repetitive dna sequence of mycobacterium tuberculosis: analysis

INTERNATIONAL JOURNAL OF LEPROSY^ Volume 56, Number 4

Printed in the US.A.

Repetitive DNA Sequence of Mycobacterium tuberculosis:Analysis of Differential Hybridization

Pattern with Other MycobacterialPrabhakara P. Reddi, G. P. Talwar, and Pramod S. Khandekar2

Tuberculosis is a global problem of enor-mous dimensions. Mycobacterium tuber-culosis, the etiological agent of tuberculosis,cannot be easily differentiated from othermembers of the tuberculosis complex andfrom the atypical mycobacteria of the non-tuberculosis complex. Conventional meth-ods used in identifying mycobacteria, suchas acid-fast stains and culture, are often timeconsuming, insensitive, and nonspecific ( 20).Phage typing has been a useful tool in epi-demiological investigations but there arelimitations (3, 10. 13, 21 ).) The nonavailabilityof a specific antigen and variation in thereproducibility of ELISA results make sero-diagnosis of tuberculosis difficult ( 16. 7 ).

There is, therefore, a pressing need for therapid and specific diagnosis of tuberculosis.Recombinant DNA methods and deoxyri-bonucleic acid probe technology can facil-itate the diagnosis of tuberculosis. Thistechnique utilizes a specific DNA sequenceas a probe to detect only the nucleic acidsof the target organisms, even in the presenceof excess nucleic acids from other organ-isms. To be useful as a probe for the diag-nosis of tuberculosis, the DNA fragmentshould be of a repetitive nature and specificto Al. tuberculosis, with very little or no crosshybridization to other mycobacteria of thetuberculosis as well as the nontuberculosiscomplex, which could infect humans.

Restriction endonuclease analysis has re-cently been applied to generate molecularmarkers that could distinguish between dif-ferent strains of mycobacterial species

' Received for publication on 6 October 1987; ac-cepted for publication in revised form on 22 July 1988.

= P. P. Reddi, M.Sc., Senior Research Fellow (CSIR);G. P. Talwar, D.Sc., Director; P. S. Khandekar, Ph.D.,Principal Scientific Officer, Recombinant DNA Lab-oratory, National Institute of Immunology, Shahid JeetSingh Marg, New Delhi 110067, India.

Reprint requests to Dr. Khandekar.

(4, 5, 12, Iv) Eisenach, et al., used randomlypicked recombinant clones from a libraryof Al. tuberculosis H37Rv to probe restric-tion digests of mycobacterial DNA to dif-ferentiate laboratory strains from wild-typestrains of Al. tuberculosis ( 7 ).

In our study, we included M. tuberculosisH37Rv, Al. tuberculosis H37Ra, Al. bovisBCG, and Al. microti from the tuberculosiscomplex; Al. avium, Al. intracellulare, andM. scronelaceum from the MAIS complex;and some of the cultivable mycobacteriasuch as Al. smegmatis, AI. vaccae, Al. che-loni, and Al. kansasii. We analyzed myco-bacterial DNA with restriction endonu-cleases Ilindlll, Baml-II, EcoRI, Sall, Pstl,Alul, and Sau3A, and looked for a species-specific banding pattern. Among the var-ious enzymes tested, the restriction endo-nuclease /1/uI gave specific bands in Al.tuberculosis H37Rv and M. tuberculosisH37Ra DNA digests which were totally ab-sent in corresponding DNA digests ofthe other mycobacteria tested. In the pres-ent communication, evidence is presentedon the specificity and repetitive nature ofone of these fragments. This DNA fragmentwas also used to isolate genomic sequencesfrom a Xgt 1 1 library of Al. tuberculosisH37Rv ( 23 ).

MATERIALS AND METHODSBacterial strains and growth conditions.

Al. tuberculosis H37Rv, M. tuberculosisH37Ra, Al. boils BCG, M. microti, M. avi-um, AI. intracellulare, M. scrqfulaceum, Al.cheloni, M. smegmatis, and Al. vaccae wereobtained from the standard collection of TheTrudeau Institute, Saranac Lake, New York,U.S.A., and grown in Middlebrook 7H9medium at 37°C.

DNA extraction. Mycobacterial culturesgrown to logarithmic phase were treated withglycine to destabilize the cell wall, centri-

592

56, 4^Reddi, et al.: Repetitive DNA Sequence of M. tuberculosis^593

fuged, and the DNA was isolated as de-scribed previously ( 12. 14).

