A peripheral blood diagnostic test for acute rejection in renal transplantation.

Monitoring of renal graft status through peripheral blood (PB) rather than invasive biopsy is important as it will lessen the risk of infection and other stresses, while reducing the costs of rejection diagnosis. Blood gene biomarker panels were discovered by microarrays at a single center and subsequently validated and cross-validated by QPCR in the NIH SNSO1 randomized study from 12 US pediatric transplant programs. A total of 367 unique human PB samples, each paired with a graft biopsy for centralized, blinded phenotype classification, were analyzed (115 acute rejection (AR), 180 stable and 72 other causes of graft injury). Of the differentially expressed genes by microarray, Q-PCR analysis of a five gene-set (DUSP1, PBEF1, PSEN1, MAPK9 and NKTR) classified AR with high accuracy. A logistic regression model was built on independent training-set (n = 47) and validated on independent test-set (n = 198)samples, discriminating AR from STA with 91% sensitivity and 94% specificity and AR from all other non-AR phenotypes with 91% sensitivity and 90% specificity. The 5-gene set can diagnose AR potentially avoiding the need for invasive renal biopsy. These data support the conduct of a prospective study to validate the clinical predictive utility of this diagnostic tool.

A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis.

We developed a modified allele-specific PCR procedure for assaying single nucleotide polymorphisms (SNPs) and used the procedure (called SNAP for single-nucleotide amplified polymorphisms) to generate 62 Arabidopsis mapping markers. SNAP primers contain a single base pair mismatch within three nucleotides from the 3' end of one allele (the specific allele) and in addition have a 3' mismatch with the nonspecific allele. A computer program called SNAPER was used to facilitate the design of primers that generate at least a 1,000-fold difference in the quantity of the amplification products from the specific and nonspecific SNP alleles. Because SNAP markers can be readily assayed by electrophoresis on standard agarose gels and because a public database of over 25,000 SNPs is available between the Arabidopsis Columbia and Landsberg erecta ecotypes, the SNAP method greatly facilitates the map-based cloning of Arabidopsis genes defined by a mutant phenotype.

High-accuracy DNA sequence variation screening by DHPLC.

Genetic maps based on biallelic single-nucleotide polymorphisms amenable to microarray-based genotyping have significantly accelerated the mapping of mono- and multigenic traits in model organisms such as Saccharomyces cerevisiae and Arabidopsis thaliana. This advance needs to be matched by highly accurate, inexpensive and robust methodology for fine-structure mapping of the candidate region(s) and the eventual identification of the causative mutation(s). To establish the usefulness of denaturing high-performance liquid chromatography (DHPLC) for those purposes, we have amplified 476 fragments from two A. thaliana ecotypes with an average length of 563 bp covering various candidate regions on chromosomes 1, 2 and 4. Parallel analysis by DHPLC and dye terminator sequencing showed that DHPLC detected 165 out of 166 polymorphic fragments with only four false positives, amounting to a sensitivity, specificity and accuracy of 99.4%, 98.7% and 99%, respectively. It proved beneficial to analyze the fragments not only at the highest but also at the lower temperatures recommended by the algorithm freely available at http:¿insertion.stanford.edu/melt.html.

Cloning of the Arabidopsis RSF1 gene by using a mapping strategy based on high-density DNA arrays and denaturing high-performance liquid chromatography.

Mapping genes by chromosome walking is a widely used technique applicable to cloning virtually any gene that is identifiable by mutagenesis. We isolated the gene responsible for the recessive mutation rsf1 (for reduced sensitivity to far-red light) in the Arabidopsis Columbia accession by using classical genetic analysis and two recently developed technologies: genotyping high-density oligonucleotide DNA array and denaturing high-performance liquid chromatography (DHPLC). The Arabidopsis AT412 genotyping array and 32 F(2) plants were used to map the rsf1 mutation close to the top of chromosome 1 to an interval of approximately 500 kb. Using DHPLC, we found and genotyped additional markers for fine mapping, shortening the interval to approximately 50 kb. The mutant gene was directly identified by DHPLC by comparing amplicons generated separately from the rsf1 mutant and the parent strain Columbia. DHPLC analysis yielded polymorphic profiles in two overlapping polymorphic amplicons attributable to a 13-bp deletion in the third of five exons of a gene encoding a 292-amino acid protein with a basic helix-loop-helix (bHLH) domain. The mutation in rsf1 results in a truncated protein consisting of the first 129 amino acids but lacking the bHLH domain. Cloning the RSF1 gene strongly suggests that numerous phytochrome A-mediated responses require a bHLH class transcription factor.

