High-resolution molecular discrimination by RNA.

Species of RNA that bind with high affinity and specificity to the bronchodilator theophylline were identified by selection from an oligonucleotide library. One RNA molecule binds to theophylline with a dissociation constant Kd of 0.1 microM. This binding affinity is 10,000-fold greater than the RNA molecule's affinity for caffeine, which differs from theophylline only by a methyl group at nitrogen atom N-7. Analysis by nuclear magnetic resonance indicates that this RNA molecule undergoes a significant change in its conformation or dynamics upon theophylline binding. Binding studies of compounds chemically related to theophylline have revealed structural features required for the observed binding specificity. These results demonstrate the ability of RNA molecules to exhibit an extremely high degree of ligand recognition and discrimination.

Multilevel regulation of surface antigen gene expression in Paramecium tetraurelia.

A family of genes is responsible for production of surface antigenic components of Paramecium tetraurelia. These surface proteins are expressed in a mutually exclusive manner. Individuals rarely display more than one type. However, changes in environmental conditions can cause different surface proteins which replace preexisting types to be expressed. We investigated the nature of regulation of the genes for the A, C, and H surface antigens of P. tetraurelia. A system for in vitro run-on transcription was developed from crude Paramecium extracts and used in this analysis. The genes for surface antigens A and H were controlled at the level of transcription. However, the gene for surface antigen C demonstrated both transcriptional and posttranscriptional control, depending on the serotype being expressed. When animals expressed serotype A, the gene for surface antigen C was not transcribed. However, when animals expressed serotype H, the gene for surface antigen C was actively transcribed and stable surface antigen C mRNA was present in the cells, although surface antigen C was not detectable by serotype testing or by a salt-alcohol extraction method. The kinetics of transformation from serotype H to serotype C were determined by using the in vitro transcription system and monitoring steady-state RNA levels. During the transition, serotype A transcription was detected in run-on transcription experiments, although this RNA did not accumulate. The results indicate that serotype expression is controlled at several levels and that not all serotype genes are controlled in the same manner.

Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei.

Paramecium tetraurelia can be transformed by microinjection of cloned serotype A gene sequences into the macronucleus. Transformants are detected by their ability to express serotype A surface antigen from the injected templates. After injection, the DNA is converted from a supercoiled form to a linear form by cleavage at nonrandom sites. The linear form appears to replicate autonomously as a unit-length molecule and is present in transformants at high copy number. The injected DNA is further processed by the addition of paramecium-type telomeric sequences to the termini of the linear DNA. To examine the fate of injected linear DNA molecules, plasmid pSA14SB DNA containing the A gene was cleaved into two linear pieces, a 14-kilobase (kb) piece containing the A gene and flanking sequences and a 2.2-kb piece consisting of the procaryotic vector. In transformants expressing the A gene, we observed that two linear DNA species were present which correspond to the two species injected. Both species had Paramecium telomerelike sequences added to their termini. For the 2.2-kb DNA, we show that the site of addition of the telomerelike sequences is directly at one terminus and within one nucleotide of the other terminus. These results indicate that injected procaryotic DNA is capable of autonomous replication in Paramecium macronuclei and that telomeric addition in the macronucleus does not require specific recognition sequences.

Interference-based detection of nucleic acid targets on optically coated silicon.

Sequence-specific detection of polynucleotides typically requires modified reporter probes that are labeled with radioactive, fluorescent, or luminescent moieties. Although these detection methods are capable of high sensitivity, they require instrumentation for signal detection. In certain settings, such as clinical point of care, instrumentation might be impractical or unavailable. Here we describe a detection approach in which formation of a nucleic acid hybrid is enzymatically transduced into a molecular thin film that can be visually detected in white light. The system exploits a flat, optically coated silicon-based surface to which capture oligonucleotides are covalently attached. The optimized system is capable of detection of nucleic acid targets present at sub-attomole levels. To supplement visual detection, signals can be quantitated by a charge-coupled device. The design and composition of the optical surface, optimization of immobilization chemistry for attachment of capture probes, and characterization of the efficiency of the hybridization process are presented. We describe the application of this system to detection of a clinically relevant target, the mecA gene present in methicillin-resistant Staphylococcus aureus.

