Publications

the amino terminus of bacteriophage lambda integrase is involved in protein-protein interactions during recombination.	bacteriophage lambda integrase (int) catalyzes at least four site-specific recombination pathways between pairs of attachment (att) sites. protein-protein contacts between monomers of int are presumed to be important for these site-specific recombination events for several reasons: int binds to the att sites cooperatively, catalytic int mutants can complement each other for strand cleavage, and crystal structures for two other recombinases in the int family (cre from phage p1 and int from haemop ...	2000	10648529
mammalian genomes contain active recombinase recognition sites.	recombinases derived from microorganisms mediate efficient site-specific recombination. for example, the cre recombinase from bacteriophage p1 efficiently carries out recombination at its loxp target sites. while this enzyme can function in mammalian cells, the 34bp loxp site is expected to be absent from mammalian genomes. we have discovered that sequences from the human and mouse genomes surprisingly divergent from loxp can support cre-mediated recombination at up to 100% of the efficiency of ...	2000	10689186
molecular organization of the mouse gastrin-releasing peptide receptor gene and its promoter.	the murine gastrin-releasing peptide receptor (mgrp-r) is a member of the g protein-coupled receptor family and mediates important physiological actions of its specific ligand, the gastrointestinal hormone/neurotransmitter grp, including mitogenic properties in the mouse swiss 3t3 fibroblasts. glucocorticoids and increases in intracellular camp are reported to alter grp-r gene transcription, but the molecular basis for these effects is unknown. to begin to identify possible gene regulatory mecha ...	2000	10689196
cre-mediated cerebellum- and hippocampus-restricted gene mutation in mouse brain.	using the phage p1-derived cre/loxp recombination system, we have created a line of cre-transgenic mice in which the cre-mediated gene deletion is restricted to granule cells of cerebellum and dentate gyrus of hippocampus. low levels of deletion were also present in pyramidal cells of hippocampal ca1 and ca3 fields. the cre/loxp recombination occurred prenatally. the recombination efficiencies in the granular layer of the cerebellum, the granular layer of the dentate gyrus, and the ca1 and ca3 p ...	2000	10694492
genomic organization and chromosomal mapping of elks, a gene rearranged in a papillary thyroid carcinoma.	we recently isolated a novel cdna, designated elks, that was fused to ret cdna in a papillary thyroid carcinoma. its encoded polypeptide sequence was rich in glutamic acid (e), leucine (l), lysine (k), and serine (s), and was characterized by the presence of nine alpha-helical coiled-coil domains consisting of periodic heptad repeats. we have now cloned the entire structure of the human elks gene from within a 700-kb genomic region represented by overlapping bacteriophage p1-derived artificial c ...	2000	10697956
synthesis of a new cre recombinase gene based on optimal codon usage for mammalian systems.	the origin of the cre recombinase gene is bacteriophage p1, and thus the codon usages are different from in mammals. in order to adapt this codon usage for mammals, we synthesized a "mammalian cre recombinase gene" and examined its expression in chinese hamster ovarian tumor (cho) cells. significant increases in protein production as well as mrna levels were observed. when the recombination efficiency was compared using cho cell transfectants having a cdna containing loxp sites, the "mammalian c ...	2000	10731707
a family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of escherichia coli and other bacteria.	modifications of microbial genomes often require the use of the antibiotic-resistance (anb(r))-encoding genes and other easily selectable markers. we have developed a set of such selectable markers (cm(r), km(r) and gm(r)), which could easily be inserted into the genome and subsequently removed by using the cre/loxp site-specific recombination system of bacteriophage p1. in this manner the same marker could be used more than once in the same background, while the resulting strain could or would ...	2000	10773465
a sequence-ready physical map of the region containing the human natural killer gene complex on chromosome 12p12.3-p13.2.	we developed a sequence-ready physical map of a part of human chromosome 12p12.3-p13.2 where the natural killer gene complex (nkc) is located. the nkc includes a cluster of genes with structure similar to that of the ca(2+)-dependent lectin superfamily of glycoproteins that are expressed on the surface of most natural killer (nk) cells and a subset of t cells. these killer cell lectin-like receptors (klr) are involved in nk target cell recognition, leading to activation or inhibition of nk cell ...	2000	10783260
drosophila rosa gene, which when mutant causes aberrant photoreceptor oscillation, encodes a novel neurotransmitter transporter homologue.	the drosophila receptor oscillation a (rosa) mutations, which cause electroretinogram (erg) defects, including oscillations, were localized to the 24f4-25a2 region of chromosome 2l. genomic fragments from this region, isolated from bacteriophage p1 clones, included those that detect transcriptional defects in rosa mutants in rna blot experiments. one of these genomic fragments was used to screen a head cdna library. the largest cdna clone (3.6 kb) isolated was shown to rescue a rosa mutant in p ...	1996	10876650
the applications of fish in tumor pathology.	the current fish technology was greatly improved during the past 10 years. a large number of cosmids and yeast (yacs), bacterial (bacs), phage p1 derived (pacs) artificial chromosomes have been rapidly mapped and are useful as probes. in parallel, methods were established to specifically "paint" entire chromosomes or chromosome segments. using these chromosome libraries as probes, complex rearrangements and marker chromosomes can be identified irrespective of their banding pattern. ripetitive dn ...	1999	10936888
in vivo and in vitro function of groel mutants with impaired allosteric properties.	escherichia coli cells that produce only plasmid-encoded wild-type or mutant groel were generated by bacteriophage p1 transduction. effects of mutations that affect the allosteric properties of groel were characterized in vivo. cells containing only groel(r197a), which has reduced intra-ring positive cooperativity and inter-ring negative cooperativity in atp binding, grow poorly upon a temperature shift from 25 to 42 degrees c. this strain supports the growth of phages t4 and t5 but not phage la ...	2000	10973985
