The use of 16S and 16S-23S rDNA to easily detect and differentiate common Gram-negative orchard epiphytes.

The identification of Gram-negative pathogenic and non-pathogenic bacteria commonly isolated from an orchard phylloplane may result in a time consuming and tedious process for the plant pathologist. The paper provides a simple "one-step" protocol that uses the polymerase chain reaction (PCR) to amplify intergenic spacer regions between 16S and 23S genes and a portion of 16S gene in the prokaryotic rRNA genetic loci. Amplified 16S rDNA, and restriction fragment length polymorphisms (RFLP) following EcoRI digestion produced band patterns that readily distinguished between the plant pathogen Erwinia amylovora (causal agent of fire blight in pear and apple) and the orchard epiphyte Pantoea agglomerans (formerly E. herbicola). The amplified DNA patterns of 16S-23S spacer regions may be used to differentiate E. amylovora at the intraspecies level. Isolates of E. amylovora obtained from raspberries exhibited two major fragments while those obtained from apples showed three distinct amplified DNA bands. In addition, the size of the 16S-23S spacer region differs between Pseudomonas syringae and Pseudomonas fluorescens. The RFLP pattern generated by HaeIII digestion may be used to provide a rapid and accurate identification of these two common orchard epiphytes.

A gene associated with filamentous growth in Ophiostoma novo-ulmi has RNA-binding motifs and is similar to a yeast gene involved in mRNA splicing.

The COL1 gene was isolated from Ophiostoma novo-ulmi using the techniques of insertional mutagenesis and plasmid rescue. Sequence analyses suggest that the COL1 gene encodes a unique protein of 826 amino acids with consensus-type RNA-binding domains, most similar to a putative protein from Schizosaccharomyces pombe which resembles the C-terminus of the Saccharomyces cerevisiae U4/U6 splicing factor PRP24. Disruption of the COL1 gene produced the yeast-like col1 mutant. The inability of the mutant to synthesize the COL1 gene product was confirmed by transcript analysis. Transformation of the col1 mutant with the COL1 gene restored the wild phenotype and production of the 4.0-kb mRNA. The results from this study demonstrate that the COL1 RNA-binding protein is associated with filamentous growth in O. novo-ulmi.

Ribosomal variation in six species of Fusarium.

Eighteen isolates representing six Fusarium species from diverse hosts and geographical origins were evaluated to determine ribosomal DNA variation using polymerase chain reaction and restriction fragment length polymorphisms. No length variation was observed for amplified 18S and 28S regions. However, amplification of the ITS region showed one isolate, a F. oxysporum, to be about 120 bp larger than the remaining 17. Restriction digestions in the 18S region revealed polymorphisms within species of F. oxysporum and F. solani. An amplified variable stretch of the 28S gene showed restriction site differences between F. avenecum, F. sambucinum and F. sporotrichioides. A large degree of polymorphism was observed both between and within species in the ITS region. Therefore, entire sequences of the ITS and the 5.8S subunit were obtained for 17 of the 18 isolates. These sequences, along with those from eight additional isolates, were analysed using PAUP to assess the occurrence of DNA sequence divergence within the ITS region. The lack of correlation between molecular-based relationships and species affinities inferred from morphology for some isolates indicates that species designation can be unreliable using morphological data alone. Possible reasons for the discordance of the sequence and morphological data are discussed.

Lack of association between cerato-ulmin production and virulence in Ophiostoma novo-ulmi.

Cerato-ulmin (CU), a hydrophobin produced by Ophiostoma novo-ulmi, has been implicated in the pathogenicity of this fungus on elm. We have generated a CU- mutant by transformation-mediated gene disruption of a highly virulent (aggressive) strain of O. novo-ulmi. The inability of the mutant to synthesize CU was confirmed by transcript analysis as well as turbidity and immunological measurements. Bioassay of the CU- strain in highly susceptible elm trees indicated no difference in the virulence parameters, percent vascular discoloration, and percent foliar wilting, when compared with the wild type. Our results indicate that the inability to produce CU had no measurable effect on the ability of O. novo-ulmi to produce symptoms of Dutch elm disease on inoculated elms.

