FEMS MicrobiologyLetters 96 (1992) 179-186 © 1992 Federation of European MicrobiologicalSocieties0378-1097/92/$05.00 Published by Elsevier

179

FEMSLE 05040

Characterisation of a novel repetitive D N A sequence from Mycobacterium boris T.J. Doran a.b, A.L.M. Hodgson a, J.K. Davies b and A.J. Radford a a CSIRO Dicision of Animal Health, Animal Health Research Laboratory. Parkcille.Australia and h Microbiology Department. Monash Unicersity. Clayton. Australia

Received 19 May 1992 Revision received24 June 1992 Accepted 24 June 1992 Key words: Mycobacterium boris; Repetitive DNA; Pathogenicity 1. S U M M A R Y We report charaeterisation of three copies of a novel repeat sequence isolated from a Mycobacterium boris genomic library. The repeat occurs within open reading frames, potentially encoding a conserved tandem array of a pentapeptide sequence with the consensus X-Gly-Asn-X-GIy. The tandem array is present up to five times in M. boris and it is proposed that they may occur in a family of genes expressing functionally related proteins. We postulate that these proteins may play a role in binding of M. bot'is to host cell receptors.

2. I N T R O D U C T I O N Mycobaeterial disease represents a major health problem for both humans and animals.

Correspondence to: T.J. Doran, CSIRO Division of Animal

Health, Animal Health Research Laboratory, Private Bag No. 1, Parkville,Victoria 3052, Australia.

This problem is largely the result of a poor understanding of mycobacterial pathogenicity, which in turn has hampered the development of effective diagnostic and treatment programmes. One direction of research aimed at overcoming such obstacles has been the study of mycobacterial repetitive DNA. Repeated D N A sequences have been identified in a range of mycobacterial species, including pathogens. In a number of cases, analyses of these repeats have revealed insertion (IS) elements such as IS6110 [1] and IS986 [2], which were identified in Mycobacterium tuberculosis. In addition, IS1081 has been isolated from M. bm'is and is also present in M. tuberculosis [3]. M. paratuberculosis and pathogenic strains of the closely related M. avium have multiple copies of an atypical IS element, IS900 and IS901 respectively [4,5]. Martin et al. [6] have identified four copies of IS6100 from M. fortuitum FC1, two of which flank a sulphonamide resistance gene as part of the compound transposon Tn610. However, mycobacterial repeats not characteristic of IS-like elements have also been reported, namely the family of RLEP elements identified in M. leprae [7-9], a 1016-bp sequence of two 507- and

180

509-bp direct repeats identified in M. tuberculosis [10] and multiple direct repeats of 36 bp identified in M. boris BCG [11]. Repetitive DNA sequences have been primarily used as templates in polymerase chain reactions for rapid detection of mycobacteria from clinical specimens [10,12-15]. Furthermore, mycobacterial DNA repea~ promise to enhance our understanding of mycdbacterial pathogenicity. Both IS901 and IS900 have been implicated in the increased pathoge~ficit.v of M. avium type A / 1 and M. paratube~culosis from a M. avium type A background [4;5]. Elucidation of the role of these elements in this ,evolutionary process may provide valuable information on pathogenicity. In addition, since both IS900 and Tn6lO transpose in M. smegmatis [6,16], it may now be possible to use transposon mutagenesis to study mycobacterial virulence determinants. We report characterisation of three copies of a novel repeat sequence isolated from a M. boris genomic library. The repeat appears to be contained within open reading frames (ORFs), encoding a conserved tandem array of a pentapeptide sequence with the consensus X-Gly-Asn-XGly. The tandem array occurs up to five times in M. boris and it is proposed that it is associated with a family of genes that are functionally related.

