Two-bottle preference testing of inbred strains (129/J, BALB/cByJ, C3HeB/FeJ, C57BL/6J, and DBA/2J), a replicated half-diallele of Intercross (F1) progeny from these strains, (C57X129)X129 Backcross progeny, and (C57X129) X (C57X129) Intercross (F2) progeny were used to examine phenotypes for sodium saccharin (10$\sp{-3}$M) and d-phenylalanine (10$\sp{-1}$M). The genetic correlation from the inbred strains (0.80 $\pm$ 0.012) indicated shared genetic variance between the two phenotypes. Broad and narrow sense heritabilities were calculated for sodium saccharin (0.95 and 0.75) and for d-phenylalanine (0.96 and 0.74). The sodium saccharin phenotype was found to be monogenic in two segregating populations. Lack of significant linkage for this phenotype with the Mup-1 locus on chromosome four has been taken to indicate that Sac and dpa (on chromosome four) are independent loci. The d-phenylalanine phenotype appears to be determined largely by the Sac locus. / Source: Dissertation Abstracts International, Volume: 56-08, Section: B, page: 4631. / Thesis (Ph.D.)--The Florida State University, 1995.

Genetic and environmental contributions to the ingestion of sweet tastants (sucrose, glucose + saccharin) were examined using two complete diallel crosses of four inbred mouse strains, each replicated four times. Diallel sets differed only in regard to prior gustatory experience. One diallel set had a life history of 10$\sp{-1}$M sucrose solution as its sole source of fluid and the second set had water. In adulthood and while maintained on their respective treatment regimens, food and fluid intakes were measured for individuals of both sets. Mice were then subjected to a descending concentration series of sucrose solution versus water 48-h, two-bottle preference tests, followed by a single preference test using glucose + saccharin. Results indicated significant effects of both rearing history and genetic constitution on a variety of ingestive phenotypes. Relative to the number of food calories ingested by water-reared mice, those ingested by sucrose-reared mice decreased in direct compensation for calories obtained from fluid. Across multiple sucrose concentrations, sucrose-reared mice ingested and preferred sucrose significantly more than water-reared mice. This treatment effect, apparent for sucrose concentrations not yet reported as detectable by mice, also generalized to a novel sweet tastant, glucose + saccharin. Treatment effects also largely depended on mouse genotype. Genetic influences on sweet fluid ingestion were indicated as predominantly additive. For sweet preferences, low levels of dominance were detected among sucrose-reared mice only; for glucose + saccharin intake, dominance levels were higher for both treatment groups. Indications of large additive genetic effects, in combination with no, low, and/or ambidirectional dominance are consistent with an interpretation of an evolutionary history of stabilizing selection for sweet fluid / preferences and ingestions. Results indicating environmental modification of genetic predilections for sweets emphasizes the interactive contribution of genotype and environment to sweet ingestion phenotypes. / Source: Dissertation Abstracts International, Volume: 50-03, Section: B, page: 0866. / Major Professor: Glayde Whitney. / Thesis (Ph.D.)--The Florida State University, 1988.

The rabbit provides a unique model for the study of the expressed antibody gene repertoire. Polymorphic serological determinants, called the VHa allotypes, are expressed on the VH regions of the majority of serum antibody molecules, and are usually inherited in rabbits as Mendelian codominant alleles. However, recent evidence suggests that these molecules are encoded by a small subset of the rabbit immunoglobulin VH gene family. Also, genetically unexpected alleles, called latent allotypes, are expressed upon immunization with anti-allotype antibody. These data suggest highly selective VH gene expression in the rabbit. / The purpose of this study was to investigate the genetic basis for latent VH allotype expression in the rabbit. VH region cDNA libraries were produced from spleen mRNA of a homozygous ${\it a\sp2 a\sp2}$ rabbit expressing latent al allotype, and cloned into the vector lambda gtll. These sequences derived from this library were compared to those from a homozygous nominal ${\it a\sp1 a\sp1}$ rabbit. VHal-homologies were identified by hybridization with oligonucleotide probes for VHal allotype-specific segments the VH region. / DNA sequence comparisons reveal that some latent VHal genes encode a framework one region that is completely homologous to the nominal al allotype. Other latent VHal genes, however, have blocks of sequence identical to nominal VHal in the first or third framework regions that are flanked by VHa2 or VHa-negative sequences. These latent cDNA sequences, which are composites of more than one allotype, may be directly encoded by composite germline VH genes. Alternatively, they may be the products of somatically generated recombination or gene conversion between genes encoding nominal allotypes. The latent sequences do not appear to be the result of extensive modification by somatic point mutation. These results support the hypothesis that the rabbit germline VH gene family encodes sequences for more than one VHa allotype, and that the expressed VH repertoire is regulated and highly selective. Models for the mechanism of this regulation of allotype expression are discussed. / Source: Dissertation Abstracts International, Volume: 48-12, Section: B, page: 3501. / Thesis (Ph.D.)--The Florida State University, 1987.

