which are associated with a variety of cellular functions including membrane transport, cell division, nodulation in Rhizobium and haemolysin export. These proteins comprise a family of structurally and functionally related subunits which share a common evolutionary origin, bind ATP and probably serve to couple ATP hydrolysis to each of these biological processes. This finding suggests a specific role for ATP in cell division, nodulation during nitrogen fixation and protein export, and allows us to assign a probable function to one of the protein components from each of these systems.

An enzymic method of preparing plant chromosomes for in situ hybridization.

A method of preparing chromosomes from plant root tips for in situ hybridization with tritiated DNA is described. The technique relies on the enzymic hydrolysis of plant cell walls with a pectinase-cellulase mixture. It is shown that, despite the enzymic mixture possessing nuclease activity, there is no detectable degradation of DNA within fixed root tips. To demonstrate the suitability of this method of preparing plant chromosomes for in situ hybridization, a cloned repetitive DNA sequence has been hybridized to Allium sativum chromosomes. Chromosomes prepared using this technique also can be readily C-banded.

The nodI gene product of Rhizobium leguminosarum is closely related to ATP-binding bacterial transport proteins; nucleotide sequence analysis of the nodI and nodJ genes.

The nucleotide sequence of a 2-kb fragment immediately downstream of the nodABC genes of the Rhizobium leguminosarum symbiotic plasmid pRL1JI has been determined. Genes corresponding to the two open reading frames identified are named nodI and nodJ. Tn 5 insertions into these genes result in a "nodulation-delayed" phenotype. The predicted amino acid sequence of the nodI gene shows considerable homology to inner-membrane-located gene products involved in active transport systems in Escherichia coli and Salmonella typhimurium. The predicted product of the nodJ gene is very hydrophobic, suggesting that it may be an integral membrane protein.

Organization and evolution of repeated DNA sequences in closely related plant genomes.

In common with many other eukaryotic species, the genomes of species in the genus Allium contain a high proportion of repeated DNA sequences, which may be implicated in the considerable differences in genome size that are seen between even very closely related species. The gross organization of repetitive sequences within the genome of Allium sativum and of some other related species has been investigated using DNA/DNA hybridization studies. Such studies show that there has been much modulation in the amounts of different repeated DNA families during the evolution of the genus Allium; these repetitive elements are interspersed in all species with sequences of low repetition. The organization and distribution of one particular repetitive family within the genus has been examined using a cloned hybridization probe. Hybridization of this probe to DNA from related genomes reveals that this element is present in all Allium species examined, but with large-scale modulation of its abundance, and some considerable changes in its sequence environment. The evolution of such genome-specific arrangements of common repetitive elements and the possible mechanisms by which they might be maintained are discussed.