Gene finding in eukaryotic genomes is an essential part of a comprehensive approach to modern systems biology. Most methods developed in the past rely on a combination of computational prediction and external information about gene structures from transcript sequences and comparative genomics. In the past, external sequence information consisted of a combination of full-length cDNA and expressed sequence tag (EST) sequences. Much improvement in prediction of genes and gene isoforms is promised by availability of RNA-seq data. However, productive use of RNA-seq for gene prediction has been difficult due to challenges associated with mapping RNA-seq reads which span splice junctions to prevalent splicing noise in the cell. This work addresses this difficulty with the development of methods and implementation of two new pipelines: 1/ a novel pipeline for accurate mapping of RNA-seq reads to compact genomes and 2/ a pipeline for prediction of genes using the RNA-seq spliced alignments in eukaryotic genomes. Machine learning methods are employed in order to overcome errors associated with the process of mapping short RNA-seq reads across introns and using them for determining sequence model parameters for gene prediction. In addition to the development of these new methods, genome annotation work was performed on several plant genome projects.

Mutations in the clk-1, clk-2, clk-3 and gro-1 genes in Caenorhabditis elegans show alterations in developmental and behavioral timing and lifespan, collectively termed the Clk phenotype. While the clk-1, clk-2, and gro-1 genes have been cloned, clk-3 gene has not been identified. Gene expression changes in clk-3 mutant worms were determined using microarray expression data. I examined genes in the region to which clk-3 gene maps, for strongly reduced expression in the clk-3 mutants and identified thirteen clk-3 candidate genes. RNAi feeding vectors for all these candidate genes were picked and cultured from the RNAi library. Knock-down worm strains were generated by feeding RNAi and analyzed for Clk phenotypes. Of all the candidate genes tested, the Y48E1B.5 gene showed the most similar phenotypic profile to the clk-3 mutants. The Y48E1B.5 gene shows weak homology to a mammalian mitochondrial ribosomal protein. Primers were designed to amplify all 9 exons of the Y48E1B.5 gene. Sequence analysis was carried out on the resulting PCR products from clk-3 mutants. An amino acid change was found in exon 4.

<p>With easy access to technology to generate expressed sequence tags (ESTs), several groups have sequenced from thousands to several thousands of ESTs. These ESTs benefit from consolidation and organization to deliver significant biological value. A number of EST projects are underway to extract maximum value from fragmented EST resources by constructing gene indices, where all transcripts are partitioned into index classes such that transcripts are put into the same index class if they represent the same gene. Therefore a gene index should ideally represent a non-redundant set of transcripts. Indeed, most gene indices aim to reconstruct the gene complement of a genome and their technological developments are directed at achieving this goal. The South African National Bioinformatics Institute (SANBI), on the other hand, embarked on the development of the sequence alignment and consensus knowledgebase (STACK) database that focused on the detection and visualisation of transcript variation in the context of developmental and pathological states, using all publicly available ESTs. Preliminary work on the STACK project employed an approach of partitioning the EST data into arbitrarily chosen tissue categories as a means of reducing the EST sequences to manageable sizes for subsequent processing. The tissue partitioning provided the template material for developing error-checking tools to analyse the information embedded in the error-laden EST sequences. However, tissue partitioning increases redundancy in the sequence data because one gene can be expressed in multiple tissues, with the result that multiple tissue partitioned transcripts will correspond to the same gene.</p> <p><br /> Therefore, the sequence data represented by each tissue category had to be merged in order to obtain a comprehensive view of expressed transcript variation across all available tissues. The need to consolidate all EST information provided the impetus for developing a STACK human gene index, also referred to as a whole-body index. In this dissertation, I report on the development of a STACK human gene index represented by consensus transcripts where all constituent ESTs sample single or multiple tissues in order to provide the correct development and pathological context for investigating sequence variation. Furthermore, the availability of a human gene index is assessed as a diseasecandidate gene discovery resource. A feasible approach to construction of a whole-body index required the ability to process error-prone EST data in excess of one million sequences (1,198,607 ESTs as of December 1998). In the absence of new clustering algorithms, at that time, we successfully ported D2_CLUSTER, an EST clustering algorithm, to the high performance shared multiprocessor machine, Origin2000. Improvements to the parallelised version of D2_CLUSTER included: (i) ability to cluster sequences on as many as 126 processors. For example, 462000 ESTs were clustered in 31 hours on 126 R10000 MHz processors, Origin2000. (ii) enhanced memory management that allowed for clustering of mRNA sequences as long as 83000 base pairs. (iii) ability to have the input sequence data accessible to all processors, allowing rapid access to the sequences. (iv) a restart module that allowed a job to be restarted if it was interrupted. The successful enhancements to the parallelised version of D2_CLUSTER, as listed above, allowed for the processing of EST datasets in excess of 1 million sequences. An hierarchical approach was adopted where 1,198,607 million ESTs from GenBank release 110 (October 1998) were partitioned into &quot / tissue bins&quot / and each tissue bin was processed through a pipeline that included masking for contaminants, clustering, assembly, assembly analysis and consensus generation. A total of 478,707 consensus transcripts were generated for all the tissue categories and these sequences served as the input data for the generation of the wholebody index sequences. The clustering of all tissue-derived consensus transcripts was followed by the collapse of each consensus sequence to its individual ESTs prior to assembly and whole-body index consensus sequence generation. The hierarchical approach demonstrated a consolidation of the input EST data from 1,198607 ESTs to 69,158 multi-sequence clusters and 162,439 singletons (or individual ESTs). Chromosomal locations were added to 25,793 whole-body index sequences through assignment of genetic markers such as radiation hybrid markers and g&eacute / n&eacute / thon markers. The whole-body index sequences were made available to the research community through a sequence-based search engine (http://ziggy.sanbi.ac.za/~alan/researchINDEX.html).</p>