A rRNA gene cluster containing the isoleucine-alanine tRNA-type spacer was cloned and sequenced from ten genera of the Enterobacteriaceae and one outgroup (Xanthomonas maltophilia) resulting in 27,000 bases of new sequence data. Primary and secondary structure analyses of homologous coding and noncoding sequences revealed the following: (1) variable regions in the rRNA genes are present which may prove to be informative in inferring relationships within a rapidly evolving group; (2) a highly variable region was discovered in the 23S rDNA which contains a 120 base-pair insert (IVS) in Providencia stuartii; (3) extra sequences are present in the 16S-23S rDNA spacer region which appear to be related and form long, stable, stem-loop structures which may serve as alternate recognition sites for RNase III processing; (4) phylogenetic analyses for both the rDNA and spacer regions were highly congruent; and (5) these results are in general agreement with those based on DNA-DNA hybridization data, but further reveal a closer relationship of Salmonella with Escherichia and Shigella and a distinct Klebsiella-Citrobacter-Enterobacter clade. / Source: Dissertation Abstracts International, Volume: 52-03, Section: B, page: 1209. / Major Professor: Robert H. Reeves. / Thesis (Ph.D.)--The Florida State University, 1991.

<p> Gene expression pathways exhibit many "twists and turns," with theoretically numerous ways in which the pathways can be regulated by both negative and positive feedback mechanisms. A key step in gene expression is RNA maturation (RNA processing), which in the bacterial cell can be accomplished through RNA binding and enzymatic cleavages. The well-characterized bacterial protein Ribonuclease III (RNase III), is a conserved, double-stranded(ds)-specific ribonuclease. In the gram-negative bacterium <i>Escherichia coli</i>, RNase III catalytic activity is subject to both positive and negative regulation. A recent study has indicated that an <i>E. coli</i> protein, YmdB, may negatively regulate RNase III catalytic activity. It has been proposed that YmdB inhibition of RNase III may be part of an adaptive, post-transcriptional physiological response to cellular stress. </p><p> In <i>E. coli</i>, the model organism in this study, YmdB protein is encoded by the single <i>ymdB</i> gene, and has a predicted molecular mass of &sim;18.8 kDa. YmdB has been classified as a macrodomain protein, as it exhibits a characteristic fold that specifically provides an ADP-ribose (ADPR) binding site. While YmdB can bind ADPR with good affinity, there may be additional ligands for the binding site. Thus, YmdB protein may interact with other components in the cell, which in turn could modulate the interaction of YmdB with RNase III. </p><p> In previous research conducted within the Nicholson laboratory at Temple University, affinity-purified <i>Escherchia coli(Ec)</i> YmdB and <i> Aquifex aeolicus (Aa)</i> YmdB were found to exhibit ribonucleolytic activity. This observation initiated the long-term goal of learning how YmdB regulates RNase III, and how the ribonucleolytic activity of YmdB may be involved in this process. The specific goal of this thesis project was to further characterize the ribonucleolytic activity of <i>Ec-</i>YmdB through site-specific mutational analysis. Mutations were introduced into a proposed adenine-binding pocket previously identified by crystallography and by molecular modeling. The adenine-binding pocket is a region within the macrodomain fold where ADP-ribose could bind. The mutations were examined for their effect on <i>Ec-</i>YmdB cleavage of a model RNA, R1.1. The results of this study will contribute to the development of a model describing how the ribonucleolytic activity of YmdB is regulated.</p><p>

The human genome contains nearly 1.1 million Alu elements (11% of its total DNA content). Alu elements use a copy and paste retrotransposition mechanism that can result in de novo disease insertion alleles. However, the majority of these copies are inactive. There are nearly 900,000 old Alu elements from subfamilies S and J and about 200,000 from subfamily Y or younger, which include a few thousand copies of the Ya5 subfamily which makes up the majority of current activity. Only Alu elements belonging to the younger subfamilies are currently amplifying in the human genome. Given the much higher copy number of older Alu subfamilies, it is not known why the active Alu elements belong to the younger subfamilies. We present a systematic analysis evaluating the impact of the observed sequence variation in the different sections of an Alu on retrotransposition. We have identified several sequence regions of the Alu that contribute to the relative activity levels between old and young elements. We have determined that the length of the longest number of uninterrupted adenines in the A-tail, the degree of A-tail heterogeneity, the length of the 3' unique end after the A-tail and before the RNA polymerase III terminator, and random mutations found in the right monomer all modulate the retrotransposition efficiency. These changes occur over different time frames, where some contribute to the inactivation of an Alu soon after insertion, others are likely to occur at a later evolutionary time point. The combined impact of sequence changes in all of these regions allows us to explain why young Alus are currently causing disease through retrotransposition, and the old Alus have lost their ability to retrotranspose. We present a predictive model to evaluate the retrotransposition capability of individual Alu elements and successfully applied it to identify the first putative source element for a disease-causing Alu insertion in a patient with cystic fibrosis / acase@tulane.edu

