The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers

Vieira, Jeffrey; Messing, Joachim

doi:10.1016/0378-1119(82)90015-4

Cited by 6,058 publications

(2,345 citation statements)

References 16 publications

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“…Moloney murine leukemia virus reverse transcriptase (Mo-MLV-RT) was purchased from Bacterial strains and media M. xanthus FB (DZF1) was grown at 30ЊC in CYE medium and supplemented with streptomycin (500 g ml ¹1 ) or kanamycin (40 g ml ¹1 ) when necessary (Campos et al, 1978). E. coli JM83 (Vieira and Messing, 1982) CL83 (Lerner and Inouye, 1990), SB221 (Nakamura et al, 1982) and DH5␣ (Hanahan, 1983) were used as recipient strains for transformations. E. coli BL21(DE3) was used for the T7 RNA polymerase expression system (Studier et al, 1990).…”

Section: Methodsmentioning

confidence: 99%

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Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus

Hanlon

Inouye

1997

Molecular Microbiology

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SummaryThe Myxococcus xanthus gene, pkn9, encodes a protein that contains significant homology with eukaryotic Ser/ Thr protein kinases. The pkn9 gene was singled out of a previously identified family of kinase genes by amplification techniques that displayed differences in kinase gene expression during selected periods of the M. xanthus life cycle. Pkn9 was constitutively expressed during vegetative growth and upregulated during the aggregation stage of early development. It consists of 589 amino acids, and its N-terminal 394 residues show 38% identity with both Pkn1 and Pkn2 of M. xanthus. This region also shows 29, 25 and 29% identity with myosin light-chain kinase, protein kinase C, and cAMP-dependent protein kinase, respectively. A 22-residue hydrophobic transmembrane domain separates the kinase domain from the 173-residue C-terminal domain that resides on the outside of the inner membrane. The C-terminal domain contains two sets of tandem repeats of 13 and 10 residues which have no known function. When expressed in Escherichia coli under the T7 promoter, Pkn9 was found to be phosphorylated on serine and threonine residues. Disruption of the pkn9 kinase catalytic subdomains I-III by the insertion of a kanamycin-resistance gene resulted in slightly delayed, smaller and more-crowded fruiting bodies, while spore formation was normal. Total deletion of the pkn9 gene caused severely reduced progression through development resulting in light loose mounds that become slightly more compact over time. Development progressed further at the centre than at the edge of the spot, and spore formation was significantly reduced. Twodimensional gel analysis revealed that both the disruption and the deletion of pkn9 prevented the expression of five membrane proteins (KREP9-1-4). These results suggest that the loss of Pkn9 kinase activity caused altered fruiting-body formation, the absence of the KREP9 proteins in the membrane, and reduced spore production.

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Section: Methodsmentioning

confidence: 99%

“…2). The 2.9 kb Sal I(a)-Sal I(b) fragment hybridized with the same probe as described above and was cloned into the Sal I site of pUC9 (Vieira and Messing, 1982) and designated pWAH01. The 2.9 kb Sal I(a)-Sal I(b) fragment was further subcloned and sequenced.…”

Section: Introductionmentioning

confidence: 99%

Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus

Hanlon

Inouye

1997

Molecular Microbiology

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“…Purified mtDNA and puc8 DNA (Vierra and Messing, 1983) were cleaved to completion with Hind III and ligated. The resultant ligated DNA was transformed into E. coli JM83 by the method of Maniatis et a!.…”

Section: Materials and Methods (I) Flies And Fly Stocksmentioning

confidence: 99%

Mitochondrial DNA variability in natural populations of Hawaiian Drosophila. I. Methods and levels of variability in D. silvestris and D. heteroneura populations

1986

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We describe techniques by which mitochondrial DNA (mtDNA) restriction site information can be obtained for up to 16 different restriction endonucleases on individual Hawaiian Drosophila, in particular D. silvestris and D. heteroneura. We have constructed mtDNA restriction site maps for a total of forty-eight wild caught genomes of both species, from seven major collecting sites on the island of Hawaii. Levels of variability are, in general, high in D. silvestris (p of Ewens et al., 1981 = 0.0486) and lower in D. heteroneura (p = 0.0327). Measures of population subdivision using Nei's G, indicate that about 50-60 per cent of the observed variability is due to interdemic subdivision. In accordance, populations within a species show a much lower level of variability, however some populations harbour individuals that are very divergent from the rest of their conspecifics at the same locality. We review two possible mechanisms that could explain the presence of these divergent individuals, hybridisation and the effects of stochastic branching processes.

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“…For construction of plasmids p3X1428 and pf3X1429, the Pst I m-fragment of HCMV was cloned in both orientations in pUC8 (Vieira and Messing, 1982), excised as Hind III-Sal I fragments, and recloned between the Hind III and Xho I sites of pf3X14. For construction of the recombinant viruses carrying deletions in the enhancer region, recombinant viruses C2 and C4, purified from the plasmid vector, were subjected to partial digestion with Aha II, and the partial digestion products were isolated.…”

Section: Construction Of Recombinant Dna Clonesmentioning

confidence: 99%

A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus

Boshart

Weber

Jahn

et al. 1985

Cell

1,092

648

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SummaryA strong transcription enhancer was identified in the genomic DNA (235 kb) of human cytomegalovirus (HCMV), a ubiquitous and severe pathogen of the herpesvirus group. Cotransfection of enhancerless SV40 DNA with randomly fragmented HCMV DNA yielded two SV40-HCMV recombinant viruses that had incorporated overlapping segments of HCMV DNA to substitute for the missing SV40 enhancer. Within HCMV, these enhancer sequences are located upstream of the transcription initiation site of the major immediate-early gene, between nucleotides -118 and -524. Deletion studies with the HCMV enhancer, which harbors a variety of repeated sequence motifs, show that different subsets of this enhancer can substitute for the SV40 enhancer. The HCMV enhancer, which seems to have little cell type or species preference, is severalfold more active than the SV40 enhancer. It is the strongest enhancer we have analyzed so far, a property that makes it a useful component of eukaryotic expression vectors.

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The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers

Cited by 6,058 publications

References 16 publications

Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus

Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus

Mitochondrial DNA variability in natural populations of Hawaiian Drosophila. I. Methods and levels of variability in D. silvestris and D. heteroneura populations

A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus

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