Proteins of uniform sequence and stereochemistry can be produced in bacterial cells for use in fundamental studies of polymer morphology. In earlier work, a copolypeptide with repetitive sequence 1, designed to adopt a lamellar architecture upon crystallization, was synthesized using genetic engineering techniques. Solid state studies indicated that the material did not adopt the desired architecture, perhaps because the odd number of amino acids in the repeat unit prevented the formation of the appropriate chain trajectory for chain folding, disrupting the normal hydrogen bond pattern associated with $\beta$-sheets (a key component of the desired chain-folded assembly). It was asserted that this energetic liability would be offset by adding hydrogen-bonding pairs to the bulk structure, by insertion of additional alanylglycine dyads in sequence 1.$$\lbrack\rm (AlaGly)\sb3ProGluGly\rbrack\sb{\rm m}\eqno{\bf 1}$$ To test this premise, solid state analysis of a series of repetitive copolypeptides with increasing numbers of alanylglycine dyads (sequences 2) was accomplished.$$\lbrack\rm (AlaGly)\sb{\rm n}ProGluGly\rbrack\sb{\rm m}\quad n = 3, 4, 5, 6\eqno{\bf 2}$$In a preliminary study, a copolypeptide with repetitive sequence ((AlaGly)$\sb4$ProGluGly) $\sb{14}$ was synthesized. X-ray diffraction and infrared analysis of this material indicated an increase in solid state order over that of polypeptides with sequence 1. Mass analysis of this material using matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry prompted the discovery of sequence errors in the DNA code for the protein. This analysis and the analysis of other chain-length variants of sequence 2 (n = 4, m = 10, 12, 16, and 20) highlighted the utility of the MALDI technique for the assessment of protein sequence, molecular weight and purity. Analysis of proteins with sequence 2 (m = 16, n = 3, 4, 5, 6) using differential scanning calorimetry, infrared spectroscopy, electron microscopy and X-ray diffraction was consistent with an increase in solid-state order with an increase in the number of alanyl-glycine dyads in the repetitive sequence. A model is presented featuring folded chains, which is rationalized on the basis of comparison of the X-ray scattering patterns of the materials with those of poly(L-alanylglycine).

The objective of this dissertation was to study B cell development and the mechanism of immunoglobulin gene diversification in cattle. The bovine immune system differs from that of the mouse and human in several respects. First, the bovine have an unique lymphoid tissue, the ileal Peyer's patch (IPP). Secondly, at least 90% of the immunoglobulin molecules produced by bovine are of the lambda ($\lambda$) isotype. Northern blot and FACS analysis were performed in order to document the distribution of B cells found in calves of various ages. B cells were located in the IPP, jejunal Peyer's patch, spleen and peripheral blood. It was demonstrated that $>$90% of the IPP lymphoid cells were IgM$\sp+$ B cells. In contrast to the IPP, bone marrow of young calves did not appear to be a site for B cell development. To determine the mechanism by which bovine create a diverse repertoire of antibody molecules, cDNA clones encoding $\lambda$ light chains from IPP B cells were sequenced. A comparison of these sequences indicated that all of the V$\sb{\lambda}$ regions were very homologous to one another, and there appeared to be three different V$\sb{\lambda}$ regions expressed. This correlated with Southern blot analyses which showed a limited number of V$\sb{\lambda}$ gene segments in the germline. The bovine $\kappa$ light chain gene was also investigated. Based on mRNA expression and cDNA sequence analysis, it was found that cattle appear to possess a functional $\kappa$ locus. Preliminary studies suggest that the bovine $\kappa$ locus may have a limited number of V$\sb{\kappa}$ gene segments in the germline DNA. These data suggest the following: (1) IPP tissue, not bone marrow, is the major site of B cell development in a young calf. (2) Bovine B cells do not depend upon a large number of V$\sb{\lambda}$ gene segments to produce their immunoglobulin repertoire. Thus, diversity must be generated by another mechanism, perhaps somatic mutation or gene conversion. (3) Cattle are able to transcribe the $\kappa$ light chain gene, although the number of V$\sb{\kappa}$ gene segments may be limited in number.

