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Biomedical subjects

J Renaud

Publications and source records attributed to J Renaud.

At least 19 recordsLinked to original sources
PubMed1994

Total synthesis of taxol.

Taxol, a substance originally isolated from the Pacific yew tree (Taxus brevifolia) more than two decades ago, has recently been approved for the clinical treatment of cancer patients. Hailed as having provided one of the most significant advances in cancer therapy, this molecule exerts its anticancer activity by inhibiting mitosis through enhancement of the polymerization of tubulin and consequent stabilization of microtubules. The scarcity of taxol and the ecological impact of harvesting it have prompted extension searches for alternative sources including semisynthesis, cellular culture production and chemical synthesis. The latter has been attempted for almost two decades, but these attempts have been thwarted by the magnitude of the synthetic challenge. Here we report the total synthesis of taxol by a convergent strategy, which opens a chemical pathway for the production of both the natural product itself and a variety of designed taxoids.

Models, Molecular↗
PubMed1993

Transcription of the histone H5 gene is regulated by three differentiation-specific enhancers.

Histone H5, an early marker of the avian erythroid lineage, is expressed at low levels in early erythroid precursors and at higher levels in more mature cells. We show that the increase in H5 expression is due to transcriptional activation of the H5 gene following differentiation of precursor CFU(E). We have found and characterized two upstream enhancers, E1 (between -2233 and -1878 from the site of transcription initiation, +1) and E3 (between -1321 and -1163), and confirmed the presence of a downstream enhancer (C. D. Trainor, S. J. Stamler, and J. D. Engel, Nature [London] 328:827-830, 1987) E7 (between +846 and +1181) which are responsible for the increase in H5 gene transcription. The enhancers had a weak effect in nondifferentiated CFU(E) but a strong effect when the cells were induced to differentiate. Cooperation among the three enhancers, however, was not required for H5 gene activity in the differentiated cells. The enhancers contain binding sites for several ubiquitous and erythroid cell-specific nuclear proteins, including GATA-1, as demonstrated with GATA-1-specific antibodies. Although the GATA sites were required for enhancer function, the concentration of GATA-1, GATA-2, and GATA-3 decreased during cell differentiation, and overexpression of these factors had little effect on H5 transcription. Hence, the differentiation-specific effect of the enhancers is not mediated by changes in relative levels of the GATA factors. Functional analysis of the H5 promoter indicated that the requirement of several elements, including a GC box necessary for transcription enhancement, did not change during the early stages of CFU(E) differentiation. However, the UPE, a positive element in proliferating CFU(E) recognized by the transcription factor H4TF2, was dispensable in the differentiated cells. These results suggest that as the cells enter the final stages of differentiation, there is a reprogramming of the regulatory factors that control H5 transcription and that the enhancers rescue and increase the activity of the promoter.

Amino Acid Sequence↗
PubMed1992

Repression of the H5 histone gene by a factor from erythrocytes that binds to the region of transcription initiation.

Expression of histone H5, like that of other erythrocyte specific proteins, declines during the latter stages of erythroid maturation because of a decrease in the rate of gene transcription. Here, we report the isolation of cIBR (chicken initiation binding repressor), a 75 kDa DNA binding glycoprotein from mature chicken erythrocytes that recognizes sequences spanning the transcription start sites of the H5 gene. cIBR was found to repress transcription from the H5 promoter in vitro and this effect could be relieved by mutations that lowered the affinity of the factor for its cognate sequence. cIBR inhibited transcription by interfering with assembly of the initiation complex, but it did not affect transcription from pre-assembled complexes. Consistent with this, binding of bacterially expressed human TFIID to the TATA element prevented subsequent binding of cIBR, although the opposite was not true. This, and the fact that cIBR had no effect when bound in a location upstream from the promoter, suggests that binding of cIBR to the start site region causes repression by direct interference with general transcription factors other than TFIID, possibly TFIIB. cIBR was found in mature and relatively late erythrocytes but not in early erythroid cells which actively transcribe the H5 gene; the transcriptionally active cells contain instead cIBF (chicken initiation binding factor). Purified cIBF is a non-glycosylated 68-70 kDa DNA binding protein(s) which also recognizes the region of transcription initiation of the H5 gene.

Animals↗
PubMed1990

[Focal infection? Did you say focal infection ... focal infection and uveitis].

