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

Previous research has indicated that biotypes A and B of Colletotrichum 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. gloeosporioides 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.

Application of inter simple sequence repeat (ISSR) markers to plant genetics

Microsatellites or simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Single-locus SSR markers have been developed for a number of species, although there is a major bottleneck in developing SSR markers whereby flanking sequences must be known to design 5'-anchors for polymerase chain reaction (PCR) primers. Inter SSR (ISSR) fingerprinting was developed such that no sequence knowledge was required. Primers based on a repeat sequence, such as (CA)(n), can be made with a degenerate 3'-anchor, such as (CA)(8)RG or (AGC)(6)TY. The resultant PCR reaction amplifies the sequence between two SSRs, yielding a multilocus marker system useful for fingerprinting, diversity analysis and genome mapping. PCR products are radiolabelled with P-32 or P-33 via end-labelling or PCR incorporation, and separated on a polyacrylamide sequencing gel prior to autoradiographic visualisation. A typical reaction yields 20-100 bands per lane depending on the species and primer. We have used ISSR fingerprinting in a number of plant species, and report here some results on two important tropical species, sorghum and banana. Previous investigators have demonstrated that ISSR analysis usually detects a higher level of polymorphism than that detected with restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) analyses. Our data indicate that this is not a result of greater polymorphism genetically, but rather technical reasons related to the detection methodology used for ISSR analysis.

Characterization of baboon (Papio hamadryas) milk proteins

The major proteins of baboon milk were identified as beta -lactoglobulin (beta LG), alpha -lactalbumin (alpha LA), lysozyme, lactoferrin, casein, and albumin by immobiline isoelectric focusing, SDS-PAGE, immunoblotting of gels with rabbit antisera to human alpha LA, lysozyme, and albumin and bovine beta LG and casein, and N-terminal sequencing of proteins blotted from gels. The first 30 N-terminal residues of baboon polymorphism at residue 2. The complete cDNA sequence and derived amino acid composition of beta LG were elucidated using RT-PCR amplification of poly(A)(+) mRNA purified from lactating mammary gland. Baboon beta LG identified to date. beta LG and alpha LA polymorphisms with three (A, B, and C) and two (A and B) variants, respectively, were detected by immobiline IEF, pH 4-6, of individual baboon milk samples at varying stages of lactation.

Mutation analysis of the CDKN2A promoter in Australian melanoma families

Approximately 50% of all melanoma families worldwide show linkage to 9p21-22, but only about half of these have been shown to contain germ line CDKN2A mutations. It has been hypothesized that a proportion of these families carry mutations in the noncoding regions of CDKN2A. Several Canadian families have been reported to carry a mutation in the 5' UTR, at position -34 relative to the start site, which gives rise to a novel AUG translation initiation codon that markedly decreases translation from the wild-type AUG (Liu et al., 1999). Haplotype sharing in these Canadian families suggested that this mutation is of British origin. We sequenced 1,327 base pairs (bp) of CDKN2A, making up 1,116 bp of the 5' UTR and promoter, all of exon 1, and 61 bp of intron 1, in at least one melanoma case from 110 Australian families with three or more affected members known not to carry mutations within the p16 coding region. In addition, 431 bp upstream of the start codon was sequenced in an additional 253 affected probands from two-case melanoma families for which the CDKN2A mutation status was unknown. Several known polymorphisms at positions -33, -191, -493, and -735 were detected, in addition to four novel variants at positions 120, -252, -347, and -981 relative to the start codon. One of the probands from a two-case family was found to have the previously reported Q50R mutation. No family member was found to carry the mutation at position -34 or any other disease-associated mutation. For further investigation of noncoding CDKN2A mutations that may affect transcription, allele-specific expression analysis was carried out in 31 of the families with at least three affected members who showed either complete or indeterminate 9p haplotype sharing without CDKN2A exonic mutations. Reverse transcription polymerase chain reaction and automated sequencing showed expression of both CDKN2A alleles in all family members tested. The lack of CDKN2A promoter mutations and the absence of transcriptional silencing in the germ line of this cohort of families suggest that mutations in the promoter and 5' UTR play a very limited role in melanoma predisposition. (C) 2001 Wiley-Liss, Inc.