Assessment of quantitative models for plasmid ColE1 copy number control.

Two quantitative models of plasmid ColE1 copy number control are compared with respect to mathematical logic of derivation and application to experimental observations. Explanatory background material and clarifications are supplied for selected aspects of each model. Contrasting features are emphasized and experiments are suggested to distinguish between predictions of the models.

Analysis of establishment phase replication of the plasmid ColE1.

The replication regulatory mechanisms by which the small, multicopy plasmid ColE1 maintains a constant steady-state copy number have been extensively characterized by a combination of in vivo genetics and in vitro biochemistry. We have extended the analysis of replication control into the "establishment" phase of replication, when ColE1-directed replicons replicate more than once per cell generation and the intracellular concentrations of plasmid-encoded replication regulatory elements are changing. To study establishment phase replication, in which plasmid-directed replicons amplify from an initially low concentration to the characteristic, steady-state concentration, bacteriophage-plasmid hybrids, termed phasmids, were constructed. Phasmids were shown to exhibit stability, segregation, and incompatibility properties similar to those of the parent plasmid. Establishment phase replication was analyzed by measuring the number of phasmids per cell as a function of time after infection. We observed a linear increase in phasmid concentration until the steady-state concentration characteristic of the ColE1 plasmid component of the hybrid was reached. The number of cell doublings required for the phasmid concentration to reach steady-state was inversely related to cell growth rate. The observed amplification kinetics imply that the frequency of replication initiation per phasmid continually decreases until steady-state is reached. Kinetics of establishment phase amplification were sensitive to rate of expression of RNA II. A phasmid containing an up mutation in the RNA II promoter amplified at a 15-fold faster rate than the wild-type phasmid. Concentration of the ColE1 replication negative regulator (RNA I) was proportional to phasmid concentration throughout the amplification phase. These results suggest that the same elements that regulate steady-state replication also control establishment phase replication.

RNA1 is sufficient to mediate plasmid ColE1 incompatibility in vivo.

The multicopy plasmid ColE1 specifies a small RNA designated RNA1 that has been implicated in copy number control and incompatibility. We have inserted a 148 base-pair ColE1 DNA fragment containing a promoter-less RNA1 gene into a plasmid vector downstream from the tryptophan promoter of Serratia marcesens . The ColE1 RNA1 produced by this plasmid is not functional in vivo due to the presence of 49 nucleotides appended to the 5'-terminus of the wild-type RNA1 sequence. Deletions of these sequences by Bal3l nuclease in vitro and genetic selection for ColE1 incompatibility function in vivo permitted isolation of a plasmid expressing wild-type ColE1 RNA1 initiated properly from the S. marcesens trp promoter. These experiments demonstrate that RNA1 is sufficient to mediate ColE1 incompatibility in vivo. In addition, several plasmids were isolated that contain altered RNA1 genes. These alterations consist of additions or deletions of sequences at the 5'-terminus of RNA1. Analysis of the ability of these altered RNA1 molecules to express incompatibility in vivo suggests that the 5'-terminal region of RNA1 is crucial for its function.

Isolation and characterization of mutants affecting functional domains of ColE1 RNAI.

The control of DNA replication initiation in the plasmid ColE1 is mediated by RNAI, a 108 nucleotide plasmid-encoded RNA that is entirely complementary to the 5'-terminal region of the replication primer RNA. RNAI acts in trans to inhibit primer maturation. Previously, we constructed a plasmid in which the ColE1 RNAI was separated from the primer and placed under transcriptional control of the Serratia marcesens tryptophan promoter. This plasmid provides RNAI in trans in vivo and mediates ColE1-type incompatibility. To determine the critical structural and functional domains of RNAI, we have undertaken a mutational analysis of the RNAI gene carried by this plasmid. We have selected mutants that no longer mediate ColE1-type incompatibility in trans. From the DNA sequences of 18 mutants we have identified mutations at nine new sites in RNAI. In addition, we have determined the secondary structural features of several mutant RNAI species and compared them to wild-type RNAI. Analysis of these mutations has revealed several key features of RNAI secondary structure and function. The domains of RNAI identified in this work which are essential for its function are: the single-stranded loop regions; the integrity of the double-stranded stems; and the single-stranded 5' terminus.

