László Dorgai

Tissue- and stressor-specific differential expression of two hsc70 genes in carp

Two genes expressing 70 kDa heat shock proteins were identified in Cyprinus carpio. The sequence similarities and the intron-interrupted structure of the coding regions indicate that carp Hsc70-1 and Hsc70-2 belong to the Hsp70 cognate subfamily. The expressions of the two hsc70 genes were followed by semi-quantitative RT-PCR. Both genes are expressed under unstressed conditions in a characteristic tissue-specific manner. Inducibility of the response to elevated temperature, cold shock, and Cd treatment was investigated in the liver and muscle, in whole-animal experiments. Both genes were insensitive to or only weakly induced by the stressors, with two exceptions: Cd treatment resulted in an 11-13-fold enhanced induction of hsc70-1 in the liver and cold shock enhanced induction of hsc70-2 in the muscle by 7.5-10-fold.

The bacterial attachment site of the temperate Rhizobium phage 16-3 overlaps the 3′ end of a putative proline tRNA gene

Bacteriophage 16-3 inserts its genome into the chromosome of Rhizobium meliloti strain 41 (Rm41) by site-specific recombination. The DNA regions around the bacterial attachment site (attB) and one of the hybrid attachment sites bordering the integrated prophage (attL) were cloned and their nucleotide sequences determined. We demonstrated that the 51 by region, where the phage and bacterial DNA sequences are identical, is active as a target site for phage integration. Furthermore it overlaps the 3′ end of a putative proline tRNA gene. This gene shows 79% similartiy to the corresponding proline tRNA-like genomic target sequence of certain integrative plasmids in Actinomycetes.

Orientation of the genetic and physical map of Rhizobium meliloti temperate phage 16-3

SummaryThe attachment site, the C cistron of Rhizobium meliloti temperate phage 16-3, and the insertion of the host cys46+ gene in the phage genome were localized on the HindIII and EcoRJ restriction endonuclease maps, as well as mapped genetically. The strategy employed included restriction analysis and Southern in situ hybridization of plasmid pGY1, which carries the bacterial chromosome region containing the integration site of 16-3, plasmid pGY2, which carries the 16-3 prophage, deletion and inversion mutants, and the cys46+ transducing 16-3 particles. The colinear array of genetic and physical data was possible. The possibility of isolation of a replacement phage vector for Rhizobium is discussed.

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

SummaryRestriction cleavage maps for enzymes EcoRI, BamHI, PstI, PvuII, XbaI and EcoRV of Rhizobium meliloti temparate 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 the conventional techniques a computer program (PMAP) for physical mapping of linear DNA has been employed which made the experimentation, in several cases, extremely efficient.

On the site specific recombination of phage 16-3 of Rhizobium meliloti: identification of genetic elements and att recombinations

SummaryThe genome segment carrying the activities int and xis, responsible for integration and excision of phage 16-3, have been identified and cloned. Mutants were isolated, permitting the investigation of int, xis and att sites (attP, attR, attB) in trans arrangements. The efficiency and role of int- and xis-promoted reactions and of homologous recombination in the formation of lysogenic cells are established. The possible use of the cloned int-attP chromosomal segment in the manipulation of Rhizobium meliloti is discussed.

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).

Lysogenic control of temperate phage 16-3 of Rhizobium meliloti 41 is governed by two distinct regions

SummaryIn addition to the regulator gene C of temperate phage 16-3 of Rhizobium meliloti 41, a second repressor function, called immune X, was identified and cloned into the low copy number cosmid vector pLAFR1. Both repressor functions are necessary to establish complete immunity against superinfecting non-virulent 16-3 strains, but either of the two alone decreases the efficiency of plating (e.o.p.) dramatically. It was shown that the primary target of gene product immune X was the avirT operator locus. The coding region of immune X was localized to the left arm of the phage genome, inside the EcoRI L and H fragments. This region maps about 14 kb from cistron C and had been thought to be genetically silent.

Recombination deficient mutants of Rhizobium meliloti 41

SummaryTwo 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.