MRC Biochemical Parasitology Unit, The Molteno Institute, Downing Street, Cambridge CB2 3EE and *Department of Pathology, University of Cambridge, ...
Volume 10 Number 21 1982
Volume 10 Number 211982
Nucleic Acids Research
Nucleic Acids Research
Ribosomal RNA genes of Trypanosoma brucei. Cloning of a rRNA gene containing a mobile element
G.Hasan, M.J.Turner and J.S.Cordingley MRC Biochemical Parasitology Unit, The Molteno Institute, Downing Street, Cambridge CB2 3EE and *Department of Pathology, University of Cambridge, Downing Street, Cambridge CB2 1 QP, UK Received 4 August 1982; Revised and Accepted 12 October 1982
An ordered restriotion Wp of the ribosomal RIlA gens of Trypanosoma bruoei bruoei is presented. Bgl II fr t of T.b.bruoei genocic MlA were closed into pAT 153, and the olones cotaing rNAk identified. Restriction maps were established and the sense strands identified. One close was shown by heterodaplex mapping to contain a 1.1 kb inserted soquence which was denostrated to be widely distributed thraougot the genoss of members of the subeas T oo. However, in two other oubgsnera of laomona Mo and Sohisotia-m the sequenoe is far le andant. Anauiif the genomio M from two serodemes of T.b.brqoei showed that the sequnce wa presnt in te rENA of only ou of them, implying that the sequeno is a mobile element and that its appearance in rIA is a cosparitively reoent oo rne.
IDrNMUCTIc T.b.br3oei is a paraitic protoson that lives in the bloodstream and tissue spes of its lian hosts. Earlier studies from this laboratory d elsewhere (1,2,3) hae shown that the rElAs (4) of Trnosomatids differ comiderably from those of other wckarotes studied to date. The rMTA of the lrge subunit is nicked to produce two fragmats of 2300k100 and 1800+100 bases respectively. Within the large subnit, these two fragments are hydrogen bonded to each other. This pheomenon has also been described in other protosoa and insects (5,6). The small submit contain a single rIEA, 3000f100 bases in length. In general, the large subnits of eukaryotio ribosoes contain two small MAs designated the 5.8 S and 5 S BRAs. Exceptiom exit, as in Drosophila, where an additional 2 S HA has been observed (7). The ribosomes of rp ooatids are exceptional, in that they contain five or six small ribosomal BNAs desigated in cur laboratory an aRA 1-6 (2,3). This interesting and unal pattern of RIAs led us to investigate their arr sent in the genome. Ribosomal RMA. ge are known to be repetitive in all eukaryotic orguism studied so far (8)e. Th repeats are often all identioal. Howevr, in C I RL Press Limited, Oxford, England. 0305-1048/82/1021-67478 2.00/0
6747
Nucleic Acids Research Droopila and some other d<eraa imects a fraction mW be interrupted in their coding region by the preesene of inserted DNA s*equenes (8) . Our investigation of rENA gene structre sugests that a similar phenomenon oours in T,b,biuoei ad re present evidenoo for a ribosomal gene with an inserted sequnce belongi to a repeated family. Recently there has been a great deal of interest in the surace antigen gens of T.b.brucei whose expression is realated by tranposition to an expression site (9). The data presented an other than surfae antigen below indicate that trAsWposition of DNA s gens is probably a como event during the evolution of the Trypanosome genoem as it ha proved to be in other Ote in which it has been soaught
(10). The ribosomal gene inerted sequence, referred
to as RINE
(Ribosonal
Mobile Element) would appear to be such a tapossable soequne.
