Limb regeneration in urodeles is a complex process that involves the reinduction of a number of genes that are expressed during limb development. Notch is an essential developmental gene that is likely to play a pivotal role in the genetic control of forelimb regeneration in the red-spotted newt, Notophthalmus viridescens. In this thesis, a partial cDNA of the newt homologue of notch1 was cloned, as well as a complete cDNA containing only 5 EGF repeats and a NIDO domain which also showed homology to notch1. This truncated version of notch1 is suspected, based on its functional domains, to act as a secreted dominant negative regulator of Notch signaling. Real time RT-PCR detection of expression of both genes revealed that notch is expressed at relatively high levels in both the 24 hour and the 2 week regenerate. This may point towards a functional role for Notch signaling in the wound healing process, through activation of cell migration and mobilization, and in the regeneration blastema, through induction of cell proliferation and repression of differentiation. The truncated notch isoform is expressed at low levels in all the tissues that express notch, but whether this results in dominant negative regulation of Notch signaling is currently unknown. Together, these data point towards a functional role for notch in epimorphic limb regeneration.

Secretoneurin (SN) is a 33--34 amino acid neuropeptide derived from secretogranin II (SgII), a protein belonging to the chromogranin family. In static incubation studies, SN (10--500 nM) had a direct effect on pituitary fragments to increase luteinizing hormone (LH) release after 3-hour treatment. SN also induced a specific 2.6-fold increment of LHbeta subunit messenger RNA levels after 6-hour treatment. Using a western blot analysis with a polyclonal rabbit anti-SN antibody, two intermediate proteins (∼57 kDa and ∼30 kDa) likely processed from the 69.6-kDa SgII precursor were detected in the goldfish pituitary. Levels of the ∼57-kDa SN-immunoreactive protein were ∼5-fold higher in the pars distalis than the neurointermediate lobe. In summary, SN has a direct stimulatory action on LH release and synthesis. High production of SN-containing proteins in the pars distalis of the pituitary suggests the existence of a local SN-mediated mechanism to regulate LH in goldfish.

Bicuspid aortic valve (BAV) affects approximately 1% of the general population and its etiology remains unknown. Based on literature reports, we hypothesized that congenital BAV (cBAV) was a developmental defect inherited as an autosomal dominant trait with variable penetrance. We evaluated the NOS3 candidate gene based on the reported phenotype of a mouse mutant, and the FBLN2 and TIMP4 genes based on their role in heart development. DNA sequencing of these genes in our BAV families did not reveal the presence of any potential disease-causing mutations. We also examined the 3p25 region, which showed suggestive linkage at the D3S1259 marker locus in a previous 20 cM genome-wide screen. Linkage analysis with additional markers in the region suggested that the initial hit at D3S1259 had probably been obtained by chance in most families. We are currently recruiting additional families and plan to undertake a 10 cM genome screen to uncover new loci. This work represents the first effort to investigate the genetic nature of cBAV.

Dilated cardiomyopathy (DCM) is a cardiac muscle disorder characterized by ventricular dilatation and impaired systolic function. LMNA, one of fifteen autosomal genes implicated in this disease, encodes for two alternatively spliced nuclear intermediate filaments proteins, lamins A and C. Two novel missense mutations, D192G and R541 S, were identified in highly conserved regions of the LMNA gene. Electron micrographs of cardiac tissue containing the D192G mutation demonstrated dramatic morphologic alterations of the nucleus. By contrast, cardiac samples from the R541 S carrier were almost indistinguishable from those of transplanted DCM patients without LMNA mutations. Expression levels of total LMNA mRNA in this individual were comparable to those found in end-stage DCM patients without LMNA mutations. Moreover, the mutated allele was expressed in the heart tissue of this patient. Functional studies to determine the impact of these mutations on cellular models are ongoing.

This thesis reports the identification and characterization of a novel gene, named 7/2, from soybean (Glycine max L. Merrill). Genomic clones of 7/2 from two different cultivars, cvs Maple Arrow and Resnik were isolated and sequenced, and the sequences were compared to each other. These sequences were 100% identical. Alignment with a cDNA sequence from cv. Maple Arrow revealed the structure of the gene as having 6 exons and 5 introns. The full length cDNA contains an ORF of 798 by encoding a novel protein of 265 amino acids with a calculated molecular weight of 29.98 kDa. The conceptual 7/2 protein is similar in structure to Myb-like transcription factors in that it has an N-terminal DNA binding domain of 52 amino acid and a potential acidic activation domain. Unlike most known plant Myb transcription factors it has one, not two, N-terminal DNA binding repeats. As determined by RT-PCR, the7/2 message is expressed in most soybean tissues but is most highly expressed in nodules. These characteristics suggest that 7/2 may represent a novel soybean regulatory protein.