Variation in Alcohol Pharmacokinetics as a Risk Factor for Alcohol Dependence

Background: The significant association between alcohol dehydrogenase (ADH)-2 genotype and alcohol-dependence risk, demonstrated in both Asian and non-Asian populations, suggests a link between the metabolism of alcohol (ethanol) and individual differences in susceptibility to dependence. Methods: We tested this hypothesis by following up on subjects who took part in the Alcohol Challenge Twin Study conducted in 1979-1981 and comparing the blood and breath alcohol results in that study between subjects who subsequently did or did not meet diagnostic criteria for lifetime alcohol dependence in 1992-1993. Results: Subjects who met DSM-III-R criteria for lifetime alcohol dependence at follow-up had higher blood and breath alcohol values after alcohol challenge than never-dependent subjects. Multivariate analysis showed independent effects of susceptibility to alcohol dependence and smoking status on blood alcohol concentrations, whereas habitual alcohol intake at the time of the initial study had marginally significant effects. The risk of alcohol dependence was 2-fold higher in men and 3-fold higher in women with blood or breath alcohol concentrations in the highest quartile than in the lowest quartile. Conclusions: In view of this association and the known genetic influences on both alcohol pharmacokinetics and alcohol dependence, it is probable that part of the heritability of dependence is mediated by genes (other than the known ADH2 and ADH3 polymorphisms) affecting alcohol metabolism.

Increasing relevance of pharmacogenetics of drug metabolism in clinical practice

Much of the individual variation in drug response is due to genetic drug metabolic polymorphisms. Clinically relevant examples include acetylator status; cytochrome P450 2D6, 2C9 and 2C19 polymorphisms; and thiopurine methyltransferase deficiency. It is important to be aware of which drugs are subject to pharmacogenetic variability. In the future, population-based pharmacogenetic testing will allow more individualized drug treatment and will avoid the current empiricism.

Development of ligase-assisted spacer addition for the measurement of microsatellites

Conventional methods for detecting differences in microsatellite repeat lengths rely on electrophoretic fractionation on long denaturing polyacrylamide gels, a time-consuming and labor-intensive method. Therefore, there is a need for the development of new and rapid approaches to routinely detect such length polymorphisms. The advent of techniques allowing the coupling of DNA molecules to solid surfaces has provided new prospects in the area of mutation. We describe here the development and optimization of the ligase-assisted spacer addition (LASA) method, a novel and rapid procedure based on an ELISA format to measure microsatellite repeat lengths. The LASA assay was successfully applied to a set of 11 bird samples to assess its capability as a genotyping method.

A single nucleotide polymorphism in the 5' untranslated region of RAD51 and risk of cancer among BRCA1/2 mutation carriers

RAD51 colocalizes with both BRCA1 and BRCA2, and genetic variants in RAD51 would be candidate BRCA1/2 modifiers. We searched for RAD51 polymorphisms by sequencing 20 individuals. We compared the polymorphism allele frequencies between female BRCA1/2 mutation carriers with and without breast or ovarian cancer and between population-based ovarian cancer cases with BRCA1/2 mutations to cases and controls without mutations. We discovered two single nucleotide polymorphisms (SNPs) at positions 135 g-->c and 172 g-->t of the 5' untranslated region. In an initial group of BRCA1/2 mutation carriers, 14 (21%) of 67 breast cancer cases carried a c allele at RAD51:135 g-->c, whereas 8 (7%) of 119 women without breast cancer carried this allele. In a second set of 466 mutation carriers from three centers, the association of RAD51:135 g-->c with breast cancer risk was not confirmed. Analyses restricted to the 216 BRCA2 mutation carriers, however, showed a statistically significant association of the 135 c allele with the risk of breast cancer (adjusted odds ratio, 3.2; 95% confidence limit, 1.4-40). BRCA1/2 mutation carriers with ovarian cancer were only about one half as likely to carry the RAD51:135 g-->c SNP. Analysis of the RAD51:135 g-->c SNP in 738 subjects from an Israeli ovarian cancer case-control study was consistent with a lower risk of ovarian cancer among BRCA1/2 mutation carriers with the c allele. We have identified a RAD51 5' untranslated region SNP that may be associated with an increased risk of breast cancer and a lower risk of ovarian cancer among BRCA2 mutation carriers. The biochemical basis of this risk modifier is currently unknown.

