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.     

Interference with viral infection by RNA replicase deleted at the carboxy-terminal region

Escherichia coli cells harboring an altered Q beta RNA replicase which has amino acid substitutions of the glycine residue at position 357 in the conserved sequence Tyr356-Gly357-Asp358-Asp359 of the beta-subunit protein lost the replicase activity but interfered with proliferation of Q beta phage [Inokuchi and Hirashima (1987) J. Virol. 61, 3946-3949]. To examine the mechanism of the interference, we further analyzed various mutants lacking the carboxy-terminal region of the beta-subunit protein. The cells expressing the beta-subunit gene with up to 17% deletion from the carboxy-terminus of the protein prevented the proliferation of Q beta phage. However, in the case that the deletion extended beyond 25% from the carboxy-terminus, the cells showed no interference. In addition, when the interference took place, the phage coat protein synthesis was inhibited. These results indicate that the region between amino acids 440 and 487 of the beta-subunit protein is involved in the interference and suggest that the defective replicase inhibits the phage coat protein synthesis by competing with the ribosomes at the initiation site of the coat gene.     

Efficient templates for Q beta replicase are formed by recombination from heterologous sequences.

A very efficient replicase template has been isolated from the products of spontaneous RNA synthesis in an in vitro Q beta replicase reaction that was incubated in the absence of added RNA. This template was named RQ135 RNA because it is 135 nucleotides in length. Its sequence consists entirely of segments that are homologous to ribosomal 23 S RNA and the phage lambda origin of replication. The sequence segments are unrelated to the sequence of Q beta bacteriophage genomic RNA. Nonetheless, this natural recombinant is replicated in vitro at a rate equal to the most efficient of the known Q beta RNA variants. Apparently, the structural properties that ensure recognition of an RNA template by Q beta replicase are not confined to viral RNA, but can appear as a result of recombination among other RNAs that usually occur in cells.     

On the nature of spontaneous RNA synthesis by Q beta replicase.

Numerous RNA species of different length and nucleotide sequence grow spontaneously in vitro in Q beta replicase reactions where no RNA templates are added deliberately. Here, we show that this spontaneous RNA synthesis by Q beta replicase is template directed. The immediate source of template RNA can be the laboratory air, but there are ways to eliminate, or at least substantially reduce, the harmful effects of spontaneous synthesis. Solitary RNA molecules were detected in a thin layer of agarose gel containing Q beta replicase, where they grew to form colonies that became visible upon staining with ethidium bromide. This result provides a powerful tool for RNA cloning and selection in vitro. We also show that replicating RNAs similar to those growing spontaneously are incorporated into Q beta phage particles and can propagate in vivo for a number of phage generations. These RNAs are the smallest known molecular parasites, and in many aspects they resemble both the defective interfering genomes of animal and plant viruses and plant virus satellite RNAs.     

The bacteriophage T4 insertion/substitution vector system. A method for introducing site-specific mutations into the virus chromosome

A bacteriophage T4 insertion/substitution vector system has been developed as a means of introducing in vitro generated mutations into the T4 chromosome. The insertion/substitution vector is a 2638-base pair plasmid containing the pBR322 origin of replication and ampicillin resistance determinant, a T4 gene 23 promoter/synthetic supF tRNA gene fusion, and a polylinker with eight unique restriction enzyme recognition sites. A T4 chromosomal "target" DNA sequence is cloned into this vector and mutated by standard recombinant DNA techniques. Escherichia coli cells containing this plasmid are then infected with T4 bacteriophage that carry amber mutations in two essential genes. The plasmid integrates into the T4 chromosome by recombination between the plasmid-borne T4 target sequence and its homologous chromosomal counterpart. The resulting phage, termed "integrants," are selectable by the supF-mediated suppression of their two amber mutations. Thus, although the integrants comprise 1-3% or less of the total phage progeny, growth on a nonsuppressing host permits their direct selection. The pure integrant phage can be either analyzed directly for a possible mutant phenotype or transferred to nonselective growth conditions. In the latter case, plasmid-free phage segregants rapidly accumulate due to homologous recombination between the duplicated target sequences surrounding the supF sequence in each integrant chromosome. A major fraction of these segregants will retain the in vitro generated mutation within their otherwise unchanged chromosomes and are isolated as stable mutant bacteriophage. The insertion/substitution vector system thereby allows any in vitro mutated gene to be readily substituted for its wild-type counterpart in the bacteriophage T4 genome.     