illegitimate cre-dependent chromosome rearrangements in transgenic mouse spermatids.	the bacteriophage p1 cre/loxp system has become a powerful tool for in vivo manipulation of the genomes of transgenic mice. although in vitro studies have shown that cre can catalyze recombination between cryptic "pseudo-loxp" sites in mammalian genomes, to date there have been no reports of loxp-site infidelity in transgenic animals. we produced lines of transgenic mice that use the mouse protamine 1 (prm1) gene promoter to express cre recombinase in postmeiotic spermatids. all male founders an ...	2000	11087830
the assembly of large bacs by in vivo recombination.	we have developed a method for recombining bacterial artificial chromosomes (bacs) and p1 artificial chromosomes (pacs) containing large genomic dna fragments into a single vector using the cre-lox recombination system from bacteriophage p1 in vivo. this overcomes the limitations of in vitro methods for generating large constructs based on restriction digestion, ligation, and transformation of dna into escherichia coli cells. we used the method to construct a human artificial chromosome vector o ...	2000	11112344
identification of a new human cyp2a gene fragment with close linkage to cyp2gp1.	human genomic libraries were screened to identify cyp2g-related cytochrome p450 genes. a genomic fragment comprising exons 7 through 9 of cyp2gp1 and exons 6 through 9 of a previously unidentified cyp2a gene, designated cyp2a7p2, was isolated from an embl3 library; the two genes were arranged in outward opposite directions with about 8 kbp of intervening sequence. the same structure was also detected in a bacteriophage p1 clone, which contained a full-length cyp2gp1 gene, exons 6 through 9 of cy ...	2001	11124222
inter-chromosomal recombination of mll and af9 genes mediated by cre-loxp in mouse development.	chromosomal translocations are crucial events in the aetiology of many leukaemias, lymphomas and sarcomas, resulting in enforced oncogene expression or the creation of novel fusion genes. the study of the biological outcome of such events ideally requires recapitulation of the tissue specificity and timing of the chromosomal translocation itself. we have used the cre-loxp system of phage p1 to induce de novo mll-af9 chromosomal recombination during mouse development. loxp sites were introduced i ...	2000	11265751
efficient gene activation in cultured mammalian cells mediated by flp recombinase-expressing recombinant adenovirus.	a recombinant adenovirus (rad) expressing cre recombinase derived from bacteriophage p1 has already been extensively used for the conditional gene activation and inactivation strategies in mammalian systems. in this study, we generated axcaflp, a rad expressing flp recombinase derived from saccharomyces cerevisiae and carried out quantitative comparisons with cre-expressing rad in both in vitro and in cultured cells to provide another efficient gene regulation system in mammalian cells. in the i ...	2001	11266575
translocation pathway of protein substrates in clpap protease.	intracellular protein degradation, which must be tightly controlled to protect normal proteins, is carried out by atp-dependent proteases. these multicomponent enzymes have chaperone-like atpases that recognize and unfold protein substrates and deliver them to the proteinase components for digestion. in clpap, hexameric rings of the clpa atpase stack axially on either face of the clpp proteinase, which consists of two apposed heptameric rings. we have used cryoelectron microscopy to characterize ...	2001	11287666
interruption of coding sequences by heterologous introns can enhance the functional expression of recombinant genes.	sustained expression of recombinant proteins is a critical factor for the effectiveness of numerous applications in the biomedical sciences including the treatment of human disease by gene therapy, the large scale production of therapeutic proteins, as well as the investigation of gene function by transgenesis or cell type specific mutagenesis. although much attention has been paid to the optimisation of regulatory sequences such as promoters, untranslated regions and polyadenylation signals, ef ...	2001	11320412
a structural view of cre-loxp site-specific recombination.	structural models of site-specific recombinases from the lambda integrase family of enzymes have in the last four years provided an important new perspective on the three-dimensional nature of the recombination pathway. members of this family, which include the bacteriophage p1 cre recombinase, bacteriophage lambda integrase, the yeast flp recombinase, and the bacterial xercd recombinases, exchange strands between dna substrates in a stepwise process. one pair of strands is exchanged to form a h ...	2001	11340053
intein-mediated rapid purification of cre recombinase.	cre recombinase produced by bacteriophage p1 catalyzes site-specific recombination of dna between loxp recognition sites in both prokaryotic and eukaryotic cells and has been widely used for genome engineering and in vitro cloning. recombinant cre has been overproduced in escherichia coli and its purification involves multiple steps. in this report, we used an "intein" fusion system to express cre as a c-terminal fusion to a modified protein splicing element, i.e., intein. the modified intein co ...	2001	11388811
smallest region of overlapping deletion in 1p36 in human neuroblastoma: a 1 mbp cosmid and pac contig.	in human neuroblastomas, the distal portion of 1p is frequently deleted, as if one or more tumor suppressor genes from this region were involved in neuroblastoma tumorigenesis. earlier studies had identified a smallest region of overlapping deletion (sro) spanning approximately 23 cm between the most distally retained d1s80 and by the proximally retained d1s244. in pursuit of generating a refined delineation of the minimally deleted region, we have analyzed 49 neuroblastomas of different stages ...	2001	11391793
isolation and characterization of the murine transforming growth factor-beta2 promoter.	this report describes the isolation and characterization of the 5' flanking region of the murine transforming growth factor beta-2 (tgf-beta2) gene. a genomic clone containing the promoter region of the gene was isolated after screening a bacteriophage p1 genomic library. the resulting clone was sequenced and compared to promoters for the human and chicken tgf-beta2 genes. the sequence located near the transcription start site is highly conserved. it includes a tata box, an e-box, and a largely ...	2001	11404017
directional resolution of synthetic holliday structures by the cre recombinase.	the cre recombinase of bacteriophage p1 cleaves its target site, loxp, in a defined order. recombination is initiated on one pair of strands to form a holliday intermediate, which is then resolved by cleavage and exchange of the other pair of strands to yield recombinant products. to investigate the influence of the loxp sequence on the directionality of resolution, we constructed synthetic holliday (chi) structures containing either wild-type or mutant lox sites. we found that cre preferentiall ...	2001	11406627
