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D Christophe

Publications and source records attributed to D Christophe.

At least 55 records · Page 3Linked to original sources
PubMed1989

Identification of a cAMP-responsive region in thyroglobulin gene promoter.

The DNA sequences involved in transcription control by a cAMP-dependent mechanism have been localized in the thyroglobulin gene promoter region by a functional assay. The proximal 5'-flanking sequences from the bovine thyroglobulin gene were linked to the bacterial chloramphenicol acetyl-transferase gene. Transient expression of this reporter gene was studied in dog thyrocytes in primary culture in the presence, or absence, of cAMP stimulation. Deletion analysis showed that the cAMP-responsive region is contained within the first 250 base-pairs of the promoter, and suggests that it could correspond to a sequence conserved between species. These DNA sequences do not bear significant homology with cAMP-responsive elements (CRE) described previously. By contrast, some similarities were found with the fat-specific element (FSE2) of genes under cAMP control in adipocytes and with DNA elements mediating cAMP-dependent regulation of expression of two different genes in the lower eukaryote Dictyostelium discoideum. This suggests that control of Tg gene transcription by cAMP could involve a mechanism different from the one mediated by a classical CRE.

Adipose Tissue↗
PubMed1989

Control of thyroperoxidase and thyroglobulin transcription by cAMP: evidence for distinct regulatory mechanisms.

The expression of the genes coding for thyroglobulin (TG), and thyroperoxidase (TPO), are regulated by TSH. These effects are mediated by cAMP as they are reproduced by forskolin. In vitro run-on transcription assays performed on nuclei isolated from dog thyrocytes in culture or from dog thyroid slices, indicate that the forskolin-induced transcriptional stimulation of TG and TPO genes are very different. For the TG gene, the kinetics of transcriptional activation vary according to the experimental model: it is rapid (1 h) in thyroid slices and slow (8 h) in primary cultures. In contrast, TPO induction is rapid in both cases. In primary cultures, insulin is responsible for the basal level and for a part of forskolin-induced TG transcription, whereas TPO transcription is not affected by insulin. The forskolin-induced increase of TG transcription requires ongoing protein synthesis, as it is blocked by cycloheximide. TPO gene transcription is unaffected by cycloheximide. Taken together with previous data on the two genes, our results suggest that while TPO regulation corresponds to the classical model of genes in which the promoter is regulated directly via cAMP regulatory elements, TG gene regulation involves the synthesis of an intermediary, rapid turnover trans-acting protein.

Animals↗
PubMed1988

Thyroid-specific and cAMP-dependent hypersensitive regions in thyroglobulin gene chromatin.

Two regions hypersensitive to DNase I digestion were found in a 7-kb segment of thyroglobulin gene 5'-flanking sequences in the chromatin from bovine thyroid. The most upstream site (-2000 to -1600 bp relative to the transcriptional start) was found in thyroid chromatin only, but independently of actual expression of the gene. It therefore represents a tissue-specific characteristic which may be associated with the commitment of the thyroglobulin gene to transcriptional activity. The very 5' end of the gene and the proximal promoter sequences (-100 to +60 bp relative to transcriptional start), constitute the second site, the hypersensitive character of which could be directly correlated with transcriptional activity. The structural changes occurring in this region of the chromatin were dependent on cAMP stimulation of the thyroid cells.

Animals↗
PubMed1988

Transcriptional regulation of the thyroperoxydase gene by thyrotropin and forskolin.

The expression of the gene coding for thyroperoxydase, the main enzyme involved in the synthesis of the thyroid hormones, is controlled by thyroid stimulating hormone. In vitro transcription assays performed on nuclei isolated from dog thyroid cells in primary culture showed that this control is at the transcription level, takes place rapidly (1 h) and is cyclic AMP-dependent as it is mimicked by forskolin. Insulin does not seem to be an important modulator of the thyroperoxydase gene expression.

Animals↗
PubMed1987

Structural organization of the 5' region of the thyroglobulin gene. Evidence for intron loss and "exonization" during evolution.

