Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 1980 / Vita. / Includes bibliographical references. / by James F. Gusella. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Biology

No description available.

This investigation reorganizes and identifies chromosomal anomalies and delineates the associated clinical findings. The present investigation involved 37 individuals with anomalies of chromosome 22. The clinical profile with the corresponding cytogenetic anomalies was studied.

Cell cultures were examined for percentage of fragile sites seen in straight-stained, Q-stained and reverse fluorescent-stained preparations. In all cases, percentage of fragile site expression was decreased when compared to straight-stained preparations. However, fragile sites seen in Q- and RF-stain could be identified as on X chromosomes.

Ciliates are important model organisms that have been used to study many aspects of cellular biology, including telomeres, histone modifications, and ribozymes. These unicellular eukaryotes house both a germline genome and a somatic genome in distinct nuclear structures within a single cell. One of their most unique features is their ability to undergo complex programmed genome rearrangements, during which their germline genome is fragmented and rearranged to form a new somatic genome. This rearrangement process results in a highly specialized somatic genome with many polyploid short chromosomes that are rich with genes. While all ciliates can undergo this developmental process, Oxytricha trifallax experiences particularly complex rearrangements that result in a more radically unconventional structure in its somatic genome. Much of the previous work studying Oxytricha has been focused on the complex rearrangements that it undergoes during sexual development and the mechanisms that allow it to perform these genome rearrangements events at the level of accuracy required for proper somatic function afterwards. Due to this particular focus on Oxytricha sexual development, the rest of Oxytricha’s unique biology has not been studied to the same degree. For my thesis I examined two aspects of Oxytricha biology that have not been well understood. In Chapter 1 I report the results of a proteomic survey of both types of nuclei found within the vegetative cell, the somatic macronucleus and the germline micronucleus. We performed mass spectrometry on enriched samples of both nuclear types and analyzed the enrichment of proteins between the two. Despite some mitochondrial contamination, we found that many categories of functional proteins were enriched in one of the two nuclei. We validated the appropriate nuclear localization of specific proteins from each subcategory through imaging Our results confirmed many previously predicted aspects of the two nuclei and provide a valuable resource for further studies on nuclear proteins in Oxytricha. In Chapter 2 I describe various features of a comparative analysis between the somatic genomes of multiple strains of Oxytricha trifallax. Previous work from the lab has focused primarily on the reference strains JRB310 and JRB510, which are most commonly used due to their ability to mate. We generated four new draft assemblies of the somatic genomes of strains JRB27, JRB39, SLC89, and SLC92. Many metrics demonstrate that these new assemblies are largely complete. Our analyses of these new strains revealed that there are numerous strain-specific chromosomes in Oxytricha that can encode genes. While they do not seem to encode core genes that would be missing otherwise, they are prime candidates for further examination to identify mating type-related genes.

No description available.

