{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,27]],"date-time":"2026-10-27T07:18:22Z","timestamp":1793085502609,"version":"4.5.0"},"reference-count":38,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5419","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[1999,5,28]]},"abstract":"<jats:p>\n                    Bacille Calmette-Gue\u0301rin (BCG) vaccines are live attenuated strains of\n                    <jats:italic>Mycobacterium bovis<\/jats:italic>\n                    administered to prevent tuberculosis. To better understand the differences between\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    ,\n                    <jats:italic>M. bovis<\/jats:italic>\n                    , and the various BCG daughter strains, their genomic compositions were studied by performing comparative hybridization experiments on a DNA microarray. Regions deleted from BCG vaccines relative to the virulent\n                    <jats:italic>M. tuberculosis<\/jats:italic>\n                    H37Rv reference strain were confirmed by sequencing across the missing segment of the H37Rv genome. Eleven regions (encompassing 91 open reading frames) of H37Rv were found that were absent from one or more virulent strains of\n                    <jats:italic>M. bovis<\/jats:italic>\n                    . Five additional regions representing 38 open reading frames were present in\n                    <jats:italic>M. bovis<\/jats:italic>\n                    but absent from some or all BCG strains; this is evidence for the ongoing evolution of BCG strains since their original derivation. A precise understanding of the genetic differences between closely related Mycobacteria suggests rational approaches to the design of improved diagnostics and vaccines.\n                  <\/jats:p>","DOI":"10.1126\/science.284.5419.1520","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T05:37:46Z","timestamp":1027748266000},"page":"1520-1523","source":"Crossref","is-referenced-by-count":1230,"title":["Comparative Genomics of BCG Vaccines by Whole-Genome DNA Microarray"],"prefix":"10.1126","volume":"284","author":[{"given":"M. A.","family":"Behr","sequence":"first","affiliation":[{"name":"Division of Infectious Diseases, Department of Medicine, McGill University Health Centre, Montreal H3G 1A4, Canada."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M. A.","family":"Wilson","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University, Stanford, CA 94305, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"W. P.","family":"Gill","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University, Stanford, CA 94305, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"H.","family":"Salamon","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University, Stanford, CA 94305, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"G. K.","family":"Schoolnik","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University, Stanford, CA 94305, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"S.","family":"Rane","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University, Stanford, CA 94305, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"P. M.","family":"Small","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University, Stanford, CA 94305, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","unstructured":"Centers for Disease Control and Prevention Morb. Mortal. Wkly. Rep. 47 (RR-13) 1 (1998)."