The pattern of inactivation in the DXS8237E-UBE1-PCTK1 region is of particular interest, since the mechanisms of X chromosome inactivation and the escape from inactivation are, as yet, not fully understood. The inactivation status of the DXS8237E and PCTKl gene differ: the first undergoes normal inactivation and the second escapes this process. The status of the UBEl gene has been controversial, although it is widely excepted that it does escape X chromosome inactivation. Physical mapping of the region employing YACs and subsequently P ACs has been undertaken, but was restricted in scope by the high frequency of rearrangements occurring. DNA sequences between DXS8237E, UBE1, PCTKl and the distal gene, UHX1, have been investigated with regard to LINEI elements, which are thought to playa role in X-inactivation. The results obtained strongly suggest a link between LINE1 elements and X chromosome inactivation. Sequence analysis results also contributed to the understanding of difficulties with restriction mapping of the region. Further, this work includes the first reported establishment of the UBEl exonintron boundaries. Additionally, genomic sequence analysis showed that only 46kb separate DXS8237E from UHX1, which confirms that this region is extremely gene rich.

<p>Alternative RNA splicing is one of the leading mechanisms contributing towards transcript and protein diversity. Several alternative splicing surveys have confirmed the frequent occurrence of exon skipping in human genes. However, the occurrence of exon skipping in mouse genes has not yet been extensively examined. Recent improvements in mouse genome sequencing have permitted the current study to explore the occurrence of exon skipping in mouse genes orthologous to human genes on chromosome 22. A low number (5/72 multi-exon genes) of mouse exon-skipped genes were captured through alignments of mouse ESTs to mouse genomic contigs. Exon-skipping events in two mouse exon-skipped genes (GNB1L, SMARCB1) appear to affect biological processes such as electron and protein transport. All mouse, skipped exons were observed to have ubiquitous tissue expression. Comparison of our mouse exon-skipping events to previously detected human exon-skipping events on chromosome 22 by Hide et al.2001, has revealed that mouse and human exon-skipping events were never observed together within an orthologous gene-pair. Although the transcript identity between mouse and human orthologous transcripts were high (greater than 80% sequence identity), the exon order in these gene-pairs may be different between mouse and human orthologous genes.<br /> <br /> Main factors contributing towards the low detection of mouse exon-skipping events include the lack of mouse transcripts matching to mouse genomic sequences and the under-prediction of mouse exons. These factors resulted in a large number (112/269) of mouse transcripts lacking matches to mouse genomic contigs and nearly half (12/25) of the mouse multi-exon genes, which have matching Ensembl transcript identifiers, have under-predicted exons. The low frequency of mouse exon skipping on chromosome 22 cannot be extrapolated to represent a genome-wide estimate due to the small number of observed mouse exon-skipping events. However, when compared to a higher estimate (52/347) of exon skipping in human genes for chromosome 22 produced under similar conditions by Hide et al.2001, it is possible that our mouse exon-skipping frequency may be lower than the human frequency. Our hypothesis contradicts with a previous study by Brett et al.2002, in which the authors claim that mouse and human alternative splicing is comparable. Our conclusion that the mouse exon-skipping frequency may be lower than the human estimate remains to be tested with a larger mouse multi-exon gene set. However, the mouse exon-skipping frequency may represent the highest estimate that can be obtained given that the current number (87) of mouse genes orthologous to chromosome 22 in Ensembl (v1 30th Jan. 2002) does not deviate significantly from our total number (72) of mouse multi-exon genes. The quality of the current mouse genomic data is higher than the one utilized in this study. The capture of mouse exon-skipping events may increase as the quality and quantity of mouse genomic and transcript sequences improves.</p>

