Single molecules and single cells are the fundamental building blocks in biology. Facilitated by the advancement of technology, quantitative single-molecule and single-cell measurements provide a unique perspective toward many biological systems by revealing individual stochasticity and population heterogeneity. Taking advantage of these approaches, we studied chromosome organization and gene expression in bacteria and discovered new biophysical mechanisms: chromosome organization by a nucleoid-associated protein in live bacteria, and transcriptional bursting by the regulation of DNA supercoiling in bacteria.

The 2 micron plasmid of Saccharomyces cerevisiae is a multi-copy circular DNA genome that resides in the nucleus and exhibits nearly chromosome-like stability in host populations. Several host factors are required for equal plasmid segregation during cell division. One of them is cohesin (a multi-subunit protein complex) which mediates sister chromatid cohesion, a crucial mechanism for faithful segregation of replicated chromosomes in eukaryotes. The 2 micron plasmid mimics chromosomes in assembling cohesin at its partitioning locus. Studies on minichromosomes (centromere containing plasmids) reveal that cohesin forms a ring that embraces replicated sister centromeres topologically rather than physically. The functional similarities between chromosome and plasmid segregation prompted us to examine whether the topological mechanism proposed for centromere-mediated replicative cohesion is also true in the case of the plasmid. In the present study, we have characterized the nature and stoichiometry of cohesin's association with the 2 micron plasmid. Another host factor required for equal plasmid segregation is the CenH3 histone variant Cse4, so far considered to be uniquely associated with centromeric nucleosomes. Cse4 provides an epigenetic landmark at centromeres, and is required for assembly of the kinetochore complex. Surprisingly, Cse4 also interacts with the 2 micron plasmid partitioning locus. We have now functionally characterized this interaction, which can be preserved even in an ectopic, chromosomal context. The steady state level of Cse4 is highly limiting in yeast due to ubiquitin-mediated proteolysis. Only centromere-associated Cse4 is protected from this regulatory turnover control. We find that, in contrast to the situation with centromeres, association of Cse4 with the 2 micron plasmid is highly sub-stoichiometric but still promotes equal plasmid segregation. We also find that Cse4 induces an unusual right handed DNA writhe at the plasmid partitioning locus, as it does at the centromere. Our findings suggest that the plasmid has designed strategies to minimize the utilization of host factors that are in short supply. They signify the advantage of clustering and group behavior in the evolutionary success of a multi-copy selfish genome. Finally, they also suggest the possible emergence of the yeast centromere and the plasmid partitioning locus from a common ancestral sequence. / text

In order to combine previous observations from different sources on sex determination, and to identify sex chromosomes including the major sex determination locus in Nile tilapia, physical and genetic maps based on sex-linked markers and genes (such as sex-linked AFLPs, microsatellites, ovarian aromatase and DMO genes) were integrated and anchored. An accurate physical map using FISH techniques on mitotic cells was developed based on a previous map and 23 tilapia BAC clones previously assigned to linkage groups (LGs) 1, 3, 6, 7, 10 and 12; and on meiotic cells, 2 BAC clones containing the SLAM OniY227 and the dmrt4 gene were mapped. The six linkage groups were then assigned to different chromosomes, but surprisingly, the putative sex LG1 was located to a small submetacentric chromosome and not to the larger subtelocentric chromosome 1, where LG3 was assigned instead. The other LGs were assigned to different chromosomes and oriented with respect to the centromeres. A detailed comparison of the physical distribution of markers on chromosome 1 with respect to LG3 revealed a suppression in recombination in the subtelomeric region of the q arm between the marker GM354 (0 cM) and clcn5 (29 cM) and an abrupt increment of recombination between clcn5 (29 cM) and GM128 (77 cM) close to the centromere (Flpter=0.2). The unpairing region (20% of the total length) observed on the larger bivalents of XY fish during early pachytene in meiotic cells has been confirmed by DAPI staining and FISH to be at the terminal part of the q arm, opposite to the centromere. Comparison with six other tilapia species (2n=44) revealed a well conserved karyological distribution of the suspected LGs associated with sex determination (1 and 3). Besides, in O. karongae (2n=38) it was shown by SATA and UNH995/UNH104 marker hybridisation that LG1 has been re-arranged into the subtelomeric chromosome 2 as a result of a telomere-telomere fusion. A pool of 15 tilapia BAC clones previously localised on chromosome 1 and containing sex-linked AFLPs, dmrt1, dmrt4 and several SINEs were screened for new microsatellites; BACs were digested with SAU3AI and TC, GT, ATCT and CTGT probes radio-labelled with 32P. The high abundance of repetitive sequences in the BACs used led to only one useful polymorphic and co-dominant marker being obtained, associated to a BAC clone containing a copy of the dmrt1 gene on chromosome 1 (Flpter=0.85). Four linkage maps were constructed from an XY male, XY neofemale, XX neomale and XX female, mapping 4 and 8 markers on LG1 and LG3 (including the dmrt1 associated microsatellite) respectively. A specific sex-determination locus was identified on LG1 clearly linked with UNH995. However there appeared to be different allelic strengths for this sex determination locus, as shown by different sex ratios associated with different UNH995 genotypes. Additionally, one of the two XX fish mapped, showed the location of the recessive black blotching trait on LG3 (chromosome 1) between the markers GM128 and GM526, close to the centromere (Flpter=0.14). The results presented suggest a nascent Y chromosome in early stage of differentiation in Nile tilapia and with a functional master gene on LG1 close to the marker UNH995 (Flpter=0.67) located on the q arm of a small submetacentric chromosome. The potential influences of the autosomal LG3 (chromosome 1) in sex differentiation are also discussed.

