Isolation and characterization of ribosomal RNA genes from opisthorchis viverrini
Issued Date
2023
Copyright Date
1991
Language
eng
File Type
application/pdf
No. of Pages/File Size
ix, 145 leaves : ill.
Access Rights
restricted access
Rights Holder(s)
Mahidol University
Bibliographic Citation
Thesis (Ph.D. (Microbiology))--Mahidol University, 1991
Suggested Citation
Sunee Korbsrisate Isolation and characterization of ribosomal RNA genes from opisthorchis viverrini. Thesis (Ph.D. (Microbiology))--Mahidol University, 1991. Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/89667
Title
Isolation and characterization of ribosomal RNA genes from opisthorchis viverrini
Alternative Title(s)
การแยกและวิเคราะห์ไรโบโซมยีนส์ของพยาธิใบไม้ตับ
Author(s)
Abstract
The ribosomal DNA (rDNA) of the liver fluke Opisthorchis viverrini has been cloned and characterized by Southern blot analysis and sequencing. The results demonstrated that the total length of this unit was approximately 13 kb, containing 4.2 kb of large subunit (LSU) DNA, 2.0 kb of small subunit (SSU) rDNA, 1.0 kb of transcribed spacer (TS) DNA and 5.8 kb of non-transcribed spacer + external-transcribed spacer (NTS+ETS) regions. The 5.8S rRNA gene was also found to localize between the LSU and SSU rRNA genes (TS). The rRNA genes in this unit are organized as 5NTS+ETS - SSU rRNA - 5.8S - LSU rRNA NTS+ETS 3 and are tandemly repeated in the genome of O. viverrini. Examination of the NTS region between different rDNA units showed variation in restriction sites rather than in length. There are approximately 1,230 copies (6.1%) of the rDNA unit per haploid genome as determined by hybridization of the rDNA plasmid to restriction endonuclease digested of the O. viverrini genomic DNA. At the RNA level, the LSU rRNA of 0. viverrini and F. gigantica has been shown to contain a hidden break. The molecule consisted of two fragments (28S and 28S ) co-migrated with the SSU rRNA when electrophoresis was carried out under denaturing conditions, resulting in 1 fragment at position 19S when compared with other organisms examined. In addition to the study of organization of the rRNA gene and the nature of rRNA, nucleotide sequence 381 bp at the 5terminal of LSU rRNA gene was also determined. Taken together with those of species previously reported by other investigators, the nucleated sequence of 0. viverrini was used to construct the phylogenetic tree. As to be expected, the data obtained clearly illustrated that 0. viverrini is more closely related to its close relative S. mansoni than to H. diminuta but more distantly related to nematode. This demonstrated the usefulness of rRNA gene sequence for the systematic phylogenetic classification of parasitic animals. Furthermore, the characterization of 0. wiverrini SSU rRNA gene was also done at the nucleotide (nt) level. The result demonstrated that the coding sequence is 1,992 nt long and contains 50.94% G+C content. Comparisons of the nucleotide sequence with those of Caenorhabditis elegans, Trypanosoma brucei, Dictyostelium discoideum, Euglena gracilis and human indicated that the 5and 3end sequences of SSU rRNA gene are highly conserved. The 5end and 3 sequence of SSU rRNA in most eukaryotes are 5 -A-CT GGTTG ATCCT GCCAG 3 and 5 GGTGA ACCTG CGGM GGATC AT 3, respectively. Another import ant feature is that the 0. viverrini SSU rRNA gene is made up of alternated constant and variable regions that are similar to the gene organization of other eukaryotes. There are 8 variable regions (V1-V9, V6 is missing in eukaryotic SSU rRNA) in the SSU rRNA gene, in which V2, V4, V5, V7 and V8 account for the length and nucleotide difference of SSU gene among organisms examined. However, comparison of the nt sequence demonstrated that there is an unexpected high degree of sequence homology between the 0. viverrini and the human rRNA genes. This was unexpected in view of a marked difference between the two organisms on evolutionary tree. It should be interesting to know whether the SSU rRNA gene of other trematodes also shares this high degree of homology with the human gene. It is important to note that DNA probe has made great strides in providing a new method for identifying parasites and their vectors. The technique is suitable for clinicians and field epidemiologists. Using rRNA sequence to develop probes has been performed in many parasites. When nt sequence of 0. viverrini SSU rRNA gene and 5 terminal LSU rRNA gene sequence were compared with other organisms examined, the variable regions (V2, V4 and V7) of SSU rRNA and nucleotide positions 273-286 of 0. viverrini LSU rRNA gene have been observed to contain sequence specific to 0. viverrini. Such sequence could be used to construct specific diagnostic probes for this organism
Degree Name
Doctor of Philosophy
Degree Level
Doctoral Degree
Degree Department
Faculty of Science
Degree Discipline
Microbiology
Degree Grantor(s)
Mahidol University