A comparison of the nucleotide sequence of the cerato-ulmin gene and the rDNA ITS between aggressive and non-aggressive isolates of Ophiostoma ulmi sensu lato, the causal agent of Dutch elm disease.

Little genetic information exists comparing aggressive and non-aggressive isolates of the causal agent of Dutch elm disease, Ophiostoma ulmi. Two genetic elements were compared between the subgroups. The ceratoulmin cu gene product has been associated with disease symptoms. Nucleotide-sequence analysis of cu and the internal transcribed spacer (ITS) region of the rDNA were made from three aggressive and three non-aggressive isolates of the pathogen. Our results suggested uniformity within, and unique differences between, subgroups. Differences were detected for cu in the promoter, coding, and transcription termination regions. Sequence data for the ITS clearly distinguish the subgroups.

RFLP Analysis of PCR Amplified ITS and 26S Ribosomal RNA Genes of Selected Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae).

This study examined the polymerase chain reaction (PCR) amplified internal transcribed spacer (ITS) and 26S ribosomal DNA (rDNA) regions of 15 entomopathogenic nematode isolates including Steinernema feltiae syn. bibionis, S. glaseri, seven strains of S. carpocapsae, four strains of Heterorhabditis bacteriophora, and two field isolates. RDNA length variation was not observed among the isolates examined. Restriction fragment length polymorphisms (RFLP) of PCR amplified ITS and 26S regions provided specific banding patterns for all isolates but S. feltiae syn. bibionis and S. glaseri. These two species were separated by zymograms of esterase and tetrazolium oxidase. A field trapping method retrieved two isolates of naturally occurring nematodes. One field isolate collected (F1) displayed banding patterns identical to those of S. carpocapsae DD136 released in the same location 1 year earlier. The second field isolate (F2) had unique PCR-RFLP profiles compared with all other strains. This study provides a rapid molecular taxonomic method to more fully establish species relationships among members of Steinernema and Heterorhabditis.

Restriction fragment length polymorphisms in polymerase chain reaction amplified ribosomal DNAs of three Trichogramma (Hymenoptera: Trichogrammatidae) species.

Restriction fragment length polymorphisms from PCR amplified ribosomal DNAs of three Trichogramma species, T. minutum, T. brassicae, and T. near sibiricum, were studied. Length variation in the internal transcribed spacer (ITS) region was observed. The ITS region of T. brassicae is about 1350 base pairs (bp) in length and those of T. minutum and T. near sibiricum are 1300 bp. These three species also differ in the size of their ITS1 and ITS2 regions. Restriction enzyme digestions of these regions showed unique banding patterns for each species. The amplified 18S region of ribosomal DNA is about 1800 bp in length and showed no length variation between the three species of Trichogramma. Restriction enzyme digestion of this region by BamHI differentiated T. brassicae from the other two species. Restriction site maps of the ITS and 18S regions were constructed for each species. The amplified 28S region is about 1700 bp for these three species. Restriction of this region by RsaI and SacII differentiates these three species. The reported results indicate that these species of Trichogramma can be clearly differentiated from one another by nuclear ribosomal DNA markers.

Differentiation of poplar and willow clones using RAPD fingerprints.

The technique of random amplified polymorphic DNA (RAPD) fingerprinting was used to differentiate species and identify clones of 15 poplar and 15 willow clones (ramets of three poplar clones were also included to verify the stability of the results). Four random DNA primers (Deca-11, Chl-1, Chl-4 and Chl-10) and an M13 universal primer were used to determine DNA polymorphism among the clones. Based on the DNA banding pattern obtained with the four DNA primers by polymerase chain reaction (PCR) amplification we conclude that RAPD fingerprinting with properly selected primers can be used for clonal and species differentiation of poplar and willow. The technique is simple, accurate and inexpensive.

Isolation and characterization of the cerato-ulmin toxin gene of the Dutch elm disease pathogen, Ophiostoma ulmi.

The hydrophobic protein cerato-ulmin (CU), produced by Ophiostoma ulmi, has been implicated in the pathogenicity of this fungus on elm. Primers were designed based on the nucleotide sequence deduced from the published CU amino-acid sequence, and a DNA fragment of the cu gene was amplified using the polymerase chain reaction. The amplified cu fragment was used as a hybridization probe to identify and isolate the cu gene from a genomic DNA library of an aggressive isolate of O. ulmi ( = O. novo-ulmi). The cu coding region is interrupted by two introns and encodes a 100 amino-acid prepro-CU polypeptide that is processed to a 75 amino-acid mature protein upon secretion. CU shows significant sequence similarity to hydrophobins secreted by certain other fungi.

