1. Molecular and Biochemical Parasitology, 52 (1992) 75-84 75
© 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00
MOLBIO 01719
Characterization of the rDNA 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 CenterJbr 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 RNA gene unit of the protozoan parasite Tritrichomonasfoetus has been cloned and analyzed. Southern
blot analysis of the genomic DNA showed that the ribosomal RNA 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 RNA gene unit. The lengths of the small subunit ribosomal RNA 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 rRNA 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 para-
site Tritrichomonasfoetus is the causative agent
of bovine trichomoniasis, a contagious sexu-
ally transmitted disease characterized by
infertility, abortion and pyometra in cattle
Correspondence address: John B. Dame, Department of
Infectious Diseases, College of Veterinary Medicine, Univer-
sity of Florida, Gainesville, FL 32611, USA.
Note. Nucleotide sequence data reported in this paper have
been submitted to the GenBankTM 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, Tris-
borate-EDTA buffer.
[1]. 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 rDNA 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 [2]. Although these features
of ribosomal RNA 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 [3] and Plasmodium with
4-8 copies [4-6]. The length of rDNA units
vary widely, from 5.5 kb in Giardia intestinalis
[7] to 44 kb in rat [8]. The SSrRNA also differs
in length to its currently known extremes
within the protozoa from 1244 bp in Vair-

2. 76
imorpha necatrix to 2319 bp in Trypanosoma
cruzi [9]. We report in this paper the cloning
and characterization of the rDNA unit from T.
,foetus. The SSrRNA in this organism is one of
the shortest reported and the copy number of
the rDNA 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 [10]. Strains Crop-I
(kindly provided by J.M. Cheney, Colorado
State University) and UT-I (American Type
Culture Collection #30233) were used through-
out these studies. Organisms were harvested in
the mid-log growth phase.
Genomic DNA and total RNA were
routinely isolated by extracting cells with a
solution containing 4 M guanidinium isothio-
cyanate/5 mM sodium citrate/ 10 mM EDTA/
0.5% N-lauryl sarcosine /1 mM 2-mercapto-
ethanol, followed by fractionation via isopyc-
nic gradient centrifugation in cesium trifluoro-
acetate (CsTFA) [11]. 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 [12]. Intact chromosomal DNA
was prepared in 1% agarose blocks as
described [13]. Plasmid DNA was isolated by
the boiling method [12]. Genomic and plasmid
DNAs were restriction enzyme digested, elec-
trophoresed on agarose gels, and blotted on
nylon membranes as described [12]. Total
RNA was fractionated in formaldehyde agar-
ose gels [12] 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 rDNA cistron in
T..foetus, genomic DNA samples from 2
different isolates, Crop-1 and UT-1, were
digested to completion with various restric-
tion 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 DNA fragments were
electrophoresed onto DEAE cellulose mem-
brane. The isolated fragments were ligated into
the XhoI site of pBluescript SK(+) followed
by transformation into XL-1 blue cells.
Transformed colonies were screened with 32p_
labeled total RNA probe prepared as described
[14]. The DNA was isolated from positive
colonies, digested with XhoI and probed with
5'-end labeled RNA probes for further con-
firmation. A positive clone containing the 6-kb
fragment, designated as p433, was used in
further studies. A physical map of the T. foetus
rDNA 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 [9]. 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 rRNA precursor is long enough to be

3. the primary transcript, suggesting that the non-
transcribed 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 rRNA sequencing primers [15]
did not work, the information from the
restriction map was used to prepare exonu-
clease Ill deletion subclones. In addition
specific sequencing primers were prepared
using the sequence obtained from the sub-
1 2 3 4 5 6 7 8
6kb~
77
clones. The nucleotide sequence of the
SSrRNA and 5.8S coding regions was deter-
mined 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 fea-
tures of the secondary structure are similar to
those established for the Escherichia coli 16S
rRNA [16], and form an overall structure that
is common to eukaryotic SSrRNAs. Nucleo-
tides for which comparative sequence analysis
can not discern a common structure are left
5.8kb
4.0kb
2.5kb
-.,1-- 1.6kb
L S 5.8 L S
S A
E CX RSm A CBSt EH CS't E
I II II I II I II II I
D
I 1kb I
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.

4. 78
unstructured. The only region left unstructured
is between bases 557 and 677. Regions outside
the core structure are smaller compared with
most other eukaryotic SSrRNAs [9,16].