This dissertation was to study the sites of B cell development, the mechanisms of Ig diversification, the time when diversification occurs, the B cell repertoire during development and usage of Ig light chains in cattle. The expression of TdT, RAG-1 and RAG-2 was detected in spleen in young animals but not in old animals indicating that gene rearrangement of B cells takes place in spleen and is restricted to a short period of time during early development in cattle. To determine the mechanisms by which cattle create a diverse Ig repertoire, genomic rearrangement patterns and V$\lambda$ sequences of cDNA and germline genes were examined. Few rearrangements, potential donor sequences in germline pseudogenes and point mutations in the cDNAs were found. These data suggest that cattle primarily use gene conversion to create a diverse Ig repertoire and somatic hypermutation may be used to refine the Ig repertoire upon antigen challenge. To investigate when diversification occurs, V$\lambda$ IPP cDNA were compared between young and old animals. Many diverse nucleotides were found in young animals as that in old animals indicating that the diversification takes place in the very early stage of development. To determine the peripheral Ig repertoire during development, expression of peripheral light chains of an animal at different ages was examined. Diversity clustered in the CDRs was increased and different clones which were present in the periphery at a low level in neonatal animals were found to be expanded in older animals. These data indicate that the peripheral Ig repertoire is ligand-selected and clonally expanded by positive selection. To investigate the control of $\kappa$/$\lambda$ usage, expression and germline $\kappa$/$\lambda$, V$\kappa$ and C$\kappa$ repertoire were examined. The ratio of expressed $\kappa$/$\lambda$ was high in IPP and spleen, but it was low in the periphery. Additionally, similar numbers of $\kappa$/$\lambda$ genes were found in the germline and V$\lambda$ cDNA sequences were found to be as diverse as that of V$\lambda$. In contrast, C$\kappa$ possesses a low homology to other primarily $\kappa$ expressing mammals. These data suggest that a post-transcriptional control may govern the usage of $\kappa$/$\lambda$ in cattle.

Mesophilic cellulolytic clostridia play an important role in the global carbon cycle because they are able to degrade abundantly-produced cellulosic material. Many strains may also take part in the nitrogen cycle inasmuch as they are able to satisfy their nitrogen requirement for growth by fixing dinitrogen (N$\sb2).$ The first objective of this research was to characterize nitrogenase systems of mesophilic cellulolytic clostridial species using physiological and molecular approaches. DNA probes, nifDK, vnfDGK, and anfDGK, constructed from structural genes representing three different nitrogenase systems in Azotobacter sp., were used to detect and clone potential nitrogenase genes from cellulolytic clostridia. A 7-kb genomic DNA fragment, which hybridized the nifDK probe, was cloned from Clostridium cellobioparum ATCC 18532, and a 2.1-kb subfragment was found to contain a nifH sequence. Northern analyses suggested this sequence was expressed under nitrogen-fixing conditions. Based on phylogenetic analyses involving 34 known nifH gene sequences, nifH from C. cellobioparum was most closely related to the multiple nifH genes of Clostridium pasteurianum. In addition, a 3-kb genomic DNA fragment was cloned from Clostridium hungatei B3B and found to have a high sequence identity to anfDGK from Azotobacter vinelandii. However, in studies of nitrogen fixation by C. hungatei B3B under Mo-deficient conditions, ethane was not detected during acetylene reduction assays, suggesting that the putative anfDGK genes were, if expressed in C. hungatei, part of a nitrogenase system that differed from that of A. vinelandii. The second objective of this research was to isolate plasmids indigenous to cellulolytic clostridia that may be used as vectors in strain modification. Several antibiotic resistant strains were screened for the presence of plasmids and a small plasmid (2450 bp), designated pMCF1, was isolated from strain MCF1 and ligated to the Hind III site of the vector pBluescript II KS. DNA sequence analyses identified an open reading frame encoding a peptide with homology to plasmid replication proteins. It is proposed that pMCF1 belongs to a subfamily of the single-strand DNA plasmids, which replicate via a rolling circle mechanism.

