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J A Irwin

Publications and source records attributed to J A Irwin.

14 recordsLinked to original sources
PubMed1999

Myoglobin-induced oxidative damage: evidence for radical transfer from oxidized myoglobin to other proteins and antioxidants.

Reaction of equine Fe(III) myoglobin with H2O2 gives rise to an Fe(IV)-oxo species at the heme center and protein (globin)-derived radicals. Studies have shown that there are two (or more) sites for the protein-derived radical: at tyrosine (Tyr-103) or tryptophan (Trp-14). The latter radical reacts rapidly with oxygen to give a Trp-derived peroxyl radical. The formation of both the tyrosine phenoxyl radical and the tryptophan-derived peroxyl species have been confirmed in the present study; the latter appears to be the major initial radical, with the phenoxyl radical appearing at longer reaction times, possibly via secondary reactions. We have investigated, by EPR spectroscopy, the reactivity of the Trp-14 peroxyl radical with amino acids, peptides, proteins, and antioxidants, with the aim of determining whether this species can damage other targets, i.e., whether intermolecular protein-to-protein radical transfer and hence chain-oxidation occurs, and the factors that control these reactions. Three amino acids show significant reactivity: Tyr, Trp, and Cys, with Cys the least efficient. Evidence has also been obtained for (inefficient) hydrogen abstraction at peptide alpha-carbon sites; this may result in backbone cleavage in the presence of oxygen. The myoglobin Trp-14 peroxyl radical has been shown to react rapidly with a wide range of proteins to give long-lived secondary radicals on the target protein. These reactions appear to mainly involve Tyr residues on the target protein, although evidence for reaction at Trp has also been obtained. Antioxidants (GSH, ascorbate, Trolox C, vitamin E, and urate) react with the myoglobin-derived peroxyl radical; in some cases antioxidant-derived radicals are detected. These reactions are only efficient at high antioxidant concentrations, suggesting that protein-to-protein damage transfer and protein chain-oxidation may occur readily in biological systems.

Amino Acids↗
PubMed1998

Transfer of a supernumerary chromosome between vegetatively incompatible biotypes of the fungus Colletotrichum gloeosporioides.

Two biotypes (A and B) of Colletotrichum gloeosporioides infect the tropical legumes Stylosanthes spp. in Australia. These biotypes are asexual and vegetatively incompatible. However, field isolates of biotype B carrying a supernumerary 2-Mb chromosome, thought to originate from biotype A, have been reported previously. We tested the hypothesis that the 2-Mb chromosome could be transferred from biotype A to biotype B under laboratory conditions. Selectable marker genes conferring resistance to hygromycin and phleomycin were introduced into isolates of biotypes A and B, respectively. A transformant of biotype A, with the hygromycin resistance gene integrated on the 2-Mb chromosome, was cocultivated with phleomycin-resistant transformants of biotype B. Double antibiotic-resistant colonies were obtained from conidia of these mixed cultures at a frequency of approximately 10(-7). Molecular analysis using RFLPs, RAPDs, and electrophoretic karyotypes showed that these colonies contained the 2-Mb chromosome in a biotype B genetic background. In contrast, no double antibiotic colonies developed from conidia obtained from mixed cultures of phleomycin-resistant transformants of biotype B with biotype A transformants carrying the hygromycin resistance gene integrated in chromosomes >2 Mb in size. The results demonstrated that the 2-Mb chromosome was selectively transferred from biotype A to biotype B. The horizontal transfer of specific chromosomes across vegetative incompatibility barriers may explain the origin of supernumerary chromosomes in fungi.

Ascomycota↗
PubMed1997

Transfer of an autonomously replicating vector between vegetatively incompatible biotypes of Colletotrichum gloeosporioides.

Previous research has indicated that biotypes A and B of Collectotrichum gloeosporioides that infect Stylosanthes spp. in Australia are asexual and vegetatively incompatible. Selectable marker genes conferring resistance either to hygromycin or phleomycin were introduced into isolates of these biotypes. Vectors conferring resistance to hygromycin and carrying telomeric sequences from Fusarium oxysporum replicated autonomously in C. gloesoporioides and gave frequencies of transformation 100-times higher than vectors that integrated into the genome. Monoconidial colonies resistant to both antibiotics were recovered when hygromycin-resistant biotype-A transformants carrying an autonomously replicating vector were paired in culture with a phleomycin-resistant biotype-B transformant carrying integrative vector sequences. Molecular analysis of double antibiotic-resistant progeny indicated that they contained the autonomous vector in a biotype-B genetic background. Results indicate that transfer of the autonomous vector had occurred from biotype A to biotype B, demonstrating the potential for transfer of genetic information between these biotypes.

