Effects of palindrome size and sequence on genetic stability in the bacteriophage phi X174 genome

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.     

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.     

Isolation and characterization of the smallest bacteriophage P4 derivatives packaged into P4-size head in bacteriophage P2-P4 system

Bacteriophage P4, a satellite phage of coliphage P2, is a very useful experimental tool for the study of viral capsid assembly and cos-cleavage. For an in vitro cos-cleavage reaction study of the P2-P4 system, new shortened and selectable markers containing P4 derivative plasmids were designed as a substrate molecules. They were constructed by swapping the non-essential segment of P4 DNA for either the kanamycin resistance (kmr) gene or the ampicillin resistance (apr) gene. The size of the genomes of the resulting markers were 82% (P4 ash8 delRI:: kmr) and 79% (P4 ash8 delRI:: apr) of the wild type P4 genome. To determine the lower limit of genome size that could be packaged into the small P4-size head, these shortened P4 plasmids were converted to phage particles with infection of the helper phage P2. The conversion of plasmid P4 derivatives to bacteriophage particles was verified by the heat stability test and the burst size determination experiment. CsCl buoyant equilibrium density gradient experiments confirmed not only the genome size of the viable phage form of shortened P4 derivatives, but also their packaging into the small P4-size head. P4 ash8 delRI:: apr turned out to be the smallest P4 genome that can be packaged into P4-sized head.     

Two regions of an avian hepadnavirus RNA pregenome are required in cis for encapsidation.

We have constructed a series of deletion mutants spanning the genome of duck hepatitis B virus in order to determine which regions of the viral genome are required in cis for packaging of the pregenome into capsid particles. Deletion of sequences within either of two nonadjacent regions prevented replication of the mutant viral genomes expressed in a permissive avian hepatoma cell line in the presence of functionally active viral core and P proteins. Extraction of RNA from cells transfected with these replication-defective mutants showed that the mutants retained the capacity to be transcribed into a pregenomic-size viral RNA, but that these RNA species were not packaged into viral capsids. The two regions defined by these deletions are located 36 to 126 (region I) and 1046 to 1214 (region II) nucleotides downstream of the 5' end of the pregenome and contain sequences which are required in cis for encapsidation of the duck hepatitis B virus pregenome.     

Restriction analysis of DNA from phage SM of Pseudomonas aeruginosa

The DNA of temperate phage SM P. aeruginosa has one PvuII site, two BamHI sites, three HindIII sites and five EcoRI sites. Using these restrictases the physical map of the phage genome has been constructed. The DNA of phage SM has in their structure cohesive ends similar to cos-sites of phage lambda DNA. EcoRI-fragments with cohesive ends have molecular masses 2.9 and 4.9 MDa.     

A helper-dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging.

Adenoviruses (Ads) are intermediate-sized mammalian DNA viruses with a double-stranded linear genome of 36 kb. The icosohedral virion has been shown to accommodate up to 105% of the wild-type genome length, and genomes larger than this size are either unpackageable or extremely unstable, frequently undergoing DNA rearrangement. Here we show that the Ad virion also has a lower packaging limit of approximately 75% of the wild-type genome length. We have constructed a series of vectors with sizes ranging from 15.1 to 33.6 kb and used these to show that in our Cre/loxP helper-dependent system (R. J. Parks, L. Chen, M. Anton, U. Sankar, M. A. Rudnicki, and F. L. Graham, Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996), vectors with genomes greater than or equal to 27.7 kb are packaged with equal efficiencies, whereas vectors with smaller genomes are inefficiently packaged. A 15.1-kb vector, approximately half the size of the wild-type adenovirus genome, was packaged with an efficiency intermediate between that of the small (21.3- to 25.7-kb) and large (27.7- to 33.5-kb) vectors. Analysis of vector DNA after amplification in helper virus-infected cells showed that vectors below 75% of the Ad genome had undergone DNA rearrangements, whereas larger vectors were unaltered. The 15.1-kb vector was recovered primarily as a mix of head-to-tail and tail-to-tail covalent dimers, with a size of 30 kb. We conclude that the Ad virion can efficiently accommodate viral DNA of greater than 75% of the viral genome but that smaller viral genomes tend to undergo rearrangement, resulting in a final size of greater than approximately 27 kb before they can be efficiently packaged. Knowledge of the lower limit to Ad DNA packaging should allow for the design of better and more stable vectors.     

