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MOLECULAR BIOLOGY 9:00 AM SATURDAY, MARCH 31, 2001 KOLENBRANDER-HARTER ROOM 011 KERRY CHEESMAN-PRESIDING.

09:00 DIFFERENTIAL EXPRESSION OF ALPHA CRYSTALLIN IN THE ZEBRAFISH, DANIO RERIO. Stephanie A. Runkle, srunkle@ashland.edu, Julie Hill, Mason Posner, mposner@ashland.edu, Ashland University, Department of Biology/Toxicology, Ashland OH 44805.

Crystallins are a diverse group of proteins that produce the refractive properties of the ocular lens. One of these, alpha crystallin, is a member of the small heat shock protein family. In mammals, alpha crystallin functions as a molecular chaperone. Whereas alpha crystallin has been extensively studied in the endothermic mammals, little is known about this protein in ectothermic vertebrates. Previously, we cloned and sequenced the alpha B-crystallin subunit. Here, we used the reverse-transcriptase polymerase chain reaction (RT-PCR) to clone alpha A-crystallin from the zebrafish. Semi-quantitative RT-PCR was used to examine possible heat-induced expression of zebrafish alpha B-crystallin. There was a high level of amino acid sequence similarity between zebrafish alpha A-crystallin and the alpha crystallins of other vertebrates. Zebrafish alpha A- and alpha B-crystallin were only expressed in the lens. Mammalian alpha crystallins, in contrast, are expressed in various tissues. Zebrafish alpha B-crystallin expression was not induced after a 2-hour heat shock, even though we found increased expression of HSP 70. This lens specific expression and lack of heat shock induction suggests that zebrafish alpha crystallin plays less of a role as a molecular chaperone than its mammalian orthologue. Differences in chaperone activity may be linked to the shorter life span and cooler body temperature of the zebrafish.

09:15 PARTIAL SEQUENCE OF 16S RIBOSOMAL RNA GENE OF AMERICAN MASTODON. Anice Sabag, aasabag@cc.owu.edu, Jerry Goldstein, jegoldst@cc.owu.edu, Erin Wagner, ekwagner@cc.owu.edu, Jennifer M. LaPlante, jmlaplan@cc.owu.edu, Ohio Wesleyan University, Botany/Microbiology Department, Delaware OH 43015.

In 1989 the skeleton of an American mastodon, Mammut americanum, was unearthed in Newark, Ohio. The skeleton is the most complete and most well preserved collection of mastodon bones ever discovered. Along with the bones, material that proved to be the remains of its intestinal tract was also discovered. Intestinal contents of mammals contain significant amounts of epithelial cells derived from the tissues lining the intestinal tract. In 1998 DNA of an extinct sloth was successfully purified from epithelial cells present in its petrified dung and was subsequently amplified by polymerase chain reaction (PCR). Using similar procedures, samples of the intestinal contents of the mastodon were prepared for DNA isolation and PCR amplification. Primer pairs were designed for several known genes of the African elephant, Loxodonta africana, and PCR reactions were performed. DNA amplification was obtained with several primer pairs as noted on agarose gels. Recombinant plasmids of several successfully cloned DNA fragments were purified and the sequence of the cloned DNA was determined. The DNA sequences, with the aid of a computer, were compared with genes present in the GenBank Database. One of the gene fragments successfully amplified contains 598 nucleotides that is composed of 4 sections that were calculated to be 95%, 92% identical, 83% identical, and 85% identical to homologous regions of the 16S ribosomal RNA gene of the African elephant. To date, only one other gene fragment, a 228 base sequence of the cytochrome C gene of the American mastodon, has been obtained.

09:30 EXAMINATION OF FOCAL ADHESION KINASE (FAK) IN CELL-TO-CELL ADHESION. Melissa N. Donnelly, donnel m@denison.edu, (Catherine Smith, smithc@denison.edu), Denison University, Slayter Box 716, Granville OH 43023.