Materials The balk of the experiments were performed with T.b.bruoei of -two serodames, i.e. isolates from different geogaphioal looales, expressing different reperteires of VSCh (4). The history of the NITAR 1 (427) serodeme is desin (11), that of the TIIfAR 1 (IuuP 227) serodeme in (12). Tv qWlense strain TE 1290 wa obtained frm Dr A.RoOrmy of the Centre for Tropical Veterinar Medicine, Eiburgh. T.evansi and T.b.rhodesiense INA were the kind gifts of Dr R.O.Williams, I.LR.A.D., Nairobi, Keya. T.or,osi IZA was isolated from a culture of T.orusi. geerously provided by Dr Michael Miles of the London School of Hgiene and Tropioal Medioine. Riboomal Sk and nme1la DNA from T.bbruoei were prepard acoording to published es (1,13). Restriotion encomoalxea were obtained from CL, Jew Egland Biiolabe and DEL. Polynaleotide knasw obtainme from PL Diochomioals, exouclease from New Engla Diolabs, T4 IA ligas and DNA polymere I from DCL. Cellulose nitrate paper usd for Soutern an Northern blotting was obtained from Sohleioher and sohuell End ibel was performed using p chased from bo-th Amrshas International and. mN. All chemieals were of Analar grade or better. Southern blott+iu Nuclear NA was digested with restriotion endomoleases under a riato conditions and frationated o 0.8% a egels in TM (90 mM Tris, 90 ml Borio acid, 3 m ERNA# pH 8.2). Each well reoeived I ug of INA. Iels were run for 12-16 hours at 2 V/os, and Southern blotting was performd acoording e to pablished Procedures (14). All gels were calibrated with sars ro
oribed
Y[32p]
6748
Nucleic Acids Research by digestion of x MA with Boo RI and Hind III. Ribosomal RNA wa 5 endlabelled, after partial base cleavage, using y [2PJ A!P and polyucleotide kinase (15). Where plasuds containing ifJA gene inserts were used as probeg, they were radiolabelbii by nick tanslati0n (16). (enomic Bgl II
fragments
oloned into the Baa HI site of pAT 153 (17). Following li3gtion of Bgl II digested molear DINA with Barn HI-cleaved pAT 153, the ligation mixture wa extracted with phenol and ohlorofors and the aqueoas Phase ethuoL-preoipiated. The pellet wa dissolved in a small volume of water and was re-digested with Baa HI. More than 500 AapTet- clones were obtained by transformtion of E. coli strain BB 101. Clones were screened for the presence of rENA gne inerts by ooloiy bybridisation (18) with radiolabelled zMA. Restriction Emina Restriction maps of genomic rHIrA genes were constnwted by single and double digests, and Southern blotting using the apprpriate labelled probes. Clones were mapped by the en-labolling patial digestion tecbnique of Smith and Birnateil (19). Dot blots Exoimolease III digestions and "dot blots" were both performed using published prooedures (20,21). were
Northern blottinr KrA from the small andt large ribosomal subunits was dissolved at a concentration of 10 pag/jl in distilled water, and Ip1 was added to 10pl of PEA buffer (20 mM M PS-aetate, PR 7.0, 1 mI EITA, 5 ml sodium aoetate) oontaining 50% formaldebyde, 10% sucrose and 0.2% PB (4). Samples were heated at 650C for 5 minutes and oooled to room temperature before loading on a 1.2% agarose gel in SEA buffer otaini 3.8% formaldehyde. The gel wa run overnight at 2 V/cm, with recirculation of NSA buffer containing 1.9% forwAldehyde and one track of each of the large subunit ENA and small subunit ENA was stained in 5 )1g/1 Aoridine orange (22). The remainder of the gel wa used for tranfer to nitrooellulose filters and bybridisation to DNA probes
(22). Electron microsoos Analysis of INA/INA heteroduplex molecules in the electron microscope was carried out using standard published prooedures (23). So 3a as fent from aINS Pre2pGEltion of 3 if was Plsuidl PCH 330 digested with Son 3a, and the fragments were separated 6749
Nucleic Acids Research on a
1.2% apros gel in TME. The el
was blotted onto
IE81 paper and the
specific fragent eluted (24).
