After preliminary serologic data demonstrated elevated antibody titers to human herpesvirus (HHV) 6 in patients with sinus histiocytosis with massive lymphadenopathy (SHML) or Rosai-Dorfman disease, tissues were examined from 9 patients with classical SHML to search for evidence of HHV-6 infection. Involved tissues from 7 of the 9 patients had detectable HHV-6 by in situ hybridization: Tissue from 1 had detectable Epstein-Barr virus genome but no HHV-6 and tissue from another had no detectable HHV-6 or Epstein-Barr virus. These studies suggest that HHV-6 and, to a lesser extent, Epstein-Barr virus may be involved in the etiology of SHML.
We have developed a fluorescence resonance energy transfer (FRET)-based assay to detect ciprofloxacin resistant (Cp r ) mutants of the biothreat agent Yersinia pestis. We selected spontaneous mutants of the attenuated Y. pestis KIM 5 strain that were resistant to a ciprofloxacin (CIP) concentration of at least 1 g/ml. DNA sequencing of gyrA encoded by 65 of these mutants revealed that all isolates contained one of four different point mutations within the quinolone resistance-determining region of gyrA. We developed a FRET-based assay that detected all of these mutations by using a single pair of fluorescent probes with sequences complementary to the wild-type Y. pestis gyrA sequence. Melting peak analysis revealed that the probe-PCR product hybrid was less stable when amplification occurred from any of the four mutant templates. Resistance to antibiotics has become a major concern for the medical community over the past several years (13, 14, 16). Many organisms have become resistant to the common "drug of choice" used to treat the disease. A few examples are methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci (18), and multiple-drug-resistant Mycobacterium tuberculosis (19), as well as organisms causing many enteric diseases. One of the current antibiotics that is effective in treating bacterial infectious diseases is ciprofloxacin (CIP), a fluorinated quinolone that blocks DNA replication through inhibition of gyrase activity (2, 24). Resistance to CIP does occur and is usually mediated by point mutations in DNA gyrases or, less commonly, through membrane alterations that reduce drug entry into the bacteria (28).A critical piece of information necessary for the treatment of any bacterial disease is the antibiotic sensitivity profile of the infectious agent. Classically the sensitivity profile has been determined by growth of the organism in the presence of the antibiotic either in agar diffusion assays or by incubation of the organism in various concentrations of the drug for determination of the MIC. Both of these methods depend on growth of the bacterium after its initial isolation and are therefore timeconsuming. DNA probe-based detection of antibiotic resistance offers the potential for increased speed. Among DNAbased techniques, PCR offers the best opportunity for speed, sensitivity, and specificity.Recently it has become possible to couple PCR with realtime detection of the amplification product by use of fluorescent probes, thus eliminating the necessity to analyze the reaction product by gel electrophoresis. Fluorescence resonance energy transfer (FRET) is one of the available chemistries that can be used to detect the PCR product in these reactions. Roche Diagnostics has adopted this chemistry for its "Hybridization Probes" technology (5). Two DNA probes are used to bind to the amplification product when FRET chemistry is used to specifically detect the amplification product. The two light-activated molecules are positioned in close proximity at the 3Ј and 5Ј termini of the ...
To define the morphological transforming region II (mtrl) of human cytomegalovirus (HCMV), a series of subclones of the Xba I/BamHI fragment EM was constructed in vitro and tested for focus-forming activity and tumorigenicity. A 980-base-pair subclone of fragment EM was identified, and its nucleotide sequence revealed three small open reading frames (ORFs), encoding 79, 83, and 34 amino acid residues. Si nuclease analysis of HCMV-infected cells DNA of human cytomegalovirus (HCMV) contains three transforming fragments, which have been mapped in the long unique region of the viral genome (see Fig. 1 Left). A minimal region of 558 base pairs (bp) (pCM4127) was localized in the Xba 1/HindIII fragment of HCMV strain AD169 (map unit 0.123-0.140) and designated morphological transforming region I (mtri). pCM4127 DNA was reported to cause one-step focal transformation of primary Wistar rat embryo cells and NIH 3T3 mouse cells (1, 2). This sequence was noncoding and contained a stem-loop structure (3) analogous to an insertion-like element.Two distinct transforming regions were mapped in the 20-kilobase (kb) Xba I fragment E (XbaI-E) of HCMV Towne DNA (map unit 0.680-0.770). Cloned XbaI-E will immortalize diploid Syrian hamster embryo cells and induce the neoplastic transformation of established rodent cell lines (4). The left-hand 3-kb Xba I/BamHI EM segment of XbaI-E, designated mtrII, and a right-hand 7.6-kb Xba I/BamHI EJ segment, designated mtrIII, were independently capable of inducing tumorigenic transformation of established rodent cells (5). Cell lines transformed individually by EJ or EM and their tumor derivatives retained the EM but not the EJ sequence (5). Similarly, only EM sequences were retained in the transformed and tumor-derived cells induced by cotransfection of cloned EJ and EM plasmids (unpublished data). These data suggested that EM sequences were required both to initiate and to maintain the transformed phenotype. In contrast, EJ sequences were required only for the initiation of transformation, perhaps by a hit-and-run mechanism analogous to the BglII-N fragment of herpes simplex virus 2 or pCM4127 of HCMV AD169 (3).In the present study we localized a minimal 980-bp transforming Ban II-Xho I region within the HCMV Towne Xba I/BamHI EM fragment and determined its nucleotide sequence. ¶ Nucleotide sequence analysis identified three potential open reading frames (ORFs) within the 980-bp fragment, and S1 nuclease protection assay revealed the presence of "early" RNA transcripts in infected cells. In addition, noncoding DNA sequence elements were observed with the potential to form stem-loop structures.
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