Autocatalytic replication of a recombinant RNA

We demonstrate that a heterologous RNA sequence can be copied in vitro by Q beta replicase when it is inserted into a naturally occurring Q beta replicase template. A recombinant RNA was constructed by inserting decaadenylic acid between nucleotides 63 and 64 of MDV-1 (+) RNA, using phage T4 RNA ligase. The insert was located away from regions of the template known to be required for the binding of the replicase and for the initiation of product strand synthesis. To minimize the disruption of template structure, we inserted the heterologous sequence into a hairpin loop on the exterior of the molecule. Q beta replicase copied this recombinant RNA in vitro, and the complementary product strands served as templates for the synthesis of additional copies of the original recombinant RNA. The reaction was therefore autocatalytic and the amount of recombinant RNA increased exponentially. A 300-fold amplification of the recombinant RNA occurred within nine minutes. Insertion of biologically significant RNAs into the MDV-1 RNA sequence should allow them to be replicated autocatalytically.     

Structural consensus of RNA templates replicated by Q beta replicase.

Q beta replicase replicates a variety of enzyme-specific small RNAs in addition to the phage genomic RNA. The sequence analysis has revealed that all these RNAs are potentially capable of forming a consensus secondary structure element. It represents a stalk which is formed by the 5'-GGG ... and ... CCCA-3' complementary stretches at the termini of the replicating RNA molecules and adjacent 5'- and 3'-hairpins, which may form a stacking with the stalk. The structure found is rather similar to the analogous structure in the tRNA molecule. The genomic RNA of the Q beta phage and other related phages can also form a similar structural element.     

Interference with viral infection by defective RNA replicase.

RNA-dependent RNA and DNA polymerases have a conserved segment, Tyr-X-Asp-Asp (G. Karmer and P. Argos, Nucleic Acids Res. 12:7269-7282, 1984). To investigate the function of this segment, we changed the Gly residue at position 357 in the conserved sequence Tyr-356-Gly-357-Asp-358-Asp-359 of the replicase of RNA coliphage Q beta to Ala, Ser, Pro, Met, or Val and examined the replicase activity in vivo. Cells carrying the variant plasmids lost the replicase activity and severely inhibited the proliferation of phage Q beta (group III) and related phage SP (group IV) by suppressing phage RNA synthesis. In contrast, substitution of the Gly residue at 390 showed only a slight inhibitory effect, although replicase activity was also lost. These results suggest that the cells harboring an altered replicase at the conserved segment can interfere specifically with the wild-type phage and different but related phage infections.     

Structural Constraints and Mutational Bias in the Evolutionary Restoration of a Severe Deletion in RNA Phage MS2

A 4-nucleotide (nt) deletion was made in the 36-nt-long intercistronic region separating the coat and replicase genes of the single-stranded RNA phage MS2. This region is the focus of several RNA structures conferring high fitness. One such element is the operator hairpin, which, in the course of infection, will bind a coat-protein dimer, thereby precluding further replicase synthesis and initiating encapsidation. Another structure is a long-distance base pairing (MJ) controlling replicase expression. The 4-nt deletion does not directly affect the operator hairpin but it disrupts the MJ pairing. Its main effect, however, is a frame shift in the overlapping lysis gene. This gene starts in the upstream coat gene, runs through the 36-nt-long intercistronic region, and ends in the downstream replicase cistron. Here we report and interpret the spectrum of solutions that emerges when the crippled phage is evolved. Four different solutions were obtained by sequencing 40 plaques. Three had cured the frame shift in the lysis gene by inserting one nt in the loop of the operator hairpin causing its inactivation. Yet these low-fitness revertants could further improve themselves when evolved. The inactivated operator was replaced by a substitute and thereafter these revertants found several ways to restore control over the replicase gene. To allow for the evolutionary enrichment of low-probability but high-fitness revertants, we passaged lysate samples before plating. Revertants obtained in this way also restored the frame shift, but not at the expense of the operator. By taking larger and larger lysates samples for such bulk evolution, ever higher-fitness and lower-frequency revertants surfaced. Only one made it back to wild type. As a rule, however, revertants moved further and further away from the wild-type sequence because restorative mutations are, in the majority of cases, selected for their capacity to improve the phenotype by optimizing one of several potential alternative RNA foldings that emerge as a result of the initial deletion. This illustrates the role of structural constraints which limit the path of subsequent restorative mutations. [Reviewing Editor: Dr. John Hulsenbeck]     

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.     