using an in vivo phagemid system to identify non-compatible loxp sequences.	the site-specific recombination system of bacteriophage p1 is composed of the cre recombinase that recognizes a 34-bp loxp site. the cre/loxp system has been extensively used to manipulate eukaryotic genomes for functional genomic investigations. the creation of additional heterologous loxp sequences potentially expands the utility of this system, but only if these loxp sequences do not recombine with one another. we have developed a stringent in vivo assay to examine the degree of recombination ...	2001	11418130
dual regulatory control of a particle maturation function of bacteriophage p1.	a unique arrangement of promoter elements was found upstream of the bacteriophage p1 particle maturation gene (mat). a p1-specific late-promoter sequence with conserved elements located at positions -22 and -10 was expected from the function of the gene in phage morphogenesis. in addition to a late-promoter sequence, a -35 element and an operator sequence for the major repressor protein, c1, were found. the -35 and -10 elements constituted an active escherichia coli sigma(70) consensus promoter, ...	2001	11418548
definition of a 1-mb homozygous deletion at 9q32-q33 in a human bladder-cancer cell line.	we performed detailed molecular analyses of a suspected homozygous deletion on chromosome 9q32-q33 in a bladder-cancer cell line (kybtds) derived from a superficial papillary transitional cell carcinoma (tcc). we examined 13 sequence-tagged site (sts) markers mapped along 9q32-q33 by polymerase chain reaction (pcr), and used 13 bacterial artificial chromosome (bac)/bacteriophage p1-derived artificial chromosome (pac) genomic clone probes representing these sts markers as probes for dual-color fl ...	2001	11450846
altered directionality in the cre-loxp site-specific recombination pathway.	the site-specific recombinase cre must employ control mechanisms to impose directionality on recombination. when two recombination sites (locus of crossing over in phage p1, loxp) are placed as direct repeats on the same dna molecule, collision between loxp-bound cre dimers leads to excision of intervening dna. if two sites are placed as inverted repeats, the intervening segment is flipped around. cre catalyzes these reactions in the absence of protein co-factors. current models suggest that dir ...	2001	11492999
delivery of the cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo.	the cre recombinase (cre) from bacteriophage p1 is an important tool for genetic engineering in mammalian cells. we constructed lentiviral vectors that efficiently deliver cre in vitro and in vivo. surprisingly, we found a significant reduction in proliferation and an accumulation in the g(2)/m phase of cre-expressing cells. to minimize the toxic effect of cre, we designed a lentiviral vector that integrates into the host genome, expresses cre in the target cell, and is subsequently deleted from ...	2001	11553794
using an in vivo phagemid system to identify non-compatible loxp sequences.	the site-specific recombination system of bacteriophage p1 is composed of the cre recombinase that recognizes a 34-bp loxp site. the cre/loxp system has been extensively used to manipulate eukaryotic genomes for functional genomic investigations. the creation of additional heterologous loxp sequences potentially expands the utility of this system, but only if these loxp sequences do not recombine with one another. we have developed a stringent in vivo assay to examine the degree of recombination ...	2001	11576551
detection of illegitimate rearrangement within the immunoglobulin locus on 14q32.3 in b-cell malignancies using end-sequenced probes.	translocation involving the immunoglobulin heavy chain (igh) locus is a recurring event in b-cell oncogenesis. the aim of this study was to characterize clones from bacterial artificial chromosome (bac) libraries and/or bacteriophage p1 artificial chromosome libraries spanning the igh locus for detection of illegitimate rearrangement within the region by fluorescence in situ hybridization (fish). in silico analysis of the igh variable (ighv) dna sequence (nt_001716.v1) was performed to identify ...	2001	11579466
targeted somatic mutagenesis in mouse epidermis.	gene targeting in the mouse is a powerful tool to study mammalian gene function. the possibility to efficiently introduce somatic mutations in a given gene, at a chosen time and/or in a given cell type will further improve such studies, and will facilitate the generation of animal models for human diseases. to create targeted somatic mutations in the epidermis, we established transgenic mice expressing the bacteriophage p1 cre recombinase or the tamoxifen-dependent cre-er(t2) recombinase under t ...	2000	11595821
application of cre-loxp system to the urinary tract and cancer gene therapy.	the bacteriophage p1-derived cre-loxp system is a powerful and versatile tool for in vivo dna recombination. it is widely used in gene targeting research. the combination of the cre-loxp system and a specific promoter enables conditional "knockout" of a target gene in a particular tissue or cell type. it has also made it possible to delete genes in adult animals that are essential for embryogenesis. this system is also used to enhance tissue- or tumor-specific promoter activities that are useful ...	2001	11690553
dna substrates influence the recombination efficiency mediated by flp recombinase expressed in mammalian cells.	the flp recombinase derived from saccharomyces cerevisiae mediates precise site-specific recombination between a pair of flp recognition targets (frts). like the cre/loxp system derived from bacteriophage p1, the flp/frt system has recently been applied to gene regulation systems using an flp-expressing recombinant adenovirus (rad) (nakano et al, nucleic acids res. 29: e40, 2001). in an attempt to improve the flp/frt system by altering its dna substrates, we compared the recombination efficiency ...	2001	11694078
controlled expression in klebsiella pneumoniae and shigella flexneri using a bacteriophage p1-derived c1-regulated promoter system.	the utility of promoters regulated by the bacteriophage p1 temperature-sensitive c1 repressor was examined in shigella flexneri and klebsiella pneumoniae. promoters carrying c1 operator sites driving lacz expression had induction/repression ratios of up to 240-fold in s. flexneri and up to 50-fold in k. pneumoniae. the promoters exhibited remarkably low basal expression, demonstrated modulation by temperature, and showed rapid induction. this system will provide a new opportunity for controlled ...	2001	11698385
