An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions.

Ohtsuka, Eiko; Matsuki, Shigeru; Ikehara, Morio; Takahashi, Y.; Matsubara, Kazuo

doi:10.1016/s0021-9258(18)89400-5

Cited by 299 publications

(40 citation statements)

References 26 publications

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“…Screening of 20,000 plaques failed to yield any reactive plaques. Therefore, the library was screened by hybridization with a family of end-labeled oligonucleotides corresponding to reverse translation of the N-terminal amino acid sequence of purified P2 from strain 5657 (39,42). A mixture of 16 oligonucleotides was used (Fig.…”

Section: Resultsmentioning

confidence: 99%

Molecular analysis of the P2 porin protein of nontypeable Haemophilus influenzae

Sikkema

Murphy

1992

Infect Immun

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The P2 porin protein is the most abundant outer membrane protein (OMP) of nontypeable Haemophilus influenzae (NTHI) and shows extensive antigenic heterogeneity among strains. To study the molecular basis of this heterogeneity, the DNA sequences of the genes encoding the P2 proteins of three unrelated strains of NTHI were determined, and restriction fragment length polymorphisms around the P2 genes of 35 strains were analyzed. The deduced amino acid sequences of the P2 genes from the three strains of NTHI revealed four major (12 to 35 amino acids long) and several smaller (2 to 7 amino acids) hypervariable regions in each protein. The major variations occurred in identical portions of the genes, and these regions showed a high antigenic index and surface exposure probability in computer modeling analysis. Differences in the molecular mass of the P2 protein correlate with differences in the size of the variable region in each strain. Oligonucleotide primers suitable for amplification of the P2 genes by polymerase chain reaction were developed. Restriction fragment length polymorphism analysis showed marked heterogeneity in and around the ompP2 locus of 35 NTHI strains. These results contrast with the high degree of conservation of the P2 genes in H. influenzae type b strains. We conclude that the molecular mass and antigenic heterogeneity of the P2 molecule of NTHI is due to variations in gene sequence that are clustered primarily in four large hypervariable regions of the gene.

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Section: Resultsmentioning

confidence: 99%

Molecular analysis of the P2 porin protein of nontypeable Haemophilus influenzae

Sikkema

Murphy

1992

Infect Immun

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“…The 16S rRNA gene was analysed as described previously (Sakamoto et al, 2002). The partial hsp60 gene was amplified by PCR using primers H729 (59-CGC CAG GGT TTT CCC AGT CAC GAC GAI III GCI GGI GAY GGI ACI ACI AC-39) and H730 (59-AGC GGA TAA CAA TTT CAC ACA GGA YKI YKI TCI CCR AAI CCI GGI GCY TT-39) (Brousseau et al, 2001); inosine was used to reduce the degeneracy of the sequences (Ohtsuka et al, 1985). These primers were derived from the previously described H279A and H280A primers (Goh et al, 1997) by addition of the sequences for commercially available M13 24 bp sequencing primers (underlined nucleotides).…”

Section: Methodsmentioning

confidence: 99%

hsp60 and 16S rRNA gene sequence relationships among species of the genus Bacteroides with the finding that Bacteroides suis and Bacteroides tectus are heterotypic synonyms of Bacteroides pyogenes

