Gerald F. Vovis

Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori

Helicobacter pylori, one of the most common bacterial pathogens of humans, colonizes the gastric mucosa, where it appears to persist throughout the hosts life unless the patient is treated. Colonization induces chronic gastric inflammation which can progress to a variety of diseases, ranging in severity from superficial gastritis and peptic ulcer to gastric cancer and mucosal-associated lymphoma. Strain-specific genetic diversity has been proposed to be involved in the organisms ability to cause different diseases or even be beneficial to the infected host, and to participate in the lifelong chronicity of infection. Here we compare the complete genomic sequences of two unrelated H. pylori isolates. This is, to our knowledge, the first such genomic comparison. H. pylori was believed to exhibit a large degree of genomic and allelic diversity, but we find that the overall genomic organization, gene order and predicted proteomes (sets of proteins encoded by the genomes) of the two strains are quite similar. Between 6 to 7% of the genes are specific to each strain, with almost half of these genes being clustered in a single hypervariable region.

Complementary action of restriction enzymes endo R · DpnI and endo R · DpnII on bacteriophage f1 DNA

Abstract Bacteriophage f1 duplex DNA was isolated from Escherichia coli strains containing different DNA methylases and assayed for its sensitivity to endonucleolytic cleavage by the enzymes endo R · DpnI and endo R · DpnII. The former enzyme is specific for methylated DNA, the latter for unmethylated DNA (Lacks & Greenberg, 1975). The E. coli dam methylase was found to be responsible for making f1 resistant to endo R · DpnII and sensitive to endo R · DpnI. Endo R · DpnI cleaved f 1 DNA from dam+ cells at four sites. Additional methylation by enzymes other than the dam methylase gave no further cleavage. Endo R · DpnII cleaved f1 DNA from dam− cells also at four sites to give restriction fragments identical to those obtained with endo R · DpnI cleavage. Thus, the two enzymes are complementary in that they recognize and cleave within the same DNA sequence, one if the DNA is methylated, the other if it is unmethylated. DNA duplexes containing one methylated strand (dam +) and one unmethylated strand (dam−) were prepared in vitro. These methylated hybrids were refractory to endonucleolytic cleavage by both endo R · DpnI and endo R · DpnII. Neither enzyme, therefore, appears to make even a single strand break at a methylated/unmethylated hybrid site.

Cleavage map of bacteriophage f1: Location of the Escherichia coli B-specific modification sites☆

Abstract Replicative form DNA of bacteriophage f1 was cleaved into specific fragments by two endonucleases isolated from Hemophilus aegyptius and an endonuclease isolated from H. influenzae . The fragments were ordered so as to construct a circular map of the phage f1 genome by: (1) digesting the isolated restriction fragments with a second restriction enzyme; and (2) testing in a transfection system for the ability of the fragments to rescue amber mutations contained on single-stranded viral DNA that was hybridized to a particular fragment. The genome of bacteriophage f1 contains two SB sites, genetic sites which confer upon a DNA molecule susceptibility to the restriction-modification system of Escherichia coli B. Each of these sites was located on a specific restriction fragment by transfection experiments. In vitro modification of replicative form DNA of f1 and its SB mutants by endonuclease R · Eco B and the subsequent cleavage of the DNA by the Hemophilus endonucleases showed that the B-specific methylation occurs within at least 200 nucleotide pairs of the SB site.

The Filamentous Phage Genome: Genes, Physical Structure, and Protein Products

In this article we review the organization of the filamentous phage genome and the phage-specific protein products. A great deal of work has been done on the filamentous phages specific to strains of Escherichia coli carrying an F factor. Included in this category are f1 (Loeb 1960; Zinder et al. 1963), fd (Marvin and Hoffmann-Berling 1963), M13 (Hofschneider 1963), and ZJ/2 (Bradley 1964). These phages are so closely related that we will not usually distinguish among them. Other filamentous phages, including the I-specific phages of E. coli (Meynell and Lawn 1968) and the filamentous phages of Pseudomonas (Minamishima et al. 1968), have been described but are not included in this discussion. We describe the properties of the various mutants of the filamentous phages, the organization of the genes on the genetic and physical maps, and the methylatable bases in the genome. We also list the gene products and describe some of their properties, as well as discuss aspects of the control of gene expression. MUTANTS Conditional Lethals Initial genetic studies of the filamentous phages were carried out by Pratt and his colleagues (1966 colleagues (1969), who collected amber and temperature-sensitive mutants and classified them by standard complementation analysis into eight complementation groups. No additional genes have been identified since. In the Pratt collection, gene VIII is represented by only one conditionally lethal mutation, am 8H1, and none of the other groups working with the filamentous phages have identified any other conditionally lethal mutations in gene VIII. Since the map is not saturated,...