Physical mapping of the virion and the prophage DNAs of a temperate Lactobacillus phage phi FSW

We analysed the physical structure of the DNA of phi FSW, which is a temperate phage of Lactobacillus casei S-1. A circular restriction map of the virion DNA has been constructed with three restriction endonucleases, BamHI, SalI and XhoI. Other data indicated that the phage genome was circularly permuted. In lysogens, the DNA of the prophage was found to be linearized at a specific site and integrated into a specific locus of the host genome, with the same orientation in each case, as evidenced by Southern filter hybridization. We compared the physical structure of phi FSW with its three virulent mutants. One of them had a restriction map indistinguishable from that of phi FSW and two of them contained host-derived DNA sequence(s) in a specific region of the phi FSW genome (V-region). The prophage integration site was mapped on a different segment of the phage genome to the V-region. Derivation of virulent mutants from phi FSW is discussed in relation to the physical structure of the phage genome.     

Dimeric RNA Recognition Regulates HIV-1 Genome Packaging

How retroviruses regulate the amount of RNA genome packaged into each virion has remained a long-standing question. Our previous study showed that most HIV-1 particles contain two copies of viral RNA, indicating that the number of genomes packaged is tightly regulated. In this report, we examine the mechanism that controls the number of RNA genomes encapsidated into HIV-1 particles. We hypothesize that HIV-1 regulates genome packaging by either the mass or copy number of the viral RNA. These two distinct mechanisms predict different outcomes when the genome size deviates significantly from that of wild type. Regulation by RNA mass would result in multiple copies of a small genome or one copy of a large genome being packaged, whereas regulation by copy number would result in two copies of a genome being packaged independent of size. To distinguish between these two hypotheses, we examined the packaging of viral RNA that was larger (≈17 kb) or smaller (≈3 kb) than that of wild-type HIV-1 (≈9 kb) and found that most particles packaged two copies of the viral genome regardless of whether they were 17 kb or 3 kb. Therefore, HIV-1 regulates RNA genome encapsidation not by the mass of RNA but by packaging two copies of RNA. To further explore the mechanism that governs this regulation, we examined the packaging of viral RNAs containing two packaging signals that can form intermolecular dimers or intramolecular dimers (self-dimers) and found that one self-dimer is packaged. Therefore, HIV-1 recognizes one dimeric RNA instead of two copies of RNA. Our findings reveal that dimeric RNA recognition is the key mechanism that regulates HIV-1 genome encapsidation and provide insights into a critical step in the generation of infectious viruses.     

In Vitro Packaging of UV Radiation-Damaged DNA from Bacteriophage T7

When DNA from bacteriophage T7 is irradiated with UV light, the efficiency with which this DNA can be packaged in vitro to form viable phage particles is reduced. A comparison between irradiated DNA packaged in vitro and irradiated intact phage particles shows almost identical survival as a function of UV dose when Escherichia coli wild type or polA or uvrA mutants are used as the host. Although uvrA mutants perform less host cell reactivation, the polA strains are identical with wild type in their ability to support the growth of irradiated T7 phage or irradiated T7 DNA packaged in vitro into complete phage. An examination of in vitro repair performed by extracts of T7-infected E.coli suggests that T7 DNA polymerase may substitute for E. coli DNA polymerase I in the resynthesis step of excision repair. Also tested was the ability of a similar in vitro repair system that used extracts from uninfected cells to restore biological activity of irradiated DNA. When T7 DNA damaged by UV irradiation was treated with an endonuclease from Micrococcus luteus that is specific for pyrimidine dimers and then was incubated with an extract of uninfected E. coli capable of removing pyrimidine dimers and restoring the DNA of its original (whole genome size) molecular weight, this DNA showed a higher packaging efficiency than untreated DNA, thus demonstrating that the in vitro repair system partially restored the biological activity of UV-damaged DNA.     

Molecular characterization of lactococcal bacteriophage Tuc2009 and identification and analysis of genes encoding lysin, a putative holin, and two structural proteins.

