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F Georges

Publications and source records attributed to F Georges.

At least 19 recordsLinked to original sources
PubMed1995

Promoters from kin1 and cor6.6, two homologous Arabidopsis thaliana genes: transcriptional regulation and gene expression induced by low temperature, ABA, osmoticum and dehydration.

The Arabidopsis thaliana genes kin1 and cor6.6 belong to the same family and were expressed at higher levels following low temperature and ABA treatments. In an attempt to elucidate the mechanism of gene regulation by low temperature, the relationship between low-temperature- and abscisic acid (ABA)-induced gene expression and possible differential expression of the two genes, we have cloned a 5.3 kb genomic fragment harboring kin1 and cor6.6 and their respective 5' sequences. The putative promoters of both genes were fused to the beta-glucuronidase (GUS) coding sequence and GUS expression was analysed in transgenic tobacco and Arabidopsis plants. The cor6.6 promoter produced a higher basal level of expression than the kin1 promoter in transgenic tobacco. Enzyme assays of inducible GUS activity in transgenic Arabidopsis and tobacco plants showed that GUS activity directed by both kin1 and cor6.6 promoters was significantly induced by ABA, dehydration and osmoticum, but not by low temperature. Northern analysis revealed, in contrast, that GUS mRNA was significantly induced in these transgenic plants by low temperature. Further analysis showed that, at low temperature, GUS protein synthesis from the induced GUS mRNA was inhibited. Together these results reveal induction of kin1 and cor6.6 transcription by low temperature, exogenous ABA and dehydration. However, low-temperature expression is dramatically reduced at the translational level.

Abscisic Acid↗
PubMed1995

Domains in human interferon alpha-1 gene containing tandems of arginine codons AGG play the role of translational initiators in E. coli.

The AGG and AGA are the least used arginine codons in E. coli but they are the most preferable ones in eukaryotes. The low expression of some eucaryotic genes (such as human alpha-1 interferon gene) which contain clusters of AGG codons is explained either by the limited pool of the tRNA(AGG) (Varenne and Lazdunski, 1986) or by the competition of these clusters with the Shine-Dalgarno (SD) sequence (Ivanov et al., 1992). The aim of the present study is to demonstrate the in vivo capacity of AGG tandems to bind to bacterial ribosomes. The two tandems of AGG codons (Arg12 Arg13 and Arg163 Arg164) of hIF alpha 1 with their surrounding nucleotides were cloned in a bacterial expression plasmid containing a strong promoter and a reporter gene (chloramphenicol acetyltransferase, CAT) devoid of a ribosome binding site. The results obtained showed that both AGG tandems initiated translation of the CAT mRNA with an efficiency equal to that of the consensus SD sequence and several fold higher than the native SD sequence of the CAT gene.

Amino Acid Sequence↗
PubMed1993

Deamidation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system, involves asparagine 38 (HPr-1) and asparagine 12 (HPr-2) in isoaspartyl acid formation.

Histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, when incubated at elevated temperatures forms many species of protein. The two major species are HPr-1 and HPr-2, which have been shown to lack one or two amides, respectively (Anderson, B., Weigel, N., Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 7023-7033). The formation of HPr-1 and HPr-2 is shown to be pH-dependent and does not occur readily below pH 6. Investigation of the identities and properties of the two residues that deamidate involved creation of site-directed mutants at the 6 glutamine and 2 asparagine residues of HPr; description of their deamidation species by isoelectric focusing; determination of their relative antibody binding properties; assay of their phosphoacceptor and phosphodonor activities; characterization of tryptic and V8-protease peptides; obtaining two-dimensional nuclear magnetic resonance spectra of HPr, HPr-1, and several mutants. It was determined that the sequential deamidation of Asn-38 and Asn-12 yields HPr-1 and HPr-2. Both residues exist as Asn-Gly pairs, and both deamidations probably form isoaspartyl acid. HPr from Bacillus subtilis and Staphylococcus carnosus which also have Asn-Gly at residues 38 and 39 form HPr-1 species presumably by deamidation. HPr from Streptococcus faecalis which does not have Asn-38 does not form a HPr-1 species. The E. coli mutant HPrs, N12D and Q51E, residues that may be involved in the active site, had impaired phosphohydrolysis properties and decreased phosphoenolpyruvate:sugar phosphotransferase system activity.

