The protein phosphatase inhibitor okadaic acid (OA) previously has been shown to induce hyperphosphorylation of p53 protein both grossly and at specific tryptic peptide sites. However, the consequences of OA induced phosphorylation (and phosphorylation in general) on p53 function in vivo remain unclear. The focus of this study was to determine if hyperphosphorylation wrought by OA or expression of human p53 in protein phosphatase-deficient yeast strains could indeed regulate the interaction between p53 and a physiological downstream target, the cdk inhibitor, p21waf1. In S. pombe, one strain containing a mutant p53 (arg->his 175) and a type 1 protein phosphatase gene knockout was unable to grow whereas both parental strains were both able to thrive, indicating a possible gain of function related to p53 phosphorylation. Rat embryonic fibroblasts harboring a highly expressed mouse p53 transgene and a p53 null control cell line were treated with 50nM doses of OA. This treatment resulted in: (1) the formation and retention of acidic p53 protein isoforms, and, more specifically, phosphorylation of tryptic peptide sites in the transactivation domain, (2) an increase in p53 affinity for a p21waf1 promotor oligonucleotide, (3) an increase in cellular steady state levels of p21waf1 message, (4) an increase in p53-dependent transcriptional activity from a waf1 reporter construct, and (5) a G2/M cell cycle blockage that is associated with intact p53. These results demonstrate for the first time that hyperphosphorylation of p53 induced by OA may regulate a critical downstream affector of cell growth suppression in an intact cellular environment.

A follow-up study of 139 women and 105 men for whom genetic counseling had been provided was conducted to assess the effectiveness of the genetic counseling service at the Arizona Health Sciences Center. Adequate recall of all of the genetic information was demonstrated by less than 2% of the respondents. Significantly more women than men were able to recall the diagnosis, whereas no difference was found between men and women in the ability to recall the recurrence risk or mode of inheritance. Eighty-seven percent of the respondents described the counseling experience as favorable. A direct relationship was noted between the degree of satisfaction and the counselees¹ 1) understanding of the counseling and 2) satisfaction with the answers to their questions. Women were significantly more concerned about pain associated with amniocentesis than were men, whereas men were significantly more concerned about injury to the mother during amniocentesis than were women.

<p> I studied the population genetic structure of abyssal grenadier: <i> Coryphaenoides armatus</i> and <i>Coryphaenoides yaquinae</i> of the Pacific and Atlantic Ocean basins, testing the hypothesis that population genetic structure is detectable by microsatellite allele analysis. I determined allele frequencies at six microsatellite loci, assessing genetic structure using Jost&rsquo;s <i>D_est.</i> </p><p> Pacific versus Atlantic <i>C. armatus</i> yielded <i> D_est</i> comparable to Pacific C. armatus caught 170 km apart. Given the subspecies designation of North Pacific versus Atlantic C. armatus, it was unexpected that <i>D_est</i> of the former comparison would approximate the latter; or that the latter would be significant at all. Pacific fish may exhibit depth-driven genetic structure. Coryphaenoides yaquinae of the eastern Pacific, CCZ, and central North Pacific were also compared. Only the CCZ versus eastern Pacific yielded unambiguously significant <i> D_est</i>. Similar values among <i>D_est</i> in both species suggest horizontal distances among sampling sites are not the principal driver of population genetic structure.</p>

<p> Fat tissue is involved in many aspects of biology such as appetite regulation, vascular diseases, diabetes, hypertension, and obesity. It plays an important role in these processes through its endocrine factors and other secretory products. Thus, there is a need to understand better the mechanisms and molecules that control the formation of adipocytes and the expansion of adipose tissue. WNT signaling is one of several important factors that plays a crucial role in development, and may also be important for adipogenesis. The activity of WNT signaling is modulated by a plethora of extracellular modulators that mostly antagonize WNT signaling. The extracellular WNT antagonists consist of four conserved families: Wnt-inhibitory factor 1 (WIF1), secreted frizzled related protein (SFRP), Cerberus, and Dickkopf (Dkk). It has been found that WIF1 is upregulated in abdominal fat tissue in chickens during early development. Thus, we hypothesize that WIF1 plays a role in adipose tissue development by inhibiting WNT signaling, and thereby stimulating adipogenic gene expression. The objective of this research is to examine the in vitro regulation of adipogenesis by WIF1. Mouse WIF1 expression vector (pCMV6-ENTRY-WIF1) was prepared, and used to transfect the mouse pre-adipocyte cell line 3T3-L1.The mRNA levels for WIF1, PPAR&gamma; and C/EBP&alpha; were then examined with real time RT-qPCR. Results indicate that the transgene was expressed in the transfected cells within 30 hours after transfection, and the mRNA level of WNT target genes and CEBP&alpha; were affected. However, the mRNA level of PPAR&gamma; was not affected. In conclusion, exogenous WIF1 was expressed in 3T3-L1 cells, at least at the mRNA level. The exogenous WIF1 expression caused an elevation of CEBP&alpha; mRNA. Future studies should examine other genes, and more investigation should take place to better understand the mechanisms of adipogenesis.</p>

Source: Dissertation Abstracts International, Volume: 41-07, Section: B, page: 2480. / Thesis (Ph.D.)--The Florida State University, 1980.