},{"key":"e_1_3_1_3_2","first-page":"553","volume":"34","author":"Calmette A.","year":"1920","unstructured":"Calmette A., Gu\u00e9rin C., Ann. Inst. Pasteur 34, 553 (1920).","journal-title":"Ann. Inst. Pasteur"},{"key":"e_1_3_1_4_2","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1038\/38151","volume":"389","author":"Behr M. A.","year":"1997","unstructured":"Behr M. A., Small P. M., Nature 389, 133 (1997).","journal-title":"Nature"},{"key":"e_1_3_1_5_2","doi-asserted-by":"crossref","first-page":"915","DOI":"10.1016\/S0264-410X(98)00277-1","volume":"17","author":"___","year":"1999","unstructured":"___, Vaccine 17, 915 (1999).","journal-title":"Vaccine"},{"key":"e_1_3_1_6_2","unstructured":"Strains used were M. tuberculosis H37Rv BCG-Russia (ATCC 35740) BCG-Moreau BCG-Japan BCG-Sweden BCG-Prague BCG-Phipps (ATCC 35744) BCG-Denmark 1331 (ATCC 35733) BCG-Tice (ATCC 35743) BCG-Frappier (ATCC 35735) BCG-Connaught BCG-Birkhaug (ATCC 35731) BCG-Glaxo (ATCC 35741) and BCG-Pasteur 1173."},{"key":"e_1_3_1_7_2","doi-asserted-by":"publisher","DOI":"10.1038\/31159"},{"key":"e_1_3_1_8_2","unstructured":"Second Workshop on Methods and Applications of"},{"key":"e_1_3_1_8_3","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1038\/ng0398-195","volume":"18","author":"Microarray Technology DNA","year":"1998","unstructured":"Microarray Technology DNA, Nature Genet. 18, 195 (1998).","journal-title":"Nature Genet."},{"key":"e_1_3_1_9_2","unstructured":"Using the annotated sequence of M. tuberculosis H37Rv made available by the Sanger Centre () we designed PCR primer pairs to amplify 250- to 1000-bp internal fragments of predicted ORFs (details about primers used are available at ). Primer design was automated using Primer 3 [release 0.6 S. Rozen and H. J. Skaletsky (1996 1997) code available at www-genome.wi.mit.edu\/genome_software\/other\/primer3.html]. PCR reactions were prepared in 65-\u03bcl reactions in 96-well plates; amplicons were evaluated to ensure they were the expected size and then robotically applied to poly- l -lysine\u2013coated microscope slides as described (protocols available at ). After printing free amino groups of the poly- l -lysine\u2013coated slides were acetylated with 5.5 g of succinic anhydride in 350 ml of borate-buffered 1-methyl-2-pyrrolidinone (pH 8)."},{"key":"e_1_3_1_10_2","unstructured":"Bacteria listed above were grown in 7H9 broth with ADC enrichment and DNA was extracted after 14 to 21 days' growth as described ["},{"key":"e_1_3_1_10_3","doi-asserted-by":"crossref","first-page":"2578","DOI":"10.1128\/jcm.29.11.2578-2586.1991","volume":"29","author":"van Soolingen D.","year":"1991","unstructured":"van Soolingen D., Hermans P. W., de Haas P. E., Soll D. R., van Embden J. D., J. Clin. Microbiol. 29, 2578 (1991);","journal-title":"J. Clin. Microbiol."},{"key":"e_1_3_1_10_4","unstructured":"]. Whole genomic DNA from either H37Rv or BCG strain (2 \u03bcg) was used as templates for direct incorporation of fluorescent nucleotide analogs (Cy3 and Cy5 dUTP Amersham) by a randomly primed polymerization reaction. In brief 50-\u03bcl labeling reactions contained 2 \u03bcg of template DNA 5 \u03bcl of 10\u00d7 buffer 1.5 \u03bcl of fluorescent dUTP 0.5 \u03bcl each of 5 mM dATP dCTP and dGTP 1 \u03bcl of random hexamers and decamers and 2 \u03bcl of Klenow ( Escherichia coli DNA polymerase 3\u2032 to 5\u2032exo- New England Biolabs). Labeled DNAs from the strains being compared were mixed together to 14.75 \u03bcl total volume in a solution with final concentration of 4\u00d7 SSC buffer tRNA (0.7 \u03bcg\/\u03bcl) and 0.3% SDS. The hybridization mixture was denatured at 98\u00b0C for 2 min cooled to 65\u00b0C applied to the microarray and covered with a 22-mm 2 cover slip. The slide was then placed in a waterproof hybridization chamber for hybridization in a 65\u00b0C water bath for 3 hours. After hybridization slides were washed 2 min in 1\u00d7 SSC buffer with 0.06% SDS followed by 2 min in 0.06\u00d7 SSC buffer."},{"key":"e_1_3_1_11_2","doi-asserted-by":"crossref","first-page":"9869","DOI":"10.1073\/pnas.94.18.9869","volume":"94","author":"Sreevatsan S.","year":"1997","unstructured":"Sreevatsan S., et al., Proc. Natl. Acad. Sci. U.S.A. 94, 9869 (1997).