HE5s1VPS
M!E2pin
of ribosomal RIA enes in the T.b.bruoi WenoMe b Southern blotting lnoadi MlA from .b.bruoei (NITat 1.6) wa digested with various restrtd on agaroe gels and were iction endouaaoleases, the fragmnts were blotted onto nitrooelbalose filters. The filters were then bybridised to 5' 32p end-labelled total zNA from T.b.bruoei. A single band of hybridisation Sal I (21 kb) was visible on digestion with the restriction endomolea whereas d III produoed two bands (11 kb and 10 kb) as did Boo RI (8 kb and 7 kb) and Bgl I (5 kb and 3 kb) (FiHrel). To eluoidate where the lage and smal sunmit rENA were located an the Bgl II and EBo RI fnragmnts, [32p] Ap a from EMA-dissooiated riboso_al subtmits was ybridised to Southern blots of geomio 13A digested with Boo RI, Bgl II and oeo RI + Bgl ITI. Small ubunt [32P] rNA bybridised to: 1) the 8 kb Eoo RI band; 2) the 5 kb Bgl 1: band; 3) the 4 kb Boo RI + Bgl II bad (Figure 1 tracks 8-10), The faint bybridisation to the 7 kb Boo RI fr nt d tho 3 kb Bgl II fragment in tracks 8-10 ms reflect sequence homologies between the subunit rERAs, but is more probably due to slight cross-contaInaftion of the ElYTA subunits. The t
2
3
4
5
6
7
8
9
10
21 0--s-
78O
-
_-s 4.0--
30U
Fiare 1. Autoradiogra of Southe ,lots of nuolear IA from T.b.buoei (NITat 1.6) bybridised to i) total rUP]rA (tracks 1-4); ii) [32P]rRA f the large ribooo_al subunit (tr 5-7) and iii) 32p rmNA from the small subuit (tracks 8-10). The 1A was digested with Boo RI (tracks 1 7,10); Bgl II (tracks 4,6,9); Boo RI + Bgl II (tracks 5,8); Hind III (track 2$ and Sal I (track 3). In this Figur and in Figures 3 and 4, the sises of major bands, in kilobase pairs, are indicated at the side. 6750
Nucleic Acids Research
32p-labelled large subit ENA hybridised
to the same rstriotion fragments as total iSNA. However, clos e nation of the origlnal autoradiograph revealed that in track 5, the 3 kb fragent hybridised more strongly tha the 4 kb f nt and in track 7, the 7 kb fragment hybridised. acre strongly than the 8 kb fragment. These features were reprodible in seeral expriments. From these data a restriotion map of the zkA gone was onstructed (Figure 2). Thus the mafl subit is ooded entirely within the 4.0 kb Boo El + Bgl II fragment. The large isubit 3IJA is 4100t20 bases long, and is coded by part of the 4.0 kb Zo ER + Bgl TI fragment, all of the 1.0 kb Boo RI + Bgl II fragmnt and at least 2.2 kb of the 3.0 kb Bgl II fragment. All eukaryotio rEA genes studied so far ontain a trascribed spacer betwee the genes for the large and sall submit rWA.lThis spacer contain the sequence coding for the 5.8 S zEJA (8) . The poition of the tcribed spacer in T.b.bruoi w d .bi b o g hybridisation of sNA (equivalent to the 5.8 S RA of other Akaryotes, our unpiblishod data and reference 25) to the relevant restriotion fragments of the rENA clones (data not show). This g.nio mc p took into aount all the strong bads of hybridisation that were observed. However, whenL using [32p] t§NA of high spoific activity ( > 10 pCi/pg) frm the lage submit, additiaal hybridisation to high molecular weight ban and to a few other bands at a lower intensity, was also obsorred. It ha been established in other systens that not all gsne sequenes presmed to belong to a tandemly r ated sltigene family are nessarily present within the repeat. Some of them q be dipersed in other parts of the gsoume (26).o The additional hybridisation observed m be due to such a phenoomeon. Specifically, the faint bon at 4.1 kb in th Bgl IT digest (Figure 1, track 6) was
ine"tigmted further.
rENA genes in different serodemes of Tob.b3mcei INk from two variants of th TAR I sroe and two Variants of the ILIAR I serode was analysed. by Southern blotting. From Figure 3, it is apparent that the majority of rREA genes are invariant in eaoh of the four tpaosome clone, bit that the umnaual 4.1 kb rRNA gene fragment is present
sas.RNA
l.s.RNA
transcribed spacer with sRNA3
1k
Figur 2. Restriction map of the rEA gens. - Boo RI sites; v sites; n.s.8A - small submit rESA; 1.s.EnA - large submit rENA.
-
Bgl II
6751
Nucleic Acids Research a
b
b
a
*_
a
b
b
a
5
4W
S O -.t
0
-c-S O1 .--04 -40
S.
