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Biomedical subjects

A Landy

Publications and source records attributed to A Landy.

At least 55 records · Page 3Linked to original sources
PubMed1986

Mutations in an integration host factor-binding site: effect on lambda site-specific recombination and regulatory implications.

The manner in which integration host factor (IHF) regulates lambda site-specific recombination has been analyzed by examining the behavior of both wild-type and mutant DNAs in integrative and excisive recombination as well as in protein binding. While integrative recombination of an attP with two base changes in the H1 site required 8-fold more IHF than did wild type, binding to this site was lowered at least 500-fold, suggestive of cooperative interactions. A mutant attP with nine base changes did not integrate at all in vitro, with the defect being less severe in vivo. IHF inhibition of excisive recombination was relieved by both mutations in vitro and in vivo. These results imply that occupancy of the H1 site is critical for determining the direction of recombination. It is proposed that IHF inhibition of excision provides a monitor of the strength of the induction stimulus and the nutritional state of the cell; this would allow the prophage to excise selectively in conditions which favor successful completion of the lytic cycle.

Bacterial Proteins↗
PubMed1985

Control of directionality in lambda site specific recombination.

The simple relation between the substrates and products of site-specific recombination raises questions about the control of directionality often observed in this class of DNA transactions. For bacteriophage lambda, viral integration and excision proceed by discrete pathways, and DNA substrates with the intrinsic property of recombining in only one direction can be constructed. These pathways display an asymmetric reliance on a complex array of protein binding sites, and they respond differently to changes in the concentrations of the relevant proteins. The Escherichia coli protein integration host factor (IHF) differentially affects integrative and excisive recombination, thereby influencing directionality. A four- to eightfold increase in intracellular IHF coincides with the transition from exponential to stationary phase; this provides a mechanism for growth phase-dependent regulation of recombination that makes the cellular physiology an intrinsic part of the recombination reaction.

Bacterial Proteins↗
PubMed1985

The phi 80 and P22 attachment sites. Primary structure and interaction with Escherichia coli integration host factor.

Although the lambdoid bacteriophage phi 80 and P22 possess site-specific recombination systems analogous to bacteriophage lambda, they have different attachment (att) site specificities. We have identified and determined the nucleotide sequences of the att sites of phi 80 and P22 and have examined the interaction of these sites with purified Escherichia coli integration host factor (IHF). The sizes of the homologous core regions of the att sites vary greatly: P22 has a 46-base pair core, while phi 80 and lambda have 17- and 15-base pair cores, respectively. The core sequences of the three phage show no significant homology, although dispersed regions of homology in arm sequences indicate that the three phage att sites are related. All three att sites have a high A + T composition, and restriction fragments carrying these sites migrate anomalously upon polyacrylamide gel electrophoresis. IHF binds to a site to the left of the common core in the phi 80 and P22 phage att sites (attP) and to a site to the right of the core in P22 attP and attB (the bacterial att site). In the lambda system, IHF interacts with three regions on attP (designated H1, H2, and H') and none on attB (Craig N., and Nash, H.A. (1984) Cell 39, 707-716). Alignment of the IHF sites of all three phage results in a consensus sequence for IHF binding, Pyr-AANNNNTTGATAT. Among the three phage, the number of IHF sites differs; however, the location and orientation of the binding sites in relation to the respective core regions are well conserved. An IHF site analogous to lambda H2 is present in both phi 80 and P22 attP, while a site analogous to lambda H' is present in P22 attP. This conservation suggests that IHF plays a very similar role in the site-specific recombination pathways of all three phage, and that the flanking arm sequences are necessary for phi 80 and P22 attP function, as is the case for lambda attP function. These structural similarities presumably reflect a conservation of the mechanism of site-specific recombination for the three phage.

Bacteriophage lambda↗
PubMed1985

Generation of single base-pair deletions, insertions, and substitutions by a site-specific recombination system.

The sequence analysis of both products of individual phi 80 site-specific recombination events in vivo shows that recombination with a secondary attachment (att) site generates several different novel joints at the mismatched position: one recombination event resulted in a single base-pair deletion and two other recombination events resulted in two different single base-pair substitutions. The characterized products of recombination can be straightforwardly interpreted as the outcome of strand exchange involving staggered nicks bracketing the heterology within an overlap region of five to nine base pairs. In comparison, more complex segregation patterns have been observed in previous studies of lambda recombination between nonidentical att sites; the nature of the overlap region heterology may have a significant effect on the segregation patterns. To recover both products of a single recombination event, we used a plasmid that carries the phi 80 int and xis genes and both att sites. Because the two att sites are situated in opposite orientation, intramolecular recombination between them inverts rather than deletes the intervening segment of DNA. Although subsequent reinversion restores the original gross genetic arrangement, single base-pair insertions, deletions, and substitutions are introduced at the sites of recombination. One of the mutations improves the recombination efficiency of the secondary att site and thereby converts a formerly "stable" sequence to an efficient target for rearrangement, and other mutations are predicted to alter the specificity of recombination. These pathways may also provide useful models for the efficient generation of localized sequence diversity on a development (as well as evolutionary) time scale.

