Mesenchymal stem cells (MSCs) are best characterized by their capacity to differentiate into connective tissue cell lineages. Many aspects of their basic biology including the molecular mechanisms that regulate lineage commitment are poorly described. We developed a method to enrich MSCs from mouse bone marrow and reported that FGF2 exposure induced proliferation but reversibly inhibited their multi-lineage differentiation. Herein, kinetic studies performed via real-time PCR revealed that FGF2 down regulated expression of soluble frizzled-related receptors 1 and 2 (sfrp1, 2), but induced expression of twist 2 and sprouty 4 (spry4), respectively. In contrast, Wnt3a exposure up-regulated expression of twist1 and had no effect on spry4 expression. Consistent with these differential effects on gene expression, FGF2 was shown to inhibit osteogenic differentiation of MSCs, while Wnt3a modestly inhibited osteogenesis but promoted chondrogenic differentiation Efforts to localize Twist and Spry4 in cells revealed that the proteins were expressed in a small percentage (∼7 %) of MSCs but both proteins were co-expressed in the majority (∼80 %) of these cells. Exposure to FGF2 afforded a significant enrichment of Twist/Spry4 double positive cells, which represent a novel progenitor population in bone marrow. To study the nature of this subpopulation, we developed a Twist-promoter GFP-reporter expression vector and showed that it afforded significant enrichment of Twist-expressing Neuro2A cells in vitro. An HIV-1 based expression vector was constructed to ectopically express Twist1 in human MSCs, which completely blocked the capacity of cells to differentiate into osteoblasts FGF2 was shown to promote growth of MSCs, but even exposure cells to FGF2 failed to prevent their growth arrest after prolonged culture in vitro. Culture of MSCs in hypoxia (5% O2) increased their proliferative capacity and had no effect on FGF2 or Wnt3a induced cell signaling, indicating that effects on growth were not related to Twist regulation. However, MSCs cultured in normoxia (21% O2) exhibited rapid increasing p53 levels, which were down regulated after cells were switched to low oxygen condition. These data indicates that the poor growth of MSCs in room air (21% O2) is related to oxygen-induced toxicity that results in cell cycle arrest due to p53 induction / acase@tulane.edu

A pilot study of body shape variation in three species of darters (Etheostoma caeruleum, E. nigrum, and E. stigmaeum) showed significant variation among species and among populations within species, and suggests that conclusions regarding darter body shapes drawn from interspecific comparisons are robust to significant intraspecific variation. The remaining studies addressed interspecific variation in body shapes and reproductive life-history traits as they relate to body size (S), phylogeny, and each other, based on data collected from 1,089 reproductive female darters representing 32 taxa (species and subspecies) Within taxa, clutch size (CS) was found to be significantly associated with S for 28 of the 32 taxa, as was ovum mass (OM) for 10 and clutch mass (CM) for 13. All three were significantly correlated with S for all individuals as a group, and were therefore size-adjusted in subseqent analyses. Using four recent phylogenies, I tested for phylogenetic effects on S, size-adjusted life-history traits, and the slopes of the regressions of CS on S. Body size showed no evidence of phylogenetic effects, but conflicting results were found for the remaining variables, highlighting these tests' sensitivity to differences among phylogenies In contrast, geometric morphometric analysis showed consistent evidence of phylogenetic effects on body shapes. Transformation grids revealed qualitative similarities within most higher taxa, and this observation was confirmed with ordination and cluster analyses. Mantel tests showed significant positive association between body shapes and each of the four phylogenies---a surprising result, given the large differences among them. To examine this result further, I reanalyzed the data including only a single representative species per subgenus, which rendered the correlations insignificant for three of the four phylogenies Associations between body shape and life-histories were tested with phylogenetic generalized least squares multiple regression. The results indicated that body shape explains relatively little (13--24%) of the observed variation in size-adjusted CS or OM, but explains much (39--58%) of the variation in size-adjusted CM. Subsequent examination revealed that the best predictors of life-history variation were those measures associated with body depth / acase@tulane.edu