Purification of DNA from low-meltingagarose gel. An agarose gel slice was meltedat 65°C for 20 min in the presence of onehalf the volume of TE (10 mM Tris, pH 8.0,1.0 mM EDTA). To this an equal volumeof TE-saturated phenol was added, and theextraction was carried out for 3-5 min atroom temperature. The upper aqueous phasewas reextracted with phenol followed bythree times with chloroform : isoamyl al-cohol (24:1) and two times with ether. Tracesof ether were removed by evaporation at60°C for 10 min. DNA was precipitated fromthe aqueous phase by the addition of 2.5volumes of chilled ethanol in the presenceof 1/11ill volume of 3 M sodium acetate, pH5.2. The procedure was repeated twice toensure the purity of the fragment.

Radiolabeling of DNA. DNA was radio-labeled with [ 32 13] deoxycytidine triphos-phate (Amersham Corp., Arlington Heights,Illinois, U.S.A.) by nick translation ( 18) to aspecific activity of 10 6 cpm/pg of DNA.

Restriction endonuclease digestion. Vary-ing amounts of mycobacterial DNA (see fig-ure legends) were digested with 20 units eachof the following restriction endonucleases,EcoRI, BamHI, Pstl, HindIll„Cau3A, and/1/tr I, for a period of 18 hr according to con-ditions specified by the manufacturer (Be-thesda Research Laboratories, Gaithers-burg, Maryland, U.S.A.). To ensurecomplete digestion, 10 units more of eachenzyme were added, and the incubation pe-riod was continued for a further 4-6 hr.

Hybridization. Denatured DNA wastransferred from agarose gels onto nitrocel-lulose filters (Schleicher & Co., Keene, NewHampshire, U.S.A.) by the methods ofSouthern ( 22). After transfer, the filter wasair dried and baked in vacuo at 80°C for 2hr followed by prehybridization at 42°C for2 hr with a solution containing 50% form-amide, 6X standard saline citrate (SSC), 5XDenhardt's solution (6), 100 µg/ml of calfthymus DNA. The [ 32 P]-labeled probe DNAwas denatured in a boiling water bath for 5min, and was added to the filters (2 x 10 6

cpm/filter) in the same mixture, and hy-bridization was continued for 24 hr. Afterhybridization, the filters were washed threetimes with 2X SSC and 0.1% sodium duo-

decyl sulfate (SDS) (100 ml each) at 55°Cfollowed by three more washings with 0.1XSSC and 0.05% SDS at 65°C, air dried, andexposed to X-ray film with a duPont inten-sifying screen at —70°C.

Screening of Xgtl 1 gene libraries of M.tuberculosis. From the genomic libraries ofM. tuberculosis made in Xgtll ( 23 ), recom-binant phages were screened by plaquehybridization ( 15) using the ["P]-labeled 5.6-kb DNA fragment as probe. The recombi-nant phages picked on the basis of a positivehybridization signal were purified to ho-mogeneity through a second and thirdscreening.

RESULTSDNA from M. tuberculosis H37Rv, M.

tuberculosis H37Ra, Al. boris BCG, Al. mi-croti, Al. avium, Al. intracellulare, M. .ccro-fielaceum, M. kansasii, Al. cheloni,smegmails, and M. vaccae was digested tocompletion with restriction endonucleasesrecognizing both hexanucleotide and te-tranucleotide sequences, and the bandingpattern of the restricted fragments was stud-ied by agarose gel electrophoresis. The re-striction patterns obtained with EcoRI, Sall,Pstl, HindIll, BamHI, and Sau3A did notreveal any species-specific differences amongmembers of the tuberculosis complex butshowed intense bands over the backgroundsmear (data not presented). However, in thecase of digestion with AhtI, which recog-nizes the sequence AGCT, the restrictionpatterns had distinct features. The overallsize of the DNA fragments generated withit/u1 ranged from 0.2 kb to 3 kb, and ap-peared as a smear on staining with ethidiumbromide. In addition, in the high-molecu-lar-weight region, a specific banding patternwas observed (Fig. 1, A and B). Two bands5.6 kb and 4.8 kb in size were observedwhich were only seen in Al. tuberculosisH37Rv and Al. tuberculosis H37Ra and wereabsent in the ilheI digests of M. boris BCG(Fig. IA). The 4.8-kb and 5.6-kb Ahrl frag-ments seen only in Al. tuberculosis were alsofound to be absent in Al. mieroti, A1. avian!,M. intracellulare, M. scrofulaceum, M.smegmatis, M. vaccae, M. cheloni, and M.kansasii (Fig. 1 B). The structural organi-zation of these /1/tt fragments in the Al. tu-berculosis H37Rv genome and their relat-

