A diagnostic hybridization assay for detecting human cytomegalovirus (HCMV) DNA in urine specimens was developed by using cloned viral DNA and in vitro-synthesized RNA probes. Both probes detected 3 pg of homologous DNA and hybridized with DNA of HCMV but not with other viral or human cellular DNA tested. In 95 urine specimens simultaneously tested by cell culture, the sensitivity of hybridization was at least 83%, and the specificity was at least 92%. This assay will be useful for rapid viral diagnosis with wide clinical applications such as screening of immunocompromised patients and quantitation of viral shedding in patients with primary or reactivated HCMV infection who may be receiving antiviral therapy.

1. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from Saccharomyces cerevisiae was purified 9400-fold by affinity chromatography giving rise to an electrophoretically homogeneous preparation.2. The molecular weight of the enzyme was determined by gel filtration with Sephadex G-100 and by sodium dodecylsulfate gel electrophoresis. Both methods reveal a molecular weight of 51 000.3. The enzyme requires Mg2+ and has its pH optimum at 8.5. 4. Isoelectric focussing as well as gel electrophoresis of the purified extract reveals a single band which exhibits enzyme activity. The isoelectric point of the enzyme is 5.1.5. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine and phosphoribosylpyrophosphate of 23 pM, 18 pM, and 50 pM respectively.The yeast Saccharomyces cerevisiae has a common transport system for the purine bases adenine, guanine, hypoxanthine, and the pyrimidine base cytosine [l -41. In recent publications we studied the recognition step of the transport system [ 5 ] and discussed a possible mechanism of energy coupling [6,7] with the purine-cytosine transport system being active as a proton symporter and a potassium antiporter.These results are in contrast to the findings of Hochstadt-Ozer and Stadtman [8 -121 for the purine and pyrimidine transport in Escherichia coli. They support the concept that purines and pyrimidines are transported by a group-translocating process involving the appropriate membrane-bound phosphoribosyltransferase enzyme and 5-phosphoribosyl 1-diphosphate as cofactor.However, reports from Burton [13,14], RoyBurman and Visser [15] question this concept. Their data support a transport without direct involvement of the phosphoribosyltransferases dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases.Abbreviation. PRib-PP, 5-phosphoribosyl l-diphosphate. Enzymes. Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8); adenine phosphoribosyltransferase (EC 2.4.2.7); orotidine-5'-phosphate pyrophosphorylase (EC 2.4.2.10); inorganic pyrophosphatase (EC 3.6.1.1).Housset and Nagy [16], who investigated the transport of purines in Schizosaccharomyces strains devoid of phosphoribosyltransferase activities, report a reduced uptake for these mutants. However, their results do not support the hypothesis of a grouptranslocation mechanism.We found a reduced uptake in Saccharomyces cerevisiae after prefilling the cells with hypoxanthine but a prefilling with cytosine had no effect (unpublished results). This supports the assumption that the hypoxanthine-guanine phosphoribosyltransferase might have a regulatory function on the purine-cytosine transport system. To gain more information about this enzyme we isolated, purified and characterized the hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae. MATERIALS AND METHODS Chemicals14C-labelled guanine and hypoxanthine were obtained from Amersham Buchler (Braunschweig). The unlabelled purines were purchased from Pha...

Three brothers from one family died of Hodgkin disease and a fourth brother from a diffuse malignant non-Hodgkin lymphoma. This patient exhibited a constant deficiency of serum immunoglobulins and elevated antibody titers to Epstein-Barr viral antigens. Epstein-Barr virus DNA sequences were detected in DNA isolated from lymph node biopsies from two of the patients. Initially, no abnormalities in the numbers of B and T cells could be detected. Peripheral blood lymphocytes of the patients did not react in the mixed lymphocyte culture assay. We suggest that an immune deficiency to Epstein-Barr virus may favor the proliferation of malignant lymphocytes after Epstein-Barr viral infection. Monoclonal lymphoid B cell lines established spontaneously in vitro from a lymph node biopsy specimen and from peripheral blood specimens from two of the patients. The cells harbor Epstein-Barr viral DNA sequences in multiple genome equivalents and express Epstein-Barr nuclear antigen. The cells contain a tenfold increased level of c-fgr-related RNA transcripts compared with peripheral blood lymphocytes of healthy adults. No obvious amplifications or translocations of the c-myc, c-abl, or c-fgr gene could be detected.

A diagnostic hybridization assay for detecting varicella zoster virus (VZV), herpes simplex virus (HSV), and Epstein-Barr virus (EBV) in different clinical specimens was developed using cloned viral DNAs as probes. All probes detected at least 5 pg of homologous DNA and did not cross-react with other viral or cellular DNA. Results of cell culture, serology, and DNA assay were highly concordant. Using a simple standardized protocol for preparation of specimens, hybridization, and washing procedures, this sensitive and specific assay appears to be useful for screening clinical specimens and may be helpful in confirming the serological diagnosis of HSV encephalitis and persistent EBV infections or EBV-associated diseases.

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