Incomplete primer extension during in vitro DNA amplification catalyzed by Taq polymerase; exploitation for DNA sequencing.

Polyacrylamide gel electrophoresis of DNA fragments obtained by the polymerase chain reaction using Taq polymerase revealed the presence of multiple fragments shorter than the expected product. These abortive extension products were observed even when analysis by agarose gel electrophoresis showed only a single band. The production of prematurely terminated fragments can be exploited for the sequencing of PCR products if phosphorothioate groups are incorporated base specifically during the reaction in the presence of two oligonucleotide primers, one of which is 5'-32P-labeled. The addition of snake venom phosphodiesterase to the reaction mixture after completion of the amplification cycles digests each fragment from the 3'-end to a phosphorothioate group so that the sequence can be read by polyacrylamide gel electrophoresis.     

The use of nuclease P1 in sequence analysis of end group labeled RNA.

A method is described for the direct sequence analysis of 20-25 nucleotides from the termini of 5'- or 3'-end-group [32P] labeled RNA. The method involves partial endonucleolytic digestion of the labeled RNA with nuclease P1 (from Penicillium citrinum) followed by separation of the partial digestion products by two-dimensional homochromatography, the nucleotide sequence being determined by mobility shift analysis. This procedure has been applied to the sequence analysis of the terminal regions of tRNAs and of high molecular weight RNA, such as messenger RNA or viral RNA. A further application involves its use in conjunction with snake venom phosphodiesterase to determine the sequence of 5'-end group labeled oligonucleotides, containing modified bases, derived from T1 or pancreatic RNase digestion of tRNA.     

Conformational changes of yeast tRNAPhe and E. coli tRNA2Glu as indicated by different nuclease digestion patterns.

The susceptibility of yeast tRNAPhe and Escherichia coli tRNA2Glu to digestion by nucleases Tl and Sl are examined in a variety of environments, and the results are interpreted in view of the available three-dimensional structural information. Significant differences are found in the digestion pattern of the two tRNAs using the guanosine-specific Tl nuclease. In particular, differences are seen due to varying the type of salts in the environment. However, the Sl nuclease results on the two tRNAs do not differ greatly. E. coli tRNA2Glu is known to exist in two different conformations. Nuclease digestion results are presented revealing differences which make it possible to draw some inferences about the structural differences in these two conformations. In carrying out these analyses, the tRNA molecules are labeled either by putting 32P at the 5'-end of the molecular or by adding 32P-labeled pCp at the 3'-end. It is found that both yeast tRNAPhe and E. coli tRNA2Glu have modified Tl nuclease digestion patterns when pCp is added at the 3'-end of the molecule.     

32P-postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and phosphoglycolate termini.

A 32-P-postlabeling assay has been developed that permits detection of several radiogenic base and sugar lesions of DNA at the femtomole level. The technique is based on the inability of DNase I and snake venom phosphodiesterase to cleave the internucleotide phosphodiester bond immediately 5' to the site of damage so that complete digestion of irradiated DNA with these nucleases and alkaline phosphatase yields lesion-bearing "dinucleoside" monophosphates. Because these fragments contain an unmodified nucleoside at the 5'-end of each molecule, they can be readily phosphorylated by T4 polynucleotide kinase and [gamma-32P]ATP and analyzed by polyacrylamide gel electrophoresis and reverse-phase HPLC. We observed a linear induction of total damage in DNA irradiated with 5-50 Gy. Virtually no damage was detected when the DNA was irradiated in solution containing 1 M DMSO, implicating hydroxyl radicals in the formation of these lesions. Evidence for the presence of thymine glycols and phosphoglycolate groups came from (i) a comparison of the radiation-induced products with those produced by OsO4 and KMnO4 and (ii) incubation of irradiated DNA with Escherichia coli endonuclease III and exonuclease III before analysis by the postlabeling procedure. This was confirmed by comigration of the radiogenic products with chemically synthesized markers. G values of 0.0022 and 0.0105 mumol J-1 were obtained for thymine glycol and phosphoglycolate production, respectively. The identity of the 5'-nucleotide of each isolated compound was obtained by nuclease P1 digestion. This analysis of nearest-neighbor bases to thymine glycols and phosphoglycolates indicated a nonrandom interaction between radiation-induced hydroxyl radicals and DNA.     