Methylation patterns in bovine satellite I DNA were studied by direct sequence analysis. Satellite I DNA was prepared as a discrete restriction fragment from genomic DNA of various bovine tissues and subjected to sequencing by the technique of Maxam and Gilbert. Sequence was obtained for a region of 350 nucleotide pairs in the satellite fragments from calf thymus and bull sperm DNA's. Comparison of sequence ladders from methylated and unmethylated DNA's (thymus and sperm, respectively) facilitated identification of methylated cytosines. / Methylation in thymus DNA was shown to be confined to the configuration 5' ('m)CpG 3', and there was no indication that these sites were methylated in the sperm sequence. In thymus DNA, there were a few occurrences of the CpG doublet in which cytosines were apparently not methylated. In regions where sequence was obtained for both strands, methylation was shown to be symmetrical. When the analysis was extended to DNA from other tissues, the unmethylated pattern was demonstrated only in chorion DNA. All other tissues showed the thymus methylation pattern. / Sequence was also obtained from a cloned satellite I fragment. This sequence was compared by computer with sequence from genomic DNA and with other known bovine satellites. A computerized homology search demonstrated degenerate repetitions within the satellite I sequence and degenerate homologies with other bovine satellites. / The results are discussed in light of other recent findings, and in terms of their possible implications for evolution and development. / Source: Dissertation Abstracts International, Volume: 42-12, Section: B, page: 4681. / Thesis (Ph.D.)--The Florida State University, 1982.

Ribosomes and transfer ribonucleic acids (tRNAs) are integral parts in the cells protein synthesis machinery. tRNAs are small polymers of ca. 77 RNA residues in length. The ribosome is a ribonucleo-protein, a large heterologous complex comprised of ca. 55 proteins and three species of ribosomal RNAs (rRNA). The ribosomal RNAs (and quite often the tRNAs) are organized on the chromosome in a close linear array known as the rRNA operons. The organization of these genes into multi-gene clusters ultimately facilitates the cell's regulation of the protein synthesis apparatus. / Isoleucine and alanine tRNAs are encoded tandemly within the 16S-23S intergenic spacer of some eubacterial rRNA operons. A previous study has demonstrated that the intergenic spacer in the eubacterium S. pneumoniae is unconventional. The isoleucine tRNA is not tandemly encoded with the alanine tRNA. Rather it appears to be encoded at the distal end of the operon near the 5S rRNA gene. This unique organization, the presence of numerous direct repeats in the non-coding regions of the 16S-23S intergenic spacer, and the potential for stem-loop formation of the isoleucine tRNA gene suggest that it might have been rearranged intrachromosomally via an illegitimate recombination. / Reported herein is a characterization of the distal spacer regions of the rRNA operons on the S. pneumoniae chromosome. Dot-blot hybridization analysis shows that the number of 16S rRNA genes on the S. pneumoniae chromosome is four. This is consistent with a previous study wherein the results suggested a minimum copy number of four for the rRNA operons. The distal spacer region from each of the four ribosomal RNA operons has been cloned and sequenced. An isoleucine tRNA is encoded on two of the four cloned fragments. DNA sequence analysis of the regions flanking the cloned isoleucine tRNA genes revealed motifs that are consistent with one of the popular models for illegitimate recombination. / Source: Dissertation Abstracts International, Volume: 53-11, Section: B, page: 5529. / Major Professor: Robert Hunter Reeves. / Thesis (Ph.D.)--The Florida State University, 1992.

Whole mount spread preparations of Tradescantia nuclei were used to examine synaptonemal complex (SC) formation. Tradescantia spreads have the standard SC morphology. Kinetochores and recombination nodules were not regularly observed. Thickenings of the lateral elements were observed and increased in numbers between mid- and late-zygotene. The thickenings were distributed evenly amongst the chromosomes, but were nonrandomly distributed within a chromosome. They were underrepresented in unsynapsed regions and overrepresented in synapsed regions, especially in synapsed regions near the junction with an unsynapsed region. / Lateral component material is well formed along the chromosome axis before SC formation begins. In early- and mid-zygotene the telomeres occur in a restricted region and have modifications for attachment at or near the nuclear envelope. Initiation of SC occurred at many sites along the chromosomes; regions nearer the telomeres had a greater tendency to form SC first. Between 29 and 106 initiation sites were observed in the early-zygotene nuclei. The average distance between initiations ranged from 7.3 to 11.2 (mu)m. The total number of potential initiation sites was estimated and ranged from 250-299. / Foldback synapsis was observed in all nuclei of Clone 02. The extent of the foldback synapsis was fairly constant at all stages examined. / The results from Tradescantia and other organisms was considered, and a model of SC formation was proposed. The key aspect of the model is that SC formation does not require homology. Synthesis of zygotene DNA is required for SC formation and is regulated such that homologous regions are in close proximity as their zygotene DNA is synthesized. This proximity, just as homologous regions have been made competent to form SC, makes it highly likely that homologous SC formation will ensue. / Source: Dissertation Abstracts International, Volume: 45-01, Section: B, page: 0061. / Thesis (Ph.D.)--The Florida State University, 1984.