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), 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.

The yeast mitochondrial genome encodes seven hydrophobic subunits of oxidative phosphorylation enzymes and Var1p, an essential protein in the small ribosomal subunit. Expression of the membrane proteins is dependent on nuclear, mRNA-specific regulatory genes, several of which specify translational activators that recognize sites within the 5$\sp\prime$-untranslated leaders (UTLs) of their target mRNAs. At the onset of this work, it was not known if the expression of the Var1p also requires mRNA-specific regulatory genes. To investigate this and other aspects of Var1p synthesis and function, I developed a novel system in which Var1p is supplied from a recoded gene in the nucleus (VAR1$\sp{u})$ and the VAR1 coding sequence in mtDNA is replaced by a recoded nuclear gene for an arginine biosynthetic enzyme (Arg8p), thus creating a reporter gene designated $var{\it 1\/}{:}{:}ARG{\it 8\/}\sp{m}.$ This system has been used to address the following objectives: (1) Genetic screens were conducted to identify nuclear mutants defective in $var{\it 1\/}{:}{:}ARG{\it 8\/}\sp{m}$ expression. One such gene, SOV1, was identified and cloned. SOV1 is specifically required for the stable accumulation of VAR1 mRNA. (2) To determine whether the targeting information for mitochondrial membrane proteins is contained in UTLs of their mRNAs, I examined the ability of chimeric mRNAs containing the VAR1 UTL to direct expression of COX2 and COX3. Although cells expressing these chimeric mRNAs synthesized both proteins, they were deficient in the accumulation of Cox2p and Cox3p. These data suggest that translation of Var1p is different from that of the membrane proteins, and support the physiological importance of interactions between the translational activators and the 5$\sp\prime$-UTLs of the COX2 and COX3 mRNAs for localizing synthesis of hydrophobic proteins to the inner membrane. (3) Heretofore, it has not been possible to produce respiratory competent $\lbrack rho\sp+\rbrack$ diploids by mating two $\lbrack rho\sp-\rbrack$ haploid petites. An explanation for this lack of functional complementation is that $\lbrack rho\sp-\rbrack$ cells are devoid of small ribosomal subunits and translation cannot be restored without a source of Var1p. I have shown that respiratory competent diploids can be obtained in crosses between two complementing $\lbrack rho\sp-\rbrack$ strains, but only when Var1p is supplied from $VAR{\it 1\/}\sp{u}.$

Genetic and physical mapping of human and animal genomes has been greatly facilitated by the use of chromosome specific DNA libraries. Mapping with libraries specific to a chromosome or chromosomal region increases marker saturation by reducing the gaps resulting from a purely random shotgun approach. This study was undertaken to construct a genetic and physical map of microsatellites on the chicken Z chromosome. This chromosome is the fifth largest in the chicken genome, comprising about 8% of the total, yet very few microsatellites have been mapped to it. DNA originating from the chicken Z chromosome was previously isolated and reported. This was used to construct a small insert library in Lambda ZAP Express, representing 14 chromosome equivalents. This library was screened for microsatellites with an (AC)12 oligo, and positive clones were isolated. Confirmation of the presence of the microsatellite, as well as its approximate location in the insert was accomplished by PCR amplification. Clones with adequate flanking regions were sequenced, and primers for 19 microsatellites were developed. These primers were used to genotype individuals from the East Lansing poultry reference population and a linkage map was constructed. Thirteen markers were scorable and polymorphic in the population. These were combined with 64 existing markers, and the resulting map spans 220 cM with an average spacing of 2.7 cM between markers. The physical location of selected markers were established by fluorescent in situ hybridization (FISH.) Hybridization results enabled the anchoring and orientation of the linkage group along the length of the Z chromosome.

