Nucleotide sequence of the SrRNA gene of Entamoeba gingivalis: applications for construction of a species-specific DNA probe and phylogenetic analysis.
The small subunit ribosomal RNA (SrRNA) gene of Entamoeba gingivalis was amplified by PCR and the product of 1.9-kbp sequence was cloned into a plasmid vector pUC18. Four clones were isolated and sequenced. The insert DNAs were 1918- to 1921-bp long and A+T rich (65.5%). The four SrRNA sequences of E. gingivalis were found to be aligned with those of nine related protozoans while searching for E. gingivalis-specific sequences. A sequence of 28 oligonucleotides was chosen, chemically synthesized, and labeled with digoxigenin for use as a DNA probe. The probe thus constructed was shown to hybridize only with either the SrRNA-coding DNAs or the cells of the two E. gingivalis strains and not with those of other protozons or oral fungi tested. A representative SrRNA-sequence was analyzed and a phylogenetic tree was constructed by the neighbor-joining (NJ) method. Among the protists examined, E. gingivalis was placed next to Entamoeba histolytica as expected from the traditional taxonomy.
Yamamoto
A
A
Department of Oral Microbiology, Showa University School of Dentistry, Tokyo, Japan.
Kikuta
N
N
Hashimoto
T
T
Oyaizu
H
H
Goto
N
N
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics*; D016054:DNA, Protozoan / Q000032:analysis / Q000737:chemistry; D004747:Entamoeba / Q000145:classification / Q000235:genetics*; D017125:Genes, Protozoan; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids; D016133:Polymerase Chain Reaction; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics*; D013045:Species Specificity
D017931:DNA Primers; D015342:DNA Probes; D016054:DNA, Protozoan; D016053:RNA, Protozoan; D012335:RNA, Ribosomal