The examination of type and non-type specimens and of original descriptions for several nominal species of the genus Solea allowed me to determine that among the 97 nominal species, only eight species are valid. They are Solea solea, S. senegalensis, S. aegyptiaca, S. turbynei, S. ovata, S. stanalandi, S. elongata and S. heinii. I provide a new description for the genus with comments on its phylogenetic position, and a new description for each species. I also resurrect the genus Barnardichthys Chabanaud, 1927b for the species Solea fulvomarginata Gilchrist, 1904. I provide an identification key to species of Solea. The second part of the work is a revision of the genus Dagetichthys Stauch and Blanc, 1964, including the species that were traditionally described as Synaptura sensu Chabanaud, 1928, with a description and the phylogeny of these species. I first determined that the five species in the genus Synaptura sensu Chabanaud, 1928, were forming a monophyletic group with Dagetichthys lakdoensis as its sister group. As Synaptura has never been a valid genus name, I assigned the Synaptura sensu Chabanaud, 1928, species to the genus Dagetichthys. Its sister group is Buglossidium luteum (Risso, 1810). From the most plesiomorphic to the most apomorphic, the species are D. lakdoensis Stauch and Blanc, 1964, D. marginatus Boulenger, 1900, D. lusitanicus Brito Capello, 1868, D. commersonnii (Lacepede, 1802), D. albomaculatus Kaup, 1858, and D. cadenati Chabanaud 1948. I discuss a biogeographic scenario explaining the distribution of species and I give an identification key to species.

We studied ectopic gene conversions, i.e., gene conversions between duplicated genes located at different chromosomal positions, in eukaryotic genomes. In the first part we examined the factors affecting ectopic gene conversions in the human genome and compared their characteristics to those observed in other eukaryotic and prokaryotic species. In the second part, we examined the effect that ectopic conversions have on the GC-content of the duplicated genes found in yeast and Arabidopsis genomes. Using Stanley Sawyer's method implemented in his GENCONV program, we identified and characterized the ectopic gene conversions of the human genome. The human gene families containing 3 or more members contained 483 pairs of converted genes. The average length of conversions is 371+/-752 (+/- standard deviation) nucleotides long with the smallest conversions being 10 nucleotides long and the largest 6011 nucleotides long. Larger gene conversions are found between sequences that are more similar and the frequency of intra-chromosomal gene conversion increases as the distance between genes decreases. Pairs of intra-chromosomal genes sharing the same transcriptional orientation convert more often than intra-chromosomal genes in opposite transcriptional orientation. The excess of conversions in the 3'-end suggest incomplete cDNA molecules are often involved in gene conversions with chromosomal gene copies. Allelic recombination has previously been shown to increase the GC-content of the sequences of a wide variety of eukaryotic species. Ectopic recombination between clustered tandemly repeated genes has also been shown to increase their GC-content. Here we show that gene conversions between the dispersed genes found in the duplicated regions of the yeast and Arabidopsis genomes also increases their GC-content when these genes are more than 88% similar.

Brachydactyly type A1 (BDA1) belongs to a group of rare, congenital disorders whereby normal bone development and patterning is adversely affected, leading to shortened and malformed digits. BDA1 has the unique feature of being the first human trait described in terms of autosomal dominant Mendelian inheritance. In 2001, missense mutations within the Indian Hedgehog (IHH) gene, specifically codons 95, 100 and 131, were identified in three unrelated families affected with BDA1. Our laboratory recently identified linkage of BDA1 to a second locus on chromosome 5p13.3 in a large Canadian kindred. The critical region on chromosome 5p has been refined to 7 cM by genotype and haplotype analysis of family members. One goal of this thesis was to further reduce the 7 cM critical region, which was successful, given that a 40 kb region was eliminated. Throughout the early 1900s, William Farabee and Harry Drinkwater identified, and subsequently detailed, several families affected with BDA1 (Farabee, 1903 and Drinkwater, 1907/8, 1912, 1915). At the time of his reports, Drinkwater proposed that the Pennsylvania family, identified by Farabee, may be related to his kindreds. For years the question of whether the two Drinkwater families and the Farabee family were related through a common founder remained a mystery. This thesis provides evidence and support for a common ancestry between Drinkwater's two BDA1 families and the BDA1 family of Farabee's. In addition, this thesis also investigates IHH's role in BDA1 as numerous BDA1 families underwent IHH mutational analysis in the search for novel IHH mutations leading to BDA1.

