The SEN1 gene of Arabidopsis is regulated by signals that link plant defence responses and senescence

Plant defence and senescence share many similarities as evidenced by extensive co-regulation of many genes during these responses. To better understand the nature of signals that are common to plant defence and senescence, we studied the regulation of SEN1 encoding a senescence-associated protein during plant defence responses in Arabidopsis. Pathogen inoculations and treatments with defence-related chemical signals, salicylic acid and methyl jasmonate induced changes in SEN1 transcript levels. Analysis of transgenic plants expressing the SEN1 promoter fused to uidA reporter gene confirmed the responsiveness of the SEN1 promoter to defence- and senescence-associated signals. Expression analysis of SEN1 in a number of defence signalling mutants indicated that activation of this gene by pathogen occurs predominantly via the salicylic and jasmonic acid signalling pathways, involving the functions of EDS5, NPR1 and JAR1 In addition, in the absence of pathogen challenge, the cpr5/hys1 mutant showed elevated SEN1 expression and displayed an accelerated senescence response following inoculation with the necrotrophic fungal pathogen Fusarhan oxysporum. Although the analysis of the sen1-1 knock-out mutant did not reveal any obvious role for this gene in defence or senescence-associated events, our results presented here show that SEN1 is regulated by signals that link plant defence and senescence responses and thus represents a useful marker gene to study the overlap between these two important physiological events. (c) 2005 Elsevier SAS. All rights reserved.

Analysis of distinct tartrate-resistant acid phosphatase promoter regions in transgenic mice

The tartrate-resistant acid phosphatase (TRAP) is present in multiple tissues, including kidney, liver, lung, spleen, and bone. Recent study of (TRAP) gene expression has provided evidence for distinct promoters within the (TRAP) gene, suggesting that the gene has alternative, tissue-preferred mRNA transcripts. Examination of endogenous (TRAP) exon 1B and 1C mRNA transcripts revealed tissue-preferred transcript abundance with increased exon 1B transcripts detected in liver and kidney and increased exon 1C transcripts detected in bone and spleen. In this investigation, we have made transgenic mice that express a marker gene driven by two candidate promoters, designated BC and C, within the (TRAP) gene. The BC and C promoters are 2.2 and 1.6 kb, respectively, measured from the translation initiation site. Evaluation of BC transgenic lines demonstrated robust expression in multiple tissues. In contrast, significant transgene expression was not detected in C transgenic lines. Evaluation of transgene mRNAs in BC transgenic lines revealed that virtually all expression was in the form of B transcripts, suggesting that the tissue-preferred pattern of endogenous (TRAP) was not replicated in the BC transgenic line. Likewise, osteoclastogenic cultures from BC, but not C, transgenic bone marrow cells expressed the transgene following receptor activator of NFkappaB ligand/macrophage colony-stimulating factor stimulation. In conclusion, when compared with the 2.2-kb BC portion of the (TRAP) promoter region, the 1.6-kb C portion does not account for significant gene expression in vivo or in vitro; production of the bone- and spleen-preferred (TRAP) C transcript must depend on regulatory elements outside of the 2.2-kb promoter. As the majority of currently investigated transcription factors that influence transcriptional regulation of osteoclast gene expression bind within the 1.6-kb C portion of the (TRAP) promoter, it is likely that transcription binding sites outside of the 2.2-kb region will have profound effects on regulation of the gene in vivo and in vitro.

Mapping the I-3 gene for resistance to Fusarium wilt in tomato: application of an I-3 marker in tomato improvement and progress towards the cloning of I-3

Fusarium wilt of tomato, caused by the fungal pathogen, Fusarium oxysporum f. sp. lycopersici (Fol), is an economically damaging disease that results in huge losses in Australia and other countries worldwide. The I-3 gene, which confers resistance to Fol race 3, has been described in wild tomato, Lycopersicon pennellii, accessions LA716 and PI414773. We are pursuing the isolation of I-3 from LA716 by map-based cloning. We have constructed a high-resolution map of the I-3 region and have identified markers closely flanking I-3 as well as markers co-segregating with I-3. In addition, construction of a physical map based on these markers has been initiated. This review describes the context of our research and our progress towards isolating the I-3 gene. It also describes some important practical outcomes of our work, including the development and use of a PCR-based marker for marker-assisted selection for I-3, and the finding that the I-3 gene from LA716 is different to that from PI1414773, which we have now designated I-7. Tomato varieties combining I-3 and I-7 have been developed and are currently being introduced into commercial production to further safeguard tomato crops against Fusarium wilt.

