Molecular and Biochemical Parasitology, 52 (1992) 75-84
© 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00 MOLBIO 01719
Characterization of the r D N A unit and sequence analysis of the small subunit rRNA and 5.8S rRNA genes from Tritrichomonasfoetus* Debopam Chakrabarti 1'2, John B. Dame 1., Robin R. Gutell 3, and Charles A. Yowell 1 1Department of lnfectious Diseases, College of Veterinary Medicine, and 2Interdisciplinary Center Jbr Biotechnology Research, University ~?fFlorida, Gainesville, FL, USA; and 3MCD Biology, University of Colorado, Boulder, CO, USA (Received 6 September 1991 ; accepted 26 November 1991)
The ribosomal R N A gene unit of the protozoan parasite Tritrichomonasfoetus has been cloned and analyzed. Southern blot analysis of the genomic DNA showed that the ribosomal R N A gene unit is organized as a tandem head to tail repeat with a unit length of 6 kb. By Northern analysis a primary transcript of 5.8 kb was detected. Copy number analysis showed the presence of 12 copies of the ribosomal R N A gene unit. The lengths of the small subunit ribosomal R N A and 5.8S ribosomal RNA are 1571 bp and 159 bp, respectively, as determined by sequence analysis. The T. foetus small subunit ribosomal RNA sequence is one of the shortest eukaryotic small subunit r R N A sequences, similar in length to those from 2 other amitochondrial protists. Although shorter than the majority of the eukaryotic small subunit ribosomal RNAs, this sequence maintains the primary and secondary structure common to all eukaryotic small subunit ribosomal RNA structures, while truncating sequences found within the eukaryotic variable regions. The length of the large subunit ribosomal RNA was measured at 2.5 kb. Key words: Trichomonad; Ribosomal RNA; 16S rRNA; 18S rRNA; Eukaryote; Protozoa
Introduction The flagellated, anaerobic protozoan parasite Tritrichomonasfoetus is the causative agent of bovine trichomoniasis, a contagious sexually transmitted disease characterized by infertility, abortion and pyometra in cattle Correspondence address: John B. Dame, Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611, USA.
Note. Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M81842. This paper is part of the University of Florida Agricultural Experiment Station Journal Series, No. R-01860.
Abbreviations." rRNA, ribosomal RNA; rDNA unit, ribosomal RNA gene unit; SSrRNA, small subunit rRNA; ITS, internal transcribed spacer; CHEF, contour-clamped homogeneous electric field; CsTFA, cesium trifluoroacetic acid; TBE, Trisborate-EDTA buffer.
. Basic to control of the disease is a sensitive and specific diagnostic test. We have initiated a study of the rRNA genes of this organism in an effort to develop molecular markers for the diagnosis of infection. The r D N A units in most eukaryotes studied to date encode 18S, 5.8S and 28S rRNAs and are tandemly repeated with the number of copies varying from approx. 100-5000 . Although these features of ribosomal R N A gene units are well conserved in most eukaryotes, the most notable exceptions to the general rule of the supramolecular organization are found in protozoa. In some protozoan parasites the number of rDNA repeats are often remarkably low and are not tandemly arranged such as Babesia with 3 copies  and Plasmodium with 4-8 copies [4-6]. The length of r D N A units vary widely, from 5.5 kb in Giardia intestinalis  to 44 kb in rat . The SSrRNA also differs in length to its currently known extremes within the protozoa from 1244 bp in Vair-
imorpha necatrix to 2319 bp in Trypanosoma cruzi . We report in this paper the cloning and characterization of the r D N A unit from T. ,foetus. The SSrRNA in this organism is one of the shortest reported and the copy number of the r D N A repeating unit is low, but tandemly arranged.