strong minor groove base conservation in sequence logos implies dna distortion or base flipping during replication and transcription initiation.	the sequence logo for dna binding sites of the bacteriophage p1 replication protein repa shows unusually high sequence conservation ( approximately 2 bits) at a minor groove that faces repa. however, b-form dna can support only 1 bit of sequence conservation via contacts into the minor groove. the high conservation in repa sites therefore implies a distorted dna helix with direct or indirect contacts to the protein. here i show that a high minor groove conservation signature also appears in sequ ...	2001	11726698
the p1 phage replication protein repa contacts an otherwise inaccessible thymine n3 proton by dna distortion or base flipping.	the repa protein from bacteriophage p1 binds dna to initiate replication. repa covers one face of the dna and the binding site has a completely conserved t that directly faces repa from the minor groove at position +7. although all four bases can be distinguished through contacts in the major groove of b-form dna, contacts in the minor groove cannot easily distinguish between a and t bases. therefore the 100% conservation at this position cannot be accounted for by direct contacts approaching in ...	2001	11726699
genetic organization of the francisella plasmid pfnl10.	we report here the molecular characterization of pfnl10, a 3990-bp cryptic plasmid of francisella novicida-like f6168. the plasmid was maintained in f. novicida utah 112 and f. tularensis lvs strains. we sequenced the entire plasmid and found six open reading frames (orfs)-orf1, orf2, orf3, orf4, orf5, and orfm. orf3, orf4, orf5, and orfm are located on the same strand, and we designated it the plus strand. orf1 and orf2 are on the complementary strand. the orfs appear to be arranged in two oper ...	2001	11735370
molecular approaches to chemo-radiotherapy.	although radiotherapy is used to treat many solid tumours, normal tissue tolerance and inherent tumour radioresistance can hinder successful outcome. cancer gene therapy is one approach being developed to address this problem. however, the potential of many strategies are not realised owing to poor gene delivery and a lack of tumour specificity. the use of treatment-, condition- or tumour-specific promoters to control gene-directed enzyme prodrug therapy (gdept) is one such method for targeting ...	2002	11803140
is2 insertion is a major cause of spontaneous mutagenesis of the bacteriophage p1: non-random distribution of target sites.	insertion mutations arising spontaneously in the p1 prophage and affecting vegetative phage reproduction were screened for the presence of insertion sequence 2 (is2). filter hybridization identified 28 out of 44 independent insertions as is2. their target specificity is not random. a region that amounts to < 2% of the phage genome had trapped 15 of the 28 is2 elements. however, precise mapping of nine mutants in this hot spot segment revealed no preferred insertion site. rather, the nine is2 are ...	1983	11894911
directed evolution of the site specificity of cre recombinase.	cre recombinase from bacteriophage p1 recognizes a 34-bp recombination site, loxp, with exquisite sequence specificity and catalyzes the site-specific insertion, excision, or rearrangement of dna. to better understand the molecular basis of protein-dna recognition and generate recombinases with altered specificities, we have developed a directed evolution strategy that can be used to identify recombinases that recognize variant loxp sites. to be selected, members of a library of cre variants pro ...	2002	11904359
development of a p1 phagemid system for the delivery of dna into gram-negative bacteria.	the inability to transform many clinically important gram-negative bacteria has hampered genetic studies addressing the mechanism of bacterial pathogenesis. this report describes the development and construction of a delivery system utilizing the broad-host-range transducing bacteriophage p1. the phagemids used in this system contain a p1 pac initiation site to package the vector, a p1 lytic replicon to generate concatemeric dna, a broad-host-range origin of replication and an antibiotic-resista ...	2002	11932441
efficient recombination in diverse tissues by a tamoxifen-inducible form of cre: a tool for temporally regulated gene activation/inactivation in the mouse.	in recent years, the cre integrase from bacteriophage p1 has become an essential tool for conditional gene activation and/or inactivation in mouse. in an earlier report, we described a fusion protein between cre and a mutated form of the ligand binding domain of the estrogen receptor (cre-er) that renders cre activity tamoxifen (tm) inducible, allowing for conditional modification of gene activity in the mammalian neural tube in utero. in the current work, we have generated a transgenic mouse li ...	2002	11944939
tight regulation and modulation via a c1-regulated promoter in escherichia coli and pseudomonas aeruginosa.	we describe the development and analysis of a novel promoter system regulated by the bacteriophage p1 temperature-sensitive c1 repressor. using transcriptional fusions to the lacz reporter gene to monitor gene expression, we show that the ratio of induction/repression can be up to 1500-fold in escherichia coli. the promoters exhibited extremely tight repression and could be modulated over a range of temperatures. the utility of the promoter system was tested in pseudomonas aeruginosa. c1 effecti ...	2002	12000993
e-cadherin is a survival factor for the lactating mouse mammary gland.	the ca(++)-dependent cell adhesion molecule e-cadherin is expressed throughout mouse development and in adult tissues. classical gene targeting has demonstrated that e-cadherin-deficient embryos die at the blastocyst stage. to study the involvement of e-cadherin in organogenesis, a conditional gene inactivation scheme was undertaken using the bacteriophage p1 recombinase cre/loxp system. mice with homozygous loxp sites in both alleles of the e-cadherin (cdh1) gene were generated and these mice w ...	2002	12049767
unmodified cre recombinase crosses the membrane.	site-specific recombination in genetically modified cells can be achieved by the activity of cre recombinase from bacteriophage p1. commonly an expression vector encoding cre is introduced into cells; however, this can lead to undesired side-effects. therefore, we tested whether cell-permeable cre fusion proteins can be directly used for lox-specific recombination in a cell line tailored to shift from red to green fluorescence after loxp-specific recombination. comparison of purified recombinant ...	2002	12060697
murine peripherin gene sequences direct cre recombinase expression to peripheral neurons in transgenic mice.	spatially and temporally regulated somatic mutations can be achieved by using the cre/loxp recombination system of bacteriophage p1. to develop a cell type-specific system of gene targeting in the peripheral nervous system, we generated the transgenic mouse lines expressing cre recombinase under the control of the mouse peripherin gene promoter. the activity of the cre recombinase during embryonic development was examined by mating the peripherin-cre transgenic mice to the knock-in cre-mediated ...	2002	12123806