Methylation of f1 DNA by a restriction endonuclease from escherichia coli B.

Abstract Bacteriophage f1 duplex DNA containing hybrid SB sites, the genetic sites which confer upon DNA sensitivity to Escherichia coli B-specific restriction and modification, were prepared in vitro . The hybrid SB sites (modified and mutant) were tested for their ability to be methylated in vitro by endonuclease R · Eco B, the enzyme responsible for both B-specific restriction and modification in vivo . DNA containing hybrid (modified) SB sites can be methylated. One methyl group is added to the DNA per hybrid (modified) SB site. On the other hand, DNA containing hybrid (mutant) SB sites is refractory to modification. The nature and the function of the SB site as well as the implications of these observations for f1 recombination are discussed.

Organization of chimeras between filamentous bacteriophage f1 and plasmid pSC101.

Two recombinants formed in vivo between the filamentous phage f1 and the tetracycline-resistance-conferring plasmid pSC101 are capable of transducing sensitive cells to Tetr. These chimeric filamentous phage, VO-1 and VO-2, were previously shown to contain the entire f1 and pSC101 genomes (Vovis et al., 1977; Ohsumi et al., 1978). The genomes of VO-1 and VO-2 are unstable in vivo; VO-1 breaks down to yield a molecule similar to pSC101 and an f1-like species, f1′. f1′ was previously shown to differ from f1 by the presence of 209 additional nucleotides inserted in the carboxy-terminal portion of gene IV (Ravetch et al., 1979). We have found by hybridization analysis and direct DNA sequencing that this 209-nucleotide segment is present in one copy in pSC101, and that it has properties similar to known transposable elements. Therefore, we have called this sequence IS101. We have characterized the structures of both VO-1 and VO-2 in greater detail by restriction mapping and DNA sequence analysis. Both chimeras contain two copies of IS101, which are present as direct repeats and form the junctions between the f1 and pSC101 genomes. The IS101 elements in VO-1 and VO-2 are flanked by a five-base direct repeat of f1 sequence that is not repeated in wild-type f1. The junction between f1 and pSC101 in VO-1 is located at the same point as the IS101 element in f1′, while in VO-2 the junction between the two genomes is at a point in f1 located between the promoter and ribosome binding site for gene VIII. The pSC101-like molecules derived from the breakdown of VO-1 in vivo are identical to the original pSC101 in the region of IS101. The IS101 elements in the original and derived pSC101 plasmids are not flanked by any repeated sequence. Attempts to regenerate VO-1 from f1′ and pSC101, both of which contain one IS101 element, indicate that the breakdown of VO-1 is irreversible. These results are discussed in terms of current models for transposition, which postulate structures similar to VO-1 and VO-2 as intermediates in transposition.

The isolation and characterization of an in vivo recombinant between the filamentous bacteriophage f1 and the plasmid pSC101.