The nucleotide sequences of 5.8S rRNA,
ITS l, and ITS2 are shown in Fig. 3. The
U~AU
c 1150
u u%' AC 750 ~ ~u Ao,, •
U C Q A GAG = G ~ I I I''1 ( I*l CCAAUUA A A UACUCGUUUCUGUu
AAuGu CC AUGAGA AGA A GC A I I I I I I I ¢ I I 4 " I • I ( ( I U
G • l r i p 1 4 ~ i • r ° l ~ i a iAA U • (k UG C UG Q,AUOG O GGUUAA U C U UGAGAUAGAGACk
GUGGC AA G cOGUA CUU UA UCUA UA CG C_ O O G C A GA
^ u G oA c
~u G.u A........ 6SO 700 A~ . . . . .
O U 6 0 C .C ° -- C~
u Uu ~,-,.)u .,. u u .,, - 1200
G G • A C ~U.G
o G A~U CC G A U G.U
U U ~ ~ C ~ l a ~ ~ -- C
AA~UCAAUGA U mU G * U UA . A C UQ _ C
G ~ 800 A-U AUuuuu// UUuO.U
U U ~ l C ~ U U u
G ~ l C U U
, % ~,:,~ ? ', "O,c~-oo,
U GUCCGUCAAGAc// UCAA o-CCU U 0U % (i A C =
c c u_" G i, A UC ,,*d ~ ~.u 12S0
A A .GA w~ A--U
c ~ u c / Co A uu ° c A cuu c a
% ~ 550 % UA~850 ~ oG ,, ~ ~ ....
^: oo ", ..... ~^o°.,~' .~ ~ '~.~
uA A cu~ A ~111= • Uu 1000 a%'- uC-G ccu
AA Gc A G ~C A U CGGCUUG ~ AC~" UUAA u G--C A U GU )AA
c^ ~A C G u u Gu, A G C (;U ~--C U Gc ~ ~G
60o .,% ,u ,c ~ • ~ ~:c u o,/,% ~, , ~.~ -^ ^ ,,,,:%c
SUc . _uGU ~ b¢¢.0 A U'~uu C -- O ~UU ~U A CU/' U • G c" A~G ~ Co
^CUU(kCu * ~ CC A*' a G AA U C U ~ U U
Cc O.U u G C-e GA uc CA
A U U 0U GGAACUACGACC G G~C AG ~
/G G--C U • II c A--uAC A GuU % CA
UU / uo a~c u A 0 ~.U uc u~.~.c
C // C U--A A U CA UC~aA (~ x A
Ac//AG AGUC -- G U ~JA AAG Q~t~ nGUk ii GCA
°o,'~°', ~..... ~ "t ....... ~o,,.... o,o,co,,=.,oo:
• ~C ~U CC~ AccGUcc/! II II. IIIIIli u C¢ G u
C ~-u u A ~, u ac UOU UUCCC(IU U g u
A AG--C uU., ~ U u UO A C •
u u =u, ~u ~c c c c u ~ .
uUO~uuo u u A~ x A A C U A ~ " U
U I)lll ~11~ xAC U a 0 A u
UuCCAAC ACAU u ~:A Uc C A A C //~U A U
°°°~ ,, ,'o°° "o, %° oo
UUCGGA OGUO G ~ u C A UAACOQUAG~U G O A A~
Ue,(,. Ill" G: G A .......... A ~cA CU UG
A~'=CUc cCC~U u uCZA =uu=cc=ucc, 1~-00 AA
• ,,~° Y, ~-o 1c-(~
a - c~.50 c - o •C-G
c= 350 A-u uA ~I c-~
U--AUg,u
u~u/ ~ u ~ G - C
AGAUA~cGAc A GU~ U GUUC GO A--UU--A
U C
GA Ill*it I* II=I A G*UA--U
300 .¢c aucC=C==A c= UCA AA GcA C--G
• ;'."~t% "= ,° ~ ,~
uooG,o,.~9, <,~,o°,o" °-°¢ou
u - A
< 2' , - u • 1500
u-A
C~ o x CA Ac" u-^
u c • c O-C A--U
A U c-(; u.o
u CuP' o-c I00 oc-a,
OGu O-c o • A
• C U-A
=~', ~,; u.o, %-~"
• ,>.uo .250 ,-u~ c:~
u~x,d ~ o-c u
u a~ A-U c c ~
cU c--o (L.~U
c-oc c 1~0A -- u uu
~GC A e 0 uUuo AU CA CAGUA
~$$$ , II.II I~ Ill A
UCCC A ~uACAUA OUGGUCuU
o-c au¢
U-A
O.u
Aa ~"U{i
A--U
200 a -u,o.u
0-¢
(;-¢
A--U
AC -- 0.~
A~A oUU
Fig. 2. TI Joetus SSrRNA primaryand secondarystructure.The higherorderstructureof the SSrRNA was determinedby
comparativemethodsas described[16].

