Gene to Phenotype Integrative Science and Modelling Applied to Pioneer elite

This project aims to use simulatiion modelling to improve our understanding of the genetics and physiology of complex traits with a view to increasing the rate of genetic gain in plant breeding programs.

Trait physiology and crop modelling as a framework to link phenotypic complexity to underlying genetic systems

New tools derived from advances in molecular biology have not been widely adopted in plant breeding for complex traits because of the inability to connect information at gene level to the phenotype in a manner that is useful for selection. In this study, we explored whether physiological dissection and integrative modelling of complex traits could link phenotype complexity to underlying genetic systems in a way that enhanced the power of molecular breeding strategies. A crop and breeding system simulation study on sorghum, which involved variation in 4 key adaptive traits-phenology, osmotic adjustment, transpiration efficiency, stay-green-and a broad range of production environments in north-eastern Australia, was used. The full matrix of simulated phenotypes, which consisted of 547 location-season combinations and 4235 genotypic expression states, was analysed for genetic and environmental effects. The analysis was conducted in stages assuming gradually increased understanding of gene-to-phenotype relationships, which would arise from physiological dissection and modelling. It was found that environmental characterisation and physiological knowledge helped to explain and unravel gene and environment context dependencies in the data. Based on the analyses of gene effects, a range of marker-assisted selection breeding strategies was simulated. It was shown that the inclusion of knowledge resulting from trait physiology and modelling generated an enhanced rate of yield advance over cycles of selection. This occurred because the knowledge associated with component trait physiology and extrapolation to the target population of environments by modelling removed confounding effects associated with environment and gene context dependencies for the markers used. Developing and implementing this gene-to-phenotype capability in crop improvement requires enhanced attention to phenotyping, ecophysiological modelling, and validation studies to test the stability of candidate genetic regions.

Trait physiology and crop modelling to link phenotypic complexity to underlying genetic systems

New tools derived from advances in molecular biology have not been widely adopted in plant breeding because of the inability to connect information at gene level to the phenotype in a manner that is useful for selection. We explore whether a crop growth and development modelling framework can link phenotype complexity to underlying genetic systems in a way that strengthens molecular breeding strategies. We use gene-to-phenotype simulation studies on sorghum to consider the value to marker-assisted selection of intrinsically stable QTLs that might be generated by physiological dissection of complex traits. The consequences on grain yield of genetic variation in four key adaptive traits – phenology, osmotic adjustment, transpiration efficiency, and staygreen – were simulated for a diverse set of environments by placing the known extent of genetic variation in the context of the physiological determinants framework of a crop growth and development model. It was assumed that the three to five genes associated with each trait, had two alleles per locus acting in an additive manner. The effects on average simulated yield, generated by differing combinations of positive alleles for the traits incorporated, varied with environment type. The full matrix of simulated phenotypes, which consisted of 547 location-season combinations and 4235 genotypic expression states, was analysed for genetic and environmental effects. The analysis was conducted in stages with gradually increased understanding of gene-to-phenotype relationships, which would arise from physiological dissection and modelling. It was found that environmental characterisation and physiological knowledge helped to explain and unravel gene and environment context dependencies. We simulated a marker-assisted selection (MAS) breeding strategy based on the analyses of gene effects. When marker scores were allocated based on the contribution of gene effects to yield in a single environment, there was a wide divergence in rate of yield gain over all environments with breeding cycle depending on the environment chosen for the QTL analysis. It was suggested that knowledge resulting from trait physiology and modelling would overcome this dependency by identifying stable QTLs. The improved predictive power would increase the utility of the QTLs in MAS. Developing and implementing this gene-to-phenotype capability in crop improvement requires enhanced attention to phenotyping, ecophysiological modelling, and validation studies to test the stability of candidate QTLs.

