The Smyth line chicken (SL), a model for autoimmune human vitiligo is characterized by spontaneous, posthatch, destruction of melanocytes. Morphological and immunological changes accompanying the SL vitiligo has been well characterized, but information on the mode of inheritance of the disease is limited. In this study, a comprehensive genetic analysis was conducted to get a better picture of the genetics of this animal model. The DNA fingerprint analysis has revealed moderate level of inbreeding within the SL and BL parental sublines. 5-AzaC treatment increased the incidence of vitiligo in BL controls, while no changes were noticed in the unrelated LBL controls, providing the evidence that the BL sublines are genetically susceptible controls. High Embryonic mortality and low incidence of vitiligo were observed when the SL was used as the female parent in SL/BL matings. The number of affected females were higher, when SL was used as the male parent, suggestive of sex-linked inheritance. However, a model involving polygenic inheritance and genomic imprinting, better explain these data. Based on the results of the above experiments, SL101 and BL101 were selected to produce an F$\sb2$ population for the mapping of vitiligo genes. Genome scan was conducted with 156 microsatellite (MS) markers and 75 polymorphic markers were selected for the gene mapping. The results of the linkage analysis showed that MS markers on chromosome 1, 2 and linkage group 36 are linked to vitiligo. Candidate gene analysis revealed linkage disequilibrium between vitiligo and endogenous virus (EV) genes. EV genes were found to be expressed in SL chicken and 5-Azacytidine treated vitiliginous BL chickens. In situ hybridization experiments revealed one EV locus in LBL chickens (1q14) and 3 in SL101 and BL101 birds (1p25, 2q26 and a microchromosome). The results from this study suggests the possible effect of EV genes on SL vitiligo. One of the loci mapped on chromosome 2 is most likely an EV gene or one that is linked to it. The genes mapped at the other two loci are not identified at this time and a detailed positional cloning strategy would be necessary to identify the genes from these regions.

Although over 20 different neuron ally-expressed genes have been implicated in the regulation of body weight, the transcriptional mechanisms controlling expression of these genes remain unclear. Evidence from our laboratory suggested that the basic helix-loop-helix transcription factor Nhlh2, plays a key role in the regulation of these body weight control genes. Nhlh2 knockout mice are obese after maturation. We found that Nhlh2 is expressed in hypothalamic areas that control body weight such as arcuate nucleus (ARC), paraventricular nucleus (PVN), dorsal and ventral medial hypothalamus (DMH and VMH), and lateral hypothalamus (LHA). In ARC, we showed that pro-opiomelanocortin (POMC) neurons co-expressed Nhlh2 mRNA in a rostral-caudal pattern. In these neurons, the POMC pro-peptide is processed into several bioactive signaling peptides that regulate food intake and energy expenditure. We found that while POMC mRNA levels are comparable in normal and Nhlh2 knockout mice, levels of hypothalamic POMC derived peptides such as β-endorphin and α-MSH were dramatically reduced in the preobese KO mice. These findings suggested that Nhlh2 KO animals could have a defect that prevents production of bio-active POMC derived peptides post-transcriptionally. Since POMC is processed by prohormone convertases such as pro-hormone convertase I (PC1) and prohormone convertase II (PC2), we set out to investigate if differential expression of POMC derived peptides in wild type and Nhlh2 KO mice are regulated via regulation of PC1 and PC2 expression. Indeed, levels of PC1 and PC2 mRNAs were reduced over 50% in the KO animals. Other signaling molecules such as TRH showed the similar expression change to POMC expression that confirms our hypothesis that PC1 and/or PC2 are regulation target candidates of Nhlh2. We identified motifs in the PC1 promoters that could act as direct binding sites for Nhlh2. Thus it is likely that these genes are direct regulatory targets of the Nhlh2 transcription factor, and that decreased expression of PC1 and PC2 contribute to the maturity onset obesity in Nhlh2 KO mice.

Cuticular collagen is a major structural protein of nematode cuticles encoded by a large multigene family. The cuticle of nematode parasites provides a barrier between the worm and its hosts. This study is focused on the collagen genes expressed in the third larval (L3) and the molting third larval (3M) developmental stages of a human filarial parasite, Onchocerca volvulus , the major causative agent of onchocerciasis, or African River Blindness, one of the four leading causes of blindness worldwide. Based on the River Blindness Genome Project launched in Steven Williams' laboratory, EST analysis has identified 45 distinct cuticular collagen clusters derived from L3 and 3M stages, showing differential expression patterns through the life cycle stages. The collagens appear to be the most abundant gene family in the O. volvulus genome, reflecting their critical role in parasite development. The genomic structures of the three most highly expressed cuticular collagen genes (Ov-col-1, Ov-col-9, and Ov-col-10) have been characterized. These three genes are similar in the size of the genomic copy of the gene, transcripts and open reading frame, but belong to three distinct subfamilies based upon the feature of conserved cysteine residues. Due to its extremely high expression level and 3M-stage upregulation, Ov-col-9 is considered a putative candidate for vaccine design. The gene is 2.6 kb long, has 4 introns, a 1017 by transcript, and encodes a protein 289 amino acids in length. Western blot analyses identified the native COL-9 protein at a mass between 64 kDa and 80 kDa, about 3-fold the predicted size, indicating a nonreducible convalent crosslink between the COL-9 and other polypeptides under reducing conditions. Immunogold EM localized the COL-9 to the L3 body channel, to the 3M hypodermis, and to uterine microfilariae in the adult female. Analysis of the human IgG and IgG subclass responses to COL-9 demonstrated statistically similar levels between putatively immune (PI) and infected (INF) group except slightly elevated in IgG1 by the INF group.

