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J A Shapiro

Publications and source records attributed to J A Shapiro.

At least 73 records · Page 4Linked to original sources
PubMed1983

Insertion and replication of the Pseudomonas aeruginosa mutator phage D3112.

D3112 is a temperate bacteriophage of P. aeruginosa with heterogeneous sequences at one extremity of the virion DNA molecule. Infection of strain PAOl with phage D3112 results in a 40- to 65-fold increase in the frequency of ami mutants resistant to fluoroacetamide. Nine ami::D3112 prophages have been mapped to distinct sites within the ami locus by Southern blotting experiments with a cloned ami+ probe. All prophages have the same restriction map as the D3112 genome extracted from phage particles. The position of D3112 insertions correlates with the phenotype and reversion behavior of the ami mutants. Induction of D3112cts prophages results in amplification of internal prophage segments as discrete restriction fragments before the terminal viral fragments are visible as sharp hybridizing species. This indicates that D3112 replication is accompanied by recombination of prophage termini to numerous sites in the bacterial genome. Chromosomal junction fragments of an ami::D3112cts prophage are maintained through most of the replication cycle but are cleaved shortly before cell lysis, apparently by the viral encapsidation system.

Amidohydrolases↗
PubMed1983

Recombination involving transposable elements: role of target molecule replication in Tn1 delta Ap-mediated replicon fusion.

Donor DNA molecules carrying Tn1 or Tn3 deletion mutants do not need to replicate in order to participate in replicon fusion recombination events during which the Tn1/Tn3 element is duplicated. We have assayed Tn1 delta Ap-mediated replicon fusion events involving plasmid R388 and the bacteriophage lambda-derived plasmid p lambda CM, and we find that the role of the recipient molecule is distinct. When p lambda CM carries Tn1 delta Ap, replicon fusion occurs in more than 1% of all cells assayed, whether or not p lambda CM::Tn1 delta Ap can replicate. In contrast, when R388 carries Tn1 delta Ap, replicon fusion occurs only when the p lambda CM target can replicate. Blocks to p lambda CM replication by prophage repressor or amber mutations of the O and P cistrons reduce replicon fusion so that it occurs in less than 1 out of 10(5) cells assayed.

Bacteriophage lambda↗
PubMed1983

p lambda CM system: observations on the roles of transposable elements in formation and breakdown of plasmids derived from bacteriophage lambda replicons.

Transduction with phage derived from a 2-year-old lysate of lambda cam105 (lambda::Tn9) gave rise to chloramphenicol-resistant (Cm(r)) transductants harboring a plasmid (plambdaCM1) formed from lambda cam105 by a Tn9-mediated adjacent deletion to position 36.07 kilobases in the N cistron of lambda. The plambdaCM element can replicate as a plasmid, insert into the bacterial genome, or reproduce lytically as a phage on cells that provide N function. The feasibility of obtaining high titers in encapsidated form and the ease of synchronous introduction into and recovery from bacterial populations make plambdaCM very suitable for quantitative studies of recombination involving transposable elements. Replicon fusions between plambdaCM1 and RSF1596 (pMB8::Tn3Delta596) occur by duplication of either IS1 (at low rate in the absence of TnpA activity) or Tn3Delta596 (in the presence of TnpA activity). At 24 or 32 degrees C, the rate of increase of TnpA-mediated fusions per plambdaCM is about 2% per cell doubling. RSF103 contains the deleted Tn1DeltaAp (which lacks intact beta-lactamase and TnpR resolvase coding sequences) adjacent to a streptomycin resistance (Sm(r)) determinant. We observed that Tn1DeltaAp mediates insertions of external RSF103 sequences into the R388 plasmid. R388::Tn1DeltaAp plasmids show transposition immunity in cells lacking TnpR activity. Using the plambdaCM system, we isolated adjacent transpositions of the RSF103 Sm(r) determinant. The resulting plambdaCM-Sm cosmids contain Sm(r) genetic material flanked by direct repeats of Tn1DeltaAp, and all are deleted for some RSF103 or plambdaCM sequences. The plambdaCM-Sm constructs will fuse into R388 by duplication of a single Tn1DeltaAp element. In the presence of tnpR(+) (but not tnpR) Tn1 or Tn3 elements, all Tn1DeltaAp-mediated complex replicons break down completely and rapidly to simple Tn1DeltaAp inserts. The equilibrium for resolution is at least 10(5):1, and resolution is more than 90% complete after 40 min of exposure to a tnpR(+) cytoplasm. In the absence of TnpR, Rec, and Red activities, Tn1DeltaAp-mediated complex replicons yield simple Tn1DeltaAp inserts at a lower rate. The presence of intact RSF103 replication determinants between direct Tn1DeltaAp repeats appears to accelerate this precise TnpR- and Rec-independent breakdown.