The Anopheles gambiae complex is comprised of the most important vectors of malaria in Sub-Saharan Africa. Most current control methods involve the use of chemicals that help to limit potential contact with these mosquitoes. However, these control methods still have risks that include insect resistance, environmental toxicity, human health, as well as animal health. In order to develop new strategies that either produce novel targeted insecticides or transgenic mosquitoes that can replace current mosquito populations, it is important to acquire as much biological information about the vector as possible. The reduction in cost and speed of high-throughput sequencing has brought forth many new sequenced genomes that can provide a wealth of information about individual populations as well as their respective evolutionary histories. However, in order to fully understand a genome, these sequences must be assembled properly. One of the largest challenges toward fully assembling a genome is the abundance of repetitive sequences. These sequences, typically part of gene poor regions known as heterochromatin, are generally left as unassembled scaffolds that are neglected in many genomic studies. Heterochromatin is a biologically important chromatin state that has roles in gene regulation and genome stability. Exclusion of these chromatin domains from experimental assays can provide an incomplete picture in regards to organismal biology. A lack of information regarding heterochromatin, even in An. gambiae, necessitates further understanding and characterization of this chromatin type that can provide valuable information about the mosquito's biology. Heterochromatin is organized differently amongst different species. Some species with compact genomes, like Drosophila melanogaster, exhibit rigid organization of heterochromatin, with repetitive elements being confined to peri-centromeric and sub-telomeric regions of the chromosome. Larger genomes such as Aedes aegypti, have a much less structured heterochromatin pattern, with repetitive elements being dispersed across the genome. However, An. gambiae's genome is more intermediate in size as well as transposable element content. These factors may have an impact in controlling how heterochromatin is organized within the An. gambiae genome. Does An. gambiae compensate for the increased genome size by expanding past the peri-centromeric heterochromatin into new intercalary compartments? In An. gambiae, heterochromatin had yet to be identified separately from euchromatin. Morphologically, some regions of An. gambiae chromosomes exhibited characteristics similar to transcriptionally active puffs or peri-centromeric heterochromatin. We characterize these regions, as well as the rest of the genomic landscape, by using morphological and genetic features to identify various chromatin types. Peri-centromeric heterochromatin and new regions of intercalary heterochromatin were identified. Genomic coordinates representing the transition from euchromatin to heterochromatin were also identified. By finding these heterochromatin-euchromatin boundaries, various genetic features could be assigned to either heterochromatin or euchromatin. Critical genes associated with heterochromatin formation and basic genomic functions were identified. These data help to better understand features that are associated with the different environments created by chromatin compaction. This study also looks at the Piwi-interacting RNA (piRNA) pathway and its role in An. gambiae. The piRNA pathway is associated with transposable element (TE) suppression in many species, where clusters of vestigial TEs provide some of the RNA necessary for the pathway to function. These clusters are primarily associated with heterochromatin in Drosophila melanogaster. We identify piRNA clusters in An. gambiae and see a similar shift from primarily peri-centromeric compartmentalization toward the presence of intercalary regions located within the euchromatin. Transposable elements are maintained in secondary heterochromatin regions that exhibit similar morphology and features to peri-centromeric heterochromatin. The piRNA pathway also has implications in gene regulation, germline development, and anti-viral immunity. Three candidate genes associated with spermatogenesis and embryogenesis have been identified. These genes showed piRNA enrichment, and upon further analysis show up-regulation after a blood meal is taken. These genes could potentially prove useful in vector control as targets of transgenic experiments. Heterochromatin is an important, yet neglected aspect of the genome. These studies attempt to provide data to stimulate the study of heterochromatin through characterization of heterochromatin-related genomic features. / Ph. D.

Nine phenotypically normal purebred Hampshire and crossbred Hampshire x Yorkshire gilts were mated to purebred Hampshire boars to produce 25-day embryos for chromosomal analysis. Blood samples were drawn from five of these gilts and one of the boars for leucocyte cultures and a comparison was made between karyotypes prepared from the dam with those prepared from her 25-day embryos. The mean ovulation rate of the nine gilts was 11.8 ± 0.4. A total of 75 embryos were recovered, of which six were degenerating. In addition to the regressing embryos, several sites of complete embryo reabsorption were found. Prenatal losses were calculated to be 35.9%. The diploid number of 38 chromosomes were found in 85.9% and 90.2% of the cells counted in the leucocyte and embryo cultures, respectively. A normal chromosomal complement was displayed in all cells examined from the leucocyte culture obtained from the boar. Karyotype analyses showed only one abnormal embryo, which was monosomic for group 16. Other variations in chromosome number were described as the mechanical limitations of the technique used. If chromosomal abnormalities are constant in nature, these results would indicate that most abnormal embryos are unable to survive implantation, which is in agreement with findings of other researchers. Although the monosomic embryo had survived implantation, it is highly unlikely that it would have survived through parturition. / Master of Science