1
2
3
4
Fisar 3. Antoradiogra. of Southern blot of maolear IA from 1) T.b.bruoei IlIRat 1.26; 2) T.b.bruoei Mat 1.2; 3) T.b.bruoei IITfat 1.21; 4) T.b.bruoei raIt 1.6, digeted with a) Zoo RI + BIg II at b) Bgl f. Total t3P]A w used as probe.
only In rat 1 .2 (FPir 3, trak 2b) ad rTat 1 .6 (Pigr 3, track 4b). This variant gene fragmnt wouil therefore sem to be of relatively reoent origin a is probably speiflo for the 1AR serodLem of T.b.bruoei. Oloai of the SEA ess T!o stAdY the rEEk gnes from rat 1.6 of T.b.bruoei , Bgl II fragmnts were cloed into the aM EI site of PA 153 as desribed in Materials and Methods. A nmber of rMif-positive clones were obtained: pc 174 (5.0 kb insert) pci 331 azt 332 (3.0 kb insert), at pcE 330 (4.1 kb insort). F both tbh sines of the inserts and the Southern blotting experiment shon in t Figur 4, it wa conoludd that pi 174 contained the 5.0 kb Bgl II f atd pc 331 contained the 3.0 kb Bgl II fragment. Plasmid Pc 330, in addition to hybridiing to the 3.0 kb Bgl II at 7.0 kb Zoo RI bed, bywbridised to a large aber of other bends, sggesting that it contained sequences in addition to rEk*. Restriction mepn of the clon showed that Pc 331 atd Pc 332 catained the 3.0 kb Dgl IT fraent in oppoite orientations. The restriotion map of PM 174, 330 and 331 ar shown in Figur 5. Oriatation of the asnomi Ml II framse in ciM 174 at c 331 Orientation of the 5.0 kb Bgl II fr nt pc 174 wa indioated by the position of the Zoo RI site. Tha comfirmed by determining the sense 6752
Nucleic Acids Research 1
2
3
4
5
6
3800-w
Figure 4. Autoradiogras of Scuthera blot of nuclear MA from T.b.bzuoei (NITat 1.6), digested with Bgl II (tracks 1,3,5) and Eco RI (tracks 29496). The blots were hybridised to probes labelled by nick transltion of pCU 174 (tracks 1,2); pM 331 (tracks 3,4) and pCE 330 (tracks 5,6). strand for the small suma it rINA in p(E 174. For this purpose, p(M 174 was digested with a) Eco RI and b) Sal I. Half of each digest wa then treated with ezomo lease 1III. The Zoo RI/ezo III digest mast roduce four single stranded fragmnts as shown in Figre 6,1, designated 174 El-4, Teatment with Sal I sad exo III mast produoe two single stranded fragmnts, named 174 S1 and 174 S2 (Fig. 6,2). All the digested DNA was denatured, serially dil-
Al1
J
v
sRRA3 I.s.RNA2.3kb
s.s.RNA B I. RNA 1.8kb
t. * t ,I.
ci 1 kb
Figur 5. Restriction maps of A) p(H 174; B) p(N 331; and C) pGE 330, showing the location of the three lage rNA molecules from the two ribosomal sumits. The hatched region on p(E 330 indicates te presoee of an isertd seqaenoe. Ban Key to rostriction eooe sites: | 1 I/Bgl II bybrid site, oonstituting a Sau 3a site in the bybrid plid; 4, Sau 3a; V , Hid III; V Pvu II; C, :Boo RI; * Hino II., The Sa 3a site marked with an asterisk denotes a site in p(N 331 which is absent from pM 330. .