The use of molecular markers to identify quantitative trait loci ( QTL ) affecting economically important traits has become a key approach in animal genetics, both for understanding the genetic basis of these traits and to help design novel breeding programs. The general goal of the present work was to map QTL for economically important traits in beef cattle. Because of the practical limitations of phenotypic selection for meat quality, these traits are ideal candidates for the use of marker - assisted selection. Thus, the thesis specifically focused on carcass and beef quality traits. Six half - sib families were generated by mating six Limousin x Jersey crossbred sires to purebred Jersey or Limousin cows, producing 784 backcross progeny ( 366 and 418 progeny in Australia and New Zealand, respectively ). The six crossbred sires and all the backcross progeny were genotyped for 285 microsatellite markers ( on average 189 informative loci per sire family ) spread across the 29 bovine autosomes. A large number of traits were recorded on backcross progeny. In the first phase of the research, a single - QTL model based on regression interval mapping was used to map QTL for a wide range of economically important traits in the beef industry. Chromosome - wise significant evidence for linkage was found on BTA12 ( P < 0.01 ) and BTA16 ( P < 0.05 ) for age at puberty. Thirteen QTL were found to affect calving ease related traits ( birth weight, pelvic area and gestation length ). BTA11, 14 and 22 were most significant linkage groups affecting calving ease traits. Several genomic regions were linked to the carcass and beef quality traits. The results revealed a major QTL on BTA2 close to the map position of myostatin gene, affecting yield, carcass fatness and beef quality traits. In the second phase, the pleiotropic effects of a myostatin functional SNP on beef traits were studied. There was no association between this myostatin variant and birth weight and growth traits. However, the variant decreased overall fatness, increased muscle mass and improved meat tenderness, thus providing an intermediate and more useful phenotype than the more severe double - muscling phenotype caused by a major deletion in the myostatin gene described by others. In the third phase, a multiple marker analysis approach in the framework of the mixed - effects model was developed, allowing all markers of the entire genome to be included in the analysis simultaneously. Further, exploiting a factor analytic covariance structure for modeling trait by marker or family by marker interaction terms, the approach was extended to the multi - trait and multiple family situations. The simulation study showed that modeling multiple phenotypes and multiple families in a single linkage analysis simultaneously can markedly increase the power to detect QTL, compared to modeling each phenotype or family separately. Finally, the multi - trait multiple QTL approach developed herein was applied to map QTL influencing carcass and meat quality traits. Several pleoitropic QTL and also traitspecific QTL affecting beef traits were mapped, resulting in a useful resource from which fine mapping can be launched for subsequent gene discovery and marker - assisted selection. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?DB=local&Search_Arg=koshkoih+thesis+adelaide&Search_Code=GKEY%5E*&SL=None&CNT=50 / Thesis (Ph.D.)--University of Adelaide, School of Agriculture, Food and Wine, 2007.