A chronic seat of infection more often than not situated in the E.N.T. area is responsible for remote pathological symptoms commonly called focal infection. The eye may be a target organ, where focal infection appears in the form of a uveitis. Four clinical observations are reported as demonstration. The transmission mechanism operates through an immune phenomenon from bacterial antigens which engender reactions of hypersensitivity. The E.N.T. specialist must be aware of these symptoms and seek a pharyngeal, sinusal or latent buccodental infection.

Adult↗
PubMed1989

Basal expression of the histone H5 gene is controlled by positive and negative cis-acting sequences.

Sequences from -3500 to +1365 of the chicken histone H5 gene have been analyzed for the presence of cis-acting elements in H5 expressing (transformed CFU-E) and non-expressing cells (fibroblasts). The region from -3500 to -115 had little effect on transcription. Proximal upstream sequences contain a negative element (UNE, -115 to -95), capable to also repress the activity of the heterologous HSV tk promoter, and two positive elements, a consensus GC-box (-83 to -74) and a proximal element (UPE, -54 to -38). The sequence of the UPE is highly related to the histone H4 subtype-specific element and it has been conserved in the duck H5 and the human and mouse H1(0) genes at equivalent positions. Although the effect of the UNE, GC-box and UPE was not tissue-specific, sequences from -38 to +77 appear to confer a degree of tissue specificity to the promoter. An activating erythroid-specific element (DE) was found downstream of the H5 gene (+1042 to +1185). The activity of the DE was modest but independent of position and orientation and required the presence of the promoter proximal elements. The DE harbors the sequence AGATAA that is recognized by a protein factor, presumably the same that binds to other erythrocyte-specific enhancers. The low activity of DE in the CFU-E may be related to the low concentration of the AGATAA-binding factor in the differentiation-blocked cells.

Animals↗
PubMed1989

Recognition of (dG)n.(dC)n sequences by endonuclease G. Characterization of the calf thymus nuclease.

We report the purification of endonuclease G (Ruiz-Carrillo, A., and Renaud, J. (1987) EMBO J. 6, 401-407) from calf thymus nuclei and whole tissue. The enzyme has been enriched 29,000-fold, and the activity was unambiguously identified with a 26-kDa protein after renaturation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native nuclease behaves as a 50-kDa species by gel filtration, suggesting that it is composed of two subunits, presumably identical. In terms of absolute amounts, endonuclease G (endo G) is a nuclear enzyme although it was also detected in purified mitochondria. Endo G is highly specific for (dG)n.(dC)n tracts in DNA, nicking either strand of relaxed substrates with similar kinetics. The sensitivity of the homopolymer tracts is proportional to their length (from n = 8 to 29), insofar as the flanking sequences are constant. However, the overall rate of cleavage is influenced by the composition of the flanking DNA. Minor cleavage sites contain shorter (dG)n.(dC)n clusters (n = 3-7). Endo G efficiently cleaves (dC)n but not (dG)n runs in single-stranded DNA, suggesting that it may recognize an asymmetric strand conformation of the homopolymer tracts. Endo G does not recognize other homo(co)-polymer sequences or cruciform structures in DNA.

Animals↗
PubMed1987

Endonuclease G: a (dG)n X (dC)n-specific DNase from higher eukaryotes.

An endonuclease activity (termed endonuclease G) that selectively cleaves DNA at (dG)n X (dC)n tracts has been partially purified from immature chicken erythrocyte nuclei. Sites where n greater than or equal to 9 are cleaved in a manner that resembles types II and III restriction nucleases. The nicking rate of the G-strand is 4- to 10-fold higher than that of the C-strand depending on the length of the (dG)n X (dC)n tract and/or nucleotide composition of the flanking sequences. Endonuclease G hydrolyzes (dG)24 X (dC)24 of supercoiled DNA in a bimodal way every 9-11 nucleotides, the maxima in one strand corresponding to minima in the opposite, suggesting that it binds preferentially to one side of the double helix. The nuclease produces 5' phosphomonoester ends and its activity is dependent on Mg2+ or Mn2+. The wide distribution and high relative activity of endonuclease G in a variety of tissues and species argues for a general role of the enzyme. The striking correlation between genetic instability and poly(dG) X poly(dC) tracts in DNA suggests that these sequences and endonuclease G are involved in recombination processes.

Animals↗
PubMed1987

Regulation of histone and beta A-globin gene expression during differentiation of chicken erythroid cells.