Mouse Zn-16 was sequenced and compared to rat Zn-15. Amino acids in the DNA-binding zinc fingers and linker regions were conserved with 97% identity, but an unreported zinc finger was found in rat ZF V resulting in four closely-spaced tandem zinc fingers, that may be an additional functional region. The Zn-16 proximal promoter was sequenced, identifying a total of four translational start sites. Several GATA-2 and Oct-1 sites were identified by sequence homology, and may be important in regulating Zn-16 expression during pituitary organogenesis. An overlapping GATA-2/Pit-1 site was identified, similar to one found in the TSHbeta promoter. These structural findings are suggestive evidence that the Zn-16 gene is regulated during gonadotroph, thyrotroph, and somatotroph differentiation and development A quantitative RT-PCR assay using fluorescent-labeled dUTP was developed to determine mRNA copies in a single pituitary. Assay parameters of linearity, reproducibility, kinetic efficiency, and target specificity were found to be within acceptable limits. The assay was used to investigate the expression of mRNAs for four hormones (GH, PRL, POMC, and alphaGSU) and four transcription factors (Pit-1, Zn-16, TEF, and Prop-1) in the pituitaries of four types of Growth Hormone-affected mice, with or without concomitant deficits in other pituitary hormones. Snell (dw/dw) and Ames (df/df) panhypopituitary dwarf mice displayed similar patterns; GH was absent, Pit-1 (<1%), and Zn-16 (4%) were dramatically reduced, but an extremely small amount of PRL mRNA was detected in the df/df mice. In lit/lit GH-deficient mice, there was only a 50% reduction of PRL, POMC, and alphaGSU, but GH, Pit-1, Zn-16 and Prop-1 were all reduced to 1.5%, reflecting the specificity of GHRH stimulation for only somatotroph cells. Transgenic giant GHRH-excess mice demonstrated a 290% increase in expression for GH, Pit-1, and Zn-16, but not PRL or other non-somatotroph-specific factors. Thus, as would be predicted if Zn-16 has a physiological role in GH regulation, Zn-16 mRNA expression is highly correlated with GH and Pit-1, predicting localization to at least somatotrophs. Taken together, these data suggest that Zn-16 expression in the pituitary is intricately involved in controlling GH expression through a variety of processes / acase@tulane.edu

The formation of the 3'-end of messenger RNA and termination of transcription by RNA polymerase II are events that are essential for gene expression; however, these processes have incompletely understood mechanisms that involve both cis-acting and trans -acting factors. As part of an effort to examine the mechanisms of transcription termination by RNA polymerase II, a large-scale genetic screen for strains defective in 3'-end formation was conducted. Following creation of a battery of mutant strains by random mutagenesis, a temperature sensitive strain exhibiting several phenotypes consistent with a role in transcription termination was isolated. First, readthrough of a terminator increases significantly in the mutant strain. Accordingly, RNA analysis indicates a decrease in the level of terminated transcripts, both in vivo and in vitro. Moreover, a plasmid stability assay and transcription run-on analysis also demonstrate defective termination of transcription. Examination of polyadenylation and cleavage by the mutant strain indicates these processes are not affected. These results represent the first example of a transcription termination factor in Saccharomyces cerevisiae that affects transcription termination independent of 3'-end processing of mRNA Once a temperature sensitive mutant strain with phenotypes consistent with a defect in transcription termination was identified, cloning by complementation of the temperature sensitivity identified GRS1 , an aminoacyl-tRNA synthetase, as the complementing gene. Sequence analysis of grs1-1 in the mutant strain revealed that nucleotides 1656 and 1657 were both C to T transitions, resulting in a single amino acid change of proline to phenylalanine. Further studies revealed GRS1 is essential, and the grs1-1 allele confers the temperature sensitive growth defect associated with the mutant strain Several observations support the idea that the Grs1p is directly involved in 3' end formation, including that synthetases are involved in a variety of non-aminoacylation reactions, notably RNA splicing. Additionally, a suppressor of grs1-1, YMK1, was identified that is related to a previously identified 3'-end formation factors. Finally, structures with some similarity to tRNA molecules were observed within the 3'-end of various yeast genes. Based on these results, it is suggested that Grs1p is a transcription termination factor that interacts with the 3'-end of pre-mRNA to promote 3'-end formation / acase@tulane.edu