1 23 45 6 7 8 91011

2 3 4^kb

594^ International Journal of Leprosy^ 1988

kb23.1-,9.46.54.3

2.32.0

0.5

AFIG. I. For restriction analysis of mycobacierial DNAs with Alul, 2 pg each of DNA from different myco-

bacteria were digested to completion with restriction endonuclease z1/u 1. DNA fragments were separated byagarose gel electrophoresis (1.2%) and visualized by ethidium-bromide staining. A. Lanes 1-4. Ilind111 digestof DNA (lane 1); diul digests of M. boris BCG (lane 2)„11. tuberculosis H37Ra (lane 3)„11. tuberculosis H37Rv(lane 4); arrows indicate 5.6-kb and 4.8-kb diul fragments of M. tuberculosis. B. Lanes 1-11. A/u1 digests of 5pg each of DNA of M. tuberculosis H37Rv, ,11. boris BCG, .11. tnicroti„il. annuli, M. intmcellulare, M. scro-fitlaceuni; Ilind111 digest of XDNA; dial digests of M. cheloni. M. kansasii„11. sines:mails. and M. raccac,respectively.

edness to other mycobacterial DNAs werestudied by Southern blot hybridization ex-periments. The 5.6-kb /1/u1 fragment,purified on low-melting agarose gel electro-phoresis (see Methods), was labeled with[32 P]-dCTP by nick translation (' 8). Hybrid-ization of the zl/uI digests oil/. tuberculosisH37Rv with the 5.6-kb fragment gave sev-eral bands which indicated the repetitivenature of this sequence in the Al. tubercu-losis H37Rv genome. Although ethidium-bromide staining did not reveal the presenceof a 5.6-kb fragment in the Alul digest ofAl. boris BCG DNA (Fig. 1A, lane 1) upon

hybridization, a banding pattern was ob-tained which was comparable to that of AI.tuberculosis H37Rv (Fig. 2A, lane 1). Onthe contrary, no hybridization signal wasdetected even after 8 days of exposure inthe case of mycobacterial DNA digests ofthe nontuberculosis complex (Fig. 2A, lanes5-7) with the only exception being Al. kan-sasii where a hybridization signal with an8-10-kb fragment was obtained (Fig. 2A,lane 4). Similar studies carried out with the4.8-kb /1/reI yielded a multiple hybridizablebanding pattern with M. tuberculosis and M.boris BCG, and also showed cross hybrid-


A

0.2 pg

0.1pg

0.0514

1 2 3 4 5 6 7^ 1^2^3^4^5^6

FIG. 2. Hybridization of difkrent mycobacterial DNAs with 5.6-kb ..I/p1 fragment of M. tuberculosis H37Rv.A. Three pg each of mycobacterial DNA were digested to completion with restriction endonuclease .-1/ul,fractionated on 1.2% agarose gel, and hybridized with 2 x 10' cpm of the radiolabeled, purified 5.6-kb A/ufragment under high-stringency conditions as described in Methods. Lanes I = M. boris BCG, 2 = control DNA,3 = Al. tuberculosis H37Rv, 4 = AI. kansasii, 5 = Al. smegmatis, 6 = Al. vaccae, 7 = M. avium; arrow indicatesposition of 5.6-kb ..I/t/ fragment in Al. tuberculosis Alul DNA digest. B. DNA was dotted on nitrocellulose filterand hybridized with the ['PI-labeled 5.6-kb .1/u fragment. Concentration of DNA dotted is marked on the left.Lanes 1-6 are .11. tuberculosis H37Rv, Al. boris BCG, M. kansasii, Al. smegmatis, M. vaccae. and .11. avium,

respectively.

ization with the other mycobacteria testedwhich were the same as for the 5.6-kb Ahtlfragment (data not presented).

The apparent specificity of the 5.6-kbprobe, evident in Southern blot, is also re-flected in the DNA dot hybridization. Uponhybridization of the mycobacterial DNAsdotted on nitrocellulose membrane with the5.6-kb A/ui fragment, it was possible to dif-ferentiate between mycobacterial species ofthe tuberculosis complex from the othermycobacteria tested. M. alarm, M. smeg-matis, and Al. vaccae did not give any hy-bridization signal with the 5.6-kb ,11u probeat 50 ng concentration of DNA (Fig. 2B).AI. kansasii, on the other hand, gave a hy-bridization of similar intensity on dot blotwhen compared to M. tuberculosis H37Rv.

