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Title: Rolling circle amplification of metazoan mitochondrial genomes

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Publication Date:
Research Org.:
Joint Genome Institute (JGI)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
Resource Type:
Journal Article
Resource Relation:
Journal Name: Molecular Phylogenetics and Evolution; Journal Volume: 39; Journal Issue: 2
Country of Publication:
United States

Citation Formats

W. Brian,Simison, D.R.,Lindberg, and J.L.,Boore. Rolling circle amplification of metazoan mitochondrial genomes. United States: N. p., 2006. Web. doi:10.1016/j.ympev.2005.11.006.
W. Brian,Simison, D.R.,Lindberg, & J.L.,Boore. Rolling circle amplification of metazoan mitochondrial genomes. United States. doi:10.1016/j.ympev.2005.11.006.
W. Brian,Simison, D.R.,Lindberg, and J.L.,Boore. Mon . "Rolling circle amplification of metazoan mitochondrial genomes". United States. doi:10.1016/j.ympev.2005.11.006.
title = {Rolling circle amplification of metazoan mitochondrial genomes},
author = {W. Brian,Simison and D.R.,Lindberg and J.L.,Boore},
abstractNote = {},
doi = {10.1016/j.ympev.2005.11.006},
journal = {Molecular Phylogenetics and Evolution},
number = 2,
volume = 39,
place = {United States},
year = {Mon May 01 00:00:00 EDT 2006},
month = {Mon May 01 00:00:00 EDT 2006}
  • Here we report the successful use of rolling circle amplification (RCA) for the amplification of complete metazoan mt genomes to make a product that is amenable to high-throughput genome sequencing techniques. The benefits of RCA over PCR are many and with further development and refinement of RCA, the sequencing of organellar genomics will require far less time and effort than current long PCR approaches.
  • Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling-circle replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substratemore » DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers.« less
  • Using in situ hybridization and immunocytochemistry, the authors studied a muscle biopsy sample from a patient with Kearns-Sayre syndrome (KSS) who had a deletion of mitochondrial DNA (mtDNA) and partial deficiency of cytochrome-c oxidase. They sought a relationship between COX deficiency and abnormalities of mtDNA at the single-fiber level. COX deficiency clearly correlated with a decrease of normal mtDNA and, conversely, deleted mtDNA was more abundant in COX-deficient fibers, especially ragged-red fibers. The distribution of mtRNA has a similar pattern, suggesting that deleted mtDNA is transcribed. Immunocytochemistry showed that the nuclear DNA-encoded subunit IV of COX was present but thatmore » the mtDNA-encoded subunit II was markedly diminished in COX-deficient ragged-red fibers. Because the mtDNA deletion in this patient did not comprise the gene encoding COX subunit II, COX deficiency may have resulted from lack of translation of mtRNA encoding all three mtDNA-encoded subunits of COX.« less
  • Using quantitative PCR, we have determined that a human oocyte contains {approximately}100,000 mitochondrial genomes (mtDNAs). We have also found that some oocytes harbor measurable levels (up to 0.1%) of the so-called common deletion, an mtDNA molecule containing a 4,977-bp rearrangement that is present in high amounts in many patients with {open_quotes}sporadic{close_quotes} Kearns-Sayre syndrome (KSS) and progressive external ophthalmoplegia (PEO). This is the first demonstration that rearranged mtDNAs are present in human oocytes, and it provides experimental support for the supposition that pathogenic deletions associated with the ontogeny of sporadic KSS and PEO can be transmitted in the female germ line,more » from mother to child. The relevance of these findings to the accumulation of extremely low levels of deleted mtDNAs in both somatic and germ-line tissues during normal human aging is also discussed. 42 refs., 6 figs., 1 tab.« less
  • Although genome sequencing projects have made significant progress in the analysis of large amounts of DNA sequence information, little is known regarding primary DNA sequence organization and its determinants. Primary sequence organization was characterized for 15 different regions of the human nuclear genome (each 36 to 180 kb in length and totaling 965 kb), the complete human mitochondrial genome and several viral genomes that infect the human species. Recognition of previously unknown patterns in the organization of large DNA sequences was accomplished using chaos representation and analysis of short-sequence representation. Primary DNA sequence organization was found to have a globalmore » structure characterized by a non-random composition and order of nucleotides. Global structure was identified as a higher-order sequence organization that is independent of the functional properties and the length of the DNA sequence. Global sequence organization is similar in the human nuclear genome and viral genomes capable of integration within that genome, supporting the hypothesis of genome-type specific constraints upon primary sequence organization. Human DNA sequence organization was compared with that of four other vertebrate species and four species representing a broad phylogenetic range. Global DNA sequence organization was similar among closely-related species but different in the nuclear and mitochondrial genomes of the same species. Biases in single nucleotide and dinucleotide representation were found to be the major determinants of global structure in mitochondrial and nuclear genomes, respectively. Analysis of DNA sequence evolution must therefore incorporate both gene and genome-type specific mutational and selective constraints.« less