Mannose-binding lectin gene polymorphism predicts hospital admissions for COPD infections

Infection frequently causes exacerbations of chronic obstructive pulmonary disease (COPD). Mannose-binding lectin (MBL) is a pattern-recognition receptor that assists in clearing microorganisms. Polymorphisms in the MBL2 gene reduce serum MBL levels and are associated with risk of infection. We studied whether the MBL2 codon 54 B allele affected serum MBL levels, admissions for infective exacerbation in COPD and disease susceptibility. Polymorphism frequency was determined by PCR-RFLP in 200 COPD patients and 104 smokers with normal lung function. Serum MBL was measured as mannan-binding activity in a subgroup of 82 stable COPD patients. Frequency of COPD admissions for infective exacerbation was ascertained for a 2-year period. The MBL2 codon 54 B allele reduced serum MBL in COPD patients. In keeping, patients carrying the low MBL-producing B allele had increased risk of admission for infective exacerbation (OR 4.9, P-corrected = 0.011). No association of MBL2 genotype with susceptibility to COPD was detected. In COPD, serum MBL is regulated by polymorphism at codon 54 in its encoding gene. Low MBL-producing genotypes were associated with more frequent admissions to hospital with respiratory infection, suggesting that the MBL2 gene is disease-modifying in COPD. MBL2 genotype should be explored prospectively as a prognostic marker for infection risk in COPD.

A role for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of arabidopsis

The PDF1.2 gene of Arabidopsis encoding a plant defensin is commonly used as a marker for characterization of the jasmonate-dependent defense responses. Here, using PDF1.2 promoter-deletion lines linked to the beta-glucoronidase-reporter gene, we examined putative promoter elements associated with jasmonate-responsive expression of this gene. Using stably transformed plants, we first characterized the extended promoter region that positively regulates basal expression from the PDF1.2 promoter. Second, using promoter deletion constructs including one from which the GCC-box region was deleted, we observed a substantially lower response to jasmonate than lines carrying this motif. In addition, point mutations introduced into the core GCC-box sequence substantially reduced jasmonate responsiveness, whereas addition of a 20-nucleotide-long promoter element carrying the core GCC-box and flanking nucleotides provided jasmonate responsiveness to a 35S minimal promoter. Taken together, these results indicated that the GCC-box plays a key role in conferring jasmonate responsiveness to the PDF1.2 promoter. However, deletion or specific mutations introduced into the core GCC-box did not completely abolish the jasmonate responsiveness of the promoter, suggesting that the other promoter elements lying downstream from the GCC-box region may also contribute to jasmonate responsiveness. In other experiments, we identified a jasmonate- and pathogen-responsive ethylene response factor transcription factor, AtERF2, which when overexpressed in transgenic Arabidopsis plants activated transcription from the PDF1.2, Thi2.1, and PR4 (basic chitinase) genes, all of which contain a GCC-box sequence in their promoters. Our results suggest that in addition to their roles in regulating ethylene-mediated gene expression, ethylene response factors also appear to play important roles in regulating jasmonate-responsive gene expression, possibly via interaction with the GCC-box.

Epidermal Growth Factor Gene (EGF) Polymorphism and Risk of Melanocytic Neoplasiay

A common single nucleotide polymorphism (SNP) in the 5' untranslated region (5'UTR) of the epidermal growth factor (EGF) gene modulates the level of transcription of this gene and hence is associated with serum levels of EGF. This variant may be associated with melanoma risk, but conflicting findings have been reported. An Australian melanoma case-control sample was typed for the EGF+61A>G transversion (rs4444903). The sample comprised 753 melanoma cases from 738 families stratified by family history of melanoma and 2387 controls from 645 unselected twin families. Ancestry of the cases and controls was recorded, and the twins had undergone skin examination to assess total body nevus count, degree of freckling and pigmentation phenotype. SNP genotyping was carried out via primer extension followed by matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectroscopy. The EGIF+61 SNP was not found to be significantly associated with melanoma status or with development of nevi or freckles. Among melanoma cases, however, G homozygotes had thicker tumors (p=0.05), in keeping with two previous studies. The EGF polymorphism does not appear to predispose to melanoma or nevus development, but its significant association with tumor thickness implies that it may be a useful marker of prognosis.