Bacteriophage lambda↗
PubMed1985

Interaction of the lambda site-specific recombination protein Xis with attachment site DNA.

Nuclease protection experiments show that Xis protein of bacteriophage lambda specifically binds attachment (att) site DNA. The region of Xis binding, present in both the phage att site and the right prophage att site, extends from position -102 to position -62 in the P arm. The sequence of this region, the positions of purines protected by Xis against methylation, and the binding of Xis to a resected att site indicate the presence of two binding sites. The postulated recognition elements, contained in 13-base-pair direct repeats separated by 7 base pairs, are situated on the same face of the DNA helix. Protection experiments performed with DNase I suggest that the DNA wraps around (or along the surface of) the bound Xis protein. The Xis binding data presented here establishes that Xis, like the other two proteins involved in lambda site-specific recombination, interacts specifically with att DNA. This rules out that class of models in which the profound effects of Xis on the directionality of site-specific recombination are mediated solely through protein-protein interactions or modification of another protein. In addition, nuclease protection experiments with pairwise combinations of the proteins show that Xis and integration host factor (IHF), or Xis and Int, can bind simultaneously to either the phage or right prophage att sites, and the DNA sequences protected are the sum of those protected with each protein alone. It is therefore unlikely that the effect of Xis on the direction of recombination is exerted by directly blocking the binding of Int or IHF to one or more of their respective binding sites.

Attachment Sites, Microbiological↗
PubMed1984

Determinants of directionality in lambda site-specific recombination.

The DNA structural features governing directionality in lambda site-specific recombination are shown to reside in regions of the phage attachment site more than 70 bp to the left and more than 40 bp to the right of the cross-over region. Disposition of these sequences on the same attachment site in integration, and on different attachment sites in excision, determines the opposite effects of Xis protein upon the two reactions (stimulation of excision and inhibition of integration). The binding of Xis to two adjacent directly repeated sequences in the left phage arm is shown to occur in a highly cooperative manner, to alter the conformation of the DNA, and to produce a 32-fold stimulation of Int binding to an adjacent locus.

Bacteriophage lambda↗
PubMed1983

Role for DNA homology in site-specific recombination. The isolation and characterization of a site affinity mutant of coliphage lambda.

Site-affinity (or saf) mutations change the specificity of prophage insertion. We have isolated a saf mutation of the bacteriophage lambda attachment site by inserting the phage chromosome into and then excising it from a secondary host attachment site. This causes reciprocal exchange of two seven base-pair segments (the overlap regions) that lie within the cores of the two sites. Since the two overlap regions differ from each other in nucleotide sequence, the recombinant sites are mutants. We have determined the effect of overlap region homology on recombination. We found that homology promotes integrative and excisive recombination. This suggests that the two overlap regions interact directly during recombination. The pattern of segregation of the saf mutation during site-specific recombination shows that it lies to the right of the point of genetic exchange about 95% of the time. This is a surprising result because lambda integrative recombination normally occurs by two staggered, reciprocal single-strand exchanges, one at each edge of the overlap region (Mizuuchi et al., 1981). Since saf lies within the overlap region, we might have expected that the point of genetic exchange would occur to the left of saf as often as to the right. We offer two models to account for this. (1) The mutation alters the location of one of the single-strand exchange points. (2) Efficient and strand-specific processing of mismatched base-pairs changes the expected segregation pattern.

Attachment Sites, Microbiological↗
PubMed1983

Patterns of lambda Int recognition in the regions of strand exchange.

Int protein has two classes of binding sites within the phage att site: the arm-type recognition sequences are found in three specific sites that are distant from the region of strand exchange; the junction-type recognition sequences occur as inverted pairs around the crossover region in both attP and attB. During recombination between attP and attB each of the four DNA strands is cut at a homologous position within each of the junction-type Int binding sites. In all four junction-type sites Int protein interacts primarily with the same face of the DNA helix, as determined by those purine nitrogens that are protected against methylation by dimethylsulfate. Efficient secondary attachment sites for lambda contain sequences with partial homology to the junction-type binding sites. In addition, the sequence between, but not part of, the two junction-type sites (the overlap region) is strongly conserved in secondary att sites. Thus, in the vicinity of strand exchange, attP and a recombining partner, such as attB, are very similar; each comprises two junction-type Int recognition sites and an overlap (crossover) region.

Bacteriophage lambda↗
PubMed1982

Bacteriophage lambda int protein recognizes two classes of sequence in the phage att site: characterization of arm-type sites.