The expression of the genes for several histones and beta A-globin was examined in the chicken erythroid cells lineage. During the transition from CFU-(E) to the mature erythrocyte, histone H5 gradually increased fourfold in nuclei with little concomitant displacement of the H1 histones. This resulted in a 70% net increase in linker histone (H1 plus H5) content. The differential accumulation of H5 reflected (i) an increase in the transcriptional activity of the H5 gene occurring at the erythroblast stage, (ii) an apparent longer half-life of H5 mRNA, and (iii) a higher stability of the protein. Although the transcriptional activity of the histone genes (except H5) decreased with cell age, it was not tightly coupled to the S phase. On the other hand, the mRNA levels for these histones were tightly regulated during the cell cycle. Use of protein and DNA synthesis inhibitors indicated that the content of H5 mRNA was regulated at the posttranscriptional level by a control mechanism(s) differing from those for the other histones. Although the transcription rates of the H5 and beta A-globin genes were comparable, differential accumulation of beta A-globin mRNA led to a 30- to 170-fold-higher copy number of the beta A-globin mRNA as the cell matured.

Animals↗
PubMed1986

Fine analysis of the active H5 gene chromatin of chicken erythroid cells at different stages of differentiation.

We have analyzed the chromatin structure of a region that encompasses 14.4 X 10(3) base-pairs of the chicken histone H5 locus in adult erythroid cells at different stages of maturation. Seven of eight major lineage-specific DNase I-hypersensitive sites, some of which show complex substructure, were found in the flanking regions of the gene. The hypersensitivity of some of these sites is modulated during erythrocyte maturation in a way that parallels the transcriptional activity of the gene. DNase I, micrococcal nuclease, and S1 nuclease recognize the same regions, which differ from those cleaved by S1 on supercoiled plasmid DNA. This suggests that hypersensitivity of DNA in chromatin reflects a greater accessibility of the DNA rather than its altered conformation. The DNA sequence of some of the DNase I target sites contains repeated motifs, (T-C-C-C)2, (T-C-C)2, (T-G-G-G-G)2, which are found in the hypersensitive sites of other genes. Detailed analysis across sections of the H5 gene and flanking sequences revealed differences in the DNase I sensitivity of the different regions examined. Notably, the first one-third of the gene is more sensitive than the rest. The sequences downstream from the region where most RNA polymerases terminate transcription were found to be the most resistant.

Animals↗
PubMed1983

Genomic organization of the genes coding for the six main histones of the chicken: complete sequence of the H5 gene.

The organization of the genes coding for histones in the chicken has been examined, with special reference to that coding for the tissue-specific, developmentally regulated histone H5. Two recombinant phages containing sequences complementary to cloned H5 cDNA have been isolated from a genomic chicken library. The clones have been characterized by heteroduplex formation, restriction nuclease analysis, hybridization to cloned homologous histone gene probes, and DNA sequencing. Hybridization to genomic DNA has shown that there is only one copy of the H5 gene per haploid genome, whereas there are six to 11 copies of the genes for the other histones. Examination of 29 X 10(3) base-pairs of DNA sequences flanking the H5 gene has revealed the absence of any other histone genes which, although not tandemly reiterated, for the most part appear to reside in loosely organized clusters. The complete DNA sequence of the H5 gene and flanking regions, as well as the mapping of the 5'-end of its messenger RNA by primer extension with AMV reverse transcriptase, has shown that the gene has no introns and little homology to other histone genes, including those for H1.

Animals↗
PubMed1983

Histone synthesis by lymphocytes in G0 and G1.

Peripheral blood lymphocytes are a naturally occurring population of G0 cells which can be activated in vitro to grow and divide. Upon activation with phytohemagglutinin (PHA), they enter G1 and, after a 24-h lag, begin DNA replication (S phase). Using radioisotope labeling and gel electrophoresis of acid-soluble chromatin proteins, we investigated histone synthesis in G0, G1, and S phase cultures of human and pig lymphocytes. In G0 and G1 cultures, which have less than 0.1% S phase cells, all five histones are synthesized and are incorporated into chromatin in equimolar amounts. In G0 lymphocytes histone synthesis accounts for at least 6% of nuclear protein radioactivity, and the rate of synthesis is about 2-3% of that of S phase lymphocytes. In contrast to histone synthesis by S phase cultures, G0 and G1 histone synthesis was completely resistant to treatment with hydroxyurea.

Animals↗