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Biomedical subjects

F Brunel

Publications and source records attributed to F Brunel.

At least 37 records · Page 2Linked to original sources
PubMed1989

Interaction of DNA-binding proteins with the tissue-specific human apolipoprotein-AII enhancer.

The identification of the binding sites for liver nuclear proteins present in the enhancer that control the cell specific transcription of the human apolipoprotein AII gene is reported. Five adjacent binding sites (motifs I to V) were identified. The motifs III, IV and V can be occupied differently by liver or HeLa nuclear proteins. Two hypersensitive zones (between motifs II-III and IV-V) are present only when liver nuclear extracts were tested. A first characterization of the factors reveal that motif IV interacts with the hepatic transcription factors Tf-LF1 (29) and LF-A1 (28, 30). A CCAAT binding protein, different from CTF/NF1, appears to bind to the motif II. The different binding sites share specific DNA sequences principally with 5' regulatory regions of other apolipoprotein genes.

Apolipoprotein A-II↗
PubMed1989

Different liver nuclear proteins binds to similar DNA sequences in the 5' flanking regions of three hepatic genes.

The proximal promoter region of the human transferrin gene contains an hepatocyte-specific cis-element (PRI, nucleotides -76 to -51) whose DNA sequence is homologous to a sequence (nucleotides -89 to -68) present in the transcriptionally essential 5' region of the human antithrombin III gene and to another hepatocyte-specific sequence (A domain) of the human alpha 1-antitrypsin gene promoter. The results reported here lead to the conclusion that the liver trans-acting factor Tf-LF1, binding to the transferrin PRI cis-element interacts with the homologous antithrombin III region, but is different from the transcription factor LF-A1 interacting with the A domain of the alpha 1-antitrypsin promoter. The distal region DRI (nucleotides -480 to -454) of the human transferrin gene promoter presents in its core the same 10 nucleotide-long sequence as the PRI cis-element. We have previously shown that the liver protein Tf-LF2, binding to the DRI element is different from the Tf-LF1 trans-acting factor. In this paper we also show that Tf-LF2 is different from the transcription factor LF-A1 interacting with the alpha 1-antitrypsin promoter. The results allow us to conclude that at least three distinct liver nuclear proteins bind to different subsets of 5' DNA regions containing similar sequences. These sequences are present in genes expressed essentially in liver.

Antithrombin III↗
PubMed1988

Interactions of DNA-binding proteins with the 5' region of the human transferrin gene.

We have established by transient expression experiments that the 620 base pairs upstream of the cap site of the human transferrin gene contain the information necessary for efficient expression of the gene in hepatoma cells HepG2 or Hep3B but not in HeLa cells. DNase I footprint analysis reveals that at least five distinct factors present in human or rat liver nuclear extracts interact with different sites of this region. One of these factors, binding to nucleotides -193 to -162, is closely related to or identical with the eukaryotic factor CCAAT-binding transcription factor/nuclear factor I; another one, binding to nucleotides -103 to -83 seems to be related to the CCAAT-binding protein. The binding sites of two other factors, not recognized by HeLa nuclear proteins, each contain an identical 10-nucleotide-long sequence (5' TCTTTGACCT 3') in reverse orientation, separated by 400 base pairs. Results of gel retardation assays, cross-competition experiments, and heat inactivation strongly suggest that the proteins binding to these sites are different. One of these sequences and the binding site of the CCAAT-binding protein related factor are located in the region between nucleotides -119 and -45. We have shown by transient expression experiments with 3' deleted vectors that this region is functionally essential for human transferrin gene expression.

Base Sequence↗
PubMed1988

Cloning and sequencing of Pseudomonas genes encoding vanillate demethylase.

A 2,598-base-pair (bp) SalI-HincII DNA fragment has been cloned which codes for vanillate demethylase, the enzyme responsible for the demethylation of vanillate (3-methoxy-4-hydroxybenzoate) to protocatechuate (3,4-dihydroxybenzoate). Complementation and insertional inactivation experiments have shown that this fragment carries two genes (vanA and vanB) which are predominantly cotranscribed from a promoter upstream of vanA. Nucleotide sequencing of the SalI-HincII fragment confirmed the genetic data: two open reading frames of 987 and 942 bp were present in the transcribed orientation. These had a very high G + C content in the third base of each codon, which is characteristic of Pseudomonas chromosomal genes. Expression of the genes in Escherichia coli with the T7 RNA polymerase-promoter system gave rise to two polypeptides of 36 and 33 kilodaltons which could be identified by deletion analysis as the products of vanA and vanB, respectively. A search of the protein sequence data bank indicated that the vanB gene product was related to the ferredoxin family.

Amino Acid Sequence↗
PubMed1987

A 'phase-shift' fusion system for the regulation of foreign gene expression by lambda repressor in gram-negative bacteria.