Purified int protein from bacteriophage lambda binds to specific sites in DNA that are not part of the functional attachment sites (non-att DNA) as well as to specific sites in att DNA. Analysis of non-att sites protected from nucleases by int has permitted definition of two distinctly different consensus recognition sequences, one of which, the arm-type sequence, is characterized in this report. Both types of recognition sequence occur in attP; five copies of the arm-type consensus sequence are located distant from the crossover region in the P1, P2, and P' arm protected regions. The second type of recognition sequence occurs at the crossover region. Modification of int with N-ethylmaleimide selectively alters its interaction with arm-type sequences.

Bacteriophage lambda↗
PubMed1981

The tyrT locus: termination and processing of a complex transcript.

The tyrT locus of E. coli contains a 208 bp spacer region that separates two copies of sequence encoding tRNATyr1. The spacer includes a 120 bp sequence that is homologous to a sequence that is repeated three times in the distal portion of the tyrT locus. The tyrT locus possesses a graded set of transcription termination sites that are spaced at 180 base intervals, corresponding to the distal repeated gene structure. The major termination site occurs within the second repeat unit, 225 bases beyond the mature tRNA sequences. In the presence of a temperature-sensitive rho protein there is increased read-through at this site to a termination site located 180 bases downstream in the third repeat and to several termination sites even further downstream. The primary native transcript, in the region distal to the second tRNA, carries the information for a low molecular weight, extremely basic protein. Although analogous coding sequences are present in the spacer and other repeat units, because of single base substitutions these sequences are pseudogenes. The parallel between the tyrT and TyrU gene clusters is discussed in relation to dual function transcripts that specify both tRNA and protein.

Bacterial Proteins↗
PubMed1980

The lambda phage att site: functional limits and interaction with Int protein.

Site specific integrative recombination of bacteriophage lambda involves unequal partners. The minimal phage att site is composed of approximately 240-base pairs and four distinct binding sites for Int protein, at least three of which are crucial for function. This 'donor site' recombines efficiently with a smaller 'recipient site' that lacks the extensive interactions with Int protein.

Attachment Sites, Microbiological↗
PubMed1980

Site-specific recombination functions of bacteriophage lambda: DNA sequence of regulatory regions and overlapping structural genes for Int and Xis.

Site-specific recombination in bacteriophage lambda is mediated by two phage-encoded proteins, Int and Xis. The structural genes encoding these proteins are located immediately to the right of their site of action, the phage att site. The DNA sequence for both the structural and regulatory regions of these genes has been determined. The location and reading frame of the xis gene were ascertained by sequence comparisons with the b538 deletion (that ends within xis) and with the xis6 amber mutation. From the DNA sequence Xis has a molecular weight of 8630; it is rich in basic amino acids with lysine and arginine comprising 25% of the 72 amino acids. Identification of the int reading frame was also unambiguous. From the DNA sequence, Int has a molecular weight of 40,330; of the 356 amino acids, 69 are basic and 46 are acidic. In the NH(2)-terminal portion of Int, 35% of the first 20 amino acids are basic. The site-specific recombination functions form a very tight cluster (att-int-xis) on the lambda chromosome. The combined protein-encoding sequences of xis and int start 1347 base pairs, and terminate 84 base pairs, from the center of the phage att site. The two genes overlap one another by 20 base pairs (xis is upstream of int) and a possible means of controlling the relative synthesis rates of Int and Xis at the level of translation is proposed. Control at the level of transcription is also considered. The mutation intc226 leads to constitutive production of Int, independent of cII/cIII activator proteins normally required for transcription from the p(I) promoter. It is shown that this mutation is the result of a single base change (in the fMet codon of the xis gene) that generates an improved promoter heptamer sequence. This result, in conjunction with comparisons with other promoter sequences and other sequences responding to cII/cIII action, leads to a tentative identification of the p(I) promoter and site of cII/cIII action.

Bacteriophage lambda↗
PubMed1980

Site-specific recombination in bacteriophage lambda: structural analyses of reactive DNA sequences.

Site-specific integrative recombination in bacteriophage lambda involves unequal partners. The minimal phage att site is composed of approximately 240 base pairs and has four distinct Int binding sites that differ in size and response to heparin challenge. There appear to be two size classes of Int binding sites, approximately 30-35 base pairs and 15 base pairs. The sites at the common core and in the P' arm are of the former class. Two sites in the P arm are of the latter class. Thus far, three of the four sites have been shown to be necessary for att site function. In contrast, the minimal sequence required for a phage att site partner (such as the bacterial att site) may not be much larger than the 15 base pair common core. We have suggested a model in which integrative recombination involves two unequal partners; accordingly the phage att site is referred to as the "donor" and the bacterial att site, or its analogue, is referred to as the "recipient."

Bacteriophages↗