Amino Acid Sequence↗
PubMed1993

The involvement of the arginine 17 residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site His-15 that is phosphorylated to form N delta 1-P-histidine. The nearby conserved residue, Arg-17, has been replaced by: lysine, histidine, glutamate, glycine, serine, and cysteine. All mutations resulted in impairment of the phosphoacceptor function of HPr with enzyme I: kcat/Km values between 6% (Ser-17) and 0.1% (Glu-17), relative to wild type. Several sugar-specific enzymes II had different responses. Both the Vmax and Km of enzyme IIN-acetylglucosamine were altered, while for enzyme IImannose only Km was affected, except for R17E. For both enzymes, kcat/Km values were between 0.5 and 3%, with R17E being 10-fold lower. Except for R17E, minimal effects were observed for enzyme IImannitol. These results suggest that there are different rate-limiting steps in the enzymes II. Phosphohydrolysis properties and the pKa values for His-15 and phosphorylated His-15 determined by NMR for both wild type and mutant HPrs suggest that Arg-17 is partly responsible for the instability of P-His-15 and the depressed pKa values in wild type HPr. Other feature(s) of the tertiary structure influence the protonation of His-15 and the phosphohydrolysis properties of phosphorylated His-15.

Amino Acid Sequence↗
PubMed1992

Vectors with segmented multiple cloning sites (SMCS) for easy monitoring of restriction digests and post-cloning orientation reversal of genes.

A novel approach is described, through which the transcriptional orientation of cloned genes is manipulated without further subcloning. This is achieved through a restriction/ligation process (without changing the test tube) leading to approx. 1:1 mixture of both possible orientations. The plasmid containing the reversed orientation is easily distinguished from the wild type by built-in restriction-indicator sites for SpeI. Additionally, a large marker fragment is incorporated into the segmented multiple cloning site (SMCS) regions to facilitate monitoring the progress and/or efficiency of vector restrictions. The marker fragment is released upon appropriate double digestion, thus acting as an indicator for complete restriction-enzyme digestion. We have equipped some existing plasmids, commonly used in molecular biology, with these features and demonstrated their superiority to plasmids having regular MCS sequences.

Base Sequence↗
PubMed1991

Involvement of the carboxy-terminal residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site that involves His-15, which is phosphorylated to form a N delta 1-P-histidine, Arg-17, and the carboxy-terminal residue Glu-85. Mutant HPrs with alterations to the three C-terminal residues, Glu-85, Leu-84, and Glu-83, were produced by site-directed mutagenesis. The properties of these mutants were assessed by kinetic analysis of enzyme I, enzyme IImannose, enzyme IIN-acetylglucosamine, and enzyme IImannitol, and the phosphohydrolysis properties of the HPr mutants. The results show that it is the C-terminal alpha-carboxyl of Glu-85 that is involved in the active site, and this involvement may be restricted to the phosphoryl donor action of HPr. The contribution of this alpha-carboxyl group is modest as the deletion of Glu-85 resulted in the reduction of the enzyme II activity (kcat/Km) to about 33%. Removal of both Glu-85 and Leu-84 yields an HPr that is an impaired substrate of both the enzyme I and enzyme II reactions. Glu-83 appears to have no role in the active site.

Bacterial Proteins↗
PubMed1991

Epitope mapping by mutagenesis distinguishes between the two tertiary structures of the histidine-containing protein HPr.

Thirty-four of the 85 residues of the histidine-containing protein HPr of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system have been changed by site-directed mutagenesis. Many of the mutations have wild-type activity suggesting an unaltered tertiary structure but have altered binding to three monoclonal antibodies: Jel42, Jel44, and Jel323. This altered binding defines the residues that are involved in the epitopes of HPr. At present, two different three-dimensional structures have been determined for HPr, one from two-dimensional nuclear magnetic resonance spectra and the other from x-ray diffraction of HPr crystals. The epitope mapping for Jel42 does not distinguish between the tertiary structures. However, only the HPr structure derived from two-dimensional nuclear magnetic resonance spectra is consistent with a contiguous surface binding site that can be defined as the epitope for Jel44. Thus the x-ray structure may represent a partially unfolded HPr.