Expression of the cell surface mucin gene family in adenocarcinomas

Cell surface mucins are complex glycoproteins expressed on the apical membrane surface of mucosal epithelial cells. In malignant epithelial cells they are thought to influence cell adhesion, and are clinical targets for tumor immunotherapy and serum tumor marker assays. We have compared expression of MUC1, MUC3, MUC4, MUC11, MUC12 and MUC13 mRNA in epithelial cancers and/or cell lines with non-malignant tissues. In non-malignant tissues, MUC3, 4, 11, 12 and 13 were expressed at highest levels in gastrointestinal tissues, whereas MUC1 was more widely distributed. Significant down-regulation of the MUC4, MUC12 and MUC13 genes was observed in colonic cancers compared with normal tissue, whereas MUC1 was upregulated. In rectal cancers, levels of all six mucin genes were not significantly different to those in normal rectal tissues. Both MUC1 and MUC4 were down-regulated in gastric cancers, whereas cancer and normal tissue levels were similar for MUC3, 11, 12 and 13. In esophageal cancers there was a general trend toward higher levels than in normal tissue for MUC1, 3, 12 and 13. In ovarian cancers MUC1 levels were very high, whereas only low levels of all other mucins were observed. We also report expression in renal cell carcinomas, bladder carcinomas and breast cancer cell lines. The reported expression profiles of the cell surface mucin gene family will help direct biological and clinical studies of these molecules in mucosal biology, and in malignant and inflammatory diseases of epithelial tissues.

HMG box transcription factor gene Hbp1 is expressed in germ cells of the developing mouse testis

HMG box containing protein 1 (HBP1) is a high mobility group domain transcriptional repressor that regulates proliferation in differentiated tissues. We have found mouse Hbp1 to be expressed strongly in the embryonic mouse testis from approximately 12.5 days post coitum, compared with low levels of expression in the embryonic ovary. Expression of Hbp1 is maintained in the developing testis beyond the onset of spermatogenesis after birth. Whole-mount in situ hybridisation analysis showed that expression of Hbp1 in the XY gonad is localized within the developing testis cords, the precursors of the seminiferous tubules. Expression of Hbp1 is not apparent in testis cords of gonads from homozygous We mutant embryos, which lack germ cells. In situ hybridisation analysis on cryosectioned embryonic testis indicated that Hbp1 expression resembles that of the germ cell marker Oct4. We conclude that Hbp1 is up-regulated specifically in germ cells of the developing XY gonad. The expression of Hbp1 in XY germ cells appears to correlate with the onset of mitotic arrest in these cells. (C) 2004 Wiley-Liss, Inc.

Fine mapping of the tomato I-3 gene for fusarium wilt resistance and elimination of a co-segregating resistance gene analogue as a candidate for I-3

The I-3 gene from the wild tomato species Lycopersicon pennellii confers resistance to race 3 of the devastating vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici. As an initial step in a positional cloning strategy for the isolation of I-3, we converted restriction fragment length polymorphism and conserved orthologue set markers, known genes and a resistance gene analogue (RGA) mapping to the I-3 region into PCR-based sequence characterised amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers. Additional PCR-based markers in the I-3 region were generated using the randomly amplified DNA fingerprinting (RAF) technique. SCAR, CAPS and RAF markers were used for high-resolution mapping around the I-3 locus. The I-3 gene was localised to a 0.3-cM region containing a RAF marker, eO6, and an RGA, RGA332. RGA332 was cloned and found to correspond to a putative pseudogene with at least two loss-of-function mutations. The predicted pseudogene belongs to the Toll interleukin-1 receptor-nucleotide-binding site-leucine-rich-repeat sub-class of plant disease resistance genes. Despite the presence of two RGA332 homologues in L. esculentum, DNA gel blot and PCR analysis suggests that no other homologues are present in lines carrying I-3 that could be alternative candidates for the gene.

