Molecular analysis of DNA from Mycobacterium leprae, "Mycobacterium lufu," and Mycobacterium vaccae has demonstrated that the G+C (guanine plus cytosine) contents of the DNAs are 56, 61, and 65%, respectively, and that the genome sizes are 2.2 x 109, 3.1 x 109, and 3.1 x 109 daltons, respectively. Because CLARK-CURTISS ET AL. J. BACTERIOL. TABLE 1. Bacterial strains Strain Genotype Source or reference E. coli K-12 X289 F-tte-J prototroph 11 E. coli K-12 x925 F-thr-1 ara-13 leu-6 azi-8 tonA2 lacYl minAl Single colony isolate of P678-54; 1 ginV44 gal-6 A-minB2 rpsL35 malAl xyl-7 mtl-2 thi-i E. coli K-12 X1849 F-tonA53 dapD8 minAl purE41 ginV42 A(gal-17 uvrB)40 A-minB2 his-53 gyrA25 metC65 oms-i tte-J A(bioH-asd)29 ilv-277 cycB2 cycAl hsdR2 E. coli K-12 X2001 F-AaraC766 tonA53 dapD8 proA370 AlacZ39 minAl 7 A(gal-chiD)69 X-tyrT58 AgalUi83 AtrpE5 minB2 rfb-2 recA56 relAl AthyA57 endAI oms-i Aasd4 rpoB402 cycB2 cycAl hsdR2 E. coli K-12 X2819 F-lacYl ginV44 galK2 gaIT22 (A cI857 b2 red3 S7) This paper recA56 AthyA57 metBI hsdR2 M. leprae Wild type Armadillo liver #29 infected with M. leprae pooled from seven patients; received from C.

A 2.2-kilobase Mycobacterium leprae DNA insert fragment from a recombinant genomic library (pYA1065) was found to hybridize to at least 19 fragments of chromosomal M. leprae DNA by Southern hybridizations. The probe hybridized to identical fragments of chromosomal DNA from four M. leprae isolates (two from patients with leprosy, one from a naturally infected armadillo, and one from a naturally infected Mangabey monkey) whether the chromosomal DNA was digested with BamHI, BstEII, PstI, or SacI. The pYA1065 probe is specific for M. leprae; it did not hybridize to chromosomal DNA from 14 cultivable slow- and fast-growing mycobacterial species. Dot-blot hybridizations between pYA1065 and purified M. leprae chromosomal DNA indicate that the probe can detect DNA equivalent to 4 x 10(3) M. leprae cells in a spot. The probe can also hybridize to DNA in M. leprae cells spotted on a filter from homogenized skin biopsy specimens from patients with lepromatous leprosy.

Objectives: To assess whether emergency medicine residents (EMRs) could quickly perform accurate compression ultrasonography (CUS) for the detection of proximal lower extremity deep vein thromboses (PLEDVTs) with minimal training. Methods: A prospective, observational study using a convenience sample of patients presenting with signs and/or symptoms for PLEDVT. Vascular laboratory and department of radiology studies were considered the criterion standard. CUS of the femoral vessels was performed. Incompressibility or visualized thrombus was considered ''positive.'' Results: Eight residents with limited ultrasound (US) experience and no prior experience with deep vein thrombosis (DVT) US volunteered to participate in this study, enrolling 72 patients. Their average scan time was 11.7 minutes (95% CI ¼ 9.4 to 14). There were 23 true positives, 4 false positives, 45 true negatives, and 0 false negatives. The test characteristics for PLEDVT gave a sensitivity of 100% (95% CI ¼ 82.2 to 100) and a specificity of 91.8% (95% CI ¼ 79.5 to 97.4). Conclusion: Emergency medicine residents with limited US experience were able to quickly perform CUS after minimal training for the detection of PLEDVT in a select group of patients.

Genomic libraries of Mycobacterium leprae DNA partially digested with Pst I were constructed in the expression vector pYA626, which contains the promoter region from the Streptococcus mutans gene encoding aspartate asemialdehyde dehydrogenase, which is very efficiently expressed in Escherichia coli. We have detected several clones that complement a mutation in the citrate synthase gene of E. coli. Southern blot analysis demonstrated that the complementing DNA was M. leprae DNA. Sodium dodecyl sulfate/polyacrylamide gel analysis of polypeptides produced by minicells containing the citrate synthase-complementing recombinant molecules demonstrated the production of a 46-kDa polypeptide. When the citrate synthase-complementing fragment was cloned in pYA626 in the reverse orientation, the recombinant molecule was no longer able to complement the mutation in the citrate synthase gene and no longer produced the 46-kDa polypeptide. When the DNA fragment was cloned in the Pst I site of pHC79, so as to allow expression from the (3-lactamase promoter, the resulting recombinant failed to complement the mutation in the E. coli citrate synthase gene yet still produced the 46-kDa polypeptide, but in one-fourth the amounts than when expressed from the S. mutans asd promoter. This demonstrates that M. leprae translational sequences can be recognized by E. coli translational machinery. Promoter expression vectors can be used to obtain expression of protein antigens to be used for early diagnosis of leprosy or components of a vaccine and proteins that are targets of potential antileprosy drugs.Leprosy, an age-old chronic disease with a wide spectrum of manifestations, including gross skin disfigurement and peripheral nerve loss, afflicts over 15 million people in the world today (1). Its causative agent, Mycobacterium leprae, was shown to be associated with the disease by Gerhard Armauer Hansen in the early 1870s (2). Even so, M. leprae has been extremely difficult to study because of its inability to be cultivated in the laboratory. In the early 1960s, Shepard successfully cultivated M. leprae in the footpads of mice (3). Significant quantities of the organism became available for research upon the discovery that M. leprae produced a systemic infection in the nine-banded armadillo, Dasypus novemcinctus (4, 5).We had previously screened genomic libraries ofM. leprae DNA cloned in both plasmid and cosmid vectors and had not observed any complementation of a variety of mutations in amino acid, purine, and vitamin biosynthetic pathways or carbohydrate catabolic pathways in Escherichia coli K-12 (6). We cloned M. leprae DNA in the expression vector pYA626 and were able to demonstrate the expression of M. leprae polypeptides in minicells containing recombinant M. leprae molecules (6). In this manuscript, we describe the complementation of a mutation in the citrate synthase (EC 4.1.3.7) gene of E. coli K-12 by cloned M. leprae DNA that is expressed from the asd promoter of pYA626.

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