Genome-wide mapping with biallelic markers in Arabidopsis thaliana.

Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.

Use of Arabidopsis thaliana defense-related mutants to dissect the plant response to pathogens.

The plant defense response to microbial pathogens had been studied primarily by using biochemical and physiological techniques. Recently, several laboratories have developed a variety of pathosystems utilizing Arabidopsis thaliana as a model host so that genetic analysis could also be used to study plant defense responses. Utilizing a pathosystem that involves the infection of Arabidopsis with pathogenic pseudomonads, we have cloned the Arabidopsis disease-resistance gene RPS2, which corresponds to the avirulence gene avrRpt2 in a gene-for-gene relationship. RPS2 encodes a 105-kDa protein containing a leucine zipper, a nucleotide binding site, and 14 imperfect leucine-rich repeats. The RPS2 protein is remarkably similar to the product of the tobacco N gene, which confers resistance to tobacco mosaic virus. We have also isolated a series of Arabidopsis mutants that synthesize decreased levels of an Arabidopsis phytoalexin called camalexin. Analysis of these mutants indicated that camalexin does not play a significant role in limiting growth of avirulent Pseudomonas syringae strains during the hypersensitive defense response but that it may play a role in limiting the growth of virulent strains. More generally, we have shown that we can utilize Arabidopsis to systematically dissect the defense response by isolation and characterization of appropriate defense-related mutants.

The A. thaliana disease resistance gene RPS2 encodes a protein containing a nucleotide-binding site and leucine-rich repeats.

In plants, resistance to a pathogen is frequently correlated with a genetically defined interaction between a plant resistance gene and a corresponding pathogen avirulence gene. A simple model explains these gene-for-gene interactions: avirulence gene products generate signals (ligands), and resistance genes encode cognate receptors. The A. thaliana RPS2 gene confers resistance to the bacterial pathogen P. syringae carrying the avirulence gene avrRpt2. A map-based positional cloning strategy was used to identify RPS2. The identification of RPS2 was verified using a newly developed transient assay for RPS2 function and by genetic complementation in transgenic plants. RPS2 encodes a novel 105 kDa protein containing a leucine zipper, a nucleotide-binding site, and 14 imperfect leucine-rich repeats.

Expression of a bacterial ice nucleation gene in plants.

We have introduced an ice nucleation gene (inaZ) from Pseudomonas syringae pv. syringae into Nicotiana tabacum, a freezing-sensitive species, and Solanum commersonii, a freezing-tolerant species. Transformants of both species showed increased ice nucleation activity over untransformed controls. The concentration of ice nuclei detected at -10.5 degrees C in 15 different primary transformants of S. commersonii varied by over 1000-fold, and the most active transformant contained over 100 ice nuclei/mg of tissue. The temperature of the warmest freezing event in plant samples of small mass was increased from approximately -12 degrees C in the untransformed controls to -4 degrees C in inaZ-expressing transformants. The threshold nucleation temperature of samples from transformed plants did not increase appreciably with the mass of the sample. The most abundant protein detected in transgenic plants using immunological probes specific to the inaZ protein exhibited a higher mobility on sodium dodecyl sulfate polyacrylamide gels than the inaZ protein from bacterial sources. However, some protein with a similar mobility to the inaZ protein could be detected. Although the warmest ice nucleation temperature detected in transgenic plants is lower than that conferred by this gene in P. syringae (-2 degrees C), our results demonstrate that the ice nucleation gene of P. syringae can be expressed in plant cells to produce functional ice nuclei.

Plant and environmental sensory signals control the expression of hrp genes in Pseudomonas syringae pv. phaseolicola.

The hrp genes of Pseudomonas syringae pv. phaseolicola control the development of primary disease symptoms in bean plants and the elicitation of the hypersensitive response in resistant plants. We examined the expression of the seven operons located in the 22-kb hrp cluster (L. G. Rahme, M. N. Mindrinos, and N. J. Panopoulos, J. Bacteriol. 173:575-586, 1991) in planta and in vitro under different physiological and nutritional conditions by using chromosomally located hrp::inaZ reporter fusions. We show that (i) a plant signal(s) is specifically required for the induction of the seven hrp operons, during both compatible and incompatible interactions; (ii) hrpL and hrpRS are regulated by different mechanisms in planta and in vitro; and (iii) expression of individual hrp loci is differentially affected by pH, osmotic strength, and type of carbon source: hrpAB, hrpC, and hrpD were downregulated similarly by osmolarity, pH, and certain carbon sources; hrpE expression was affected strongly by pH and carbon substrate and slightly by osmolarity; and hrpF was not substantially affected by any of these factors. These findings suggest complex signaling mechanisms taking place during plant-pathogen interactions.

Induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains and by a cloned avirulence gene.

We developed a model system to study the signal transduction pathways leading to the activation of Arabidopsis thaliana genes involved in the defense against pathogen attack. Here we describe the identification and characterization of virulent and avirulent Pseudomonas syringae strains that elicit disease or resistance symptoms when infiltrated into Arabidopsis leaves. The virulent and avirulent strains were characterized by determining growth of the pathogen in Arabidopsis leaves and by measuring accumulation of mRNA corresponding to Arabidopsis phenylalanine ammonia-lyase (PAL), beta-1,3-glucanase (BG), and chalcone synthase (CHS) genes in infected leaves. The virulent strain, P. syringae pv maculicola ES4326, multiplied 10(5)-fold in Arabidopsis leaves and strongly elicited BG1, BG2, and BG3 mRNA accumulation but had only a modest effect on PAL mRNA accumulation. In contrast, the avirulent strain, P. syringae pv tomato MM1065, multiplied less than 10-fold in leaves and had only a minimal effect on BG1, BG2, and BG3 mRNA accumulation, but it induced PAL mRNA accumulation. No accumulation of CHS mRNA was found with either ES4326 or MM1065. We also describe the cloning of a putative avirulence (avr) gene from the avirulent strain MM1065 that caused the virulent strain ES4326 to grow less well in leaves and to strongly elicit PAL but not BG1 and BG3 mRNA accumulation. These results suggest that the Arabidopsis PAL and BG genes may be activated by distinct signal transduction pathways and show that differences in plant gene induction by virulent and avirulent strains can be attributed to a cloned presumptive avr gene.

Genetic and transcriptional organization of the hrp cluster of Pseudomonas syringae pv. phaseolicola.

The hrp cluster of Pseudomonas syringae pv. phaseolicola encodes functions that are essential for pathogenicity on bean plants and for the elicitation of the hypersensitive response on resistant plants. The cluster was saturated with insertions of transposon Tn3-spice that served both as a mutagen and as a sensitive reporter of the expression of the target regions. The mutations covered a 17.5-kb segment in strain NPS3121, in which seven hrp::Tn5 insertions had been previously mapped, and regions outside this segment. The cluster is organized into seven distinct complementation groups (hrpL, hrpAB, hrpC, hrpD, hrpE, hrpF, and hrpSR) on the basis of the analysis of over 100 Tn3-spice insertions in plasmids and 43 similar insertions in the chromosome; it spans nearly 22 kb and is chromosomally located. The transcriptional orientation of all genes in the cluster was established by measuring the level of ice nucleation activity of complemented merodiploids carrying chromosomal hrp::inaZ fusions after inoculation in Red Kidney bean leaves. Although all seven loci were actively expressed in Red Kidney bean leaves, none of them was substantially expressed when the bacteria were grown in King B broth medium. Mutations in all loci, except those in hrpC, greatly reduced the ability of the bacteria to multiply in bean leaves. Mutations in the hrpC locus, although preventing the bacteria from eliciting a hypersensitive reaction on tobacco, allowed the bacteria to produce delayed and attenuated symptoms in Red Kidney bean leaves and to multiply to a level 10(2)- to 10(3)-fold lower than that of the wild-type strain. This is the first comprehensive report of the genetic and transcriptional organization of the hrp gene cluster in a phytopathogenic bacterium.

Isolation and chromosomal location of putative vitelline membrane genes in Drosophila melanogaster.

cDNA clones for two Drosophila vitelline membrane genes have been identified on the basis of: (i) stage and tissue specificity of transcription and (ii) size and amino acid content of the translation product. Cross-hybridization data suggest that DmcMM99 and DmcMM115 are members of a multi-gene family which includes at least three members, all of which reside on the left arm of the second chromosome. DmcMM99 and DmcMM115 originate from polytene band positions 34C and 26A, respectively. A third, cross-hybridizing gene resides at position 32EF. Southern analysis of a genomic clone, lambda LS1, homologous to DmcMM115, indicates that two vitelline membrane genes may be clustered at the 26A site.