Sequence analysis of 5''[32P] labeled mRNA and tRNA using polyacrylamide gel electrophoresis.

Sequence analysis of 5'-[32P] labeled tRNA and eukaryotic mRNA using an adaptation of a method recently described by Donis-Keller, Maxam and Gilbert for mapping guanines, adenines and pyrimidines from the 5'-end of an RNA is described. In addition, a technique utilizing two-dimensional polyacrylamide gel electrophoresis for identification of pyrimidines within a sequence is described. 5'-[32P] Labeled rabbit beta-globin mRNA and N. crassa mitochondrial initiator tRNA were partially digested with T1- RNase for cleavage at G residues, with U2-RNase for cleavage at A residues, with an extracellular RNase from B. cereus for cleavage at pyrimidine residues and with T2-RNase or with alkali for cleavage at all four residues. The 5'-[32P] labeled partial digestion products were separated according to their size, by electrophoresis in adjacent lanes of a polyacrylamide slab gel and the location of G's, A's and of pyrimidines extending 60-80 nucleotides from the 5'-end of the RNA determined. Two-dimensional polyacrylamide gel electrophoresis was used to separate the 5'-[32P] labeled fragments present in partial alkali digests of a 5'-[32P] labeled mRNA. The mobility shifts corresponding to the difference of a C residue were distinct from those corresponding to a U residue and this formed the basis of a method for distinguishing between the pyrimidines.     

Stable tRNA precursors in HeLa cells.

Two tRNA precursors were isolated from 32P-labeled or unlabeled HeLa cells by two dimensional polyacrylamide gel electrophoresis, and were sequenced. These were the precursors of tRNAMet and tRNALeu, and both contained four extra nucleotides including 5'-triphosphates at their 5'-end and nine extra nucleotides including oligo U at their 3'-end. These RNAs are the first naturally occurring tRNA precursors from higher eukaryotes whose sequences have been determined. In these molecules, several modified nucleosides such as m2G, t6A and ac4C in mature tRNAs were undermodified. Two additional hydrogen bonds were formed in the clover leaf structures of these tRNA precursors. These extra hydrogen bonds may be responsible for the stabilities of these tRNA precursors.     

The interferon-induced enzyme 2-5A synthetase adenylates tRNA

The interferon-induced enzyme 2-5A synthetase is shown to adenylate tRNA. Yeast tRNAPhe was incubated with the enzyme in the presence of double stranded RNA (in this case polyI-polyC) and ATP or deoxyATP. The reaction products were analyzed by ribonuclease T1 digestion of the tRNA, polyacrylamide gel electrophoresis and autoradiography. Using ATP, the 2-5A synthetase adds one, two or three AMP residues to the 3'-end of the tRNA whereas when dATP is replacing ATP, only one nucleotide unit is added. It is concluded that one of the mechanisms of the interferon-induced antiviral effect may be an inhibition of the translation process caused by an inactivation of tRNA molecules by a 2-5A synthetase catalyzed 2'-adenylation of the 3'-end.     

Discontinuously synthesized mRNA from Trypanosoma brucei contains the highly methylated 5' cap structure, m7GpppA*A*C(2'-O)mU*A

Trypanosoma brucei mRNA is discontinuously synthesized via the 5' addition of a "mini-exon" sequence. The mini-exon-specific cap structure was purified from a complete RNase T2 and phosphatase digest of in vivo 32P-labeled poly(A)+RNA. The purified cap structure was sequenced by a series of partial and complete enzymatic digests by nuclease P1 and venom phosphodiesterase. This approach demonstrated that the T. brucei mini-exon cap structure consists of N7-methylguanosine linked in a conventional 5'-5' triphosphate bond to five nucleotides, in the sequence A*A*C(2'-O)mU*A (asterisks denote modifications that were not fully characterized in this work). 2'-O-methylations and other modifications appear to be present in this novel cap structure, which could have a functional role in the metabolism of the mini-exon.     

