A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors

Holton, Timothy A; Graham, Michael W.

doi:10.1093/nar/19.5.1156

Cited by 362 publications

(164 citation statements)

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“…This mutation was introduced by PCR amplification of pDG2 with oligonucleotides WS99 (5Ј-TA-XhoI-TAGGATGTTAATACTCCGAAG-3Ј) and WS22 (5Ј-TT-BglII-TATTT-CTAATGTTTGTCATGC-3Ј), which anneal to nucleotides (nt) -240 to -227, and nt ϩ 23 to ϩ 4 relative to the 5Ј end of the petD mRNA, respectively (Sakamoto et al, 1993). The PCR product was then ligated to ddT-tailed pSKϩ (Holton and Graham, 1991), and this plasmid was designated p9922. Plasmid p9922 was then digested with XhoI and BglII, and the 273 nt promoter-containing fragment was ligated to the large XhoI-BglII fragment of pDG2; the resulting plasmid was designated pDGS.…”

Section: Plasmidsmentioning

confidence: 99%

In vivo evidence for 5′→3′ exoribonuclease degradation of an unstable chloroplast mRNA

et al. 1998

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SummaryThe acetate-requiring Chlamydomonas reinhardtii nuclear mutant F16 harbors the mutation mcd1-1 and fails to accumulate the cytochrome b6/f complex. The primary defect of mcd1-1 was determined to be the instability of petD mRNA, which encodes subunit IV of the complex. Chimeric reporter genes introduced by chloroplast transformation demonstrated that the determinant of petD mRNA instability in the mcd1-1 background is located in the 5Ј untranslated region (UTR). However, when this 5Ј UTR was present downstream of other sequences in dicistronic or chimeric transcripts, the RNAs were no longer destabilized in the mcd1-1 background. Together, these results suggest that the 5Ј end of the petD 5Ј UTR interacts with the MCD1 product. The insertion of a polyguanosine sequence into the petD 5Ј UTR fused to a reporter gene allowed accumulation of the reporter gene transcript in the mutant background. Since polyguanosine forms a structure that is known to impede exonucleases, these data provide in vivo evidence that petD mRNA can be degraded by 5Ј→3Ј exoribonuclease activity. Furthermore, the data support a model in which protein binding to the petD 5Ј UTR protects the mRNA from 5Ј→3Ј degradation in wild-type cells.

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Section: Plasmidsmentioning

confidence: 99%

In vivo evidence for 5′→3′ exoribonuclease degradation of an unstable chloroplast mRNA

et al. 1998

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“…Initially PCR products were generated from the 3' half of TAP] (1300-1250-bp coding sequence) cDNAs prepared from normal controls and diabetic patients. The products were directly ligated into T-tailed pBluescript (34). Variants were confirmed by direct sequencing of PCR products from both cDNA and genomic DNA.…”

mentioning

confidence: 99%

TAP1 alleles in insulin-dependent diabetes mellitus: a newly defined centromeric boundary of disease susceptibility.

Jackson

Capra

1993

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

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It has been previously demonstrated that individuals with certain DR alleles have an increased relative risk of developing insulin-dependent diabetes mellitus (IDDM). The disease association is even stronger with certain DQ alleles but there is little association with DP providing a boundary of disease association to the 430 kb between DQ and DP. The recently described TAP (transporter associated with antigen processing) genes have been mapped approximately miiidway 6ietween DP and DQ. Therefore, it was of interest to determine if any TAP alleles were associated with IDDM. In addition to the alleles of TAP] that have been described, others were identified during this study. Diabetics and normal controls were screened for TAPI using single-stranded conformational polymorphism and relative risk was determined. In the same population group we have studied extensively in the past, we found a higher association of a TAPI allele with IDDM than with any single HLA-DP allele but the risk was lower than with HLA-DQBI*0302. These data provide new limits for IDDM susceptibility to the 190-kb interval between TAP) and HLA-DQBI.The recently identified TAP1 and TAP2 (transporter associated with antigen processing) proteins share homology with such proteins as MADR (multi-drug resistance) protein and CFTR (cystic fibrosis tra_nsmeTmbranie conductance regulator) profein ana other members of the ABC superfaimily of proteins, which transport materials across membranes (1-4). Although the actual function of the TAP genes is in question, indirect evidence has been presented to suggest that TAP proteins function as a heterodimer to transport peptides across the endoplasmic reticulum membrane, where the peptides associate with assembling class

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“…The amplified fragments were cloned into the vector pBluescript SK(-) and the cDNA sequence determined. Cloning into T-tailed pBluescript SK(-) vector was based on the procedure of Holton and Graham (31). Transformations using the pBluescript vector into DH5a competent E. coli cells plated on Luria Broth, ampicillin, 5-bromo-4-chloro-3-indolyl ␤-Dgalactopyranoside (X-gal) agar were performed as described (32).…”

Section: Methodsmentioning

confidence: 99%

Regulation of the Meiosis-inhibited Protein Kinase, a p38MAPK Isoform, During Meiosis and Following Fertilization of Seastar Oocytes

Morrison¹,

Yee²,

Paddon³

et al. 2000

Journal of Biological Chemistry

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A p38MAPK homolog Mipk (meiosis-inhibited protein kinase) was cloned from seastar oocytes. This 40-kDa protein shares approximately 65% amino acid identity with mammalian p38-␣ isoforms. Mipk was one of the major tyrosine-phosphorylated proteins in immature oocytes arrested at the G 2 /M transition of meiosis I. The tyrosine phosphorylation of Mipk was increased in response to anisomycin, heat, and osmotic shock of oocytes. During 1-methyladenine-induced oocyte maturation, Mipk underwent tyrosine dephosphorylation and remained dephosphorylated in mature oocytes and during the early mitotic cell divisions until approximately 12 h after fertilization. At the time of differentiation and acquisition of G phases in the developing embryos, Mipk was rephosphorylated on tyrosine. In oocytes that were microinjected with Mipk antisense oligonucleotides and subsequently were allowed to mature and become fertilized, differentiation was blocked. Because MipK antisense oligonucleotides and a dominant-negative (K62R)Mipk when microinjected into immature oocytes failed to induce germinal vesicle breakdown, inhibition of Mipk function was not sufficient by itself to cause oocyte maturation. These findings point to a putative role for Mipk in cell cycle control as a G-phase-promoting factor.

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A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors

Cited by 362 publications

References 1 publication

In vivo evidence for 5′→3′ exoribonuclease degradation of an unstable chloroplast mRNA

In vivo evidence for 5′→3′ exoribonuclease degradation of an unstable chloroplast mRNA

TAP1 alleles in insulin-dependent diabetes mellitus: a newly defined centromeric boundary of disease susceptibility.

Regulation of the Meiosis-inhibited Protein Kinase, a p38MAPK Isoform, During Meiosis and Following Fertilization of Seastar Oocytes

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