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Characterization of single strand origins of cryptic rolling-circle plasmids from Bacillus subtilis.

ABSTRACT: In this paper we describe the isolation and characterization of single strand origins (SSOs) of several cryptic Bacillus subtilis plasmids which use the rolling-circle mechanism of replication. The plasmids used in this study involved pTA1015, pTA1020, pTA1030, pTA1040, pTA1050 and pTA1060. The SSO of pTA1015 was isolated by shotgun cloning in a specially designed vector, pWM100, which has no SSO of its own. Sequence analysis revealed that the SSO of pTA1015 is almost identical to formerly described palT type SSOs. Also pTA1020 and pTA1060 were shown to contain SSOs highly homologous to palT. Using Southern hybridization with the palT of pTA1015 as a probe, the SSO of pTA1040 was cloned. Sequence analysis revealed a region of 200 bp which is 77% identical to the palT of pTA1015. The plasmids pTA1030 and pTA1050 contain an SSO which is highly homologous to the SSO of pTA1040. The majority of the SSOs of rolling-circle plasmids from B.subtilis seem to belong to two related families which we denote as palT1 (present on pTA1015, pTA1020 and pTA1060) and palT2 (present on pTA1030, pTA1040 and pTA1050). Both families of SSOs are highly efficient single-strand-conversion signals in B.subtilis.

SUBMITTER: Meijer WJ

PROVIDER: S-EPMC306728 | biostudies-other | 1995 Feb

REPOSITORIES: biostudies-other

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Characterization of single strand origins of cryptic rolling-circle plasmids from Bacillus subtilis.

Meijer W J WJ Venema G G Bron S S

Nucleic acids research 19950201 4

In this paper we describe the isolation and characterization of single strand origins (SSOs) of several cryptic Bacillus subtilis plasmids which use the rolling-circle mechanism of replication. The plasmids used in this study involved pTA1015, pTA1020, pTA1030, pTA1040, pTA1050 and pTA1060. The SSO of pTA1015 was isolated by shotgun cloning in a specially designed vector, pWM100, which has no SSO of its own. Sequence analysis revealed that the SSO of pTA1015 is almost identical to formerly descr ...[more]

PMID: 7899081

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Project description:Nucleic acid amplification is a hugely important technology for biology and medicine. While the polymerase chain reaction (PCR) has been highly useful and effective, its reliance on heating and cooling cycles places some constraints on its utility. For example, the heating step of PCR can destroy biological molecules under investigation and heat/cool cycles are not applicable in living systems. Thus, isothermal approaches to DNA and RNA amplification are under widespread study. Perhaps the simplest of these are the rolling circle approaches, including rolling circle amplification (RCA) and rolling circle transcription (RCT). In this strategy, a very small circular oligonucleotide (e.g., 25-100 nucleotides in length) acts as a template for a DNA or an RNA polymerase, producing long repeating product strands that serve as amplified copies of the circle sequence. Here we describe the early developments and studies involving circular oligonucleotides that ultimately led to the burgeoning rolling circle technologies currently under development. This Account starts with our studies on the design of circular oligonucleotides as novel DNA- and RNA-binding motifs. We describe how we developed chemical and biochemical strategies for synthesis of well-defined circular oligonucleotides having defined sequence and open (unpaired) structure, and we outline the unusual ways in which circular DNAs can interact with other nucleic acids. We proceed next to the discovery of DNA and RNA polymerase activity on these very small cyclic DNAs. DNA polymerase "rolling circle" activities were discovered concurrently in our laboratory and that of Andrew Fire. We describe the surprising efficiency of this process even on shockingly small circular DNAs, producing repeating DNAs thousands of nucleotides in length. RNA polymerase activity on circular oligonucleotides was first documented in our group in 1995; especially surprising in this case was the finding that the process occurs efficiently even without promoter sequences in the circle. We describe how one can encode cleavable sites into the product DNAs and RNAs from RCA/RCT, which can then be resolved into large quantities of almost pure oligonucleotides. Our Account then proceeds with a summary describing a broad variety of tools and methods built in many laboratories around the rolling circle concept. Among the important developments are the discovery of highly efficient DNA polymerases for RCA; the invention of exponential ("hyperbranched") RCA amplification made possible by use of a second primer; the development of the "padlock" process for detection of nucleic acids and proteins coupled with RCA; the use of circular oligonucleotides as vectors in cells to encode biologically active RNAs via RCT; and the use of small DNA circles to encode and extend human telomeres. Finally, we finish with some ideas about where the field may go in the future.

Dataset Information

Characterization of single strand origins of cryptic rolling-circle plasmids from Bacillus subtilis.

Publications

Characterization of single strand origins of cryptic rolling-circle plasmids from Bacillus subtilis.

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