Overproduction of cellular and activated Ha-ras proteins by mutating a synthetic gene.

Miura, Kazunobu; Kamiya, Hiroyuki; Tominaga, Mineko; Inoue, Yasuhiko; Ikehara, Morio; Noguchi, Shigeru; Nishimura, Susumu; Ohtsuka, Eiko

doi:10.1248/cpb.35.4878

Cited by 16 publications

(6 citation statements)

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“…The cDNA of mature FdI from E. arvense was amplified by reverse transcription polymerase chain reaction (PCR) from young leaves of horsetail ( E. arvense ) and an approximately 300 bp PCR product was cloned in pTrc99A vector (Amersham Pharmacia Biotech). A DNA fragment encoding FdII from E. arvense of 93 amino acids [18] was chemically synthesized according to the method described previously [23,24]. The designed fragment (Fig.…”

Section: Methodsmentioning

confidence: 99%

A ferredoxin Arg‐Glu pair important for efficient electron transfer between ferredoxin and ferredoxin‐NADP⁺ reductase

et al. 2003

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In order to elucidate the importance of a ferredoxin (Fd) Arg-Glu pair involved in dynamic exchange from intra-to intermolecular salt bridges upon complex formation with ferredoxin-NADP + oxidoreductase (FNR), Equisetum arvense FdI and FdII were investigated as normal and the pair-lacking Fd, respectively. The FdI mutant lacking this pair was unstable and rapidly lost the [2Fe^2S] cluster. The catalytic constant (k cat ) of the electron transfer for FdI is 5.5 times that for FdII and the introduction of this pair into FdII resulted in the increase of k cat to a level comparable to that for FdI, demonstrating directly that the Arg-Glu pair is important for e⁄cient electron transfer between Fd and FNR.

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Section: Methodsmentioning

confidence: 99%

A ferredoxin Arg‐Glu pair important for efficient electron transfer between ferredoxin and ferredoxin‐NADP⁺ reductase

et al. 2003

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“…Namely, oligonucleotides U8 and U9 ( Fig. 2a) (31) were phosphorylated in the presence of ATP and T4 polynucleotide kinase. The phosphorylated oligonucleotides were mixed with oligomers U7, LI1, L12 and L13 and the mixture was annealed and treated with T4 DNA ligase.…”

Section: Synthesis Of Oligonucleotides With Oh8adementioning

confidence: 99%

8-Hydroxyadenine (7, 8-dihydro-8-Oxoadenine) induces misincorporation inin vitroDNA synthesis and mutations in NIH 3T3 cells

Kamiya¹,

Miura²,

Murata‐Kamiya³

et al. 1995

Nucl Acids Res

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134

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An oligodeoxyribonucleotide containing 8-hydroxyadenine (OH8Ade) was chemically synthesized and single- and double-stranded c-Ha-ras gene fragments with OH8Ade at the second position of codon 61 were prepared. The single-stranded ras gene fragment was used as a template for in vitro DNA synthesis with the Klenow fragment of Escherichia coli DNA polymerase I, Taq DNA polymerase, rat DNA polymerase beta and mouse DNA polymerase alpha. The former two enzymes exclusively incorporated dTMP opposite OH8Ade. The DNA polymerases alpha and beta misinserted dGMP, and dAMP and dGMP, respectively. The c-Ha-ras gene was constructed using the double-stranded ras gene fragment containing OH8Ade and was transfected into NIH 3T3 cells. The gene with OH8Ade induced focus formation, indicating that OH8Ade elicited point mutations in cells. When c-Ha-ras genes present in transformed cells were analyzed, an A-->G transition and an A-->C transversion were detected. These results indicate that OH8Ade induced misincorporation in in vitro DNA synthesis and mutations in mammalian cells.

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“…The concentration of the rai(L61/l-171)-GDP complex was determined from the molecular mass of 19 kDa and the specific absorbance of A280 = 0.5 cm'1• mg-1-mL. The full-length ras(L61/1-189) protein as bound with GDP was also prepared as described (Miura et al, 1987). Purities of these ras proteins were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.…”

mentioning

confidence: 99%

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study

Ha¹,

Ito²,

Kawai³

et al. 1989

Biochemistry

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1H NMR spectra of a GDP/GTP-binding domain of human c-Ha-ras gene product (residues 1-171) in which glutamine-61 was replaced by leucine [ras(L61/1-171) protein] were analyzed. By one-dimensional and two-dimensional homonuclear Hartmann-Hahn spectroscopy and nuclear Overhauser effect (NOE) spectroscopy of the complex of the ras(L61/1-171) protein and GDP, the ribose H1', H2', H3', and H4' proton resonances of the bound GDP were identified. The guanine H8 proton resonance of the bound GDP was identified by substituting [8-2H]GDP for GDP. The dependences of the H1' and H8 proton resonance intensities on the duration of irradiation of the H1', H2', H3', and H8 protons were measured. By numerical simulation of these time-dependent NOE profiles, the conformation of the protein-bound GDP was elucidated; the guanosine moiety takes the anti form about the N-glycosidic bond with a dihedral angle of chi = -124 +/- 2 degrees and the ribose ring takes the C2'-endo form. Such an analysis of the conformation of a guanine nucleotide as bound to a GTP-binding protein will be useful for further studies on the molecular mechanism of the conformational activation of ras proteins on ligand substitution of GDP with GTP.

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Overproduction of cellular and activated Ha-ras proteins by mutating a synthetic gene.

Cited by 16 publications

References 0 publications

A ferredoxin Arg‐Glu pair important for efficient electron transfer between ferredoxin and ferredoxin‐NADP⁺ reductase

A ferredoxin Arg‐Glu pair important for efficient electron transfer between ferredoxin and ferredoxin‐NADP⁺ reductase

8-Hydroxyadenine (7, 8-dihydro-8-Oxoadenine) induces misincorporation inin vitroDNA synthesis and mutations in NIH 3T3 cells

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study

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Overproduction of cellular and activated Ha-ras proteins by mutating a synthetic gene.

Cited by 16 publications

References 0 publications

A ferredoxin Arg‐Glu pair important for efficient electron transfer between ferredoxin and ferredoxin‐NADP+ reductase

A ferredoxin Arg‐Glu pair important for efficient electron transfer between ferredoxin and ferredoxin‐NADP+ reductase

8-Hydroxyadenine (7, 8-dihydro-8-Oxoadenine) induces misincorporation inin vitroDNA synthesis and mutations in NIH 3T3 cells

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study

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A ferredoxin Arg‐Glu pair important for efficient electron transfer between ferredoxin and ferredoxin‐NADP⁺ reductase

A ferredoxin Arg‐Glu pair important for efficient electron transfer between ferredoxin and ferredoxin‐NADP⁺ reductase