Culex tarsalis is a major vector of West Nile Virus (WNV) in North America. Although the genome for this species was recently sequenced, the physical genome map has not developed. Unlike other Culex species, that have sex-determination locus on chromosome 1, the sex locus in Cx. tarsalis is located on chromosome 3, the longest chromosome. It is currently unknown if this difference is associated with chromosomal rearrangements. The objectives of this study were to develop a high-resolution map for the precise physical genome mapping in Cx. tarsalis and to compare mitotic chromosomes between three species of Culicinae mosquitoes. Using mitotic chromosomes from imaginal discs of 4th instar larvae of Cx. tarsalis, we developed idiograms based on morphology and proportions of the mitotic chromosomes. In addition, the physical mapping of ribosomal genes using fluorescence in situ hybridization was performed. The comparative analysis of Cx. tarsalis to Cx. pipiens and Cx. quinquefasciatus chromosomes showed that the total chromosome length in Cx. tarsalis is longer than the other two species suggesting the bigger genome size in this mosquito. A comparison of the relative chromosome length between the species indicated no significant differences suggesting that no large chromosomal translocation occurred between the species. Comparisons of the centromeric indexes demonstrated a significant difference in chromosome 1 between Cx. pipiens and Cx. quinquefasciatus. This difference suggests the presence of pericentric inversion between the species or amplification of ribosomal genes in Cx. pipiens. Studying mosquito chromosomes advances our understanding of Culex cytogenetics. Further comparative physical mapping of the three major mosquito genera will help us to understand the evolution of genus Culex better and to develop genome-based strategies for the vector control. / Master of Science in Life Sciences / West Nile Virus (WNV) is the most common virus transmitted to humans by mosquitoes in the United States. While many species of mosquitoes are known to carry WNV, Culex tarsalis is a major vector on the west coast of North America. However, previous research on Cx. tarsalis lack chromosome studies on this mosquito. Our study aims to develop a high-quality chromosome map for Cx. tarsalis and to compare the mitotic chromosomes of Cx. tarsalis and Cx. quinquefasciatus and Cx. pipiens in respect of chromosomal rearrangements. We used a fluorescent DNA probe to find the location of the ribosomal locus in the chromosomes of Cx. tarsalis. This study developed a cytogenetic tool for further genomic studies of Cx. tarsalis that will help to develop genome-based strategies for vector control. Comparing the physical mapping of the three major mosquito genera will help to understand the genome evolution in Culicinae mosquitoes better.

Les anomalies chromosomiques peuvent être classées en anomalies de nombre et en anomalies de structure. L’identification des remaniements chromosomiques de structure est facilitée par les techniques de caryotypage à haute résolution et de cytogénétique moléculaire. L’utilisation de ces techniques a été essentielle pour la détection et la caractérisation de deux anneaux issus du même chromosome 22 diagnostiqués en période prénatale. Une amniocentèse a été effectuée à 163/7 semaines chez une femme de 39 ans pour âge maternel avancé. Après investigation, la formule chromosomique du fœtus a été déterminée: 47, XY, r(22)(p11.1p11.2), +r(22)(q11.1q13.31). L’anomalie chromosomique d’origine maternelle est survenue de novo. Le nombre de cellules foetales circulant dans le sang maternel est de 10 cellules par ml. Ce premier cas de deux anneaux constitutionnels du chromosome 22 est un exemple exceptionnel de monosomie partielle avec très peu de manifestations cliniques, malgré la richesse en gènes du segment délété. / Objective: Cytogenetic and molecular genetic characterization of two constitutional ring chromosomes 22 identified during prenatal diagnosis. Materials and Methods: A 39 year-old woman, G4P2A1, had amniocentesis at 163/7 weeks of gestation. Conventional and molecular cytogenetic studies with microsatellite analysis of the fetal and parental cells were performed. Results: The fetus had two ring chromosomes derived from chromosome 22 with three breakpoints: one located at the centromere, another, at the p11.2 subband and the third, at the q13.31 subband. The distal part of the two derivative chromosomes was lost. Then, two rings resulted: a small and a large one. The small ring was formed by joining the end of p11.2 subband to a portion of the centromere; the other by joining the second part of the centromere to the end of q13.31 subband. The male fetus presents the following karyotype: 47, XY, r(22)(p11.1p11.2), +r(22)(q11.1q13.31). The proband’s chromosome aberration occurred de novo from the maternal chromosome. At the autopsy, the fetus showed minor clinical features. The number of fetal nucleated blood cells detected in peripheral maternal circulation, showing positive signals for Y chromosome and DiGeorge/VCF.TUPLE1 probes and absence of ARSA control signal, was 10 cells per mL. Conclusion: Despite the haploinsufficiency of many active genes, the fetus showed minor congenital malformations.