More than one third of thyroglobulin (1190 residues out of 2750) is made of one peptide motif repeated ten times in tandem. Segments unrelated to the motif interrupt this structure at various places. The corresponding gene region, which extends over 40 x 10(3) bases, was studied in detail. All exon borders and exon/intron junctions were localized precisely and sequenced, and their positions were correlated with the repetitive organization of the protein. When intron positions were compiled on a consensus sequence of all repeats, three categories of introns were observed. Except between repeats numbers 5 and 6, an intron was invariably found within the Cys codon making the limit of each motif. This category of intron most probably reflects the serial duplication events responsible for the evolution of this region of the gene. All other introns, except no. 2, are found at positions were the repetitive structure is disrupted by "inserted" peptides. We present the hypothesis that this second category of introns was already present in the original unit before the first duplication. Thereafter, they would have experienced either complete loss (some units do not contain any intron) or partial or total exonization, resulting in the slipping of intronic material into coding sequence. Intron no. 2, finally, separates motif no. 1 at a position on the boundary between two segments presenting sequence homology. This last type of intron probably reflects an initial duplication event at the origin of a primordial thyroglobulin gene motif. With all these characteristics, the thyroglobulin gene is presented as a paradigm for the analysis of the fate of introns in gene evolution.

Amino Acid Sequence↗
PubMed1987

A sequence in M13 phage detects hypervariable minisatellites in human and animal DNA.

The term "DNA fingerprint" has been used to describe the extensive restriction fragment length polymorphism associated with hypervariable minisatellites present in the human genome. Until now, it was necessary to hybridize Southern blots to specific probes cloned from human genomic DNA in order to obtain individual-specific restriction patterns. The present study describes the surprising finding that the insert-free, wild-type M13 bacteriophage detects hypervariable minisatellites in human and in animal DNA, provided no competitor DNA is used during hybridization. The effective sequence in M13 was traced to two clusters of 15-base pair repeats within the protein III gene of the bacteriophage. This unexpected use of M13 renders the DNA fingerprinting technology more readily available to molecular biology laboratories.

Animals↗
PubMed1987

Preparation of single-stranded deoxyribonucleic acid probes using an immobilized template.

A new method for the easy preparation of specific single-stranded DNA fragments is presented. Recombinant M13 DNA containing the strand complementary to the sequence of interest is made partially double-stranded by elongating a conventional M13 sequencing primer. Following linearization by enzymatic digestion downstream from the insert (relative to priming site), this DNA is coupled to diazotized paper through its single-stranded (vector) portion. Subsequent denaturation of the double-stranded region generates an immobilized template strand. Successive runs of primed syntheses of the (desired) complementary strand can be realized using the same template. The copies are easily isolated by release upon denaturation. DNA probes prepared by this method have proven to be valuable tools for gene analysis.

DNA, Recombinant↗
PubMed1987

Normal and defective expression of the thyroglobulin gene.

Molecular studies of the thyroglobulin (Tg) gene have progressed significantly in recent years. Cloning and sequencing the complete bovine Tg cDNA led to the knowledge of the primary structure of the Tg subunit. This large polypeptidic chain displays a repetitive structure, especially in its amino-terminal half, and bears a striking homology with the acetylcholinesterase molecule of Torpedo californica in its carboxy-terminal portion. The four specific domains known to be involved in the formation of the thyroid hormones have been assigned to both terminal parts of the polypeptide, a location which could play a role in the process leading to hormone release. The very large (greater than 250 kb) Tg gene has been localized on the long arm of chromosome 8 in man, in close linkage with the c-myc oncogene. The study of its structure allowed the characterization of the molecular defect responsible for a congenital flaw in Tg gene expression in a herd of South-African cattle. This work led to the unexpected finding that the Tg pre-mRNA undergoes alternative splicing in normal animals, too. A DNA segment involved in the transcriptional control of Tg gene expression by cAMP has been identified by transfecting primary cultured thyrocytes with recombinant genes.

Animals↗
PubMed1987

Control of thyroglobulin gene transcription by TSH and cAMP.

