Search PubMed⌕ Search

Biomedical subjects

F R Kramer

Publications and source records attributed to F R Kramer.

17 recordsLinked to original sources
PubMed1992

Amplifiable messenger RNA.

RNA molecules were prepared that consisted of an mRNA encoding chloramphenicol acetyltransferase embedded within the sequence of midivariant RNA, which is a template for the RNA-directed RNA polymerase Q beta replicase. These recombinant RNAs were shown to be bifunctional: they are amplified exponentially by incubation with Q beta replicase, and the replicated RNA serves as template for the cell-free synthesis of enzymatically active chloramphenicol acetyltransferase. The availability of amplifiable mRNAs will enable relatively large amounts of protein to be synthesized in vitro.

Base Sequence↗
PubMed1990

Amplifiable hybridization probes.

Amplifiable hybridization probes enable the development of extremely sensitive clinical assays. These novel molecules consist of a probe sequence embedded within the sequences of a replicatable RNA. The molecules are first hybridized to target sequences in a conventional manner. The probe-target complexes are then isolated and the probes are released from their targets. The released probes are then amplified by incubation with the RNA-directed RNA polymerase, Q-beta replicase. The replicase copies the probes in a geometrically increasing manner: after each round of copying, the number of RNA molecules is twice the previous number. The doubling process is very rapid, resulting in as many as one billion copies of each molecule in 30 minutes. The amount of RNA that is made is large enough to be measured without using radioisotopes. Theoretically, these assays should be extraordinarily sensitive, since only one probe molecule is required to start the amplification process. In practice the sensitivity of the assays is limited by the presence of non hybridized probes that persist, despite extensive washing of the probe-target hybrids. Currently, the limit of detection is about 10,000 molecules of target. However, replicatable probes are now being prepared that include a "molecular switch", which is a region of the RNA that undergoes a conformational change when the probe sequence hybridizes to its target. Protocols are being developed that link signal generation to the state of this switch. The simplicity and speed of the enzymatic steps that are required facilitate automation of the assays.

Molecular Probe Techniques↗
PubMed1989

Quantitative assays based on the use of replicatable hybridization probes.

Amplifiable hybridization probes--molecules with a probe sequence embedded within the sequence of a replicatable RNA--will promote the development of sensitive clinical assays. To demonstrate their utility, we prepared a recombinant RNA that contained a 30-nucleotide-long probe complementary to a conserved region of the pol gene in human immunodeficiency virus type 1 (HIV-1) mRNA. Test samples were prepared, each containing a different number of HIV-1 transcripts that served as simulated HIV-1 mRNA targets. Hybridizations were carried out in a solution containing the chaotropic salt, guanidine thiocyanate. Probe-target hybrids were isolated by reversible target capture on paramagnetic particles. The probes were then released from their targets and amplified by incubation with the RNA-directed RNA polymerase, Q beta replicase (EC 2.7.7.48). The replicase copied the probes in an exponential manner: after each round of copying, the number of RNA molecules doubled. The amount of RNA synthesized in each reaction (approximately 50 ng) was sufficient to measure without using radioisotopes. Kinetic analysis of the reactions demonstrated that the number of HIV-1 targets originally present in each sample could be determined by measuring the time it took to synthesize a particular amount of RNA (the longer the synthesis took, the fewer the number of targets originally present). The results suggest that clinical assays involving replicatable hybridization probes will be simple, accurate, sensitive, and automatable.

Autoanalysis↗
PubMed1986

Synthesis of an amplifiable reporter RNA for bioassays.

The replacement of reporter groups, such as fluorescent molecules or enzymes, by an amplifiable reporter should lead to bioassays of greatly increased sensitivity, since a very large number of copies of the reporter can be accumulated in a short time. Midivariant RNA is an appropriate reporter, since it is autocatalytically replicated by Q beta RNA polymerase in vitro. This RNA can be amplified exponentially, with a population doubling time of 36 seconds, resulting in the synthesis of 10(6) copies of each molecule in 12 minutes. We have used chemical methods to attach biotin to the 5' terminus of midivariant RNA via a disulfide linker. This biotinylated RNA combines with avidin to give a product that is readily purified by gel electrophoresis. The RNA-biotin-avidin adduct, and the RNA released from it by reductive cleavage of the linker arm, replicate normally. The RNA-biotin-avidin adduct should be a suitable reporter for a variety of replication-assisted bioassays involving biotinylated antibodies or biotinylated nucleic acid probes.