Bacteriophage Tuc2009 is a temperate bacteriophage with a small isometric head and is isolated from Lactococcus lactis subsp. cremoris UC509. The phage genome is packaged by a headful mechanism, giving rise to circularly permuted molecules with terminal redundancy. The unit genome size is approximately 39 kb. A map of the phage genome on which several determinants could be localized was constructed: pac, the site of initiation of DNA packaging; lys (1,287 bp), specifying the phage lysin; S (267 bp), specifying a putative holin; and mp1 (522 bp) and mp2 (498 bp), each specifying one of the phage's structural proteins. lys, S, mp1, and mp2 were further characterized. lys and S are partially overlapping and appear to be part of one operon. The lysin shows homology to the lysins of the Streptococcus pneumoniae phages Cp-9, Cp-1, and Cp-7. The putative holin, which is thought to be involved in the release of lysin from the cytoplasm, contains two strongly hydrophobic presumptive transmembrane domains and a highly charged C-terminal domain.     

Replication and packaging of choleraphage phi 149 DNA.

The intercellular replication of the circularly permuted DNA of choleraphage phi 149 involves a concatemeric DNA structure with a size equivalent to six genome lengths. The synthesis of both monomeric and concatemeric DNAs during replication of phi 149 occurred in the cytoplasm. The concatemers served as the substrate for the synthesis of mature phage DNA, which was eventually packaged by a headful mechanism starting from a unique pac site in the concatemeric DNA. Packaging of DNA into phage heads involved binding of concatemeric DNA to the cell membrane. A scheme involving sequential packaging of five headfuls proceeding in the counterclockwise direction from the pac site is proposed. After infection under high-phosphate conditions, the concatemeric DNA intermediates were not formed, although synthesis of monomeric molecules was unaffected.     

Maize centromeric chromatin scales with changes in genome size

Centromeres are defined by the location of Centromeric Histone H3 (CENP-A/CENH3) which interacts with DNA to define the locations and sizes of functional centromeres. An analysis of 26 maize genomes including 110 fully assembled centromeric regions revealed positive relationships between centromere size and genome size. These effects are independent of variation in the amounts of the major centromeric satellite sequence CentC. We also backcrossed known centromeres into two different lines with larger genomes and observed consistent increases in functional centromere sizes for multiple centromeres. Although changes in centromere size involve changes in bound CENH3, we could not mimic the effect by overexpressing CENH3 by threefold. Literature from other fields demonstrate that changes in genome size affect protein levels, organelle size and cell size. Our data demonstrate that centromere size is among these scalable features, and that multiple limiting factors together contribute to a stable centromere size equilibrium.     

Identification of cryptic lox sites in the yeast genome by selection for Cre-mediated chromosome translocations that confer multiple drug resistance.

The Cre recombinase efficiently causes site-specific DNA recombination at loxP sites placed into the eukaryotic genome. Since the loxP site of phage P1 is 34 base-pairs in size, the natural occurrence of this exact sequence is unlikely in any eukaryotic genome. However, related sequences may exist in eukaryotic genomes that could recombine at low efficiency with an authentic loxP site. This work identifies such cryptic lox sites in the yeast genome using a positive selection procedure that allows the detection of events occurring at a frequency of less than 1 x 10(-7). The selection is based on the disruption/reconstruction of the yeast gene YGL022. Disruption of YGL022 confers multiple drug sensitivity. Recombination events at a loxP site 5' to the structural gene restore expression of YGL022 and result in a multiple drug resistant phenotype. These drug resistant mutants all display chromosomal rearrangements resulting from low-frequency Cre-mediated recombination with an endogenous cryptic lox site. Ten such sites have been found and they have been mapped physically to a number of different yeast chromosomes. Although the efficiency of Cre-mediated recombination between loxP and such endogenous sites is quite low, it may be possible to redesign recombination substrates to improve recombination efficiency. Because of the greater complexity of the human and mouse genomes compared with yeast, an analogous situation is likely to exist in these organisms. The availability of such sites would be quite useful in the development of alternative strategies for gene therapy and in the generation of transgenic animals.     

