BACKGROUND:Retrotransposons have been suggested to provide a substrate for non-allelic homologous recombination (NAHR) and thereby promote gene family expansion. Their precise role, however, is controversial. Here we ask whether retrotransposons contributed to the recent expansions of the Androgen-binding protein (Abp) gene families that occurred independently in the mouse and rat genomes.RESULTS:Using dot plot analysis, we found that the most recent duplication in the Abp region of the mouse genome is flanked by L1Md_T elements. Analysis of the sequence of these elements revealed breakpoints that are the relicts of the recombination that caused the duplication, confirming that the duplication arose as a result of NAHR using L1 elements as substrates. L1 and ERVII retrotransposons are considerably denser in the Abp regions than in one Mb flanking regions, while other repeat types are depleted in the Abp regions compared to flanking regions. L1 retrotransposons preferentially accumulated in the Abp gene regions after lineage separation and roughly followed the pattern of Abp gene expansion. By contrast, the proportion of shared vs. lineage-specific ERVII repeats in the Abp region resembles the rest of the genome.CONCLUSIONS:We confirmed the role of L1 repeats in Abp gene duplication with the identification of recombinant L1Md_T elements at the edges of the most recent mouse Abp gene duplication. High densities of L1 and ERVII repeats were found in the Abp gene region with abrupt transitions at the region boundaries, suggesting that their higher densities are tightly associated with Abp gene duplication. We observed that the major accumulation of L1 elements occurred after the split of the mouse and rat lineages and that there is a striking overlap between the timing of L1 accumulation and expansion of the Abp gene family in the mouse genome. Establishing a link between the accumulation of L1 elements and the expansion of the Abp gene family and identification of an NAHR-related breakpoint in the most recent duplication are the main contributions of our study.

Broad Complex (BRC) is an ecdysone-pathway gene essential for entry into and progression through metamorphosis in D. melanogaster. Mutations of three BRC complementation groups cause numerous phenotypes, including a common suite of morphogenesis defects involving central nervous system (CNS), adult salivary glands (aSG), and male genitalia. Alternative splicing, of a protein-binding BTB-encoding exon (BTBBRC) to one of four tandemly duplicated, DNA-binding zinc-finger-encoding exons (Z1BRC, Z2BRC, Z3BRC, Z4BRC), produces four BRC isoforms. Highly conserved orthologs of BTBBRC and all four ZBRC were found in silico from Diptera, Lepidoptera, Hymenoptera and Coleoptera, indicating that BRC arose and underwent internal exon duplication before the split of holometalolous orders. Five Tramtrack subfamily members were characterized throughout Holometabola and used to root phylogenetic analyses of ZBRC exons, revealing that Z3BRC is the basal member. All four ZBRC domains, including Z4BRC which has no known essential function, are evolving in a manner consistent with selective constraint. Transgenic rescue and immunohistochemistry were used to explore how different BRC isoforms contribute to their shared tissue-morphogenesis functions at the onset of metamorphosis, when BRC is required for CNS reorganization. As predicted, the common CNS and aSG phenotypes were rescued by BRC-Z1 in rbp mutants, BRC-Z2 in br mutants, and BRC-Z3 in 2Bc mutants. However, the isoforms are required at two developmental stages, with BRC-Z2 and -Z3 required earlier than BRC-Z1. Each isoform had a unique expression pattern in the CNS, with no substantial three-way overlap among them. Z4 is strongly expressed in a novel subset of CNS neurons. The most prominent localizations of BRC-Z1, -Z2, -Z3 corresponded with glia, neuroblasts and neurons, respectively. There appears to be a switch from BRC-Z2 in proliferating cells to BRC-Z1 and BRC-Z3 in differentiating cells. The temporal-requirement and spatial-distribution data suggest that BRC-dependent CNS morphogenesis is the result of multicellular interactions among different cell types at different times. BRC-Z1-expressing glia in prepupae may mediate the final steps of CNS morphogenesis. Lastly, BRC is required for migration and programmed cell death of the ring gland, the site of ecdysone and juvenile hormone production. Therefore, BRC may function in ecdysone auto-regulation.

