Much effort has been devoted to the identification of immunologically important antigens of Mycobacterium tuberculosis and to the combination of target antigens to which antibodies from serum of tuberculous patients could react specifically. We searched for IgG antibodies specific for antigens of 45 to 6 kDa obtained after sonication of the well-characterized wild M. tuberculosis strain in order to detect differences in the antibody response to low molecular weight antigens from M. tuberculosis between patients with pulmonary tuberculosis and contacts. Specific IgG antibodies for these antigens were detected by Western blot analysis of 153 serum samples collected from 51 patients with confirmed pulmonary tuberculosis. Three samples were collected from each patient: before therapy, and after 2 and 6 months of treatment. We also analyzed 25 samples obtained from contacts, as well as 30 samples from healthy individuals with known tuberculin status, 50 samples from patients with other lung diseases and 200 samples from healthy blood donors. The positive predictive value for associated IgG reactivity against the 6-kDa and 16-kDa antigens, 6 and 38 kDa, and 16 and 38 kDa was 100% since simultaneous reactivity for these antigens was absent in healthy individuals and individuals with other lung diseases. This association was observed in 67% of the patients, but in only 8% of the contacts. The humoral response against antigens of 16 and 6 kDa seems to be important for the detection of latent tuberculosis since the associated reactivity to these antigens is
A purified, monoclonal antibody, specific for the left-handed Z-form of poly(dG-dC), was coupled covalently to Sephacryl S-1000 beads. Such an antibody column provides a convenient method to isolate and purify those plasmid DNAs that contain Z-DNA from a large excess of other DNAs, RNA, etc. From a library of Escherichia coli DNA, cloned into the vector plasmid pUC-8, several recombinant plasmids were isolated, which bind to this antibody. Thus, E. coli contains sequences, which in "natural" negatively supercoiled DNA, adopt a left-handed Z-DNAlike conformation.Physicochemical studies of poly-and oligo (dC-dG (17), were used for cell fusion (22, 23). Cell supernatants were screened for precipitating 32P-labeled poly(dGdC) in 4 M NaCl. Seven monoclonal cell lines producing antibodies to Z-DNA were established (unpublished data). Antibodies (K/y2b) from ascites fluids, by using cell line Z-DI1, were purified by protein A-Sepharose (Pharmacia) chromatography (24, 25). Antibody concentration was determined, by using A280 = 1.4 for a solution at 1 mg/ml. l"I-Labeled antibodies with -10,000 cpm/ng were prepared by the Chloramin-T method (26).Antibody Column. Sephacryl S-1000 (superfine; Pharmacia) was activated with cyanogen bromide in sodium carbonate buffer (27) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
The nucleotide sequence of the junction between the simian virus 40 early region and the adenovirus type 2 late region L4 in the hybrid virus Ad2+D2 was determined. The deduced amino acid sequence suggests that the D2-T antigen is a chimeric protein sharing 594 amino acids with the C-terminal end of the simian virus 40 T antigen and 104 amino acids with the N terminus of the adenovirus type 2 33,000-molecular-weight protein. The predicted structure of the D2-T antigen was confirmed by an immunoprecipitation analysis. Adenovirus type 2/simian virus 40 (Ad2/SV40) hybrid viruses have been invaluable tools in defining the coding region for SV40 T antigen, assigning functional domains to this pleiotropic protein, analyzing the transcription of both Ad2 and SV40, and studying recombination (for review, see references 10 and 29). Ad2+D2, a defective Ad2/SV40 hybrid, was isolated more recently (6) by plaque purification from a mixture of hybrid viruses known as Ad2+ +HEY (11). Ad2+D2 contains an almost entire copy of the SV40 genome ranging counterclockwise from about 0.54 to 0.63 map units in partial replacement of Ad2 sequences that are deleted between 73 and 98 map units (see reference 6 and Fig. 1). HeLa cells infected with this virus synthesize large quantities of an SV40 T-antigen-related protein, D2-T antigen, of a molecular weight estimated to be 107,000 (6, 26). This protein has been extensively purified and shown to have biological properties similar to those of an authentic SV40 T antigen (20, 27, 28). In particular, it binds with a specificity similar to that of T antigen to three sites on the SV40 genome comprising the origin of replication (2, 26). It has been proposed that the N-terminal end of D2-T antigen consists of part of the Ad2 100,000-molecular-weight (100K) protein (6), but this has not been demonstrated directly. Since the Ad2 DNA sequences adjacent to the
cDNA clones similar to rabbit muscle phosphatase inhibitor-2 (IPP-2) were isolated from human libraries. On Northern blots two transcripts of approximately 2kbp and approximately 4kbp were detected in all tissues tested. Analysis of cDNA sequences showed that the longer transcripts were similar to the shorter clones but contained extended 3' ends. The human nucleotide sequence was highly homologous (94% identity) to the rabbit IPP-2 sequence and encoded a peptide of 205 amino acids. IPP-2 sequences were highly conserved throughout vertebrates. Southern hybridization results were consistent with the existence of a family of related IPP-2 sequences in the human genome. Most of these are likely to be pseudogenes, since all of the cDNA clones examined could have originated from a single gene. By in situ hybridization IPP-2 sequences were mapped to several different human chromosomes. We sequenced one gene located in the major histocompatibility complex (MHC) on Chromosome (Chr) 6 that contained the entire coding region of IPP-2.
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