Avidin↗
PubMed1985

Comparison of pausing during transcription and replication.

Pausing during the transcription of MDV-1 cDNA by Escherichia coli RNA polymerase was compared with pausing during the replication of MDV-1 RNA by Q beta replicase. MDV-1 RNA is able to form many strong hairpin structures, and Q beta replicase pauses after the synthesis of each [Mills et al. (1978) Cell 15, 541-550]. Although the transcripts were virtually identical to MDV-1 RNA, the locations at which RNA polymerase paused were different and apparently were not related to sequences that can form hairpins. These results indicate that hairpin stability, per se, cannot be used to predict the occurrence of pausing during transcription. Four pauses that occur within a 5-nucleotide region were studied in detail. Insertions and deletions were made in the template DNA to determine the contribution made by the surrounding sequences to these pauses. The results indicate that some of the pauses require the presence of particular upstream sequences, while others are unaffected by the template modifications. Thus, there are at least two different transcriptional pausing mechanisms: one depends on the nature of upstream sequences, while the other is independent of upstream sequences.

Base Sequence↗
PubMed1985

Transcription from bacteriophage T7 and SP6 RNA polymerase promoters in the presence of 3'-deoxyribonucleoside 5'-triphosphate chain terminators.

RNA synthesis by T7 RNA polymerase or SP6 RNA polymerase is 100-1000 times more sensitive to the presence of the 3'-deoxyribonucleoside 5'-triphosphate chain terminators than is RNA synthesis by Escherichia coli RNA polymerase or Q beta replicase. These ribonucleotide analogues do not alter the specificity of each polymerase for its own promoters nor do they alter the site at which synthesis is initiated. Transcription by T7 RNA polymerase or SP6 RNA polymerase in the presence of relatively low concentrations of these chain terminators offers a useful route for determining the nucleotide sequence of any DNA segment that is inserted immediately downstream from a homologous bacteriophage promoter. This sequencing procedure was used to explore the effects that different dinucleotides have on the specificity of initiation at two different T7 RNA polymerase promoters.

Base Sequence↗
PubMed1979

RNA replication: required intermediates and the dissociation of template, product, and Q beta replicase.

Replication complexes containing only one molecule of Q beta replicase and one strand of midivariant RNA (MDV-1 RNA) template were prepared by incubating the replicase with an excess of MDV-1 (-) RNA. In the presence of excess minus strands, these monoenzyme replication complexes were shown to synthesize essentially pure MDV-1 (+) RNA in both the first and second cycles of replication. When an equivalent concentration of mutant MDV-1 (-) RNA was added to this reaction before completion of the first cycle of replication, only wild-type MDV-1 (+) RNA was produced in the first cycle, but both mutant and wild-type MDV-1 (+) RNA were produced in the second cycle of replication. These results demonstrate that a monoenzyme complex is competent to synthesize RNA and, therefore, that a multienzyme replication complex is not a necessary intermediate of replication. The data also imply that after the completion of chain elongation, the product strand is released from the replication complex and that the template and the replicase then dissociate.

Coliphages↗
PubMed1979

Structure-independent nucleotide sequence analysis.

Substitution of inosine for granosine in the nucleic acid fragments synthesized for the sequencing of RNA effectively prevents the formation of secondary structures during electrophoretic analysis. Consequently, the mobility of each fragment in the sequencing gel is a strict function of its molecular weight. Inosine substitution should markedly improve the resolution that can be obtained in the sequencing of DNA as well as RNA.

Base Sequence↗
PubMed1978

Template-determined, variable rate of RNA chain elongation.