Bacteriophage lambda↗
PubMed1982

Interactions of Tn7 and temperate phage F116L of Pseudomonas aeruginosa.

Tn7 insertions into the genome of F116L, a Pseudomonas aeruginosa generalized transducing phage, were isolated by repeated cycles of transduction and induction isolated by repeated cycles of transduction and induction of strains lysogenic for F116cts mutants with selection for trimethoprim resistance (Tpr). Two non-defective F116Lcts:: Tn7 phage were characterized. They have reduced plaquing ability, produced non-lysogenic Tpr transductants, and have yielded a deletion mutant of the phage genome upon selection for plaque formation in single infection. F116L DNA is circularly permuted and terminally redundant. A circular restriction map of 61.7 kb has been defined, and a cleavage site common to many enzymes has been identified at coordinate 23.3 kb on the map. It is presumed that this site represents the sequence for the initiation of DNA encapsidation by a headful packaging mode. The Tn7 insertion targets and a 13.4 kb deletion define regions of the F116L genome non-essential for either vegetative growth or lysogenization. The restriction map of Tn7 has been determined for five enzymes. Non-lysogenic Tpr transductants reveal a Tn7 insertion hot-spot in the P. aeruginosa genome.

Bacteriophages↗
PubMed1982

Broad-host-range IncP-4 plasmid R1162: effects of deletions and insertions on plasmid maintenance and host range.

R1162 is an 8.7-kilobase (kb) broad-host-range replicon encoding resistance to streptomycin and sulfa drugs. In vitro deletion of 1.8-kb DNA between coordinates 3.0 and 5.3 kb did not affect plasmid maintenance, but a Tn1 insertion at coordinate 6.3 kb led to a recessive defect in plasmid maintenance. The only cis-acting region necessary for plasmid replication appears to lie between the Tn1 insertion at coordinate 6.3 kb and a second Tn1 insertion at coordinate 6.5 kb. All R1162 sequences between position 6.5 kb and the EcoRI site at coordinate 8.7/0 kb were dispensible for replication in Escherichia coli and Pseudomonas putida. Plasmids carrying insertions in a variety of restriction sites in an R1162::Tn1 derivative were unstable in P. putida but stable in E. coli. Tn5 insertions in R1162 showed a hot spot at coordinate 7.5 kb. A Tn5 insertion at coordinate 8.2 kb appeared to mark the 3' end of the streptomycin phosphotransferase coding sequence. All R1162::Tn5 derivatives showed specific instability in Pseudomonas strains but not in E. coli. The instability could be relieved by internal deletions of Tn5 sequences. In the haloaromatic-degrading Pseudomonas sp. strain B13, introduction of an unstable R1162::Tn5 plasmid led to loss of ability to utilize m-chlorobenzoate as a growth substrate. Our results showed that alteration of plasmid sequence organization in nonessential regions can result in restriction of plasmid host range.

Base Sequence↗
PubMed1982

Changes in gene order and gene expression.

Studies on changes in gene position in germ line and somatic cell chromosomes during evolution and differentiation have led biologists to abandon the static view of chromosomes as invariant linear arrays of hereditary information. Prokaryotic cells contain several classes of DNA insertion elements which move from place to place in the genome and mediate chromosome rearrangements. Similar elements exist in a wide variety of eukaryotic organisms (yeasts, insects, plants, and vertebrates). In addition, both reversible and irreversible changes of chromosome primary structure provide developmental controls on gene activity in bacteria, bacteriophages, yeasts, trypanosomes, and mammalian lymphocytes. At least five recombination mechanisms are known to catalyze chromosome changes: 1) general homologous, 2) site-specific reciprocal, 3) illegitimate, 4) DNA splicing, and 5) replicative. Various combinations of these mechanisms can explain many different chromosome rearrangements and changes in gene dosage. Changes in gene position can alter gene expression in many ways, some of which we understand (such as insertional mutation and inversion of coding and regulatory sequences) and some of which are still unexplained. The activities of DNA insertion elements and somatic rearrangement systems are subjects to controls at several levels by specific regulatory systems, natural selection, and connection to cell lineage. Despite the recent increase in knowledge about the biological importance of changes in gene order on chromosomes, there are far more questions than answers, particularly about the mechanisms that coordinate recombination events and cell division in higher organisms.

Animals↗
PubMed1980

Genetic organization of the broad-host-range IncP-1 plasmid R751.

We have identified regions encoding conjugal transfer, plasmid maintenance, and trimethoprim resistance on the IncP-1 plasmid R751 by complementation tests with cloned deoxyribonucleic acid fragments and self-replicating derivatives constructed in vitro. The genes for replication and transfer show a scattered organization similar to that previously determined for RK2, another IncP-1 plasmid. Derivatives of RK2 are able to complement R751 derivatives defective in these functions. Restriction enzyme cleavage sites in R751 deoxyribonucleic acid are clustered in regions of the plasmid physical map. Neither region is required for plasmid maintenance or transfer, although one determines resistance to trimethoprim. A similar clustering of cleavage sites is seen with RK2, which nevertheless has a very different restriction map.