Abstract After infection with the male-specific filamentous bacteriophage f1, F+Escherichia coli harboring the tetracycline resistance- (Tetr) conferring plasmid pSC101, extruded some filamentous Tetr-transducing particles. One such particle, which was named VO-1, was cloned and characterized. The size of the VO-1 phage particle, as well as heteroduplex and restriction enzyme analyses of the replicative form DNA of VO-1, are consistent with the hypothesis that VO-1 contains the f1 and pSC101 genomes in their entirety. The site of transition from f1 to pSC101 and the site of transition of pSC101 to f1 within the VO-1 genome were determined. Comparison of VO-1 to another f1-pSC101 chimera formed in vivo (Vovis, G. F., Ohsumi, M., and Zinder, N. D. (1977). Bacteriophage f1 as a vector for constructing recombinant DNA molecules. In “Molecular Approaches to Eucaryotic Genetic Systems”, pp. 55–61. Academic Press, New York.) indicates that an insertion sequence probably exists in pSC101. VO-1 DNA as a separate entity was able to transform E. coli to Tetr. However, the VO-1 molecule segregated within E. coli to yield a molecule indistinguishable from authentic pSC101 DNA by restriction enzyme analysis and a molecule that was slightly larger than authentic f1. The latter molecule was named f1′. The f′ present in the original VO-1 lysate differs from f1 by about 300 extra nucleotides which are located at the site where pSC101 was inserted into f1 to form the VO-1 chimera. Thus, bacteria transformed or transduced to Tetr by VO-1 are found to contain VO-1, pSC101, and f1′ replicative form DNA. Such E. coli produce both f1′ and VO-1 phage particles. The existence of VO-1 demonstrates that it is possible to form filamentous phage particles containing a DNA molecule significantly greater in size than unit length f1. The implications of these observations for the use of the filamentous bacteriophage as cloning vectors are discussed.

Erratum: Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori: Correction

This corrects the article DOI: 10.1038/16495

The Filamentous Phages as Transducing Particles

An important property of the filamentous phages is that the length of the virion is determined by the size of the viral DNA molecule encapsulated within it. This conclusion is based on the discovery of miniphage particles, which were shown to contain the viral DNA of deletion mutants (Griffith and Kornberg 1974; Enea and Zinder 1975; Hewitt 1975). For example, the f1 deletion mutant MP-2, which consists of only about 20% of the f1 genome, is found in a phage particle whose length is about 20% that of the wild-type particle (Enea et al. 1977; see also Wheeler and Benzinger, this volume). Particles larger than unit length are also found in all filamentous stocks. Diploid particles constitute about 5% of a wild-type phage population (Scott and Zinder 1967; Salivar et al. 1967). In addition, under permissive conditions, certain amber mutants produce very large, multi-unit-length particles (Pratt et al. 1969). However, nearly all of these multi-unit-length particles, whether they be in wild-type or amber stocks, contain the corresponding number of unit-length DNA molecules. Nevertheless, the existence of greater-than-unit-length phages suggests that filamentous particles containing DNA molecules larger than that of the wild-type f1 genome should be possible. We set out to prove this supposition and, in addition, to determine whether there is a practical limit on how large a DNA molecule can be and yet still be replicated efficiently and extruded as a filamentous phage particle. In our experiments we took advantage of the observation that recombination between apparently nonhomologous DNA...

Mapping cDNAs by Hybridization to Gridded Arrays of DNA from YAC Clones

A physical map of overlapping clones covering the human genome will provide a substrate for rapid, high-throughput, high resolution mapping of genes. Such a map of megaYAC clones is currently under development in several laboratories. To permit utilization of this resource, we are developing technology for mapping of cDNAs by hybridization to gridded arrays of DNA from megaYAC clones. Included in this approach are methods and instrumentation for reducing false negatives and false positives by pooling of megaYAC DNAs, for reducing the number of hybridizations by pooling of cDNA probes, and for automating the hybridization and detection steps. Results from a pilot project involving megaYACs representing about one-quarter of the human genome are described. Total yeast DNA was prepared from 730 megaYACs, diluted to a uniform concentration, and pooled with a representation of three and a pool size of three. Various amounts of pooled DNAs from megaYAC clones were gridded onto nylon filters in a medium density array. PCR amplified inserts from cDNA libraries were radiochemically labeled by random priming and used as probes. The sensitivity of detection was adequate even at the lowest megaYAC quantity per dot, i.e., about 0.08 μg of total yeast DNA from each YAC in each dot. Most signals were triplets as expected from the pooling strategy, and the signal intensity of dots was quite uniform. Filters could be re-probed at least five times with no detectable degradation of signal to noise ratio. Application of a “two out of three” rule for validity of “real” signals appears adequate since it permitted accurate identification of all control YACs on the filters. These results suggest that such a hybridization-based approach will permit accurate, rapid, high throughput mapping of cDNAs to intervals in the emerging YAC contig map.