Bacteriophage phi X174 mutants with insertions of palindromic DNA sequences are rapidly outgrown by competing wild-type phage (Müller & Turnage, J. Mol. Biol. 189: 285). The basis for this defect was investigated and found to be due to an exclusion event early in the infectious cycle, in which phage genomes with palindromic inserts were preferentially excluded by wt phage. In addition, we have obtained further evidence for a palindrome induced genetic instability. Both defects are dependent on palindrome size and sequence, consistent with a model which involves formation of cruciforms, or cruciform-like structures. We propose that formation of unusual DNA secondary structures reduces the effectiveness of replicative form (RF) DNA to interact with limiting replication factors or membrane binding sites, possibly because of interaction with the host recombination system.

By inserting palindromes of varying length and sequence into a non-essential region of the bacteriophage phi X174 genome we have investigated the effect of palindrome size and sequence on their genetic stability. Multimers of increasing size of the EcoRI linker CCGAATTCGG (E), the BamHI linker CCGGATCCGG (B) or mixtures of both (E, B) were inserted into the PvuII site of a previously constructed bacteriophage strain phi X174 J-F ins6. The largest inserts that could be maintained in the genome without significant loss of genetic stability were 2B, 4E, and 4(E, B), respectively. Polymers exceeding this size could be inserted but resulted in rapid and precise deletion from the phage genome, whereby nB was more unstable than nE, and nE was more unstable than n(E, B). Analysis of the resulting deletion mutants provided evidence for two different types of deletions. The more frequent deletion arose from either type palindrome and removed nucleotides in blocks of ten base-pairs (one linker unit), but only from the palindromic sequence, and always left at least an 18 base-pair long palindrome (one linker plus 8 neighboring base-pairs) behind. The less frequently occurring deletions arose only from nB type palindromes, removing the complete palindromic sequence plus adjacent nucleotides. At least the first type of deletion occurred in the absence of recA activity. Our results show a correlation between the sequence, as well as size, and the genetic stability of palindromes, i.e. sequences that could decrease the stability of a cruciform increased their genetic stability. This supports the theory that palindrome deletion occurs via extrusion of the palindrome into a cruciform or cruciform-like structure.

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 φX174 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.

We have computed the expected distribution of the potential for hairpin-like secondary structures with small loops (3-20 bases) and uninterrupted stems and compared that to the distribution observed in the complete genomes of seven DNA viruses from animals, plants and bacteria, as well as a bacterial plasmid. The formation of G-T mismatches in the stems of these structures was allowed. Furthermore we have analyzed the distribution of the potential for such structures along the genetic maps of these genomes, specifically around the start sites of known genes. Our data reveal that the potential for mismatch containing structures with stem length exceeding eight base pairs is over-represented and non-randomly distributed, but to a much lesser degree than that for perfect structures of equal size. Moreover, the potential for both types of structures is preferentially located near functional start codons. From this we deduce that in general G-T/G-U containing nucleic acid secondary structures are biologically relevant, though possibly less significant than perfect ones.

The extracellular form of bacteriophage φX174 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 φX174 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 °C and 25 °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 φX174 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 φX174 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 φX174 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.

The J-F intercistronic region of the genomes of bacteriophages phi X174 and G4 is transcribed but not translated. It contains the potential for a perfectly base-paired hairpin structure, which has been proposed to act as a terminator of transcription or as a regulatory region for mRNA turnover (or both). We measured phage-specific protein synthesis in mutants with modifications of the hairpin sequence and found a relative decrease in the expression of the upstream gene D as compared to the downstream genes F, G, and H in all mutants. The mutations also appeared to affect the efficiency of the gene F ribosome-binding site. These data strongly support the regulatory significance of the J-F intercistronic region and the putative hairpin structure therein.

A hairpin-like secondary structure in the intercistronic region between genes J and F of bacteriophages, phi X174 and G4 has been postulated to act as a transcription termination signal. We analyzed the in vivo transcripts of both phages and mutants derived from them with modifications of this hairpin structure. The phi X174 mutants appeared to fall into two groups with respect to the stability of two mRNA species. Class 1 mutants showed an mRNA profile very similar to the parental strain, whereas class 2 mutants lacked two major mRNA species normally terminated near the J-F region. The G4 mutants behaved like class 2 mutants of phi X174. Analysis of the stability of phi X174 mRNA revealed that messages specific for the genes upstream of the hairpin turn over more rapidly in class 2 mutants than in class 1 mutants. In class 1 mutants, the mRNA decay rates are similar but not identical to those of the wild-type strain. These data suggest a role for the nucleotide sequence within the J-F intercistronic region in mRNA degradation. They further imply that transcription termination occurs downstream from this site.

The J-F intercistronic region of bacteriophage G4 has the potential to form a perfectly base-paired hairpin structure, thought to act as a terminator of transcription. To investigate this proposed structure-function relationship, viable mutants were constructed by site-specific mutagenesis with small deletions of 2 to 4 base pairs in the center of the corresponding palindromic sequence. These sequence modifications had a small positive effect on the growth efficiency of the phage. The approach of biochemical rather than biological selection of these mutant phages is generally applicable to the construction of virus and plasmid vectors.

The chapter discusses the role of deoxyribonucleic acid (DNA) structure in gene regulation. It also considers the properties and conformations of various DNAs and review static structure as well as dynamic transitions. Some of the goals of current research in this area are the following: (1) determination of the properties of regions of DNA along the high molecular-weight chromosomes; (2) identification of the interactions between the neighboring regions of DNA; (3) determination of how the properties of DNA influence the specificity or affinity of regulatory proteins that interact with specific regions of DNA; (4) identification of how the interaction of regulatory proteins, with DNA, modifies the properties of the DNA target site; (5) investigation of the presumed correlation between the physical properties of a region of DNA and its genetic function. A complete knowledge of the kinetic and equilibrium properties of the interaction of specific DNA target sites, with important regulatory proteins, is fundamental for the eventual comprehension of gene regulation. Cellular differentiation is the orderly and programmed expression of a family of genes. Alternatively, the malignant transformation of a cell will eventually be recognized as a faulty interaction between one or more key proteins and their DNA receptor sites.

øX174 mutants with altered J-F intercistronic regions, which were constructed as described in the preceding paper (Müller & Wells, 1980), were characterized biochemically and biologically. Inserts of AluI fragments of pBR322 into this region were identified by restriction mapping; deletions of parts of the J-F intercistronic region were characterized by DNA sequencing. The biological consequences of deleting 27 base-pairs from or inserting 163 base-pairs into the J-F intercistronic region were determined by one-step growth curves and by competitive growth experiments of phages in mixed infections. A small reduction was observed in growth efficiency for the insertion mutant whereas no growth deficiencies were found for the mutant, which had a deletion of 75% of the J-F intercistronic region.