5. 79
T. foetus rDNA: ITS1, 5.8S, and ITS2
1 TTCGTTAATA ATTACAAACA TATTTTTTTA ATTTCTATAA CTATTTATAC
51 AAAATTAAAC ACATAATCTA AAAAATTTAG ACCTTAGGCA ATGGATGTCT
101 TGGCTTCTTA CACGATGAAG AACGTTGCAT AATGCGATAA GCGGCTGGAT
151 TAGCTTTCTT TGCGACAAGT TCGATCTTTG AATGCACATT GCGCGCCGTT
201 TTAGCTTGCT AGAACACGCA TATATGTTAC AGTAACCCAT ATTAATTTAA
251 TACCAAATTC TCTTTTTAAG CAAAAGAGCG AAAAACAAAT ATGTATTAAC
301 AA
Fig. 3. The nucleotide sequence of the ITSI, 5.8S and ITS2
of T. foetus rRNA. DNA sequence analysis of both DNA
strands was performed using Sequenase 2.0 (US Biochem-
icals) or modified T7 DNA polymerase (Pharmacia) by the
dideoxy nucleotide chain termination method as described
[12]. Primers used were either universal [15] or specific
oligonucleotide primers synthesized on an Applied Biosys-
terns 380B DNA synthesizer at the ICBR DNA synthesis
Core at the University of Florida. Some sequences were
generated from progressively deleted clones obtained by
treatment with exonuclease III/S1 nuclease using Erase-a-
base kit (Promega). Sequence analysis was performed with
various University of Wisconsin Genetics Computer Group
sequence analysis programs [17].
boundaries of each of these regions were
extrapolated from comparisons with eukaryo-
tic SSrRNA sequences in the GenBank and
EMBL data bases. The length of the 5.8S
rRNA is 159 bp with a G-C content of 44%.
The ITSs are very short at 80 and 64 bp,
respectively.
The 5'-end of the mature SSrRNA of T.
foetus was confirmed by the primer extension
analysis of the total RNA using reverse
transcriptase (Fig. 4, panel 2). The Fig. 4,
panel 1 shows the dideoxy DNA sequence
analysis of p433 using the same primer. The
sequence at the 5' end of the molecule starts
with ATGAA. Since the primer used for
sequencing is a reverse primer, the 5' end of
the coding strand sequence of the SSrRNA is 5'
UACUU .... 3'. In addition, the 5' end sequence
determined by homology comparison of the
sequence, using GAP or BESTFIT programs
of the University of Wisconsin Genetics
Computer Group [17], to other eukaryotic
SSrRNAs present in GenBank and EMBL
databases was consistent with this result. The
primer extension experiment also enabled us to
determine the 5' end of the primary transcript.
Several pauses in the 5' upstream region
1
GATC
2
GATC
A
C ......~ "
if
5OO
250
2OO
175
Fig. 4. Primer extension analysis of the T.foetus SSrRNA.
The rDNA transcription initiation sites were determined by
oligonucleotide-directed primer extension. The 17-mer
primer, 5'-GCCCGGAGTCAACTTTT, was extended at
42°C using avian myeloblastosis virus reverse transcriptase
in a dideoxy sequencing reaction [12]. The primer-extended
product was analyzed on a 6% polyacrylamide sequencing
gel adjacent to the products of a dideoxy chain termination
sequencing reaction performed on cloned rDNA using the
same sequencing primer. Panel 1, dideoxy sequencing
reaction of p433. Panel 2, reverse transcriptase extension
of the total rRNA.

6. 80
A
2000
1000
0
0.0
• i
200 400 600 800 ng
0.5 1.0 1.5 2.0 ng
B
78 kb-
39 kb-
23 kb-
1 2 3 4
9.4 kb
6.6 kb I
Fig. 5. (A) Gene copy number analysis of the T.Jbetus rDNA. Genomic DNA 750,400, 200, and 100 ng) and P433 DNA (1.6,
0.8, 0.4, 0.2 and 0.1 ng) was digested with Xho! and copy number analysis was determined by hybridization analysis of
genomic DNA using nick-translated cloned rDNA (p433) probe. Decreasing amounts of genomic and plasmid DNA digested
with XhoI were separated on a 0.8% agarose gel, blotted, and hybridized with the labeled p433 probe. The hybridized blot was
scanned in an AMBIS radioanalytic imaging system.., genomic DNA; [5], cloned rDNA. (B) Southern blot analysis of
partially digested genomic DNA of T. foetus. Samples of T. foetus total nucleic acids containing 1 /zg genomic DNA were
partially digested with different dilutions of Xhol in a 20/A reaction for 10 min. DNA fragments were separated on a 1%
agarose gel using a CHEF apparatus [34]. Size standards were a mixture of HindIII-digested 2 DNA and the ), DNA
concatamer Delta 39 (Promega, Madison, WI). The electrophoresis conditions were: 2.5 second pulse time, 200 V, 12°C, 0.5 x
32TBE [12], and 16 h run time. Blots were prepared and P-labeled p433 was used as a probe. Lane 1, undigested genomic
4 14 l 3 I
DNA; lane 2, 6.6 x 10 units XhoI /A ;lane3,2 x 10 units XhoI /d ;lane4,6.6 x 10 unitsXhollA .
