Molecular and Biochemical Parasitology, 35 (1989) 67-72 67 Elsevier

MBP 01150

C h a r a c t e r i z a t i o n of the r i b o s o m a l D N A f r o m Trichinella spiralis

David Boyd l, Theo deVos 1, Glen Klassen 2 and Terry Dick 1 I Department of Zoology and 2Department of Microbiology, University of Manitoba, Winnipeg, Manitoba. Canada

(Received 30 August 1988; accepted 9 January 1989)

Ribosomal DNA (rDNA) from three isolates of Trichinella was cloned into phage and subcloned into the plasmid pBR322. The basic repeat unit of rDNA was variable in size, with the mapped clones ranging from 10-12 kb. There were differences in restric- tion sites within the genic region among the three isolates which were due to variations in the internal transcribed region (ITS) and the intergenic spacer (IGS). Three RsaI sites were mapped to the IGS repeat unit of the isolate AF1, and one RsaI site was mapped to the IGS repeat unit of Trichinella spiralis pseudospiralis (isolate Tp). The number of repetitive units in the IGS region varied markedly within and between the isolates. It was estimated that the basic repeat unit for the rDNA of isolate P1 was 10.6--28 kb, for AF1 10.7-37 kb, and for Tp it was 11-14.9 kb. There appeared to be a greater frequency of some sizes of the basic repeat unit in each of the populations, based on the relative intensity with which certain bands hybridize to the probe.

Key words: Trichinella spiralis; rDNA; Internal transcribed spacer; Intergenic spacer; Restriction mapping; Repeat unit

Introduction

The r D N A of mos t e u k a r y o t e s consists of tan- d e m l y r e p e a t e d units compr i s ing , f rom 5 ' - t o -3 ' , the i n t e rgen ic space r ( I G S ) , smal l r R N A coding sequences , the in te rna l t r a n s c r i b e d spacer ( ITS, inc luding 5.8S r D N A ) , and large r R N A coding sequences [1,2]. A l t h o u g h r D N A is highly con- se rved , m u t a t i o n s can occur in all reg ions [2,3]. Poin t mu ta t i ons , va r i ab i l i ty in ' expans ion seg- men t s ' [4], p r e s e n c e of in t rons in r R N A coding sequences [5], and inser t ions and /o r de l e t i ons in the space r reg ions , a re all r e spons ib l e for r D N A p o l y m o r p h i c var ian ts . The I G S of ten shows in- t e rna l r epe t i t i ons and a va r i ab le n u m b e r of these r epea t s p r o d u c e d i f fe rences in the overa l l length of the en t i r e r e p e a t unit .

This p o l y m o r p h i s m in r D N A has been ex- p lo i t ed successful ly to d i f f e ren t i a t e b e t w e e n spe-

Correspondence address: Terry Dick, Dept. of Zoology, University of Manitoba, Winnipeg, Manitoba, Canada, R3T 2N2.

Abbreviations: ITS, internal transcribed spacer; IGs, inter- genic spacer; P1, pig isolate 1 of Trichinella; AF1, arctic fox isolate of Trichinella; Tp, Trichinella spiralis pseudospiralis

cies, s t ra ins and sex in the t r e m a t o d e Schisto- soma, using c loned segmen t s of the r D N A as p r o b e s [6-8]. These same p r o b e s have been used to de tec t in ter - and int raspecif ic d i f fe rences in the ces tode Echinococcus [9].

In our s tudy we have i so la ted c lones con ta in ing r D N A f rom genomic l ib rar ies of Trichinella iso- la tes (P1, A F 1 , TP, ) and used t h e m to examine the o rgan iza t ion of r D N A in the g e n o m e s of the th ree isolates .