A 'phase-shift' translation fusion vector was constructed in which mutually compatible restriction sites BamHI, BclI and BglII are positioned in such a manner that the cut point is in a different reading frame, immediately following the ATG start codon and ribosome-binding site of the lambda cro gene. The lambda cro gene is expressed from promoter pR and controlled by a thermosensitive (cI857) lambda repressor. The usefulness of the expression vector was demonstrated using a galK gene lacking the ATG start codon and fusing this to the pR promoter and ATG start codon of the lambda cro gene, resulting in cI857-regulated expression of galactokinase. The vector is of general use for foreign gene expression in Escherichia coli when the target gene has a compatible cohesive end (5'-GATC-3') at the N terminus (provided, for example, by a BamHI linker). The lambda cI857-pR-cro-galK cassette was cloned into pJRD215, a wide-host-range plasmid and transferred by conjugation to a variety of Gram-negative bacteria. In all cases, thermosensitive regulation of galactokinase could be demonstrated, though the levels of induction varied considerably. These results show that the powerful lambda pR promoter and the efficient lambda repressor can be used to regulate expression of foreign genes in Gram-negative organisms other than E. coli.

Bacteriophage lambda↗
PubMed1987

Vectors with restriction site banks. V. pJRD215, a wide-host-range cosmid vector with multiple cloning sites.

The construction of a new wide-host-range, restriction-site bank, cosmid-cloning vehicle (pJRD215) is described. The wide-host-range properties and the ability to be transferred by conjugation, extend genetic engineering to those Gram-negative species that cannot be transformed. The vector permits the cloning of genes from Gram-negative bacteria using a complementation screening procedure in a mutant host. This procedure is simplified by the possibility of construction of a cosmid gene bank so that only a few hundred clones need to be screened. Subsequent subcloning of the gene of interest is facilitated by the presence of at least 23 unique cloning sites.

Base Sequence↗
PubMed1985

Localisation and characterization of a new rho-dependent transcription terminator from bacteriophage T5.

Relatively few rho-dependent terminators have been described in the literature. This manuscript describes another such terminator, isolated from phage T5. Functional analysis, involving the generation of deletion subclones, has permitted the localization of the terminator on a 413 bp fragment. Attempts to further reduce the size of this fragment resulted in loss of terminator activity. DNA sequence analysis of the terminator region supports the model whereby a rho-dependent terminator is composed of a long region of non-translated unstructured DNA, which permits rho binding, followed by RNA polymerase pausing sites where termination (in the presence of rho) may occur. The results agree with the currently held hypothesis that, despite the many similarities found between various rho dependent termination sequences, no consensus can be defined for either the rho binding or the rho termination sites (1,2).

Base Sequence↗
PubMed1985

Structure of the galactokinase gene of Escherichia coli, the last (?) gene of the gal operon.

We present the nucleotide sequence of the galactokinase gene (galK) of Escherichia coli including its 5' and 3' flanking regions. This DNA sequence derives from the lambda gal8 transducing phage and is identical to the sequence present in the galK gene fusion vectors, pKO and pKG, commonly used to study transcriptional regulatory elements. We define the precise 3' junction between the bacterial and phage sequences in lambda gal8 and demonstrate that this junction probably results from a homologous recombination event between identical 9 bp sequences common to the gal operon and phage lambda. Moreover, we examine the 300 bp region located immediately beyond galK for transcription termination function and find no gal operon terminator. Lastly, we compare the galK genes of E. coli and the yeast S. cerevisiae and find several regions of strong homology among which is a potential ATP-binding site homology shared by a variety of ATP-binding proteins including protein kinases encoded by mammalian oncogenes.

Adenosine Triphosphate↗
PubMed1984

Vectors with restriction-site banks. I. pJRD158, a 3903-bp plasmid containing 28 unique cloning sites.

A DNA fragment has been constructed that contains many unique cloning sites not present in currently used Escherichia coli plasmid cloning vehicles. Insertion of this fragment into a modified version of pBR322 results in an AmpRTetR vector (pJRD158) of 3903 bp containing 28 unique cloning sites, four "almost unique" cloning sites, and eight unassigned unique 6-bp palindromes. The plasmid has the additional advantages of very high copy number and altered incompatibility. The latter permits it to be stably maintained in the same host as pBR322.

Base Sequence↗
PubMed1983

Transcription regulatory elements in the late region of bacteriophage T5 DNA.

Transcription promoters and terminators have been cloned from the late region of bacteriophage T5 DNA and their strengths determined in vivo in plasmid derivatives. DNA sequence analysis shows these transcription signals to be remarkable in that, in all four cases studied in detail, the promoters and terminators overlapped or were very close together.

Base Sequence↗
PubMed1983

Gene rearrangements leading to the expression of an insertion-inactivated tetracycline resistance gene in pBR322.

Cloning into the HindIII site of plasmid pBR322 inactivates the tetR promoter and usually prevents the expression of the tetR gene. The corresponding clones revert to tetracycline resistance at a low frequency. Such reversion is caused by gene rearrangement within the plasmids. DNA sequence analysis reveals three classes of revertants. The first class contains plasmids with partial duplications, which result in the fusion of the promoter of the RNA I species to the tetR gene. The event itself destroys the region encoding the RNA primer for replication and thus the plasmids would be replication defective if the duplication did not also include this region of the molecule. The plasmids from the second class are simple deletions which again fuse the tetR region to the RNA I promoter. In one case, the junction takes place at the end of the RNA I transcript, leaving RNA I and the RNA primer virtually intact. However, it removes the promoter of the RNA primer, the latter now being read from the cloned material. The second member of this class has fused the tetR gene well upstream of the RNA I region so that the RNA primer is still read from its own promoter. The low-level tetracycline resistance is probably due to partial read-through of the RNA I terminator. The third class of revertants differs from the previous two by the acquisition of foreign DNA in the form of an IS2-type insertion element which is known to promote transcription.

Base Sequence↗