Reciprocal chromosomal translocations involving the Mixed-Lineage Leukemia (MLL) gene at locus 11q23 are common in infant acute leukemias, occurring in up to 80% of cases of AML and ALL. MLL translocations result in the in-frame fusion of MLL to a variety of different genes, which leads to the expression of a chimeric protein with transforming properties. To date, more than 50 different fusion proteins have been identified. One of the more common, AF9 fuses with MLL as a consequence of the t(9;11)(p22;q23) translocation that is detected in acute and secondary leukemias The function of AF9 in normal and diseased cells is not completely clear. It is known, however, that the &sim;90 amino acids carboxy terminus of AF9 contains a putative transcription activation domain functional in reporter gene assays. Moreover, when fused to MLL, the carboxy-termini of the AF9 and ENL proteins are required for transformation of hematopoietic stem cells. In previous experiments we identified AF4, another common MLL fusion partner, as well as two other proteins involved in transcription repression, Pc3 and BCoR, as a binding partner of the AF9 carboxy terminus. Importantly, both AF4 and AF9 binding sites are retained in the MLL fusions suggesting that the interaction plays a critical role in leukemogenesis. The carboxy terminus of AF9 is comprised of approximately 90 amino acids that are predicted to adopt a structure containing two putative alpha-helices. Deletions limited to either helix prevent the binding of AF4, Pc3 and BCoR. To gain some insights into the higher order structure of AF9 C-terminus, this dissertation utilized a yeast two-hybrid selection and screen to identify the residues that mediate binding between AF9 and AF4, Pc3 and BCoR. Additionally, a method for expression and purification of the AF9 C-terminus was developed and preliminary characterization of the AF9 C-terminus by circular dichroism was initiated. Given the importance of the AF9 C-terminus in the development of acute leukemias, these studies provide the groundwork for future investigations into the overall structure of the AF9 C-terminus for the ultimate goal of drug design and targeting / acase@tulane.edu

Strain variation ('degeneration') in fungi is characterized by changes in phenotype, attenuated virulence, reduced sporulation, and/or loss of secondary metabolite production. In this study, wild type and mutant strains of Aspergillus parasiticus (designated sec+) making aflatoxin and/or pigmented pathway intermediates were subjected to a protocol of serial transfer of mycelial macerates in a defined medium. Variant forms (designated sec$-$) exhibiting altered morphology, scanty sporulation, and lack of detectable amounts of secondary metabolite production were isolated after 5-12 transfers of mycelia. In contrast, when spores rather than mycelia were serially transferred, no $sec-$ forms were generated. The $sec-$ forms were stable and did not revert to sec+ phenotype upon subculturing on complete medium for 10 weeks. The DNA methylation patterns and, with one exception, the dry weights of the sec+ and $sec-$ strains were similar To conduct parasexual cycle analysis, heterokaryons between sec+ and $sec-$ forms with contrasting auxotrophic and spore color markers were produced. Spore color, auxotrophic markers, and the sec+ or $sec-$ phenotype failed to segregate in the heterokaryon test, suggesting that degeneration was not under cytoplasmic control. Qualitative and quantitative assays of heterozygous diploids showed similar aflatoxin production (155-157 $\mu$g/100 ml defined medium) in sec+/sec+ and sec+/sec$-$ diploids, indicating dominance of the sec+ phenotype Haploids were isolated from diploids using benomyl as the haploidization agent. The appearance of a few revertants of the $sec-$ forms from the sec+/sec$-$ crosses indicated that the aflatoxin pathway genes were present but turned off in these variants. To investigate the nature of non-expression, biotransformation experiments were conducted, where both sec+ and sec$-$ forms were fed with 200 $\mu$g of sterigmatocystin, an aflatoxin precursor. All the sec+ forms successfully biotransformed sterigmatocystin to aflatoxin. All the sec$-$ forms did not convert the precursor to detectable amounts of aflatoxin. This phenomenon of non-expression of aflatoxin pathway genes in the experimentally induced sec$-$ forms suggests that this fungus could be developed as a model system to understand the poorly understood process of strain degeneration in filamentous fungi / acase@tulane.edu

