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Biomedical subjects

P Van de Putte

Publications and source records attributed to P Van de Putte.

16 recordsLinked to original sources
PubMed1996

Repair of rDNA in Saccharomyces cerevisiae: RAD4-independent strand-specific nucleotide excision repair of RNA polymerase I transcribed genes.

Removal of UV-induced pyrimidine dimers from the individual strands of the rDNA locus in Saccharomyces cerevisiae was studied. Yeast rDNA, that is transcribed by RNA polymerase I(RNA pol I), is repaired efficiently, slightly strand-specific and independently of RAD26, which has been implicated in transcription-coupled repair of the RNA pol II transcribed RPB2 gene. No repair of rDNA is observed in rad1,2,3 and 14 mutants, demonstrating that dimer removal from this highly repetitive DNA is accomplished by nucleotide excision repair (NER). In rad7 and rad16 mutants, which are specifically deficient in repair of non-transcribed DNA, there is a clear preferential repair of the transcribed strand of rDNA, indicating that strand-specific and therefore probably transcription-coupled repair of RNA pol I transcribed genes does exist in yeast. Unexpectedly, the transcribed but not the non-transcribed strand of rDNA can be repaired in rad4 mutants, which seem otherwise completely NER-deficient.

DNA Repair↗
PubMed1996

Diagnostic and therapeutic implications of transesophageal echocardiography in medical ICU patients with unexplained shock, hypoxemia, or suspected endocarditis.

OBJECTIVE: To evaluate the diagnostic and therapeutic implications of transesophageal echocardiography (TEE) in intensive care patients. DESIGN: Comparative study. SETTING: A 10-bed general intensive care unit. PATIENTS: Between 1 January 1992 and 31 May 1993, 61 patients prospectively identified with shock (n = 14), severe, unexplained hypoxemia (Partial pressure of oxygen in arterial blood/fractional inspired oxygen < 200) (n = 31), or suspected endocarditis (n = 16) underwent a TEE examination to supplement transthoracic echocardiography (TTE) examination. INTERVENTIONS: The results of each TEE examination were compared with the clinical findings and TTE data. TEE examinations were classified as follows: 0, TEE results were similar to TTE results; 00, TEE examination resulted in exclusion of suspected abnormalities; 1, TEE revealed a new but minor diagnosis compared to the TTE diagnosis; 2, TEE revealed a new major diagnosis not requiring a change of treatment; 3, TEE results revealed a new major diagnosis requiring an immediate change of treatment. RESULTS: Intraobserver reliability of the TEE classification was confirmed by a 100% concordance and interobserver reliability was evaluated as an 84% concordance. Results of the TEE classification were: class 0, n = 21 (34%); class 00, n = 13 (21%); class 1, n = 7 (12%); class 2, n = 8 (13%); class 3, n = 12 (20%). Therapeutic implications of TEE in class 3 patients were cardiac surgery in 5 patients (2 cases of acute mitral regurgitation, 2 valvular abscesses, and 1 hematoma compressing the left atrium), discontinuation of positive end-expiratory pressure in 1 ventilated patient with an atrial septal defect, weaning off mechanical ventilation in 1 patient with an atrial septal defect, prescription of antimicrobial therapy in 3 patients with endocarditis, and prescription of anticoagulant therapy in 2 patients with left atrial thrombus. No difficulty inserting the transducer was observed in any of the 61 patients studied. The only noteworthy complication was a case of spontaneously resolving atrial fibrillation. CONCLUSION: TEE is safe and well tolerated and is useful in the management of patients in the intensive care unit with shock, unexplained and severe hypoxemia, or suspected endocarditis when TTE is inconclusive.

Aged↗
PubMed1987

Transfer of human genes conferring resistance to methylating mutagens, but not to UV irradiation and cross-linking agents, into Chinese hamster ovary cells.

Chinese hamster ovary cells were transfected by human DNA ligated to the bacterial gpt (xanthine-guanine-phosphoribosyltransferase) gene which was used either in its native form or after partial inactivation with methylnitrosourea. The gpt+ transfectants were screened for resistance to high doses of N-methyl-N'-nitro-N-nitrosoguanidine. Using this approach, we showed that Chinese hamster ovary cells can acquire N-methyl-N'-nitro-N-nitrosoguanidine resistance upon transfection with DNA from diploid human fibroblasts, that this resistance is transferable by secondary transfection and is specific for methylating mutagens, and that it is not caused by increased removal of O6-methylguanine, 3-methyladenine, and 7-methylguanine from DNA.