Materials and Methods

Trichinella genomic D N A isolation. Biological and b iochemica l da t a on the i so la tes ; Trichinella spir- alis P1 (from domest ic pig), AF1 (from arctic fox) and Tp (T. spiralis pseudospiralis) worms has been pub l i shed e l sewhere [10,11]. Bulk D N A was iso- la ted f rom 30-day-o ld la rvae by a p ro t e ina se K / p h e n o l e x t r a c t i o n / R N a s e m e t h o d , as desc r ibed

[121.

Blotting, hybridization, and labelling o f D N A . Res t r i c t ion digests of D N A s were b lo t t e d [13] on to G e n e s c r e e n Plus ( N E N R e s e a r c h p roduc t s ) . Plaque blott ing was onto nitrocellulose filters [14].

0166-6851/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

68

Hybridizations were performed at 55°C or 65°C in 1 M NaC1, 1% SDS containing a heat-dena- tured probe that had been labelled with 32p by nick translation [15].

Cloning of rDNA. The construction of a P1 gen- omic library in EMBL3 has been described [16]. Similarly, AF1 and Tp genomic libraries have been constructed in EMBL3. Plaques for screen- ing were produced by infection of Escherichia coli P2392 (Stratagene) by standard methods [14].

The P1 genomic library was screened with sub-

(A) hTsP1- r15

P G P

/(( large ;

(B) ~.TsAF1 - r 12,5

E

~4

G S S P G P

small ' large i :

pTsP1- GS5.2 ~ ' : :¢ ~; pTsPI-ITSI.4

S G S S P G

l k b

pTsAF1- IGS5.7

(C) ZTsTp- r16

P G P

J

i,

I kb

pTsTp-IGS5.8

P G S S p G P

; : 1

" ! . .÷~1 .8 '1 k~'

Fig. 1. Location of restriction enzyme sites for the ribosomal genes of three strains of Trichinella. IGS, Intergenic spacer; ITS, internal transcribed spacer. (A) hTsPl-rl5, a lambda clone of the P1 ribosomal unit. The positions of the large and small subunits were mapped by hybridization with specific gene probes (pCe 2.3 and pCel.8) from C. elegans. The IGS and ITS regions of hTsPl-rl5 were subcloned into pBr322 to form the plasmids pTsPI-IGS5.7 and pTsPI-ITS1.4. (B) hTsAF1- r12.5, a lambda clone of the AF1 ribosomal unit. The posi- tions of the large and small subunits were determined by com- parison with the P1 map. The IGS region was subcloned into pBr322 to form the plasmid pTsAFI-IGS5.7. (C) hTsTp-rl6, a lambda clone of the Tp ribosomal unit. The position of the large and small subunits were mapped by comparison with the P1 map, and the IGS and ITS regions were subcloned into the plasmids pTsTp-IGS5.81, and pTsTp-ITS1.8. B, BamHI; E, EcoRI; G, BgllI; H, HindlII; O, XhoI; P, PstI; S, SalI; X,

XbaI.

clones of the plasmid pCe7 which contains a 7-kb BamHI fragment representing a single rDNA re- peat unit from Caenorhabditis elegans [17]. Du- plicate plaque blots were hybridized (at 55°C) with a subclone containing approximately 90% of the 18S rRNA gene of C. elegans, and with a sub- clone containing approximately 65% of the 3' end of the 26S rRNA gene of C. elegans. A plaque which hybridized to both probes was picked, the phage amplified and its DNA isolated [18]. This recombinant phage was designated hTsPl-rl5 (Fig. 1A). The ll.4-kb BamHI fragment from the insert in hTsPl-rl5 was purified from an agarose gel with Geneclean (Bio 101) and used as a probe to screen the AF1 and Tp genomic libraries. Hy- bridization was at 65°C. A positive plaque was picked from both libraries and the phage DNA isolated as above. The AF1 clone was designated hTsAFI-rl2.5 and the Tp clone designated hTsTp- r16 (Fig. 1B,1C). Fragments were subcloned from each phage into pBR322 as indicated in Fig. 1.