Onchocerciasis, or African River blindness caused by the filarial parasite Onchocerca volvulus is the second leading cause of infectious blindness world-wide, with over 18 million people infected. The parasite has a complex life cycle with developmental stages in both the black fly vector (L1, L2 and L3) and human host (infective L3, L4, adult, microfilaria). The parasite is protected by an external exoskeleton called the cuticle, which is a complex structure composed primarily of two classes of proteins: collagens and cuticlins. Of these two types of proteins, only the cuticlins are present in the parasite and not in the human host. Because the cuticle defines the host-parasite interface, characterization of these molecules is important in understanding the first line of parasite defense. More specifically, the composition of the O. volvulus cuticle during the dynamic process of the L3 to L4 molt may elucidate a mechanism for targeting the disruption of the infection cycle. Expressed sequence tag (EST) analysis of an O. volvulus molting L3 cDNA library reveals three distinct cuticlin gene family members present in the River Blindness Genome Project dataset. Although the three genes are most abundantly expressed in the molting L3 stage, PCR analysis indicates that they are differentially expressed in other O. volvulus life cycle stage cDNA libraries (L2, L3, adult female, adult male and microfilaria). Genomic copies of these three cuticlin gene family members have been obtained and the intron-exon structure determined for each gene. The genes contain between 5 and 9 exons ranging in size from 72 to 647 base pairs. The intron sizes of the three genes range from 84–1660 base pairs, with some of the largest O. volvulus introns represented. The cDNA copies of each gene, which range in size from 42–48 kDA, have been cloned in pieces (12–15 kDA) into a plasmid expression vector, and the highly insoluble protein fragments have been expressed. The proteins have been used for antibody production and patient serum ELISAs using both infected and putatively immune sera. Additionally, the proteins were identified in O. volvulus worms using immuno-light microscopy and immuno-gold electron microscopy localization.

About 400 million individuals worldwide are currently infected with filarial parasites. To find a cure or prevention for the infections, studies of the mechanisms of pathogenesis and the survival stratagies of the parasites are necessary. At this early stage of investigation, the lack of an animal model and an in vitro culture system make it impossible to define the important genes for the parasites by any functional analysis (forward genetics). For the purpose of unraveling the survival strategies of O. volvulus, the best approach is to gain a broad knowledge of the genes that are expressed in the parasite and then select those that may be important for survival (reverse genetics). cDNA libraries of infective third stage larvae of the two major filarial parasites, Onchocerca volvulus and Brugia malyai, were constructed. EST (Expressed Sequence Tag) analysis was perfomed on randomly selected clones. 200 ESTs were analyzed from each cDNA library. Seven identical ESTs out of the first 200 ESTs from the cDNA library of Onchocerca volvulus infective stage larvae were found to be very similar to a newly discovered antioxidant enzyme: Thioredoxin Peroxidase (TPx). Two genes that match with TPx were also identified from the cDNA library of Brugia malayi infective third stage larvae through more EST analysis of the ongoing Brugia malayi genome project. The detoxification ability of parasites has always been interesting to the parasitologist because it may be a parasite survival strategy in the face of host generated oxidative stress. In this thesis, I report my success in characterizing the TPx gene and protein of Onchocerca volvulus and I discuss its possible role in parasite survival. The complete sequence of the TPx cDNA of O. volvulus reveals an ORF (Open Reading Frame) that encodes a polypeptide of 199 amino acid residues with a calculated molecular weight of 21,890 daltons. The TPx mRNA represents roughly 2% of total transcripts in the infective third stage larvae of Onchocerca volvulus. The TPx gene was expressed in the pRSET prokaryotic gene expression system and the expressed product was shown to have antioxidant activity. The antiserum raised against the expressed TPx gene product recognized a native protein from both the infective third stage larvae and adult extract that has a molecular weight of 22 kD, in agreement with the calculated molecular weight. The localization of the O. volvulus TPx protein at different stages of parasite development was detected immunochemically using the O. volvulus TPx specific antiserum. It was found to be surface located in infective third stage larvae, microfilariae and probably young adult worms. Therefore, TPx protein in O. volvulus may protect the parasites from being damaged by host generated oxidative stress. The results of immunohistochemistry combined with the results of in situ hybridization for mRNA detection, support the hypothesis that microfilariae are protected by a layer of TPx protein on their surface which is provided by the adult female worms. Evidence suggests that the expression of the TPx is induced initially inside of the intermediate host (blackfly).