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_12_2","doi-asserted-by":"crossref","first-page":"3135","DOI":"10.1099\/13500872-142-11-3135","volume":"142","author":"Philipp W. J.","year":"1996","unstructured":"Philipp W. J., et al., Microbiology 142, 3135 (1996).","journal-title":"Microbiology"},{"key":"e_1_3_1_13_2","unstructured":"Slides were scanned with a custom-built scanning laser microscope as described ["},{"key":"e_1_3_1_13_3","doi-asserted-by":"publisher","DOI":"10.1126\/science.278.5338.680"},{"key":"e_1_3_1_13_4","unstructured":"]. Fluorescent spot intensities were quantified using custom software as described by DeRisi and available at  each spot background fluorescence was subtracted from the average spot fluorescence to produce a channel-specific value. Spots were excluded from analysis when background intensity was greater than the spot intensity. The ratio of Cy3\/Cy5 hybridization was calculated and log-transformed for representation according to genomic location implementing ProbeBrowser software (designed and written in our laboratory and available at ). This software integrates microarray experimental results with the genomic positions of the hybridization targets and displays corresponding ORF annotations (see Fig. 1 B and C)."},{"key":"e_1_3_1_14_2","unstructured":"For any single experimental spot a mismatched hybridization could represent (i) a repeated element more numerous in H37Rv such as IS 6110 (ii) an element present in H37Rv and absent from BCG or (iii) a false positive result that may occasionally occur during thousands of simultaneous hybridizations. We observed that spots representing IS 6110 reliably resulted in high ratios of H37Rv:BCG corresponding to their known 16:1 genomic presence. For assigning deletions we restricted our investigation to regions where probes from at least two adjacent ORFs exhibited mismatched hybridization. This strategy was invoked to maximize the likelihood of detecting true deletions at the expense of decreased resolution. For putative deletions identified by the array experiments we performed Southern (DNA) hybridization using probes independent of those on the array to confirm their absence in BCG strains. Then to characterize confirmed deletions we designed PCR primers to approach the predicted edges of each deletion so that the amplified region would span the missing genomic locus for those strains in which it is deleted. These amplicons were sequenced using the PCR primers at the Protein and Nucleic Acid (PAN) Core Facility Stanford University by ABI dye terminator chemistry. Sequences obtained were aligned to the known sequence of H37Rv by BLAST search ["},{"key":"e_1_3_1_14_3","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/25.17.3389"},{"key":"e_1_3_1_14_4","unstructured":"] to determine the precise site of each deletion (details available at ). Based on the site and the predicted ORFs from the H37Rv we documented which reading frames were partially or completely missing from BCG strains. To classify deleted regions as BCG-specific or as differences between H37Rv and virulent M. bovis we repeated PCR with the primers spanning the missing genetic region using DNA from virulent M. bovis (four human and four bovine isolates)."},{"key":"e_1_3_1_15_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.178.5.1274-1282.1996"},{"key":"e_1_3_1_16_2","doi-asserted-by":"crossref","first-page":"2221","DOI":"10.1128\/IAI.66.5.2221-2229.1998","volume":"66","author":"Brosch R.","year":"1998","unstructured":"Brosch R., et al., Infect. Immum. 66, 2221 (1998).","journal-title":"Infect. Immum."},{"key":"e_1_3_1_17_2","doi-asserted-by":"crossref","unstructured":"L. M. O'Reilly and C. J. Daborn Tuberc. Lung. Dis. 76 (suppl. 1) 1 (1995).","DOI":"10.1016\/0962-8479(95)90591-X"},{"key":"e_1_3_1_18_2","doi-asserted-by":"crossref","first-page":"3259","DOI":"10.1128\/iai.64.8.3259-3266.1996","volume":"64","author":"Johansen K. A.","year":"1996","unstructured":"Johansen K. A., Gill R. E., Vasin M. L., Infect. Immun. 64, 3259 (1996).","journal-title":"Infect. Immun."