This study reports the development of chromosome-specific libraries by chromosome microdissection and microcloning and its utility in developing high density linkage map for particular chromosomes. Chromosome-specific painting probes were prepared for bovine (Bos taurus) autosomes 11 and 23 using two different translocation cell lines. Chromosome painting probe for swine chromosome 6 was developed using chromosomes from primary swine fibroblast cultures. The purity and specificity of the painting probes was verified by fluorescent in situ hybridization (FISH) on bovine and swine metaphase chromosomes. Bovine painting probes were used on sheep (Ovis aries) and goat (Capra hircus) metaphases to identify their corresponding homologs. BTA 11 and SSA 6-specific DNA fragments were cloned in Lambda Zap Express vector to develop high titer chromosome-specific libraries. BTA 11 library was screened for microsatellite-containing clones using (AC)$\sb{12}$ oligos. Primer pairs developed for 17 microsatellites yielded successful amplifications with bovine genomic DNA. Three markers were binned on Illinois Reference/Resource family (IRRF) and 14 were mapped on the USDA-MARC resource family. Two point analysis was done on MARC population to generate a preliminary linkage map for BTA 11. A bovine YAC library was screened with BTA 11-specific microsatellite primers. Four YACs were identified and physically mapped by FISH. Two YAC clones that were mapped to BTA 11 by linkage and by FISH helped to orient and anchor the linkage map on bovine chromosome 11. BTA 11-specific DNA was subjected to subtractive hybridization using bovine Cot4 DNA and the subtracted product was used to screen a bovine cosmid library. Positive clones were pooled and mapped to bovine metaphases by FISH. All the clones showed very strong hybridization on the centromeres of all autosomes in the bovine chromosome complement. However, they failed to hybridize to the sex chromosome centromeres suggesting that the sequences are specific to autosomal centromeres. The same probes failed to hybridize to sheep and goat metaphases suggesting species specificity of these probes. An answer to the exact function of these DNA sequences need to be investigated.

The Smyth line chicken (SL), an avian model for autoimmune vitiligo, is characterized by a postnatal loss of melanin pigmentation in feathers and the choroid of the eye. Earlier studies supported a hypothesis that an inherent pigment cell defect is necessary for the amelanosis which is the consequence of the selective destruction of melanocytes by the immune system. Data collected during the last decade has demonstrated that the MHC plays a major role in the development and severity of the SL vitiligo. The major objective of this study was to determine the factors contributing to the variable expression of the disease in the SL102 subline, selected by serotyping to be homozygous for the 102 MHC haplotype. The homogeneity of the MHC loci among SL102 and BL (parental control) birds was evaluated by serological, mixed lymphocyte response (MLR) and restriction fragment length polymorphism (RFLP) techniques. The results of these analyses were then studied in respect to their effects on the variation in incidence and severity of autoimmune vitiligo. All SL102 birds used in these studies appeared to carry the same, or nearly identical Ea-B haplotype as determined by serology as well as by MLR. Surprisingly, only 15 out of 22 BL birds were homozygous for Ea-B102, possibly due to a previous pedigreeing error. RFLP genotype assignment based on 4 different restriction enzymes and MHC class I and class II probes did not appear to have any influence on the incidence of vitiligo. No polymorphisms were found among SL102 birds using class II probe, while 2 RFLP genotypes were revealed with the class I probe. One of these loci was shown (P $<$.05) to be associated with the severity of the vitiligo. Peripheral blood lymphocytes obtained from the serologically haplotype matched SL, BL and an unrelated control (LBL) birds were analyzed for their mitogenic responses as well as their lymphocyte subset populations. SL102 birds had a higher proportion of CD4+ and CD8+ T-cells and a lower proportion of TCR1+ T-cells; but, they were also the poorer responder to a T-cell mitogen, ConA. One of the variant B-F RFLP polymorphisms was associated with a low ConA response, although these birds had a higher proportion of CD8+ T-cells than controls. From this study it is hypothesized that MHC class II locus is involved in the incidence of autoimmune vitiligo where as the severity of the disease is associated with MHC class I genes. The variation in mitogenic response and lymphocyte subset populations are possibly associated with subline differences that may not be MHC-related.