Mutations affecting RNA-DNA hybrid formation of the ColE1 replication primer RNA. Restoration of RNA I sensitivity to a copy-number mutant by second-site mutations.

Certain high copy-number mutants of the ColE1 plasmid produce a primer RNA that, unlike the wild-type, is resistant to inhibition by the plasmid-encoded replication inhibitor RNA I. We show that this resistance is associated with the ability of mutant primer RNA to hybridize to the DNA template strand more efficiently than does the wild-type transcript in vitro. We have isolated two second-site intramolecular suppressor mutations that partially restore wild-type copy number behavior to the high copy-number mutant in vivo. Each of these mutations alters a second base in primer RNA near the original mutation. We show that the primer RNA made by the pseudo-revertants regained wild-type-like sensitivity to RNA I in vitro. Also, the efficiency of RNA-DNA hybrid formation by the pseudo-revertant primer RNAs is restored to a level similar to that of wild-type primer. Using non-denaturing gel electrophoresis as an indication of RNA conformation, we identified two primer RNA conformers, each of 550 nucleotides, whose equilibrium distribution differs between wild-type and the mutant plasmid. The pseudo-revertant plasmids have a conformer distribution similar to that of wild-type, indicating that these primer sequence changes have long-range effects on primer conformation. An oligonucleotide complementary to the primer domain containing the mutation reduced hybrid formation when present during primer elongation. These results indicate that the copy-number behavior of these plasmids is a consequence of conformational alterations in primer RNA that alter its hybridization efficiency with the DNA template strand and its sensitivity to inhibition by RNA I.

Identification of DNA segments capable of rescuing a non-mendelian mutant in paramecium.

The non-Mendelian mutant d48 of Paramecium tetraurelia contains micronuclear wild type A genes, but at autogamy and conjugation proper processing fails and new macronuclei lack A genes. When cloned A genes are injected into the macronucleus of d48, proper processing is restored at the next autogamy; d48 is rescued, becoming permanently wild type. In the present study we have injected portions of the A gene into d48. We find that the ability to rescue extends over a large portion of the gene, with highest activity near a series of 221-bp repeat units in the middle of the gene. Regions outside the A gene are inactive.

Mutants affecting processing of DNA in macronuclear development in paramecium.

In Paramecium tetraurelia, stock 51, the A surface protein is coded by the wild type A51 gene, present in micronuclei in two copies and in macronuclei in about 1500 copies. DNA processing, comprised of DNA cleavage, copy number amplification and telomere addition occurs at autogamy and conjugation when old macronuclei degrade and new macronuclei are formed from micronuclei. In this paper we characterize mutants with macronuclear A gene deletions. These mutants are notable in three respects. First, the mutants do not appear to be simple micronuclear deletions. Although genetic analysis shows that the d12 mutant d12(-1300) is homozygous for the allele A-1300 and the mutant d12(+1) for A+1, analysis by the polymerase chain reaction indicates that the micronuclei in these two mutants contain intact, but presumably altered, micronuclear A genes. They undergo deletion during DNA processing when new macronuclei are formed. Second, the position of the deletions in these alleles has been shown to change. The deficiency present in the d12 allele A-1300 was originally determined to extend from position -1300 (relative to the start of translation of the A gene) to the end of the chromosome. Later, a derivative of this strain, homozygous for the d12 allele A+1 was isolated in which the start site of the deletion was found to have moved from -1300 to +1. Third, a surprising interaction occurs in crosses between a line homozygous for the d12 allele and one homozygous for the wild-type A51 allele. Previous work on the non-Mendelian d48 mutant (which has intact A51 genes in its micronucleus, but has truncated A51 genes in its macronucleus) has shown that intact A51 alleles must be present in the old macronucleus in order for A51 alleles to undergo proper processing. We find that d12 alleles act on A51 alleles in heterozygotes such that intact macronuclear A genes are no longer required for proper processing of A51. Thus, in crosses of 51 x d12 (either +1 or -1300) d12 exconjugants, as well as 51 exconjugants, give rise to clones carrying both intact A51 and truncated d12 alleles. Remarkably the d12 alleles, which are themselves deleted during processing, are capable in the heterozygote of fostering normal processing of the A51 allele.