Variations in gene expression have long been hypothesised to be the major cause of individual differences. An initial focus of this research thesis is to elucidate the genetic regulatory architecture of gene expression. Expression quantitative trait locus (eQTL) mapping analyses have been performed on expression levels of over 22,000 mRNAs from three tissues of a panel of recombinant inbred mice. These analyses are "single-locus" where "linkage" (i.e. significant correlation) between an expression trait and a putative eQTL is considered independently of other loci. Major conclusions from these analyses are: 1. Gene expression is mainly influenced by genetic (sequence) variations that act in trans rather than in cis; 2. Subsets of genes are controlled by master regulators that influence multiple genes; 3. Gene expression is a polygenic trait with multiple regulators. Single-locus mapping analyses are not designed for detecting multiple regulators of gene expression, and so observation of multiple-linkages (i.e. one expression trait mapped to multiple eQTLs) formed the basis of the second objective of this research project: to investigate the relationship between multiple-linkages and genotype pattern-association. A locus-pair is said to have associated genotype patterns if they have similar inheritance pattern across a panel of individuals, and these are attributed to one of fours sources: 1. linkage disequilibrium between loci located on the same chromosome; 2. non-syntenic association; 3. random association; 4. un-associated. To understand the validity of multiple-linkages observed in single-locus mapping studies, a newly developed method, bqtl.twolocus, is applied to confirm two-locus effects for a total of 898 out of 1,233 multiple-linkages identified from the three studies mentioned above as well as from seven publicly available eQTL-mapping studies. Combining these results with information of genotype pattern-association, a subset of 478 multiple-linkages has been deduced for which there is high confidence to be real.

Copies of author's previously published works inserted. / Bibliography: leaves 321-370. / xv, 370, [21] leaves : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of the project presented in this thesis is to isolate microsatellite markers and to construct a high resolution genetic map of the human X chromosome using these and pre-existing microsatellite markers. AC dinucleotide repeat markers are isolated from a bacteriophage library for application to the genetic localisations of X-linked disease genes, particularly those responsible for non-specific mental retardation (MRX). The genetic map is used to refine the location of the disease gene segregating in five families affected with X-linked mental retardation. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1997