purification of the c1 repressor of bacteriophage p1 by fast protein liquid chromatography.	a fast protein liquid chromatographic method is described for the purification of the c1 repressor of bacteriophage p1 and its truncated form c1*. by using one crude extract, both repressor proteins were purified in parallel to homogeneity and were shown to interact specifically with p1 operator dna in vitro. the method involves an affinity chromatographic step on heparin-sepharose, followed by a combination of ion-exchange chromatography on q sepharose and s sepharose. the availability of a hom ...	1992	12126108
phylogenetic and functional analysis of the bacteriophage p1 single-stranded dna-binding protein.	bacteriophage p1 encodes a single-stranded dna-binding protein (ssb-p1), which shows 66% amino acid sequence identity to the ssb protein of the host bacterium escherichia coli. a phylogenetic analysis indicated that the p1 ssb gene coexists with its e. coli counterpart as an independent unit and does not represent a recent acquisition of the phage. the p1 and e. coli ssb proteins are fully functionally interchangeable. ssb-p1 is nonessential for phage growth in an exponentially growing e. coli h ...	2002	12208948
interaction of the dnak and dnaj chaperone system with a native substrate, p1 repa.	dnak, the hsp70 chaperone of escherichia coli interacts with protein substrates in an atp-dependent manner, in conjunction with dnaj and grpe co-chaperones, to carry out protein folding, protein remodeling, and assembly and disassembly of multisubunit protein complexes. to understand how dnaj targets specific proteins for recognition by the dnak chaperone system, we investigated the interaction of dnaj and dnak with a known natural substrate, bacteriophage p1 repa protein. by characterizing repa ...	2002	12237299
minimization of the escherichia coli genome using a tn5-targeted cre/loxp excision system.	an increasing number of microbial genomes have been completely sequenced, and functional analyses of these genomic sequences are under way. to facilitate these analyses, we have developed a genome-engineering tool for determining essential genes and minimizing bacterial genomes. we made two large pools of independent transposon mutants in escherichia coli using modified tn5 transposons with two different selection markers and precisely mapped the chromosomal location of 800 of these transposons. ...	2002	12244329
[expression of cre gene in escherichia coli and bioassay its expression product].	the cre recombinase from bacteriophage p1 can recognize specific dna sequences, cleave dna at specific target sites, and then ligate it to the cleaved dna of a second site. in this study, cre gene was cloned into the pgem-t easy vector via pcr procedure. then the cre gene was inserted into an expression vector pet-29a and expressed in e. coli bl21 (de3). a 38 kd soluble protein was expressed and named cre. cre was purified by deae-52 chromatography. bioassay of the partially purified product sho ...	2002	12385251
doc-mediated cell killing in shigella flexneri using a c1/laci controlled expression system.	in this report we describe the development of a highly stringent and dually regulated promoter system for shigella flexneri. dual regulation was provided by utilizing a promoter susceptible to control by the bacteriophage p1 temperature-sensitive c1 repressor that in turn was under the transcriptional control of laci. the level of induction/repression ratios observed was up to 3700-fold in s. flexneri. the general utility of this promoter system was evaluated by demonstrating that the bacterioph ...	2002	12399040
the p1 plasmid in action: time-lapse photomicroscopy reveals some unexpected aspects of plasmid partition.	the prophage of bacteriophage p1 is a low copy number plasmid in escherichia coli and is segregated to daughter cells by an active partition system. the dynamics of the partition process have now been successfully followed by time-lapse photomicroscopy. the process appears to be fundamentally different from that previously inferred from statistical analysis of fixed cells. a focus containing several plasmid copies is captured at the cell center. immediately before cell division, the copies eject ...	2002	12460532
transgenic mice with hematopoietic and lymphoid specific expression of cre.	bacteriophage p1 cre/loxp based systems can be used to manipulate the genomes ofmice in vivo and in vitro, allowing the generation of tissue-specific conditional mutants. we have generated mouse lines expressing cre recombinase in hematopoietic tissues using the vav regulatory elements, or in lymphoid cells using the hcd2 promoter and locus control region (lcr). the r26r-eyfp cre reporter mouse line was used to determine the pattern of cre expression in each line and enabled the assessment of cr ...	2003	12548562
[progress in the study of the structure and function of cre recombinase].	the cre recombinase, an integrase from bacteriophage p1, catalyzes site-specific recombination between 34-bp repeats termed loxp sites, in the absence of any additional cofactors and energy. mediated by cre recombinase, specific dna fragments can be excised, inversed or integrated depending on the orientation or position of loxp sites in vitro or in vivo. because of its simplicity and high efficiency, cre/loxp site-specific recombination system has been widely used in gene deletion and function ...	2002	12561193
productive interaction between the chromosome partitioning proteins, para and parb, is required for the progression of the cell cycle in caulobacter crescentus.	in caulobacter crescentus the partitioning proteins para and parb operate a molecular switch that couples chromosome partitioning to cytokinesis. homologues of these proteins have been shown to be important for the stable inheritance of f-plasmids and the prophage form of bacteriophage p1. in c. crescentus, parb binds to sequences adjacent to the origin of replication and is required for the initiation of cell division. additionally, parb influences the nucleotide-bound state of para by acting a ...	2003	12603730
development of a thermally regulated broad-spectrum promoter system for use in pathogenic gram-positive species.	selectively regulating gene expression is an essential molecular tool that is lacking for many pathogenic gram-positive bacteria. in this report, we describe the evaluation of a series of promoters regulated by the bacteriophage p1 temperature-sensitive c1 repressor in enterococcus faecium, enterococcus faecalis, and staphylococcus aureus. using the lacz gene to monitor gene expression, we examined the strength, basal expression, and induced expression of synthetic promoters carrying c1 operator ...	2003	12788740