In vitro methylation of yeast tRNAAsp by rat brain cortical tRNA-(adenine-1) methyltransferase.

Rat brain cortices from young animals contain large amounts of tRNA (adenine-1)methyltransferase(s). The enzyme(s) can methylate E. coli tRNA and to a lower degree yeast tRNA. Among yeast tRNA species which can be methylated we have selected tRNAAsp as a substrate for the brain enzyme. The digestions of in vitro methylated [Me-3H]-tRNAAsp with pancreatic and/or T1 ribonucleases followed by chromatographies on DEAE-cellulose, 7 M urea, suggested that the methylation of tRNAAsp occurred at a single position within the D-loop. Further digestion of the radioactive oligonucleotide recovered after DEAE-cellulose chromatography by phosphomonoesterase and snake venom phosphodiesterase enzymes followed by bidimensional thin layer chromatography enabled us to determine the location of the adenine residue which becomes methylated by the brain enzyme. This one resulted to be the adenine 14 in the D-loop of yeast tRNAAsp.     

Properties of mixed backbone oligonucleotides containing 3'-O-methyl ribonucleosides.

Oligonucleotides containing 3'-O-methyl ribonucleosides were synthesized, and their thermal stabilities and global conformations with RNA and DNA have been studied. The duplexes displayed lower T(m) values as compared to the unmodified ones, and adopted A-conformations. Furthermore, they are not a substrate for RNase H, are slightly resistant to snake venom phosphodiesterase, and are not cleaved by nuclease S 1.     

Determination of the sequences of 18 nucleotides from the 5''-end of the 1-strand and 15 nucleotides from the 5''-end of the r-strand of T7 DNA.

The sequences of 18 nucleotides from the 5'-end of the 1-strand and 15 nucleotides from the 5'-end of the r-strand of T7 bacteriophage DNA have been determined to be pT-C-T-C-A-C-A-G-T-G-T-A-C-G-T-C-C-C (1-strand) and pA-G-G-G-A-C-A-C-A-G-C-G-C-T-C (r-strand). The 5'-termini of whole DNA or separated strands were kinased using polynucleotide kinase and (gamma-32-P) rATP. The DNA was partially digested with pancreatic DNase and the fragments were separated by two dimensional electrophoresis and homochromatography. To complete the sequence, snake venom phosphodiesterase digestions of these fragments were carried out. The relationship of these sequences to the proposed cleavage of concatemeric DNA during DNA replication is discussed.     

Methionyl-tRNA synthetase induced 3'-terminal and delocalized conformational transition in tRNAfMet: steady-state fluorescence of tRNA with a single fluorophore

Five species of tRNAfMet labeled with a single fluorophore are prepared to analyze the conformational changes at the 3'-end, at dihydrouridine, and at thiouridine in tRNAfMet upon binding of methionyl-tRNA synthetase. The emission and excitation spectra, anisotropy, and solvent accessibility of the fluorophore in each of the modified tRNAfMet's are determined in the absence and presence of methionyl-tRNA synthetase. The results are consistent with the following. The probes at the 3'-end are in a nonpolar environment, mobile relative to the tRNA molecule, and fully exposed to the solvent. The probes at dihydrouridine are partially stacked over the neighboring bases, nearly immobile, and relatively inaccessible. The S8-C13 cross-linked product is rigid. Upon binding of methionyl-tRNA synthetase, the probes at the 3'-terminus become localized in a less polar environment, highly immobilized, and effectively shielded against solvent access, while the probes at dihydrouridine appear to be partially unstacked from the neighboring base and become slightly more accessible for solvent. Singlet-singlet energy transfer between the intrinsic protein fluorescence and the fluorophores in modified tRNA's was observed by sensitized emission for tRNAfMet modified at the 3'-end and for S8-C13 but not for tRNAfMet's modified at dihydrouridine. These results suggest that dihydrouridine in tRNAfMet is oriented away from methionyl-tRNA synthetase in the tRNA-enzyme complex.     