Sakamoto

Suzuki

Benno

2010

International Journal of Systematic and Evolutionary Microbiology

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hsp60 gene sequences were determined for members of the genus Bacteroides and sequence similarities were compared with those obtained for the 16S rRNA gene. Among the 29 Bacteroides type strains, the mean sequence similarity of the hsp60 gene (84.5 %) was significantly less than that of the 16S rRNA gene (90.7 %), indicating a high discriminatory power of the hsp60 gene. Species of the genus Bacteroides were differentiated well by hsp60 gene sequence analysis, except for Bacteroides pyogenes JCM 6294 T , Bacteroides suis JCM 6292 T and Bacteroides tectus JCM 10003 T . The hsp60 gene sequence analysis and the levels of DNA-DNA relatedness observed demonstrated that these three type strains are a single species. Consequently, B. suis and B. tectus are heterotypic synonyms of B. pyogenes. This study suggests that the hsp60 gene is an alternative phylogenetic marker for the classification of species of the genus Bacteroides. INTRODUCTIONOrganisms of the genus Bacteroides are part of the indigenous microbiota of human and animal gastrointestinal tracts, but different species in this group are commonly associated with a variety of human and animal infections. The genus Bacteroides is important in clinical bacteriology. Although species of the genus Bacteroides are isolated frequently from hospitalized patients, it can be difficult to identify every clinical isolate to the species level. In the past, because of poor definition of the genus, more than 50 species of the genus Bacteroides have been included in this group. The taxonomy of the genus Bacteroides has undergone significant changes in the last two decades (Dewhirst et al., 1990;Moore & Moore, 1994;Rautio et al., 2003;Sakamoto et al., 2002Sakamoto et al., , 2007Sakamoto & Benno, 2006;Shah & Collins, 1988. It has also been reported that 16S rRNA gene sequencing is useful for the identification of isolates of clinically significant species of the genus Bacteroides (Song et al., 2005).To date, a multilocus sequence typing (MLST) approach has been widely used to infer the phylogeny of organisms (Alexandre et al., 2008; Itoh et al., 2006;Shah et al., 2007;Yamamoto & Harayama, 1995). However, only limited information has been accumulated for species of the genus Bacteroides. It has been reported that housekeeping gene sequences, e.g. rpoB, could be used to clarify interspecies phylogenetic relationships within the genus Bacteroides (Ko et al., 2007). The ad hoc committee for the re-evaluation of the species definition in bacteriology has recommended evaluation of protein-coding gene sequence analysis for its applicability to genomically circumscribe the taxon species and differentiate it from neighbouring species detected by 16S rRNA gene sequence analysis (Stackebrandt et al., 2002).Hsp60, also known as Cpn60 or GroEL, is a member of the heat-shock protein (Hsp) family. The hsp60 gene has been shown to be more discriminative than the 16S rRNA gene for the identification of Streptococcus suis serotypes (Brousseau et al., 2001) and species of the genus Prevotella (Saka...

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“…The most extensively studied example is 2′-deoxyinosine (S.26; Fig. 1.4.8), which has been in use as a putative universal nucleoside in oligonucleotide probes and primers since 1985 (Ohtsuka et al, 1985). Structural studies on deoxyinosine-modified oligonucleotides show that dI can base pair to dC, dA (Corfield et al, 1987;Uesugi et al, 1987), T (Cruse et al, 1989;Carbonnaux et al, 1990), and dG (Oda et al, 1991).…”

Section: Polar Hydrogen Bonding Nucleobase Analogsmentioning

confidence: 99%

Unnatural Nucleosides with Unusual Base Pairing Properties

Bergstrom

2001

CP Nucleic Acid Chemistry

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Synthetic modified nucleosides designed to pair in unusual ways with natural nucleobases have many potential applications in nucleic acid biochemistry. This overview lays the foundation for future protocol units on synthesis and application of unnatural bases, with particular emphasis on unnatural base analogs that mimic natural bases in size, shape, and biochemical processing. Topics covered including base pairs with alternative H-bonding schemes, hydrophobic base pairs, degenerate bases, universal nucleosides, and triplex constituents.

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An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions.

Cited by 299 publications

References 26 publications

Molecular analysis of the P2 porin protein of nontypeable Haemophilus influenzae

Molecular analysis of the P2 porin protein of nontypeable Haemophilus influenzae

hsp60 and 16S rRNA gene sequence relationships among species of the genus Bacteroides with the finding that Bacteroides suis and Bacteroides tectus are heterotypic synonyms of Bacteroides pyogenes

Unnatural Nucleosides with Unusual Base Pairing Properties

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