A genetic survey of the pathogenic fungus Ophiostoma ulmi across a Dutch elm disease front in western Canada.

The natural population structure of the Dutch elm pathogen Ophiostoma ulmi was determined from isolated collected from across a Western Canadian disease front through an analysis of restriction-site polymorphisms in the ribosomal DNA repeat, length mutations in the mitochondrial genomes, and through DNA fingerprinting of the nuclear genomes using a minisatellite DNA probe. The 8.8-kbp rDNA repeat was selected from a genomic library, and restriction-site and genic maps were constructed for the nonaggressive and aggressive subgroups of O. ulmi. There were only three restriction-site differences that distinguished these two subgroups and no intrasubgroup variation was detected. All of the isolates collected from the disease front were of the aggressive subgroup and were represented by two distinct nuclear and four mitochondrial genotypes. The minority of the isolates were of a single genotype (type A nuclear DNA; type I mtDNA), indicating the presence of a single very large clone extending across much of Manitoba and into Saskatchewan.

Human placental beta-galactosidase. Characterization of the dimer and complex forms of the enzyme.

GM1 ganglioside beta-galactosidase (beta-Gal) is deficient in the autosomal recessive disorder GM1 gangliosidosis. A portion of the enzyme occurs in a complex with neuraminidase and an additional glycoprotein, protective protein, but the nature of the interactions conferring the stability of the complex is unknown. Affinity chromatography of beta-Gal on p-aminophenylthiogalactose-Sepharose (PATG-Sepharose) at pH 4.3, the pH optimum of beta-Gal, resulted in a 260-fold enrichment of beta-Gal, but the major protein in the fraction had an M(r) value of 74,000. Affinity chromatography on PATG-Sepharose at pH 5.2 showed substantial enrichment (4000-fold) of beta-Gal, and the mature form of the enzyme (M(r) 64,000) was the major protein in the preparation. Using h.p.l.c. molecular-sieve chromatography, we found that about 15% of the total beta-Gal occurred in a high-M(r) form (greater than 600,000), the presumptive complex, with 85% eluting at M(r) 150,000, suggestive of a dimer. This distribution was independent of both high (60 mg/ml) and low (5 mg/ml) protein concentration and the pH (pH 4.3 or 5.2) of the sample applied to the column. Furthermore, incubation for 90 min at 37 degrees C, conditions which had previously been suggested as optimal for formation of the complex, had no effect on this distribution. Further fractionation by anion-exchange chromatography and a second affinity column step yielded a beta-Gal preparation that contained a single polypeptide chain (M(r) 64,000), was devoid of neuraminidase and protective protein (absent carboxypeptidase activity), and when injected into rabbits gave rise to monospecific rabbit antisera. We conclude that the protein composition of the complex is variable (i.e. it is different when isolated at pH 4.3 and 5.2) and that the amount of beta-Gal tightly associated with the complex constitutes a small fraction of the total beta-Gal activity. The more prevalent form of the enzyme is a beta-Gal homodimer that is stable and devoid of either neuraminidase activity or protective protein.

Characteristics of the beta-galactosidase-carboxypeptidase complex in GM1-gangliosidosis and beta-galactosialidosis fibroblasts.