3. MATERIALS AND METHODS 3.1. Bacterial strains, plasmids and media M. bov/s AN5 and M. bov/s BCG variant CSL were obtained from CSL, Parkville, Australia. Escherichia coli strains DH5 alpha and MVll90 were hosts for plasmids pUC18 and p U C l l 8 [17]. E. coli strains were grown in Luria broth [17] and M. bov/s strains in Dubos broth (Difco) or on 7 H l l plates [18]. 3.2. DNA techniques Except where otherwise mentioned, all DNA manipulations were conducted as previously described [17]. M. bov/s genomic DNA extraction was performed as follows: M. bovis cells were

harvested from 400 ml of Dubos broth by centrifugation and resuspended in 3 ml of TE buffer (10 mM Tris, 1 mM EDTA). The bacterial suspensions were then transferred to liquid nitrogen in a mortar pre-cooled in a bath of liquid nitrogen and crushed to a fine powder using a precooled pestle in a biohazard hood. Frozen, crushed cells were added to 15 ml of lysis buffer (10 mM Tris, 40 mM EDTA, 1.2% (w/v) sodium lauroylsarkosinate). Five mg of proteinase K was added and the mixture incubated for 90 min at 37°C. The mix was then centrifuged and the resulting supernatant precipitated by adding 1/10 volume of 3 M sodium acetate and an equal volume of isopropanol. The pellet was dissolved in 800/~! of TE and extracted with phenol, chloroform, and then water-saturated ether prior to ethanol precipitation and resuspension in TE. Genomic DNA from three New Zealand isolates of M. boris, M. boris BCG French (Pasteur seedlot 1173P2)and M. tuberculosis H37Rv was kindly supplied by Dr. Desmond Collins (Central Animal Health Laboratory, Wallaceville Animal Research Centre, Upper Hutt, New Zealand). 3.3. Construction of an M. boris AN5 genomic library Genomic DNA from M. bovis AN5 was partially digested with Sau3A1. Fragments approximately 3-8 kb in size were purified (Geneclean, Bresatech, Adelaide) from a 1% agarose gel and cloned into a dephosphorylated BamH1 cleaved pUC18 vector (Pharmacia LKB). The ligation mix was electroporated (Biorad) into E. coli DH5 alpha and recombinants selected on LB plates supplemented with 50 /~g of ampicillin (Sigma) per ml. 3.4. Colony and Southern-blot hybridization From the genomic library, 200 E. coli recombinants were transferred to Hybond N nylon filters (Amersham) [19]. Digests of both chromosomal and plasmid DNA were electrophoreticaUy separated on 1.0% agarose gels and transferred to Biorad Zeta-Probe nylon filters [20]. Filters were hybridized in 50% formamide, 1.0% SDS, 0.5% skim milk powder and 0.5% SSPE [17] overnight at 37°C with probes labelled with 32p using ran-

gave strong hybridization signals and were presumed to possess repeated DNA sequences. The 12 recombinants were probed against Southern blots of BstEll digested genomic DNA from three virulent M. boris strains, M. bo~,is BCG and M. tuberculosis H37Rv. All 12 hybridized with multiple BstEll fragments from each strain, producing complex banding patterns and confirming thei: r~=petitive nature (data not shown). Three clones, designated pTB189, pTB 190 and pTB191 had similar banding patterns to each other within the five test organisms, suggesting that the three cloned repeated sequences may be closely related.

dom primers [21]. Filters were washed at 65°C in 0.1 x SSC/0.1% SDS and exposed to x-ray film (Fuji RX). 3.5. DNA sequencing Nucleotide sequence was determined from single-stranded templates by the dideoxynucleotide chain termination method [22] using modified T7 DNA polymerase (Pharmacia LKB). Singlestranded DNA was produced using the phagemid system [23]. Sequence data was stored, assembled and analysed using DNASIS software (Pharmacia LKB).

4. RESULTS 4.1. Isolation of repetitive sequences from an M. boris AN5 genomic library To identify repeated DNA sequences on the M. boris chromosome, a gene library was probed using M. boris AN5 genomic DNA. Twelve clones

517~ OU

4.2. Sequozcing of repeated DNA To determine the nucleotide sequence of the repeated DNA, pTB189, pTBIg0 and pTB191 were digested simultaneously with Sinai and Sacll, Southern blotted and probed with total M. boris AN5 genomic DNA. A fragment of approximately 1 kb from pTBI89, 1.6 kb from pTB190