Methods for engineering mice to express polyclonal repertoires of human antibodies are well established and their use to produce human monoclonal antibodies of predefined specificity has been widely demonstrated (Ishida, et al., 2002; Lonberg, et al., 1994; Mendez, et al., 1997; Nicholson, et al., 1999). Although such engineered mice do expresses diverse repertoires of human antibodies and are immunophysiologically similar to humans; due to their small size, they are not suitable for the production of significant quantities of human polyclonal antibodies (hPAbs). Currently, hPAbs are in wide clinical use for prophylaxis and therapy in immunocompetent and immunodeficient patients [Keller, et al., 2000). Because these antibodies are obtained from human sources their supply is limited and their titers are often low because immunization protocols to raise pathogen-specific antibodies in donors are optimized for safety rather than for magnitude and duration of antibody response. Given these limitations, a technology for the production of antigen-specific hPAbs in large nonhuman hosts is novel and has significant biomedical and biodefense interest. Considering the differences in the mechanisms and strategies used by bovines and humans to diversify their antibody repertoires (Butler, 1998; Flajnik, 2002; Reynaud, et al., 1991; Meyer, et al. , 1997), questions arise about the capacity of HACs to sponsor the generation of functional human Ig repertoires. This prompted the following critical questions to be addressed: Can a large and viable population of human Ig-producing cloned HAC-Tc cattle be produced? Does human Ig synthesis persist as the animals mature? Is any portion of the human Ig assembled as fully human antibodies free of bovine heavy or light chains? Do rearranged human heavy chain loci undergo class switching in bovine cells? Does the HAC construct encode a broad diversity of human immunoglobulins in cattle? Most importantly, does immunization induce any fully human, antigen-specific polyclonal antibodies in cloned, HAC-Tc cattle, and effect protective functions? Resolving these questions is necessary to determine if the immunological divergence of bovines and humans prevent the use of HAC-bovines as suitable bioreactors for production of human antibodies for therapy. The availability of cloned HAC-Tc cattle that are imrnunologically mature has enabled the conduct of studies to address these questions, and the following results have been obtained. Biochemical and serological studies determine that fully human Ig isolated from HAC-Tc cattle is polyclonal and is comprised of both human μ and γ isotypes, demonstrating that the HAC-borne human IgH locus undergoes class switching within bovine cells. (Abstract shortened by UMI.)

Cattle represent an economically important species whose immune system seems to depart from the standard human and murine models. Little has been documented about bovine lymphocyte activation in vitro, such work is needed for comparative immunology and for us to understand bovine immunology. In general, lymphocyte activation is accompanied by many molecular changes at the mRNA level. The unique characteristics of lymphocyte activation imply that a unique set of genes is associated with this biological process. However, there is very little known (in any species) about lymphocyte-specific genes that are differentially up-regulated after activation. In this thesis, bovine lymphocyte activation was first stimulated in vitro. Secondly, representational difference analysis (RDA) method was used to clone mRNAs that are exclusively present in activated bovine lymphocytes. Subsequently, the cDNAs were analyzed by DNA sequencing and homology search against the Genbank database. Clone E8 was identified as a potential G-protein-coupled receptor. E8 is up-regulated in activated bovine lymphocytes at 2 hours post stimulation. When up-regulated, E8 mRNA level remains constant from 2 hours to at least 72 hours post stimulation. Similar kinetic expression of E8 is observed following either LPS or Con A stimulations. Expression of E8 was also detected in murine lymphocytes upon activation with LPS or Con A and with similar kinetic expression. E8 showed increased level of expression when human T and B lymphocytes were activated by cross-linking of antigen receptors along with costimulatory molecules. E8 expression was found not to be associated with resting non-lymphoid tissues, activated non-lymphoid cell lines, nor activated macrophages and neutrophils. Therefore, E8 represents an early gene specific to lymphocyte activation. The size of the full-length transcript of clone E8 was estimated at about 2.2 kb. A full-length cDNA was obtained by the RACE procedure. Sequence alignment revealed that E8 is homologous to EB11, a human gene induced by EBV; CXCR1, as well as human CCR4 and CCR5 genes. Potential biological functions of this gene are discussed.

At fertilization, the sperm induces a series of species-specific intracellular Ca2+ rises ([Ca2+]i) that are important for oocyte activation and embryonic development in all mammalian species studied to date. These have not been investigated in the horse, where in vitro assisted reproduction techniques generally provide suboptimal results. In this study, we characterize the activity of equine sperm extracts (sperm factor; eSF) in mouse oocytes, and demonstrate their ability to induce [Ca 2+]i transients and parthenogenetic activation in equine oocytes. However, sperm injected (ICSI) horse oocytes do not consistently mount [Ca2+]i responses, and this failure is not the result of inadequate sperm factor release into the oocyte. This research may explain suboptimal results reported for ICSI in the equine and suggests potential differences in fertilization induced-signaling mechanisms amongst different mammalian species.