Studies of somatic cell genetics in larches (Larix spp.) were carried out using somatic hybridization, cytogenetics as well as fluorescence in situ hybridization. Haploid embryogenic protoplasts are ideal sources for somatic hybridization if they possess stable chromosome complements. In my protoplast fusion experiments, I used diploid embryogenic protoplasts because genetic variation was detected in the haploid lines available. Cytogenetics coupled with fluorescence in situ hybridization was used to reveal genetic instabilities in haploid embryogenic lines as well as to produce a standard karyotype of Larix decidua. A diploid embryogenic culture of tamarack (Larix laricina, line L2) was used as one of the fusion partners while the other partner used was one of the two hybrid larches (Larix x leptoeuropaea, line L5 and Larix x eurolepis, line L6 ). The selection system was based on complementation of metabolic inhibition (with sodium iodoacetate) of tamarack and the lack of ability to produce mature embryos of the hybrid larches. Ideally, only the heterofused cells would have been able to regenerate. The vital fluorescent dyes, DiOC₆ and R6 , were used to stain protoplasts of each parent to determine fusion events and frequencies. I compared fusion firequency as well as cell division between fusion mediated by PEG or electric pulses. PEG-mediated fusion resulted in 14-18 % of heterofused cells. All electrofusion treatments gave much lower fusion frequencies, at only 4-8 %. Although the percentages of cell division after 4d of PEG-fusion (17-24%) and electrofusion (19.3%) were about the same, PEG-fusion was found to be a more efficient means than electrofusion. Sodium iodoacetate at a concentration of 4-5 mM was found to efficiently inactivate the protoplasts of tamarack. All control-treated protoplasts as well as mixed cultures (unfused protoplasts) died. Tamarack protoplasts produced mature single embryos, whereas protoplasts of hybrid larches never completed embryogenesis. Some post-fusion products produced colonies and mature embryos. RAPD was used to verify the hybridity of those fusion-derived colonies and mature embryos. Of thirty-one fusion experiments between lines L2 and L5, only one produced individual colonies. Of the thirteen colonies which developed in that experiment, none yielded mature embryos. RAPD analysis of the colonies picked out from L2/L5 fusion showed DNA banding characteristics of L5. From twenty four experiments fusing L2 and L6 , there were five experiments which produced colonies. A total of two hundred and thirty nine individual colonies and nineteen single mature embryos were picked out from those L2/L6 fusions. RAPD banding profiles of eighty seven colonies and nineteen mature embryos showed DNA banding characteristics of L2 only. Tested haploid embryogenic lines (total of 6 lines; n=12) of Larix decidua initiated from megagametophyte tissue were maintained on half-strength Litvay’s medium without growth regulators. All lines had been verified as being haploid by chromosome squashes when they were initiated. Some lines have been stably haploid for only a short period of time while others have been stable for many years. Variations in chromosome numbers increased proportionately with the age of the culture. Haploids doubled their chromosome numbers. Aneuploidization occurred because of unequal separation of the chromosomes. Unusual events during mitosis such as formation of anaphase bridges, fragmentation of chromosomes, and development of long kinetochores were detected. There was a tendency of rising chromosome numbers in all lines tested over the years. Fluorescence in situ hybridization (FISH) was used to physically map highly repetitive sequences of genes coding for 18S-5.8S-26S rDNA on Larix decidua chromosomes. A karyotype of L. decidua (2n=24) was created from average relative lengths derived from the six best squashes with strong probe-target FISH signals. Hybridization of 18S-26S rDNA onto L. decidua chromosomes gave very precise locations of secondary constriction as well as unexpressed nucleolar organizer regions. In L. decidua, there were 6 major 18S-26S rDNA loci detected in 60.53% of cells (23 out of 39 cells). Five I8S-26S rDNA loci were also found but at a lower rate of 39.47%. All loci were expressed and located at the sites of secondary constriction on chromosomes 2, 4 and 7. Two extra locations of 18S-26S rDNA were mapped on aneuploid chromosomes (30 chromosomes) derived from cells of an aneuploid line (line 2110) of L. decidua. Chromosome measurement resulted in a preliminary karyotype of this line. The relative total lengths and locations of I8S-26S rDNA of standard (2n=24) chromosomes and aneuploid (2n=30) chromosomes was compared. / Graduate

This study was initiated after finding two chromosomal types of Peromyscus maniculatus north and south of the Red River in Texas and Oklahoma. The problem was to explain the chromosomal variations and their implications to the systematics of the grassland subspecies of P. maniculatus in this region.