: CCGCr,GC~r,C ~XTGATr,r,CC GTCGCM¢CC AC;TACCTAGC c r~CTCA(;¢ ACC,r.:CCr.,CAG Cr.,CA~Cr,r.~ Ar.JU~CG~¢ (;CACAC(,CC~ r G ~ r . ~ C

pT819"5 DHA pn1197 ONA

r,ACCr,CGTTC r.,~r~r.,~,cr, c Tr,r.,Cr.,C*CC.~ C(;Tr,C~,CCG I ~

................................................................................................ T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -C-- -GC-GC-TTG CCAT-GTGG- --C-GGC-TT GC--GG-CTG C---G-TTG- G T - - - - T - - C CAGC---ATT T-CGGC-GTG C---T-SCAT -TTT--CGC- CCA*----TG C-A-A--GG - T--CI~C - - -

5171tll ONA : GCAGTGGTAGCAG¢CAATCGTGGCCTCATG CAGCTGCTGGCAGCTACGAACTGGTTTGGG ~ T C

CGGCC-CTCAT ~,ACGTCGAG CCGGCATACG/I~JU~ATGTG GGCATTGGACG T ~

TGGCTGGTTA 280

pTB193 ONA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pTS195 H A : - / ~ : C~-TG-C~C p T | l g T ONA : C-G-C-C-GA ACA-GGG--T G--GAAT--T GGCAGCTG-A A¢¢TGGGTGG -G-AAAC-TC GGTTTGTTGA ACTT-GG--G CoGGAA-TTT -GCAGCCTGA -C-TGGGTG- C-GCAACACo -GCMT--C- ACCTG--CGG $17e40NA

: CCA~TCC,AT r~GTCTGCGG CCGTCGCACAACTGGC~CC TGGC,J~./~G TCTTGCC-GAACCTCGGCATC GACATCGGCA~/b~CCACCA GATC4ATCTG ~wt~CTTCG ~ A C

CGGCAGCGGCAACN*ATCG

I~O

pTll193 DIM : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . m ............ m ............... m .... pTll'195 ONA : AGCTGCT--G IGTCT---C- --CGT-GTG" C-GCCGCG-- G-C-G-TGC- -GGG'GTCCC AGCA-Cr~-- -CG---CCGG CCCTG--*GT CGAG--CA-C m - - - G - G "G°*'TTCTT G - - - - T . . . . . . . m .... pT|lg;r DIM : AGGNUt-TGG -GC-T---CA A-C-G-GGAG CGGCAACATT G-TA-CACCA A---CG-C-- -GG-AA-CAA -G--ATCT-- -CTTT---AG CGGG--C*-C CTC---/L4t-- *A---m~'TT - - - T T T - - - A ---(iCCT--5 1 7 u DIM

: GCAAC, AACAA CAII~-ACAAC AACAACATCGGCAACAACAACATCGGCAGCGGCJUtCACCGGCACCGGCAACATCGGCAGCGGr.AACACCGGr.AGCGGr.AACCTGGC-CCTCGGC/kqCCTCG~

TATC(;GCTTC $60

p r s l ~ ONA : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~'~'=~'~ ~ ............................................................................................. pTB19S DIM : . . . . . . . . . . . G-G----G- G G - - - - - C - - - - G - - T . . . . . T . . . . . . T- --G--T-T . . . . . A - - C - - - -C . . . . . . A. . . . . . . . T . . . . . AT-CA. . . . . . . . . AG- --A---GC-o --TT-TTC-- C T - - - - - & ~ pTB19F ONA : -TG--GGA-- - C - G " T ' G " G G - - - - G - - - *T-ST-CA-- - C - - - - T . . . . . G---TTT- -TT-GTT " ' o -G-G--T . . . . . . . . . . TG- -G-TGA'T o" TA-C--GT . . . . . . . . . . . . . IA--AACo- CC-G-----T §1tom ONA : CACAACACCGGTAGCC-GGAATATCGGGTTC GGGATCACCGGTGATCACCA~TGGGTTTC GGCGG~TTCA ACTCCAGTAGCt~,CAACATC GGCTTCGGGAACTCAGGCACCGGCAACGTCGGCI'TATTCA ACTCI:GGC, AG 700 pTS193 ONA : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pX|lgS DIM : o - A - - - G A - - -C-A-ACC-- ¢T----CAG- " ' A ' A T G " " " C * ' I ' n T T C¢-CAACA. . . . . A-TGGA- --GAA---TC T - - A - - - C . . . . . . . . . . pTlllgT ONA : - - G - - . - A - -

-C-ATAAC-- C - - - - - T - - T

51"~41 ONA : C ~ T C

CIGCATCGGGAATTCGGGCTC CCTGAACAGC GGCATCGGG* * * ~ A C

pTl1193 ONA p T | l ~ DNA

---C ..........

