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Biomedical subjects

K Randerath

Publications and source records attributed to K Randerath.

At least 181 records · Page 10Linked to original sources
PubMed1977

Use of specific endonuclease cleavage in RNA sequencing-an enzymic method for distinguishing between cytidine and uridine residues.

The extracellular ribonuclease I of the common slime mold physarum polycephalum (RNase Phy1), which has recently been purified to homogeneity, has been used to distinguish between C and U residues in 3'-end-labeled oligoribonucleotides. As shown by Bargetzi and coworkers, this enzyme exhibits strong cleavage preference for U-N over C-N and N-C over N-U bonds. In the present paper, conditions are being detailed, which enable one to deduce the sequences of rather large, pyrimidine-rich, terminally labeled oligonucleotides by partial digestion with RNases U2, A, and Phy1, followed by resolution of the cleavage products by size. The techniques described in this and a previous communication provide a direct means for identifying A, G, C, and U residues in end-labeled polyribonucleotides.

Base Sequence↗
PubMed1977

Use of specific endonuclease cleavage in RNA sequencing.

Nonradioactive RNA fragments may be sequenced by incorporation of (3H)-label into 3'-terminal positions, controlled digestion with specific ribonucleases, and separation according to size of the digestion products on polyethyleneimine- (PEI-) cellulose thin layers. This combination of techniques allows one to measure accurately distances of specific cleavage sites from the labeled terminal positions. The cleavage specificities of RNases T1, U2, and A are utilized to identify the positions of G, A, and pyrimidine residues respectively. C and U may be distinguished by mobility differences on PEI-cellulose thin layers at ph 2.6. The procedure is simple, rapid, and highly sensitive; as little as 0.5 - 1 microgram of a RNA of the size of tRNA will be needed to sequence all fragments in a complete RNase digest.

Base Sequence↗
PubMed1976

Effects of 5-fluorouridine on modified nucleosides in mouse liver transfer RNA.

Administration of the pyrimidine antimetabolite, 5-fluorouridine, to mice was found to cause a marked specific reduction of the amounts of 5-methyluridine, pseudouridine, and dihydrouridine but not of 3-(3-amino-3-carboxypropyl)uridine in tRNA from the livers of the treated animals. The data presented indicate that this effect is not simply due to the incorporation of 5-fluorouridine into tRNA; the drug appears to interfere directly with the enzymic reactions involved in the modification of the 5-position of uridine. 5-Fluorouridine was found to have no effect on the modification of adenosine, guanosine, and cytidine in mouse liver tRNA.

Animals↗
PubMed1976

Base composition studies on mitochondrial 4 S RNA from rat liver and Morris hepatomas 5123D and 7777.

The major and modified base composition of mitochondrial 4 S RNA from rat liver and from Morris hepatomas 5123D and 7777 has been determined for 16 constituents using a chemical tritium-derivative method. The base composition of these mitochondrial 4 S RNA preparations was compared with the base composition of cytoplasmic and bacterial (Escherichia coli B and Bacillus subtilis) 4-S RNAs. The results of these studies are: 1. When compared with cytoplasmic 4 S RNA, the liver and hepatoma mitochondrial 4-S RNAs are characterized by high (A + U)/(G + C) ratios and low overall degrees of base methylation and modification. 2. The mammalian mitochondrial 4-S RNAs are qualitatively even more different from the bacterial 4-S RNAs than from their cytoplasmic counterparts. Thus, several modified constituents found in both cytoplasmic and mitochondrial 4 S RNA are absent from the bacterial 4-S RNAs. 3. Mitochondrial 4S RNA from both hepatomas was found to be under-methylated and undermodified when compared with normal liver mitochondrial 4S RNA. This trend is more pronounced for the rapidly growing hepatoma 7777 (i.e., 17% undermethylation) than for the more slowly growing hepatoma 5123D (i.e., 8% undermethylation). These findings are discussed in relationship to (1) results of other authors on composition of mitochondrial 4 S RNA, (2) special features of structure and biosynthesis of mitochondrial 4 S RNA, (3) the possible evolutionary origin of mitochondria and (4) the possible role played by aberrant mitochondrial 4 S RNA in altered mitochondrial protein synthesis in tumors.

