Two of the Sal I fragments and all of the internal BamHI fragments (with the exception of BamHI c, a 0.6 X 106 dalton fragment) of Epstein-Barr virus (EBV) DNA (2); DNA isolated from virus particles that have aged in the culture medium prior to harvest has frequent single-strand nicks and probably gaps arising from proteolytic degradation of virus and endonucleolytic degradation of the DNA (4-6). (iv) At both ends of the DNA there are from 1 or 2 to as many as 10-12 tandem repeats of a 500-base-pair sequence (5,6); the sequences at both ends of EBV DNA are direct repetitions of each other (6). (v) Beginning approximately 1.5 X 104 base pairs from one end of the DNA, there are from 1 or 2 to as many as 10 or 11 tandem reiterations of a 3000-base-pair sequence (7,8); these internal tandem reiterations connect the short unique region of 1.5 X 104 base pairs with the long unique region of 12.5 X 104 base pairs. (vi) The order of fragments generated by cleavage of the DNA with EcoRI, HindIII, Sal I, Xba I, Kpn I, and Hpa I is known in full or in part (5,8,9).More precise knowledge of the restriction enzyme sites in the DNA is needed for ongoing studies of EBV-specified mRNAs in growth-transformed human B lymphocytes (10) and in human Burkitt tumor tissue (11), for analysis of the more complex nuclear RNAs of growth-transformed cells (12, 13), for the mapping of virus-specified proteins in transformed and productively infected cells, for determination of whether EBV DNA is integrated into cellular DNA, and for comparison of EBV DNAs from different epidemiologic niches (5,14). For all of these purposes, it is necessary to obtain large quantities of specific viral DNA fragments. We therefore undertook to clone EBV DNA fragments in two vectors: the Col E1/pSC101The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad-

A continuous lymphoblastoid cell line, IB-4, was established by infection and growth transformation of normal neonatal B lymphocytes with the B95-8 isolate of Epstein-Barr virus (EBV). The IB-4 cells contained the intranuclear antigen, EBNA, but not early antigen, EA. The fragments produced by the digestion of intracellular episomal viral DNA (density, 1.700 to 1.720 g/cm3) with EcoRI restriction endonuclease were identical in size to the A, B, C, E, F, G, and H fragments of virion DNA. As expected from the previous observation that episomal intracellular DNA is circular, the fragment containing the rightward terminal sequences of EBV DNA in IB-4 cells was larger than the corresponding fragment of linear viral DNA, probably as a consequence of covalent linkage to the leftward terminal fragment. Also, two fragments, EcoRI-I and -J, which were adjacent to each other in the virion DNA, were absent from the intracellular DNA. The labeled EcoRI-J of viral DNA hybridized instead to a new fragment equal in size to EcoRI-I and -J combined. Analysis of viral RNA in IB-4 cells showed that RNAs encoded by more than 30% of the viral DNA comprised approximately 0.06% of the nuclear RNA, whereas RNAs encoded by 20% and 10% of the viral DNA comprised approximately 0.06% and 0.003% of the polyadenylated and polyribosomal RNAs, respectively. Viral mRNA (polyribosomal RNA) was encoded by DNA which mapped at 0.05 x 10(8) to 0.36 x 10(8) daltons and to a lesser extent by DNAs which mapped at 0.62 x 10(8) to 0.67 x 10(8), 0.70 x 10(8) to 0.73 x 10(8), and 1.13 x 10(8) to 1.15 x 10(8) daltons in the B95-8 genome. The most agundant nuclear viral RNAs were encoded primarily by DNA which mapped at the same loci; but RNAs encoded by many other fragments of viral DNA could also be detected among nuclear RNAs. Viral mRNA(s) (polyribosomal) was encoded by about 40% of the internal reiteration and by 25% of the BamHI-H fragments which mapped from 0.32 x 10(8) to 0.36 x 10(8) daltons, nuclear RNAs were encoded by at least 57% of the internal reiteration and 40% of BamHI-H. These data indicate that there is selective accumulation of some viral RNAs within the nucleus of IB-4 cells and that there is selective post-transcriptional processing of these RNAs. Finer mapping of the DNA which encodes mRNA (polyribosomal) in IB-4 cells indicated that some of this DNA is deleted in the DNA of the P3 HR-1 virus, the only isolate of EBV which cannot initiate growth transformation. These data, therefore, support the hypothesis that expression of this region of EBV genome is important for growth transformation or for the maintenance of restrigent infection.

Mutagenesis was studied in cultured F9 embryonal carcinoma cells infected with a variant of Moloney murine leukemia virus. Proviral insertion induced the inactivation of the hypoxanthine phosphoribosyltransferase locus, and the virus was used to isolate the mutated genes rapidly. Mutagenesis by these methods may be useful for the genetic dissection of the various mammalian cell phenotypes.

The P3HR-1 subclone of Jijoye differs from Jijoye and from other Epstein-Barr virus (EBV)-infected cell lines in that the virus produced by P3HR-1 cultures lacks the ability to growth-transform normal B lymphocytes (

We report on a rapid, sensitive, Q-Beta replicase-amplified nucleic acid hybridization assay for the detection of Mycobacterium tuberculosis directly from spiked human sputum. Specimens were processed by either an N-acetyl-L-cysteine-NaOH or a 2% NaOH digestion-decontamination method and then washed to neutralize the pH of the cell pellet. The washed sputum pellets were heated at 100؇C to inactivate the M. tuberculosis organisms. The heat-inactivated samples were mechanically lysed at 5,000 rpm for 6 min in the GENE-TRAK Sample Processing Instrument in the presence of zirconium oxide beads and a buffer containing guanidine thiocyanate. The released nucleic acid was subjected to the GENE-TRAK Q-Beta replicase-amplified, dualcapture assay. The assay sensitivity was 10 3 purified rRNA targets or 1 CFU of M. tuberculosis spiked into M. tuberculosis-negative human sputum. There was a low level of noise because of the limitations of performing a signal amplification assay in an open system. High levels of other mycobacterial rRNA (approximately 10 7 organisms), including rRNAs of Mycobacterium avium and Mycobacterium gordonae, did not interfere with the sensitivity of the assay.