escherichia coli sspa is a transcription activator for bacteriophage p1 late genes.	the stringent starvation protein a (sspa), an escherichia coli rna polymerase (rnap)-associated protein, has been reported to be essential for lytic growth of bacteriophage p1. unlike p1 early promoters, p1 late promoters are not recognized by rnap alone. a phage-encoded early protein, lpa (late promoter activator protein, formerly called gp10), has been shown to be required for p1 late transcription in vivo. here, we demonstrate that sspa is a transcription activator for p1 late genes. our resu ...	2003	12791143
identification of cre residues involved in synapsis, isomerization, and catalysis.	the cre protein of bacteriophage p1 is a tyrosine recombinase and catalyzes recombination via formation of a covalent protein-dna complex and a holliday junction intermediate. several co-crystal structures of cre bound to its target lox site have provided novel insights into its biochemical activities. we have used these structures to guide the mutagenesis of several cre residues that contact the lox spacer region and/or are involved in intersubunit protein-protein interactions. none of the muta ...	2003	12851389
bacteriophage p1 ban protein is a hexameric dna helicase that interacts with and substitutes for escherichia coli dnab.	since the ban gene of bacteriophage p1 suppresses a number of conditionally lethal dnab mutations in escherichia coli, it was assumed that ban protein is a dna helicase (dnab analogue) that can substitute for dnab in the host replication machinery. we isolated and sequenced the ban gene, purified the product, and analysed the function of ban protein in vitro and in vivo. ban hydrolyses atp, unwinds dna and forms hexamers in the presence of atp and magnesium ions. since all existing conditionally ...	2003	12853607
escherichia coli single-stranded dna-binding protein mediates template recycling during transcription by bacteriophage n4 virion rna polymerase.	coliphage n4 virion rna polymerase (vrnap), the most distantly related member of the t7-like family of rna polymerases, is responsible for transcription of the early genes of the linear double-stranded dna phage genome. escherichia coli single-stranded dna-binding protein (ecossb) is required for n4 early transcription in vivo, as well as for in vitro transcription on super-coiled dna templates containing vrnap promoters. in contrast to other dna-dependent rna polymerases, vrnap initiates transc ...	2003	12876194
crystal structure of a wild-type cre recombinase-loxp synapse reveals a novel spacer conformation suggesting an alternative mechanism for dna cleavage activation.	escherichia coli phage p1 cre recombinase catalyzes the site-specific recombination of dna containing loxp sites. we report here two crystal structures of a wild-type cre recombinase-loxp synaptic complex corresponding to two distinct reaction states: an initial pre-cleavage complex, trapped using a phosphorothioate modification at the cleavable scissile bond that prevents the recombination reaction, and a 3'-phosphotyrosine protein-dna intermediate resulting from the first strand cleavage. in c ...	2003	12954782
studies on lysogenesis. ii. the effect of temperature on the lysogenization of shigella dysenteriae with phage p1.		1954	13129214
[behavior of the temperate phage p1 on its host, staphylococcus s2].		1953	13167489
transduction of linked genetic characters of the host by bacteriophage p1.		1955	13267987
effect of chloramphenicol on lysogenization by temperate phage p1.		1957	13456369
the effects of ultraviolet light and ionizing radiation on the transduction of escherichia coli by phage p1.		1960	13845040
the specific gravity of transducing particles of bacteriophage p1.		1962	13921337
mapping of the galactose genes of escherichia coli by transduction with phage p1.		1963	14011089
the control of host-induced modification by phage p1.		1964	14187915
macromolecular syntheses in the initiation of bacteriophage p1 induction.		1964	14202270
drug resistance of enteric bacteria. iv. active transducing bacteriophage p1 cm produced by the combination of r factor with bacteriophage p1.	kondo, eiko (gunma university, maebashi, japan), and susumu mitsuhashi. drug resistance of enteric bacteria. iv. active transducing phage p1 cm produced by the combination of r factor with phage p1. j. bacteriol. 88:1266-1276. 1964.-during an investigation of the transduction of r factors with phage p1, a phage lysate capable of transducing the character of chloramphenicol resistance (cm(r)) in extremely high frequency was obtained. the transduction of the cm(r) character with the lysate was con ...	1964	14234780
cre/lox-mediated marker gene excision in transgenic maize (zea mays l.) plants.	after the initial transformation and tissue culture process is complete, selectable marker genes, which are used in virtually all transformation approaches, are not required for the expression of the gene of interest in the transgenic plants. there are several advantages to removing the selectable marker gene after it is no longer needed, such as enabling the reuse of selectable markers and simplifying transgene arrays. we have tested the cre/ lox system from bacteriophage p1 for its ability to ...	2003	14513214
high efficiency, site-specific excision of a marker gene by the phage p1 cre-loxp system in the yellow fever mosquito, aedes aegypti.	the excision of specific dna sequences from integrated transgenes in insects permits the dissection in situ of structural elements that may be important in controlling gene expression. furthermore, manipulation of potential control elements in the context of a single integration site mitigates against insertion site influences of the surrounding genome. the cre-loxp site-specific recombination system has been used successfully to remove a marker gene from transgenic yellow fever mosquitoes, aede ...	2003	14602940
characterization of a highly polymorphic marker adjacent to the slc4a1 gene and of kidney immunostaining in a family with distal renal tubular acidosis.	mutations in the human slc4a1 (ae1/band 3) gene are associated with hereditary spherocytic anaemia and with distal renal tubular acidosis (drta). the molecular diagnosis of ae1 mutations has been complicated by the absence of highly polymorphic genetic markers, and the pathogenic mechanisms of some drta-associated ae1 mutations remain unclear. here, we characterized a polymorphic dinucleotide repeat close to the human ae1 gene and performed an immunocytochemical study of kidney tissue from a pat ...	2004	14736961
phage multiplication on two hosts. isolation and activity of variants of staphylococcus phage p1.		1952	14949003
conditional, recombinase-mediated expression of genes in plant cell cultures.	in plant cells, overexpression of critical genes can be hampered by deleterious effects on development that results in a counterselection of transgenic cells harboring the gene of interest. inducible expression systems have been reported, but many of them show unwanted leaky expression. to circumvent this potential problem, a novel inducible system was developed based on two previously characterized systems: the cre-loxp site-specific recombination system of bacteriophage p1 and the subcellular ...	2004	14996220