Ethanol and gene expression in brain

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Izuru Matusmoto and Peter A. Wilce. The presentations were (1) GABA receptor subunit expression in the human alcoholic brain, by Tracey Buckley and Peter Dodd; (2) NMDAR gene expression during ethanol addiction, by Jorg Puzke, Rainer Spanagel, Walther Zieglgansberger, and Gerald Wolf; (3) Differentially expressed gene in the nucleus accumbens from ethanol-administered rat, by Shuangying Leng; (4) Expression of a novel gene in the alcoholic brain, by Peter A. Wilce; and (5) Investigations of haplotypes of the dopamine Da-receptor gene in alcoholics, by Hans Rommelspacher, Ulrich Finckh, and Lutz G. Schmidt.

Relationships between non-occupational cadmium exposure and expression of nine cytochrome P450 forms in human liver and kidney cortex samples

This study was undertaken to assess associations between age, gender, cigarette smoke and non-workplace cadmium exposure, and liver pathology and inter-individual variation in cytochrome P450 (CYP) expression in human tissues. Autopsy specimens of twenty-eight Queensland residents whose ages ranged from 3 to 89 years were analyzed for the presence of nine CYP protein isoforms by immunoblotting. All subjects were Caucasians and their liver cadmium contents ranged from 0.11 to 3.95 kg/g wet weight, while their kidney cadmium contents were in the range of 2 to 63 mug/g wet weight. CYP1A2, CYP2A6, CYP2D6, CYP3A4, and CYP3A5 were detected in liver but not in kidney, and CYP1A1 and CYP1B1 were not found in liver or kidney. Lowered liver CYP2C8/19 protein contents were found to be associated with liver pathology. Importantly, we show elevated levels of CYP2C9 protein to be associated with cadmium accumulation in liver. No mechanism that explains this association is apparent, but there are two possibilities that require further study. One is that variation in CYP2C9 protein levels may be, in part, attributed to an individual's non-workplace exposure to cadmium, or an individual's CYP2C9 genotype may be a risk factor for cadmium accumulation. A positive correlation was found between liver CYP3A4 protein and subject age. Levels of liver CYPIA2 protein, but not other CYP forms, were increased in people more exposed to cigarette smoke, but there was no association between CYPIA2 protein and cadmium. CYP2A6 protein was found in all liver samples and CYP2A6 gene typing indicated the absence of CYP2A6 null allele (CYP2A6(D)) in this sample group, confirming very low prevalence of homozygous CYP2A6(D) in Caucasians. CYP2A6 gene types W/W, WIC, and CIC were not associated with variations in liver microsomal CYP2A6 protein. CYP2D6 protein was absent in all twenty-five kidney samples tested but was detectable in liver samples of all but two subjects, indicating the prevalence of the CYP2D6 null allele (CYP2D6(D)) in this sample group to be about 7%, typical of Caucasian populations. (C) 2001 Elsevier Science Inc. All rights reserved.

A DNA fingerprinting procedure for ultra high-throughput genetic analysis of insects

Existing procedures for the generation of polymorphic DNA markers are not optimal for insect studies in which the organisms are often tiny and background molecular Information is often non-existent. We have used a new high throughput DNA marker generation protocol called randomly amplified DNA fingerprints (RAF) to analyse the genetic variability In three separate strains of the stored grain pest, Rhyzopertha dominica. This protocol is quick, robust and reliable even though it requires minimal sample preparation, minute amounts of DNA and no prior molecular analysis of the organism. Arbitrarily selected oligonucleotide primers routinely produced similar to 50 scoreable polymorphic DNA markers, between individuals of three Independent field isolates of R. dominica. Multivariate cluster analysis using forty-nine arbitrarily selected polymorphisms generated from a single primer reliably separated individuals into three clades corresponding to their geographical origin. The resulting clades were quite distinct, with an average genetic difference of 37.5 +/- 6.0% between clades and of 21.0 +/- 7.1% between individuals within clades. As a prelude to future gene mapping efforts, we have also assessed the performance of RAF under conditions commonly used in gene mapping. In this analysis, fingerprints from pooled DNA samples accurately and reproducibly reflected RAF profiles obtained from Individual DNA samples that had been combined to create the bulked samples.