........................ -ACC----¢G --'GG*TTC" -C-----GGA . . . . . . . .

pTi1197' ONA : T--C---G-G - - - T - - T T T -

CA-T-T -G---*CA-T --T-CG--G . . . . . . . .

C. . . . . . . . .

A-T - - - G - - - - - G

T--G-A - M - - - - A - T

. . . . . C. . . . . . . . . . . . .

A

CTCGGGGACAATC.~.ACGCGGGCCTCGGCAGCC-CAGGCAGCCTCAACACCAGCTTCTGG~A CGCGGGGATG~

CA GCCCGGTC-- TCAA--CGTC GCG---T . . . . .

-C--C--GGA -GGC----C- - - - T - - - - ¢ t ~ * - A

A - --G-C*--GA --A----ACG --A-GGGGT- -T--G---GA

-oT . . . . . . . . . . .

T. . . . . . X - A ' G " " C "

8~0

GGTC°'T'GG . . . . . . TT-- -T-C--¢-AT AGTGG'T" ' o

-G----CGAT G--

51Tram OHA : CCTTAGGAAGCGCGGCTGGCTCGGAGGCGGCCTTGGTCAG CTCCCK:CGGCTACC-CCACCGGC~GCATGAGCACGGCGGCT TTGAG pTS|9~J 0NA : G---CCAG-A -TT--GCAA- GGCAGCT-C" G---C-GA-A -GoGAG--A - ACTAG-T. . . . . TT-CA--A TG-A-GC,A'C GC-TT

980

Fig. 1. Nucleotide sequence alignments. Sequence of the 699-bp Sacll-EcoRl fragment of pTB193, 653-bp Hincll fragment of pTB195 and 758-bp Sacll-Hincll fragment of pTB197, are aligned with sequence fTom the central domain (nucleotides 266-1172) of Shinnicks [24] 517-amino-acid ORF. Residues identical to those of the 517-amino-acid O R F are given by dashes. Asterisks are used to represent gaps introduced in the sequence during alignment.

182

and 1.7 kb from pTBI91 hybridized very strongly to the genomic probe, indicating that the repetitive sequence was localized on these fragments (data not shown). The three fragments were subcloned into p U C l l 8 and designated pTBI92, pTB194 and pTB196 respectively. To localize the repeats to sr~a!ler fragments, pTB192 was digested with SacII and EcoR| and both pTB194 and pTB196 with Sacll and HinclI. Southern blotting and probing with total M. bot'is AN5 genomic DNA revealed a fragment of approximately 700 bp from pTB192 and pTB196 and 650 bp from pTB194 that hybridized strongly to the genomic probe (data not shown). These fragments wele sabcloned into pUC118 and designated pTB193, pTB197 and pTB195 respectively. The nucleotide sequence of the cloned DNA in pTB193, pTB195 and pTB197 was then determined from both strands (Fig. 1). 4.3. Analysis of nucleotide sequence The cloned fragment in pTB193 was observed to share considerable homology with part of a 517-amino-acid ORF downstream of the 65-kDa antigen gene of M. tuberculosis [24] (Fig. 1). The

region of homology includes the central domain of the 517-amino-acid ORF, which putatively encodes 29 tandemly arranged perfect and imperfect repeats of a pentapeptide sequence with the consensus X-Gly-Asn-X-Gly. Translation of the 699-bp fragment in pTBI93, revealed a single ORF encoding a similar tandem array of pentapeptides (Fig. 2). The ORFs differed by the occurrence of an extra 15 bp at position 453 in the 517-amino-acid O R F , encoding an additional copy of the pentapeptide (Fig. 1). Restriction endonuclease analysis indicated that the cloned fragment of pTB193 was contiguous with the downstream region of the 65-kDa gene on the M. bocis chromosome. Sequencing of pTB195 and pTB197 revealed the respective cloned fragments to be 653 and 758 bp in size. When both sequences were compared with the nucleotide sequence of the 517amino-acid ORF [24], a large degree of homology was observed around its central domain (Fig. 1). Computer translation of pTB195 and pTB197 DNA revealed single continuous ORFs, with the region of homology corresponding to a tandem array of the pentapeptide consensus X-Gly-Asn-