a signal-arrest-release sequence mediates export and control of the phage p1 endolysin.	the lyz endolysin of bacteriophage p1 was found to cause lysis of the host without a holin. induction of a plasmid-cloned lyz resulted in lysis, and the lytic event could be triggered prematurely by treatments that dissipate the proton-motive force. instead of requiring a holin, export was mediated by an n-terminal transmembrane domain (tmd) and required host sec function. exported lyz of identical sds/page mobility was found in both the membrane and periplasmic compartments, indicating that per ...	2004	15090650
transgenic mice as a model to study the regulation of human transferrin expression in sertoli cells.	understanding the regulation of proteins secreted by human sertoli cells is important for identifying the causes of infertility in men. however, experiments with sertoli cells purified from healthy testes are difficult to perform, for obvious ethical reasons. therefore, experiments with transgenic mouse models could provide an alternative approach to study the function and regulation of a human gene in sertoli cells.	2004	15105395
in vivo excision and amplification of large human genomic segments using the cre/loxp-and large t antigen/sv40 ori-mediated machinery.	in vivo excision and amplification of pre-determined large genomic segments, directly from the genome of a natural host, can be a powerful tool for obtaining the genomic sequences with minimum rearrangements. in this study, an in vivo excision and amplification system in human bjab cells was devised by combining the cre/loxp system of bacteriophage p1 and the large t antigen/sv40 ori system of simian virus 40. two loxp sequences, each of which serves as a recognition site for recombinase cre, we ...	2004	15163513
functional mapping of cre recombinase by pentapeptide insertional mutagenesis.	cre is a site-specific recombinase from bacteriophage p1. it is a member of the tyrosine integrase family and catalyzes reciprocal recombination between specific 34-bp sites called loxp. to analyze the structure-function relationships of this enzyme, we performed large scale pentapeptide insertional mutagenesis to generate insertions of five amino acids at random positions in the protein. the high density of insertion mutations into cre allowed us to identify an unexpected degree of functional t ...	2004	15218019
a new family of conditional replicating plasmids and their cognate escherichia coli host strains.	we constructed a family of conditionally replicating plasmids, the ptx1 family, which are based on the incpalpha oriv origin of replication that is dependent on the trfa-encoded protein. we constructed several escherichia coli derivatives expressing trfa from different chromosomal loci, which can be transduced by phage p1 to any e. coli strain. the ptx1 plasmids also carry the oritrp4 origin of transfer, and can be conjugated to e. coli, vibrio cholerae and likely to a broad range of bacteria fr ...	2004	15249062
escherichia coli mazef-mediated cell death as a defense mechanism that inhibits the spread of phage p1.	the escherichia coli gene pair mazef is a regulatable chromosomal toxin-antitoxin module: mazf encodes a stable toxin and maze encodes for a labile antitoxin that overcomes the lethal effect of mazf. because maze is labile, inhibition of maze expression results in cell death. we studied the effect of mazef on the development of bacteriophage p1 upon thermoinduction of the prophage p1cm c1ts and upon infection with virulent phage particles (p1vir). in several e. coli strains, we showed that the d ...	2004	15316771
utility of the flp-frt recombination system for genetic manipulation of rice.	to develop an flp-frt recombination system- (derived from 2 mu plasmid of saccharomyces cerevisiae) based marker gene removal application for rice, we introduced the gene for flp recombinase, under the control of the maize ubiquitin-1 promoter, into the rice genome. flp activity was monitored in callus and regenerated plants by an assay based on the deletion of the frt-flanked dna fragment, leading to the activation of the beta-glucuronidase gene. flp activity was detected both in the callus and ...	2005	15480685
bacteriophage p1 in retrospect and in prospect.		2004	15489415
genome of bacteriophage p1.	p1 is a bacteriophage of escherichia coli and other enteric bacteria. it lysogenizes its hosts as a circular, low-copy-number plasmid. we have determined the complete nucleotide sequences of two strains of a p1 thermoinducible mutant, p1 c1-100. the p1 genome (93,601 bp) contains at least 117 genes, of which almost two-thirds had not been sequenced previously and 49 have no homologs in other organisms. protein-coding genes occupy 92% of the genome and are organized in 45 operons, of which four a ...	2004	15489417
mutually exclusive recombination of wild-type and mutant loxp sites in vivo facilitates transposon-mediated deletions from both ends of genomic dna in pacs.	recombination of wild-type and mutant loxp sites mediated by wild-type cre protein was analyzed in vivo using a sensitive phage p1 transduction assay. contrary to some earlier reports, recombination between loxp sites was found to be highly specific: a loxp site recombined in vivo only with another of identical sequence, with no crossover recombination either between a wild-type and mutant site; or between two different mutant sites tested. mutant loxp sites of identical sequence recombined as e ...	2004	15494454
selecting transpositions using phage p1 headful packaging: new markerless transposons for functionally mapping long-range regulatory sequences in bacterial artificial chromosomes and p1-derived artificial chromosomes.	new tn10 minitransposons were constructed to functionally map long-range transcription regulatory sequences in bacterial artificial chromosomes (bacs) and p1-derived artificial chromosomes (pacs). each contained a wild-type loxp site but, significantly, contained no mammalian or bacterial genes and/or promoter elements within the transposed portion of dna. in contrast to loxp transposons described previously, the new ones do not introduce transcription regulatory elements capable of interfering ...	2004	15556570
phage like it hot: solution structure of the bacteriophage p1-encoded hot protein, a homolog of the theta subunit of e. coli dna polymerase iii.	dna polymerase iii, the main replicative polymerase of e. coli, contains a small subunit, theta, that binds to the epsilon proofreading subunit and appears to enhance the enzyme's proofreading function--especially under extreme conditions. it was recently discovered that e. coli bacteriophage p1 encodes a theta homolog, named hot. the (1)h-(15)n hsqc spectrum of hot exhibits more uniform intensities and less evidence of conformational exchange than that of theta; this uniformity facilitates a de ...	2004	15576035
