Comparative mapping for positional cloning and defining homology regions between the mouse and human genomes

The goal of this dissertation project was to utilize genetic and physical mapping methods as a means to define genomic homology between human and mouse genomes, as well as to use this information to define functional relationships between the two species. The Comparative mapping studies were designed to expand upon the current knowledge of comparative mapping and homology regions between mouse and man, and to begin to study the homology region borders. Positional cloning research was initiated to localize the translocation breakpoints in a mutant mouse associated with a neurological defect as a first step toward isolation of genes that could be involved in the phenotype of this animal.Comparative mapping of human chromosome 19 and related regions of the mouse genome represents one major focus of this research. Human chromosome 19 was a good target for comparative studies due to the extensive physical mapping of the chromosome,and availability of conserved, mapped gene markers to use for these studies. Comparative Studies involved both the 19q- and 19p-arms of the chromosome, and helped lay the foundation of a chromosome-wide comparative map. One region, 19pl3.1, was investigated in detail. This region is shown to be prone to rearrangements during evolution, as indicated by the homology groups associated within both the mouse and human genomes. These studies revealed the need for more fine mapping of the genome for both species, demonstrating that examination of specific homology groups at a higher resolution reveals that both similarities and surprising differences between related mouse and human regions. These studies provided data suggesting that repeated sequences are associated with at least some homology region borders, a concept that may serve as a guide for future comparative mapping between human, mouse, and other species.One application of comparative mapping is its ability to link functional information derived from mouse mutations to specific genes and disease within the human genome. As part of this effort, research focused upon localizing and physical mapping a region surrounding the breakpoint in IGso, a mouse mutation associated with a reciprocal translocation. Homozygosity for the translocation causes developmental lethality. Animals That are heterozygous display neurological defects including the inability to swim and abnormal startle responses. A genetic map of the region surrounding one of the translocation breakpoints was established on mouse chromosome 2. Fluorescence in situ hybridization (FISH) techniques were used to localize translocation breakpoints to a small interval less than IcM in length. This region shows remarkable linkage conservation with a well-characterized region of human chromosome lip 13, which harbors genes responsible for the WAGR syndrome (Wilms tumor, aniridia, genitourinary malformations, and mental retardation). Comparative mapping information was used to narrow down the translocation interval, and establish a contig consisting of yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and Pl-derived artificial chromosomes (PACs), which covered approximately 600 kb throughout the breakpoint region. This allowed the breakpoint to be isolated within a single PAC clone of 150 kb in length.Together these studies have set the stage for future investigations of genes located on chromosome 19, and for the cloning of gene(s) associated with the IGso phenotype in mice

Molecular and Cell Biology of Infantile (CLN1) and variant Late Infantile (CLN5) Neuronal Ceroid Lipofuscinoses

Myös verkossa; väitöskirja, ohj. Leena Peltonen-Paloti

Molecular and cell biology of infantile (CLN1) and variant late infantile (CLN5) neuronal ceroid lipofuscinoses

Myös verkossa; väitöskirja, ohj. Leena Peltonen-Paloti

A Single Molecule Scaffold for the Maize Genome

About 85% of the maize genome consists of highly repetitive sequences that are interspersed by low-copy, gene-coding sequences. The maize community has dealt with this genomic complexity by the construction of an integrated genetic and physical map (iMap), but this resource alone was not sufficient for ensuring the quality of the current sequence build. For this purpose, we constructed a genome-wide, high-resolution optical map of the maize inbred line B73 genome containing >91,000 restriction sites (averaging 1 site/∼23 kb) accrued from mapping genomic DNA molecules. Our optical map comprises 66 contigs, averaging 31.88 Mb in size and spanning 91.5% (2,103.93 Mb/∼2,300 Mb) of the maize genome. A new algorithm was created that considered both optical map and unfinished BAC sequence data for placing 60/66 (2,032.42 Mb) optical map contigs onto the maize iMap. The alignment of optical maps against numerous data sources yielded comprehensive results that proved revealing and productive. For example, gaps were uncovered and characterized within the iMap, the FPC (fingerprinted contigs) map, and the chromosome-wide pseudomolecules. Such alignments also suggested amended placements of FPC contigs on the maize genetic map and proactively guided the assembly of chromosome-wide pseudomolecules, especially within complex genomic regions. Lastly, we think that the full integration of B73 optical maps with the maize iMap would greatly facilitate maize sequence finishing efforts that would make it a valuable reference for comparative studies among cereals, or other maize inbred lines and cultivars