In order to study factors affecting homologous recombination in P. falciparum, two constructs were prepared containing the Toxoplasma gondii dhfr-ts gene as the selectable marker. In the first construct (pDHD-Tg23), the T. gondii dhfr-ts was flanked by 5' and 3' untranslated flanking regions (UTFRs) from dhfr-ts (chr 4), as well as the 5 ' UTFR from histidine rich protein 3 (hrp3, chr 13). In the second construct (pHSP-Tg23), the T. gondii dhfr-ts gene was flanked by the 5' and 3' UTFRs from heat shock protein 86 (hsp86, chr 7). After transfection of the pyrimethamine-susceptible 3D7 and Haiti 135 strains of P. falciparum by electroporation and selection with pyrimethamine, individual pyrimethamine-resistant clones were selected by limiting dilution. In contrast to the parental 3D7 and Haiti 135 parasites, these pyrimethamine-resistant clones contained both the P. falciparum and T. gondii dhfr-ts genes. Based on chromosomal electrophoresis and hybridization, the P. falciparum dhfr-ts gene was consistently localized to chr 4 in both the parental strains and the transfectants. In contrast, the T. gondii dhfr-ts gene was found on chr 7, and chr 13, after transfection with pHSP-Tg23, and pDHD-Tg23, respectively. Southern blotting and PCR amplification suggest that deletions occurred in the transfected constructs. Two or three copies of the pDHD-Tg23 DNA are present on chromosome 13 (3D7) and 1 copy of the pHSP-Tg23 DNA is on chromosome 7 (Haiti 135) of the pyrimethamine-resistant transfectants. These results suggest that it is possible to direct genes to specific chromosomes in P. falciparum, based on the UTFRs flanking the marker in the construct used for electroporation and selection / acase@tulane.edu

We report the first detailed karyotypic examination of untreated lymphoblastoid cell lines (LCLs) from patients with the ICF syndrome (immunodeficiency, centromeric region instability, and facial anomalies), a DNA hypomethylation disease usually due to the DNA methyltransferase DNMT3B gene mutations. These LCLs displayed distinctive cytogenetic abnormalities specifically targeted to chromosomes (Chr) 1 and 16, namely decondensation of the juxtacentromeric heterochromatin (qh), and pericentromeric rearrangements (PRs). Since these abnormalities are apparently continuously generated, the study of ICF LCLs provides an excellent model for the generation of Chr1 and Chr16 aberrations, which are also overrepresented in different types of cancer We also present the first report that extremely high levels of ICF-like 1qh or 16qh abnormalities are present in all examined, untreated chorionic villus (CV) and amniotic fluid (AF) cultures from normal individuals at late passage, but not in early passage cultures. Their frequency increased with passage number, but CV cultures had significantly more aberrations at lower passages than the AF cultures. Regardless of passage number, CV DNA, but not AF DNA, was hypomethylated at satellite 2 and globally. We propose that hypomethylation of satellite 2 DNA in CV cultures increases the frequency of 1qh decondensation, and favors Chr1 PRs. However, hypomethylation of satellite 2 DNA is not always necessary for 1qh decondensation, as observed in the late passage AF cultures Our findings on ICF LCLs have additionally given us insight into a complex 3.3-kb repeat (D4Z4). Approximately 95% of fascioscapulohumeral muscular dystrophy (FSHD) patients have <10 copies of D4Z4 on one Chr4 homologue at 4q35. We found that many restriction endonuclease sites in D4Z4 are highly methylated in normal and FSHD cell lines and somatic tissues. However, in sperm and in ICF LCLs, D4Z4 was undermethylated, as found for heterochromatic satellite 2 DNA repeats. Our data indicate that even if hypomethylation of D4Z4 does not accompany the FSHD deletion of D4Z4 repeat copies at 4q35, its highly methylated status may help repress inappropriate expression of the postulated FSHD genes in normal skeletal muscle in conjunction with a high copy number. This may occur by D4Z4 methylation helping to maintain a heterochromatin structure / acase@tulane.edu

