Lawrence E. Pelcher

Modified binary plant transformation vectors with the wild-type gene encoding NPTII ☆

The defective gene encoding neomycin phosphotransferase (NPTII) present in the binary plasmid vector, pBin19, was replaced with the wild-type (wt) gene. Plasmid vectors analogous to pBin19, pBI121 and pBI101 were constructed carrying the gene encoding the wt NPTII enzyme activity.

Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism

Elicitor-induced sanguinarine accumulation in opium poppy (Papaver somniferum) cell cultures provides a responsive model system to profile modulations in gene transcripts and metabolites related to alkaloid biosynthesis. An annotated expressed sequence tag (EST) database was assembled from 10,224 random clones isolated from an elicitor-treated opium poppy cell culture cDNA library. The most abundant ESTs encoded defense proteins, and enzymes involved in alkaloid metabolism and S-adenosylmethionine-dependent methyl transfer. ESTs corresponding to 40 enzymes involved in the conversion of sucrose to sanguinarine were identified. A corresponding DNA microarray was probed with RNA from cell cultures collected at various time-points after elicitor treatment, and compared with RNA from control cells. Several diverse transcript populations were coordinately induced, with alkaloid biosynthetic enzyme and defense protein transcripts displaying the most rapid and substantial increases. In addition to all known sanguinarine biosynthetic gene transcripts, mRNAs encoding several upstream primary metabolic enzymes were coordinately induced. Fourier transform-ion cyclotron resonance-mass spectrometry was used to characterize the metabolite profiles of control and elicitor-treated cell cultures. Principle component analysis revealed a significant and dynamic separation in the metabolome, represented by 992 independent detected analytes, in response to elicitor treatment. Identified metabolites included sanguinarine, dihydrosanguinarine, and the methoxylated derivatives dihydrochelirubine and chelirubine, and the alkaloid pathway intermediates N-methylcoclaurine, N-methylstylopine, and protopine. Some of the detected analytes showed temporal changes in abundance consistent with modulations in the profiles of alkaloid biosynthetic gene transcripts.

Comparison of the 5.8s rDNA and internal transcribed spacer sequences of isolates of Leptosphaeria maculans from different pathogenicity groups

The regions coding for the 5.8s rRNA and the flanking internal transcribed spacers (ITS1 and ITS2) from nine isolates of the blackleg pathogen Leptosphaeria maculans and one isolate of Sclerotinia sclerotiorum were amplified by the polymerase chain reaction and sequenced. Five of the L. maculans isolates were highly virulent to Brassica plants, two were weakly virulent and two were isolated from the cruciferous weed Thlaspi arvense. The 5.8s DNA sequences of all L. maculans isolates were identical. However, there were major differences in both ITS1 and ITS2 sequences that correlated with the pathogenicity grouping. Phylogenetic analysis of the ITS sequences by both parsimony and maximum-likelihood methods indicated that each pathogenicity group was statistically different from each other with the weaklyvirulent isolates being more closely related to the Thlaspi than to the highly-virulent isolates. The relationships of L. maculans to other fungi, based on a comparison of the 5.8s rDNA sequences, are discussed.

Unique composition of plastid chaperonin-60: α and β polypeptide-encoding genes are highly divergent.

Abstract Molecular chaperones of the chaperonin family occur in prokaryotes and in plastids and mitochondria. Prokaryotic and mitochondrial chaperonin-60 oligomers (Cpn-60) are composed of a single subunit type (p60cpn-60). In contrast, preparations of purified plastid Cpn-60 contain approximately equal quantities of two polypeptides, p60cpn-60α and p60cpn-60β, with slightly different electrophoretic mobilities. We have isolated cDNA clones encoding plastid p60cpn-60α and p60cpn-60β polypeptides from Brassica napus and Arabidopsis thaliana. The unexpected degree of sequence divergence observed between p60cpn-60α and p60cpn-60β raises questions concerning the structure of the oligomer and the functions of these polypeptides. We have also found an amino acid sequence motif within all p60cpn-60 sequences which resembles the p10cpn-10 sequences.

Comparison of nuclear ribosomal DNA sequences from Alternaria species pathogenic to crucifers

The sequences coding for the nuclear 18 s rRNA, 5·8 s rRNA, and the internal transcribed spacers (ITS1 and ITS2) were amplified by the polymerase chain reaction and sequenced for one isolate each of Alternaria brassicae, A. brassicicola, A. raphani, A. alternata and Pleospora herbarum . The 5·8 s rDNA sequences from the four Alternaria species were identical and differed at only one base pair from that of P. herbarum . The internal transcribed spacer sequences, especially ITS1, were very variable in both base composition and length. The 18 s rDNA sequences were highly conserved, but enough variability was present to distinguish genera clearly. Phylogenetic analysis of the sequence data sets by both parsimony and maximum likelihood methods clearly separated genera and species. All of the Alternaria species were closely related. Pleospora also appeared to be more closely related to Alternaria than to Leptosphaeria .