Ascomycota↗
PubMed1996

CgT1: a non-LTR retrotransposon with restricted distribution in the fungal phytopathogen Colletotrichum gloeosporioides.

Two genetically distinct biotypes (A and B) of Colletotrichum gloeosporioides that cause different anthracnose diseases on the legumes Stylosanthes spp. have been identified in Australia. A DNA sequence that was present in biotype B and absent in biotype A was isolated by differential hybridisation of a genomic library using total genomic DNA of each biotype as hybridisation probes. This sequence also failed to hybridise to DNA of three biotypes of C. gloeosporioides from other host species and to DNA of three other species of Colletotrichum. This clone was used to isolate two cosmid clones of biotype B. Sequence analysis of these clones revealed a repetitive element of approximately 5.7 kb in length. This element, termed CgT1, was dispersed in the genome and present in about 30 copies. The element contained open reading frames encoding deduced sequence motifs homologous to gag-like proteins, reverse transcriptase and RNase H domains of non-LTR retrotransposons. The termini of CgT1 lacked long terminal repeats (LTRs) but contained a 3' A-rich domain. The insertion site of one copy of the element was flanked by short 13-bp direct repeats. These characteristics of the termini, taken together with the overall structure and sequence homologies, indicate that CgT1 belongs to the non-LTR, LINE-like retrotransposon class of elements that are present in many eukaryotes. PCR primers designed to amplify regions of CgT1 can be used to distinguish biotypes A and B in Australia. DNA fingerprinting analysis of genomic DNA using hybridisation probes derived from the terminal regions of CgT1 revealed that Australian isolates of biotype B are monomorphic. CgT1 was not detected in some isolates causing Type B disease from other countries and when CgT1 was present there was considerable polymorphism in CgT1 organisation in the genome. CgT1 is the first transposon-like element to be identified in the genus Colletotrichum and has considerable potential as a tool for the study of population structure, genome dynamics and evolution in C. gloeosporioides.

Amino Acid Sequence↗
PubMed1994

Evidence for outcrossing in Phytophthora sojae and linkage of a DNA marker to two avirulence genes.

Two genetically different isolates of the homothallic Oomycete, Phytophthora sojae, were demonstrated to outcross and form hybrid oospores after co-culturing in vitro. Random amplified polymorphic DNA (RAPD) markers revealed ten hybrids among 354 oospores analysed. One F1 hybrid was allowed to self fertilise and produce an F2 population of 247 individuals. Among 53 F2 individuals, selected at random, 18 polymorphic RAPD markers were observed to segregate at near 3:1 Mendelian ratios, consistent with segregation for dominant alleles at single loci. Segregation of virulence against soybean resistance genes Rps1a, 3a, and 5 revealed that the avirulence genes Avrla, 3a and 5 were dominant to virulence. Avirulence against these three resistance genes appeared to be conditioned by one locus for Avr1a and two independent, complementary dominant loci for both Avr3a and Avr5. Segregation of virulence against Rps6 was in the ratio of 1:2:1 (avirulent:mixed reaction:virulent), suggesting a semi-dominant allele at a single locus. Two avirulence genes and one RAPD marker formed one linkage group, in the order Avr3a, OPH4-1, Avr5, each separated by approximately 5 cM. Our results confirm that outcrossing occurred between the parental isolates, and that sexual recombination under field conditions may play an important role in generating and maintaining genetic diversity in populations of P. sojae.

Base Sequence↗
PubMed1993

A strain-specific cyclin homolog in the fungal phytopathogen Colletotrichum gloeosporioides.

The fungus, Colletotrichum gloeosporioides, which infects the tropical pasture legume, Stylosanthes guianensis, contains highly variable mini-chromosomes. The transcription of strain-specific genomic DNA clones previously isolated from one variable mini-chromosome was investigated by using these clones to screen a cDNA library prepared from the fungus grown in liquid medium. A cDNA clone was obtained with one of the genomic clones and was sequenced. A single long open reading frame of 259 amino acids (aa) was detected with significant homology to cyclin proteins in other organisms. Northern blot analysis indicated that the cDNA corresponded to a low-abundance mRNA (approximately 0.001% of poly(A)+RNA). Southern blot analysis indicated that genes encoding this mRNA were discontinuously distributed in this fungal species, indicating it encodes a dispensable function. This result suggests that natural populations of fungi may have variable complements of cyclin-encoding genes.