The hypomethylating agent 5-azacytidine (5-azaC) has been shown to reactivate certain X-linked genes. In order better to understand the cellular responses to this cytosine analog, the replication pattern of the inactive X chromosome was studied using the terminal bromodeoxyuridine technique. The replication pattern of the inactive X was altered from late to early in normal female lymphocytes exposed to various concentrations of 5-azaC. There was a direct correlation between the exposure time and the frequency of X chromosomes with variant replication patterns when different exposure schemes were analyzed. There was no apparent difference in the response to 5-azaC between B- and T-lymphocytes. Lymphocytes from an individual with multiple X chromosomes in the presence of a Y (48,XXXY) were also exposed to 5-azaC and demonstrated distinct alterations in the timing of X chromosome replication. Female lymphocytes were also exposed to other S-specific agents (L-ethionine and cytosine arabinoside) and the response was comparable to that seen with 5-azaC. No gross alteration was observed in the replication timing of the autosomes of cells with variant X's Studies were performed on the incidence of earlier replicating X chromosomes in lymphocytes from normal women throughout the menstrual cycle and definite cyclic fluctuation patterns were observed. The replication pattern of the inactive X in human tumor cell lines with multiple X chromosomes was analyzed after 5-azaC treatment and was much greater than that seen in normal cells. In order to determine alterations in methylation, DNA from tumor cells before and after 5-azaC treatment was digested with the isoschizomers, MspI and HpaII. Two oncogenes were analyzed and their methylation status had not changed due to 5-azaC These studies have provided cytogenetic data concerning 5-azaC-induced changes in replication timing and are important for clarifying the mechanism controlling X inactivation and its possible link to tumor progression / acase@tulane.edu

Tumor necrosis factor alpha (TNF-alpha) is an important mediator of interstitial lung disease, but the exact mechanisms by which it promotes lung inflammation and fibrosis are unknown. In order to gain more information about the molecular mechanisms of TNF-alpha induced lung disease, transgenic mice were generated that express TNF-alpha in the lung under the control of a doxycycline inducible promoter. DNA constructs containing a mouse TNF-alpha cDNA downstream from tetracycline operator and minimal cytomegalovirus promoter sequences (TRE-TNF) and the reverse tetracycline transactivator downstream from the human surfactant protein-C promoter (SPC-rtTA) were prepared. Linear SPC-rtTA and TRE-TNF fragments were coinjected, resulting in 14 founder mice carrying both constructs. Offspring from founder animals were screened for inducible TNF-alpha expression by administering doxycycline at 0.5 mg/mL in the drinking water for 10 days and collecting lung tissue for Northern blot and histological analyses. Six of 12 lines examined exhibited TNF-alpha transgene expression in the lung that was inducible after treatment with doxycycline. Upregulation of the endogenous TNF-alpha message could be detected, suggesting activation of the wild type protein by the TNF-alpha transgene. The 320-1 line was selected for further experiments, because of its high level of transgene expression 320-1 transgenic mice exhibit perivascular, subpleural, and alveolar inflammation 10 days after induction of the transgene. An increase in neutrophils and lymphocytes in bronchoalveolar lavage fluid was seen seven days after TNF-alpha induction. Lung histology, after one month of transgene induction is characterized by massive inflammatory infiltrate, forming nodules concentrated in the vicinity of the airways and pleura. Alveolar septa in these lesions are thickened, with enlarged airspaces. At three months the phenotype becomes more severe, with focal fibrotic lesions that show thickened septa, enlarged airspaces, and architectural remodeling. Trichrome staining indicated an increase in peribronchiolar collagen. Due to the focal nature of the response, analysis for hydroxyproline, a major component of collagen, showed no significant difference between the nontransgenic and 320-1 mice at any time point. The inflammatory response in the 320-1 mice diminishes with greater lengths of TNF-alpha induction, with fewer nodules and less severe phenotypes at six and nine months In order to determine the potential interactions between TNF-alpha and transforming growth factor beta (TGF-beta), 320-1 mice were infected with an adenoviral vector carrying active TGF-beta sequences. In this model, it was hypothesized that TNF-alpha would promote inflammation, while TGF-beta caused deposition of extracellular matrix, two important events in the pathogenesis of interstitial pulmonary fibrosis. The 320-1 mice given the TGF-beta adenovirus show more severe inflammation and earlier onset of focal fibrotic lesions. However, this treatment is not sufficient to cause a widespread phenotype approaching the severity of disease seen in human patients The studies presented here indicate that TNF-alpha expression alone, or in conjuction with TGF-beta expression, is insufficient to cause severe interstitial lung disease. Although the focal areas of fibrosis resemble the phenotype seen in human patients, much of the lung remains unaffected, and the phenotype diminishes with time. The severe pathology seen in human patients is probably due to genetic interactions between many different factors / acase@tulane.edu