Development of an efficient PCR-based diagnosis protocol for the identification of the pinewood nematode, Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae)

Nematology ◽  
2004 ◽  
Vol 6 (2) ◽  
pp. 279-285 ◽  
Author(s):  
Jae Soon Kang ◽  
Kwang Sik Choi ◽  
Sang Chul Shin ◽  
Il Sung Moon ◽  
Sang Gil Lee ◽  
...  
Keyword(s):  
Intergenic Spacer ◽  
Nematode Species ◽  
Bursaphelenchus Xylophilus ◽  
5S Rrna ◽  
Rrna Gene ◽  
Pinewood Nematode ◽  
Pine Wood ◽  
5S Rrna Gene ◽  
Primer Sets ◽  
Species Specific

Abstract Pine wood wilt disease caused by the pine wood nematode, Bursaphelenchus xylophilus , has been a serious problem in the southern regions of Korea. Efficient diagnosis of B. xylophilus from infected pine wood specimens is critical for the management of this pest. Traditional microscopic examination often results in an erroneous identification because a closely related non-pathogenic species, B. mucronatus, has a great degree of morphological similarity to B. xylophilus. In an attempt to search for reliable molecular markers for the discrimination of these species, we have cloned the 5S rRNA genomic DNA fragments containing both coding and intergenic spacer (IGS) regions from B. xylophilus and B. mucronatus through a homology-probing PCR strategy. Sequence analyses revealed that coding sequences of the 5S rRNA gene from the two species are almost identical (98.3% homology) but that the IGS sequences differ substantially between the species. Based on the IGS sequence differences (69.7% homology), we designed species-specific primer sets and developed a PCR-based diagnosis protocol for the identification and discrimination of the two nematode species on a molecular basis.

scholarly journals Cloning and Sequence Analysis of 5S Ribosomal RNA Gene(s) and Associated Intergenic Spacer Regions in Carica Species

2021 ◽  
Vol 13 (1) ◽  
pp. 1
Author(s):  
Mahipal Singh ◽  
Pushpa Yadav ◽  
Anand K. Yadav
Keyword(s):  
Sequence Analysis ◽  
Ribosomal Rna ◽  
Carica Papaya ◽  
Intergenic Spacer ◽  
5S Rrna ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Ribosomal Rna Gene ◽  
5S Ribosomal Rna ◽  
5S Rrna Gene

The 5S ribosomal RNA gene(s) and their associated intergenic spacer regions were amplified from Carica papaya and Carica quercifolia by polymerase chain reaction. Both Carica species exhibited differently sized amplification products. Sequence analysis of these PCR products revealed that the 5S rRNA genes are arranged as tandem repeats in these regions. Sequence data revealed that the 5S rRNA gene from Carica quercifolia was 119 bp in length. Sequence variation was observed in various 5S rRNA gene copies cloned from Carica quercifolia. Only truncated 5S rRNA gene but with its full spacer region was recovered from Carica papaya. Interestingly, intergenic spacer sequence cloned from Carica papaya contained two specific domains, a 30bp “CT” rich domain exhibiting 95-100% homology to several human chromosomes and a domain matching with mitrocomin precursor, a photo-protein from Mitrocoma cellularia. The role of 5S rRNA gene and their spacer regions in discerning the germplasm and in adaptation of the species is discussed.

scholarly journals Molecular and biochemical characterization of an endo-β-1,3-glucanase from the pinewood nematode Bursaphelenchus xylophilus acquired by horizontal gene transfer from bacteria

2005 ◽  
Vol 389 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Taisei KIKUCHI ◽  
Hajime SHIBUYA ◽  
John T. JONES
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Biochemical Characterization ◽  
Glycosyl Hydrolase ◽  
Functional Characterization ◽  
Nematode Species ◽  
Bursaphelenchus Xylophilus ◽  
Pinewood Nematode ◽  
Sequence Comparisons