Epigenetic differences between maternally inherited and paternally inherited chromosomes, such as CpG methylation, render the maternal and paternal genome functionally inequivalent, a phenomenon called genomic imprinting. This functional inequivalence is exemplified with imprinted genes, whose expression is parent-of-origin specific. The dosage of imprinted gene expression is disrupted in cells with uniparental disomy (UPD), which is an unequal parental contribution to the genome. I have derived mouse embryonic stem (ES) cell sub-lines with maternal UPD (mUPD) for mouse chromosome 6 (MMU6) to characterize regulation and maintenance of imprinted gene expression. The main finding from this study is that maintenance of imprinting in mitotic UPD is extremely variable. Imprint maintenance was shown to vary from gene to gene, and to vary between ES cell lines depending on the mechanism of loss of heterozygosity (LOH) in that cell line. Certain genes analyzed, such as Peg10, Sgce, Peg1, and Mit1 showed abnormal expression in ES cell lines for which they were mUPD. These abnormal expression levels are similar to that observed in ES cells with meiotically-derived full genome mUPD (parthenogenetic ES cells). Imprinted CpG methylation at the Peg1 promoter was found to be abnormal in all sub-lines with mUPD for Peg1. Two cell sub-lines which incurred LOH through mitotic recombination showed hypermethylation of Peg1, consistent with the presence of two maternal alleles. Surprisingly, a cell sub-line which incurred LOH through full chromosome duplication/loss showed hypomethylation of Peg1. The levels of methylation observed in these sub-lines correlates with expression, as the first two sub-lines showed a near-consistent reduction of Peg1, while the latter showed Peg1 levels close to wild-type. Altogether these results suggest that certain imprinted genes, like Peg1 and Peg10, have stricter imprinting maintenance, and as a result show abnormal expression in UPD. This strict imprint maintenance is disrupted, however, in UPD incurred through full chromosome duplication/loss, possibly because of the trisomic intermediate stage which occurs in this mechanism.