Animals↗
PubMed1976

A double-labeling procedure for sequence analysis of picomole amounts of nonradioactive RNA fragments.

A double-labeling procedure for sequence analysis of nonradioactive polyribonucleotides is detailed, which is based on controlled endonucleolytic degradation of 3'-terminally (3H)-labeled oligonucleotide-(3') dialcohols and 5"-terminal analysis of the partial (3H)-labeled fragments following their separation according to chain length by polyethyleneimine- (PEI-)cellulose TLC and detection by fluorography. Undesired nonradioactive partial digestion products are eliminated by periodate oxidation. The 5'-termini are assayed by enzymic incorporation of (32p)-label into the isolated fragments, enzymic release of (32p)-labeled nucleoside-(5') monophosphates, two-dimensional PEI-cellulose chromatography, and autoradiography. Using this procedure, as little as 0.1 - 0.3 A260 unit of tRNA is needed to sequence all fragments in complete ribonuclease T1 and A digests, whereas radioactive derivative methods previously described by us1-4 required 4 - 6 A260 units.

Base Sequence↗
PubMed1976

An improved separation procedure for nucleoside monophosphates on polyethyleneimine-(PEI-)cellulose thin layers.

A procedure is described for the two-dimensional separation of the 4 major and 16 modified nucleoside-(5') monophosphates on anion-exchange thin layers of polyethyleneimine- (PEI-)cellulose. The method, which is simple and less time-consuming than existing partition chromatographic methods, may be used for the identification of 5'-termini of RNA and RNA fragments.

Chromatography, Ion Exchange↗
PubMed1976

Effects of DL-ethionine on mouse liver tRNA base composition.

Treatment of mice with DL-ethionine and adenine causes a reduction of all methylated bases of liver tRNA. This effect is dose-dependent and specific for the methylated bases. Individual methylated components are affected to different extents, m22G being most sensitive to inhibition.

Adenine↗
PubMed1974

Tritium sequence analysis of oligoribonucleotides: a combination of post-labeling and thin-layer chromatographic techniques for the analysis of partial snake venom phosphodiesterase digests.

A tritium derivative method for sequence analysis of polyribonucleotides is detailed, which is based on borotritide reduction of oligonucleotide-3' dialdehydes generated by controlled snake venom phosphodiesterase/alkaline phosphomonoesterase digestion and periodate treatment of time point aliquots of the incubation mixture. Radioactive oligonucleotide derivatives are resolved according to chain length by PEI-cellulose(1) anion-exchange TLC and their 3'-termini identified by techniques described in the preceding paper of this series(2). The present tritium derivative method is compared with the one described previously(2).

Borohydrides↗
PubMed1974

Sequence analysis of nonradioactive RNA fragments by periodate-phosphatase digestion and chemical tritium labeling: characterization of large oligonucleotides and oligonucleotides containing modified nucleosides.

A tritium derivative method for sequence analysis of polyribonucleotides is detailed, which is based on borotritide reduction of oligonucleotide dialdehydes generated by treatment of polyribonucleotides with alkaline phosphatase and excess periodate at pH 8 (borate buffer; no primary amine present in the reaction mixture). While neither phosphatase nor periodate possess any intrinsic exonuclease activity their combination mimics an RNA-specific exonuclease ("pseudo-exonuclease"). Procedures are described for separation and characterization of tritiumlabeled oligonucleotide derivatives. The sequence is deduced by identification of labeled 3'-termini following separation of the reduced nucleotide intermediates according to chain length. The sensitivity of the method is indicated by the fact that as little as 0.01 O.D.260 unit of a nonradioactive decanucleotide is sufficient for sequence determination.

Alkaline Phosphatase↗