The optimization of preparations of competent cells for transformation of<i>E.coli</i>

Tang, Xiaoou; Nakata, Yasuhiko; Li, Haiou; Zhang, Mingdi; Gao, Hui; Fujita, Akiko; Sakatsume, Osamu; Ohta, Tomohiko; Yokoyama, Kazushige

doi:10.1093/nar/22.14.2857

Cited by 61 publications

(36 citation statements)

References 3 publications

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“…Either E. coli TG1 or E. coli BL21(DE3) cells were grown in 0.5 liter of LB medium to an OD600 Of 0.90-0.95 (20). The cells were collected by centrifugation at 4500 x g, and the cell pellet was resuspended in 0.5 liter of ice-cold 10% (vol/vol) glycerol solution.…”

Section: Methodsmentioning

confidence: 99%

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The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine.

Schuster

Rétey

1995

Proc. Natl. Acad. Sci. U.S.A.

127

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Phenylalanine ammonia-lyase (EC 4.3.1.5) from parsley is posttranslationally modified by dehydrating its Ser-202 to the catalytically essential dehydroalanine prosthetic group. The codon of Ser-202 was changed to those of alanine and threonine by site-directed mutagenesis. These mutants and the recombinant wild-type enzyme, after treatment with sodium borohydride, were virtually inactive with L-phenylalanine as substrate but catalyzed the deamination of L-4-nitrophenylalanine, which is also a substrate for the wild-type enzyme. Although the mutants reacted about 20 times slower with L-4-nitrophenylalanine than the wild-type enzyme, their V... for L-4-nitrophenylalanine was two orders of magnitude higher than for L-phenylalanine. In contrast to L-tyrosine, which was a poor substrate, DL-3-hydroxyphenylalanine (DL-m-tyrosine) was converted by phenylalanine ammonia-lyase at a rate comparable to that of L-phenylalanine. These results suggest a mechanism in which the crucial step is an electrophilic attack of the prosthetic group at position 2 or 6 of the phenyl group. In the resulting carbenium ion, the 18-Hsi atom is activated in a similar way as it is in the nitro analogue. Subsequent elimination of ammonia, concomitant with restoration of both the aromatic ring and the prosthetic group, completes the catalytic cycle.Phenylalanine ammonia-lyase (PAL) is an important plant enzyme that converts L-phenylalanine into trans-cinnamic acid, which in turn is the precursor of various phenylpropanoids, such as lignins, flavonoids, and coumarins (1, 2). It has been known for a long time that PAL contains a catalytically essential dehydroalanine residue (3). Although the latter occurs in a number of natural peptides (4, 5) and has been postulated as an intermediate in the conversion of L-serine residues into D-alanine (6), it is shared only by one related enzyme, histidine ammonia-lyase (HAL) (7-10).The electrophilic property of the prosthetic dehydroalanine has been demonstrated by addition of various nucleophiles, such as nitromethane (7-11), cyanide (12), and sodium borohydride (3,8). All these reagents caused inactivation and, using radiolabel, incorporation of the radioisotope into the expected product. Twenty-five years ago a mechanism was proposed for the PAL and HAL reactions involving as an initial step the nucleophilic attack at the dehydroalanine residue by the amino group of the substrate (3, 7). Such a reaction should enhance the leaving ability of the amino group. Recently, this laboratory has identified the precursor of the dehydroalanine in HAL from Pseudomonas putida and PAL from parsley to be Ser-143 and Ser-202, respectively (9, 10, 13). Heterologous expression allowed the production of mutants by site-directed mutagenesis. The PAL mutant S202A and the HAL mutants S143A and S143T were virtually inactive with L-phenylalanine and L-histidine as substrates, respectively (9,10,13,14). Surprisingly, L-5-nitrohistidine, known to be a moderately good substrate of HAL, reacted with the dehydroalanine-less m...

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Section: Methodsmentioning

confidence: 99%

“…Either E. coli TG1 or E. coli BL21(DE3) cells were grown in 0.5 liter of LB medium to an OD600 Of 0.90-0.95 (20). The (16).…”

Section: Methodsmentioning

confidence: 99%

The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine.

Schuster

Rétey

1995

Proc. Natl. Acad. Sci. U.S.A.

127

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“…The resultant fragments were ligated overnight at 14°C in a 500-l volume with T4 DNA ligase (2,400 U; New England Biolabs). Ligations were concentrated by centrifugation through a Microcon-100 concentrator at 500 ϫ g for 14 min, and the entire concentrated ligation was transformed into 1 ml of XL1-Blue MRFЈ cells (Stratagene), which were made to competencies of Ͼ1 ϫ 10 8 transformants/g (35,73). One to 15 transformants were visible after overnight growth at 37°C on LB agar supplemented with 50 g/ml kanamycin.…”

