Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli.

Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli. Research Abstract Details 

Research Abstract Table of Contents

Jump to the:

Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli. Abstract Text:

M Hamada,S Fujita,H Kise,Y Jigami,K Taira,

If ribozymes are to be exploited in vivo, it is necessary to select ribozymes that are functional in the intracellular environment. Ribozymes selected in the intracellular environment should retain their function in vivo as well as in vitro. We have devised a novel system for selection of active ribozymes from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In our first attempt, a sequence encoding either an active or an inactive ribozyme was connected upstream of the gene for DHFR. Each plasmid was designed such that, when the ribozyme was active, the ribozyme would cleave the target site and, as a result, the rate of production of DHFR would be high enough to endow resistance to trimethoprim (TMP). However, a critical defect may be associated with introduction of a ribozyme upstream of the DHFR gene because, during actual screening for active ribozymes on the 5' side from a pool of random sequences, there is the danger of selecting sequences that are not related to the activity of ribozymes. Indeed, some upstream linker sequences affected the level of expression of the DHFR protein and, as a result, the resistance of Escherichia coli to TMP. Therefore, we newly constructed a 3'-connected ribozyme system, and activities in vivo of 5'-connected and 3'-connected ribozymes were compared. We found that the cleavage efficiencies in vivo were nearly identical for the two types of ribozyme, 24% for the 5'-side ribozyme and 23% for the 3'-side ribozyme, indicating that polysomes did not seem to inhibit the action of the 3'-connected ribozyme. In both cases, when cells were transformed with a 1 : 1 mixture of active and inactive ribozyme-coding plasmids, it was mainly the cells that harbored the active ribozyme that survived in the presence of TMP.

Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli. Publishing Authors By Initials

M Hamada,S Fujita,H Kise,Y Jigami,K Taira,

For similar enzymes and coenzymes: enzymes: oxidoreductases: oxidoreductases acting on ch-nh group donors: tetrahydrofolate dehydrogenase research abstracts see: enzymes and coenzymes: enzymes: oxidoreductases: oxidoreductases acting on ch-nh group donors: tetrahydrofolate dehydrogenase research

PUBMED ID PMID:

MEDLINE DATE:

Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: Journal of biochemistry

VOLUME: 123

Page Numbers: 684-92

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: Apr

YEAR: 1998

Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 376600

Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli. Keywords Mesh Terms:

KEYWORDS: Tetrahydrofolate Dehydrogenase

MESH TERMS: genetics

Chemical & Substance for Abstract: Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli. Information

Substance Name: Tetrahydrofolate Dehydrogenase

Registry Number: EC 1.5.1.3

Grant and Affiliation Information for Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli.

AFFILIATION: National Institute for Advanced Interdisciplinary Research, Tsukuba Science City 305-8562.

Country: JAPAN

AGENCY:

GRANT:

ACRONYM:

MEDLINETA: J Biochem

REFSOURCE:

DATABASENAME:

ACCESSION NUMBER:

Number Hits: 0

Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase DHFR as a selective marker in Escherichia coli Related Publications

• A normalized lexical lookup approach to identifying UMLS concepts in free text.
• Broadband quasi-phase-matched second-harmonic generation in MgO-doped periodically poled LiNbO_3 at the communications band.
• Cardiac sympathetic denervation in bradykinesia-dominant Parkinson's disease.
• Cleavage of an inaccessible site by the maxizyme with two independent binding arms: an alternative approach to the recruitment of RNA helicases.
• Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli.
• Functional analysis of the individual oligosaccharide chains of sendai virus fusion protein.
• Gene expression pattern analysis of the tight junction protein, Claudin, in the early morphogenesis of Xenopus embryos.
• High-energy, narrow-bandwidth periodically poled Mg-doped LiNbO3 optical parametric oscillator with a volume Bragg grating.
• Involvement of angiotensin-(1-7) in the hypothalamic hypotensive effect of captopril in sinoaortic denervated rats.
• Kinetics of interactions of sendai virus envelope glycoproteins, F and HN, with endoplasmic reticulum-resident molecular chaperones, BiP, calnexin, and calreticulin.
• Laser emission in highly doped Nd:YAG crystals under (4)F(5/2) and (4)F(3/2) pumping.
• MR imaging of olfactory bulbs and tracts.
• Ribozymes: applications to functional analysis and gene discovery.
• Ribozymes: from mechanistic studies to applications in vivo.
• The roles of individual cysteine residues of Sendai virus fusion protein in intracellular transport.
• [Acute purulent pericarditis followed by early constriction--report of three cases]
• [Cardiac valve replacement in patients over 60 years of age]
• [Cold blood cardioplegia in heart valve surgery with prolonged aortic cross-clamping]
• [Cold blood cardioplegia: five-year experience with 400 consecutive patients]
• [Disseminated intravascular coagulopathy associated with thromboexclusion for a DeBakey IIIb dissecting aneurysm: a case report]
• [Effect of the vein bypass graft diameter on late graft patency and flow volume]
• [Intestinal infarction without mesenteric vascular occlusion after open heart surgery: a case report]
• [Intravascular hemolysis after prosthetic valve replacement]
• [Multiple-valve surgery: a comparison with single valve surgery]
• [Primary malignant cardiac tumors: a report of four cases]
• [Severe mechanical hemolysis after valve replacement with St. Jude Medical valve--a report of five cases]
• [Surgical treatment of infective endocarditis]
• [Surgical treatment of tricuspid regurgitation in combined valvular diseases]
• [The optional usage of blood cardioplegia through eleven-year experience of open-heart surgery]
• [Valve surgery in aortic regurgitation and evaluation of results]