Other methods. Plasmid DNA was isolated by al- kaline extraction [19]. The RsaI sites in pTsP1- IGS5.2 and the RsaI and PstI sites in pTsAF1- IGS5.7 were mapped by the indirect end labell- ing method [20].

Results

Organization of the rDNA. Physical maps of the inserts in hTsPl-rl5, hTsAFl-rl2.5, and hTsTp- r16 were constructed using several restriction en- zymes (Fig. 1). The small and large rRNA coding sequences were mapped to the physical map of hTsPl-rl5 by hybridization with C. elegans rDNA subclones containing 18S (pCel.8) or 26S (pCe2.3) coding sequences (Fig. 1). The small and large rRNA coding sequences in hTsAFI-rl2.5 and hTsTp-rl6 were positioned by comparing their restriction maps to that of hTsPl-rl5 (Fig. 1). hTsPl-rl5 and hTsTp-r16 contain repeated large rRNA coding sequences. In addition both hTsAFl-rl2.5 and hTsTp-rl6 contain a number of short tandemly-repeated sequences character- istic of those found in the IGS of eukaryotic rDNA [2]. The restriction maps of the genic re- gions (small rRNA gene-ITS-large rRNA gene) of the three strains are almost identical, but there

1 A 2 3

B 1 2

C 3 1 2 3

-5D

i

4 m m 4.6

-0.5

Fig. 2. Hybridization of an 11.4-kb BamHI fragment of ~TsP1-r15 to digests of genomic D N A from (1) P1, (2) AF1, and (3) Tp. Digested D N A was run on a 0.8% agarose gel at 35 V for 16 h, and blotted on Genescreen Plus. The restric- tion enzymes used were (A) EcoRI; (B) HindIII; and (C) XbaI. Sizes are in kb, and were determined with the BRL 1-

kb ladder.

are a few restriction site differences (Fig. 1). To determine the organization of the rDNA in

the genome the clones were hybridized against blots of single and double digests of the respec- tive genomic DNAs (data now shown). Fig. 2 shows side-by-side single digests of the genomic DNAs probed with the ll.4-kb BamHI band from kTsP1-r15. Analysis of the genomic hybridization patterns showed that the organization of rDNA in the genic regions in the genomes of the three strains, is the same as in the genic regions in the respective k clones. The genomic hybridizations also revealed that in the AF1 genome, as in the P1 genome, there is a 0.4-kb EcoRI fragment at the 3' end of the large rRNA coding sequences, which is not represented in ~TsAFl-r12.5 (Fig. 1). Most genomic digests from any strain (except AF1 PstI or TP SalI digests), when probed with the respective ~ clone, showed in addition to bands originating in the genic regions, a hybridizing high-molecular weight smear (5-20 kb), but in Tp, the range of the smear was less than in either P1 or AF1 (Fig. 2).

69

Heterogeneity in genic region. Restriction map- ping of the rDNA clones revealed both intra and interstrain differences for a number of restriction enzyme sites in the genic regions. The distribu- tion of HindlII sites differs between the two large genes within the P1 clone kTsPl-rl5 (Fig. 1A). HindIII digestion of this clone produces 1.25 and 1.35 kb fragments. However, probing of the HindIII genomic DNA reveals only the 1.25-kb band, indicating that this is the more common or- ganization. HindIII, PstI and EcoRI sites for the large gene also differed between strains (Fig. 1A-C; Fig. 3A).