Phylogenetic relationship among several Leptosphaeria species based on their ribosomal DNA sequences

The sequences coding for the 18 s and 5·8 s ribosomal RNAs and their internal transcribed spacers (ITS1 and ITS2) from isolates of five Leptosphaeria species were amplified by PCR and sequenced. Phylogenetic analysis of these sequences indicated that, with the exception of L. bicolor, Leptosphaeria species are closely related. There were two distinct lineages among species of Leptosphaeria , one represented by two weakly virulent isolates of L. maculans and the other by L. microscopica, L. nodorum and L. doliolum . The highly virulent isolate of L. maculans was not placed definitively in either lineage and may represent an additional branch of the genus. The sugarcane pathogen L. bicolor was found to be less closely related to the other Leptosphaeria species than to Alternaria alternata and Pleospora herbarum , and is likely to be a representative of a different genus among the Pleosporales.

Probing carotenoid biosynthesis in developing seed coats of Bixa orellana (Bixaceae) through expressed sequence tag analysis

Abstract Bixin is a commercially important diapocarotenoid derived from the seed coats of the tropical shrub Bixa orellana . Based on available chemical data and by analogy to abscisic acid biosynthesis, a biosynthetic pathway for bixin starting from lycopene is proposed. This putative pathway was investigated through expressed sequence tag (EST) analysis using a subtracted cDNA library. This analysis identified ESTs corresponding to most of the enzymes of the carotenoid biosynthesis pathway including the 1-deoxy- d -xylulose 5-phosphate pathway of plastids. ESTs corresponding to a number of genes encoding dioxygenases, aldehyde dehydrogenases and carboxyl methyltransferases were also identified. These represent candidate enzymes for the last stage of the bixin biosynthetic pathway.

Evidence forCis- andTrans-acting element coevolution of the 2-μm circle genome inSaccharomyces cerevisiae

SummaryWe compared the DNA sequence of the yeas 2-μm plasmidcis-actingSTB andtrans-actingREP1 partition loci of laboratory haploid and industrial amphiploid strains. Several industrial strains had a uniqueSTB sequence (type 1) sharing only 70% homology with laboratorySTB (type 2). Type 1 plasmids had a REP1 protein with 6–10% amino acid substitutions when compared to REP1 of type 2 plasmids. All 2-μm variants that shared a similarSTB consensus sequence exhibited a high degree ofREP1 nucleotide and amino acid sequence conservation. These observations suggest molecular coevolution oftrans-acting elements with cognate target DNA structure. Based on DNA sequencing and Southern hybridization analyses, we classified 2-μm variants into two main evolutionary lineages that differ atSTB as well asREP1 loci. The role of molecular coevolution in yeast intra- and interspecies plasmid evolution was discussed.

Sequence diversity of yeast 2 μm RAF gene and its co-evolution with STB and REP1

Abstract Despite the extensive study of yeast 2 μm plasmid, the exact function of plasmid-encoded RAF gene is not clear. Variants of 2 μm plasmids from industrial Saccharomyces cerevisiae yeasts were isolated and characterized. Sequencing of RAF alleles revealed about 8 % nucleotide and 10% amino acid diversities between 2 μm variants of closely related strains. RAF sequence variations were correlated with STB-REP1 sequence diversity. We also used restriction fragment length polymorphism linkage to screen a large number of yeast strains from different fermentation industries. The results clearly show a tight linkage of STB-REP1-RAF variations. Thus, our observations suggest that plasmid-borne cis- and tram-acting elements co-evolved to form an optimal molecular parasite and that RAF may play a role in active plasmid partitioning.

The effects of heterologous and homologous coat protein on alkaline disassembly of tobacco and tomato isolates of tobacco mosaic virus.

Tobacco (Tb) and tomato (Tm) isolates of tobacco mosaic virus (TMV) disassemble in dilute alkaline solutions (pH > 8.0). Tm-TMV is more sensitive to alkali than is Tb-TMV. Tb protein is, however, able to partially stabilize both Tm-TMV and Tb-TMV, thus retarding the rate of alkaline disassembly. Tm protein is unable to stabilize Tm-TMV and also increases the rate of alkaline disassembly of Tb-TMV.