Amino Acid Sequence↗
PubMed1991

Effect of low temperature stress on the expression of sucrose synthetase in spring and winter wheat plants. Development of a monoclonal antibody against wheat germ sucrose synthetase.

A monoclonal antibody against wheat germ sucrose synthetase is developed and characterized. Its use in studying the effect of cold acclimation on the expression of sucrose synthetase in winter and spring wheat plants is described. The antibody shows cross-reactivity with sucrose synthetase from maize and pea plants, as well as carrot cells. A gradual accumulation of the enzyme as a function of time spent at 2 degrees C is observed in both wheat varieties. In contrast, an initial sharp rise in the mRNA level is observed, which agrees with the previously reported response of maize plants subjected to anaerobic stress.

Antibodies, Monoclonal↗
PubMed1990

Design and cloning of a synthetic gene for the flounder antifreeze protein and its expression in plant cells.

A synthetic gene coding for the winter flounder antifreeze protein (AFP) has been constructed. A new strategy for the synthesis has been employed such that one strand of the duplex was chemically synthesized and the other was produced enzymatically by chain extension. The chemically synthesized blocks were constructed so that the second strand was self-priming. The resulting DNA fragment was incorporated into the vector, pGCS1, which contained a translational fusion of the sequence encoding AFP and the N terminus of cat (encoding chloramphenicol acetyltransferase, CAT), under the control of the cauliflower mosaic virus 35S promoter. This plasmid was introduced into protoplasts of corn (var. Black Mexican Sweet) by electroporation. Production of the fusion peptide was monitored by CAT assay and Western blotting with antisera to AFP and CAT.

Animals↗
PubMed1989

[Tamponade disclosed by functional right-left shunt reversible after surgical drainage].

The authors report a case of tamponade revealed by the discovery, during emergency radioisotope scanning of the lung, of an acquired right-to-left shunt which was reversed by surgical drainage. The patient was a 60-year old woman treated by anticoagulants for suspected pulmonary embolism who had sudden worsening of her symptoms on the 7th day of the disease.

Cardiac Tamponade↗
PubMed1988

Enhancing the sensitivity of DNA detection and recovery from agarose gels.

By inserting nitrocellulose strips into agarose gels alongside the electrophoresed lanes and passing an electric current perpendicularly in the direction of the strips, highly efficient transfer of DNA bands onto the membrane in the form of concentrated dots is achieved. DNA detection limits by this technique are enhanced, at least three times as visualized by ethidium bromide fluorescence and at least twice more by radiolabeling.

Chromatography, High Pressure Liquid↗
PubMed1987

[Is the theoretical blood volume the optimum for chronic respiratory insufficiency? Hemodynamic study of chronic bronchitis patients as a function of the hematocrit].

We have studied oxygen transport and haemodynamics in 19 clinically stable chronic bronchitis split into two groups with comparable blood gases but different haematocrits (Group I = 33%, Group II = 51.5%). The aim of the study was to assess the suitability of bringing the blood volume down to the predicted normal value by repeated venesection. The respiratory function was moderately altered in these patients. The pulmonary and systemic vascular resistance were significantly more elevated in Group II. The arterial oxygen transport was comparable in the two groups. On the other hand for Group I the systolic work of the right ventricle was greater on account of the increased cardiac output. We have not observed any difference in blood volume between the two groups of patients. These results suggest that a return to the predicted blood volume is not recommended without some doubts in chronic bronchitis with polycythaemia.

Blood Pressure↗
PubMed1985

[Exertion isotope tests in coronary insufficiency. Comparison with isotopic ventriculography and myocardial scintigraphy].

