PubMed Health⌕ Search

Biomedical subjects

F Buxton

Publications and source records attributed to F Buxton.

6 recordsLinked to original sources
PubMed2005

Development of comprehensive functional genomic screens to identify novel mediators of osteoarthritis.

OBJECTIVE: The aim of this study was to develop high-throughput assays for the analysis of major chondrocyte functions that are important in osteoarthritis (OA) pathogenesis and methods for high-level gene expression and analysis in primary human chondrocytes. METHODS: In the first approach, complementary DNA (cDNA) libraries were constructed from OA cartilage RNA and full-length clones were selected. These cDNAs were transferred into a retroviral vector using Gateway Technology. Full-length clones were over-expressed in human articular chondrocytes (HAC) by retroviral-mediated gene transfer. The induction of OA-associated markers, including aggrecanase-1 (Agg-1), matrix metalloproteinase-13 (MMP-13), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), collagen IIA and collagen X was measured by quantitative real-time polymerase chain reaction (QPCR). Induction of a marker gene was verified by independent isolation of 2-3 clones per gene, re-transfection followed by QPCR as well as nucleotide sequencing. In the second approach, whole cDNA libraries were transduced into chondrocytes and screened for chondrocyte cluster formation in three-dimensional agarose cultures. RESULTS: Using green fluorescent protein (eGFP) as a marker gene, it was shown that the retroviral method has a transduction efficiency of >90%. A total of 40 verified hits were identified in the QPCR screen. The first set of 19 hits coordinately induced iNOS, COX-2, Agg-1 and MMP-13. The most potent of these genes were the tyrosine kinases Axl and Tyro-3, receptor interacting kinase-2 (RIPK2), tumor necrosis factor receptor 1A (TNFR1A), fibroblast growth factor (FGF) and its receptor FGFR, MUS81 endonuclease and Sentrin/SUMO-specific protease 3. The second set of seven hits induced both Agg-1 and MMP-13 but none of the other markers. Five of these seven genes regulate the phosphoinositide-3-kinase pathway. The most potently induced OA marker was iNOS. This marker was induced 20-500 fold by seven genes. Collagen IIA was also induced by seven genes, the most potent being transforming growth factor beta (TGFbeta)-stimulated protein TSC22, vascular endothelial growth factor (VEGF) and splicing factor 3a. This screening assay did not identify inducers of collagen X. The second chondrocyte cluster formation screen identified 14 verified hits. Most of the genes inducing cluster formation were kinases. Additional genes had not been previously known to regulate chondrocyte cluster formation or any other chondrocyte function. CONCLUSIONS: The methods developed in this study can be applied to screen for genes capable of inducing an OA-like phenotype in chondrocytes on a genome-wide scale and identify novel mediators of OA pathogenesis. Thus, coordinated functional genomic approaches can be used to delineate key genes and pathways activated in complex human diseases such as OA.

Cartilage, Articular↗
PubMed1998

Position-independent expression of a human nerve growth factor-luciferase reporter gene cloned on a yeast artificial chromosome vector.

Two yeast artificial chromosomes containing the entire human nerve growth factor gene were isolated and mapped. By homologous recombination a luciferase gene was precisely engineered into the coding portion of the NGF gene and a neomycin selection marker was placed adjacent to one of the YAC telomeres. Expression of the YAC-based NGF reporter gene and a plasmid-based NGF reporter gene were compared with the regulation of endogenous mouse NGF protein in mouse L929 fibroblasts. In contrast to the plasmid-based reporter gene, expression and regulation of the YAC-based reporter gene was independent of the site of integration of the transgene. Basic fibroblast growth factor and okadaic acid stimulated expression of the YAC transgene, whereas transforming growth factor-beta and dexamethasone inhibited it. Although cyclic AMP strongly stimulated production of the endogenous mouse NGF, no effect was seen on the human NGF reporter genes. Downregulation of the secretion of endogenous mouse NGF already occurred at an EC50 of 1-2 nM dexamethasone, but downregulation of the expression of NGF reporter genes occurred only at EC50 of 10 nM. This higher concentration was also required for upregulation of luciferase genes driven by the dexamethasone-inducible promoter of the mouse mammary tumor virus in L929 fibroblasts.