The availability of the sequence of Tg promoter from three different species enabled a fruitful comparison to be made from which a general picture of the organization of the Tg gene promoter region emerged. Chromatin structure studies identified tissue-specific modifications at the level of DNA protein interactions. A DNA element has been shown to be involved in transcription control by cAMP. This sequence includes a highly conserved region of the Tg gene promoter, overlaps a domain of the promoter which becomes hypersensitive to DNase I when the gene is expressed and displays specific protein-DNA interaction. These observations are consistent with the possibility that this sequence is a target element for trans-acting factor(s) which control(s) the transcriptional activity of the gene.

Animals↗
PubMed1986

Methylation and expression of the human thyroglobulin gene.

The DNA methylation pattern at the 5'end of the human thyroglobulin gene has been determined in different tissues. Out of the four HpaII/MspI sites (5'-CCGG-3') present in this region, three were found to be non-methylated in thyroid DNA, while full methylation was observed in liver, salivary gland and sperm DNA. This demethylation therefore correlates with expression of the thyroglobulin gene. However, all four sites were found to be non-methylated in placental DNA, regardless of the activity of the gene.

Base Sequence↗
PubMed1985

An unusually long poly(purine)-poly(pyrimidine) sequence is located upstream from the human thyroglobulin gene.

A region of human genomic DNA encompassing the 5' end of the thyroglobulin gene has been sequenced and the position of the transcriptional start site has been determined. The 5' non-translated portion of the mRNA displays a quasi-palindromic sequence which could allow this region to adopt a hairpin structure. The first exon of the gene encodes a 19 amino-acids signal peptide and the 3 first amino acids of the mature protein. Apart from the canonical TATA-Box and from a CAAT-Box homology, the promoter region contains a 209 bp-long poly(purine)-poly (pyrimidine) sequence located between positions-512 and -304 relative to the transcription start. When contained in a supercoiled plasmid, this sequence exhibits sensitivity to S1 nuclease at two distinct positions. A precise mapping of the borders of the sensitive regions was achieved by extending primers from both ends of the sequence after digestion by the enzyme. The resulting data can be explained by a model involving the formation of a triple helix structure.

Base Sequence↗
PubMed1984

Structural organization of the 5' region of the human thyroglobulin gene.

Sequence analyses of bovine and human thyroglobulin (Tg) cDNA have demonstrated that the 5' region of the mRNA encodes a domain responsible for thyroid hormone synthesis and exhibits striking internal repetition. Knowledge of the organization of the corresponding chromosomal DNA region would provide insight as to how such a structure has evolved. A human genomic DNA library was screened by hybridization in situ, using a bovine Tg cDNA probe corresponding to 2.8 X 10(3) base pairs at the 5' end of the mRNA. Out of 3 X 10(5) phage plaques, four were scored as positive and yielded three different phages containing thyroglobulin sequences. Selected human Tg cDNA probes were used to order the phages and to identify overlapping regions. Electron microscopy of hybrids between human Tg mRNA and the phage DNA was performed to determine the intron/exon organization of this region. The following conclusions were reached. (a) About 4 X 10(4) base pairs corresponding to the 5' region of the gene have been isolated as three overlapping recombinant phages. (b) The three phages cover altogether 2.9 X 10(3) base pairs of exonic sequence at the 5' end of the mRNA. (c) Out of the 11 exons identified in this region, 9 were of a size similar to that of the 3' exons characterized previously (less than or equal to 200 base pairs); exons 9 (1.12 X 10(3) base pairs) and 10 (0.56 X 10(3) base pairs) were exceptions to this rule. (d) The phage nearest the 5' end contains about 9 X 10(3) base pairs of sequence located upstream from the gene. The availability of clones covering the region upstream from the thyroglobulin gene will provide the basis for the identification of sequences involved in its transcriptional control by thyroid-stimulating hormone (thyrotropin).

Bacteriophage lambda↗
PubMed1983

Molecular cloning of complementary DNA: preparation of a plasmid vector with low transformation background.

A simple method that allows the rapid preparation of oligo dG-tailed plasmid vectors is presented. The procedure involves purification of the tailed molecules by hybridization to oligo dC-cellulose followed by a stepwise thermal elution. The resulting plasmid is virtually devoid of transformation activity in the absence of oligo dC-tailed DNA fragments. It allows construction of cDNA libraries with as low as 1% of colonies harboring wild-type plasmids.

Cellulose↗