Assembly and Compositional Analysis of Human Genomic DNA - Doctoral Dissertation, August 2002

In 1990, the United States Human Genome Project was initiated as a fifteen-year endeavor to sequence the approximately three billion bases making up the human genome (Vaughan, 1996).As of December 31, 2001, the public sequencing efforts have sequenced a total of 2.01 billion finished bases representing 63.0% of the human genome (http://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsProgress.shtml&&ORG=Hs) to a Bermuda quality error rate of 1/10000 (Smith and Carrano, 1996). In addition, 1.11 billion bases representing 34.8% of the human genome has been sequenced to a rough-draft level. Efforts such as UCSC\u27s GoldenPath (Kent and Haussler, 2001) and NCBI\u27s contig assembly (Jang et al., 1999) attempt to assemble the human genome by incorporating both finished and rough-draft sequence. The availability of the human genome data allows us to ask questions concerning the maintenance of specific regions of the human genome. We consider two hypotheses for maintenance of high G+C regions: the presence of specific repetitive elements and compositional mutation biases. Our results rule out the possibility of the G+C content of repetitive elements determining regions of high and low G+C regions in the human genome. We determine that there is a compositional bias for mutation rates. However, these biases are not responsible for the maintenance of high G+C regions. In addition, we show that regions of the human under less selective pressure will mutate towards a higher A+T composition, regardless of the surrounding G+C composition. We also analyze sequence organization and show that previous studies of isochore regions (Bernardi,1993) cannot be generalized within the human genome. In addition, we propose a method to assemble only those parts of the human genome that are finished into larger contigs. Analysis of the contigs can lead to the mining of meaningful biological data that can give insights into genetic variation and evolution. I suggest a method to help aid in single nucleotide polymorphism (SNP)detection, which can help to determine differences within a population. I also discuss a dynamic-programming based approach to sequence assembly validation and detection of large-scale polymorphisms within a population that is made possible through the availability of large human sequence contigs

Construction of a BAC/PAC contig of SSC 6q1.2 and comparative analysis of this genome region

[no abstract

Genomic analysis of sorghum by fluorescence in situ hybridization

The reliability of genome analysis and proficiency of genetic manipulation in vivo and in vitro are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, orientation with respect to telomeres, and linear alignment with respect to chromosomal features and dimensions. I undertook five studies aimed at integrating sorghum genomics and cytogenetics at several levels. The results help establish an entirely new "cyto-genomics" resource, impacts of which are likely to be broad. In the first study, I developed a FISH-based karyotyping system for Sorghum bicolor Moench. I used integrated structural genomic resources, including linkage maps and large-insert clonal libraries of sorghum genomic DNA to develop a 17-locus probe cocktail for simultaneous fluorescent in situ hybridization (FISH). This probe enabled facile identification of all chromosome pairs in mitotic chromosome spreads. Perhaps just as important, I established time-efficient means to select sorghum BAC clones for multi-probe FISH. Thus, an integrated cyto-genomics system for sorghum can be constructed without need of chromosome flow sorting or microdissection, both of which are difficult and costly. In the second study, hybridization of DNA clones from 37 different genomic regions enabled the assignment of linkage groups and orientation of linkage maps to chromosomes. Comparisons between genetic and physical distances throughout the genome enabled a new nomenclature for linkage group designation in sorghum. The results provide an integrated nomenclature system of Sorghum bicolor chromosomes and linkage groups. In the third study, I created high-resolution maps by FISH to pachytene bivalents for two linkage groups (B and H), and defined relationships between pericentromeric heterochromatin, centromeres, mapped markers and recombination rates. These relationships will help guide the development and use of sorghum genomics. In the fifth study, I used FISH in two ongoing gene-targeted efforts. For the maturity gene ma5 and fertility restoration gene rfl, I estimated physical lengths between currently available flanking molecular markers. This enables estimation of recombination densities in these regions and assessment of the applicability of map-based and -assisted cloning