A class of Neurospora crassa mutants that display the phenotype of ultraviolet light sensitivity, histidine sensitivity, accelerated mitotic recombination, and meiotic self-incompatibility were found to have impaired zinc accumulation. This may result in the formation of the pleiotropic phenotype. Since the original description of the mutant class several additional characteristics have been added including: extra-cellular nuclease deficiency, radiomimetic chemical sensitivity, amino acid transport deficiency, cell surface (gamma)-glutamyl transpeptidase deficiency, and extracellular azo reductase amplification. / The extreme divergence exhibited by the mutants implicated some fundamental mechanism of cellular control. This investigation grew from the observation that many of the enzymes involved with the phenotype were associated with zinc or copper metabolism. In comparison to wildtype the mutant was found to have a 50% reduction in ('65)zinc accumulation. The affected transport system apparently also transports Cu('+2). Zinc binding proteins in wildtype and mutant cells were examined. Two Zn('+2) binding proteins of similar low molecular weight and charge were purified from the wildtype and the mutant strain. Copper failed to bind to the zinc metallothionein-like proteins. The kinetics of zinc and copper accumulation suggest that high concentrations of copper inhibit zinc accumulation. One of the binding proteins, zinc metallothionein II, was not expressed in the mutant at physiological concentrations of zinc in media, but could be induced with high concentrations of the metal. The two metallothioneins exhibit amino acid compositions that are cysteine rich and lack aromatic amino acids and histidine, have a molecular weight of approximately 6,500; and therefore, they are similar to mammalian metallothioneins. / A model is proposed whereby zinc metallothionein II in uvs-6 is not expressed at physiological concentrations of zinc which results in impaired zinc metabolism. The enzymes of nitrogen metabolism are particularly affected by the change in zinc homeostasis. / Source: Dissertation Abstracts International, Volume: 45-04, Section: B, page: 1112. / Thesis (Ph.D.)--The Florida State University, 1984.

An amino acid transport deficient strain of Neurospora crassa grown under nitrogen limitation in the presence of an amino acid produces extracellular L-amino acid oxidases. The enzymes were purified by a protocol consisting of affinity chromatography followed by molecular sieve chromatography which results in the purification to electrophoretic homogeneity of three L-amino acid oxidases. / The native molecular weight of each of the three enzymes is approximately 83,000 daltons and the subunit molecular weight is estimated to the 85,000 daltons for L-amino acid oxidase(,1) and 81,000 daltons for L-amino acid oxidase(,2) and L-amino acid oxidase(,3). / The three L-amino acid oxidases exhibit the same pH optimum, similar heat inactivation curves, similar substrate specificities and possess a flavin prosthetic group. / Tryptic digestion of each L-amino acid oxidase followed by reverse phase high performance liquid chromatography of the resulting peptides differentiates the enzymes from each other. Each enzyme displays a unique tryptic fingerprint and appears to be a distinct L-amino acid oxidase. / In addition, L-amino acid oxidases produced in response to a number of other stress conditions were isolated and compared to the oxidases induced by nitrogen limitation. The conditions included (1) reduced biotin concentration, (2) the presence of the amino acid analogue, para-fluorophenylalanine, or (3) reduced nitrogen plus D-methionine in the growth medium. Under each of these conditions, three oxidases are produced which display isolation characteristics identical to those of the three oxidases induced by nitrogen limitation. / A comparison of tryptic digestion fingerprints of the various L-amino acid oxidases led to the formation of three distinct classes. The tryptic fingerprints of members of Class 3 display almost complete homogeneity. It is likely that these enzymes are the same polypeptide. / Limited heterogeneity in the tryptic fingerprints among the members of Class 2 prevents such a definitive statement regarding their relationship to each other, but in light of the limited number of differences, it can be stated that the homology among the enzymes is extensive. The same conclusion can be reached regarding the four enzymes in Class 1. / Source: Dissertation Abstracts International, Volume: 45-08, Section: B, page: 2436. / Thesis (Ph.D.)--The Florida State University, 1984.