Q beta replicase polymerizes MDV-1 RNA at a markedly variable rate. Electrophoretic analyses of partially synthesized strands showed that a few of the elongation intermediates are much more abundant than others, reflecting a variable rate of chain elongation. Our data suggest that at a relatively small number of specific sites in the sequence of this RNA, the progress of the replicase is temporarily interrupted, and then resumes spontaneously, with a finite probability. Since the time spent between these pause sites is negligible compared with the time spent at pause sites, the mean time of chain elongation is well approximated by the sum of the mean times spent at each pause site. Nucleotide sequence analysis of the most prominent elongation intermediates indicated that they all have the potential to form a 3' terminal hairpin structure. This suggests that the marked variability in the rate of chain elongation is due to the formation of terminal hairpins in the product strand, or the reformation of hairpins in the template strand. A survey of the literature shows that this phenomenon occurs with most, if not all, nucleic acid polymerases. Structure-induced pauses may play a role in the regulation of nucleic acid synthesis.

Base Sequence↗
PubMed1978

RNA sequencing with radioactive chain-terminating ribonucleotides.

A rapid method for determining nucleotide sequences in RNA is described. It employs the 3'-deoxy analogues of the ribonucleoside triphosphates as specific chain terminators during RNA synthesis. For example, the inclusion of 3'-deoxyuridine 5'-triphosphate in an RNA synthesis reaction in addition to the four usual ribonucleoside triphosphate precursors results in the synthesis of a set of different-length product strands that terminate in a 3'-deoxyuridine that has been incorporated in place of uridine. To sequence an RNA, four separate reactions are run, each employing a different 3'-deoxy terminator. Parallel electrophoretic analysis of the resulting four sets of specifically terminated product chains leads to a direct reading of the nucleotide sequence. We tested this method by sequencing MDV-1 (-) RNA, a molecule that is synthesized in vitro by phage Qbeta replicase. The sequence read from the resulting gels agreed completely with the known sequence of MDV-1 (-) RNA. The bands in some regions of the sequencing gels were unusually close to one another, as has also been observed in other rapid sequencing procedures, making order assignment in these regions very difficult. Because the secondary structure of MDV-1 (-) RNA was known, it was shown that the compression of the bands is due to the persistence of secondary structures during electrophoresis. Thus, structured regions of nucleic acids may introduce difficulties for sequencing techniques that employ the currently available methods of gel electrophoresis.

Base Sequence↗
PubMed1975

Nucleotide sequence of microvariant RNA: another small replicating molecule.

Microvariant RNA, a small self-replicating molecule (114 nucleotides long), has been isolated from Qbeta replicase reactions incubated in the absence of exogenous template. Its complete nucleotide sequence has been determined. Comparison with MDV-1 RNA, a somewhat larger endogenous Qbeta replicase product (220 nucleotides long) that had previously been characterized, revealed no significant sequence similarity. Since Qbeta replicase can mediate the synthesis of both of these disparate RNA molecules, primary sequence cannot be the sole determining factor in the processes of enzyme recognition and replication. This implies that the key is to be found in the secondary or tertiary structures. The availability of two different replicating molecules of defined sequence should aid in identifying these critical structural features.

Base Sequence↗
PubMed1972

A replicating RNA molecule suitable for a detailed analysis of extracellular evolution and replication.

The aim of the present study is to make available a replicating molecule of known sequence. Accordingly, we sought a molecule that has the following properties: (a) replicates in vitro in a manner similar to phage Qbeta RNA; (b) produces antiparallel complementary strands that can be separated from one another; and (c) is small enough to yield its sequence with reasonable effort. We report here the isolation of a replicating RNA molecule that contains 218 nucleotides and possesses the other features desired for a definitive analysis of the replicating mechanism. Despite its small size, this molecule can mutate to previously determined phenotypes. It will, therefore, permit the precise identification of the base changes required to mutate from one phenotype to another in the course of extracellular Darwinian selection experiments.

Base Sequence↗