Insertions of palindromic DNA sequences into the J-F intercistronic region of bacteriophage phi X174 interfere with normal phage growth

The effect of hairpin (cruciform) size on the regulation of gene expression was investigated by cloning a series of palindromic sequences into the non-essential J-F intercistronic region of the bacteriophage phi X174 ins6 genome. Genetic stability of the insert sequence and its effect on the growth efficiency of the phage was used as an initial measure of the biological consequence of hairpin insertions. Multimers of increasing size of the BamHI linker sequence C-C-G-G-A-T-C-C-G-G were inserted into the PvuII site of the parental strain ins6. The largest hairpin that could be constructed and maintained in the phi X174 genome had a stem length of 22 base-pairs and a loop size of four nucleotides (linker tetramer). However, this structure proved to be disadvantageous to the phage and was rapidly deleted from its genome. Trimer inserts were more stable, but were eventually deleted also. Monomer and dimer inserts, though genetically stable, decreased the growth efficiency of the phage as judged by competitive growth experiments and measurements of burst size. The physical formation of these hairpins was shown by restriction digests of single-stranded DNA with BamHI and HpaII. We argue that these secondary structures form in vivo, at least in the single-stranded genome and the polycistronic mRNAs, and were responsible for the observed growth defects.     

Construction of bacteriophage luminal diameterX174 mutants with maximum genome sizes.

The bacteriophage phi X174 strain ins6 constructed previously was used to investigate the maximum genome size that could be packaged into the icosahedral phage without concomitant loss of phage viability. The J-F intercistronic region of ins6, which already contains an insert of 117 base pairs with a unique PvuII site, was enlarged further by insertion of HaeIII restriction fragments of the plasmid pBR322 into that PvuII site. By using a biochemical approach for the site-specific mutagenesis as well as selection of mutant genomes, a series of mutants was isolated with genomes of up to 5,730 nucleotides, 6.4% larger than that of the wild-type DNA. Phages with genomes larger than 5,550 nucleotides were highly unstable and were rapidly outgrown by spontaneously occurring deletion mutants. The data predict that genomes of at least 6,090 nucleotides could be constructed and, most likely, packaged, but the resulting phages would not grow well. We speculate that the volume of the phage capsid is not the limiting factor of genome size or is not the only limiting factor.     

Isolation and characterization of phi X174 mutants carrying lethal missense mutations in gene G.

A previously constructed Escherichia coli transformant carrying a functional copy of bacteriophage phi X174 gene G on a plasmid, p phi XG, was used to isolate gene G mutants carrying temperature sensitive and lethal missense mutations. Two of the mutations have been characterized by sequencing: one carries a G --> A transition at residue 2821 producing a Gly --> Ser change in codon 143 of the G spike protein; the other carries an A --> G transition at residue 2678 producing Glu --> Gly change in codon 95. Sequencing DNA from 2 other mutants carrying lethal mutations that are rescued with p phi XG did not reveal any changes in the coding sequence. The lesion is believed to be in the intercistronic region between genes F and G. The adsorption kinetics for these mutants appear to be normal. Their burst size is about 25% that of wild type phi X174 on the host carrying p phi XG. These results along with previous results from the senior author's laboratory demonstrate that p phi XG can be used to rescue any gene G mutant of phi X174 regardless of the nature of the mutation involved.     

Eclipse kinetics as a probe of quaternary structure in bacteriophage phi X174

The extracellular form of bacteriophage phi X174 consists of single-stranded DNA within an icosahedral capsid, which has short spikes at each of its vertices. Each spike is composed of gene G and H proteins, while the capsid itself consists of gene F protein. Since several molecules of gene H protein are injected into the cell along with the DNA, specific protein--protein and DNA--protein interactions must be broken when the genome exits and leaves an intact capsid structure at the receptor site. To demonstrate this we examined the eclipse (DNA ejection) reaction with two types of phi X174 mutants. The first contains missense mutations in a capsid or spike protein gene, and the second involves insertions or deletions in non-coding regions of the DNA. Using an improved procedure, the eclipse rate in vivo of the eclipse mutants Fcs70 has been redetermined over a larger temperature range than in previous studies. The three- to fivefold decrease in rate between 37 degrees C and 25 degrees C is due to an increase in both the enthalpy and entropy of activation when compared to the wild-type values of these kinetic parameters. This missence mutation also confers an increase in virus stability in 2 to 3 M-urea. In contrast to this, inserting 163 bases into the length of DNA packaged within the phi X174 capsid does not lead to a detectable change in eclipse rate over the same temperature range. yet this insertion into the J--F intercistronic region imparts a significant decrease in virus stability in urea. These results suggest that a specific set of non-covalent interactions is involved in phi X174 DNA ejection. This is supported by the small (50%), but significant, increase in eclipse rate that occurs when 27 bases are deleted from the J--F intercistronic region. The latter effect must be base-sequence-specific since no change in rate is observed when only seven of the 27 bases are deleted. Thus, the kinetics of the phi X174 eclipse reaction can be used as a sensitive probe of quaternary structure by correlating the change in reaction rate with alterations in amino acid and base sequences in the structural components of the virus.     

