Rapid and sensitive detection of Chlamydia trachomatis using a ligatable binary RNA probe and Q beta replicase.

A simple assay format was developed for the direct detection of C. trachomatis rRNA utilizing ligation of recombinant MDV-1 probe RNA fragments hybridized to 23S rRNA after capture and release from a solid support. Assay background (equivalent to 10(4) targets) was suppressed by blocking sequences in the 5' MDV reporter probe fragment complementary to the 3' fragment by prehybridization of a DNA oligonucleotide. A pair of reporter fragments bearing a deletion within the region, obtained by a hydrid-selection-amplification protocol, yielded a low level of assay background which was reduced to < 2% with a blocker directed against the remaining pairing sequence. This probe set showed a sensitivity of 10(3) molecules of 23S rRNA (> 95% responding) and could detect a single elementary body (EB) of Chlamydia trachomatis or 1-10 EB added to a clinical matrix of pooled negative human cervical swab samples. The time of first appearance of amplification products by real-time fluorescence detection showed a linear response to log increases in the target level over a 10(5)-fold range, permitting the determination of target level within an order of magnitude. The assay showed approximately 10(9)-fold discrimination over Chlamydia pneumonae (TWAR) rRNA. High levels of cultured C. albicans, E. coli, S. aureus, or N. gonorrhoeae had no detectable effect on assay background or the ability to detect a single elementary body.

Real-time fluorescence detection of RNA amplified by Q beta replicase.

Amplification of RNA probes by Q beta replicase can be used to detect a wide range of analytes with a potential sensitivity of a single molecule. A system has been developed in which Q beta amplification of midivariant-(MDV)-based RNA is measured in real time by fluorescence. This was accomplished by including a fluorescent intercalating dye, propidium iodide, in the reactions and monitoring the fluorescence change using a custom fluorometer. The time at which fluorescence is detectable above background is referred to as the "response time" and is calculated using curve-fitting algorithms. A response time is inversely and linearly proportional to the logarithm of the number of template RNA molecules which initiated the reaction. Therefore, this system permits an unknown amount of input RNA probe to be quantified through 11 orders of magnitude when compared to a standard curve. Under the described conditions with MDV RNA, the response time occurs when about 3 x 10(11) RNA molecules are synthesized and occurs within the exponential phase of the reaction, before the number of active enzyme molecules are saturated with RNA templates. This system has been used to determine the replication properties of MDV RNA reporter molecules bearing specific probe sequences and to develop hybridization assays for the clinical diagnostic field.

Amplified detection of viral nucleic acid at subattomole levels using Q beta replicase.

A sensitive, amplified assay for HIV-1 pol region RNA was developed using RNA probes which are replicated by the RNA-dependent RNA polymerase, Q beta replicase. A synthetic target RNA was hybridized in cell lysates prepared with guanidine thiocyanate with an RNA reporter probe and four deoxyoligonucleotide "capture" probes. The RNA reporter probe was a recombinant MDV RNA molecule generated by transcription from a cloned cDNA template. Capture probes are synthetic oligonucleotides that are complementary to the target nucleic acid and that bear 3' poly d(A) tails. The ternary hybrids (of target RNA with capture probe and reporter probe) were captured on oligo d(T)-derivatized paramagnetic particles by hybridization with the d(A) tails of the capture probes. Non-hybridized reporter probes were removed by washing and successively eluting and recapturing the ternary hybrids on fresh particles. After three cycles of elution and capture, the hybrids were eluted in a low ionic strength buffer and the MDV RNA reporter probes were amplified directly by Q beta replicase. Amplified product RNA was detected by fluorescence using propidium iodide. The assay detects one femtogram (600 molecules) of a synthetic target RNA containing the pol region of HIV-1. The complete assay takes about 2.5 hours.

Assembly of a polyadenylation-specific 25S ribonucleoprotein complex in vitro.