Adenine↗
PubMed1986

Location and cloning of the ultraviolet-sensitizing function from the chromosomally associated IncJ group plasmid, R391.

The IncJ plasmid R391, which specifies a uv-sensitizing function, has been shown to be associated with chromosomal DNA. Deletions originating from Tn10 insertion into the kanamycin-resistance determinant of plasmid R391 gave rise to uv-resistant derivatives. This apparent linkage between the kanamycin-resistance determinant and the uv-sensitizing gene(s) was used to clone the uv-sensitizing function from plasmid R391 into pUR222. A recombinant plasmid containing both functions (KanR and Uvs+) was obtained. The uv-sensitizing function was mapped to a 4-kb EcoRI fragment.

Chromosome Mapping↗
PubMed1984

Analysis of the methylation-regulated Mu mom transcript.

The Mu mom gene is transcribed only when DNA is methylated by Dam; this methylation dependence is determined by a region upstream of the gene containing three Dam-methylation sites. The mom operon contains two open reading frames, one of which codes for Mom. Experiments were done to find a function for the other reading frame; it is shown that it does not affect phage immunity or the regulation of mom expression. Deletion mapping and S1 nuclease mapping experiments identify the +1 of the mom mRNA: it is 45 bp downstream of the three Dam-methylation sites. Possible models for the short-distance transcription regulation by methylation are discussed. The mom promoter is surrounded by sequences that are shown to be homologous to the oriC region of E. coli. This suggests a relation between replication and methylation-dependent mom expression. It is shown however, that replication is not essential for mom expression.

Bacteriophage mu↗
PubMed1983

Transcription initiation of Mu mom depends on methylation of the promoter region and a phage-coded transactivator.

The product of the bacteriophage Mu gene mom modifies adenine residues of DNA within the consensus sequence CGAGCNPy, providing protection against various restriction endonucleases (ref. 1 and D. Kamp, personal communication cited in ref. 2). The mom gene is only expressed during lytic development of the phage. It is known that mom is nonfunctional in Escherichia coli host mutants in a gene (dam) which itself encodes an adenine methylation system. We show here that the E. coli dam gene is essential for transcription initiation of the mom gene, and that this dependence on dam seems to lie in a short segment preceding the mom coding region, which also contains the mom promoter. The sequence of this segment reveals the presence of dam methylation sites (GATC), and suggests a model for the regulation of mom gene expression based on DNA secondary structure, which may explain why mom is only expressed during phage lytic development. We also show that expression of phage-coded proteins (A, B and C) is needed for transactivation of mom transcription.

Bacteriophage mu↗
PubMed1983

Site-specific recombination by Gin of bacteriophage Mu: inversions and deletions.

A 3000-bp invertible segment in the DNA of bacteriophage Mu determines the host range of the phage. The inversion is catalyzed by the phage-coded protein Gin; the recombination sites are short inverted repeats. Gin protein is only made in low amounts by Mu. To further investigate the Gin-mediated recombination reaction a Gin overproducing strain was constructed. The gin gene was cloned on a plasmid behind the PL-promotor of phage lambda. This results in a 100-fold higher inversion frequency of a Mu gin phage as compared to the situation when Gin is expressed from its own promoter. A test system was developed suitable for the detection of Gin action in vivo and in vitro: the lacZ gene of E. coli was cloned within the invertible region in such a way that it is only expressed when the region is in one specific orientation. Thus inversions can be detected or selected as a switch from Lac- to Lac+. This system was used to determine the inversion frequency under different experimental conditions. The ability of Gin to catalyze deletions was investigated by inverting in vitro one of the two recombination sites using restriction enzymes and genetically marking the DNA between those sites. Deletions do occur, although at a lower frequency than inversions.

Bacterial Proteins↗
PubMed1982

Bacteriophage Mu DNA replication is stimulated by non-essential early functions.

The replication of a spontaneous Kil- mutant of bacteriophage Mu has been investigated. The Kil- mutation (Mucts62-13/4), which was introduced into a defective prophage, is pleiotrophic, leading to the loss of also the Gam, Cim and Sot functions. The mutation is caused by an insertion with the characteristics of IS1, located just outside the B gene. Mucts62-1 3/4 phages form extremely small plaques on wildtype indicator strains. The replication of the insertion mutant as compared to Mucts62 is strongly reduced. Normal replication could be restored by relieving the polarity of the insertion or by complementation with defective prophages which express all early functions. Apparently, early genes other than A and B are involved in normal Mu DNA replication.