Role of the intercistronic region in post-transcriptional control of gene expression in the histidine transport operon of Salmonella typhimurium: involvement of REP sequences

The high-affinity histidine permease of Salmonella typhimurium is encoded by a four-gene operon containing a large intercistronic region located between the first gene (hisJ) and the three distal genes (hisQ, hisM, hisP). The level of expression of hisJ is 30-fold greater than that of hisP. In order to investigate the role of the intercistronic region in intra-operonic control of gene expression, we have isolated MudII-mediated lacZ gene fusions to hisQ, hisM and hisP. We have used these fusions to isolate and analyse mutants that have altered levels of expression of the hisQ gene, the first gene downstream from the intercistronic region. The results indicate that intra-operonic regulation is due to a combination of factors including efficiency of translational initiation, mRNA degradation, and retroregulation of hisJ expression. They also suggest that the REP (Repetitive Extragenic Palindromic) sequences, which are located in the hisJ-hisQ intercistronic region, may interfere with translation of the hisQ gene and affect upstream messenger RNA stability by protecting it from 3' to 5' nuclease degradation (in agreement with data presented by Newbury et al., 1987).     

The complete nucleotide sequence of the group II RNA coliphage GA

The complete nucleotide sequence of the RNA coliphage GA, a group II phage, is presented. The entire genome comprises 3466 bases. Three large open reading frames were identified, which correspond to the maturation protein gene (390 amino acids), the coat protein gene (129 amino acids) and the replicase beta-subunit protein gene (531 amino acids). In addition, untranslated regions occur at the 5' (135 bases) and 3' (122 bases) ends of the molecule. Two intercistronic untranslated regions occur between the cistrons for the maturation and coat proteins, and between the coat and beta-subunit proteins. We have compared the nucleotide sequence of GA RNA with the published sequence of MS2 RNA, and show that they are related. The comparative structures of two important regulatory regions are presented; the coat protein binding site which is involved in translational repression of the replicase beta-subunit protein gene, and a hairpin in a region proximal to the lysis protein gene.     

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]     

Conditional RAG-1 mutants block the hairpin formation step of V(D)J recombination

Hairpin formation serves an important regulatory role in V(D)J recombination because it requires synapsis of an appropriate pair of recombination sites. How hairpin formation is regulated and which regions of the RAG proteins perform this step remain unknown. We analyzed two conditional RAG-1 mutants that affect residues quite close in the primary sequence to an active site amino acid (D600), and we found that they exhibit severely impaired recombination in the presence of certain cleavage site sequences. These mutants are specifically defective for the formation of hairpins, providing the first identification of a region of the V(D)J recombinase necessary for this reaction. Substrates containing mismatched bases at the cleavage site rescued hairpin formation by both mutants, which suggests that the mutations affect the generation of a distorted or unwound DNA intermediate that has been implicated in hairpin formation. Our results also indicate that this region of RAG-1 may be important for coupling hairpin formation to synapsis.     

Nucleotide sequence of the J gene and surrounding untranslated regions of phage G4 DNA: Comparison with phage φX174

A 290 nucleotide long region of the bacteriophage G4 genome including the end of the overlapping genes D and E, the entire gene J and the untranslated region between genes J and F has been sequenced and compared with the same region in bacteriophage φX174. Deletions, insertions, duplications and single base changes in G4 relative to φX174 have resulted in the following changes: the loss of the φX174 overlapping gene D termination and gene J initiation codons, resulting in their separation by 32 untranslated nucleotides; the deletion of one third of the gene J coding region, so that the G4 J protein is only 24 amino acids long compared with 37 amino acids in φX174; and the establishment of a longer untranslated region between G4 genes J and F, which despite many nucleotide changes retains the ability to form a stable hairpin loop in the same place and with the same geometry as in φX174. The G4 overlapping gene E is longer than in φX174 and extends beyond gene D. Sixteen nucleotides at the end of genes D and E in φX174 are duplicated in G4 before gene J.     

A study of the thermophilic ribosomal protein S7 binding to the truncated S12-S7 intercistronic region provides more insight into the mechanism of regulation of the str operon of E. coli(1).

A study of the ability of His6-tagged ribosomal protein S7 of Thermus thermophilus to interact with the truncated S12-S7 intercistronic region of str mRNA of Escherichia coli has been described. A minimal S7 binding mRNA fragment is a part of the composite hairpin, with the termination codon of the S12 cistron on one side and the initiation codon of the next S7 cistron on the other. It has a length in the range of 63-103 nucleotides. The 63 nucleotide mRNA fragment, which corresponds to a putative S7 binding site, binds very poorly with S7. Tight RNA structure models, which behave as integral systems and link the S7 binding site with the translational regulation region of the hairpin, are suggested. This observation provides more insight into the mechanism of S7-directed autogenous control of translational coupling of str mRNA.     

Novel point mutations in mitochondrial 16S rRNA gene of Chinese hamster cells

To know the nature and mechanisms of spontaneous mutations in mitochondrial DNA (mtDNA), we determined, by direct cycle sequencing, the nucleotide sequence of the 3' terminal region of the mitochondrial 16S rRNA gene from chloramphenicol-resistant (CAP-R) mutants isolated in Chinese hamster V79 cells. Four different base substitutions were identified in common for the six CAP-R mutants. All mutations were heteroplasmic. One A to G transition was mapped at a site within the putative peptidyl transferase domain, the target region for chloramphenicol, and one G to A transition and two T to G transversions were located within the two different segments which form the stems of the hairpin loop structures attached to this key domain in the predicted secondary structure of 16S rRNA. The mutations detected in this study do not map to the same sites where CAP-R mutations were found previously in mammalian cells. Allele specific-PCR analyses revealed that all four mutations occurred on a single mutant-DNA molecule, but not on several ones independently. Together with the other previous reports, our data suggest that spontaneous mtDNA mutations may not be caused exclusively by oxidative DNA damage at least in 16S rRNA gene.