Phosphorylated threonine as the covalent intermediate in snake venom phosphodiesterase

The covalent intermediate of snake venom phosphodiesterase has been isolated using thymidine 5'-[alpha-32P]triphosphate as substrate. Phosphoamino acid analysis of the labeled enzyme demonstrates that threonine is the active site residue forming the covalent intermediate. 5'-Nucleotide phosphodiesterase is the first enzyme reported to have an active site threonine forming a covalent intermediate.     

Enzymatic analysis of isomeric trithymidylates containing ultraviolet light-induced cyclobutane pyrimidine dimers. I. Nuclease P1-mediated hydrolysis of the intradimer phosphodiester linkage

Our recent findings suggest that enzymatic hydrolysis of the intradimer phosphodiester bond may constitute the initial step in the repair of UV light-induced cyclobutane pyrimidine dimers in human cells. To examine the susceptibility of this phosphodiester linkage to enzyme-mediated hydrolysis, the trinucleotide d-Tp-TpT was UV-irradiated and the two isomeric compounds containing a cis-syn-cyclobutane dimer were isolated by high performance liquid chromatography and treated with various deoxyribonucleases. Snake venom phosphodiesterase hydrolyzed only the 3'-phosphodiester group in the 5'-isomer (d-T less than p greater than TpT) but was totally inactive toward the 3'-isomer (d-TpT less than p greater than T). In contrast, calf spleen phosphodiesterase only operated on the 3'-isomer by cleaving the 5'-internucleotide bond. Kinetic analysis revealed that (i) the activity of snake venom phosphodiesterase was unaffected by a dimer 5' to a phosphodiester linkage, (ii) the action of calf spleen phosphodiesterase was partially inhibited by a dimer 3' to a phosphodiester bond, and (iii) Escherichia coli phr B-encoded DNA photolyase reacted twice as fast with d-T less than p greater than TpT as with d-TpT less than p greater than T. Mung bean nuclease, nuclease S1, and nuclease P1 all cleaved the 5'-internucleotide linkage, but not the intradimer phosphodiester bond, in d-TpT less than p greater than T. Both phosphate groups in d-T less than p greater than TpT were refractory to mung bean nuclease or nuclease S1. Incubation of d-T less than p greater than TpT with nuclease P1, however, generated the novel compound dT less than greater than d-pTpT containing a severed intradimer phosphodiester linkage. Accordingly, nuclease P1 represents the first purified enzyme known to hydrolyze an intradimer phosphodiester linkage.     

Ribonuclease activity of rat liver perchloric acid-soluble protein, a potent inhibitor of protein synthesis.

Rat liver perchloric acid-soluble protein (L-PSP) is a potent inhibitor of cell-free protein synthesis; however, its mechanism of action is not known. Here we show that the protein is a unique ribonuclease and that this activity is responsible for the inhibition of translation. The addition of perchloric acid-soluble protein to a rabbit reticulocyte cell-free system at a concentration of 6.2 microM led to an almost complete inhibition of protein synthesis. The kinetics are unlike those of hemin-controlled inhibitor, a protein that acts at the initiation step. The inhibition appears to be due to an endoribonucleolytic activity of perchloric acid-soluble protein because L-PSP directly affects mRNA template activity and induces disaggregation of the reticulocyte polysomes into 80 S ribosomes, even in the presence of cycloheximide. These effects were observed with authentic as well as recombinant L-PSP. Analysis by thin-layer chromatography of [alpha-32P]UTP-labeled mRNA incubated with the protein showed production of the ribonucleoside 3'-monophosphates Ap, Gp, Up, and Cp, providing direct evidence that the protein is an endoribonuclease. When either 5'- or 3'-32P-labeled 5 S rRNA was the substrate, L-PSP cleaved phosphodiester bonds only in the single-stranded regions of the molecule.     

Synthesis and enzymatic characterization of P1-thio-P2-oxo trideoxynucleoside diphosphates having AZT, FdU, or dT at the 3'-position

Model compounds for oligonucleotide-prodrugs, P1-thio-P2-oxo-trideoxyribonucleoside diphosphates: d[G(s)C(o)X] and d[T(s)A(o)X] (X = AZT, FdU or dT) have been prepared, and their hydrolyses by snake venom phosphodiesterase and nuclease S1 are described.     

