Interest in improving the speed of DNA analysis via capillary electrophoresis has led to efforts to integrate DNA amplification into microfabricated devices. This has been difficult to achieve since the thermocycling required for effective polymerase chain reaction (PCR) is dependent on an effective contact between the heating source and the PCR mixture vessel. We describe a noncontact method for rapid and effective thermocycling of PCR mixtures in electrophoretic chip-like glass chambers. The thermocycling is mediated through the use of a tungsten lamp as an inexpensive infrared radiation source, with cooling effected with a solenoid-gated compressed air source. With temperature ramping between 94 and 55 degrees C executed in glass microchambers as rapidly as 10 degrees C/s (heating) and 20 degrees C/s (cooling), cycle times as fast as 17 s could be achieved. Successful genomic DNA amplification was carried out with primers specific for the beta-chain of the T-cell receptor, and detectable product could be generated in a fraction of the time required with commercial PCR instrumentation. The noncontact-mediated thermocycling format was not found to be restricted to single DNA fragment amplification. Application of the thermocycling approach to both quantitative competitive PCR (simultaneous amplification of target and competitor DNA) and cycle sequencing reactions (simultaneous amplification of dideoxy terminated fragments) was successful. This sets the stage for implementing DNA thermocycling into a variety of microfabricated formats for rapid PCR fragment identification and DNA sequencing.

We have developed 21 specific nucleic acid probes which target the large subunit rRNA genes from Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus niger, Aspergillus terreus,

The presence of KatG(S315T), a mutation frequently detected in clinical isolates of Mycobacterium tuberculosis, has been associated with loss of catalase-peroxidase activity and resistance to isoniazid therapy. Wild-type KatG and KatG(S315T) were expressed in a heterologous host (Escherichia coli) and purified to homogeneity, and enzymatic activity was measured. The catalase activity for KatG(S315T) was reduced 6-fold, and its peroxidase activity was decreased <2-fold, compared with the activities for wild-type KatG. Pyridine hemochrome analysis demonstrated 1.1 +/- 0.1 hemes/subunit for wild-type KatG and 0.9 +/- 0.1 hemes/subunit for KatG(S315T), indicating that the difference in enzymatic activity is not the result of incomplete heme cofactor incorporation in KatG(S315T). High-performance liquid chromatography analysis showed that wild-type KatG was more efficient than KatG(S315T) at converting isoniazid to isonicotinic acid. These results demonstrate that KatG(S315T), as expressed in E. coli, is a competent catalase-peroxidase that exhibits a reduced ability to metabolize isoniazid.

The F sex factor plasmid of Escherichia coli contains a pair of genes, ccdA and ccdB, whose protein gene products are involved in an unusual feature of plasmid maintenance. The CcdB protein is a cytotoxin that becomes activated when the F plasmid is lost, thereby killing the F-segregant cells. In F+ cells, the CcdA protein protects against the lethal effects of CcdB. In the present study we show that ccdA and ccdB expressions are negatively autoregulated at the level of transcription. Genetic studies showed that repression required at least ccdB; ccdA alone was without effect, and ccdB alone was not examined because it is lethal. Ccd-operator complexes were purified and contained a mixture of both CcdA and CcdB proteins; however, we could not conclude from our results whether CcdA was necessary for DNA binding or autorepression. By using restriction fragments of the promoter-operator region, we obtained results indicating that at least two DNA-binding sites existed for the Ccd protein(s). Subsequent footprinting of the binding sites showed protection over about a 113-base-pair region encompassing the putative promoter-operator and the beginning of the cedA gene.An F+ culture maintains its F plasmids not only by periodic plasmid replication and equipartitioning but also by destruction of F-segregants. The plasmid-encoded ccdA and ccdB genes are involved in the latter process (7-9, 19). Interestingly enough, at least the ccdB gene product, the CcdB protein (Mr 11,700), also participates in F DNA replication initiated at origin V (13) (Fig. 1).Very little is known about how the ccd proteins affect any of their functions. With respect to destruction of F-segregants, nascent F-cells grow and divide normally for two or three generations and then filament and lose their ability to act as colony-forming cells (7,8). The CcdB protein is necessary for filamentation and is lethal in the absence of CcdA protein (Mr 8,700), which implies that the latter neutralizes CcdB (19). CcdA and CcdB form a complex of about 69,000 Mr in vitro (Tam and Kline, submitted for publication). A comparable interaction in vivo could be relevant to neutralization of the lethal effects of CcdB.The ccd locus has been sequenced (20), and the proteins have been expressed and partially characterized by twodimensional gel electrophoresis (1). In these earlier publications, the proteins were referred to as H(CcdA) and G(CcdB), but we now refer to them according to the widely employed convention mimicking the name of the genetic locus. Both the sequence and expression data suggested that ccdA, ccdB, and the repD locus might form an operon. The latter locus encodes a site-specific resolvase (12).Our purpose in the present work has been to define control of ccd expression. Our results indicate that expression of ccdA and ccdB is negatively controlled at the level of transcription. At least the CcdB protein is required for repression, and perhaps CcdA is required as well. The CcdA protein by itself is not regulatory. We have cloned the ccdA and ccdB genes ...

The ccd operon of plasmid F produces three proteins, CcdA, CcdB, and RepD. Prior research has established that the operon is autorepressed and that at least CcdB, but not RepD, is required for autorepression. A role for CcdA in autorepression was suggested but not clearly shown. We now present a series of biochemical experiments which show that both CcdA and CcdB proteins are required for maximal formation of protein-ccd operator complexes. We also show that CcdA and CcdB are present in a complex whether or not ccd operator is present. The clear implication is that autorepressor is a complex of CcdA and CcdB. We also map the start site of the ccd transcript thus providing the first experimental evidence for the location of the ccd promoter.

The complete catalase-peroxidase (katG) gene DNA sequence was determined for 15 strains of Mycobacterium tuberculosis with a wide range of susceptibility to isoniazid. Five of 9 strains with isoniazid MICs > or = 1.0 microgram/mL had one or more missense mutations and all 5 strains had a common G-->T transversion in codon 463, causing the replacement of arginine with leucine and the loss of an NciI or MspI restriction site. None of 6 strains with an isoniazid MIC < 1.0 microgram/mL had mutations affecting codon 463. Restriction analysis of 43 strains with isoniazid MICs > or = 1.0 microgram/mL showed that 19 (44.2%) had lost the NciI-MspI restriction site at the locus of codon 463 while only 1 of 32 strains with isoniazid MICs < or = 1.0 microgram/L had this restriction polymorphism. These results indicate that the mutation arginine-->leucine in codon 463 of the catalase-peroxidase gene occurs in a significant fraction (44.2%) of M. tuberculosis strains with isoniazid MICs > or = 1.0 microgram/mL.

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The 16S rRNA sequences of enterococcal species E. faecium, E. faecalis, E. gallinarum, E. casseliflavus/flavescens, E. dispar,E. pseudoavium, E. sulfureus,E. malodoratus, E. raffinosus,E. cecorum, E. hirae, E. saccharolyticus, E. seriolicida, E. mundtii, E. avium, E. durans,E. columbae, and E. solitarius are presented herein. These data were utilized to confirm the species identification of two nonmotile E. gallinarum isolates which had been previously phenotypically identified asE. faecium. The implications of this finding are discussed.