<i>Clonorchis sinensis</i> tropomyosin: cloning and sequence of partial cDNA amplified by PCR

, Hong, S J

doi:1993.31.3.285

Korean J Parasito > Volume 31(3):1993 > Article

Original Article


Korean J Parasitol. 1993 Sep;31(3):285-292. English. Published online Mar 20, 1994. http://dx.doi.org/10.3347/kjp.1993.31.3.285
Copyright © 1993 by The Korean Society for Parasitology


Clonorchis sinensis tropomyosin: cloning and sequence of partial cDNA amplified by PCR
Sung-Jong Hong^*
Department of Parasitology, Gyeongsang National University College of Medicine, Chinju, Kyongnam 660-280, Korea.

Received June 28, 1993; Accepted July 20, 1993.
Abstract
C. sinensis total RNA was containing large amount of 18S rRNA but little 28S rRNA. The size of the double-stranded cDNA synthesized from poly (A)⁺ mRNA was 0.4-4.2 kb long with tapering upto 9.5 kb. Degenerated oligonucleotides (as 2 sense and 3 antisense primers) were designed on the conserved regions of the known tropomyosin amino acid sequences. From one out of the PCR amplifications using total cDNA and matrix of primers, a specific gene product, 580 bp in size, was produced. Upon Southern hybridization of the PCR products with Schistosoma mansoni tropomyosin (SMTM) cDNA, only one signal appeared at the band of 580 bp product. This 580 bp product was considered to encode C. sinensis tropomyosin (CSTM) and cloned in pGEM-3Zf(-) for DNA sequencing. CSTM cDNA was 575 bp containing one open reading frame of 191 predicted amino acids, which revealed 86.3% homology with SMTM and 51.1% with Trichostrongylus colubriformis tropomyosin. CSTM cDNA obtained will serve as a probe in the studies of molecular cloning of CSTM.

Figures


	Fig. 1 Degenerated oligonucleotides used as primers in PCR amplification of C. sinensis tropomyosin cDNA were designed on the base of amino acid sequences of tropomyosin of S. mansoni and the others (Xu et al., 1989).


	Fig. 2 Total RNA 10 µg each electrophorosed in 1% formaldehyde gel. C. sinensis total RNA (lane 2) revealed a thick 18S rRNA band but not 28S rRNA. Rat liver total RNA (lane 1) extracted simultaneously as an RNA extraction control was containing thick and undegraded 18S rRNA bands.


	Fig. 3 Autoradiograph of double-standed cDNA synthesized (see MATERIALS AND METHODS) was showing major product ranged in 1.4-4.2 Kb in size. Lambda phage DNA digested with Hind III endonuclease was end-labeled by using Klenow fragment, dATP, dGTP and [α-³²P] dCTP, and run as DNA size marker.

Fig. 4
Panel A; PCR amplification was carried out on C. sinensis total double-standed cDNA using degenerate oligonucleotide primers and the products were electrophorosed on 1.4% agarose gel. A specific band, 580 bp in size, appeared in a lane where sense primer #2 and antisense primer #2 were used. On land Ⓑ, cDNA fragment of C. mansomi tropomyosin (SMTM), 730 bp in size, was run as a positive control. Panel B; The gel was dried and hybridized with radiolabeled SMTM cDNA. A strong signal appeared at 580 bp band.


	Fig. 5 DNA sequence and deduced amino acid sequence of PCR-amplified C. sinensis tropomyosin cDNA (CSTMP-1).


	Fig. 6 Comparison of homologies between the amino acid sequences of tropomyosin for C. sinensis (CSTM), C. mansomi (SMTM) and T. colubriformis (TCTM). Identities between the two species are idicated by (*).


	Fig. 7 Northern hybridization of C. sinensis total RNA (A) probed with radiolabeled CSTMP-1 cDNA showing a signaling band at approximately 1.3 kb in size (B).

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