The past two decades have witnessed an accelerated effort to understand the nature of schizophrenia and related psychotic disorders, but no causative gene(s) has been discovered yet. Family, twin, and adoption studies indicate that genetic factors are clearly implicated in the etiology of these disorders (Cardno and Gottesman, 2000; Cardno et al., 2002; McGuffin et al., 2003; Weinberger, 2005). Several aspects of 22q11 Deletion Syndrome (22qDS) - the most common chromosomal microdeletion found in humans - create a unique opportunity for susceptibility gene identification. For instance, the reported risk of psychotic disorders in 22qDS is 25-fold higher than in the general population (Murphy et al., 1999) and genome-wide linkage studies in families with schizophrenia without 22qDS indicate that the 22q11.2 region is a strong susceptibility locus for psychosis (Badner and Gershon, 2002; Lewis et al., 2003). This thesis aims to identify genetic factors associated with the development of psychosis in 22qDS by i) investigating the relationship between the length of the 22q11.2 deletions and the presence of a psychotic disorder in patients with 22qDS; ii) studying diagnostic molecular methods that improve detection of 22q11.2 deletions and duplications; and iii) exploring the relationship between 22qDS-psychotic phenotype and gene expression patterns. The central hypothesis was that psychosis in 22qDS would not be associated with haploinsufficiency (having one copy of the gene), but rather, it would be associated with distinct 22q11.2 gene expression profiles. Chapter 2 showed that 22q11.2 deletion size did not appear to be associated with the development of psychosis in adults with 22qDS. In Chapter 3, a molecular method that detects and size 22q11.2 deletions and duplications of various sizes was shown to be superior to the traditional molecular diagnostic technique used for molecular diagnostic of 22qDS. Finally, in Chapter 4, decreased gene expression of three genes located in the 22q11.2 region (SNAP29, COMT and BID) was significantly associated with psychosis in adults with 22qDS. Focusing on genes located in the 22q11.2 region has helped revealing genetic alterations associated with the frequent development of psychosis in 22qDS. Future studies focusing on investigating the heterogeneity of the psychotic presentation in 22qDS and further elucidating potential genetic mechanisms likely to explain the gene expression changes in the 22q11.2 region demonstrated here will help advance the scientific understanding of the etiology of psychosis.

The past two decades have witnessed an accelerated effort to understand the nature of schizophrenia and related psychotic disorders, but no causative gene(s) has been discovered yet. Family, twin, and adoption studies indicate that genetic factors are clearly implicated in the etiology of these disorders (Cardno and Gottesman, 2000; Cardno et al., 2002; McGuffin et al., 2003; Weinberger, 2005). Several aspects of 22q11 Deletion Syndrome (22qDS) - the most common chromosomal microdeletion found in humans - create a unique opportunity for susceptibility gene identification. For instance, the reported risk of psychotic disorders in 22qDS is 25-fold higher than in the general population (Murphy et al., 1999) and genome-wide linkage studies in families with schizophrenia without 22qDS indicate that the 22q11.2 region is a strong susceptibility locus for psychosis (Badner and Gershon, 2002; Lewis et al., 2003). This thesis aims to identify genetic factors associated with the development of psychosis in 22qDS by i) investigating the relationship between the length of the 22q11.2 deletions and the presence of a psychotic disorder in patients with 22qDS; ii) studying diagnostic molecular methods that improve detection of 22q11.2 deletions and duplications; and iii) exploring the relationship between 22qDS-psychotic phenotype and gene expression patterns. The central hypothesis was that psychosis in 22qDS would not be associated with haploinsufficiency (having one copy of the gene), but rather, it would be associated with distinct 22q11.2 gene expression profiles. Chapter 2 showed that 22q11.2 deletion size did not appear to be associated with the development of psychosis in adults with 22qDS. In Chapter 3, a molecular method that detects and size 22q11.2 deletions and duplications of various sizes was shown to be superior to the traditional molecular diagnostic technique used for molecular diagnostic of 22qDS. Finally, in Chapter 4, decreased gene expression of three genes located in the 22q11.2 region (SNAP29, COMT and BID) was significantly associated with psychosis in adults with 22qDS. Focusing on genes located in the 22q11.2 region has helped revealing genetic alterations associated with the frequent development of psychosis in 22qDS. Future studies focusing on investigating the heterogeneity of the psychotic presentation in 22qDS and further elucidating potential genetic mechanisms likely to explain the gene expression changes in the 22q11.2 region demonstrated here will help advance the scientific understanding of the etiology of psychosis.

No description available.