Materials and Methods
T. foetus cultures were grown in TYM medium, pH 7.0, supplemented with 5% heat inactivated calf serum . Strains Crop-I (kindly provided by J.M. Cheney, Colorado State University) and UT-I (American Type Culture Collection #30233) were used throughout these studies. Organisms were harvested in the mid-log growth phase. Genomic D N A and total RNA were routinely isolated by extracting cells with a solution containing 4 M guanidinium isothiocyanate/5 mM sodium citrate/ 10 mM EDTA/ 0.5% N-lauryl sarcosine /1 m M 2-mercaptoethanol, followed by fractionation via isopycnic gradient centrifugation in cesium trifluoroacetate (CsTFA) . Total nucleic acids were isolated by phenol-chloroform extraction from cells incubated at 65°C for 2 h in extraction buffer containing 0.1 M NaC1/ 0.025 M sodium EDTA/ 0.05 M Tris-HC1, pH 8.0/ 1% sodium dodecyl sulfate/ 1 mg ml 1 proteinase K . Intact chromosomal DNA was prepared in 1% agarose blocks as described . Plasmid D N A was isolated by the boiling method . Genomic and plasmid DNAs were restriction enzyme digested, electrophoresed on agarose gels, and blotted on nylon membranes as described . Total R N A was fractionated in formaldehyde agarose gels  adjacent to a 0.24-9.5-kb RNA ladder (Gibco/BRL, Gaithersburg, MD), and blotted on nylon membranes. Results
In an effort to identify the r D N A cistron in T..foetus, genomic D N A samples from 2
different isolates, Crop-1 and UT-1, were digested to completion with various restriction enzymes. Southern blots of these digests were probed with 5'-end labeled total RNA. As can be seen from Fig. 1A, digestion of T.foetus genomic DNA, derived from both Crop-1 and UT-1, with PvulI, Smal and XhoI gave rise to a single 6-kb band indicating the presence of a single site in a repeating unit whereas digestion with SstI resulted in 2 bands of 5.2 kb and 0.8 kb. The faint bands around 1.1 kb in the Smal digest, and > 10 kb in the PvulI and Sstl digests, may represent the end fragment of the repeat. End fragments were not identified in the XhoI digest indicating that the flanking fragments may be very large or they both coincidentally have sizes similar to the rDNA unit length. The organization of rDNA repeating unit is shown in Fig. 1C. T. foetus genomic DNA was digested to completion with XhoI, separated on a 0.8% agarose gel, and 6-kb D N A fragments were electrophoresed onto DEAE cellulose membrane. The isolated fragments were ligated into the XhoI site of pBluescript S K ( + ) followed by transformation into XL-1 blue cells. Transformed colonies were screened with 32p_ labeled total RNA probe prepared as described . The DNA was isolated from positive colonies, digested with XhoI and probed with 5'-end labeled RNA probes for further confirmation. A positive clone containing the 6-kb fragment, designated as p433, was used in further studies. A physical map of the T. foetus r D N A unit was constructed by digestion with different restriction enzymes alone or in various combinations and also from sequence analysis (Fig. 1D). Northern blot analysis of the T. Joetus total RNA with labeled p433 (Fig. 1B) indicates that the length of the large and small subunit rRNAs are approximately 2.5 and 1.6 kb, respectively. This suggests that the T../oetus rRNAs are smaller than the majority of eukaryotes studied to date . Two larger molecular length bands of 5.8 kb and 4 kb were also detected upon overexposure of the film which are thought to be precursor rRNAs. The 5.8-kb r R N A precursor is long enough to be
clones. The nucleotide sequence of the SSrRNA and 5.8S coding regions was determined from both strands. The T. Joetus SSrRNA is 1571 bp long with a G + C content of 48.5%, and it is presented in a secondary structure format in Fig. 2. The sequence of the part of the unit containing the SSrRNA, ITS1, 5.8S, and ITS2 regions is available in the GenBank database (M81842). The core features of the secondary structure are similar to those established for the Escherichia coli 16S r R N A , and form an overall structure that is common to eukaryotic SSrRNAs. Nucleotides for which comparative sequence analysis can not discern a common structure are left
the primary transcript, suggesting that the nontranscribed spacer is approximately 200 bp long. The 4-kb transcript is most likely generated from the primary transcript by processing cleavages but the processing sites which give rise to it are not known. A combination of sequencing strategies were used to obtain the nucleotide sequence of the SSrRNA coding region. Since many of the 'universal' 18S r R N A sequencing primers  did not work, the information from the restriction map was used to prepare exonuclease Ill deletion subclones. In addition specific sequencing primers were prepared using the sequence obtained from the sub1
Fig. 1. (A) Southern blot analysis of the restriction enzyme digested T. Jbetus genomic DNA (1 #g) probed with 32P-labeled total RNA. DNA samples from 2 different strains, Crop-1 (lanes 1,3,5, and 7) and UT-1 (2,4,6, and 8), were used. Lanes 1 and 2, PvuIl; 3 and 4, Sstl; 5 and 6, Sinai; 7 and 8, XhoI. (B) Northern blot analysis of total RNA (2/~g) probed with 32p-labeled p433. (C) Organization of the rDNA unit. (D) Restriction map of T. Jbetus rDNA unit. E, EcoRl; C, ClaI; X, Xhol; R, EcoRV; Sm, Sma|; A, Accl; S, SaII; B, BstX I; St, Sstl; H, HindlIl.
most other eukaryotic SSrRNAs [9,16]. The nucleotide sequences of 5.8S rRNA, ITS l, and ITS2 are shown in Fig. 3. The
unstructured. The only region left unstructured is between bases 557 and 677. Regions outside the core structure are smaller compared with
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