~17aaONF : A A A M M % V A T Q Y L A W ~ S T A A A ~ A E Q A A A Q A M A T A T A ~ E A A L A A T V Q P A V V A A N pTB193ORF:AAAMMAVATQYLAWL TAARQAEgAAA~AHI ATAFEAALAATV~PAVVAAN

S2

517aaORF :RGLM~LLAATNWFG~NAPALNDVE&AYE~MWALDVAAHaGYHFDASAAVA~L ~51930RF:RGLN LLR~TNWFG NAPALMDVER&YE~HWRLDV&AMAGYHFD~SAAVA~L ~B1960~: VDPVAAAAECLA~JNARG~R pTBlgTORF=L~NZGSWNLGGGNVGLLNL~SGNF~SLNLGGGNTGN~NL~GGNWGF LGSG

617aao~

104

: ~ . P W Q Q V L R N L G I D Z G X N G Q I N L - O F G N - T G S G N - I G N ~ N I G N N N Z O N N N I O S

I~TB193ORF pTBI950~F

APWQQVLRNLGIDIGKNGQINL-GFGN-TOSGN-IGNNNZGNNNZG RaGRCV[~JGVPAALAI ° N -o °N ° GN-I(~NNN

S2?aa ORF : G N T O T G N ~ B 1 9 3 ORF GNTGTGN

I OSGNTOSGNLOLGNLODG I GSGNTOS GNLGLGNLGDGN

N Z QFGNTQSGN ]; ~ F G N T G S G N

156 s

OSGN

X GFG I OF°

°

I TODH~MG FGG I TGDH MGFGG

208

Frszsv o~r =o ~jrl~Is r .~jvla s o .I.~M s~.Izl~Jrla. L al.. Ul.~Hor..9 .l.lal. "l" z o r GILT~_.~_.~JRLV~JZI_~IA -,,,~ pTBI93

~

,.,o.~.~

ORr ~ rN

L~rB195 ORF

L F

S GSG

SA NT

N I

o,~.~o~.,,o~,.~o~.~o~.~_~sI-L--~l~D)~i~]---~I~l~,~ GF

SLNT8 TGHSG

O N S ~¢0

W F

A S

N V GL

r N S ~S

G N I GI

260

GN

H g N ~'. P. L G S A ^ G 6 E A ~, L V S S P, G £ R T NSGSGFQNLGNGS SGFGNASDTSS

GMST/~/~LS FQNAGT&L

312

Fig. 2. Amino acid sequence alignments. The ORFs encoded on pTBI93, pTBI95 and pTB197 are aligned with the central domain (amino acids 90-391) of Shinnicks [24] 517-amino-acid ORF. The glycine residue at the fifth position of all the pentapeptide sequences is italicised. Residues identical to those of the 517-amino-acid ORF are boxed. Dashes are used to represent gaps introduced in the sequence during alignment.

183 X-Gly. Alignment of the three amino acid sequences with the 517-amino-acid ORF, showed an increase in amino acid over DNA homology for the core residues of the pentapeptide consensus (Fig. 2). For the pentapeptides encoded on all three clones, 56% had glycine as the second residue, 90% had asparagine as the third residue and 98% had glycine as the fifth residue. However, for these three residues, only 46%, 88% and 69% respectively, were conserved at the nucleotide level. Codon usage for the three amino acid sequences was observed to comply with that of nine genes from the M. tuberculosis complex [251.

