McrA and McrB restriction phenotypes of some<i>E.coli</i>strains and implications for gene cloning

Raleigh, Elisabeth A.; Murray, Noreen E.; Revel, Helen R.; Blumenthal, Robert; Westaway, David; Reith, A. D.; Rigby, P. W. J.; Elhai, Jeff; Hanahan, Douglas

doi:10.1093/nar/16.4.1563

Cited by 334 publications

(139 citation statements)

References 41 publications

Supporting

Mentioning

137

Contrasting

Order By: Relevance

“…CC118.1 is a derivative of CC118 (Manoil and Beckwith, 1985) that contains FЈ lacI q1 lacZ ::Tn5; it was used as a host strain for the B. burgdorferi genomic library and identification of clones expressing haemolytic activity. E. coli MM294 (Meselson and Yuan, 1968) was routinely used as the host strain for pDP1 and its derivatives; DH5␣ (Raleigh et al, 1988) was used routinely for plasmid DNA manipulation; and BL21(DE3), BL21(DE3)(pLysE), BL21(DE3)(pLysS) (Studier et al, 1990), and MM294(DE3) (this work) were used for expression of plasmids containing the T7 promoter. Plasmid pET11b (Studier et al, 1990) was used for cloning and expression of blyA and blyB.…”

Section: Methodsmentioning

confidence: 99%

Cloning and analysis of a Borrelia burgdorferi membrane‐interactive protein exhibiting haemolytic activity

Guina

Oliver

1997

Molecular Microbiology

View full text Add to dashboard Cite

SummaryWe cloned the gene encoding a membrane-interactive protein of Borrelia burgdorferi by means of its haemolytic activity in Escherichia coli. The haemolytic activity was erythrocyte-species specific, with progressively decreasing activity for erythrocytes from horse, sheep, and rabbit, respectively. Genetic analysis of the haemolytic determinant revealed two borrelia haemolysin genes, blyA and blyB, that are part of a predicted four-gene operon which is present in multiple copies on the 30 kb circular plasmid(s) of B. burgdorferi B31. blyA encodes a predicted ␣-helical 7.4 kDa protein with a hydrophobic central region and a positively charged C-terminus, which is structurally homologous to a large group of pore-forming toxins with cytolytic activity. blyB encodes a soluble protein which stabilized BlyA and enhanced haemolytic activity. While the majority of BlyA in E. coli was membrane-associated, only soluble protein was haemolytically active. The haemolytic activity was shown to be highly protease sensitive, heat labile, independent of divalent cations, and extremely dependent on protein concentration, consistent with a requirement for oligomerization as the mechanism of action. BlyA was highly purified from E. coli in a single step utilizing Triton X-114 phase partitioning. Genetic analysis of blyA and blyB mutants indicated that the stability, membrane association, and activity of BlyA was dependent on subtle changes in its sequence and on the BlyB protein. The bly genes were found to be expressed at a very low level in cultured B. burgdorferi.

show abstract

Section: Methodsmentioning

confidence: 99%

Cloning and analysis of a Borrelia burgdorferi membrane‐interactive protein exhibiting haemolytic activity

Guina

Oliver

1997

Molecular Microbiology

View full text Add to dashboard Cite

show abstract

“…The DNA was dialysed against distilled water on a micropore nitrocellulose filter for an hour and 1 lg was used for a single electro-transformation of competent NM554 cells (Raleigh et al, 1988) at 20 kV/cm using a Gene Pulser machine (Bio-Rad). The transformation mixture was plated on LB medium containing 35 mg/l chloramphenicol and the plasmid content of resistant clones was characterized by restriction analysis.…”

Section: Detecting Circular Excised Dnamentioning

confidence: 99%

Size Does Matter: Cre-mediated Somatic Deletion Efficiency Depends on the Distance Between the Target lox-Sites

et al. 2005

View full text Add to dashboard Cite

Cre/lox recombination in vivo has become an important tool to induce chromosomal rearrangements like deletions. Using a combination of Ds transposition and Cre/lox recombination in two independent experiments on chromosomes 6 and 7 of tomato, two sets of somatic deletions up to a size of 200 kb were obtained. The efficiency of somatic deletion decreased with increasing deletion size. The largest germinally transmitted deletion had a size of only 55 kb. The results show that Cre-mediated deletion in somatic cells is less efficient when the lox sites are separated over larger distances. A further drop of the deletion efficiency after germinal transmission of the larger deletions can be explained by the probable loss of genes that are of vital importance to gametophyte function. Plasmid rescue of an 8.4 kb circularised deleted DNA showed that the Cre-mediated deletion takes place in tomato as expected. Since the circular Cre-deleted DNA could only be PCR amplified in plant cells where the deletion was not complete, the double-stranded DNA circle is assumed to be instable.

show abstract

“…The bacterial strains used were Escherichia coli K12 derivatives NM554 (recA13 araD139 D(araABC-leu)7679 DlacX74 galU galK rpsL thi hsdR mcrB) (Raleigh et al 1988), JE6638 (polA1 purE trp lys proC leu thi lacZ xyl ara mtl mal man gal mel tonA tsx str rif nalA) (National Institute of Genetics collection), and BW313 ] dut1 ung1 thi1 relA1) (Kunkel et al 1987). BW313 was used for site-directed mutagenesis according to Kunkel et al (1987).…”

Section: E Coli Strainsmentioning

confidence: 99%

Inhibition of transpositional recombination by OrfA and OrfB proteins encoded by insertion sequence IS3

et al. 1997

View full text Add to dashboard Cite

Background: An insertion element IS3 is flanked by terminal inverted repeat (IR) sequences. IS3 encodes two, out-of-phase, overlapping open reading frames, orfA and orfB, from which three proteins are produced. OrfAB is a transframe protein produced by ¹1 translational frameshifting between orfA and orfB, and it is known to be IS3 transposase. OrfA and OrfB are the proteins produced without frameshifting, but their functions have not been elucidated.

show abstract

McrA and McrB restriction phenotypes of someE.colistrains and implications for gene cloning

Cited by 334 publications

References 41 publications

Cloning and analysis of a Borrelia burgdorferi membrane‐interactive protein exhibiting haemolytic activity

Cloning and analysis of a Borrelia burgdorferi membrane‐interactive protein exhibiting haemolytic activity

Size Does Matter: Cre-mediated Somatic Deletion Efficiency Depends on the Distance Between the Target lox-Sites

Inhibition of transpositional recombination by OrfA and OrfB proteins encoded by insertion sequence IS3

Contact Info

Product

Resources

About