Animals↗
PubMed1994

Nitrogen, carbon, and pH regulation of extracellular acidic proteases of Aspergillus niger.

Aspergillus niger secretes a number of enzymes, including proteases, into its culture fluid. The regulation of the two major acidic extracellular proteases, pepA and pepB, was investigated using Northern analyses. Our data suggest that the regulation of pepA and pepB expression occurs predominantly at the level of mRNA content and that, while they are regulated in a similar manner, differences are also clear in their expression. Both genes were found to be under complex regulatory control. The expression of the two genes could be turned off by the presence of good nitrogen or carbon sources in the media, and external protein sources did not induce expression of either gene under conditions of carbon and nitrogen repression. The pH of the medium also played a major role in their regulation as the expression of both genes was completely turned off under alkaline conditions, even when grown in media lacking good nitrogen and carbon sources but containing proteins. We isolated clones containing 5' non-coding sequences of the pepA gene from a lambda genomic library with a pepA specific probe. Analysis and comparison of the promoter sequences of the pepA and pepB genes revealed that both contain several putative AREA- and CREA-binding sites and they also share an 18-bp-long sequence which is 83% identical in these two genes.

Aspartic Acid Endopeptidases↗
PubMed1983

Point mutations and DNA rearrangements 5' to the inducible qa-2 gene of Neurospora allow activator protein-independent transcription.

Expression of the qa-2 gene of Neurospora crassa normally requires a functional activator protein encoded by qa-1F. Twelve transcriptional mutants of the qa-2 gene have been isolated in qa-1F- strains, and these allow partial expression of qa-2 (1-45% of induced wild type) in the absence of functional activator protein. All 12 mutants have been characterized by genomic (Southern) blot hybridization and the DNAs of 5 have been cloned and sequenced. Eight mutations consist of large DNA rearrangements within a 500-base-pair region 5' to the qa-2 gene. One large rearrangement mutation, located 378 base pairs before the normal site of transcription initiation, causes exceptional levels of qa-2 transcription (45% of induced wild type) from near the normal initiation site. Two of the other four mutations cloned involve tandem duplications (68 and 84 base pairs) of the same upstream region (centered at nucleotide - 145), and two involve "point" mutations (at nucleotides -200 and -95) that closely flank the duplicated region. With one possible exception, none of the mutations appears to involve changes directly associated with RNA polymerase II binding and hence they differ from analogous mutations in comparable prokaryotic systems. The overall results suggest that at least some of the large DNA rearrangement mutations may be acting as upstream activator elements, possibly by juxtaposing enhancer-like sequences, whereas the duplications and point mutations may define a region of qa-2 regulation, for instance at the level of RNA polymerase II access.

Base Sequence↗
PubMed1982

5'-Untranslated sequences of two structural genes in the qa gene cluster of Neurospora crassa.

The coding regions of two genes (qa-2 and qa-3) in the qa gene cluster of Neurospora crassa have been localized by nucleotide sequence analysis combined with data on previously determined NH2-terminal amino acid sequences for the proteins that these genes encode. The start point of transcription for each of these genes has been determined by nuclease S1 mapping experiments with poly(A)+RNA isolated from quinic acid-induced cultures of N. crassa. The sequences of approximately 200 nucleotides 5' to the start point of transcription have been compared with each other and with those of other eukaryotes. The results show that neither of these regions for the qa-2 nor the qa-3 genes share any significant homology with sequences apparently conserved in higher eukaryotic promoters (-25 and -70 regions). However, the qa-2 and qa-3 sequences do show homology with each other in these regions. Comparison of the 5'-flanking regions of these Neurospora genes with those of several Saccharomyces cerevisiae genes reveals a number of similarities in the region preceding the translation initiation codons.

Base Sequence↗