pvx-cre-mediated marker gene elimination from transgenic plants.	cre recombinase gene from bacteriophage p1 was transiently expressed by a potato virus x (pvx)-based vector in transgenic lox -target nicotiana benthamiana plants to remove the selectable marker gene. the target construct consisted of two directly oriented lox sites flanking a bar gene located between a gfp coding region and an upstream camv 35s promoter. the cre-mediated excision of intervening sequence placed the gfp coding region under the transcriptional control of the camv 35s promoter. gfp ...	2004	15604695
the molecular chaperone, clpa, has a single high affinity peptide binding site per hexamer.	substrate recognition by clp chaperones is dependent on interactions with motifs composed of specific peptide sequences. we studied the binding of short motif-bearing peptides to clpa, the chaperone component of the atp-dependent clpap protease of escherichia coli in the presence of atpgammas and mg2+ at ph 7.5. binding was measured by isothermal titration calorimetry (itc) using the peptide, aandenyalaa, which corresponds to the ssra degradation motif found at the c terminus of abnormal nascent ...	2005	15657062
from mapping to sequencing, post-sequencing and beyond.	the rice genome research program (rgp) in japan has been collaborating with the international community in elucidating a complete high-quality sequence of the rice genome. as the pioneer in large-scale analysis of the rice genome, the rgp has successfully established the fundamental tools for genome research such as a genetic map, a yeast artificial chromosome (yac)-based physical map, a transcript map and a phage p1 artificial chromosome (pac)/bacterial artificial chromosome (bac) sequence-read ...	2005	15659433
rhodopsin-icre transgenic mouse line for cre-mediated rod-specific gene targeting.	retinal photoreceptors are highly differentiated postmitotic neurons that transduce photons into electrical signals. while the functions of many photoreceptor-specific genes can be evaluated by direct gene targeting, here we facilitate the studies of nonphotoreceptor-specific genes in these cells by developing an opsin-icre transgenic mouse line, icre-75, in which a 4-kb mouse rod opsin promoter drives the expression of bacteriophage p1 cre recombinase. immunohistochemical analysis demonstrated ...	2005	15682388
identification and characterization of a novel allele of escherichia coli dnab helicase that compromises the stability of plasmid p1.	bacteriophage p1 lysogenizes escherichia coli cells as a plasmid with approximately the same copy number as the copy number of the host chromosome. faithful inheritance of the plasmids relies upon proper dna replication, as well as a partition system that actively segregates plasmids to new daughter cells. we genetically screened for e. coli chromosomal mutations that influenced p1 stability and identified a novel temperature-sensitive allele of the dnab helicase gene (dnab277) that replaces ser ...	2005	15687186
structural basis for the function of stringent starvation protein a as a transcription factor.	stringent starvation protein a (sspa) of escherichia coli is an rna polymerase-associated transcriptional activator for the lytic development of phage p1 and is essential for stationary phase-induced acid tolerance of e. coli. we report the crystal structure of yersinia pestis sspa, which is 83% identical to e. coli sspa in amino acid sequence and is functionally complementary in supporting the lytic growth of phage p1 and acid resistance of an e. coli sspa mutant. the structure reveals that ssp ...	2005	15735307
limited use of the cre/loxp recombination system in efficient production of loxp-containing minicircles in vivo.	the cre/loxp recombination system of bacteriophage p1 is one of the most powerful tools in genome engineering. we report, however, that the activity of the cre/loxp system interferes with the stability of the multicopy loxp-bearing plasmids in escherichia coli reca bacteria. due to the predominantly unidirectional cre-mediated high-order multimer formation of these plasmids, the number of their copies (overall yield) gradually decreases. intermolecular recombination reduces the copy number of pl ...	2004	15737402
a new family of mobilizable suicide plasmids based on broad host range r388 plasmid (incw) and rp4 plasmid (incpalpha) conjugative machineries and their cognate escherichia coli host strains.	we describe the construction of the psw family of conditionally replicating plasmids which are based on the incx oriv origin (oriv(r6kgamma)) of replication that is dependent on the pir-encoded protein. we constructed several escherichia coli derivatives expressing pir from different chromosomal loci, and the pir gene could be transduced by phage p1 to any e. coli strain. these chromosomal constructions generate dapa and thya knockouts, which lead to diaminopimelate or thymidine auxotrophies, re ...	2005	15748991
sspa is required for acid resistance in stationary phase by downregulation of h-ns in escherichia coli.	the stringent starvation protein a (sspa) is a rna polymerase-associated protein and is required for transcriptional activation of bacteriophage p1 late promoters. however, the role of sspa in gene expression in escherichia coli is essentially unknown. in this work, we show that sspa is essential for cell survival during acid-induced stress. apparently, sspa inhibits stationary-phase accumulation of h-ns, a global regulator which functions mostly as a repressor, thereby derepressing multiple str ...	2005	15819627
[a simple and visible assay for cre recombinase activity in escherichia coli by using two incompatible plasmids].	the cre/loxp system derived from bacteriophage p1 is widely used to carry out complex manipulations of dna molecules both in vitro and in vivo. in order to further characterize and modify the cre/loxp system, a convenient method for assaying the recombination efficiency is needed. a simple and visible assay is described, in which two incompatible plasmids, separately carrying the cre gene and loxp-flanked gfp gene, were co-transferred into e. coli. the cre gene was inserted into a kanamycin-resi ...	2005	15847178
effect of deletion mutation on the recombination activity of cre recombinase.	cre recombinase from bacteriophage p1 is widely used in both in vitro and in vivo dna manipulations. based on a structural and functional analysis, three deleted cre mutants were constructed and expressed in escherichia coli. mutated recombinases were purified and their recombination activities were determined in vitro. our results revealed that the mutant with amino-terminal deletion retains the recombination activity as high as wild type cre; however, the carboxy-terminal deletion and the midd ...	2005	15912212