The CTNS gene encodes the lysosomal membrane transport protein cystinosin. Defects in CTNS result in the disorder cystinosis, characterized by the renal Fanconi syndrome, progressive renal failure, corneal crystals and retinopathy. A nonsense mutation (G753A) found in the CTNS gene encodes a premature termination codon (PTC; W138X), and results in severe nephropathic cystinosis. Aminoglycoside antibiotics induce faulty proofreading and allow translation of full-length protein from nonsense message. Treatment of fibroblasts containing the PTC mutation with the aminoglycoside antibiotic, gentamicin, results in cystine depletion. Transfection with PTC-GFP fusion constructs followed by treatment with gentamicin demonstrated that GFP expression was localized to lysosomes. Tissue expression of the mouse homolog of CTNS and the effect of lysosomal cystine on CTNS expression were examined To investigate the regulation of lysosome volume and number normal human fibroblasts were cultured in the presence of sucrose, which induces lysosomal swelling. Gene expression in three normal human fibroblast cell lines treated with 100 mM sucrose for 24 hours, as well as untreated time-matched controls, was analyzed by HGU95A microarray. Of the 12,500 genes thus examined, thirty-six were increased and eleven were decreased an average of 2-fold or greater, relative to matched controls. The majority of increased genes function in cholesterol biosynthesis and fatty acid metabolism or have lysosomal and intracellular vesicle related roles. The latter included lysosomal neuraminidase, CLN3, and Rab7L1 genes. Rab7L1 was examined by expression of a GFP-Rab7L1 fusion protein and found to localize primarily to the Golgi apparatus, and in some cells, to the membranes bounding vesicles Transcription factors activated in sucrose treated cells were analyzed using protein/DNA arrays. C/EBP activity was increased in nuclear extracts from cells exposed to sucrose for 6 and 12 hours, relative to matched controls, and the increased activity at 12 hours was confirmed by EMSAs Study of lysosome biogenesis and the regulation of gene expression using the sucrose-induced vacuolation model has yielded several genes of interest and a potentially relevant transcriptional regulator. A better understanding of the genes regulating lysosome function may improve our understanding of lysosomal storage disorders and yield new options for their treatment / acase@tulane.edu

Chinese hamster ovary cells were synchronized at the G$\sb1$/S boundary of the cell cycle and labeled for 10 min with $\sp{125}$I-iododeoxyuridine. Samples were frozen for decay accumulation between 15-480 min after labelling. Cells frozen within 1 h after labeling yielded a low LET survival response with a pronounced shoulder and a large D$\sb0$ (255 decays/cell). With longer chase periods the shoulder and the D$\sb0$ decreased progressively and cells harvested 5 h after labeling exhibited a high LET survival response (D$\sb0$: 65 decays/cell). If DNA is assumed to be the sole target for radiation-induced death, these results indicate that DNA maturation increases radiation damage to DNA or reduces repair. Alternatively, if radiation death involves damage to higher-order structures in the nucleus, the findings suggest that newly replicated DNA is not attached to these structures during the initial low LET period, but $\sp{125}$I starts to induce high LET effects as labeled DNA becomes associated with the target structure(s). On balance, our data favor the latter interpretation. / In related experiments, exponentially growing cells were labeled with $\sp{125}$I-iododeoxyuridine for 12 h. Mitotic cells were selected, plated for cell cycle traverse, and harvested during successive stages of the cell cycle for decay accumulation. $\sp{125}$I damage during G$\sb1$ resulted in shoulderless exponential survival curves with a D$\sb0$ of 60-65 decays/cell. Resistance to $\sp{125}$I decays increased as cells progressed through S and the survival curves of cells in late S/G$\sb2$ were characterized by a pronounced shoulder and a D$\sb0$ of 127-139 decays/cell. These findings suggest that the primary target for radiation death is duplicated during S with G$\sb1$ cells containing one set and G$\sb2$ cells two sets of targets. Dual targets, although located within a single cell, act independently, as if already distributed between separate daughter cells. Analysis of the data suggests that the emergence of a shoulder and doubling of the D$\sb0$ in late S/G$\sb2$ cells represents an artifact of the colony formation assay which systematically overstates cell survival when intracellular target multiplicity exceeds 1. / Source: Dissertation Abstracts International, Volume: 53-03, Section: B, page: 1235. / Major Professor: Kurt G. Holer. / Thesis (Ph.D.)--The Florida State University, 1992.

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