We report the cloning and functional characterization of an endo-β-1,3-glucanase from the pinewood nematode Bursaphelenchus xylophilus acquired by horizontal gene transfer from bacteria. This is the first gene of this type from any nematode species. We show that a similar cDNA is also present in another closely related species B. mucronatus, but that similar sequences are not present in any other nematode studied to date. The B. xylophilus gene is expressed solely in the oesophageal gland cells of the nematode and the protein is present in the nematode's secretions. The deduced amino acid sequence of the gene is very similar to glycosyl hydrolase family 16 proteins. The recombinant protein, expressed in Escherichia coli, preferentially hydrolysed the β-1,3-glucan laminarin, and had very low levels of activity on β-1,3-1,4-glucan, lichenan and barley β-glucan. Laminarin was degraded in an endoglucanase mode by the enzyme. The optimal temperature and pH for activity of the recombinant enzyme were 65 °C and pH 4.9. The protein is probably important in allowing the nematodes to feed on fungi. Sequence comparisons suggest that the gene encoding the endo-β-1,3-glucanase was acquired by horizontal gene transfer from bacteria. B. xylophilus therefore contains genes that have been acquired by this process from both bacteria and fungi. These findings support the idea that multiple independent horizontal gene transfer events have helped in shaping the evolution of several different life strategies in nematodes.

A natural case of RIP: degeneration of the DNA sequence in an ancestral tandem duplication

1989 ◽  
Vol 9 (10) ◽  
pp. 4416-4421
Author(s):  
W S Grayburn ◽  
E U Selker
Keyword(s):  
Dna Methylation ◽  
Tandem Duplication ◽  
De Novo ◽  
5S Rrna ◽  
Rrna Genes ◽  
Structural Basis ◽  
Rrna Gene ◽  
Oak Ridge ◽  
5S Rrna Gene ◽  
5S Rrna Genes

5S rRNA genes of Neurospora crassa are generally dispersed in the genome and are unmethylated. The xi-eta region of Oak Ridge strains represents an informative exception. Most of the cytosines in this region, which consists of a diverged tandem duplication of a 0.8-kilobase-pair segment including a 5S rRNA gene, appear to be methylated (E. U. Selker and J. N. Stevens, Proc. Natl. Acad. Sci. USA 82:8114-8118, 1985). Previous work demonstrated that the xi-eta region functions as a portable signal for de novo DNA methylation (E. U. Selker and J. N. Stevens, Mol. Cell. Biol. 7:1032-1038, 1987; E. U. Selker, B. C. Jensen, and G. A. Richardson, Science 238:48-53, 1987). To identify the structural basis of this property, we have isolated and characterized an unmethylated allele of the xi-eta region from N. crassa Abbott 4. The Abbott 4 allele includes a single 5S rRNA gene, theta, which is different from all previously identified Neurospora 5S rRNA genes. Sequence analysis suggests that the xi-eta region arose from the theta region by duplication of a 794-base-pair segment followed by 267 G.C to A.T mutations in the duplicated DNA. The distribution of these mutations is not random. We propose that the RIP process of N. crassa (E. U. Selker, E. B. Cambareri, B. C. Jensen, and K. R. Haack, Cell 51:741-752, 1987; E. U. Selker, and P. W. Garrett, Proc. Natl. Acad. Sci. USA 85:6870-6874, 1988; E. B. Cambareri, B. C. Jensen, E. Schabtach, and E. U. Selker, Science 244:1571-1575, 1989) is responsible for the numerous transition mutations and DNA methylation in the xi-eta region. A long homopurine-homopyrimidine stretch immediately following the duplicated segment is 9 base pairs longer in the Oak Ridge allele than in the Abbott 4 allele. Triplex DNA, known to occur in homopurine-homopyrimidine sequences, may have mediated the tandem duplication.

