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D R Mills

Publications and source records attributed to D R Mills.

14 recordsLinked to original sources
PubMed1991

Coliphage Q beta RNA replication: RNA catalytic for single-strand release.

We have generated 14 recombinant RNA templates for Q beta replicase, each having either an exogenous inverted repeat sequence or a sequence with no repeat. These templates were used to initiate in vitro replication by Q beta replicase in amounts that saturated the enzyme. We observed that replication rates for RNAs that putatively contained secondary structures in the recombinant sequences ranged from 33 to 69% that of a wild-type MDV-1 RNA control, regardless of the size of the inserted hairpin. Moreover, most of the newly synthesized RNA was present as single strands. Alternatively, RNAs that contained exogenous sequences not expected to form secondary structures exhibited replication rates less than 25% that of MDV-1. In each case, the reaction rate was correlated with the length of the insertion, and the majority of product RNA consisted of duplexed molecules (complementary plus and minus strands hybridized together). When these same recombinant RNAs were used in reactions in which the molar amount of RNA template was 10(6)-10(7) times lower than that of the replicase, only those that putatively contained secondary structures survived in the replication reaction. Our results are consistent with the theory that hairpin structure formation during RNA synthesis by Q beta replicase directly influences the regeneration of single-stranded RNA products.

Base Sequence↗
PubMed1990

Q beta RNA bacteriophage: mapping cis-acting elements within an RNA genome.

We have identified, for the first time, regions of cis-acting RNA elements within the bacteriophage Q beta replicase cistron by analyzing the infectivities of 76 replicase gene mutant phages in the presence of a helper replicase. Two separate classes of mutant Q beta phage genomes (35 different insertion mutants, each containing an insertion of 3 to 15 nucleotides within the replicase gene, and 41 deletion genomes, each having from 15 to 935 nucleotides deleted from different regions of the gene) were constructed, and their corresponding RNAs were tested for the ability to direct the formation of progeny virus particles. Each mutant phage was tested for plaque formation in an Escherichia coli (F+) host strain that supplied helper Q beta replicase in trans from a plasmid DNA. Of the 76 mutant genomes, 34% were able to direct virus production at or close to wild-type levels (with plaque yield ratios of greater than 0.5), another 36% also produced virus particles, but at much lower levels than those of wild-type virus (with plaque yield ratios of less than 0.05), and the remaining 30% produced no virus at all. From these data, we have been able to define regions within the Q beta replicase gene that contain functional cis-acting RNA elements and further correlate them with regions of RNA that are solely required to code for functional RNA polymerase.

Base Sequence↗
PubMed1989

Q beta replicase: mapping the functional domains of an RNA-dependent RNA polymerase.

We have localized a functional region of the RNA bacteriophage Q beta replicase following an extensive mutational analysis. Using the method of oligonucleotide linker-insertion mutagenesis, we specifically introduced mutations into a cloned DNA copy of the Q beta replicase gene so that the resulting replicase products would putatively contain small amino acid insertions. In a selective phenotypic assay, we screened mutant replicases for RNA-directed replication activity in vivo. Analysis of 37 different mutant clones indicated that Q beta replicase can accept amino acid substitutions and insertions at several sites at the amino and carboxy termini without abolishing functional activity in vivo or in vitro. However, disruption within the internal amino acid sequence resulted almost exclusively in nonfunctional enzyme. The results suggest that the central region of the replicase protein contains a rigid amino acid composition that is required for replicase function, whereas the amino and carboxy termini are much more receptive to small amino acid insertions and substitutions. These experiments should further enable us to analyze the coding function of the Q beta replicase gene independently of other phage RNA functions contained within this nucleotide region.

Amino Acids↗
PubMed1988

Engineered recombinant messenger RNA can be replicated and expressed inside bacterial cells by an RNA bacteriophage replicase.