A

B

9.4--

6.6-

4.4-

The ORFs identified on pTB193, pTB195 and pTB197 encoded 21, 35 and 39 copies of a pcntapeptide sequence respectively, that partially matched the consensus XoGiy-Asn-X-Gly. As part of these pentapeptides, two sequences on pTB197 at position 123 and 135 and another on pTB195 at position 129, were six amino acids in length, but still displayed matches to the three core residues of the consensus sequence (Fig. 2). On all three clones, the pentapeptides were arranged as two sets of tandem repeats, separated by seven amino acids on pTBI93 and pTB197 at position 201 and eight amino acids on pTB195 at position 248 (Fig. 2). The seven amino acid sequences were very similar, with three of the residues being identical and three of the remaining four being related. 4.4. Copy number of sequences encoding the tandem repeats When BstEII restricted genomic DNA from M. boris AN5 and M. bocis 13CG was Southern blotted and probed with the 653-bp fragment from pTB195, five fragments were observed to hybridize to the probe (Fig. 3). One fragment produced a stronger hybridization signal than the remaining four, and presumably corresponds to the chromosomal region from which the 653-bp fragment derived. The fact that there are no BstEIl sites within the three conserved domains, suggests there are at least five copies of this domain on the M. bm'is chromosome.

5. DISCUSSION 2.3-

~

~

Fig. 3. Copy number of repeat DNA motif on the M. bocis chromosome. Chromosomal DNA from (A) M. bm'is AN5 and (B) M. boeis BCG was digested with BstEll and probed with the 653-bp fragment of pTB195. Numbers at left indicate sizes of standard DNA fragments in kilobase pairs.

This study presents the identification of three copies of a repeat DNA sequence isolated from a M. boeis genomic library. The repeat potentially encodes a conserved tandem array of a pentapeptide sequence with the consensus X-Gly-Asn-XGly. It is proposed that the tandemly repeated amino acids form part of larger open reading frames (ORFs) that may be expressed in M. bot'is. Sequence homology and restriction analysis predicts that the sequence of one repeat, encoded on pTB193, is likely to be part of a large

184 O R F previously characterised in the closely related M. tuberculosis [24]. This 517-amino-acid O R F isolated from M. tuberculosis is located downstream of the 65-kDa antigen gene, and encodes 29 tandemly arranged perfect and imperfect repeats of the pentapeptide consensus, XGly-Asn-X-Gly. The fact that the 517-amino-acid O R F is so large, possesses distinct repeats of pentapeptide amino acids and includes both a potential ribosome binding site and transcriptional terminators, suggests that it is likely to encode a protein [24]. O u r observation that tandem repeats of the pentapeptide sequence are conserved not only within Shinnicks O R F [24] but within two o t h e r potential coding regions, may indicate that this structural feature has a specific function within the putative proteins. The amino acid sequence encoded by the repeat is structurally analagous to known virulence associated proteins, including the R T X family of toxins [26] and certain cell wall proteins identified in Gram-positive pathogens [27]. Tandemly arranged blocks of amino acids are present in the Staphylococcus aureus protein A [28] and fibronectin-binding protein [29], Streptococcus pyogenes M protein [30] and internalin protein (InlA) from Lysteria monocytogenes [31]. InlA is a protein necessary for the entry of L. monocytogenes into host cells. It has been postulated that its tandem repeats of amino acids are involved in binding to a specific mammalian cell receptor, perhaps an integrin [32,33]. Interestingly, inlA appears to be a m e m b e r of a group of related genes in L. monocytogenes [31]. It is known that phagocytic uptake of M. tuberculosis can be initiated by binding to the integrin CR3 [34] This suggests that entry of mycobacteria into host cells requires expression of a class of proteins capable of interacting with cell receptors such as integrins. T h e O R F s that contain the tandem repeats identified in this work may be part of this protein class, with the t a n d e m pentapeptide sequence involved in receptor binding, allowing attachment of M. boris to host cells. W e therefore postulate that the O R F s containing the tandem pentapeptides may be part of a family of cell receptor binding proteins. F u r t h e r work involving the expression of this proposed gene fam-

ily is required to determine a possible role in the interaction of M. boris with host cells. A n understanding of this process may provide valuable information on the pathogenicity of not only M. boris but all mycobacteria.

ACKNOWLEDGEMENT This work was supported by a Meat Research Corporation junior research fellowship.

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Characterisation of a novel repetitive DNA sequence from Mycobacterium bovis.

We report characterisation of three copies of a novel repeat sequence isolated from a Mycobacterium bovis genomic library. The repeat occurs within op...
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