Using molecular markers to assess the effect of introgression on quantitative attributes of common bean in the Andean gene pool

Progress in bean breeding programs requires the exploitation of genetic variation that is present among races or through introgression across gene pools of Phaseolus vulgaris L. Of the two major common bean gene pools, the Andean gene pool seems to have a narrow genetic base, with about 10% of the accessions in the CIAT core collection presenting evidence of introgression. The objective of this study was to quantify the degree of spontaneous introgression in a sample of common bean landraces from the Andean gene pool. The effects of introgression on morphological, economic and nutritional attributes were also investigated. Homogeneity analysis was performed on molecular marker data from 426 Andean-type accessions from the primary centres of origin of the CIAT common bean core collection and two check varieties. Quantitative attribute diversity for 15 traits was studied based on the groups found from the cluster analysis of marker prevalence indices computed for each accession. The two-group summary consisted of one group of 58 accessions (14%) with low prevalence indices and another group of 370 accessions (86%) with high prevalence indices. The smaller group occupied the outlying area of points displayed from homogeneity analysis, yet their geographic origin was widely distributed over the Andean region. This group was regarded as introgressed, since its accessions displayed traits that are associated with the Middle American gene pool: high resistance to Andean disease isolates but low resistance to Middle American disease isolates, low seed weight and high scores for all nutrient elements. Genotypes generated by spontaneous introgression can be helpful for breeders to overcome the difficulties in transferring traits between gene pools.

Genetic and economic analysis of a targeted marker-assisted wheat breeding strategy

The advent of molecular markers as a tool to aid selection has provided plant breeders with the opportunity to rapidly deliver superior genetic solutions to problems in agricultural production systems. However, a major constraint to the implementation of marker-assisted selection (MAS) in pragmatic breeding programs in the past has been the perceived high relative cost of MAS compared to conventional phenotypic selection. In this paper, computer simulation was used to design a genetically effective and economically efficient marker-assisted breeding strategy aimed at a specific outcome. Under investigation was a strategy involving the integration of both restricted backcrossing and doubled haploid (DH) technology. The point at which molecular markers are applied in a selection strategy can be critical to the effectiveness and cost efficiency of that strategy. The application of molecular markers was considered at three phases in the strategy: allele enrichment in the BC1F1 population, gene selection at the haploid stage and the selection for recurrent parent background of DHs prior to field testing. Overall, incorporating MAS at all three stages was the most effective, in terms of delivering a high frequency of desired outcomes and at combining the selected favourable rust resistance, end use quality and grain yield alleles. However, when costs were included in the model the combination of MAS at the BC1F1 and haploid stage was identified as the optimal strategy. A detailed economic analysis showed that incorporation of marker selection at these two stages not only increased genetic gain over the phenotypic alternative but actually reduced the over all cost by 40%.

Olfactory marker protein modulates primary olfactory axon overshooting in the olfactory bulb

Olfactory marker protein (OMP) is expressed by mature primary olfactory sensory neurons during development and in adult mice. In mice that lack OMP, olfactory sensory neurons have perturbed electrophysiological activity, and the mice exhibit altered responses and behavior to odor stimulation. To date, defects in axon guidance in mice that lack OMP have not been investigated. During development of the olfactory system in mouse, primary olfactory axons often overshoot their target glomerular layer and project into the deeper external plexiform layer. These aberrant axonal projections are normally detected within the external plexiform layer up to postnatal day 12. We have examined the projections of primary olfactory axons in OMP-tau:LacZ mice and OMP-GFP mice, two independent lines in which the OMP coding region has been replaced by reporter molecules. We found that axons overshoot their target layer and grow into the external plexiform layer in these OMP null mice as they do in wild-type animals. However, in the absence of OMP, overshooting axons are more persistent and remain prominent until 5 weeks postnatally, after which their numbers decrease. Overshooting axons are still present in these mice even at 8 months of age. In heterozygous mice, axons also overshoot into the external plexiform layer; however, there are fewer axons, and they project for shorter distances, compared with those in a homozygous environment. Our results suggest that perturbed electrophysiological responses, caused by loss of OMP in primary olfactory neurons, reduce the ability of primary olfactory axons to recognize their glomerular target. © 2005 Wiley-Liss, Inc.