The use of molecular markers to identify quantitative trait loci ( QTL ) affecting economically important traits has become a key approach in animal genetics, both for understanding the genetic basis of these traits and to help design novel breeding programs. The general goal of the present work was to map QTL for economically important traits in beef cattle. Because of the practical limitations of phenotypic selection for meat quality, these traits are ideal candidates for the use of marker - assisted selection. Thus, the thesis specifically focused on carcass and beef quality traits. Six half - sib families were generated by mating six Limousin x Jersey crossbred sires to purebred Jersey or Limousin cows, producing 784 backcross progeny ( 366 and 418 progeny in Australia and New Zealand, respectively ). The six crossbred sires and all the backcross progeny were genotyped for 285 microsatellite markers ( on average 189 informative loci per sire family ) spread across the 29 bovine autosomes. A large number of traits were recorded on backcross progeny. In the first phase of the research, a single - QTL model based on regression interval mapping was used to map QTL for a wide range of economically important traits in the beef industry. Chromosome - wise significant evidence for linkage was found on BTA12 ( P < 0.01 ) and BTA16 ( P < 0.05 ) for age at puberty. Thirteen QTL were found to affect calving ease related traits ( birth weight, pelvic area and gestation length ). BTA11, 14 and 22 were most significant linkage groups affecting calving ease traits. Several genomic regions were linked to the carcass and beef quality traits. The results revealed a major QTL on BTA2 close to the map position of myostatin gene, affecting yield, carcass fatness and beef quality traits. In the second phase, the pleiotropic effects of a myostatin functional SNP on beef traits were studied. There was no association between this myostatin variant and birth weight and growth traits. However, the variant decreased overall fatness, increased muscle mass and improved meat tenderness, thus providing an intermediate and more useful phenotype than the more severe double - muscling phenotype caused by a major deletion in the myostatin gene described by others. In the third phase, a multiple marker analysis approach in the framework of the mixed - effects model was developed, allowing all markers of the entire genome to be included in the analysis simultaneously. Further, exploiting a factor analytic covariance structure for modeling trait by marker or family by marker interaction terms, the approach was extended to the multi - trait and multiple family situations. The simulation study showed that modeling multiple phenotypes and multiple families in a single linkage analysis simultaneously can markedly increase the power to detect QTL, compared to modeling each phenotype or family separately. Finally, the multi - trait multiple QTL approach developed herein was applied to map QTL influencing carcass and meat quality traits. Several pleoitropic QTL and also traitspecific QTL affecting beef traits were mapped, resulting in a useful resource from which fine mapping can be launched for subsequent gene discovery and marker - assisted selection. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?DB=local&Search_Arg=koshkoih+thesis+adelaide&Search_Code=GKEY%5E*&SL=None&CNT=50 / Thesis (Ph.D.)--University of Adelaide, School of Agriculture, Food and Wine, 2007.

The initial focus of this PhD project was on comparative gene mapping. Comparative gene mapping is facilitated by consensus PCR primers which amplify homologous gene fragments in many species. As a part of an international co-ordinated programme of comparative mapping in pigs, 47 CATS (Comparative Anchor Tagged Sequence) consensus primer pairs for loci located on human chromosomes 9, 10, 20, and 22, were used for amplifying homologous loci in pigs. After optimization of PCR conditions, 23 CATS products have confirmed by comparison with homologous sequences in GenBank. A French somatic cell hybrid panel was used to physically map the 6 porcine CATS products distinguishable from rodent background product, namely ADRA1A, ADRA2A, ARSA, GNAS1, OXT and TOP1. Of these, the map location of ADRA1A and OXT showed inconsistency with the previously recognised conserved relationship between human and pig. The other four loci mapped to positions consistent with known syntenic relationships. Despite low levels of polymorphism, frequently indistinguishable rodent and porcine products in somatic hybrids and some confusion of identity of gene family members, these CATS primers have made a useful contribution to the porcine-human comparative map. The focus of the project then changed to genetic and molecular characterisation of endogenous retroviruses in pigs and their relatives. Pigs are regarded as a potentially good source of organs and tissues for transplantation into humans. However, porcine endogenous retroviruses have emerged as a possible problem as they can infect cultured human cells. Two main types of pig retrovirus, determined by envelope protein, PERV-A and PERV-B, are widely distributed in different pig breeds and a third less common type, PERV-C, has also been recognised. Endogenous retroviruses were analyzed from the Westran (Westmead transplantation) inbred line of pig, specially bred for biomedical research. Thirty-one 1.8 kb env PCR product clones were sequenced after preliminary screening with the restriction enzymes KpnI and MboI. Five recombinant clones between A and B were identified. 55% of clones (17/31) sequenced had stop codons within the envelope protein-encoding region, which would prevent the retrovirus from making full-length envelope protein recognizable by cell-surface receptors of the virus. The endogenous viruses were physically mapped in Westran pigs by FISH (Fluorescence In Situ Hybridisation) using PERV-A and PERV-B envelope clones as probes. Preliminary FISH data suggest that there are at least 22 PERVs (13 PERV-A and 9 PERV-B) and the chromosomal locations of these in the Westran strain are quite different from European Large White pigs. The sequences and mapping results of inbred Westran pig suggest that there are relatively few PERV integration sites compared with commercial pigs and further that a large proportion of clones are defective due to premature stop codons in the envelope gene. To investigate the relationship of endogenous retroviruses in peccaries and pigs, a set of degenerate primers was used to amplify peccary retroviral sequences. The sequences of two putative retroviral clones showed close homology, albeit with a 534 bp deletion, to mouse and pig retroviral sequences. Also, four non-target sequences were amplified from peccary with the degenerate retroviral primers. They are a part of the peccary cofilin gene, a SINE, and a sequence containing a microsatellite. The peccary endogenous retroviral sequences are significant in that they are the first such sequences reported in peccary species and repudiate old claims in the literature that peccaries do not have C-type retroviral sequences.