Le développement embryonnaire précoce des mammifères femelles s'accompagne de l'inactivation transcriptionnelle d'un de leurs deux chromosomes X. Cet évènement est initié suite à l'expression mono-allélique de l'ARN non codant Xist, qui est contrôlée par de nombreux éléments cis-régulateurs présents dans le centre d'inactivation du chromosome X (Xic) - tel son anti-sens répresseur Tsix. Mon travail de thèse a consisté à développer des approches permettant d'appréhender le paysage structural dans lequel s'exerce cette régulation. La caractérisation de l'architecture tridimensionnelle du Xic, par des techniques basées sur la capture de conformation chromosomique (3C) et l'hybridation in situ en fluorescence (FISH), m'a permis de mettre en évidence que les promoteurs respectifs de Xist et Tsix sont engagés dans des interactions physiques intimes avec des loci distaux, localisés au sein du Xic, et de montrer qu'au moins certaines de ces régions exercent un effets régulateurs à longue-distance. Les éléments du Xic contactés par les régions promotrices de Xist et de Tsix sont en outre fondamentalement différents, chacune engageant des associations chromosomiques sur plusieurs centaines de kilobases dans leur direction 5' respective.Ce travail a également permis de révéler des propriétés insoupçonnées de l'architecture chromosomiques. En effet, le Xic apparaît scindé en plusieurs sous-régions, couvrant chacune entre 200kb et 1Mb, à l'intérieur desquelles les interactions chromosomiques sont préférentiellement établies. L'existence de ces domaines d'interaction s'intègre avec d'autres propriétés structurales du génome, tels la composition de la chromatine sous-jacente et l'association à la lamine nucléaire, mais n'apparaît pas en dépendre directement. En étudiant la dynamique de la conformation chromosomique du Xic au cours de la différenciation cellulaire, j'ai pu constater la robustesse de cette organisation, sauf sur le chromosome X inactif, qui se distingue par la perte des contacts chromosomiques préférentiels détectables sur son homologue actif.Enfin, j'ai pu mettre en évidence que la variabilité du repliement général du chromosome X amène à un instant donné chaque allèle de Tsix à contacter physiquement des jeux de séquences distales différents, suggérant que l'environnement structural instantané de chacun de ces allèles à l'orée de l'activation mono-allélique de Xist est différent. Ce travail, combinant des approches à l'échelle de la population cellulaire d'une part et de la fibre de chromatine unique d'autre part, apporte une nouvelle vision du paysage structural et régulateur dans lequel s'inscrit le contrôle de l'activité transcriptionnelle de Xist, et fourni de nouvelles perspectives concernant les principes fondamentaux de l'organisation topologique des chromosomes chez les mammifères.

The Distal-less homeobox (Dlx) genes encode a group of transcription factors that are involved in various developmental processes including forebrain development. Dlx genes are arranged in convergently transcribed bigene clusters with enhancer sequences located in the intergenic region of each cluster. The expression patterns of Dlx1/Dlx2 and of Dlx5/Dlx6 are attributed in part to the activity of I12a/I12b and I56i/I56ii intergenic enhancers, respectively. In an effort to determine how Dlx intergenic enhancers interact with the promoter regions of each cluster, I employed the Chromosome Conformation Capture (3C) technique on developing forebrain at E13.5 and E15.5. My 3C analysis provided potential enhancer-promoter interaction, in cis, that are consistent with previously known regulatory mechanisms. Furthermore, trans interactions may exist between Dlx1/Dlx2 and Dlx5/Dlx6 clusters in the developing forebrain at E13.5, thus providing a possible novel cross-regulatory mechanism between these two loci. I have also investigated the phenotypic consequences of Dlx enhancer deletion(s) on forebrain development by characterizing mice with I56ii and I56ii/I12b enhancer deletions. Enhancer deletions significantly impair Dlx expression as well as that of Evf2, Gad2 and of the striatal markers Islet1 and Meis2. Enhancer deletion(s) also reduce the expression of ISLET1 and CTIP2 proteins and Semaphorin 3A, Slit1 and Ephrin A5 that are thought to provide guidance cues in the corridor cells. Overall, these changes may disrupt the guidance of the thalamocortical axons. The data presented here further our understanding of the interactions between Dlx intergenic enhancers and promoter regions. Enhancer deletion(s) furthers our understanding of Dlx regulatory networks necessary that ensure proper Dlx expression, which, in turn may be involved in a genetic pathway underlying the synthesis of GABA, which may be further essential in maintaining the GABAergic phenotype.

Plant breeding and the development of new or better varieties of plants are essential parts of modern agronomy, horticulture and forestry. The basis for such improvement is a knowledge of the factors and principles of genetics. A number of genetic studies have been made with barley in recent years. This is partly because of the many distinct heritable characters of barley plants. The cultivated species of barley offers the plant breeder and geneticist a wealth of material for genetic studies. Varieties differ in a great many readily distinguishable characters, species hybridize readily, and their small number of chromosomes make it good material for inheritance studies. The barley genetic work has been divided among the principal workers in the U.S., each being responsible for one linkage group. This station has been assigned group IV of which this study is a part. A study of the inheritance of other genes not located in linkage group IV, but appearing in the crosses used, has also been made. This investigation is a by-product of the cereal breeding and improvement program being carried on the Utah Agricultural Experiment Station.