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Biomedical subjects

L Orosz

Publications and source records attributed to L Orosz.

At least 19 recordsLinked to original sources

Stable incorporation of genetic material into the chromosome of Rhizobium meliloti 41: construction of an integrative vector system.

An integrative vector system has been developed from the site-specific recombination elements of temperate phage 16-3. The system can be used for highly efficient stable introduction of genetic material into the chromosome of the symbiotic nitrogen-fixing organism, Rhizobium meliloti 41 (Rm41) at the attB site. Vectors carrying the phage-borne attachment site were constructed, and helper phages providing the site-specific recombination functions in trans were isolated. Other possible applications of the system are discussed.

Bacteriophages

The isolated N-terminal DNA binding domain of the c repressor of bacteriophage 16-3 is functional in DNA binding in vivo and in vitro.

The 197 amino acid c repressor of the temperate Rhizobium meliloti phage 16-3 still regulates the OR operator of the phage after removal of its carboxyl terminal region. When cloned in the low-copy-number plasmid pGA46, a severely truncated variant (R1-77), which retains only the first 77 amino acids of the intact protein, repressed in vivo transcription from the phage promoter PR. When the R1-77 repressor was fused to E. coli beta-galactosidase, the hybrid protein bound OR operator DNA in vitro. The behavior of fusion proteins derived from a point mutant is consistent with the assignment of DNA binding specificity to the amino-terminal region. Furthermore two repressor alleles bearing ts mutations that mapped in the R1-77 region (near a helix-turn-helix motif) were also temperature sensitive for regulation of the OR site, while an 18 bp "in frame" deletion mutant, which mapped in the carboxyl terminal segment, regulated the OR operator in wild-type fashion. The carboxyl terminal region of the repressor is however necessary for the control of lysogenic development of 16-3.

Alleles

[Infectious diseases and hemostasis].

Authors survey the most significant haemostatic complications (thrombocytopenia, DIC, vasculitis, thrombotic microangiopathy) as well as their immune and non-immune pathogenesis in infectious diseases. A short summary of therapeutic facilities and the infectious hazards of blood component therapy is also given.

Blood Coagulation Disorders

[Protein C and apoprotein levels in ischemic heart disease diagnosed by coronarography].

The levels of protein C and different lipid parameters were measured in 22 patients, younger than 50 years, suffering from coronary heart disease, proved previously by angiography. The severity of coronariasclerosis showed certain correlation with the concentration of apolipoprotein B and triglycerids, while low levels of protein C were also mainly observed in those with severe coronary heart disease.

Adult

[Connection between plasma thromboxane and prostacyclin levels in the metabolic control of diabetic children].

Platelet poor plasma thromboxane and prostacyclin levels and the quantity of metabolic control, altogether with vascular complications were evaluated in 55 children diabetes mellitus. The control group consisted of 33 healthy children of the similar age. Thromboxane levels remained unchanged in diabetics, while prostacyclin proved to be significantly decreased, which resulted in greater thromboxane/prostacyclin ratio. No meaningful differences were found according to the presence or absence of vascular complications in this group of diabetics. A positive correlation could have been detected between glycosylated haemoglobin and thromboxane levels, while a negative one between glycosylated haemoglobin and prostacyclin levels. The alterations of prostaglandin metabolism may be regarded as a consequence of diabetic metabolic changes, rather than of vascular complications. Disturbed prostaglandin metabolism in diabetic children might have a role in the pathogenesis of vascular complications.

Blood Glucose

PMAP, PMAPS: DNA physical map constructing programs.

Computer programs are described, which facilitate the construction of the restriction site (physical) map of DNA molecules. By knowing the length of each fragment and its degree of error in the single and the double restriction enzyme digestions, the programs give all the possibilities for the physical map. This method is applicable to linear DNA molecules. Several examples are presented which indicate the high efficiency of the programs in constructing restriction site maps for the 62 Kb chromosome of bacteriophage 16-3. We have constructed complex maps (i.e. EcoRI map with 16 and EcoRV with 11 fragments).

Base Sequence

Recombination deficient mutants of Rhizobium meliloti 41.

Two mutants deficient in homologous genetic recombination have been isolated from Rhizobium meliloti 41 after Tn5 mutagenesis. Both mutants are defective in the induction of temperate phage 16-3 by UV-light, Mytomycin-C or Bleomycin, their UV sensitivity is more pronounced than that of the wild-type strain, and they lack the 'SOS activity' responsible for induced mutations.

Bacteriophages

The detailed physical map of the temperate phage 16-3 of Rhizobium meliloti 41.

Restriction cleavage maps for enzymes EcoRI, BamHI, PstI, PvuII, XbaI and EcoRV of Rhizobium meliloti temperate phage 16-3 have been established. Together with the earlier maps (HindIII, KpnI, HpaI, BglII) 98 restriction sites, 'evenly' distributed, have been mapped along the phage genome, including the so far unmarked silent region of the chromosome. All the restriction maps have been fitted to each other by computer optimalization. Beyond for conventional techniques a computer program (PMAP) for physical mapping of linear DNA has been employed which made the experimentation, in several cases, extremely efficient.

Bacteriophages

A control element within a structural gene: the gal operon of Escherichia coli.

The gal operon of Escherichia coli is transcribed from two overlapping promoters, PG1 and PG2. Cyclic AMP and its receptor protein (CRP) modulate the two promoters in opposite directions by binding to a single cat locus. Both the promoters are negatively regulated by a single repressor, the product of the galR gene. An operator site, defined by several mutations, has previously been located upstream from the cat locus. We have isolated and characterized a new set of cis-dominant constitutive mutations of the gal operon and determined their locations by DNA sequencing. From these studies, we propose the existence of a second functional gal operator element at an extraordinary site--within galE, the first structural gene. Both the operators, OE (exterior) and OI (interior), are involved in the repression of PG1 and PG2. This would be the first example of the presence of a functional operator element within a structural protein-coding region.