Guppies exhibit color based sexual dimorphism and females generally prefer the most colorful males. It has also recently been found that guppies possess a large opsin repertoire. As opsins are the receptors responsible for color vision, this ten gene repertoire might have contributed to the evolution of extravagant male coloration in this species. My study starts by characterizing the opsin repertoire of Jenynsia onca, a noncolorful relative of the guppy belonging to the family Anablepidae (sister group to Poeciliidae, of which the guppy is a member). A PCR based survey indicated that J. onca had a very similar opsin repertoire to the guppy; J. onca had nine genes including orthologs of all but one of the guppy opsins. To gain further insight into the origin of the guppy repertoire, a bioinformatics based survey of ray-finned fish opsins was undertaken. This revealed that large opsin repertoires are common in ray-finned fish and are the product of gene duplication events, spanning the age of the taxon Teleostei. Given that the large opsin repertoire of the guppy did not appear to be perfectly correlated with the evolution of color based sexual selection in this lineage, I turned to investigating the expression of this opsin repertoire. In situ hybridization was used to characterize the pattern of opsin expression across the surface of the retina of adult male and female guppies. In situ hybridization demonstrated that most opsin genes had distinct expression profiles. These expression patterns also indicated that sensitivity and discrimination in the dorsal retina might differ from the ventral retina; the ventral retina appears to be tuned to middle-wavelength light (green), while the dorsal retina is predicted to have exceptional wavelength discriminatory ability and broad spectral sensitivity. This expression data was then used to evaluate models of sexual selection in the context of the predicted visual capacity of the guppy. / Graduate

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [111]-128).

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 146-156).

Understanding the genetics underlying the speciation process has been a long-standing goal of evolutionary biology. Studying inter-population crosses can elucidate the genetic architecture of reproductive isolation and, ultimately, the process of speciation. Hybridization between two species is often maladaptive and results in offspring with decreased fitness compared to the parental forms. Recently, with the development of molecular and genomic tools, it has become possible to understand how and when reproductive isolation arises and what are the underlying mechanisms in the evolution of genetic incompatibilities. Heliconius is a genus of neotropical butterfly best know for their Müllerian mimicry. Here I focus on Heliconius cydno and Heliconius melpomene, two hybridising sympatric species with low levels of inter-specific hybridisation that nonetheless results in genome-wide signatures of admixture. I show that hybrids develop ovarian tissue and, occasionally, oocytes; and use genomic approaches to examine several potential mechanisms underlying post-zygotic isolation between H. cydno and H. melpomene. Firstly, I investigate evolution by gene duplication and identify loci putatively under divergent selection that may play a role in species divergence and speciation. Secondly, I quantify sexually dimorphic expression in H. melpomene, and calculate rates of molecular evolution between H. melpomene and H. erato. Thirdly, I identify differentially expressed genes in the H. cydno x H. melpomene F1 hybrids that may be involved in the species barrier. Finally, investigate whether epigenetic silencing mechanisms could underlie post-zygotic isolation between H. cydno and H. melpomene by quantifying transposable element expression and small RNAs. Overall, I identify loci that merit further investigation for their potential in maintaining reproductive barriers between these two species. I show that different regions of the genome evolve at different molecular rates but there is no faster-Z effect, and consider how might this affect evolution of reproductive isolation. Finally, I show that aberrant epigenetic silencing, a mechanism behind hybrid sterility that is common in other species, is not correlated with post-zygotic isolation between H. cydno and H. melpomene.

ix, 120 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Gene duplication underlies the evolution of many protein functions and is a known stimulus for molecular innovation. Many models exist to explain the maintenance of duplicate genes in the genome and the dynamics that drive the evolution of novel protein functions; few if any of these models, however, incorporate knowledge of how protein structures and functions actually evolve. A growing body of work on the historical mechanisms of molecular evolution and the ways in which proteins evolve in the lab has provided profound insights into the ways in which proteins respond to mutation, selection, and drift. Evolutionary models of duplicate gene evolution could greatly benefit from the knowledge gained from these mechanistic studies of protein evolution. My dissertation seeks to address this gap in knowledge by reconstructing the process by which novel steroid signaling pathways evolved after gene duplication. I focus specifically on a class of hormones called corticosteroids--critical regulators of the stress response, metabolism, and immunity--and the mineralocorticoid and glucocorticoid receptors that mediate the steroid response. Both the enzymes that synthesize corticosteroids and the hormone receptors are the result of ancient gene duplication events, and I make use of methods in phylogenetics, molecular biology, and structural biology to reconstruct the mechanisms and dynamics by which they evolved. This dissertation comprises three separate but complementary studies that illuminate the origins of corticosteroid signaling. In the first project, I show how lineage-specific steroid signaling arose in elasmobranchs as a novel hormone exploited the structural promiscuity of preexistent receptors. Next, I describe how degenerative and stabilizing mutations defined the divergence of the glucocorticoid receptor after gene duplication. And finally, I use phylogenetic and functional analyses to reconstruct the origins of corticosteroid synthesis with the duplication of enzymes in the steroid synthesis pathway. Together, I provide a comprehensive reconstruction of the evolution of corticosteroid signaling. This work also highlights specific evolutionary mechanisms--molecular exploitation, structural and functional promiscuity, degenerative mutations, and stabilizing mutations--that could drive the evolution of novel protein functions after gene duplication. This dissertation includes both previously published and unpublished co-authored materials. / Committee in charge: Patrick Phillips, Chairperson, Biology; Joseph Thornton, Advisor, Biology; William Cresko, Member, Biology; John Postlethwait, Member, Biology; Kenneth Prehoda, Outside Member, Chemistry