Permanent rescue of a non-Mendelian mutation of Paramecium by microinjection of specific DNA sequences.

The mutant Paramecium tetraurelia cell line d48 is unable to express the serotype A protein on its surface. Although the A gene is intact in the micronuclei of d48, the A gene copies in the macronucleus contain a large deletion eliminating virtually the entire coding sequence. Previous studies showed that microinjection of a plasmid containing the entire A gene into the macronucleus of d48 permanently restored A expression after autogamy. Together with other data, this result suggests that in wild type cells the A gene in the old macronucleus ensures the presence of a cytoplasmic factor that prevents A gene deletions at autogamy. In d48, where there are few, if any copies of the intact A gene in the old macronucleus, deletions occur during macronuclear formation. To elucidate the specific molecular mechanisms involved in this unusual phenomenon, we attempted to define the region(s) of the A gene necessary for rescuing d48. We show that microinjection of a 4.5-kb internal A gene fragment is sufficient for proper processing at autogamy and leads to permanent rescue of d48; i.e., the rescued strain is indistinguishable from wild type. Thus, rescue of d48 does not require upstream transcriptional control sequences, intact A mRNA or A serotype protein. We also show that various fragments of the A gene have the ability to rescue d48 to different extents, some being more efficient than others. We find no evidence to suggest that the A gene gives rise to a small stable RNA that might act as or encode a cytoplasmic factor. Molecular mechanisms that may be involved in the rescue of d48 are discussed.

High-level expression in Escherichia coli of calcium-binding domains of an embryonic sea urchin protein.

A plasmid expression vector is described having features that facilitate high-level expression of eukaryotic DNA in Escherichia coli. The vector, designated pMAM17, carries the ColE1 rop gene under the control of the thermally inducible lambda PL promoter. The rop gene product is a negative regulator of ColE1 DNA replication, and its high-level expression is lethal to cells. However, cells harboring a plasmid with an insert in the rop gene grow normally under these conditions. pMAM17 has been used to investigate the properties of a family of proteins expressed in the dorsal ectoderm of sea urchin embryos. The coding sequences of these proteins (termed Spec proteins) have homology to the troponin C superfamily. Large amounts of the Rop-Spec fusion protein were produced at 42 degrees C in E. coli. Unfractionated E. coli extracts containing the fusion protein could be used to produce antibodies that were highly specific for Spec proteins present in crude extracts of sea urchin embryos. Analysis of the Rop-Spec fusion protein on SDS-polyacrylamide gels in the presence and absence of EGTA indicated that the fusion protein bound calcium ions in a manner characteristic of proteins of the troponin C superfamily. This behavior provides biochemical evidence that the Spec proteins are functionally homologous to other members of this superfamily.

Rapid multiserotype detection of human rhinoviruses on optically coated silicon surfaces.