Very few studies have been described comparing the value of exercise myocardial scintigraphy and left ventricular angioscintigraphy. The authors designed a study comparing these two investigations with conventional exercise stress testing and coronary angiography. The isotopic investigations were carried out within 48 hours of coronary angiography. A total of 143 patients undergoing coronary angiography (35 normal, 108 coronary patients: 36 single vessel, 36 double vessel and 36 triple vessel disease) were included in this study. The lesions were located of the LAD (77 cases), left circumflex (77 cases) and right coronary arteries (62). The sensitivity and specificity of both radionuclide investigations were evaluated to assess their diagnostic value; the best results were obtained with myocardial scintigraphy (sensitivity 86 p. 100; specificity 100 p. 100); angioscintigraphy had a sensitivity of 71 p. 100 and specificity of 97 p. 100, and conventional exercise stress testing of 42 p. 100 and 70 p. 100 respectively. The sensitivity seemed to increase with the degree of stenosis; although the sensitivity of myocardial scintigraphy increased progressively, that of angioscintigraphy doubled in cases of stenosis 90 p. 100 (stenosis less than 90 p. 100, sensitivity = 37 p. 100; stenosis greater than 90 p. 100, sensitivity = 73 p. 100). The sensitivity of myocardial scintigraphy with respect to the severity of the coronary artery disease was best in cases of right coronary artery stenosis (sensitivity in cases of RCA stenosis = 74 p. 100; sensitivity in LAD stenosis = 58 p. 100; sensitivity in left circumflex stenosis = 43 p. 100). The sensitivity of left ventricular angioscintigraphy was best in LAD stenosis (RCA stenosis = 50 p. 100, LAD stenosis = 64 p. 100, left circumflex stenosis = 36 p. 100). The sensitivity of both investigations was poor in left circumflex artery stenosis even when severely diseased. The sensitivity of both investigations was better in diffuse coronary artery disease: myocardial scintigraphy (single vessel disease: 72 p. 100, double vessel disease: 92 p. 100, triple vessel disease: 94 p. 100), left ventricular angioscintigraphy (61 p. 100, 69 p. 100, and 83 p. 100 respectively). Although the association of these two radioisotopic investigations does not improve diagnostic sensitivity, it does provide more information about the localisation and extension of the coronary artery disease especially in LAD and right coronary artery stenosis. These results suggest that these investigations are complementary in the evaluation of patients with coronary artery disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Coronary Disease↗
PubMed1984

A potential stem-loop structure and the sequence CAAUCAA in the transcript are insufficient to signal rho-dependent transcription termination at lambda tR1.

It has been suggested that a sequence in the RNA transcript that can form a stem and loop structure, followed by the sequence CAAUCAA, is the signal for rho-dependent transcription termination. We tested this hypothesis by synthesizing a DNA duplex whose sequence corresponds to a region of the lambda tR1 terminator that contains these structural features. We cloned this synthetic DNA fragment under the control of the lacUV5 promoter, and showed that it does not cause rho-dependent termination in vitro. RNA polymerase pauses during in vitro transcription across the synthetic sequence, although less efficiently than at the corresponding sequence on the lambda template. No rho-mediated termination was detected even under conditions that prolonged transcriptional pausing at the synthetic site, indicating that the synthetic sequence is defective as a transcript release site. We suggest that unlike rho-independent terminators, rho-dependent terminators require sequences in addition to those immediately before the sites of termination.

Base Sequence↗
PubMed1984

Synthesis of a human insulin gene. VII. Synthesis of preproinsulin-like human DNA, its cloning and expression in M13 bacteriophage.

A 74-bp DNA sequence coding for the pre sequence of human preproinsulin and containing EcoRI termini was synthesized by the chemical enzymatic method, joined with previously synthesized proinsulin DNA, and cloned in the M 13mp8 vector. A clone pNB82 -121 was identified by DNA sequence which confirmed the correct orientation of the pre sequence to the proinsulin DNA. The EcoRI site at the junction of pre- and proinsulin DNA was eliminated by removing a triplet ATT using a synthetic 19-mer primer. To simplify preproinsulin isolation and to study its expression in the M 13 system, a 25-bp affinity leader sequence coding for (glu)7 was inserted at the remaining EcoRI site; this put the preproinsulin DNA in a correct reading frame with the AUG initiation codon of beta-galactosidase. Preproinsulin was expressed under lac promoter control as analyzed by a radioimmunoassay (RIA) against C-peptide.

Base Sequence↗