represent the presence of minor population of
transcripts at various stages of processing.
Major pause sites detected were at approx.
175, 200 and 250 bp upstream from the mature
SSrRNA. These represent processing sites at
the 5'-end of the transcript. Very faint bands
seen approx. 500 bp upstream may represent
the true transcription initiation site.
The copy number of the rDNA unit of T.
foetus was calculated as a percentage of the
genome. As shown in Fig. 5A, the 6-kb XhoI
rDNA band in 400 ng of the genomic DNA is
equivalent to 1.12 ng of the fragment cloned in
p433. Since the genome size of T. foetus is
about 2.5 x 10 7 bp [18], these results indicate
that 12 copies of rDNA units are present per
genome. The presence of only 12 copies of
rDNA unit in T. foetus is relatively low
compared to other eukaryotic organisms,
where the rDNA units are usually repeated
100-5000 times [2]. However, it was possible
that we did not detect the presence of any
extrachromosomal elements carrying rDNA
sequences since we used a CsTFA isopycnic
gradient centrifugation method to isolate
genomic DNA. Therefore, we looked for the
existence of extrachromosomal DNA elements
containing rDNA sequences using prepara-

7. tions of total nucleic acids. Southern blots of a
partial XhoI digest of T. foetus total nucleic
acids separated on pulsed field gel electrophor-
esis were probed with 32p-labeled p433 (Fig.
5B). A ladder of about 12 bands at an interval
of 6 kb was found which is consistent with the
copy number analysis. Behavior of the rDNA
in this partial digest also suggests that rDNA is
arranged as a linear, tandem repeat of 12 units
located within the chromosomal DNA. Geno-
mic DNA prepared in agarose blocks were
separated by pulsed field electrophoresis and
hybridized with the same probe. Probe hybri-
dized as a band at the position of the
unresolved intact chromosomal DNA (> 500
kb) and as a smear at 25-70 kb, which
presumably was partially degraded chromoso-
mal DNA (data not shown). Extrachromoso-
mal DNA was not detected in this experiment.
Neither was it detected by the Hirt fractiona-
tion method [19] nor by fractionation of T.
foetus genomic DNA on a Hoechst dye 33258 -
CsC1 gradient, which separates DNA based on
G-C content [20].
Discussion
The T. foetus rDNA repeating unit having a
molecular length of 6 kb is one of the smallest
reported thus far for a eukaryotic organism.
The length of the rDNA unit varies widely
among eukaryotes, ranging from 5.5 kb in
Giardia intestinalis [7] to 44 kb in rat [8].
Length variation occurs in tandemly repeated
rDNA units both within and between species
[2,8,21-23]. This is primarily due to differences
in the length of the intergenic or non-
transcribed spacer region, although length
differences in the coding region and introns
may also contribute. No rDNA restriction
fragment length polymorphism was detected
between 2 different isolates used in this study.
The sequence analysis showed that the length
of the SSrRNA is only 1571 bases, one of the
shortest of all eukaryotic sequences reported
thus far [9]. Only Vairimorpha necatrix with
1244 nucleotides and Giardia lamblia with 1453
nucleotides are shorter. Though not compel-
81
ling as an argument in light of the large size of
the T. cruzi SSrRNA, the presence of a short
SSrRNA is a feature of several eukaryotic
organisms which appear to have diverged from
most eukaryotic species early in their phylo-
genetic history. It has been proposed that all 3
organisms, G. lamblia, V. necatrix and a
related trichomonad, Trichomonas vaginalis,
have branched off early in the eukaryotic
subtree [24], although the sequence of the T.
vaginalis SSrRNA has not been reported. All
of these organisms are parasitic protozoa
lacking mitochondria. Phylogenetic distance
analyses based on a partial sequence of the
large subunit rRNA of T. vaginalis have also
placed the trichomonads on a deep branch of
the tree of eukaryotes [25].
