Quantitative competitive NASBA for measuring mRNA expression levels of the immediate early 1, late pp67, and immune evasion genes US3, US6 and US11 in cells infected with human cytomegalovirus.

Different cell types were infected with human cytomegalovirus (HCMV) and RNA expression dynamics were analyzed by quantitative NASBA assays for IE1 (UL123), pp67 (UL65) and the immune evasion genes (US3, US6 and US11). The quantitative NASBA assays gave reproducible quantification in the range of 10(3)-10(6) RNA copies for IE1 and pp67 RNA, from 3x10(3) to 1x10(6) RNA copies for US6 and US11 RNA, and from 1x10(4) to 1x10(6) RNA copies for US3 RNA. SMC, HAEC and HUVEC cells infected with an, in endothelial cells, propagated HCMV strain (VHL/E) showed similar RNA expression dynamics for the analyzed genes. Expression of all genes studied was observed within the first 4 h post-infection. The first gene for which expression could be detected was IE1, followed by US3, US11, pp67 and US6. Fibroblasts infected with HCMV strain AD169 showed a different RNA expression pattern for US3. Translation of the mRNA studied was demonstrated by detection of the proteins 48 h post-infection by immunofluorescence.

Monitoring of Chlamydia trachomatis infections after antibiotic treatment using RNA detection by nucleic acid sequence based amplification.

AIM: To investigate the value of RNA detection by nucleic acid sequence based amplification (NASBA) for the monitoring of Chlamydia trachomatis infections after antibiotic treatment. METHODS: Cervical smears (n = 97) and urine specimens (n = 61) from 25 C trachomatis positive female patients were analysed for the presence of C trachomatis 16S ribosomal RNA (rRNA) by NASBA and C trachomatis plasmid DNA by the polymerase chain reaction (PCR) before and up to five weeks after antibiotic treatment. RESULTS: Chlamydia trachomatis RNA was found in all cervical smears taken before antibiotic treatment (n = 24) and in two smears taken one week after antibiotic treatment; no C trachomatis RNA was detected after two weeks or more. In contrast, C trachomatis DNA was found in all such specimens before treatment, and 21 of 25, six of 21, and five of 20 smears were found to be positive at one, two, and three weeks after treatment, respectively. After four weeks, only one of six smears was positive, and this smear had been negative in the two preceding weeks. Of the 61 urine samples investigated, C trachomatis DNA and C trachomatis RNA were found in all before treatment (n = 15), whereas one week after treatment four of 15 were C trachomatis DNA positive and C trachomatis RNA was detected in one sample only. CONCLUSIONS: These data show that RNA detection by NASBA can be used successfully to monitor C trachomatis infections after antibiotic treatment. Furthermore, it might be possible to use urine specimens as a test of cure because neither C. trachomatis DNA or RNA could be detected two weeks or more after treatment.

Structure, chromosomal localization and evolutionary conservation of the gene encoding human U1 snRNP-specific A protein.

Three specific proteins, called A, 70K and C, are present in the U1 small nuclear ribonucleoprotein (snRNP) particle, in addition to the common proteins. The human U1 snRNP-specific A protein is, apart from a proline-rich region, highly similar to the U2 snRNP-specific protein B". To examine the homologous regions at the genomic level, we isolated and characterized the human U1-A gene. The human U1-A protein appears to be encoded by a single-copy gene and its locus has been mapped to the q arm of chromosome 19. The gene, about 14-16 kb in length, consists of six exons. The regions homologous to the U2-B" gene are not limited to single exons and are mostly not confined by exon-exon junctions in the corresponding U1-A mRNA. However, the proline-rich region of U1-A, absent in U2-B", is encoded by a single exon, suggesting a specific function for this domain of U1-A. The region of the cap site and upstream sequences contain interesting similarities to the promoter region of other snRNP protein-encoding genes and several housekeeping genes, in particular the vertebrate ribosomal protein-encoding genes. Hybridization experiments with various vertebrate genomic DNAs revealed that U1-A sequences are evolutionarily conserved in all tested vertebrate genomes, except for chicken, duck and pigeon. The divergence of these avian genomes is probably typical for the class of birds.

Small nuclear RNA associated proteins: autoantigens in connective tissue diseases.