Extracts from HeLa cell nuclei assemble RNAs containing the adenovirus type 2 L3 polyadenylation site into a number of rapidly sedimenting heterodisperse complexes. Briefly treating reaction mixtures prior to sedimentation with heparin reveals a core 25S assembly formed with substrate RNA but not an inactive RNA containing a U----C mutation in the AAUAAA hexanucleotide sequence. The requirements for assembly of this heparin-stable core complex parallel those for cleavage and polyadenylation in vitro, including a functional hexanucleotide, ATP, and a uridylate-rich tract downstream of the cleavage site. The AAUAAA and a downstream U-rich element are resistant in the assembly to attack by RNase H. The poly(A) site between the two protected elements is accessible, but is attacked more slowly than in naked RNA, suggesting that a specific factor or secondary structure is located nearby. The presence of a factor bound to the AAUAAA in the complex is independently demonstrated by immunoprecipitation of a specific T1 oligonucleotide containing the element from the 25S fraction. Precipitation of this fragment from reaction mixtures is blocked by the U----C mutation. However, neither ATP nor the downstream sequence element is required for binding of this factor in the nuclear extract, suggesting that recognition of the AAUAAA is an initial event in complex assembly.

Identification of Epstein-Barr nuclear antigen polypeptide in mouse and monkey cells after gene transfer with a cloned 2.9-kilobase-pair subfragment of the genome.

A new antigenic polypeptide was identified in mouse L cells and in monkey COS-1 cells in which Epstein-Barr nuclear antigen (EBNA) was expressed as the result of gene transfer with cloned fragments of Epstein-Barr virus (EBV) DNA. The same-size protein (Mr, approximately equal to 78,000) was seen in stably transformed mouse cells harboring only the BamHI K fragment [approximately equal to 5.2 kilobase pairs (kbp)] or its BamHI/HindIII subfragment, I1f (approximately equal to 2.9 kbp). Thus, the latter DNA fragment is sufficient to code for the protein. In transfected COS cells, a deletion mutant of the I1f fragment (approximately equal to 2.3 kbp) gave rise to a truncated protein (Mr, approximately equal to 52,000), whereas the BamHI K fragment yielded a full-sized Mr 78,000 species. This finding indicates that EBNA is encoded by viral genes. In Burkitt lymphoma lines or in immortalized lymphocytes, variation in the size of the I1f fragment correlated with the apparent molecular weight of the EBNA polypeptide. EBNA is truncated in two Burkitt lymphoma lines, Raji (Mr, 67,000) and P3JHR-1 (Mr, 70,000), which have deletion mutant I1f genes. EBNA in human lymphoid cells bearing a complete I1f fragment as part of the entire EBV genome is the same size (Mr, 78,000) as EBNA found after gene transfer of I1f alone into mouse or monkey cells. Therefore, these expression systems make an authentic EBNA after transfer of the appropriate EBV genes.

Purified lupus antigen La recognizes an oligouridylate stretch common to the 3' termini of RNA polymerase III transcripts.

The small ribonucleoproteins recognized by anti-La sera consist mainly of RNA polymerase III products complexed with an antigenic cellular protein of 50 kd. A biochemical procedure for purifying the La protein from HeLa cells is described. The interaction of the isolated protein with a collection of model tRNA precursors, generated by ligation of specific oligonucleotides to the 3' terminus of yeast tRNAPhe, was studied. The most stable complexes are formed with adducts possessing three or four terminal uridylate residues. Addition of a terminal phosphate, fragmentation of the RNA, or substitution of other nucleotides reduce the affinity for the La protein. The preferred terminal sequence recognized and bound by La protein is homologous to the transcriptional termination signal for RNA polymerase III.

Mutation-induced changes in RNA polymerase-lac ps promoter interactions.

