Nucleotide clusters in deoxyribonucleic acids. Comparison of the sequences of the large pyrimidine oligonucleotides of bacteriophages S13 and phiX174 deoxyribonucleic acids.

The large pyrimidine oligonucleotides from the DNAs of the two related bacteriophages phiX174 and S13 have been sequenced. The largest pyrimidine oligonucleotide present is unique to S13 DNA and is the undecanucleotide C5T6, sequence C-T-T-C-C-T-C-T-T-C-T. Considerable sequence homology has been found between the pyrimidine oligonucleotides of the two phage DNAs. Out of 14 oligonucleotide sequences from S13 DNA (120 bases) at least ten are identical with sequences of oligonucleotides from phiX174 DNA (92 bases) and two are closely related (17 bases), the only difference being a single thymine to cytosine transition in each sequence (a total of 107 identical bases). The pyrimidine oligonucleotides of each phage DNA show extensive internal sequence homology among each other with up to eight bases identical in sequence in pairs of different oligonucleotides. Another interesting observation is the occurrence of symmetrical sequences (true palindromes) which read the same forwards as backwards. The longest symmetrical sequence is the nonanucleotide C4T5 sequence, C-T-C-T-T-T-C-T-C, present in both S13 and phiX174 DNAs. The extensive sequence homology observed between the pyrimidine oligonucleotides of S13 and phiX174 supports the close relationship of the two phages and provides further evidence that they were derived from recent common ancestors.     

Nucleotide clusters in deoxyribonucleic acids. XII. The distribution of 5-methylcytosine in pyrimidine oligonucleotides of mouse L-cell satellite DNA and main band DNA.

Mouse L-cell DNA radioactively labeled in the 5-methylcytosine (5-MeC) residue was fractionated into satellite and main band DNA. Satellite DNA was found to contain about four times the molar concentration of 5-MeC than the main band DNA. Based on the known 5-MeC content of total L-cell DNA it was calculated that satellite DNA contains 3.5 – 4.6% 5-MeC. Both DNA fractions were depurinated and the pyrimidine oligonucleotides released separated by ionophoresis-homochromatography. In satellite DNA 5-MeC is distributed non-randomly. About 40% of the total 5-MeC is present in the sequence Pu - 5-MeC - Pu. The remainder occurs in the oligonucleotides CT, CT₃, C₂T₄, C₂T₅ and C₃T₅ only. The distribution of 5-MeC in main band DNA differs from that in satellite DNA indicating that two different fractions of the same nuclear DNA are methylated in different sequences.     

Detection of hepatitis B virus DNA in human serum samples: use of digoxigenin-labeled oligonucleotides as modified primers for the polymerase chain reaction.

Nonradioactive techniques have been used for the direct detection of hepatitis B virus DNA in human serum samples. A comparison of two different systems using digoxigenin-labeled DNA probes is presented. Furthermore, oligonucleotides containing one molecule of the hapten digoxigenin at the 5'-end were prepared and used as primers for the polymerase chain reaction. Amplified DNA can be directly analyzed with anti-digoxigenin Fab fragments labeled with alkaline phosphatase and chemiluminescent substrates.     

Amplification of the phage lambda DNA sequence by polymerase chain reaction using thermostable DNA polymerase]

The efficiency of phage DNA amplification by the method of polymerase chain reaction (PCR) with Tth DNA-polymerase was studied for optimization of PCR conditions. The effect on amplification efficiency of medium ionic strength and pH, the presence of univalent cations, detergents, gelatin, ATP, pyrophosphate, SH-reagents and ratio of concentrations of Mg and dNTPs, primers and template was studied. It has been found that a pH optimum for PCR with Tth DNA-polymerase varies from 8.5 to 9.0. An ionic strength optimum for PCR is about 0.08. The influence of univalent cations on the activity of Tth DNA-polymerase can be expressed as NH4+ greater than Na+ greater than K+. 0.01% Tween-20 significantly increases the efficiency of PCR and 0.01% gelatin inhibits it. Addition of ATP, pyrophosphate, SH-reagents to the reaction mixture did not increase the yield of PCR product. It has been also shown that for the given PCR-system an optimum Mg/dNTPs molar ratio is within the range of 1.5-2.0. An optimum concentration of each of the pair of primers for this PCR-system is about 0.3 microM. The possibility of PCR-amplification of 500-8500 b.p. DNA fragments has been demonstrated.     

Nucleotide sequence of the Escherichia coli polA gene and primary structure of DNA polymerase I

We report the nucleotide sequence of 3.2 kilobase pair region of the Escherichia coli polA gene, comprising the coding region for DNA polymerase I with about 400 base pairs of flanking sequence. The amino acid sequence for DNA polymerase I derived from our DNA sequence is largely consistent with previous protein chemical data. In the following paper, Brown et al. (Brown, W. E., Stump, K. H., and Kelley, W. S. (1982) J. Biol. Chem. 257, 1965-1972) present additional protein chemistry experiments that further confirm our sequence. Mild proteolysis of DNA polymerase I is known to produce two enzymatically active fragments (Brutlag, D., Atkinson, M. R., Setlow, P., and Kornberg, A. (1969) Biochem. Biophys. Res. Commun. 37, 982-989; Klenow, H., and Henningsen, I. (1970) Proc. Natl. Acad. Sci. U. S. A. 74, 5632-5636). We have located the site of this cleavage between residues 323 and 324 of the 928 amino acid polymerase molecule. By sequence comparison of the polA1 and wild type alleles, we have identified the polA1 mutation as a change from Trp (TGG) to amber (TAG) at residue 342.     

5'-derivatives of oligonucleotides as primers of DNA polymerization catalyzed by AMV reverse transcriptase and Klenow fragment of DNA polymerase 1.

The Km and Vmax values for d(pT)8 and its derivatives containing various 5'-end groups were estimated in the reaction of polymerization catalyzed with AMV-RT and FK. The change in affinity of modified primers was more pronounced in the case of AMV-RT than in the case of FK. Introducing in d(pT)8 of intercalators such as phenazinium, ethidium and daunomycin residues results in 2.7-, 8.7- and 11-fold increases in the primer affinity to AMV-RT, respectively. However, in the case of hemin and cholesterol derivatives the Km values were 3 and 5 times higher than those for d(pT)8. Compared to d(pT)8, the affinity of FK to all the above analogs was 2.3-3.6 times higher with the exception of cholesterol derivative to which it was 2.4-fold lower. The effect of the 5'-end residues on the Vmax values of d(pT)8 was small and ranged from 44% to 120% of that for d(pT)8. Therefore such reactive derivatives of oligonucleotides can be used as effective primers of AMV-RT and FK. Possible reasons for various effects of the 5'-end residues of the primer on its interaction with FK or AMV-RT in the presence of poly(A) are discussed.