Genetic control of the formation of plasmid F'. I. Effect of recA- and seg-2 mutations in an Hfr donor strain on the character of plasmid F' formation]

Assuming the similarity of the processes of illegitimate recombination, such as deletion formation, with the process of F' plasmid formation, we have undertaken the study of the influence of recA- and seg- alleles of Hfr donor on the F' plasmid formation. The data obtained demonstrate the strong influence of donor genotype on the frequency of F' plasmid formation and on the nature of F' plasmids formed, thus demonstrating that the most of F' plasmids have been formed via recombination in Hfr donor cells. The recA- mutation decreased the total yield of F' plasmids selected using both proximal and distal Hfr markers and affected drastically the distribution of the F' plasmids inheriting different proximal unselected markers. The existence of recA-dependent and recA-independent modes of F' plasmid formation was demonstrated. The Escherichia coli chromosome contains regions which involve preferentially in recA-dependent (between proA and gal, and clockwise from gal) or recA-independent (between leu and proA, and the region counterclockwise from argE) recombination. The seg-2 mutation causes only partial block of both recA-dependent and recA-independent recombination pathways, however it causes dramatic decrease of genetic exchanges leading to the formation of the type II F' plasmids. Both seg- and recA- mutations decrease the frequency of the formation of Tra+ F' transconjugants. The percent of Tra- transconjugants, which remain sensitive to MS2 and Q beta donor specific phages, also drops significantly under the influence of the recA- and seg- alleles. Thus, the recombination involving the F structure in wild type strains and seg- mutants occures preferentially in the points of F outside the regions essential for transfer and sensitivity to male specific phages, while in recA- and recA-ges- strains the points inside these regions (tra operon) frequently involved in F' plasmid looping out. There exist more strict correlation between the fertility and sensitivity to phage Q beta than to phage MS2.     

Differential stimulation by polyamines of phage RNA-directed synthesis of proteins

The effect of polyamines on Q beta and MS2 phage RNA-directed synthesis of three kinds of protein in an Escherichia coli cell-free system has been studied. With both phage RNAs, the degree of stimulation of protein synthesis by spermidine was in the order RNA replicase greater than A protein, while the synthesis of coat protein was not stimulated significantly by spermidine. The synthesis of RNA replicase was stimulated by 1 mM spermidine approx. 8-fold. From the results of Q beta RNA direct alanyl-tRNA and seryl-tRNA binding to ribosomes and initiation dipeptide synthesis, it is suggested that the preferential stimulation of the synthesis of RNA replicase by spermidine is due at least partially to the stimulation of the initiation of RNA replicase synthesis.     

Base-change analysis of revertants of the hisD3052 allele in Salmonella typhimurium

This report is an investigation of the specific sequence changes in the DNA of Salmonella hisD3052 revertants induced by a set of specific frameshift mutagens found in our diet. They include B[a]P, aflatoxin B1, and the cooked-food mutagens, IQ, MeIQ, and PhIP. The Salmonella DNA was cleaved with restriction enzymes Sau3A, EcoR1, and Alu1 to give a 620-bp fragment containing the hisD3052 site. The size-fractionated fragments were ligated to the bacteriophage vector M13mp8. After transformation into E. coli, the recombinants were screened with a nick-translated hisD+ gene probe, and the isolated single-stranded DNA was sequenced. All IQ (13), MeIQ (3), PhIP (5), and aflatoxin B1 (3) induced revertants isolated had a 2-base (-CG- dinucleotide) deletion situated 10 bases upstream from the original hisD3052 -C- deletion. In contrast, 9 of 24 revertants induced by B[a]P had extensive deletions varying from 8 to 26 nucleotides in length and located at various sites along a 45-base-pair sequence beginning at nucleotide 2085 of the his operon. The other 15 B[a]P-induced revertants had a -CG- deletion at the same location as the revertants induced by the other food mutagens. 7 spontaneous revertants were also analyzed; they showed 3 -CG- deletions, 1 insertion and 3 distinct deletions (varying from 2 to 11 bases in size). In total, 13 distinct base changes are described which lead to reversion of the hisD3052 mutation.     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.