Tandemly arrayed genes (TAG) play an important functional and physiological role in the genome. Most previous studies have focused on individual TAG families in a few species, yet a broad characterization of TAGs is not available. We identified all the TAGs in the genomes of human, chimp, mouse, and rat and performed a comprehensive analysis of TAG distribution, TAG sizes, TAG gene orientations and intergenic distances, and TAG gene functions. TAGs account for about 14-17% of all the genomic genes and nearly one third of all the duplicated genes in the four genomes, highlighting the predominant role that tandem duplication plays in gene duplication. For all species, TAG distribution is highly heterogeneous along chromosomes and some chromosomes are enriched with TAG forests while others are enriched with TAG deserts. The majority of TAGs are of size two for all genomes, similar to the previous findings in C. elegans, A. thaliana, and O. sativa, suggesting that it is a rather general phenomenon in eukaryotes. The comparison with the genome patterns shows that TAG members have a significantly higher proportion of parallel gene orientation in all species, corroborating Graham's claim that parallel orientation is the preferred form of orientation in TAGs. Moreover, TAG members with parallel orientation tend to be closer to each other than all neighboring genes with parallel orientation in the genome. The analysis of GO function indicate that genes with receptor or binding activities are significantly over-represented by TAGs. Simulation reveals that random gene rearrangements have little effect on the statistics of TAGs for all genomes. It is noteworthy to mention that gene family sizes are significantly correlated with the extent of tandem duplication, suggesting that tandem duplication is a preferred form of duplication, especially in large families. There has not been any systematic study of TAG genes' expression patterns in the genome. Taking advantage of recent large-scale microarray data, we were able to study expression divergence of some of the TAGs of size two in human and mouse for which the expression data is available and examine the effect of sequence divergence, gene orientation, and physical proximity on the divergence of gene expression patterns. Our results show that there is a weak negative correlation between sequence divergence and expression similarity between the two members of a TAG, and also a weak negative correlation between physical proximity of two genes and their expression similarity. No significant relationship was detected between gene orientation and expression similarity. Moreover, we compared the expression breadth of upstream and downstream duplicate copies and found that downstream duplicate does not show significantly narrower expression breadth. We also compared TAG gene pairs with their neighboring non-TAG pairs for both physical proximity and expression similarity. Our results show that TAG gene pairs do not show any distinct differences in the two aspects from their neighboring gene pairs, suggesting that sufficient divergence has occurred to these duplicated genes during evolution and their original similarity conferred by duplication has decayed to a level that is comparable to their surrounding regions. / Master of Science