Coliphages↗
PubMed1980

Elongation factor Tu isolated from Escherichia coli mutants altered in TufA and tufB.

In a previous paper we described a number of Escherichia coli mutants resistant to the antibiotic kirromycin. These mutants are altered in both tufA and tufB, the genes coding for elongation factor Tu (EF-Tu). We have now isolated EF-Tu in a homogeneous form from the mutant strains and have studied its function in polypeptide synthesis. These EF-Tu preparations were examined in renaturation studies of Qbeta RNA replicase, described in another paper. In order to characterize the factor we have inactivated the tufB gene by insertion of bacteriophage Mu or by an amber mutation. This enabled us to isolate EF-Tu as a single gene product derived from tufA (designated EF-TuA in contrast to the tufB product, which is called EF-TuB). Kirromycin-resistant EF-TuA did not respond to addition of the antibiotic in three assays: [(3)H]GDP exchange with EF-Tu-GDP at 0 degrees C, in vitro translation of poly(U), and kirromycin-induced GTPase activity of EF-Tu. In contrast, wild-type EF-TuA responded normally to the antibiotic in these assays. One of our mutants (LBE 2012) harbors the kirromycin-resistant EF-TuA and an EF-TuB that is able to bind kirromycin. This binding does not cause inhibition of protein synthesis, indicating that EF-TuB from LBE 2012 is unable to reach the ribosome under these conditions. The two types of EF-Tu from this mutant are equal in size but differ by 0.1 pH unit in isoelectric point. In the soluble fractions of LBE 2012 cells they are present in approximately equal amounts. Our results also show that the tufB gene is not necessary for bacterial growth.

Escherichia coli↗
PubMed1979

Thermo-inducible expression of cloned early genes of bacteriophage Mu.

An EcoRI fragment, containing approx. 5100 base pairs (bp) of the immunity-end of bacteriophage Mu, was inserted into the multicopy plasmid pMB9 by in vitro recombination. The expression of early Mu genes, located on the cloned fragment, is thermo-inducible because of the presence of the ts mutation in gene c. The isolation of a transformant harbouring the recombinant plasmid, pGP1, was possible only when expression of Mu genes was prevented. pGP1 can be maintained at 28 degrees C at high copy number, but at 42 degrees C the pGP1 containing cells are killed due to the expression of the kil gene of Mu. The following Mu genes are present on pGP1: the ner gene, the integration and replication genes A and B, the cim gene, and the kil gene. pGP1 containing cells do not show Gam and Sot activity at 42 degrees C, therefore the leftmost EcoRI site on the Mu DNA is located between genes kil and gam or sot, or within the gam or sot gene.

Chromosomes, Bacterial↗
PubMed1978

Mutants of Escherichia coli altered in both genes coding for the elongation factor Tu.

Genetic analysis of a mutant of Escherichia coli resistant to the antibiotic mocimycin is presented. This resistance is due to alterations in both tuf genes coding for the elongation factor Tu. Mocimycin resistance is recessive. Bacteria carryong only one tuf gene from the resistant mutant are still mocimycin sensitive. If the mutant gene is the tufA gene, the seisitive cells can be made resistant through inactivation of the tufB gene by insertion of the bacteriophage milliunits genome. Conditional mocimycin-resistant mutants ban also be isolated when the tufB gene is altered by an amber or a temperature-sensitive mutation. When only the tufB allele from the original mocimycin-resistant mutant is present, inactivation of the wild-type tufA gene fails to give viable mocimycin-resistant progeny. We conclude that the tufA mutant allele codes for a functional mocimycin-resistant EF-Tu, whereas the mutant tufB gene does not code for a functional product.

Anti-Bacterial Agents↗
PubMedDate not supplied

Expression of a bacterial repair gene in mammalian cells.

The coding sequence of the uvrA gene from Escherichia coli has been fused to the early promoter, enhancer and origin of replication of the simian virus SV40, and was supplemented with splicing and polyadenylation sites arising from the same virus. Introduction of this hybrid gene into simian cos-1 cells results in the synthesis of a full length UvrA protein (114 kD) which has retained its ability to bind to single-stranded DNA.

Animals↗