A 0.4-kb insertion was found in the ITS of Tp relative to this region in P1 and AF1 (Fig. 1). Re- striction mapping of the recombinant plasmids, pTsPI-ITS1.4 and pTsTp-ITS1.8, revealed that

(A)

P1

A F 1

rp

E S E H

, I 'i ', L : s m a l l ' l a rge '

S S E P H B H

o~47~i ,75 'o5' . . . . •

I045 075 _ ± --__J

i ]! I ' i 1053L~ . . . . 175 0 6 O 7 5 ,

1.0 kb

0 . 5 k b

(B) E

, I s m a l l

,

ERHpX

p T s P I - I T S 1 . 4 ~ /

S E H

l a r g e

RCR U S

1.0 kb

E RHp X A N R U HpR R A S

Fig. 3. Heterogeneity in the large ribosomal subunit (A), and the internal transcribed spacer (B), in Trichinella. (A) Com- parative distribution of restriction sites within the large ribo- somal subunit between P1, AF1, and Tp. B, BamHI; E, EeoRI; H, HindIII, and P, PstI. (B) Restriction site map of pTsPI-ITS1.4 and pTsTp-ITS1.8 to determine the position of the 0.4-kb insertion in the Tp ITS. A, AvaI; C, ClaI; Hp,

HpaII; N, NruI; R, RsaI; U, PvuI; S, SalI; X, XbaI.

70

the insertion is localized in a 720-bp RsaI frag- ment in Tp, which contains a PvuII site and HpaII site not found in the homologous region of P1 (Fig. 3B). Restriction mapping of pTsPI-ITS1.4 and pTsTp-ITS1.8 also revealed a number of re- striction site differences between homologous re- gions.

Organization of the IGS. Physical mapping of the plasmid subclones, pTsPI - IGS5.2 , pTsAF1- IGS5.7, and pTsTp-IGS5.8 , confirmed the re- striction sites previously mapped in these regions in the h clones and in addition sites for AvaI, HaeIII, PvuII, and RsaI were localised (Fig. 4). Mapping studies also indicated that pTsPI-IGS5.2 contains 6 copies of a 160-bp RsaI tandem re- peat, pTsAFI - IGS5 .7 contains 10 copies of a 320- bp PstI repeat , and that pTsTp-IGS5.8 contains 5 copies of a 280-bp SalI repeat . Further mapping, using indirect end-labelling [20], showed that each PstI repeat in AF1 contained 3 RsaI sites, and each SalI repeat in Tp contained one RsaI site (Fig. 4).

To determine the range of IGS sizes and

(A) pTsP1- IGS5.2

HR R RR RR RRRRRRR R R G O / S

H a E H a E C Ha A U X A

o . ~

(B)

(C)

pTsAF1 - IGS5 .7

,illllflEii llillll IlilitiitllljJ E C R = U X

o . ~

pTsTp - IGS5.8

HR R PR P R R R R R R P R G O /S

lIij, llt lllllll I l Ha EHaCA A S S S S S S U UX A

o . ~

Fig. 4. Restriction maps of the intergenic spacer regions of (A) P1, pTsPI-IGS5.2; (B) AF1, pTsAFI-IGS5.7; and (C) Tp, pTsTp-IGS5.8. Restriction enzymes as in Figs. 1 and 2, ex- cept Ha, HaelII. R*, all lines within bracket refer to RsaI sites.

A B C D E

30- 21-

75-

/

74- i 3.5-

3.6- Fig. 5. Hybridization of the IGS clones of P1, AF1, and Tp to Southern blots of EcoRI/XbaI-digested genomic DNA. Di- gested DNA was run on a 0.4% agarose gel at 30 V for 16 h and blotted on Genescreen Plus. (A-C) P1, AF1, and Tp probed with pTsPI-IGS5.2; (D) AF1 probed with pTsAF1- IGS5.7; (E) Tp probed with pTsTp-IGS5.8. Sizes (kb) were determined with the BRL 1-kb and high-molecular-weight

ladders.

whether they are due to a variable number of in- ternal repeats each IGS subclone was used as a probe against EcoRI/XbaI digests of the respec- tive genomic DNAs (Fig. 5). The genomic D N A was digested with EcoRI and XbaI as mapping of the clones showed that in each strain there was an EcoRI site near the 3' end of the large r R N A coding sequences and a XbaI site near the 5' end of the small rRNA coding sequences (Fig. 1). The EcoRI/XbaI digests of the genomic DNAs pro- duced a series of fragments spanning the IGS of the rDNA in each strain. Probing of EcoRI/XbaI genomic digests of the three strains with the IGS clones revealed heterogeneity within the three strains (Fig. 5). Hybridization of AF1 and Tp with the P1 probe (Fig. 5B,C) differed from the hy- bridization with the AF1 and Tp probes (Fig.