BACKGROUND: More than 100 immunologically distinct serotypes of human rhinoviruses (HRV) have been discovered, making detection of surface exposed capsid antigens impractical. However, the non-structural protein 3C protease (3Cpro) is essential for viral replication and is relatively highly conserved among serotypes, making it a potential target for diagnostic testing. The thin film biosensor is an assay platform that can be formatted into a sensitive immunoassay for viral proteins in clinical specimens. The technology utilizes an optically coated silicon surface to convert specific molecular binding events into visual color changes by altering the reflective properties of light through molecular thin films. OBJECTIVE: To develop a rapid test for detection of HRV by developing broadly serotype reactive antibodies to 3Cpro and utilizing them in the thin film biosensor format. STUDY DESIGN: Polyclonal antibodies to 3Cpro were purified and incorporated into the thin film assay. The in vitro sensitivity, specificity and multiserotype cross-reactivity of the 3Cpro assay were tested. Nasal washes from naturally infected individuals were also tested to verify that 3Cpro was detectable in clinical specimens. RESULTS: The 3Cpro assay is a 28-min, non-instrumented room temperature test with a visual limit of detection of 12 pM (picomolar) 3Cpro. In terms of viral titer, as few as 1000 TCID(50) equivalents of HRV2 were detectable. The assay detected 45/52 (87%) of the HRV serotypes tested but showed no cross-reactivity to common respiratory viruses or bacteria. The thin film assay detected 3Cpro in HRV-infected cell culture supernatants coincident with first appearance of cytopathic effect. Data are also presented demonstrating 3Cpro detection from clinical samples collected from HRV-infected individuals. The assay detected 3Cpro in expelled nasal secretions from a symptomatic individual on the first day of illness. In addition, 9/11 (82%) concentrated nasal wash specimens from HRV infected children were positive in the 3Cpro test. CONCLUSION: We have described a novel, sensitive thin film biosensor for rapid detection of HRV 3Cpro. This test may be suitable for the point of care setting, where rapid HRV diagnostic test results could contribute to clinical decisions regarding appropriate antibiotic or antiviral therapy.

Use of a thin film biosensor for rapid visual detection of PCR products in a multiplex format.

Rapid, sensitive assays for nucleic acid amplification products have utility for the identification of bacterial or viral infections. We have developed a nucleic acid hybridization assay utilizing thin film technology that permits visual detection of hybrids. The silicon-based biosensor detects the presence of target sequences by enzymatically transducing the formation of nucleic acid hybrids into molecular thin films. These films alter the interference pattern of light on the biosensor surface, producing a perceived color change. We have applied this technology to the development of a chip containing capture probes specific for human respiratory virus sequences including respiratory syncytial virus, influenza virus A and B, parainfluenza virus types 1 and 3, and rhinovirus. In a ten-minute assay, the biosensor permits unambiguous identification of viral-specific RT/PCR products from infected cell lysates.

Structural analysis of RNA molecules involved in plasmid copy number control.

DNA replication of the plasmid ColE1 and its relatives is controlled mainly by a small plasmid-encoded transcript known as RNA11. We have studied the conformation of RNA1 molecules from two related but compatible plasmids, ColE1 and RSF1030, using T1 ribonuclease, S1 nuclease, cobra venom nuclease, and diethyl pyrocarbonate modification as probes for secondary structure. Both RNA1 molecules contain three double-stranded stems, three single-stranded loops, and an exposed 5' tail. All loops in both molecules show similar sensitivities to the probes used, as do stems 1 and 3. The region comprising stem 2 of each RNA1 molecule contains the most sequence differences as well as the most structural changes of any region in the two molecules. The structure of the RNA1 molecule encoded by a recessive high copy number mutant of ColE1 was also investigated. Structural alterations involving the first stem and loop of the molecule are proposed to be responsible for the inability of the mutant RNA1 to function in vivo.

Location of histones on simian virus 40 DNA.

The physical location of histone molecules in a simian virus 40 DNA-histone complex isolated from purified virions was examined using site-specific restriction endonucleases. The complex contains four host histone species but lacks histone F1. Histones prevent complete cleavage of SV40 DNA by two restriction enzymes, HindIII and EcoRI. From the pattern of DNA fragments resulting from cleavage of the histone-DNA complex by the HindIII endonuclease, which makes six breaks on purified SV 40 DNA, we have concluded that histones are randomly arranged on SV40 DNA relative to restriction enzyme cleavage sites. The EcoRI endonuclease, which makes one break in SV40 NDA, was used to determine the degree of physical coverage of the SV 40 DNA molecule by histones. We observed that 80% of the EcoRI sites in the complex are accessible to the enzyme while 20% are "closed." This degree of coverage is consistent with the mass ratio of DNA:histone in the complex as revealed by the buoyant density of the formaldehyde-fixed complex. We conclude that the histones in the complex are located randomly on the SV 40 genome and cover approximatley 20% of the DNA. These results suggest that the histone species F2b, F2al, F2a2, and F3 are bound without regard to nucleotide sequence of SV 40 DNA.