Focal adhesion kinase (FAK), a 125 kD cytoplasmic protein involved in cell-to-extracellular matrix adhesion in fibroblasts, exhibits enhanced protein-tyrosine kinase activity when bound to a substratum. In addition, cell-cell contacts in epithelial cells are associated with high levels of phosphotyrosine. FAK is one candidate protein that may be involved in mediating cell-to-cell adhesion. Our hypothesis is that activated FAK is an integral part of the protein complex involved in cell-to-cell adhesion in neuronal cells. Using an adherent, spontaneously neuritogenic cell line (B103) and a suspension, cell-to-cell adherent cell line (B103-CS), we compare FAK levels and FAK activation status to clarify the role of FAK in neuronal cell-to-cell adhesion. After performing cell lysis and fractionation yielding Triton-soluble and Triton-insoluble fractions, we immunoprecipitated both fractions with an anti-FAK antibody. Western analyses followed using an anti-FAK antibody to determine FAK levels and another antibody to determine activated FAK levels in B103 and B103-CS cell lines.

09:45 DEVELOPMENT OF SATELLITE CELLS AND MITOSIS OF NEURONAL AND NON NEURONAL CELLS IN THE SUPERIOR CERVICAL GANGLION OF THE NEONATAL MALE RAT. Christine Chiappini-Williamson, Christine M. Candea, Ronald L. Salisbury, ronald2@uakron.edu, The University of Akron, Department of Biology, Akron OH 44325.

A sex difference in neuron number occurs in the superior cervical ganglion (SCG) of rats. There are approximately 25-30% more neurons in the male rat SCG compared to the female SCG. In an effort to obtain baseline data to study factors underlying this difference, we examined cell division in neuronal and non-neuronal cells obtained from male rats on postnatal day 6, and the appearance of satellite cells on postnatal days 5, 10 and 15. Satellite cells were detected by immunohistochemistry using antibodies directed against vimentin, an intermediate filament that preferentially appears in satellite cells during postnatal development. Cell division was detected by injecting rats with 5-bromo-2-deoxyuridine (BrdU, 50mg/kg body wt.) on postnatal day 5 and the rats were terminated the next day. All animals were terminated by cardiac perfusion of 4% paraformaldehyde in 0.13M phosphate buffer, pH 7.6 Ganglia were embedded in paraffin and cut at 6 [Mu]. Cells positive for vimentin increased in number, staining intensity and organization with time. Complete satellite capsules were not evident on days 5 and 10, but were complete by day 15. Neuronal and non-neuronal cells positive for BrdU were few in number and randomly spread throughout the ganglion by day 6. We conclude that on postnatal day 6 mitosis occurs prior to the formation of satellite capsules.

10:00 EFFECTS OF A COPLANAR CONGENER PCB 77 (3,3', 4,4'-TETRACHLOROBIPHENYL) ON CHOLINE ACETYLTRANSFERASE ACTIVITY AND CIRCULATING THYROXINE ([T.sub.4]) AND TRIIODOTHYRONINE ([T.sub.3]) IN 30-DAY-OLD SPRAGUE-DAWLEY RATS. Christa L. Bowen, cbowen@bgnet.bgsu.edu, Terri L. Provost, Douglas Donahue, and Lee A. Meserve, Bowling Green State University, 255 Buttonwood Ave. Bowling Green OH 43402.

Polychlorinated biphenyls (PCB) are environmental contaminants that were first observed to cause physiological problems about 40 years after the initiation of their industrial use. PCB are a concern because of their ubiquitous and lipophilic properties that allow them to concentrate through food webs by accumulating in adipose tissue. Placental and lactational PCB exposure to offspring cause metabolic and endocrine disruptions including depressed body weight, hypothyroxinemia, spatial learning and memory alterations, and neurochemical and neurobehavioral alterations. Previous work has shown that Aroclor 1254 (a mixture of PCB congeners) consumed during pregnancy and lactation resulted in smaller offspring, offspring with depressed circulating levels of [T.sub.3] and [T.sub.4], problems with spatial learning, and alterations in choline acetyltransferase (ChAT) activity. The present study examined the effect of one specific coplanar congener, PCB 77 (3,3',4,4'-tetrachlorobiphenyl). Pregnant Sprague-Dawley rats were fed either a control diet (consisting of rat chow) or a treatment diet (consisting of standard rat chow with PCB 77 added at either 1.25 ppm (LPCB) or 25 ppm (HPCB)). Rat pups were sacrificed by decapitation on postnatal day 30, and serum frozen for RIA determination of thyroid hormones. Brains were quickly removed, placed on ice and the hippocampi and basal forebrains were dissected free for the radiometrical measurement of ChAT activity. The present study found that PCB 77 at both LPCB and HPCB significantly elevated ChAT activity in the basal forebrain and hippocampus. However, circulating levels of [T.sub.3] and [T.sub.4] were significantly depressed in these 30-day-old animals. Thus the present study suggests that a coplanar PCB congener may stimulate brain-thyroid dependence, and may help explain reports of attention deficit disorders in human offspring of mothers consuming PCB during pregnancy and lactation.