Li Chung Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 110-123). / Abstracts in English and Chinese. / ABSTRACT --- p.i / ACKNOWLEDGEMENTS --- p.iv / TABLE OF CONTENTS --- p.v / LIST OF FIGURES --- p.ix / LIST OF TABLES --- p.xi / Chapter I. --- INTRODUCTION --- p.1 / Chapter I.1 --- Gastric Carcinoma --- p.1 / Chapter I.2 --- Etiology of Gastric Carcinoma --- p.2 / Chapter I.2.1 --- Environmental Factors: --- p.2 / Chapter I.2.2 --- Helicobacter Pylori Infection: --- p.2 / Chapter I.2.3 --- Genetic Factors: --- p.3 / Chapter I.2.4 --- Other Factors: --- p.4 / Chapter I.3 --- The Lauren Classification of Gastric carcinoma --- p.5 / Chapter I.3.1 --- Histolopathology of Intestinal Type of Gastric Carcinoma --- p.5 / Chapter I.3.2 --- Histopathology of Diffuse Type of Gastric Carcinoma --- p.8 / Chapter I.4 --- Cytogenetics Studies in Gastric Carcinoma --- p.10 / Chapter I.4.1 --- Cytogenetic Studies of Gastric carcinoma --- p.10 / Chapter I.5 --- Molecular Studies of Gastric Carcinoma --- p.14 / Chapter I.5.1 --- Genetic Instability --- p.14 / Chapter I.5.2 --- Amplification/ Mutation of Oncogenes --- p.15 / Chapter I.5.3 --- Alterations of Tumor Suppressor Genes --- p.20 / Chapter I.5.4 --- Cell Adhesion Molecules --- p.23 / Chapter I.5.5 --- Molecular Studies on Intestinal Metaplasia --- p.27 / Chapter II. --- LONG ARM OF CHROMOSOME 6 --- p.28 / Chapter III. --- BRIEF REVIEWS OF THE TECHNIQUES USED IN THIS STUDY --- p.34 / Chapter III.1 --- Comparative Genomic Hybridization (CGH) --- p.34 / Chapter III.2 --- Loss of Heterozygosity (LOH) --- p.37 / Chapter III.3 --- Methylation-Specific PCR (MSP) --- p.38 / Chapter IV. --- OBJECTIVES OF STUDY --- p.40 / Chapter V. --- MATERIALS AND METHODS --- p.41 / Chapter V.l --- Sample Collections --- p.41 / Chapter V.1.1 --- Patients Information for CGH Studies --- p.42 / Chapter V. 1.2 --- Patients Information for LOH Studies --- p.42 / Chapter V.2 --- Extraction of Genomic DNA for Tumor and Normal Tissues --- p.47 / Chapter V.2.1 --- Extraction of Genomic DNA from Frozen Tissues or Paraffin Embedded Sections --- p.47 / Chapter V.2.2 --- Extraction of Genomic DNA from Blood --- p.48 / Chapter V.3 --- Comparative Genomic Hybridization (CGH) of Gastric Carcinoma --- p.49 / Chapter V.3.1 --- Preparation of Normal Metaphase Slides --- p.49 / Chapter V.3.2 --- Metaphase Slide Denaturation --- p.49 / Chapter V.3.3 --- Nick Translation for DNA Labeling --- p.50 / Chapter V.3.4 --- Dot Blot Assay for Biotin and Digoxigenin-Labeled DNA --- p.51 / Chapter V.3.5 --- "Probe Preparation, Denaturation and Hybridization" --- p.51 / Chapter V.3.6 --- Post hybridization Washing and Detection --- p.52 / Chapter V.3.7 --- Image Acquisition and Analysis of CGH Images --- p.53 / Chapter V.4 --- Loss of Heterozygosity (LOH) Analysis on Chromosome 6q --- p.55 / Chapter V.4.1 --- Microsatellite Markers --- p.55 / Chapter V.4.2 --- Polymerase Chain Reaction (PCR) --- p.57 / Chapter V.4.3 --- Denaturing Polyacrylamide Gel Electrophoresis --- p.58 / Chapter V.4.4 --- SYBR Gold Nucleic Acid Gel Staining and Image Viewing --- p.58 / Chapter V.4.5 --- Assessment of Loss of Heterozygosity (LOH) --- p.59 / Chapter V.4.6 --- Statistical Analysis --- p.61 / Chapter V.5 --- Methylation Specific Polymerase Chain Reaction (MSP) --- p.62 / Chapter V.5.1 --- Bisulfite Modification of DNA --- p.62 / Chapter V.5.2 --- Mehtylation Specific PCR --- p.63 / Chapter VI. --- RESULTS --- p.66 / Chapter VI.1 --- Results of CGH --- p.66 / Chapter VI. 1.1 --- Chromosomal Copy Number Aberrations in Gastric Carcinoma --- p.66 / Chapter VI. 1.2 --- Comparison of CGH Results with Intestinal and Diffuse Type of Gastric Carcinoma --- p.67 / Chapter VI.2 --- LOH Analysis of Chromosome 6q --- p.73 / Chapter VII. --- DISCUSSIONS --- p.83 / Chapter VII.l --- Discussions on CGH --- p.83 / Chapter VII.2 --- Discussions on LOH Study --- p.89 / Chapter VII.2.1 --- Two Distinct Deletion Regions --- p.89 / Chapter VII.2.2 --- Possible Candidate Suppressor Genes in Two Deletion Regions --- p.93 / Chapter VII.2.3 --- Infrequent Loss of IGF2R Gene --- p.95 / Chapter VII.3 --- Relationship Between Intestinal Metaplasia and Gastric Carcinoma --- p.99 / Chapter VII.4 --- Microsatellite Instability --- p.100 / Chapter VII.5 --- Correlations --- p.103 / Chapter VII.6 --- Comparison Between CGH and LOH Results on Chromosome 6 --- p.104 / Chapter VII.7 --- Conclusions --- p.106 / Chapter VII.8 --- Limitations of the Study --- p.107 / Chapter VII.8.1 --- Limitation of CGH --- p.107 / Chapter VII.8.2 --- Limited Information Supply by LOH Analysis --- p.107 / Chapter VII.8.3 --- Small Sample Size --- p.107 / Chapter VII.9 --- Future Studies --- p.108 / Chapter VIII. --- REFERENCES --- p.110

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Thesis--Cornell University. / Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains xi, 94 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 78-89).

Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 119-155).

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