"November 2001". / Thesis (PhD)--Macquarie University, Division of Environmental and Life Sciences, Department of Biological Sciences, 2002. / Bibliography: leaves 136-157. / General introduction -- Molecular markers for comparative and quantitative studies in macropods -- Genetic linkage map construction in the tammar wallaby (M. eugenii) -- Intraspecific variation, sex-biased dispersal and phylogeography of the eastern grey kangaroo (M. giganteus) -- General discussion. / The analysis of DNA using molecular techniques is an important tool for studies of evolutionary relationships, population genetics and genome organisation. The use of molecular markers within marsupials is primarily limited by their availability and success of amplification. Within this study, 77 macropodid type II microsatellite loci and two type I genetic markers were characterised within M. eugenii to evaluate polymorphic levels and cross-species amplification artifacts. Results indicated that 65 microsatellite loci amplified a single locus in M. eugenii with 44 exhibiting high levels of variability. The success of crossspecies amplification of microsatellite loci was inversely proportional to the evolutionary distance between the macropod species. It is revealed that the majority of species within the Macropodidae are capable of using many of the available heterologous microsatellites. When comparing the degree of variability between source-species and M. eugenii, most were significantly higher within source species (P < 0.05). These differences were most likely caused by ascertainment bias in microsatellite selection for both length and purity. -- The production of a marsupial genetic linkage map is perhaps one of the most important objectives in marsupial research. This study used a total of 353 informative meioses and 64 genetic markers to construct a framework genetic linkage map for M. eugenii. Nearly all markers (93.7%) formed a significant linkage (LOD > 3.0) with at least one other marker. More than 70% (828 cM) of the genome had been mapped when compared with chiasmata data. Nine linkage groups were identified, with all but one (LG7; X-linked) allocated to the autosomes. Theses groups ranged in size from 15.7 cM to 176.5 cM, and have an average distance of 16.2 cM between adjacent markers. Of the autosomal linkage groups, LG2 and LG3 were assigned to chromosome 1 and LG4 localised to chromosome 3 based on physical localisation of genes. Significant sex-specific distortions towards reduced female recombination rates were revealed in 22% of comparisons. Positive interference was observed within all the linkage groups analysed. When comparing the X-chromosome data to closely related species it is apparent that it is conserved both in synteny and gene order. -- The investigation of population dynamics of eastern grey kangaroos has been limited to a few ecological studies. The present investigation provides analysis of mtDNA and microsatellite data to infer both historical and contemporary patterns of population structuring and dispersal. The average level of genetic variation across sample locations was exceedingly high (h = 0.95, HE = 0.82), and is one of the highest observed for marsupials. Contrary to ecological studies, both genic and genotypic analyses reveal weak genetic structure of populations where high levels of dispersal may be inferred up to 230 km. The movement of individuals was predominantly male-biased (average N,m = 22.61, average N p = 2.73). However, neither sex showed significant isolation by distance. On a continental scale, there was strong genetic differentiation and phylogeographic distinction between southern (TAS, VIC and NSW) and northern (QLD) Australian populations, indicating a current and / or historical restriction of geneflow. In addition, it is evident that northern populations are historically more recent, and were derived from a small number of southern eastern grey kangaroo founders. Phylogenetic comparisons between M. g. giganteus and M. g. tasmaniensis, indicated that the current taxonomic status of these subspecies should be revised as there was a lack of genetic differentiation between the populations sampled. / Mode of access: World Wide Web. / xv, 182 leaves ill

Thesis (Ph. D.)--University of Adelaide, Dept. of Paediatrics, 1994. / Errata slip inserted at back. Includes bibliographical references (leaves 159-192).