Diversity of Phytophthora clandestina isolated from subterranean clover in southern australia: Analysis of virulence and RAPD profiles

The virulence spectrum of 112 isolates of Phytophthora clandestina collected from 56 sites in four subterranean clover-growing states in southern Australia was determined using differential cultivars of subterranean clover. Five races were detected, with race 0 in all states except New South Wales, race 1 in all states, race 2 only in Victoria, race 3 only in New South Wales, and race 4 in Victoria and Western Australia. The level of genotypic diversity among the different P. clandestina populations was investigated using five RAPD primers. Among 30 bands amplified, only two were polymorphic. This enabled identification of four multilocus RAPD genotypes. Three of the four genotypes occurred in all four states. Races 2 and 3 occurred with RAPD genotypes 1 and 2 only whereas races 0 and 1 occurred in all four multilocus RAPD genotypes. These results indicate that the pathogenicity spectrum of P. clandestina can change rapidly.

Latitudinal variability in symbiont specificity within the widespread scleractinian coral Plesiastrea versipora

We examined the genetic diversity of symbiotic dinoflagellates (Symbiodinium sp.) in the widespread hermatypic coral Plesiastrea versipora from tropical/subtropical (north-eastern Australia) and temperate waters (south-eastern Australia) using restriction fragment length polymorphisms of partial 18S ribosomal DNA (rDNA), together with sequence analysis of partial 28S rDNA. This study revealed that P. versipora associates with at least two distinct genotypes of symbiotic dinoflagellates and that the presence of these genotypes varies with latitude. P. versipora colonies from subtropical and tropical waters contained symbionts belonging to Symbiodinium clade C, while P. versipora colonies at high-latitude sites contained clade B. Variability within the two groups of symbionts (clades H and C) was minimal, suggesting possible host fidelity. The geographically distinct varieties of symbionts within the tissue of this hermatypic coral are likely to be associated with algal physiological differences, which in turn may relate to changing selective pressures as a function of latitude along the eastern Australian seaboard.

Identification of causal relationships among traits related to drought resistance in Stylosanthes scabra using QTL analysis

Previous studies have shown that a negative relationship exists between transpiration efficiency (TE) and carbon isotope discrimination (Delta) and between TE and specific leaf area (SLA) in Stylosanthes scabra, A glasshouse experiment was conducted to confirm these relationships in an F-2 population and to study the causal nature of these relationships through quantitative trait loci (QTL) analysis, One hundred and twenty F-2 genotypes from a cross between two genotypes within S. scabra were used. Three replications for each genotype were maintained through vegetative propagation, Water stress was imposed by maintaining plants at 40% of field capacity for about 45 d. To facilitate QTL analysis, a genetic linkage map consisting of 151 RAPD markers was developed, Results from this study show that Delta was significantly and negatively correlated with TE and biomass production. Similarly, SLA showed significant negative correlation with TE and biomass production, Most of the QTL for TE and Delta were present on linkage groups 5 and 11. Similarly, QTL for SLA, transpiration and biomass productivity traits were clustered on linkage groups 13 and 24, One unlinked marker was also associated with these traits, There were several markers coincident between different traits, At all the coincident QTL, the direction of QTL effects was consistent with phenotypic data, At the coincident markers between TE and Delta, high alleles of TE were associated with low alleles of Delta. Similarly, low alleles of SLA were associated with high alleles of biomass productivity traits and transpiration. At the coincident markers between trans-4-hydroxy-N-methyl proline (MHP) and relative water content (RWC), low alleles of MHP were associated with high alleles of RWC, This study suggests the causal nature of the relationship between TE and Delta. Phenotypic data and QTL, data show that SLA was more closely associated with biomass production than with TE, This study also shows that a cause-effect relationship may exist between SLA and biomass production.