Conjugation, Genetic↗
PubMed1979

Molecular model for the transposition and replication of bacteriophage Mu and other transposable elements.

A series of molecular events will explain how genetic elements can transpose from one DNA site to another, generate a short oligonucleotide duplication at both ends of the new insertion site, and replicate in the transposition process. These events include the formation of recombinant molecules which have been postulated to be intermediates in the transposition process. The model explains how the replication of bacteriophage Mu is obligatorily associated with movement to new genetic sites. It postulates that all transposable elements replicate in the transposition process so that they remain at their original site while moving to new sites. According to this model, the mechanism of transposition is very different from the insertion and excision of bacteriophage lambda.

Coliphages↗
PubMed1979

Transposition of Tn7 in Pseudomonas aeruginosa and isolation of alk::Tn7 mutations.

Conjugal crosses with Pseudomonas aeruginosa donors carrying the CAM-OCT and RP4::Tn7 plasmids result in transfer of the Tn7 trimethoprim resistance (Tp(r)) determinant independently of RP4 markers. All Tp(r) exconjugants which lack RP4 markers have CAM-OCT genes and therefore must have received CAM-OCT::Tn7 plasmids formed by transposition of Tn7 from RP4::Tn7 to CAM-OCT. Most crosses yield exconjugants carrying mutant CAM-OCT plasmids which no longer determine either camphor or alkane utilization and thus appear to carry Tn7 inserts in the cam or alk loci, respectively. Transduction and reversion experiments indicated that at least 13 alkane-negative, camphor-positive, Tp(r) CAM-OCT::Tn7 plasmids carry an alk::Tn7 mutation. Determination of linkage between the alk mutation and the Tp(r) determinant of Tn7 on these plasmids is complicated by the presence of multiple copies of the Tn7 element in the genome. Generalized transduction will remove Tn7 from a CAM-OCT alk::Tn7 plasmid to yield alk(+) cells which carry no Tp(r) determinant on the CAM-OCT plasmid (as shown by transfer of the plasmid to a second strain). But the transduction to alk(+) does not remove all Tp(r) determinants from the genome of the recipient cell because the alkane-positive transductants remain trimethoprim resistant. Thus, it appears that copies of Tn7 can accumulate in the genome of P. aeruginosa (CAM-OCT alk::Tn7) strains without leaving their original site. This result is consistent with transposition models that involve replication of the transposable element without excision from the original site.

Alkanes↗
PubMed1978

Chromosomal integration of phage lambda by means of a DNA insertion element.

Phage lambdacam112, which contains the chloramphenicol resistance transposon Tn9 and has a deletion of attP and the int gene, will lysogenize Escherichia coli K-12. Prophage integration occurs at different chromosomal sites, including lacY and malB, but not at attB. All lambdacam112 prophages are excised from the chromosome after induction but with various efficiencies for different locations. Heteroduplex analysis of lambdaplacZ transducing phages isolated from a lacY::lambdacam112 prophage reveals an insertion sequence 1 (IS1) element at the joint of viral and chromosomal DNA. Two lines of evidence indicate that lambdacam112 encodes an excision activity that recognizes the IS1 element: (i) prophage derepression increases the frequency of excision from lacY to yield lac+ revertants, and (ii) lambdacam112 infection increases reversion of a galT::IS1 mutation about 50-fold. Our results indicate that the IS1 termini of TN9 can replace attP as a site for lambda insertion in the bacterial chromosome and that excision events are catalyzed by an IS1-encoded protein under lambda repressor and N gene control.

Base Sequence↗
PubMed1977

Sequence analysis of oligodeoxyribonucleotides by mass spectrometry. 1. Dinucleoside monophosphates.

The base components of underivatized oligodeoxynucleotides can be determined qualitatively by mass-spectral analysis at the nanogram level. The thermal and electron-impact conditions of the spectrometer allow the cleavage of the phosphodiester bonds of the oligonucleotide chain, resulting in fragments (I), whose mass identifies the base, and other fragments (II), which contain the purine or pyrimidine base plus portions of the deoxyribose and the phosphate moieties. A study of 16 dinucleoside monophosphates indicates that the relative intensities of the m/e values of the type II fragments are significantly and reproducibly different for sequence isomers. From the complex spectra of dinucleoside monophosphates, specific ions for each mono-nucleotide residue have been selected which reveal the location (5' or 3' terminus) of the bases in the dinucleoside monophosphate. These ions appear in spectra of deoxyribonucleic acid fragments as well as in model compounds. A simple computer program has been devised which utilizes ion ratio values to determine sequence. The method is applicable to oligonucleotides of longer length.

Base Sequence↗