In this paper we describe how an RNA replicase can be "tricked" into recognizing, binding to, and replicating host-cell message RNAs. In our system, the gene encoding Q beta replicase is constitutively expressed from a plasmid vector present in an Escherichia coli host, while a second plasmid directs the transcription of a replication-competent, phage-like template RNA. This 680 nucleotide transcript (N- RNA) contains specific sequences required for Q beta replicase function. Included within this template RNA is a 360-nucleotide sequence that is complementary to the messenger RNA for DNA bacteriophage lambda N protein. The active messenger RNA for lambda N protein is expressed only upon replication of N- RNA by Q beta replicase. By employing an E. coli host strain that requires lambda N protein for growth under specific selective conditions, we were able to select only those cells in which RNA-directed RNA replication occurred. This system provides a potential for in vivo amplification of other heterologous messenger RNAs and their protein products via RNA replication and will enable one to study the evolution of messenger RNA in the live cell under a large variety of physiological pressures.

Base Sequence↗
PubMed1987

Mutations in the adenovirus major late promoter: effects on viability and transcription during infection.

We developed an experimental system to examine the effects of mutations in the adenovirus major late promoter in its correct genomic location during a productive infection. A virus was constructed whose genome could be digested to give a rightward terminal DNA fragment extending from the XhoI site at 22.9 map units, which can be ligated or recombined with plasmid DNA containing adenovirus sequences extending from 0 to 22.9 or 26.5 map units, respectively. Mutations were made by bisulfite mutagenesis in the region between base pairs -52 and -12 with respect to the cap site at +1 and transferred to the appropriate plasmids for viral reconstruction. Of 19 mutant plasmid sequences containing single or multiple G-to-A transitions, 14 could be placed in the viral genome with no apparent change in phenotype. These mutant sequences included those which contained four transitions in the string of G residues immediately downstream of the TATA box. There were no alterations in rates of transcription from the major late promoter, sites of transcription initiation, or steady-state levels of late mRNAs. All of the five mutant sequences which could not be placed in virus contained multiple transitions both up- and downstream of the TATA box. Two of these apparently lethal mutant sequences were used in promoter fusion experiments to test their ability to promote transcription of rabbit beta-globin sequences placed in the dispensable E1 region of the virus. Both sequences showed diminished ability compared with wild-type sequences to promote transcription in this context. Comparisons between these two sequences and the viable mutant sequences suggest a role for the string of G residues located between -38 and -33 in promoting transcription from the major late promoter. The data as a whole also demonstrate that the specific nucleotide sequence of this region of the major late promoter, which overlaps transcription elements of the divergent IVa2 transcription unit and coding sequences of the adenovirus DNA polymerase, is not rigidly constrained but can mutate extensively without loss of these several functions.

Adenoviridae↗
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↗
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↗
PubMed1979

Initiation of translation with Pseudomonas aeruginosa phage PP7 RNA: nucleotide sequence of the coat cistron ribosome binding site.

Initiation complex formation between PP7 RNA and ribosomes of Pseudomonas aeruginosa and Escherichia coli has been investigated. The PP7 RNA fragments protected by both species of ribosome have been isolated, and their sequences have been determined. Only one binding sites is available on the intact PP7 RNA strand, and this site is recognized by ribosomes of both species. The PP7 RNA binding site is approximately 38 nucleotides long. It contains two AUG sequences and a purine-rich segment near the 5'-end that is complementary to segments near the 3'-ends of the 16S ribosomal RNA's of both P. aeruginosa and E. coli. In order to establish which of the AUG codons acts as the initiator, the H2N-terminal amino acid sequence of PP7 coat protein was determined. This sequence is compatible with the codon sequence following the second AUG codon. The extent of the reaction of PP7 RNA with E. coli ribosomes is greater than with P. aeruginosa ribosomes, but our results do not indicate a qualitative difference in the initial interaction between intact PP7 RNA and the ribosomes of either species.

Amino Acid 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↗