Modification of ribonucleic acid by vitamin B6. 1. Specific interaction of pyridoxal 5'-phosphate with transfer ribonucleic acid.

Whole tRNA preparation obtained from a human cell line (HT-29) of colon carcinoma and purified specific Escherichia coli tRNA were reacted with pyridoxal 5'-phosphate, reduced by sodium borohydride and digested with RNase A and snake venom phosphodiesterase. Two-dimensional chromatography of the pyridoxal 5'-phosphate treated tRNA digest showed that pyridoxal 5'-phosphate binds specifically to GMP, presumably in the form of a Schiff base with the exocyclic amino group of the purine. The reaction of pyridoxal 5'-phosphate with whole tRNA was competitively inhibited by N-acetoxy-2-acetylaminofluorene. This suggests that binding occurred primarily to the G20 base residue at the unpaired region of the dihydrouridine loop (Fujimura et al., 1972). The modification of tRNA by pyridoxal 5'-phosphate resulted in the inhibition, to varying extent (10-80%), of amino acid acceptance in the aminoacyl-tRNA synthetase reaction. Defects in codon recognition by pyridoxal 5'-phosphate modified amino acid acylated tRNAs in the presence of the corresponding guanine-containing polynucleotide triplets were observed by the ribosomal binding assay.     

The enzyme glutamine synthetase I of Drosophila melanogaster is associated with a modified RNA

Glutamine synthetase I (L-glutamate:ammonia ligase, ADP forming; EC 6.3.1.2) was purified from Drosophila melanogaster larvae. The complete enzyme has an apparent molecular weight of 380,000. The subunit of the active enzyme has an apparent molecular weight of 43,000 after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Routine preparations yield enzymes which have at least another polypeptide component of apparent molecular weight of 64,000. Several factors suggest that the 64,000-dalton polypeptide might be a transformation product of the 43,000-dalton subunit which occurs in association with enzyme inactivation. Distinct from its protein subunit, from pure glutamine synthetase I a material can be extracted which can be labeled with 32P-labeled gamma-ATP using polynucleotide kinase. After alkaline hydrolysis the majority of the radioactivity is recovered as 5'2' and 5'3' ribonucleotide diphosphates, and after venom phosphodiesterase digestion as 5' ribonucleotide. We therefore conclude that the native glutamine synthetase I enzyme contains, or at least is reproducibly associated with, an RNA component. Several characteristics of the labeled material indicate that the RNA is small in size and is bound to polymer molecules different from RNA.     

Nucleotide sequence of U-2 ribonucleic acid. The sequence of the 5'-terminal oligonucleotide.

The nucleotide sequences were determined for the 5'-oligonucleotides obtained by complete pancreatic RNase digestion (P25) and complete T1 RNase digestion (T27) of U-2 RNA. Complete digestion of oligonucleotide P25 with snake venom phosphodiesterase produced pm3 2,2,7G, pAm, pUm, and pCp in approximately equimolar ratios. Partial digestion of these oligonucleotides with snake venom phosphodiesterase produced -Um-C-Gp and pAm-Um, indicating the sequence of the 3'-terminal portion of the 5'-oligonucleotide is pAm-Um-C-Gp. The 5'-terminal oligonucleotide did not contain a 5'-phosphate and no free nucleoside was released from the 5' end by venom phosphodiesterase digestion. Since free pm3 2,2,7G was released by digestion with nucleotide pyrophosphatase and limited digestion with snake venom phosphodiesterase, this nucleotide is apparently linked to pAm in a pyrophosphate linkage. Mass spectrometry and thin layer chromatography in borate systems showed the ribose of m3 2, 2, 7G contains no 2'O-methyl residue. Moreover, the finding that the ribose of m3 2, 2, 7G was oxidized by NaIO4 and reduced by KB3H4 in intact U-2 RNA rules out other linkages involving the 2' and 3' positions. Accordingly, it is concluded that the structure of the 5'-terminal pentanucleotide of U-2 RNA is(see article).