6753
Nucleic Acids Research uted, and spotted onto nitrooellulose filters. On hybridisation of the filters to [32p] rRNA from the small submit, both the Boo RI and Sal I digests were positive before exo III treatment. After exo III digestion,, the Sal I digest oontimes to bybridise to small subwit r1iA , indicating that the antisense strand was 174 SI. lThis was confirmed by the Eoo RI + exo III digest, which showed maoh less bybridisation, In this case, a large part of the 174 SI fragmnt, corresponding to the opposite strand of the 174 S1 was removed, but some remaining antisene 8strand produced a small amount of hybridisation
(174E2). The orientation of pGH 174 with respect to pGN 331 and pGE 332 was determined in a similar experiment. Restriction mapping implied that pCE 331 and p(H 332 were in opposite orientations, Therefore either pGK 331 or 332 had to be in the same orientation, relative to the plasdd as pOE 174. In simltaneos experiments, pM 331 sad PM 332 were digested with Sal I or Cla I. Half of eaoh digest was treated with exo III. !The predicted single strand fragments are shown in Figure 6, 3-6. The denatured digests were spotted onto nitrooellulose filters as before, and the filters were probed with [2p] xA frm the large submit. From the observed hybridisation, it is clear that in paH 331, the antisense strand is 331 S1, whereas in pOE 332, it is 332 Cl. Thus the inert in pOE 331 is present in the same orientation as that in pCE
174. Position of the three are rENAs on PM 1 and pO 331 From the orientation of the two Bgl II fragmwnts in the olones, and the genomio rHIA map, a map of the small subunit RNA and the large submit RNA in POI 174 and pOE 331 was construoted. However the relative positions of the two large sbubit RNAs of 2300 bases and 1800 bases was not olear. This was resolved by Northern blotting of larg and small submit rENA from an agaroseformaldehyde gel, and using nick-translated pO 174 as probe, Of the two rRNA bands visible in the larg mabanit track (Figur 7, track 1), it is mainly the 2300 bases band whioh hybridises to pGE 174 (Figure 7, trak 3). A oomparitively small amount of hybridisation is seen to the 1800 bases band, probably due to ross-bybridisation within the rENA gene sequenoes. The small subunit rENlA band of 3000 bases also bybridises to pOE 174, as expeoted. Thus the positions of all three large rEJAs on pGE 174 and PM 331 were established as shown in Figure 5, with the restriction maps. All of the small submit EllA of 3000 bases is ooded in pOE 174. Wost of the 2300 bases large sbunit RNA is also coded by pOEX 174, but up to 500 bses of this ElA are prosent in pO 331. All of the 1800 bases large submit NRA is present in pM 331. The gap 6754
Nucleic Acids Research 1
'4
Sal1 *4 S F174 3 E 174
2
2
4
74S 2
c
ial
GN3
N)
*
'C 2
:~~~~~331S~2
6~~~~~~~~
5 dat pGH332
*~~~~~~~~~~~~~~~~~33
332~~~~~~~~~~~~~33S PGH
Ig 4) p 331 X+ Sal Tg 5) pAl3320+CaIg6
332+SalI
tth
ih
Fignre 6. Determination of sense strands in pCN 174, PMH 331 and PME 332. The predcUted single stranded fragmentis produced from each plasmid by dig-. estion with restriction endocaolease followed in each case by exo III dig-~ estion are shown. 1) p(E 174 +EFcoERI; 2) paHh174 +Sal I; 3) Pag331 + Cla I; 4) $3m331 + sal I; 5) PCE332 +Cla I; 6) pC0332 +Sal I, At the righ-t hand side of the Figr are show the dot blots produced by hybridisation of 32P-labelled small subuit rPNA (1,2) or 32P-labelled large subnit (3-6) to serial dilutions of each restriction endonulease digest followed either by eto II treatment (+) or no eto III treatment (-). between the two large subuit Elks in pG4 331 contains some of the small E assooiated with the ribosomes of T.b.brucei (manascript in preparation). niwrte !e 20330 in PW The precise relationship of pC3 331 to pG3 330 wa shown by forming a heteroduplex between the two molecules cleaved with Cla I, and observing it in the eleotron microscope (Figure 8). The two molecules form a heteroduplex for the first 1.6 kb of the insert length. At this point, the heteroduplex is interrupted by a single stranded loop of 1.1 kb, presumably derived from pCZ 330. Beyond the single stranded loop, the heteroduplex is again oomplete. 6755
Nucleic Acids Research 1 2 3
4
Fiare 7. Identifioation of the arge subunit rNRA encoded by pGR 174. 1recks I and 2, acridine orange-stained aprose-forald.eyde gal showing the large ribosonal TWAs frm T.b.brioci. Track 1
30 23
1
' r' I' 1j8
A
Vlarge subuit rRNA; track 2, small subuit rENA. Traoks 3 and 4, autoradiogram of a Northern blot of i _aduplioate gel using nick-traslated pME 174 as _probe. Trak 3. lrgs subuit riNA; trac4k ll subunit rRNA. Sise of the riNks are shown in kilobaes.
