The saliva of various animals contains praline-rich proteins which may play important roles in prevention of mineral precipitation, protection of dietary and digestive proteins from interaction with tannins, and modulation of bacterial colonization on the tooth surface. Previously, we found a segment of Escherichia coli genomic DNA in bovine tooth germ mRNA encoding the proline-rich protein P-B. To examine whether E. coli genomic DNA is present in bovine genomic DNA, we constructed a plasmid library from the bovine DNA. Although results so far have failed to indicate any such presence in the bovine nucleotides examined, experiments using the polymerase chain reaction (PCR) revealed unusual amplification of nucleotides. As an initial step of the study on possible occurrence of E. coli-derived nucleotide sequence in bovine genomic DNA of P-B, we examined the structure of the PCR products generated by unexpected amplification. The determined structure of the PCR products suggested that when the two single strand chains that grow by reading the sequence of the respective template reached a hybridizable short nucleotide structure, they became hybridized and subsequent elongation was continued by reading the sequence of the counter chain that had been elongated by reading the template. It is possible that elongation of the chain was interrupted once before the completion of amplification due to the template’s palindrome region which had formed a double strand structure during the PCR process. Such an unusual amplification made possible under certain conditions in a DNA sequence may be one of the mechanisms for the genetic recombination found in our previous study.
The saliva of various animals contains proline-rich proteins which may play important roles in prevention of mineral precipitation, protection of dietary and digestive proteins from interaction with tannins, and modulation of bacterial colonization on the tooth surface [1-4]. We have isolated a proline-rich protein termed P-B from human saliva and determined its amino acid and nucleotide sequences [
The LA PCR kit, agarose (H14 TAKARA), restriction enzymes, and SYBR Green I Nucleic Acid Gel Stain were purchased from Takara Bio Inc. (Otsu, Japan); SynerGel for use with agarose, from Diversified Biotech Inc. (Boston, MA, USA); Bovine genomic DNA and Perfectly Blunt Cloning Kits with the plasmid pT7Blue vector, from Novagen, Inc. (Madison, WI, USA); the GenElute Five-minutes Plasmid Miniprep Kit and DNA standard for gel electrophoresis, from Sigma-Aldrich Japan, Inc. (Tokyo, Japan); and the Big Dye Terminator cycle sequencing kit, Applied Biosystems Japan Ltd. (Tokyo, Japan).
Primers used were as follows:
M13-47: 5’-CGCCAGGGTTTTCCCAGTCACGAC- 3’ RV-M: 5’-GAGCGGATAACAATTTCACACAGG-3’ T7: 5’-TAATACGACTCACTATAGGG-3’ pT7Blue9: 5’- GATTACGCCAAGCTCTAATA-3’ HindR: 5’-AAGCTTGCATGCCTGCAGGT-3’ B6C2985: 5’-ACCCGGGGATCCGATATCTT-3’
M13-47, RV-M, and T7 Bos BestTM sequencing primers were obtained from Takara Bio. Inc. The others were custom-made products of Invitrogen Life Technologies (Tokyo, Japan). Their positions in the plasmid B6 are indicated in
Agarose gel (1%) was used for the electrophoresis of nucleotide sequences longer than 1000 bp. For shorter sequences, gels prepared from 0.7% agarose and 1.2% Syner gel according to the manufacturer’s instructions were used. Nucleotides were visualized by staining with SYBR Green I.
Bovine genomic DNA (50 μg) was digested with EcoRV (450 U) in 250 μl of universal buffer H (Takara) and fractionated by agarose gel electrophoresis. Using a gel section containing 3000 - 4200 bp and the vector pT7Blue, we constructed a clone-pool library in 96-well plates. From the agar culture plate containing E. coli with plasmids from each well, 10 colonies each were removed from wells A1-H12 to prepare plasmid mixtures termed a1-h12, respectively.
PCR was performed using a long and accurate (LA) PCR kit suitable for amplification of long DNA according to the manufacturer’s instructions. Plasmid mixtures, cloned plasmids or restriction enzyme digests were subjected to PCR amplification (40 cycles of denaturation at 95˚C for 30 sec, annealing at 55˚C for 30 sec, and elongation at 72˚C for 1 min) in a total volume of 10 μl (1 μl each of forward and reverse primers (2 μM), 10 ng of template, 5 μl of One Shot LAPCR Mix, and water to give a final volume of 10 μl) using MyCycler Thermal Cycler (BIO-RAD, Hercules, CA, USA).
Plasmids were digested with HindIII or EcoRI at 37˚C overnight according to the manufacturer’s instructions.
B6 was cloned from well G12 according to methods described previously [5,8].
Nucleotide sequences were analyzed by the dye terminator method on an ABI PRISM 310NT Genetic Analyzer (Applied Biosystems Japan Ltd., Tokyo, Japan) as described previously [5,8].
In this study, we prepared 96 plasmid mixtures (a1- h12) expected to contain 3000 - 4200 bp inserts derived from bovine genomic DNA from 10 colonies of E. coli with the vector pT7Blue. The plasmid mixture derived from well G12 was named g12. According to the assumed plasmid structure shown in
The nucleotide sequence of ca. 150 bp termed pn-150 thus obtained was determined as shown in
To explain this unexpected result, we cloned the plasmid which gave a 300-bp PCR product using g12, and obtained a plasmid termed B6. When we digested B6 with HindIII on the basis that pT7Blue contains a single HindIII site, we obtained a band of ca. 6000 bp (
A portion of bovine genomic DNA in B6 was sequenced and registered as “Bos taurus DNA, palindrome sequence region” in NCBI GenBank (AB511281) (see
Based on the structure of B6, we explain how the HindIII digest of g12 gave the PCR product shown in
When the primer M13-47 or RV-M was used to determine the nucleotide sequence of b6-300 (see
was determined as shown in
When PCR was performed using the EcoRI digest of B6 as the template and primers B6C2985 and M13-47, a product of ca. 110 nucleotides was amplified and termed b6-110 (
nucleotide C2926(-)-T-G-G-C2922(-) and the complementary G3002(-)-A-C-C-G3006(-) as indicated in
PCR is useful for amplifying nucleotides having a sequence expected from the use of forward and reverse primers [
preformed during PCR.
Such an unusual amplification generated under certain conditions in a DNA sequence may be one of the mechanisms for the genetic recombination found in our previous study [