The Distal-less-like genes, commonly referred to as Dlx genes, encode homeodomain transcription factors essential for the establishment of GABAergic interneurons in the ventral forebrain as well as their migration to the cortex. The cis-regulatory element URE2 (enhancer) is located upstream of Dlx1 and is thought to regulate the transcription of Dlx1 and/or Dlx2. The objective of this study was to examine a short conserved sequence within the URE2 element, corresponding to nucleotides 699-719, and to evaluate its contribution to URE2 activity in transgenic mouse forebrain. My results showed that a mutant version of the URE2 element, in which nucleotides 699-719 were modified, was active in the cortex and the ventricular zone of forebrain of the transgenic mice produced with the mutant enhancer, where Dlx1/Dlx2 are not usually expressed. This indicated a loss of binding by a protein with a transcriptional repressor function. Previous analyses suggested that mURE2nt699-719 would be a good candidate for a REST repressor binding site. However, we were not able to observe any transcriptional regulation by REST in co-transfection experiments performed in cultured cells. This, as well as revised bioinformatical information, led us to identify four new candidates possibly using mURE2nt699-719 as a binding site, namely the Nuclear- Factor-1 proteins: NFIA, NFIB, NFIC and NFIX. These genes are associated with transcriptional regulation, being repressors and/or activators of gene expression in the brain. We showed that NFIC was able to utilize the mURE2nt699-719 sequence to reduce the luciferase reporter expression in neuronal cells, which would explain the loss of repression in our transgenic mice at embryonic days E11.5 and E13.5. These preliminary results also allowed us to suggest that a heterodimer between NFIC/NFIB and NFIC/NFIX binds to the mURE2699-719 sequence at embryonic day E15.5 and postnatal day 0 (P0) of the transgenic mice since the mutated enhancer's activity seems to indicate a loss of an activator at those stages. These results suggest that these factors could possibly be required for the regulation of the Dlx genes by URE2. These results will also help clarify the role of URE2 by identifying transcription factors that contribute to the genetic program involved in the cellular organization of the developing forebrain in vertebrates.

In mice, natural resistance to infection with murine cytomegalovirus (MCMV) is controlled by a dominant chromosome 6 locus, Cmv1, which is expressed at the level of Natural killer (NK) cells. To characterize the molecular mechanism underlying MCMV-resistance, our laboratory initiated a positional cloning approach for the identification of Cmv1, and localized the host resistance locus to a 0.35 cM genetic interval, corresponding to 1.6 Mb genomic DNA. This dissertation presents the results leading to the cloning and characterization of the Cmv1 gene. First, I constructed a transcription map for the Cmv1 interval, which included 19 genes comprising the full Ly49 gene cluster and other novel candidate genes. In an attempt to narrow down the localization of Cmv1, I determined haplotypes in the vicinity of the Cmv1 interval in a panel of 17 inbred mice strains. This approach demonstrated the presence of three major classes of independent haplotypes. Close inspection of allele sharing among those haplotypes excluded Ly49e, Ly49f and Ly49d as Cmv1 candidates. As an alternative approach to cloning Cmv1, I took advantage of a recombinant inbred strain, the BXD-8 strain. The BXD-8 line was of particular interest because it is susceptible to MCMV infection while harboring an MCMV-resistance C57BL/6 haplotype at Cmv1. Using STS content and PFGE analysis, I showed the presence of a 23 kb deletion in BXD-8. Subsequent gene expression analysis of the full Ly49 gene cluster revealed that the deletion of Ly49h is associated with MCMV susceptibility in BXD-8 mice. Finally, to test the role of Ly49h in host resistance to MCMV infection, I introduced the Ly49h gene into a susceptible background. Investigation of Ly49h gene expression by RT-PCR and FRCS analysis demonstrated a strong correlation between the level of Ly49h mRNA and protein expression and the control of MCMV replication, providing conclusive evidence of the allelism between Cmv1 and Ly49h. In conclusion, I identified the Ly49h gene encoding a NK activation receptor as Cmv1 supporting a specific role for NK cells in antiviral immunity. Transgenic mice expressing Ly49H will be instrumental for understanding of mechanism of NK mediated resistance and possible pleiotropic effects of Cmv1.