Epigenetic inactivation of chalcone synthase-A transgene transcription in petunia leads to a reversion of the post-transcriptional gene silencing phenotype

Petunia plants that exhibit a white-flowering phenotype as a consequence of chalcone synthase transgene-induced silencing occasionally give rise to revertant branches that produce flowers with wild-type pigmentation. Transcription run-on assays confirmed that the production of white flowers is caused by post-transcriptional gene silencing (PTGS), and indicated that transgene transcription is repressed in the revertant plants, providing evidence that induction of PTGS depends on the transcription rate. Transcriptional repression of the transgene was associated with cytosine methylation at CpG, CpNpG and CpNpN sites, and the expression was restored by treatment with either 5-azacytidine or trichostatin A. These results demonstrate that epigenetic changes occurred in the PTGS line, and these changes interfere with the initiation of transgene transcription, leading to a reversion of the PTGS phenotype.

An antisense approach to phenotype-based gene cloning in Tetrahymena

We report a pioneering approach using Tetrahymena thermophila that permits rapid identification of genes based on their null or hypomorphic phenotypes. This technique involves cell transformation with a library of plasmids that encode 26S ribosomal subunits containing short insertions. The insertions correspond to antisense sequences for a large number of genes. The majority of cells each acquires a single antisense sequence, which silences a single genomic locus. Because the insertion site within the ribosomal sequence is known, the silenced gene is easily amplified. We demonstrate that this approach can be used to identify genes required for dense core granule exocytosis.

Plasmid DNA mediated transfer of the herpes simplex virus thymidine kinase gene to a new bromodeoxyuridine resistant variant of human primary lung carcinoma cells

A human primary lung carcinoma cell line (HPL-R1) established from the tumor biopsy of a lung cancer patient, lacking in cytochrome P1-450 [aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH)], was cloned and used to obtain variants deficient in the expression of thymidine-kinase via treatment with 5-bromo-2'-deoxyuridine, and selection for drug resistance phenotype. The variant cell line, precharacterized for thymidine kinase negative phenotype, was transfected with the thymidine kinase gene bearing p R-tk and px1-tk plasmids. Transfections from both the plasmids, demonstrated a frequency of 5.5 X 10(-5). The transfectants showed a 76-100% retention of the transferred phenotype. These data suggest that transfection in variant human cells can approach significant levels of stability observed with rodent cell recipients.

Inducible caspase 9 suicide gene to improve the safety of allodepleted T cells after haploidentical stem cell transplantation.

Addback of donor T cells following T cell-depleted stem cell transplantation (SCT) can accelerate immune reconstitution and be effective against relapsed malignancy. After haploidentical SCT, a high risk of graft-versus-host disease (GVHD) essentially precludes this option, unless the T cells are first depleted of alloreactive precursor cells. Even then, the risks of severe GVHD remain significant. To increase the safety of the approach and thereby permit administration of larger T cell doses, we used a suicide gene, inducible caspase 9 (iCasp9), to transduce allodepleted T cells, permitting their destruction should administration have adverse effects. We made a retroviral vector encoding iCasp9 and a selectable marker (truncated CD19). Even after allodepletion (using anti-CD25 immunotoxin), donor T cells could be efficiently transduced, expanded, and subsequently enriched by CD19 immunomagnetic selection to >90% purity. These engineered cells retained antiviral specificity and functionality, and contained a subset with regulatory phenotype and function. Activating iCasp9 with a small-molecule dimerizer rapidly produced >90% apoptosis. Although transgene expression was downregulated in quiescent T cells, iCasp9 remained an efficient suicide gene, as expression was rapidly upregulated in activated (alloreactive) T cells. We have demonstrated the clinical feasibility of this approach after haploidentical transplantation by scaling up production using clinical grade materials.