Small nuclear ribonucleoproteins (smRNPs) are complexes of uridylic acid (U)-rich snRNAs with at least 7 core proteins and a varying number of specific proteins. Most of these proteins act as targets for autoantibodies in autoimmune patients. In the last two years, a number of cDNAs coding for these antigens have been cloned and some specific features of the deduced amino acid sequences of these proteins are discussed. The recombinant antigens, produced in high yields by the microorganism, can be used very efficiently in qualitative and quantitative ELISA tests. It is to be expected that the availability of rather large amounts of pure antigen will be of great help in elucidating the question of why these autoantibodies are generated and what their relation to the disease might be.

Small nuclear RNA-associated proteins are immunologically related as revealed by mapping of autoimmune reactive B-cell epitopes.

Autoantibodies from a patient with systemic lupus erythematosus, which recognize U1 and U2 small nuclear ribonucleoprotein particles (snRNPs), were used to map B-cell autoepitopes on the U1 snRNP-specific A protein. This protein contains two regions that are highly similar to regions in the U2 snRNP-specific B" protein. A site termed epitope 2 maps in one such region and was found to react with antibodies cross-reactive between A and B". A second site, epitope 1, is situated in a proline-rich region that shows no homology with B". This epitope can bind three different autoantibodies with distinct specificities. Epitope 1-affinity-purified antibodies from different patients react with either (i) the A protein exclusively; (ii) proteins A, B'/B, a synthetic peptide for part of the N polypeptide, and an unidentified protein with a molecular mass of 50 kDa; or (iii) proteins A, B'/B, C, and the N-derived peptide. Comparison of the primary structures of proteins B'/B, N, and C reveals multiple epitope 1-like sequences in all of them. The possibility that these repeating regions act as immunogens in patients with autoimmune disease is discussed.

Detection of autoantibodies in a quantitative immunoassay using recombinant ribonucleoprotein antigens.

A human cDNA expression library was screened with anti-ribonucleoprotein (RNP) antibodies from patients with connective tissue diseases. Three cDNA clones were isolated encoding 70 kD, A and B" ribonucleoprotein autoantigens which were expressed as beta-galactosidase fusion proteins. Antigens were purified and used to develop sensitive ELISAs suitable for the routine screening of large series of sera from patients with connective tissue diseases. More than 400 sera were tested both by ELISA and by immunoblotting. The ELISA was found to be at least as sensitive as immunoblotting and very specific. Anti-70 kD antibodies were found in 94% of patients with mixed connective tissue disease (MCTD), in 4% of patients with other connective tissue diseases but not in normal controls. Furthermore, the use of recombinant 70 kD antigen enabled us to discriminate between anti-70 kD antibodies present in anti-Sm and in anti-(U1) RNP sera. Recombinant A antigen contained at least two autoantibody-reactive sites; one unique for the A protein and another cross-reactive with anti-B" antibodies. Antibodies reactive with the unique site were found in 83% of MCTD patients, in 4% of patients with other connective tissue diseases and not in normal controls. Antibodies against the cross-reactive B" epitope present on A and B" recombinant antigens, were found in high titres in a small percentage of patients with systemic lupus erythematosus (SLE, 5%) and rheumatoid arthritis (RA, 2%).

Molecular cloning of the cDNA for the human U2 snRNA-specific A' protein.

The A' polypeptide is one of the protein constituents of the U2 snRNP particle. A potentially full-length cDNA clone containing the complete coding sequence for this U2 snRNP-specific protein was isolated by screening of a human lambda gt11 expression vector library with an autoimmune anti-(U1,U2)RNP serum. Monospecific antibodies, eluted from the 140-150 kD fusion protein of this cDNA recombinant, specifically recognized the A' protein on immunoblots and immunoprecipitated U2 snRNP particles from nuclear extracts. The identity of the clone was confirmed by in vitro translation of hybrid-selected mRNA or an RNA transcript synthesized from the cDNA insert. RNA blot analysis showed strong hybridization to a single polyadenylated transcript of 1.3 kb in human cells. The nucleotide sequence of the 1054 bp cDNA contains an open reading frame of 756 bp encoding a polypeptide of 255 amino acids with a predicted molecular weight of 28,444 D. The coding sequence is preceded by a 49 bp 5'-untranslated region and followed by a 226 bp 3'-untranslated region containing a single polyadenylation signal. Most striking feature of the deduced primary structure for the A' protein is a leucine-rich region in the amino-terminal half of the polypeptide. In contrast to the other U2 snRNP-specific protein B", the A' protein does not contain segments homologous to the RNP consensus sequences RNP1 and RNP2, common amino acid motifs found in several RNA-binding proteins. In the A' protein, however, the extremely hydrophilic carboxy terminus may constitute an RNA-binding moiety.