10:15 PHYLOGENETIC RELATIONSHIPS AMONG EIGHT GENERA OF HETEROCYSTOUS CYANOBACTERIA USING 23S rRNA GENE SEQUENCING. Xuesong Li, lix@muohio.edu, Megan M. Smith, smithmm12@hotmail.com, Susan R. Barnum, barnumrs@muohio.edu, Daniel J. Prochaska, prochadj@muohio.edu, Linda E. Watson, watsonle@muohio.edu, Miami University, Botany Department, Oxford OH 45056.

Ribosomal RNA operons are highly expressed genes and their products have a highly conserved secondary structure. The 23S rRNA coding region is thought to be conservative and thus may be useful for determining higher level phylogeny in bacteria. Some cyanobacterial genera can fix nitrogen in specialized cells called heterocysts, which form a monophyletic lineage. A total of 24 strains representing eight heterocystous genera (Anabaena, Noatoc, Nodularia, Cylindrospermum, Chlorogloeopsis, Fischerella, Calothrix, Scytonema) of cyanobacteria was examined using nucleotide sequences of 23S rRNA genes. Sequences were amplified using PCR and cloned. A total of 72 clones from the 24 strains were sequenced and aligned, and an evolutionary tree was constructed using PAUP 4.0 soft ware. We found that the different strains have a high sequence similarity in the 23S rRNA gene region, and that Anabaena and Nostoc are the two most closely related genera.

10:30 EVOLUTION OF NITROGEN FIXATION GENES IN HETEROCYSTOUS CYANOBACTERIA. Brian J. Henson, bhenson57@yahoo.com, Susan R. Barnum, barnumsr@muohio.edu, Linda E. Watson watsonle@muohio.edu, Miami University, Department of Botany, Oxford OH 45056.

Diazotrophy, or nitrogen fixation, is the process of converting atmospheric nitrogen ([N.sub.2]) to more reduced forms (e.g., ammonium-[NH.sub.3]). Diazotrophy is unique among prokaryotes, however not all prokaryotes have the ability to fix nitrogen. Most cyanobacteria do have this ability, and many genes involved in diazotrophy have been identified within them. The most extensively studied genes are from the nifHDK operon, which is contiguous in most cyanobacteria. However, the heterocystous cyanobacteria have an 11 kb insertion element present within the coding region of nifD, which is excised by site specific recombination during heterocyst differentiation. It is unclear whether or not all of the heterocystous cyanobacteria genera h ave this genetic element. The objective of this research is to study the evolution of nitrogen fixation genes by sequencing the nifD gene from representatives of the eight core genera of the heterocystous cyanobacteria lineage, and to determine which of these genera have the 11kb insertion element using PCR and Southern blot hybridization. The n#D sequences from six strains have been compared, and they have an average similarity of 80%, ranging from 71% to 98%. The 11kb insertion element has been detected in three of the five genera examined thus far.

10:45 ISOLATING PREOPTIC REGULATORY FACTORS -1 AND -2 GENES IN 129 OLA/HSD MICE. Alisha B. Ketner, aketner@capital.edu, Capital University 473; 2199 E. Main St., Columbus OH 43209.

Gonadotropin Releasing Hormone (GnRH) has long been known to affect sexual development in mammals. Ten years ago two unique GnRH-related mRNAs, Preoptic Releasing Factors 1 and 2 (PORF-1 and PORF-2) were described. Studies done with rat brains have shown that PORF-1 and PORF-2 express regional, age and gender dependent factors. This suggests a unique role in gender-related development. A larger study was begun to identify the sequence of PORF-1 and PORF-2 in mice. As part of the larger study, DNA was isolated from the liver and spleen tissue of an eight-week-old 120 OLA/Hsd mouse and from E14TG2a mouse Embryological Stem cells. The result of this experiment was the isolation of 500ug of E14TG2a mouse Embryological Stem cell DNA. This experiment was performed to collect enough DNA in order to be later sequenced to target the PORF-1 and PORF-2 genes for knockout experiments in mice for the ultimate purpose of determining possible roles of PORF-1 and PORF-2 in mice.
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Publication:The Ohio Journal of Science
Geographic Code:1USA
Date:Mar 1, 2001
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