At this time there are over 100 eukaryotic
SSrRNA sequences known, spanning the
major phylogenetic groups. Comparative ana-
lysis of these sequences has defined a core
structure; a primary and secondary structure,
by definition, common to all sequences in the
eukaryotic data set [9,16] (R.R. Gutell, un-
published analysis). This core region contains
in it primary and secondary structure elements
found within all life forms as well as structures
common only to eukaryotes. This type of
analysis also reveals those regions that show
extensive variation, the so called variable
regions. These regions have the characteristic
features of varying in size, base composition,
with little or no primary structure conservation
across all eukaryotic sequences. However,
within these variable regions there are varying
degrees of primary and secondary structure
conservation within local phylogenetic group-
ings.
The T. foetus SSrRNA primary structure,
while reduced in size when compared with
other eukaryotic SSrRNA sequences, does
maintain the major characteristic eukaryotic
primary and secondary structure features seen
in the core structure. The smaller size of this
SSrRNA can be attributed entirely to trunca-
tions occurring within the eukaryotic variable
regions, most notably in the 2 regions
bracketed by T. foetus bases 171 219 and
557-678. A more detailed analysis of the

8. 82
trichomonad SSrRNA higher-order structure
and phylogeny, in relation to other eukaryotic
SSrRNA structures is currently in progress.
Internal transcribed spacers in T. Joetus are
very short relative to higher eukaryotes. For
example, in Xenopus laevis rDNA ITS 1 and 2
are 557 and 262 bases, respectively [26], in
contrast to 80 and 64 bases, respectively, in T.
foetus. A view on the evolutionary origin of
ITS2 suggests that it has evolved from a
segment of the large subunit rRNA of
prokaryotes [27], since prokaryotes process
out only ITS1 resulting in the large subunit
rRNA sequence containing the equivalent of
the eukaryotic 5.8S rRNA and ITS2 at its 5'
end. Eukaryotes studied to date which have
diverged early either lack ITS2 and a separate
5.8S as in V. necatrix [28], or have very short
ITSs, as in G. lamblia (ITSI: 41 bp; ITS2:55
bp) [7]. The internal transcribed spacers, ITS1
and ITS2 were very A + T-rich, 86% and 81%
respectively. This is in contrast to another
amitochondrial parasitic protozoon, G.
larnblia, where ITSs are C-rich sequences
(ITSI: 60%, ITS2: 49%) [29].
The large subunit rRNA at 2.5 kb is one of
the shortest reported so far. Even G. lamblia
and E. coli large subunit rRNAs with 3011 and
2904 nucleotides, respectively, are longer than
that of T. foetus. In Northern blots of T..foetus
total RNA probed with the rDNA clone, no
smaller rRNA species were detected, but 2
larger transcripts were identified at 5.8 kb and
4 kb. Since the 5.8-kb band is most likely to be
the primary transcript, this suggests that the
non-transcribed spacer is approximately 200
bp long. The structure of the 4-kb transcript
has not been evaluated. At this moment we are
uncertain about the sequence of processing
cleavages of the primary rRNA transcript in T.
foetus. However, the primer extension experi-
ment showed that processing sites are located
approximately 250, 200 and 175 bp upstream
of the 5' end of the SSrRNA.
Copy number analysis of the rDNA unit of
T. foetus as a percentage of genomic DNA
showed the presence of 12 copies. These copies
arranged in tandem were also resolved in a
partial restriction enzyme digestion followed
by CHEF electrophoresis. Such a tandem
arrangement of rDNA units is a feature in
common with most eukaryotes, whereas the
relatively small number of rDNA units con-
trasts with organisms such as G. lamblia with
an estimated approx. 300 copies of its rDNA
unit per genome equivalent [30]. Where a low
copy number has been reported, such as the
apicomplexan protozoans, Babesia bigemina
[3] and Plasmodium berghei [4] with only 3 and
4 copies of rDNA units, respectively, the
rDNA units are not tandemly arranged. Other
protozoan organisms such as Tetrahymena
[31], Naegleria [32], and Entamoeba [33] have
numerous copies of extrachromosomal DNA
elements carrying rDNA sequences. Upon
careful analysis by various fractionation
methods, no extrachromosomal rDNA was
identified in T. foetus.
Acknowledgements
This research was supported by grants from
the University of Florida Institute of Food and
Agricultural Sciences and the State of Florida
High Technology and Industry Council Ap-
plied Research Grants Program. We would
also like to thank the W.M. Keck Foundation
for their generous support of RNA science on
the Boulder campus. RRG is an associate in
the Evolutionary Biology Program of the
Canadian Institute for Advanced Research.
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