Section: Vol 25 2005 Resolution Of L1 Retrotransposition Intermediamentioning

confidence: 99%

Multiple Fates of L1 Retrotransposition Intermediates in Cultured Human Cells

Gilbert

Lutz

Morrish

et al. 2005

Molecular and Cellular Biology

258

375

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LINE-1 (L1) retrotransposons comprise ϳ17% of human DNA, yet little is known about L1 integration. Here, we characterized 100 retrotransposition events in HeLa cells and show that distinct DNA repair pathways can resolve L1 cDNA retrotransposition intermediates. L1 cDNA resolution can lead to various forms of genetic instability including the generation of chimeric L1s, intrachromosomal deletions, intrachromosomal duplications, and intra-L1 rearrangements as well as a possible interchromosomal translocation. The L1 retrotransposition machinery also can mobilize U6 snRNA to new genomic locations, increasing the repertoire of noncoding RNAs that are mobilized by L1s. Finally, we have determined that the L1 reverse transcriptase can faithfully replicate its own transcript and has a base misincorporation error rate of ϳ1/7,000 bases. These data indicate that L1 retrotransposition in transformed human cells can lead to a variety of genomic rearrangements and suggest that host processes act to restrict L1 integration in cultured human cells. Indeed, the initial steps in L1 retrotransposition may define a host/parasite battleground that serves to limit the number of active L1s in the genome.Long interspersed element 1 (LINE-1 or L1) is an abundant retrotransposon that comprises ϳ17% of human DNA (43, 69). Most L1s are retrotransposition defective because they are 5Ј truncated, contain internal rearrangements, or harbor mutations within their open reading frames (25, 43). However, the average human genome is estimated to contain ϳ80 to 100 retrotransposition-competent L1s (RC-L1s), and approximately 10% of these elements are classified as highly active or "hot" (6, 63).Human RC-L1s are ϳ6.0 kb and contain a 5Ј untranslated region (UTR), two nonoverlapping open reading frames (ORF1 and ORF2), and a 3Ј UTR that ends in a poly(A) tail (Fig. 1A) (13,53,66). ORF1 encodes a 40-kDa nucleic acid binding protein (30,31,33), whereas ORF2 has the potential to encode a 150-kDa protein with demonstrated endonuclease (L1 EN) and reverse transcriptase (L1 RT) activities (15,19,22,51). ORF2p also contains a cysteine-rich domain (CX 3 CX 7 HX 4 C) of unknown function (17, 54). Both proteins are required for retrotransposition in cis (54), which most probably occurs by a mechanism termed "target site primed reverse transcription" (TPRT) (19,47,54,72). However, how L1 integration is completed remains a mystery.We recently developed a plasmid-based rescue system that allows the recovery of L1 insertions in cultured human HeLa cells with minimal influence from selective pressures that occur during genome evolution. We found that L1 retrotransposition is associated with various forms of genetic instability and that the nascent L1 cDNA can undergo recombination with endogenous L1 elements, resulting in the formation of chimeric L1s. Consistent findings by Symer et al., using a colon cell line (HCT116) with an essentially normal karyotype, have led to the hypothesis that L1 retrotransposition can lead to various types of genomic instability (2...

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“…Plasmid pPRP56 (Palacios & Escalante-Semerena, 2000), which contained a wild-type prpR allele under the control of the arabinose-inducible P araBAD promoter, was introduced into the hypermutagenic E. coli strain XL1-Red (Stratagene) by transformation (Tang et al, 1994) and plated onto LB/ampicillin plates. To recover all of the transformed cells, 5 ml LB/ampicillin was added to the plate and colonies were scraped off the agar surface.…”

Section: Genetic Techniquesmentioning

confidence: 99%

2-Methylcitrate-dependent activation of the propionate catabolic operon (prpBCDE) of Salmonella enterica by the PrpR protein

Palacios

Escalante‐Semerena

2004

Microbiology

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The function of the PrpR protein of Salmonella enterica serovar Typhimurium LT2 was studied in vitro and in vivo. The PrpR protein is a sensor of 2-methylcitrate (2-MC), an intermediate of the 2-methylcitric acid cycle used by this bacterium to convert propionate to pyruvate. PrpR was unresponsive to citrate (a close structural analogue of 2-MC) and to propionate, suggesting that 2-MC, not propionate, is the metabolite that signals the presence of propionate in the environment to S. enterica. prpR alleles encoding mutant proteins with various levels of 2-MC-independent activity were isolated. All lesions causing constitutive PrpR activity were mapped to the N-terminal domain of the protein. Removal of the entire sensing domain resulted in a protein (PrpRc) with the highest 2-MC-independent activity. Residue A162 is critical to 2-MC sensing, since the mutant PrpR protein PrpRA162T was as active as the PrpRc protein in the absence of 2-MC. DNA footprinting studies identified the site in the region between prpR and the prpBCDE operon to which the PrpR protein binds. Analysis of the binding-site sequence revealed two sites with dyad symmetry. Results from DNase I footprinting assays suggested that the PrpR protein may have higher affinity for the site proximal to the PprpBCDE promoter.

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The optimization of preparations of competent cells for transformation ofE.coli

Cited by 61 publications

References 3 publications

The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine.

The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine.

Multiple Fates of L1 Retrotransposition Intermediates in Cultured Human Cells

2-Methylcitrate-dependent activation of the propionate catabolic operon (prpBCDE) of Salmonella enterica by the PrpR protein

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