Therefore pOR 330 is homologoas to pGC 331 with the exception of a 1.1 kb inserted sequence. The position of the insert in PM 330 dedued. from the heteroduplex measarements is shown on the restriction map in Figure 5. To determine whether the additional bands of hybridisation seen with paB 330 in Figure 4 were due to the presence of this insorted sequenoe, a 530 bp San 3a restriotion fragmnt derived from within the insertoed sequene (Figure 5) was isolated and used as a probe in a Southern blot of genomio lNA from XITat 1.6 digestod with Boo RI and Bgl II. The fragment hybridised to produco a complex pattern of bid (Figur 9) which would seem to be derived from a repeating
3,~ ~
.33
-.
;
3
73:
tic representations of htereFigure 8. Eleotron mioanrds dia uplexes formd between Cla I-cleavd. PM 331 and pM 330. Scale bar indioates 1 rioro. 6756
Nucleic Acids Research .t
FPima 9. 1tioradiogram of a Southern blot of nuolear INA from T.b.bruoei (Nat 1.6) digested with 1) Eco RI + Bgl II; 2) Bgl III 3) Boo RI. The blot was hybridised to probe prpared from the 530 bse pair Sm 3a frament derived
21-70-0-
entirely from
within the
inserted sequence
of
330. Position and sis5 of molecular weight MNA markers is shown at the side.
p(M
4-27_-}
0O83-w &56-o
family.
gene
tedly
to
servation
It
bybridiseed
also
fragment
a
of
sawe
the
suggested that
330
hybridised
isatios
The genome
of
in the
insert
in
Therefore,
genomio
PMN
T,bbroei
ferred that
However,
3.0 kb
Bgl
the
thus mobile
a
fragment,,
II
Bgl
a
3a
Smu 3a-digested
sand
unexpec-
fragment.
II
530 bp Sax
was
This
PMR 331,, to
ob-
also present
pan
fragment from
hybridisation observed
a
hybrid-
no
3.0 kb Bgl
to be fortuitous.
seem
seqluence
present in
from its variable presence
constitutes
it
kcb
the
of
blot would
330 is
sand
4.1
when the
Southern blot
a
observed.
fragment
II
to
as
part of the inserted sequence
some
in the normal rRNA genes.
the
to
sise
element,
>
20
copies in the
in rENA genes
referred to
as
it
is
in-
RINM (Ribosomal
Nobi le Element).Differential
presenoe
of
the
In order to establish
peated
sBequences
from RINK erent
10,
used
was
serodemes
the RINK
serodemes
within
of
as
is
a
sand
pattern of the RINK
genus Trysoo,th
probe
in Southern blots
and
of total genome
from different
mobile
the
element
pattern Close
fsmily of
of
re-
530 bp fragment derived
species
As
extensively repeated within both the
T.b.bruceij, although
would be expected for
in members of the genus
reetn
the extent
Tbbuoi
sequence
of
a
the
RINE
M3A
from diff-
shown in
IJJTAR
I
hybridisation differs
inspeetion
of
the
Figure
sand
NITAR as
original
6757
Nucleic Acids Research 1
"a
if
2170-
4.27.348-
_
2 3 4 5 6 7 b a b a b a b a b a b a b
5. :W,* L
-
..
-.
_
-
iF
*:
1 98-.
4. 4 i.
_ _ _ _
q
s
1 59-.
a
.a *t
_ _
.:
_
_
^.