Lysosomal beta-galactosidase (beta-Gal) occurs either alone in monomeric and dimeric forms, or in a high-M(r) complex with at least two additional proteins. One is neuraminidase and the second is the protective protein, which has also been shown to possess carboxypeptidase activity. beta-Gal activity is deficient in GM1-gangliosidosis as a primary defect, and is secondarily affected in galactosialidosis (GS), where the primary defect is the absence of protective protein activity. Fibroblasts from three patients with GM1-gangliosidosis, type 1, showed markedly reduced amounts of beta-Gal cross-reacting material (CRM), and a fourth appeared to have normal levels. A patient with type 2 GM1-gangliosidosis was also found to be CRM-normal. These findings demonstrate that patients with GM1-gangliosidosis type 1 are heterogeneous with respect to the level of residual beta-Gal protein. Fibroblasts from four patients with GS were strongly CRM-positive with an anti-beta-Gal antibody, as was a sample of brain from one of these patients, suggesting that the loss of beta-Gal activity is linked to a subtler change in the primary structure of the enzyme than has been previously thought. While three GS cell lines displayed reduced carboxypeptidase activity (to 32-42% of the control), one cell line was completely devoid of activity, demonstrating that while carboxypeptidase activity is a property of the protective protein this action is distinct and separate from its protective role. On direct immunoprecipitation with anti-beta-Gal antibody, a portion of the total carboxypeptidase activity co-precipitated with beta-Gal from extracts of normal and GM1-gangliosidosis cells, consistent with the presence of the complex in these cells. However, no carboxypeptidase activity was precipitable with this antibody from GS fibroblasts, suggesting the absence of complex from these cells. To examine this further, the various forms of beta-Gal were resolved by h.p.l.c. molecular-sieve chromatography. Three forms of beta-Gal activity were resolved in normal cells: a complex, a dimer and a monomer. Residual beta-Gal activity of GS cells resolved into two of these forms, the complex and the monomer. In normal and GM1-gangliosidosis cells a portion of the total carboxypeptidase activity co-chromatographed with the complex while the bulk of the activity occurred in a single 36,000-M(r) peak. Only the low-M(r) carboxypeptidase activity was detected in GS cells. This confirms our results on immunoprecipitation indicating that portions of the beta-Gal and the carboxypeptidase activities exist outside the complex in normal, GM1-gangliosidosis and GS cells. In summary, the loss of protective protein function from GS cells results in disproportionate loss of the dimeric and monomeric forms of beta-Gal activity, but does not result in the complete degradation of the protein.

Analysis of a high frequency transformation system for Ophiostoma ulmi, the causal agent of Dutch elm disease.

A transformation system for Ophiostoma ulmi (Buism.) Nannf. was developed and analyzed. Protoplasts were generated from actively budding yeastlike cells by digestion with NovoZym 234 in MgSO4 after pretreatment with 2-mercaptoethanol. Protoplast regeneration was most efficient when 0.6 M sucrose was used as the osmoticum. Several plasmids containing fusions between fungal promoters and a bacterial gene for hygromycin phosphotransferase successfully transformed O. ulmi to hygromycin resistance. One of these vectors, pPS57, which contains a promoter for isopenicillin N synthetase from Penicillium chrysogenum, consistently conferred the greatest resistance to hygromycin. Linearization of the vector and inclusion of 2-mercaptoethanol in the transformation reaction resulted in enhanced transformation efficiency. Approximately 4 x 10(3) transformants/micrograms DNA per 10(7) protoplasts were obtained using the optimized procedure. Southern hybridization after alternating field and standard electrophoresis suggested random insertion of tandem repeats (some greater than 250 kb) into the fungal chromosomes. Antibiotic resistance was stable through mitosis. However, expression of the transforming DNA after meiosis was highly variable.

The amino-terminal sequences in the pro-alpha and -beta polypeptides of human lysosomal beta-hexosaminidase A and B are retained in the mature isozymes.

The alpha- and beta-subunits of beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52) are synthesized in the rough endoplasmic reticulum as prepropolypeptides. After the loss of the signal peptide and formation of enzymatically active dimers, the pro-isoenzymes are transported through the Golgi and into the lysosome for proteolytic and glycolytic processing to their stable mature forms. Maturation includes the hydrolysis, and previously presumed loss, of small N-terminal peptides from each propolypeptide. A recent report characterizing the processing of the beta-prepropolypeptide in beta-hexosaminidase from a human fibroblast cell line [(1989) J. Biol. Chem. 264, 3380-3384] reported that the small pro-beta peptide was retained through a disulfide bond in the mature subunit, and that it was glycosylated. We have confirmed this result in normal human tissue. However, we report a different N-terminal for the mature pro-beta peptide. Furthermore, we have found that the pro-alpha peptide is similarly retained in the mature alpha-subunit through its single cysteine residue and that each pro-peptide undergoes C-terminal processing.