Screening of Xgtl 1 libraries of M. tuber-culosis I-137Rv. The 5.6-kb Ahtl fragmentwas used to isolate the genomic counter-parts from the Xgt 1 1 library of M. tuber-culosis H37Rv ( 23) by the plaque hybridiza-tion technique. A total of 10 5 phages werescreened, and 15 recombinant phages, giv-ing hybridization signals in the primary

screening, were further purified to homo-geneity (Fig. 3), and the DNA isolated wascharacterized by Southern hybridization tomycobacterial DNAs (data not presented).

DISCUSSIONDeoxyribonucleic acid probe technology

offers an alternate approach for the rapidand accurate diagnosis of tuberculosis dueto the specificity and sensitivity of molec-ular hybridization. Furthermore, the sen-sitivity of DNA probes also depends uponthe nature of the sequences, whether repet-itive or a single copy (9 ).

With a view to generate a specific DNAprobe for M. tuberculosis, we analyzed therestriction fragment profiles of the DNA di-gests of a few mycobacterial species that fallwithin the tuberculosis complex and com-pared them with the mycobacteria of theMAIS complex and some atypical myco-bacteria. The restriction endonucleasesEcoRI, Pstl, BanIHI, Sall, and Madill didnot show any species-specific differencesamong the members of the tuberculosiscomplex, which is in agreement with earlier


41.a a

I

•FIG. 3. Isolation of recombinants specific to 5.6-

kb A/it fragment from Xgt11 library of Al. tuberculosisH37Rv. Recombinant phages plated at a density of lplaque forming units per plate were transferred to ni-trocellulose membrane filter; the DNA was then dena-tured and fixed at 80°C for 2 hr under vacuum. Pre-hybridization and hybridization were carried out (seeMethods) with ID' cpm per filter of the radiolabeledfragment DNA. Autoradiograph of one of the filters isshown.

reports (4, I I, 20) . However, the restrictionendonuclease /1/ul-digested DNA of M. tu-berculosis H 37 R v and Al. tuberculosisH37Ra yielded a specific pattern with bandsof 5.6 kb and 4.8 kb which appeared to bespecies specific (Fig. 1, A and B). A species-specific banding pattern was also obtainedwith other mycobacterial DNA digests aswell (Fig. 1 B). This would distinguish M.tuberculosis from the other members of thetuberculosis complex which were studied,such as M. boris BCG and Al. microti (Fig.1B). The reactivity of the 5.6-kb fragmentwith the DNAs of other mycobacteria, asstudied in Southern blot hybridization ex-periments, revealed restriction-based poly-morphism with Al. boris BCG. These resultsalso reflect the DNA-relatedness betweenM. tuberculosis and other members of thetuberculosis complex which is reported tobe between 90%-100% (1, 2, I I , .

) It is inter-esting to note that this 5.6-kb DNA frag-ment shows a specific hybridization signalwith Al. kansasii DNA (Fig. 2A) in the rangeof 9-10 kb but not with Al. avium, althoughthe reported DNA-relatedness of these two

species with M. tuberculosis is 32% and 27%,respectively ("). The fact that no hybrid-ization signal was observed with M. smeg-matis and Al. raccae indicates that thesespecies are evolutionarily distant to Al. tu-berculosis ( 5 ).

tubereit/osis-specific DNA sequenceswith restriction endonucleases BstE11 andAthol have been reported previousl y (4• 19).

However, no information is available re-garding the nature of these fragments. Sim-ilarly, Eisenach, et al. ( 7) reported recom-binant clones from a library of Al.tuberculosis 1-137Rv which showed strain-specific hybridization signals in the Al. tu-berculosis H37Rv genome as compared toclinical isolates. But the reactivity of theseclones with the atypical mycobacteria whichcould be found in the sputum is now known.Shoemaker, et al. ( 20) used the whole ge-nome of Al. tuberculosis as a probe to dis-tinguish Al. tuberculosis from other non-mycobacteria found in sputum. The probe,however, could not distinguish Al. tuber-culosis from other mycobacterial species.This could be due to varying degrees of nu-cleotide sequence homology between dif-ferent mycobacterial species.