},{"key":"e_1_3_1_19_2","first-page":"3411","volume":"59","author":"Parra C. A.","year":"1991","unstructured":"Parra C. A., et al., ibid. 59, 3411 (1991).","journal-title":"ibid."},{"key":"e_1_3_1_20_2","unstructured":"In the previous work there were two discrepancies between genotype data and the written record; in both we have subsequently obtained new information indicating that our historical information was incorrect. During the American Indian trial Aronson obtained BCG-Pasteur (passage) 575 in 1938 ["},{"key":"e_1_3_1_20_3","first-page":"651","volume":"42","author":"Aronson J. D.","year":"1940","unstructured":"Aronson J. D., Parr E. I., Saylor R. M., Am. Rev. Tuberc. 42, 651 (1940);","journal-title":"Am. Rev. Tuberc."},{"key":"e_1_3_1_20_4","unstructured":"]; this likely became the stock from which BCG-Phipps was derived. According to Birkhaug himself BCG-Birkhaug appears to have been obtained from the Institut Pasteur in 1927 ["},{"key":"e_1_3_1_20_5","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1007\/BF01562014","volume":"21","author":"Birkhaug K.","year":"1948","unstructured":"Birkhaug K., Psychiatr. Q. 21, 453 (1948)].","journal-title":"Psychiatr. Q."},{"key":"e_1_3_1_21_2","first-page":"651","volume":"42","author":"Aronson J. D.","year":"1940","unstructured":"Aronson J. D., Parr E. I., Saylor R. M., Am. Rev. Tuberc. 42, 651 (1940).","journal-title":"Am. Rev. Tuberc."},{"key":"e_1_3_1_22_2","first-page":"281","volume":"81","author":"Gheorghiu M.","year":"1983","unstructured":"Gheorghiu M., Augier J., Lagrange P. H., Bull. Inst. Pasteur 81, 281 (1983).","journal-title":"Bull. Inst. Pasteur"},{"key":"e_1_3_1_23_2","first-page":"625","volume":"25","author":"Calmette A.","year":"1911","unstructured":"Calmette A., Gu\u00e9rin C., Ann. Inst. Pasteur 25, 625 (1911).","journal-title":"Ann. Inst. Pasteur"},{"key":"e_1_3_1_24_2","first-page":"1","volume":"20","author":"Jensen K. A.","year":"1946","unstructured":"Jensen K. A., Acta Tuberc. Scand. 20, 1 (1946).","journal-title":"Acta Tuberc. Scand."},{"key":"e_1_3_1_25_2","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/S0140-6736(00)40633-1","author":"Dreyer G.","year":"1931","unstructured":"Dreyer G., Vollum R. L., Lancet i, 9 (1931).","journal-title":"Lancet"},{"key":"e_1_3_1_26_2","unstructured":"Indian J. Med. Res. 72S 1 (1980)."},{"key":"e_1_3_1_27_2","unstructured":"The genomic hierarchy proposed by Cole et al. is available at www.sanger.ac.uk\/Projects\/M_tuberculosis\/Gene_list\/."},{"key":"e_1_3_1_28_2","first-page":"655","volume":"74","author":"Dubos R. J.","year":"1956","unstructured":"Dubos R. J., Pierce C. H., Am. Rev. Tuberc. Pulmon. Dis. 74, 655 (1956).","journal-title":"Am. Rev. Tuberc. Pulmon. Dis."},{"key":"e_1_3_1_29_2","unstructured":"For strains and DNA of M. tuberculosis M. bovis and M. bovis BCG we thank M. Gheorghiu and S. Cole Institut Pasteur Paris (BCG-Pasteur 1173); A. Ladefoged Statens Serum Institut Copenhagen (BCG-Sweden and BCG-Prague); M. Takahashi Japanese Anti-Tuberculosis Association (BCG-Japan); P. Rousseau Institut Armand Frappier Canada (BCG-Moreau); U. Kosecka Connaught Laboratories Canada (BCG-Connaught); M. D. Cave University of Arkansas; and G. Hewinson Central Veterinary Laboratories Surrey UK. For technical assistance we thank J. DeRisi and members of P. Brown's laboratory. For assistance with the gene annotation database we thank S. Cole and his laboratory at the Institut Pasteur."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.284.5419.1520","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T03:56:59Z","timestamp":1705118219000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.284.5419.1520"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1999,5,28]]},"references-count":38,"journal-issue":{"issue":"5419","published-print":{"date-parts":[[1999,5,28]]}},"alternative-id":["10.1126\/science.284.5419.1520"],"URL":"https:\/\/doi.org\/10.1126\/science.284.5419.1520","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[1999,5,28]]}}}