Base Sequence

Cyclic AMP-dependent constitutive expression of gal operon: use of repressor titration to isolate operator mutations.

When the gal operator region is present in a multicopy plasmid it binds to all ("titrates") the gal repressor and "induces" the chromosomal gal operon. To make operator mutations (Oa) with reduced affinity toward the repressor, plasmid DNA was irradiated with UV light and mutant derivatives were isolated that were unable to release the chromosomal gal genes from repression. Then with such an Oa plasmid operator revertants were isolated that had reacquired the ability to release repression. Both sets of mutations have been localized by DNA sequence analysis. When the Oa mutations were transferred from the plasmid to the chromosome by recombination these mutant operators were found to make gal expression constitutive (independent of repressor) but still dependent on cAMP, whereas the previously reported gal operator mutants (Oc) are constitutive both in the presence and in the absence of cAMP. The titration method of isolating mutants enables the isolation of strains with operator mutations that also affect normal promoter activity, and it provides an easy way to isolate revertants of operator mutations.

Base Sequence

Identification of structural proteins of Rhizobium meliloti temperate phage 16-3.

The structural proteins of Rhizobium meliloti temperate phage 16-3 have been analysed by means of polyacrylamide gel electrophoresis, isoelectric focusing and agarose gel electrophoresis. Five major and five minor proteins were identified and characterized with respect to their size, isoelectric point and their distribution between the head ad tail of the phage particle. The synthesis of structural proteins was studied by one- and two-dimensional gel electrophoresis.

Bacteriophages

[Lassa letter].

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History, 20th Century

Heterozygosis of phage 16-3 of Rhizobium meliloti: moderate level of mismatch repair or gene conversion.

Analysis of clear/turbid mottled (heterozygotic plaques) of Rhizobium meliloti temperate phage 16-3 indicates that the efficiency of repair at three sites (ti3, ti4, and ti5) in the C cistron is 2 to 20-fold less than that observed in E. coli phage lambda. In agreement with this conclusion, heterozygotic plaques were observed at similar frequency in crosses where point and small deletion mutants were combined, suggesting that in Rhizobium, DNA molecules with short single-stranded loops can escape from repair as efficiently as the simple mismatches.

Bacteriophage lambda

Correlation between map position and phenotype of CTI mutants in the c cistron of Rhizobium meliloti phage 16-3.

Nine temperature-sensitive clear mutations (Cti) in the C cistron (coding for the repressor protein) of Rhizobium meliloti temperate phage 16-3 were characterized according to the inductive temperature, the immunity of cells lysogenic for these mutant phages to superinfection by homoimmune weak virulent mutants, the phenotype of double-ti mutants and interallelic complementation. The results indicate that mutations of similar phenotypic expression are clustered on the genetic map. Furthermore, it seems probable that the C cistron of the original phage 16-3 is identical to that of the independently isolated phage strain 36.

Bacteriophages

Isolation and characterization of an R-prime plasmid from Rhizobium meliloti.

Using a simple enrichment procedure, we isolated an R-prime derivative of plasmid R68.45 carrying a 17.8-megadalton segment of the Rhizobium meliloti 41 chromosome. The chromosomal segment carried on this plasmid (pGY1) includes the markers cys-24+, cys-46+, and att16-3. Plasmid pGY1 mobilized the chromosome in a polarized way starting from the region of homology, but cannot promote chromosome transfer from other sites. The att16-3 site on pGY1 allowed the integration of phage 16-3 into pGY1, and a composite plasmid of 91.8 megadaltons was formed. This vector (pGY2) is suitable for the introduction of Rhizobium bacteriophage 16-3 into other gram-negative bacteria.

Bacteriophages

Restriction mapping of DNA of temperate Rhizobium meliloti phage 16-3: comparison of genetic and physical maps indicates a long, genetically silent chromosomal arm.

The complete restriction map of DNA (61.57 Kb) of temperate Rhizobium meliloti phage 16-3 has been constructed for enzymes BglII, HindIII, HpaI, KpnI, and a partial map for EcoRI. The strategy employed for mapping included the analysis of double, triple and partial digests; comparison of wild type and deletion mutants; and detailed analysis of subfragments, exploiting the presence of cohesive ends of the phage. Comparison of the genetic and physical maps indicates that one arm of the chromosome is genetically silent and/or contains nonessential genes.

Bacteriophages

Effect of heparin on insulin secretion in vivo and in vitro.

The effect of heparin of therapeutic dose on the basal insulin secretion and on that of induced by glucose stimulation was studied in dogs and in Langerhans islets in vitro model. The effect of heparin, given intraarterially (a. pancreaticoduodenalis sup.) and injected i.v. before i.v. glucose load, on the immuno-reactive insulin concentration of the pancreatic and peripheral venous blood as well as the blood sugar and free fatty acid levels of the peripheral venous blood was investigated. Heparin inhibited significantly both the basal insulin secretion and that of following glucose stimulation. After the injection of heparin the glucose assimilation coefficient decreased significantly. The inhibitory effect of heparin on the basal insulin secretion and the glucose stimulated insulin secretion was observed in Langerhans islets in vitro model, too. This suggests that heparin, at least partly, inhibits insulin secretion directly through its influence on the beta cells.

Animals