Nucleotide sequence of the genome of the filamentous bacteriophage I2-2: Module evolution of the filamentous phage genome

Summary The nucleotide sequence of the circular single-stranded genome of the filamentous Escherichia coli phage I2-2 has been determined and compared with those of the filamentous E. coli phages Ff(M13, fl, or fd) and IKe. The I2-2 DNA sequence comprises 6744 nucleotides; 139 nucleotides less than that of the N- and I2-plasmid-specific phage IKe, and 337 (336) nucleotides more than that of the F-plasmid-specific phage Ff. Nucleotide sequence comparisons have indicated that I2-2, IKe, and Ff have a similar genetic organization, and that the genomes of I2-2 and IKe are evolutionarily more closely related than those of I2-2 and Ff. The studies have further demonstrated that the I2-2 genome is a composite replicon, composed of only two-thirds of the ancestral genome of IKe. Only a contiguous I2-2 DNA sequence of 4615 nucleotides encompassing not only the coat protein and phage assembly genes, but also the signal required for efficient phage morphogenesis, was found to be significantly homologous to sequences in the genomes of IKe and Ff. No homology was observed between the consecutive DNA sequence that contains the origins for viral and complementary strand replication and the replication genes. Although other explanations cannot be ruled out, our data strongly suggest that the ancestor filamentous phage genome of phages I2-2 and IKe has exchanged its replication module during evolution with that of another replicon, e.g., a plasmid that also replicates via the so-called rolling circle mechanism. Offprint requests to: R.N.H. Konings     

Physical characterization of the genome of the Myxococcus xanthus bacteriophage MX-8

Summary We have constructed a restriction map for the genome of bacteriophage MX-8 from Myxococcus xanthus using the enzymes PvuII, MboI, and EcoRI. The phage genome size, as determined by restriction analysis, is 51.7±0.6 Kb. Double digestions, redigestions of isolated fragments, and crossed-contact hybridization of partial digestion products show that the restriction map is circular. Restriction analysis and Southern hybridization show that the phage DNA molecules are packaged sequentially from a concatemer starting from a specific site which we have mapped. The DNA molecules have an average terminal redundancy of approximately 8% and are circularly permuted over at least 40% of the genome.     

Rescue of abortive T7 gene 2 mutant phage infection by rifampin.

Infection of Escherichia coli with T7 gene 2 mutant phage was abortive; concatemeric phage DNA was synthesized but was not packaged into the phage head, resulting in an accumulation of DNA species shorter in size than the phage genome, concomitant with an accumulation of phage head-related structures. Appearance of concatemeric T7 DNA in gene 2 mutant phage infection during onset of T7 DNA replication indicates that the product of gene 2 was required for proper processing or packaging of concatemer DNA rather than for the synthesis of T7 progeny DNA or concatemer formation. This abortive infection by gene 2 mutant phage could be rescued by rifampin. If rifampin was added at the onset of T7 DNA replication, concatemeric DNA molecules were properly packaged into phage heads, as evidenced by the production of infectious progeny phage. Since the gene 2 product acts as a specific inhibitor of E. coli RNA polymerase by preventing the enzyme from binding T7 DNA, uninhibited E. coli RNA polymerase in gene 2 mutant phage-infected cells interacts with concatemeric T7 DNA and perturbs proper DNA processing unless another inhibitor of the enzyme (rifampin) was added. Therefore, the involvement of gene 2 protein in T7 DNA processing may be due to its single function as the specific inhibitor of the host E. coli RNA polymerase.     

Effects of DNA Heterologies on Bacteriophage λ Packaging

We have examined the impact of DNA heterologies on the packaging of λ DNA in vitro. Heterology-containing DNA molecules were constructed by denaturing and reannealing a mixture of DNA from cI(+) phage and DNA from phage carrying small insertion or deletion mutations in the cI gene. We found that molecules with heterologies of up to 19 base pairs (bp) can be packaged as viable heterozygous phage with approximately the same efficiency as molecules with a base pair mismatch. In contrast, with a heterology of 26-bp heterozygous plaque formers are rare. In principle, the absence of cI heterozygotes among packaged phage may be due either to a failure to encapsidate the DNA or a failure to inject the packaged DNA on infection. Southern blot analysis of DNA isolated from packaged phage indicates that DNA harboring a 26-bp heterology is almost completely absent in packaged phage. Thus, an upper limit has been established for the size of heterology that can be accommodated by the packaging apparatus. The size of the connector portal could be the basis for this limit.