Human U1 snRNP-specific C protein: complete cDNA and protein sequence and identification of a multigene family in mammals.

A complementary DNA clone for the human U1 snRNP-specific C protein has been isolated. The nucleotide sequence of the 733 bp cDNA insert includes a 15 bp 5'-untranslated region, an open reading frame of 477 bp corresponding to 159 amino acids (Mr = 17,373 D), and a 223 bp 3'-untranslated region. The identity of the clone was confirmed by in vitro translation of hybrid-selected mRNA or an RNA transcript synthesized from the cDNA. The in vitro synthesized C protein has a slightly greater mobility on SDS-polyacrylamide gels, indicating that the in vivo product is post-translationally modified. The deduced primary structure contains a segment of high proline and methionine content. A region homologous to the RNP consensus sequence, found in the other two U1 snRNP-specific proteins 70K and A, is absent. Analysis of genomic DNA restriction enzyme digests shows hybridizing fragments in the genome of all vertebrate classes. The results are consistent with multi-copy representation of the C protein gene in mammals, whereas in the other vertebrate classes the related protein seems to be encoded by a single-copy gene.

cDNA cloning of the human U1 snRNA-associated A protein: extensive homology between U1 and U2 snRNP-specific proteins.

Sera from patients with connective tissue diseases often contain antibodies against snRNA-associated proteins. Using one of these sera in an immunological screening of a human lambda gt11 expression vector cDNA library, two cDNA clones for the U1 snRNP-specific A protein, termed lambda HA-1 and lambda HA-2, were isolated. Monospecific antibodies, eluted from the beta-galactosidase fusion protein of either clone reacted with the U1 snRNP-specific A antigen. The identity of the clones was confirmed by in vitro translation of hybrid selected mRNA. RNA blot analysis revealed a single polyadenylated transcript of about 1.4 kb in human cells. A cDNA of 1.2 kb, isolated from the same lambda gt11 expression library by cross-hybridization with a lambda HA-2 restriction fragment, covered the complete coding sequence of the A protein as demonstrated by in vitro translation of an RNA transcript synthesized from this cDNA. The deduced amino acid sequence contains one very hydrophilic region, and internal sequence duplication and a region highly homologous to the RNP consensus sequence that seems to be common to RNA binding proteins. Sequence comparison with the recently cloned U2 snRNP-specific B" protein revealed two extremely homologous regions located in the carboxy-terminal (homology of 86%) and amino-terminal part (homology of 77%) of the proteins. This structural relationship indicates that proteins A and B", although located in different snRNP particles, may have identical functions.

Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B" antigen.

A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled us to isolate cDNA clone lambda HB''-1 from a phage lambda gt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone lambda HB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone lambda HB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the lambda HB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone lambda HB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens.

A model for the excision of introns 1 and 2 from adenoviral major late pre-messenger RNAs.

Total steady-state RNA was extracted from nuclei of HeLa cells late after infection with adenovirus serotype 2. Most of the nuclear RNA is transcribed from the major late transcription unit (16.2 to 100.0 map units). To study the cleavage reactions involved in the splicing of leaders 1 and 2, we have used the S1 nuclease mapping technique with restriction fragments located in the region of intron 1 as DNA probes. The S1 mapping data showed that in total nuclear RNA, RNA species accumulate from which the 5' part of intron 1 has been excised, but which still contain the 3' part of the intron. This indicates that intron 1 can be removed in a stepwise fashion following the 5' to 3' direction. We have compared the nucleotide sequences from the ends of the putative processing intermediates. The internal cleavage sites do not resemble the consensus 5' or 3' splice site sequences. However, they show considerable homology to the sequence 5' A-T-G-A-T-G-G-C-A-T 3', which may act as a signal for internal cleavage. The intermediates are present in both the poly(A)+ and poly(A)- RNA fractions, although with different relative intensities. Primer extension experiments have been performed in which a primer, located with its left end in leader 2, is extended into intron 1. The results show that there may be a cleavage site as short as 35 nucleotides before the 3' splice site. Cleavage at the 3' splice site seems to be rapidly followed by ligation of leader 1 to leader 2. A model for RNA splicing based on these findings and data from the literature is presented.