Amino Acid Sequence↗
PubMed1993

DNA addition or deletion is associated with a major karyotype polymorphism in the fungal phytopathogen Colletotrichum gloeosporioides.

A 1.2 Mb minichromosome resolved by pulsed-field electrophoresis was present in two independent race 3 isolates of Colletotrichum gloeosporioides causing Type B anthracnose specifically on Stylosanthes guianensis cv. Graham in Australia. This chromosome was absent in duplicate isolates representing races 1, 2 and 4 which infect other S. guianensis cultivars. A gene library was prepared specifically from the 1.2 Mb minichromosome and ten independent DNA clones unique to this chromosome were identified by differential hybridisation to whole chromosome probes. All of the ten selected probes hybridised only to the 1.2 Mb minichromosome unique to the race 3 isolates but not to any chromosome in isolates of the other races. These ten probes also hybridised only to restriction-digested DNA of race 3 and were thus both chromosome- and strain-specific for Type B C. gloeosporioides. Hybridisation analysis of NotI fragments of the 1.2 Mb minichromosome with these sequences indicated that they were not tightly clustered on the chromosome. These data demonstrate that the variation in the occurrence of the 1.2 Mb minichromosome did not arise by rearrangement of the genome of a progenitor strain but involved either large scale deletion or addition of DNA. The 1.2 Mb minichromosome did not contain a cloned high-copy-number repeat sequence present on all other mini- and maxichromosomes, suggesting addition from a genetically distinct strain. All ten chromosome-specific DNA probes hybridised to a 2.0 Mb chromosome in all races of C. gloeosporioides causing Type A anthracnose on Stylosanthes spp. including S. guianensis.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Aberrations↗
PubMed1993

Random amplified polymorphic DNA markers reveal a high degree of genetic diversity in the entomopathogenic fungus Metarhizium anisopliae var. anisopliae.

Metarhizium anisopliae isolates from several insect hosts and from various sugar cane growing areas of Queensland, Australia, were examined for genetic diversity using random amplified polymorphic DNA (RAPD) markers. Thirty isolates of M. anisopliae var. anisopliae and one isolate of M. anisopliae var. majus were examined. Ten randomly chosen 10mer or 11mer primers were used and RAPD banding patterns were compared. Thirty distinct genotypes could be distinguished amongst the 31 isolates tested on the basis of RAPD patterns. Six of the isolates classified as M. anisopliae var. anisopliae exhibited closer similarity to the M. anisopliae var. majus isolate than to other anisopliae strains tested. Isolates exhibiting similar (> 80% similarity) RAPD profiles tended to be isolated from the same geographic area and evidence for the persistence of particular fungal genotypes in specific geographical localities was obtained. Pathogenicity assays suggested that, in some instances, RAPD groupings may also indicate insect host range. The mean similarity amongst isolates measured by band sharing in all pairwise comparisons was 41% and the most distinct pair of isolates shared only 9% of their RAPD bands. We conclude that the isolates tested belonging to the species M. anisopliae, as assessed on morphological grounds, represent a very diverse genetic group. The results also suggest that RAPD markers may be useful for the tracking of specific biocontrol strains in the field.

Animals↗
PubMedDate not supplied

Phytophthora sojae avirulence genes, RAPD, and RFLP markers used to construct a detailed genetic linkage map.

Two crosses between different races of Phytophthora sojae were established using one race as a common parent in both crosses. F2 populations comprising over 200 individuals were generated for each cross. A subset of 53 F2 individuals from each cross was selected at random for genetic analysis of virulence/avirulence and molecular markers, and finally the construction of a detailed genetic linkage map. The linkage map developed for P. sojae is based on a total of 257 markers (22 RFLP, 228 RAPD, and 7 avirulence genes). The linkage map comprises 10 major and 12 minor linkage groups covering a total of 830.5 cM. Close linkage was observed between Avr4 and Avr6 (0.0 cM), Avr1b and Avr1k (0.0 cM), and Avr3a and Avr5 (4.6 cM). Coupling phase linkage of RFLP and RAPD markers to all seven avirulence genes was identified at the minimum and maximum distances of 0.0 and 14.7 cM, respectively.

Chromosome Mapping↗