A composite rate assay has been applied to investigate the rate and mechanism of formation of open promoter complexes. Seven DNA templates were studied which were related to the lac ps promoter by single base pair changes in the -10 or -35 region of promoter sequence homology. These small changes induce a nearly 3 order of magnitude variation in the rate of open complex formation. This variation persists over a wide range of concentrations of RNA polymerase. Nevertheless, all promoters direct open complex formation which proceeds through a "closed" or A polymerase:DNA complex which dissociates readily. These data, when taken together with our previous results on the lac "spacer" mutations, demonstrate that mutation of the lac ps promoter leads to changes in the rate of open complex formation predicted by the following rule. Changes which substitute a less conserved element of sequence in the -10 and -35 regions, or of length in the spacer, always decrease the rate in this homologous series of promoters.

Spacer mutations in the lac ps promoter.

Mutations have been constructed that delete either one or two base pairs near position -19 in the lac ps promoter. Deletion of either of two adjacent base pairs increases the rates of open complex formation by nearly on order of magnitude. Two promoters that have different single-base deletions are indistinguishable by either their rates of open complex formation or stability of the open complexes once formed. However, simultaneous deletion of both base pairs produces a promoter that forms complexes at a rate similar to that of the unmodified DNA sequence. The maximal rate of open complex formation is achieved at a spacer length of 17 base pairs, the most frequently occurring spacer length among promoters. These results suggest that the spacing between the two strongly conserved regions of sequence homology is an important determinant of the rate of open complex formation. A model is suggested that proposes that three important promoter elements, the -10 region, the -35 region, and the spacer region, act simultaneously to facilitate open complex formation by RNA polymerase.

Productive and abortive initiation of transcription in vitro at the lac UV5 promoter.

The rates of productive and abortive initiation of transcription in vitro at the lac UV5 promoter have been determined and compared to values determined for phage lambda and T7 promoters. The rate constants for productive initiation of lac transcript are consistently lower over a range of low to moderate concentration of initiating nucleoside triphosphate (ATP). Abortive initiation of lac dinucleoside tetraphosphate is also slower at low to moderate concentrations of ATP. These data demonstrate the existence of significant differences in initiation rate among promoters. We suggest that these differences may be a consequence of the initial mRNA sequences and extents of RNA polymerase cycling during initiation of promoter-specific transcription.

Kinetic investigation of the mechanism of RNA polymerase binding to mutant lac promoters.

The initial rates of functional complex formation between Escherichia coli RNA polymerase and a family of mutant lac promoters have been determined as a function of the independently varied concentration of both components. The initial rates are first order in polymerase and zero order in promoter concentration even under conditions of polymerase excess. These data suggest that the promoter site competes with nonspecific sites for formation of an unstable complex with RNA polymerase. Whether or not a final complex is formed depends on the probability of occurrence of a conformational conversion during the lifetime of this intermediate. An important role of DNA sequence in modulating promoter binding rates is suggested: sequence changes which provide additional contacts to polymerase stabilize the intermediate and thus accelerate the rate of functional complex formation.

Alterations in two conserved regions of promoter sequence lead to altered rates of polymerase binding and levels of gene expression.

Characterization of recombinant lac promoters highlights the importance of two regions of sequence conservation in promoters. The "Pribnow box" sequences are necessary for specific transcription in this system. This specificity is maintained when a mutated upstream sequence is introduced. However, changing the upstream DNA sequences influences both the rate of RNA polymerase binding in vitro and levels of expression in vivo.

Lac UV5 transcription in vitro. Rate limitation subsequent to formation of an RNA polymerase-DNA complex.

The kinetics of transcription of lac UV5 mRNA using purified DNA restriction fragment as template has been studied. This template, which contains only 203 base pairs, directs the formation of a 67-base lac mRNA with high specificity. The half-time for formation of a DNA-RNA polymerase complex is approximately 0.2 min. However, upon addition of 200 micron nucleoside triphosphates to this complex, RNA production proceeds with a half-time of approximately 1 min. Therefore, it is suggested that the rate-limiting step for lac UV5 mRNA production, under typical in vitro conditions, occurs subsequent to the formation of a promoter-specific complex.