DNA methylation inhibits transcription by RNA polymerase III of a tRNA gene, but not of a 5S rRNA gene

FEBS Letters ◽  
1990 ◽  
Vol 269 (2) ◽  
pp. 358-362 ◽  
Author(s):  
Daniel Besser ◽  
Frank Götz ◽  
Kai Schulze-Forster ◽  
Herbert Wagner ◽  
Hans Kröger ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
5S Rrna ◽  
Rrna Gene ◽  
Polymerase Iii ◽  
5S Rrna Gene

scholarly journals Direct Regulation of tRNA and 5S rRNA Gene Transcription by Polo-like Kinase 1

2012 ◽  
Vol 45 (4) ◽  
pp. 541-552 ◽  
Author(s):  
Jennifer A. Fairley ◽  
Louise E. Mitchell ◽  
Tracy Berg ◽  
Niall S. Kenneth ◽  
Conrad von Schubert ◽  
...  
Keyword(s):  
Gene Transcription ◽  
5S Rrna ◽  
Rrna Gene ◽  
5S Rrna Gene ◽  
Direct Regulation

Identification of a Molecular Marker and Chromosome Mapping of the 5S rRNA Gene inAllium sacculiferum

2007 ◽  
Vol 50 (6) ◽  
pp. 687-691 ◽  
Author(s):  
jun Hyung Seo ◽  
Byung Ha Lee ◽  
Bong Bo Seo ◽  
Ho-Sung Yoon
Keyword(s):  
Molecular Marker ◽  
Chromosome Mapping ◽  
5S Rrna ◽  
Rrna Gene ◽  
5S Rrna Gene

scholarly journals Detection and Discrimination of Pratylenchus neglectus and P. thornei in DNA Extracts from Soil

Plant Disease ◽  
2008 ◽  
Vol 92 (11) ◽  
pp. 1480-1487 ◽  
Author(s):  
Guiping Yan ◽  
Richard W. Smiley ◽  
Patricia A. Okubara ◽  
Andrea Skantar ◽  
Sandra A. Easley ◽  
...  
Keyword(s):  
Nematode Species ◽  
Wheat Root ◽  
Fungal Species ◽  
Parasitic Nematodes ◽  
Rrna Gene ◽  
28S Rrna ◽  
Pratylenchus Neglectus ◽  
Pcr Products ◽  
Species Specific ◽  
Plant Parasitic

A species-specific polymerase chain reaction (PCR) method was developed to detect and identify the root-lesion nematodes Pratylenchus neglectus and P. thornei from soil. A primer set was designed from Pratylenchus 28S rRNA gene sequences of the D3 expansion domain. Primer specificity was confirmed with 23 isolates of 15 nematode species and other plant-parasitic and non-plant-parasitic nematodes typically present in the soil communities, and with six fungal species commonly associated with wheat root rot. DNA obtained using a commercially available kit and a method developed in our laboratory gave comparable amplification. PCR conditions were optimized and the two species were differentiated by PCR products of 144 bp for P. neglectus and 288 bp for P. thornei. With this assay, we detected a single juvenile in 1 g of sterile, inoculated soil. Examination of 30 field soil samples revealed that this method was applicable to a range of soils naturally infested with these two pathogens in Oregon. This PCR-based method is rapid, efficient, and reliable, does not require expertise in nematode taxonomy and morphology, and could be used as a rapid diagnostic tool for commercial and research applications for disease forecasting and management.

scholarly journals The location and nucleotide sequence of the 5S rRNA gene of bunt of wheat,Tilletia cariesandT.controversa

1992 ◽  
Vol 20 (10) ◽  
pp. 2600-2600 ◽  
Author(s):  
T. Zerucha ◽  
W.K. Kim ◽  
W. Mauthe ◽  
G.R. Klassen
Keyword(s):  
Nucleotide Sequence ◽  
5S Rrna ◽  
Rrna Gene ◽  
5S Rrna Gene
Sign in / Sign up

Export Citation Format

Share Document