Our studies show the discriminatory reac-tivity of the 5.6-kb /1/u fragment with my-cobacteria from the tuberculosis complex ascompared to Al. avium, Al. smegmatis, and

vaccae (Fig. 2, A and B). The 5.6-kbprobe crossreacts with M. kansasii, whichcould be an advantage because Al. kansasiiis a known pathogen. A clear distinction isobserved in DNA dot hybridization. Thissuggests the potential utility of the 5.6-kbDNA probe in sputum analysis, where theamount of target DNA is the limiting factor.The concentration of mycobacteria foundin sputum varies from 10 3 to 10' per ml.This concentration may be much lower insamples from treated patients (100 pg of M.tuberculosis DNA corresponds to approxi-mately 104 bacteria). The sensitivity of theprobe can be increased by an order of onelog by increasing the specific activity. Ad-ditionally, the target DNA can be amplified,making use of the polymerase chain reac-tion (PCR) technique. Further, evidence ofthe species specificities and the repetitivenature of the 5.6-kb ilhtI fragment has beenfound with genomic clones obtained fromthe Agt11 library (results not presented). De-

•


tailed analysis of the insert DNA of the M.tuberculosis genomic clones, presently un-der investigation, will enable us to under-stand more regarding the nature of the re-petitive clement present in the insert.Nevertheless, the present communication isevidence of the potential utility of the 5.6-kb zlhel fragment in the development ofDNA probes for Al. tuberculosis. Althoughthe present probe has been tested for cross-reactivity with representative mycobacteriafrom the tuberculosis complex, the MAIScomplex, and atypical mycobacteria, a larg-er number of species including clinical iso-lates need to be tested.

SUMMARYIn order to generate specific DNA probes

for AI ycobacterium tuberculosis, restrictionfragment length analysis was carried out withsome of the mycobacterial species that fallwithin the tuberculosis complex. The pres-ence of specific bands of 5.6 kb and 4.8 kbwas revealed in the .-1/itI DNA digest of Al.tuberculosis. The hybridization profile of the5.6-kb r1IutI DNA sequence, as judged bythe Southern blot and dot blot hybridizationexperiments, revealed the presence of thissequence in Al. tuberculosis H37Rv, Al. tu-berculosis H37Ra, M. bovis BCG as multi-ple copy and Al. kansasii as single copy butthis sequence was not present in Al. avium,

sinegniatis, or Al. vaccae genomes. Ge-nomic clones corresponding to the 5.6-kbA/uI fragment from Al. tuberculosis H37Rvlibrary made in the Xgt 1 I expression vectorwere isolated.

RESUMENPara generar sondas especificas para el DNA del My-

cobacterium tuberculosis se hizo el analisis de frag-mentos de restricciOn con algunas de las especies mi-cobacterianas incluidas dentro del complejo detuberculosis y con aquellas fucra de este complejo. Seencontraron bandas especificas de 5.6 kb y dc 4.8 kben el digericlo Ahtl del DNA de Al. tuberculosis. Elperfil de hibridizaciOn de la secuencia del fragmento5.6 kb Ahtl, revel() la presencia de esta secuencia enM. tuberculosis 11371(v, Al. tuberculosis H37Ra y Al.bovis 13CG, como copia multiple, y en AI. kansasiicomo copia Unica pero esta secuencia no estuvo pre-sente en los genomas de Al. avium, Al. smegmatis, oM. vaccae. De Ia genoteca de M. tuberculosis H37Rvfabricada en el vector de expresiOn Xgt11 se aislaronclones genOmicos correspondientes al fragmento 5.6kb Ahtl.

RESUME

En vuc de mettre au point des sondes d'ADN spe-cificities pour Mycobacterium tuberculosis, une analysede Ia longueur des fragments de restriction a CAc pra-tiquce chez quelques unes des espèces mycobacte-riennes qui appartiennent au complexe de Ia tubercu-lose, et chez d'autres qui n'appartiennent pas a cccomplexe. On a ainsi mis en evidence la presence debandes specifiques de 5,6 kb et 4,8 kb dans les produitsde digestion de Al. tuberculosis par AhtI ADN. Le profitd'hybridisation de Ia sequence 5,6 kb AM! ADN, idlequ'elle apparaissait par le Southern-blot et par des ex-periences d'hybridisation a blot ponetuel, montraitclairement Ia presence de cette sequence chez Al. tu-berculosis I - 13712v, M. tuberculosis I-137Ra, M. bovisBCG, en copie multiple dans tous ces cas, et en simplecopie chez Al. kansasii. Par contre, cette sequence etaitabsente dans les genomes de Al. (alum, al. sozegmatisou vaccae. On a isolê des clones génomiques quicorrespondaient au fragment, 5,6-kb AhtI de la biblio-theque de .11. tuberculosis 1137Rv, constitue a partirdu vecteur d'expression Xgt11.

Acknowledgment. The writers thank Dr. RichardYoung for making available the Xgt11 library of AI.tuberculosis. This work was supported by the Depart-ment of Biotechnology, Government of India. PPR isa Senior Research Fellow of the Council for Scientificand Industrial Research, Government of India.

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repetitive dna sequence of mycobacterium tuberculosis: analysis

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