Search PubMed⌕ Search

Biomedical subjects

L Dorgai

Publications and source records attributed to L Dorgai.

9 recordsLinked to original sources
PubMed1995

Identifying determinants of recombination specificity: construction and characterization of chimeric bacteriophage integrases.

Bacteriophage integrases are members of a family of structurally related enzymes that promote recombination between DNA molecules that carry specific sites. Phages lambda and HK022 encode closely related integrases that recognize different sets of sequences within the core regions of their respective attachment sites. To locate the amino acid residues that determine this difference in specificity, we isolated recombinant phages that produce chimeric integrases and measured the ability of these chimeras to promote recombination of lambda and HK022 sites in vivo. A chimera that is of lambda origin except for one HK022 residue at position 99 and 12 HK022 residues located between positions 279 and 329 had wild-type HK022 specificity and activity for both integrative and excisive recombination. Chimeras containing certain subsets of these 13 residues had incomplete specificity. The region around position 99 is not well-conserved in other members of the integrase family, but the 279-329 segment includes residues that are highly conserved and believed to be directly involved in catalysis. Many chimeras were inactive in recombining either HK022 or lambda sites. Selection for mutants that restored activity to these chimeras revealed sets of residues that are likely to interact with each other.

Amino Acid Sequence↗
PubMed1995

Identifying determinants of recombination specificity: construction and characterization of mutant bacteriophage integrases.

The Integrases of bacteriophages lambda and HK022 promote recombination between DNA molecules that carry attachment sites. The two integrases are about 70% identical in sequence and catalyze nearly identical reactions, but recognize different sets of sites. To identify the amino acids that determine this difference in specificity, we selected mutants of lambda integrase with increased ability to recombine HK022 sites. This selection yielded eleven different amino acid substitutions at eight different positions. Three of the positions belong to a larger set that were identified as important for the lambda/HK022 specificity difference by analysis of chimeric integrases. Substitution of the HK022 for the corresponding lambda residue at each of these three positions increased recombination of HK022 sites, and one double substitution, N99D-E319R, increased recombination to nearly wild-type HK022 levels. Mutations at the other five positions changed residues that are identical in the wild-type proteins or are at positions identified by chimera analysis as unimportant for the lambda/HK022 specificity difference. All of the mutants isolated by selection for increased recombination of HK022 sites retained considerable ability to recombine lambda sites. However, we found that substitution of HK022 for lambda residues at three additional positions, S282P, G283K, and R287K, specifically reduced recombination of lambda sites. These three substitutions when combined with N99D and E319R were sufficient to change the specificity of lambda to that of HK022 integrase. The first three substitutions act principally to prevent recombination of lambda sites, and the second two to remove a barrier to recombination of HK022 sites. We suggest that many natural alterations in the specificity of protein-DNA interactions occur by multi-step changes that first relax and then restrict specificity.

Amino Acid Sequence↗
PubMed1993

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 bp 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% similarity to the corresponding proline tRNA-like genomic target sequence of certain integrative plasmids in Actinomycetes.

Attachment Sites, Microbiological↗
PubMed1992

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↗
PubMed1989

Mutations of the phage lambda attachment site alter the directionality of resolution of Holliday structures.

Integrative recombination of bacteriophage lambda occurs by two sequential, reciprocal strand exchanges at specific positions within the attachment sites. Both exchanges are promoted by the lambda Int protein; the first forms a Holliday structure, and the second resolves it to recombinant products. Recombination requires sequence homology within the 7 bp 'overlap' region that separates the two points of strand exchange. To see if homology promotes the second strand exchange, we constructed attachment site Holliday structures by annealing DNA strands and then assayed Int-promoted resolution. Holliday structures corresponding to strand exchange between sites with homologous overlap regions were efficiently resolved to give mixtures of recombinants and parents. Holliday structures corresponding to exchanges between heterologous sites fell into two classes. Members of the first class, in which heterology limited but did not completely prevent migration of the branchpoint within the overlap region, were resolved efficiently and preferentially to parental molecules. We propose that resolution to recombinants occurs only if homology allows branch migration from the first to the second exchange site. Members of the second class, in which heterology constrained the branchpoint within an Int binding site, were resolved poorly. We suggest that Holliday structures that have a branchpoint within an Int binding site are poor substrates for Int.

Attachment Sites, Microbiological↗
PubMed1984

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↗
PubMed1983

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↗
PubMed1983

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↗