0*94_-
_ F
Figure 10. Autoradiogram of a Southern blot of nuclear INA fro 1) T.b.bruoei NITat 1.2; 2) T.b.bruoei NTat 1.8; 3) T.b.bruoei IIrat 1.6; 4) T.b.rhsiense; 5) T.wansi; 6) T.congolense; and 7) T.orusi. The IA digested with a) Eao RI and b) Hino II md the 32P-labelled 530 base pair RIME-derived fragment was used as probe. was
autoradiograph suggests Bsmll differencos between different olones from the NITAR 1 serodeme, and this possibility is being investigated further. The sequence is present in approsimatoly equal abndae in the genowe of two other mebers of the subgenus TEpM In contT.b .rhdosioense and T.evansi* rast, representatives of two other subgenera, T.(N oM o ) oongolonse and T.(Sohizotry-nuu) orusi, show only a very few bands of hybridisation. oon
DISCUSSION The
organisation of ribosomal
eukaryotic organisms,
and in
all
RNA
oaes
genes has been studied so
far,
the
genes
are
in
a
found
number of to be
rep-
eated (8). The organisation of the rRNA gense in Leishmania donovani, another member of the order K i f, is of particular relevance (27). In this 6758
Nucleic Acids Research organism as well the large ubunit rRNA is niocked and consints of two molecules, suoh as in T.b.brucei. The sNAs associated with the ribosomes in Tl7Panosomatids have not been reported in L.donovani. The sequential arrangement of the variai kziow zUA& was determined in L.donovani by looking at nINAt rINA hybrids in the eleotron mioroscope. The organisation is the sam as that observed in other eukaryotes* This is i) a region of non-transoribed spacer; ii) an internal tramoribed spacer; iii) the sequence encoding the small subunit HA; iv) an internal transcribed spacr that oontains the sequenos encoding the 5.8 S rENA; v) the sequenoe oncoding the large subunit rRNA. In the oase of L.donovani the latter oonsists of two regions encoding the two large subunit rHAs separated by a small gap. In T.b.brucei, the rENA genes are arranged in the same general pattern, and particularly the two large subuit riNks are alcO separated by a gap. However, the presenoo of five as opposed to two (or three in Drosophila) siks produoes ortain unique features whioh will be presented in a seprato oomunioation. While the general organisation of sRNA genes is very similar in all oukaryotes, the sise of the actual repeating unit can vary considerably. This length heterogeneity is usually due to differenos in the extent of the nontranoribed spacer. From the data presented in Figure 1, the length of the repeating unit in T.b.brucei would appear to be at least 21 kb. This wa deduced from the Boo RI, Hind III and Sal I digests (Fig. 1, traoks 1-3). In the Boo RI track there are two bande of hybridisation at 8 kb and 7 kb, suggesting that the repeating unit was at least 15 kb long. In the Hind III track thore is one strong band at 11 kb, and a faint band at 10 kb, suggeting a repeating unit size of 21 kb, with a Hind III site olose to one end of the coding sequences (presumably the Hind III site mapped in p(H 174). The single band seen in the Sal I digest is of 21 kb, suggeting that Sal I acut the repeating unit once only. If it is "as d therefore that the repeating unit is 21 kb in length, then from the map of the AENA genes presented in this paper we can deduo that the non-trascribed spacer is approimately 13 kb (compared with onl 5.85 kb in L.dooi) . Ribosomal HA coding sequenoes internpte by non,ooding regions have been foand in y"h , Tot a---Cdoai (2) and DrosgUla, (29). In however, the interrnptions are due to inserted sequences and genes Drospyhilag then show low of levels containing very transoription (30). We have no information regarding trnsoription of the particular T.b.bruoei gene oloned in pCN 330, but the difference in at least one Sea 3a restriotion site (marked with an asterisk in Figure 5) from the typioal ribosomal gene repeat suggests that the gene ay be non-functional. 6759
Nucleic Acids Research Inserted soquences present in D rDNA are of two types, one of which (Class I insertions) shows certain features oommon to transposable elements. They have short repeats at their ends, and are also found elsewhere in the genome, interspersed with other mobile elemnts (31). The inserted sequenoe found in a rIdJA gene in T.bbruoesi is similar to the Class I insertion of Dro la in that it shows different patterns of bybridisation within two strains of T.b.bruoei, i.e. the MITAR 1 and IJJEAR I serodemes, and within different species of trypanosomes. Transposable or mobile elements seem to be ubiquitous in eukaryotio genoies (10). Prooisely what tiole they pla in gene expression is still a matter of speoulation. However, the transposition of MDA sequoe is known to alter expression of certain genes in yeast (32), maise (33) and Drosoph1la (34). In addition, the expression of VSG genes in T.b.bncei is now known to be controlled thiougi tranposition events, Specifioally in trypanosomes it has been sugeted that transposable YSG genes arose by interspersion of mobile repeated lEA sequences among ancestral VSG VSG genes genes (35). Subsequently, repetitive DA has been foand f The mobile element we have be a identified oould well suoh sequence. (9). Work urrently in progress on sequence analyis of the relevant portions of paE 330 and pGH 331 will shed further lUht on the nature of this mobile element.
We gratefully acknowledge the expert technioal assistance from Roger Williams and Bither Choolun. One of us (G.E.) is in reoeipt of support from the INLAKS Foundation, tho Welloome Trust, and a British Government Overseas Research Student Schoeme aard.
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