Alpha B-crystallin, a new independent marker for poor prognosis in head and neck cancer

Objectives. Gene expression profiling has provided many insights into tumor progression but translation to clinical practice has been limited. We have previously identified a list of potential markers by the differences of expression profiling of seven matched head and neck cancer (HNSCC) tumors with autologous normal oral mucosa (NOM). Alpha B-crystallin (CRYAB) was in the top 5% of genes identified with statistically significant differences in expression between tumor and NOM at the mRNA level. The objective was to confirm this in routine paraffin sections at the protein level. Study Design: The level of alpha B-crystallin was determined in tumors of 62 HNSCC patients whose prognosis was known for 5 years. Methods. Immunohistochemical detection of alpha B-crystallin expression was performed on HNSCC paraffin sections. Results. Univariate survival analysis identified lack of alpha B-crystallin staining as an independent prognostic marker for disease-free interval (P < 0.001) and overall survival (P < 0.002) of HNSCC patients over the 5-year observation period. Notably, all 13 patients (100%), including 5 patients with nodal disease whose tumors lacked alpha B-crystallin had no recurrences (P < 0.001). Nineteen of 27 node-negative patients stained positive for alpha B-crystallin and seven of the 19 (36.8%) had recurrences. Conclusion: Presence or absence of expression of alpha B-crystallin was a powerful marker for prognosis in this series of patients.

Replication of association of IL1 gene complex members with ankylosing spondylitis in Taiwanese Chinese

Objective: To test the association of interleukin 1 ( IL1) gene family members with ankylosing spondylitis ( AS), previously reported in Europid subjects, in an ethnically remote population. Methods: 200 Taiwanese Chinese AS patients and 200 ethnically matched healthy controls were genotyped for five single nucleotide polymorphisms ( SNPs) and the IL1RN. VNTR, markers previously associated with AS. Allele, genotype, and haplotype frequencies were compared between cases and controls. Results: Association of alleles and genotypes of the markers IL1F10.3, IL1RN. 4, and IL1RN. VNTR was observed with AS ( p < 0.05). Haplotypes of pairs of these markers and of the markers IL1RN.6/1 and IL1RN.6/2 were also significantly associated with AS. The strongest associations observed were with the marker IL1RN. 4, and with the two-marker haplotype IL1RN.4-IL1RN.VNTR ( both p = 0.004). Strong linkage disequilibrium was observed between all marker pairs except those involving IL1B-511 ( D' 0.4 to 0.9, p < 0.01). Conclusions: The IL1 gene cluster is associated with AS in Taiwanese Chinese. This finding provides strong statistical support that the previously observed association of this gene cluster with AS is a true positive finding.

Molecular classification of cutaneous malignant melanoma by gene expression profiling

The most common human cancers are malignant neoplasms of the skin(1,2). Incidence of cutaneous melanoma is rising especially steeply, with minimal progress in non-surgical treatment of advanced disease(3,4). Despite significant effort to identify independent predictors of melanoma outcome, no accepted histopathological, molecular or immunohistochemical marker defines subsets of this neoplasm(2,3). Accordingly, though melanoma is thought to present with different 'taxonomic' forms, these are considered part of a continuous spectrum rather than discrete entities(2). Here we report the discovery of a subset of melanomas identified by mathematical analysis of gene expression in a series of samples. Remarkably, many genes underlying the classification of this subset are differentially regulated in invasive melanomas that form primitive tubular networks in vitro, a feature of some highly aggressive metastatic melanomas(5). Global transcript analysis can identify unrecognized subtypes of cutaneous melanoma and predict experimentally verifiable phenotypic characteristics that may be of importance to disease progression.