The anteroventral periventricular nucleus (AVPV) of the preoptic area mediates the positive feedback effects of estradiol (E2) on LH surge in rats. Consistent with their role in female reproduction, the neurons in this region are more numerous in adult females than males. This sex difference is established due to E2-mediated cell death in the developing male AVPV. Loss of neurons in the AVPV permanently abolishes the ability of E2 to trigger LH surge release. However, the identity of the neurons lost during AVPV masculinization and the mechanism underlying E2-triggered cell death have not been clearly defined to date. This dissertation shows that, developmental exposure to E2 permanently reduces the number of dual-phenotype GABAergic/Glutamatergic (GABA/Glu) neurons, supporting the role of these neurons in female-specific LH surge release. My results identified a key role for TNFα-activated NFκB-mediated cell survival in establishing sex differences in the GABA/Glu population in the AVPV. GABAergic neurons in males had higher levels of TRAF Interacting Protein (TRIP), an inhibitor of the NFκB pathway. Thus, the male AVPV had lower levels of nuclear NFκB and its downstream target, pro-survival Bcl-2 mRNA than females. I also showed that E2 produces these sex differences by upregulating TRIP gene expression and thus reduces the number of GABA/Glu neurons in the male AVPV. Using the N42 GABA/Glu cell line as an in vitro model for the AVPV, I verified that E2 reversed TNFα-mediated effects on NFκB activation, Bcl-2 mRNA, and caspase activity. Moreover, E2 could directly upregulate TRIP mRNA levels, only in the presence of TNFα. To understand the nature of this cooperativity, I cloned the proximal TRIP promoter, identified an ERE half-site and a novel TNFRE in a region 1000 base pairs upstream of the transcription start site. Mutation of either of these sites abolished the stimulatory effect of E2 on promoter activity, suggesting that cooperative action of E2 and TNFα is required for TRIP promoter activation. To summarize, these studies provide a novel mechanism for sexual differentiation of the AVPV in which E2 acts cooperatively with TNFα to inhibit a neuroprotective pathway in GABA/Glu neurons of the male AVPV.

Many plant RNA viruses provide replication and encapsidation functions for one or more subviral RNAs that can modulate the symptoms of the associated helper virus. In this dissertation, I report my studies on symptom modulation in Arabidopsis thaliana by subviral RNAs associated with turnip crinkle virus (TCV), a single-stranded, positive-sense, plant RNA virus. Satellite RNA C (satC) is a virulent satRNA that normally intensifies symptoms of wild-type (wt) TCV but can attenuate symptoms if the TCV coat protein (CP) is either replaced with that of cardamine chlorotic fleck carmovirus (TCV-CPCCFV) (Kong et al., 1995) or if TCV contains an alteration in the CP initiation codon (TCV-CPm) (Kong et al., 1997b). I found that TCV-CPm produced reduced level of a CP (10∼20% of wt level) that contained two additional amino acids at its N terminus and did not form virions in infected protoplasts. SatC did not substantially affect the accumulation of TCV-CPm genomic RNA in protoplasts. These results, along with data reported previously (Kong, 1996), led to the conclusion that satC-mediated symptom attenuation of TCV-CPm involves a reduction in virus long-distance movement (Kong et al., 1997b). By characterizing the promoter for the CP mRNA, i.e., the 1.45-kb sgRNA promoter and defining the sequence and structural elements required for promoter activity, I was able to construct TCV variants expressing a level of wt CP similar to TCV-CPm (10∼20% of wt). I found that these mutants also have their symptoms attenuated by satC, indicating that the level of viral CP, not the mutation in the N terminus, is the crucial factor in determining whether satC is going to attenuate or exacerbate symptoms. Another normally virulent subviral RNA, namely defective interfering RNA G (diG) exhibited different symptom modulation of TCV-CPm compared with satC, and the determinants for this differential symptom modulation were previously localized to the T-terminal 100 bases of the subviral RNAs containing six positional differences (Kong et al., 1997a). In this dissertation I report the further characterization of the determinants in these six positions and the possible mechanism underlying the differential symptom modulation by these two subviral RNAs. My results revealed that two positions located in the 3′-terminal stem-loop structures of satC and diG, which also serve as promoters for complementary strand synthesis, are critical for symptom modulation. Furthermore, the hairpin CP binding capacity correlates with the symptom modulation. Several models for symptom modulation by the subviral RNAs associated with TCV are proposed.