Thesis (PhD)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Pathogenic organisms frequently utilize proteases to perform specific functions related to virulence. There is little information regarding the role of proteolysis in Mycobacterium tuberculosis and no studies on the potential involvement of these enzymes in the pathogenesis of tuberculosis. The present study initially focused on the characterization of a family of membrane anchored, cell wall associated, subtilisin-like serine proteases (mycosins-1 to 5) of Mycobacterium tuberculosis. These proteases were shown to be constitutively expressed in M. tuberculosis, to be located in the cell wall of the organism and to be potentially shed (either actively or passively) from the wall. Relatively high levels of gamma interferon secretion by T-cells in response to these proteases were observed in Mantoux positive individuals. The absence of any detectable protease activity lead to a protein sequence analysis which indicated that the mycosins are probable mycobacterial-specific proprotein processing proteases. To identify possible substrates for these proteases, the genome sequence regions surrounding the mycosin genes were analyzed. This revealed that the mycosin genes are in fact part of a cluster of 6 to 12 genes which have been duplicated multiple times in the genome of M. tuberculosis. Due to the presence of members of the previously described ESAT-6 T-cell antigen family within this duplicated region, the five gene cluster regions were named the ESAT-6 loci. In silico analysis of finished and unfinished genome sequencing data revealed the presence of orthologues of the Mycobacterium tuberculosis H37Rv ESAT-6 loci in the genomes of other mycobacteria, e.g. M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and the avirulent strain M. smegmatis. Phylogenetic analyses done on the resulting sequences have established the duplication order of the gene clusters and demonstrated that gene cluster region 4 (Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an orthologue could be found in the genomes of Corynebacterium diptheriae and Streptomyces coelicoior. Thus, the comparative genomic analyses revealed that the presence of the ESAT-6 gene cluster seems to be a unique characteristic shared by members of the high G+C gram-positive bacteria and that multiple duplications of this cluster have occurred and have been maintained only within the genomes of members of the genus Mycobacterium. The ESAT-6 gene cluster regions were shown to consist of the members of the ESAT-6 gene family (encoding secreted T-cell antigens that lack detectable secretion signals), the mycosins (secreted, cell wall-associated subtilisin-like serine proteases) as well as genes encoding putative ABC transporters, ATP-binding proteins, and other membrane-associated proteins. Thus, from the observation that members of the ESAT-6 family are secreted without the normal sec-dependent secretion signals, it was hypothesized that the membrane-associated and energy-providing proteins function together to form a transport system for the secretion of the members of the ESAT-6 protein family. Supporting this hypothesis, one of the ESAT-6 gene clusters was shown to be expressed as a single polycistronic RNA, forming an operon structure. The promoter for this operon, P e s r e g 3. was also identified and its activity characterized. Subsequent secretion analyses results have shown that secretion of members of the ESAT-6 protein family is dependent on the presence of the proteins encoded by the ESAT-6 gene cluster regions, confirming the putative transport-associated functions of the ESAT-6 gene cluster-encoded proteins. The mycobacterial ESAT-6 gene clusters contain a number of features of quorum sensing and lantibiotic operons, and an extensive review of the literature have led to the hypothesis that the members of the ESAT-6 family may be secreted as signaling molecules and may be involved in the regulation of expression of genes during intracellular residence of the bacterium. In the final part of this study, the evolutionary history of the PE and PPE gene families (members of which is found situated in the ESAT-6 gene clusters) were investigated. This investigation revealed that the expansion of these families are linked to the duplications of the ESAT-6 gene clusters, which is supported by the absence of the multiple copies of the PE and PPE families in the genome of the fast-growing mycobacterium M. smegmatis. Furthermore, dot blot analyses showed that the PPE gene present in ESAT-6 gene cluster region 5 is able to distinguish between mycobacteria belonging to the slow-growing or fast-growing species, indicating a function for the genes of these two families and/or the ESAT-6 gene clusters in the phenotypical differences distinguishing these two groups of mycobacteria. In conclusion, this study has highlighted numerous important aspects of mycobacterial genomics and has greatly contributed to the current body of knowledge concerning the role of proteases, gene duplication and mechanisms of antigen expression and secretion in M. tuberculosis. / AFRIKAANSE OPSOMMING: Sien asb volteks vir opsomming

<p>Immune cells like NK cells, T cells, neutrophils and mast cells store high amounts of <u>gr</u>anule <u>s</u>erine <u>p</u>rote<u>ases</u>, graspases. Graspases are encoded from the mast cell chymase locus. The human locus holds four genes: α-chymase, cathepsin G, and granzymes H and B. In contrast, the mouse locus contains at least 14 genes. Many of these belong to subfamilies not found in human, e.g. the Mcpt8-family. These differences hamper functional comparisons of graspases and of immune cells in the two species. Studies of the mast cell chymase locus are therefore important to better understand the mammalian immune system. </p><p>In this thesis, the evolution of the mast cell chymase locus was analysed by mapping the locus in all available mammalian genome sequences. It was revealed that one single ancestral gene founded this locus probably over 215 million years ago. This ancestor was duplicated more than 185 million years ago. One copy evolved into the α-chymases, whereas the second copy founded the families of granzymes B and H, cathepsin G, Mcpt8 and duodenases. Different subfamilies were later remarkably expanded in particular mammalian lineages, e.g. the Mcpt8- and Mcpt2-subfamilies in the rat. Four novel members of these families were identified in rat mucosal mast cells. Rat and mouse mast cells express numerous different graspases, whereas human and dog mast cells express only one graspase, chymase. To better understand mast cell functions in these species, one member of the mouse Mcpt8-family, mMCP-8, and human and dog chymase were studied. The preferred substrate sequence was analysed by substrate phage display. mMCP-8 remains yet enigmatic, although it is probably proteolytically active. Dog and human chymase, interestingly, have common preferences in certain substrate positions, but differ in others. These two chymases may have coevolved with an <i>in vivo</i> substrate that is conserved only in the positions with a common preference. We also obtained evidence that substrate positions on either side of the scissile bond influence each other. This kind of interactions can only be detected with a method investigating both sides simultaneously, such as substrate phage display.</p>