5D,E) because these probes are not homologous. In P1, 12-14 bands hybridized ranging from 3.6 to 21kb (Fig. 5A) and were not in an obvious lad- der. These represent rDNA repeat sizes of 10.6-28 kb. In AF1, >30 bands hybridized (Fig. 5B,D) ranging from 3 to >30 kb, and the bands were in a ladder with 320-bp 'rungs' which is the size of the PstI internal repeat in this strain. These represent rDNA repeat sizes of 10.7-37 kb. In Tp, 15 bands hybridized, ranging from 3.5 to 7.4 kb (Fig. 5C,E), in an obvious ladder with 280-bp rungs, the size of the SalI internal repeat in this strain. These represent rDNA repeat sizes of 11-14.9 kb.

Discussion

We have characterized the basic repeat units of rDNA for three isolates of Trichinella and they were highly variable in size both within and among isolates. Our clones ranged from 10-12 kb, and were similar to that reported for Ascaris lum- bricoides [21], but larger than that reported for C. elegans [22]. A comparison of restriction sites for the 18S and 26S ribosomal genes showed dif- ferences as well as similarities between Trichi- nella and both Ascaris and C. elegans, but the significance of these observations can only be de- termined when rDNA of Trichinella is sequenced. Nevertheless, in our study differences in restric- tion sites between the rDNAs of the three iso- lates of Trichinella indicate polymorphisms within the species.

Size variability of the rDNA was attributed to variation in the ITS due to an insertion in the Tp strain, as well as to variability in the IGS region of each strain. This is not surprising as hetero- geneity among repeat units of rDNA is charac- teristic of eukaryotic DNA and is attributed to variable numbers of internal repeats in the IGS region [3]. This variation in number of repetitive units is well illustrated in Artemia [23]. While the length of the IGS varies from about 2.5-5.0 kb in Schistosoma [8], it was not clear whether or not differences in lengths were due to repetitive units.

71

As indicated in the restriction maps (Fig. 4), a unique repetitive fragment is found within the IGS region of each strain of Trichinella. Intrastrain variability in the size of the IGS results from var- iability in numbers of these repetitive units for AF1 and Tp, as evidenced by the ladder effect produced in genomic blots of the IGS. For P1, the size variability may be due to a combination of unique and repetitive regions, since the hybrid- izing bands did not form an obvious ladder. For all strains, differences in band intensity indicated that certain IGS sizes, and therefore repeat num- bers, were more common than others.

The level of variation in the basic repeat unit of rDNA, and especially the IGS region among and within isolates of Trichinella, raises questions regarding the function of the IGS region. It has been suggested that the number of repeat units and length of the IGS region is important in the regulation of rDNA [24] but it was found in C. elegans [22] to be only a few hundred nucleotides long. Consequently the role, if any, of the large number of internal repeats in the IGS region of Trichinella remains unclear.

From the restriction data, it is apparent that the internal repeats of the IGS region vary between strains of Trichinella. Since these strains repre- sent the three main isolates of this species, dif- ferences in the IGS internal repeats may become valuable in developing probes for strain identifi- cation. The within strain variability in size of the IGS region could be generated by differences within or between individuals. Assuming the lat- ter, heterogeneity between individuals for the rDNA IGS suggests the potential for heteroge- neity in other traits as well.

Acknowledgements

This work was supported by a Manitoba Health Research Council studentship to T. deVos and an M H R C operating grant. T. Dick also thanks the Natural Sciences and Engineering Council of Canada for financial support.

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