From gene to protein: Investigating the disease etiology of retinitis pigmentosa

Retinitis pigmentosa (RP) is a name given to a group of inherited retinal dystrophies that lead to progressive photoreceptor degeneration, and thus, visual impairment. It is evident at both the clinical and the molecular level that these are heterogeneous disorders, with wide variation in severity, mode of inheritance, and phenotype. The genetics of RP are not simple; the disease can be inherited in dominant, recessive, X-linked, and digenic modes. Autosomal dominant RP (adRP) results from mutations in at least ten mapped loci, but there may be dozens of genetic loci where mutations can cause RP. To date, there are over a hundred genes known to cause retinal degenerative diseases, and less than half of these have been cloned (RetNet). Among the dozens of retinitis pigmentosa loci known to exist, only a few have been identified and the remainders are inferred from linkage studies. Today, the genes for seven of the twelve-adRP loci have been identified, and these are rhodopsin, peripherin/RDS, NRL, ROM1, CRX, RP13 and RP1. My research projects involved a combination of the continued search for genes involved in retinal dystrophies, as well the investigation into the role of peripherin/RDS and RP1 in the disease etiology of autosomal dominant RP. ^ Most of the mutations leading to inherited retinal disorders have been identified in predominately retina expressed genes like rhodopsin, peripherin/RDS, and RP1. Expressed sequence tags (ESTs) that were retina-specific were culled from sequence databases and, together with laboratory analysis, were analyzed as potential candidate genes for retinal dystrophies. Thirteen of the fifty-five identified retina-specific ESTs mapped to within candidate regions for inherited retinopathies. One of these is RP1L1, a homologue of RP1 and a potential cause of adRP. ^ Once a disease-associated gene has been identified, elucidating the role of that gene in the visual process is essential for understanding what happens when the process is defective as it is in adRP. My next projects involved investigating the role of a novel 5′ donor +3 splice site mutation on the mRNA of peripherin/RDS in adRP affected individuals, and comparative sequencing in RP1 to define conserved regions of the protein. Comparative sequencing is a powerful way to delineate critical regions of a sequence because different regions of a gene have different functions, and each region is subject to different levels of functional or structural constraints. Establishing a framework of conserved domains is beneficial not only for structural or functional studies, but can also aid in determining the potential effects of mutations. With the completion of sequencing of human genome, and other organisms such as Saccharomyces cerevisiae, Caenorhabditis elegans , and Drosophila, the facility of comparative sequencing will only increase in the future. Comparative sequencing has already become an established procedure for pinpointing conserved regions of a protein, and is an efficient way to target regions of a protein for experimental and/or evolutionary analysis. ^

Familial typical migraine: Significant linkage and localization of a gene to Xq24-28

In a previous study we found evidence for an X-linked genetic component for familial typical migraine in two large Australian white pedigrees, designated MF7 and MF14. Significant excess allele sharing was indicated by nonparametric linkage (NPL) analysis using GENEHUNTER (P=0.031 and P=0.012, respectively), with a combined analysis of the two pedigrees showing further increased evidence for linkage, producing a maximum NPL score of 2.87 (P=0.011 ) at DXS 1123 on Xq27. The present study was aimed at refining the localization of the migraine X-chromosomal component by typing additional markers, performing haplotype analysis and applying a more powerful technique in the analysis of linkage data from these two pedigrees. Results from the haplotype analyses, coupled with linkage analyses that produced a peak GENEHUNTER-PLUS LOD* score of 2.388 (P=0.0005), provide compelling evidence for the presence of a migraine susceptibility locus on chromosome Xq24-28.

Regional chromosomal assignment of human renin gene to 1q12→qter and use in linkage studies in Charcot-Marie-Tooth disease

The gene for renin, previously mapped to human chromosome 1, was further localized to 1q12 → qter using human-mouse somatic cell hybrid DNAs. The renin DNA probe used (λ HR5) could detect a HindIII restriction fragment length polymorphism. When used in studies of 12 informative families, no linkage could be found between the renin and Charcot-Marie-Tooth disease. Furthermore, an association of any renin allele with hypertension was not apparent.

PTSD and traumatic stress : from gene to community and bench to bedside

Individuals and communities are exposed to traumatic events, those that are accidents or naturally occurring and those that are intentional or human made. Although resilience is the expected response, for some, posttraumatic stress disorder may be the outcome. Brain models of PTSD require understanding the phenomenology of the disorder and the brain “break down” that occurs. Among several models, importantly, is the perspective that PTSD is a “forgetting” disorder. Other elements in the onset and triggers of PTSD can identify further models to examine at the bench. New studies of the 5-HT2A receptor, the glucocorticoid receptor, p11, mitochondrial genes and cannabinoids are bringing new perspectives to understanding brain function in PTSD. Effective treatments indicate areas for bench research on the mechanisms of the disorder.