Conservation and variation of nucleotide sequences within related bacterial genomes: enterobacteria

We have assessed the degree of relatedness of several portions of the Escherichia coli genome to the corresponding portions of the genomes of representative enteric bacteria, using the Southern transfer and hybridization technique (E. Southern, J. Mol. Biol. 98:503-517, 1975). The degree of relatedness varied among the regions examined. Judging both by the relative amounts of deoxyribonucleic acid in the various enteric genomes that are highly homologous and by the conservation of positions of restriction enzyme cleavage sites in these regions, the enteric genomes have diverged to greater extents in some parts of the genomes than in others. Portions of the genomes (including the tnaA and thyA genes, the trp operon, and one other unassigned segment) appear to have evolved in concert with the genome as a whole. By contrast, the lacZ gene and portions of the genome that are homologous to phage lambda vary more widely, perhaps reflecting a separate evolutionary origin for these segments of deoxyribonucleic acid.     

Conservation and variation of nucleotide sequences in Escherichia coli strains isolated from nature.

A group of Escherichia coli isolates from nature were compared with one another and with laboratory strains of E. coli with respect to size distribution of chromosomal restriction endonuclease fragments and differences in nucleotide sequences in selected small portions of the genomes. The estimated frequency of base substitutions in nucleotide sequences in and near the trp operons of 26 of the 28 E. coli strains examined ranged from 0.008 to 0.066. Nucleotide sequences in or near lambda prophage homologs were significantly more variable than the sequences in or near trp, tnaA, and thyA genes. Thus, the lambda-homologous regions may have a significant horizontal component in their evolutionary histories, having undergone genetic exchange, whereas the trp, tnaA, and thyA regions may have solely vertical evolutionary histories. The relatedness of the E. coli strains in the genetic regions studied indicated that laboratory strains are not more closely related to one other than they are to isolates from nature. The isolates from natural populations did not form groups related either by host taxa or by geographical region of isolation.     

Cloning and physical mapping of enteric adenoviruses (candidate types 40 and 41)

We have studied the DNAs of fastidious enteric adenoviruses recovered from the stools of infants with gastroenteritis. By endonuclease analysis, the strains examined represent candidate adenovirus types 40 and 41, which are thought to comprise new adenovirus subgroups F and G. Cloning of DNA from representative enteric adenovirus isolates, together with hybridization and subcleavage analysis, permitted the mapping of restriction enzyme cleavage sites. Although the restriction profiles are different for the two strains, they appear to have several cleavage sites in common. Cross hybridization studies show considerable homology between the subgroup F and G strains but much less homology to adenovirus 2. In addition, regions on both ends of enteric adenovirus genomes (map units, 2.9 to 11.3 and 75 to 100) possess little or no homology to adenovirus 2. Restriction enzyme digests reveal submolar fragments that map to the terminal regions of the genome. Electron micrographic studies of denatured and renatured DNA strands suggest that the submolar fragments may derive from cleavage of defective molecules. Inverted terminal repeat sequences were shown to comprise 0 to 3.2% of the length of complete (greater than or equal to 22 megadaltons) enteric adenovirus DNA molecules but 4 to 69% of incomplete-length (less than 22-megadalton) molecules.     

Common physical map of four Brucella bacteriophage genomes

Abstract DNAs isolated from four strains of Brucella bacteriophages were studied by restriction endonuclease mapping and Southern blot analysis. In all strains the genome was composed of a 38 kb (25.1 × 10⁶ dalton) double-stranded circular DNA. The physical map was the same for the four genomes and Southern blot hybridization of restriction endonuclease fragments with the Tbilissi strain DNA as a probe showed complete homology between the four DNAs. Thus, the four phage strains appear to be identical, the specific host range of each originating from minor changes in phage or Brucella receptors or both.     

Electron microscopic heteroduplex study and restriction endonuclease cleavage analysis of the DNA genomes of three lactic streptococcal bacteriophages.

Three lactic streptococcal bacteriophages were compared with one another by electron microscopic analysis of heteroduplex DNA molecules. The phages were almost identical in morphology and had been isolated over a period of 10 years on different strains of Streptococcus cremoris from cheese plants situated in different parts of New Zealand. There was a high degree of homology between the DNAs, in agreement with Southern blot hybridization data reported earlier. There were, however, distinct regions of nonhomology, mostly between 0.45 and 1.71 kilobases in length, suggestive of the occurrence of block recombination events. A deletion of 2.23 kilobases in the two more recently isolated phages, or an insertion in the first isolate, was found. All three phage DNAs showed differences in restriction endonuclease cleavage sites. Alignment of the restriction endonuclease maps with the heteroduplex maps showed that differences in cleavage sites occurred most frequently in regions of nonhomology. However, differences in cleavage sites in regions of apparent homology were also detected, indicating that point mutations may have occurred in addition to block recombination events.     

Electron microscopic heteroduplex study and restriction endonuclease cleavage analysis of the DNA genomes of three lactic streptococcal bacteriophages

Three lactic streptococcal bacteriophages were compared with one another by electron microscopic analysis of heteroduplex DNA molecules. The phages were almost identical in morphology and had been isolated over a period of 10 years on different strains of Streptococcus cremoris from cheese plants situated in different parts of New Zealand. There was a high degree of homology between the DNAs, in agreement with Southern blot hybridization data reported earlier. There were, however, distinct regions of nonhomology, mostly between 0.45 and 1.71 kilobases in length, suggestive of the occurrence of block recombination events. A deletion of 2.23 kilobases in the two more recently isolated phages, or an insertion in the first isolate, was found. All three phage DNAs showed differences in restriction endonuclease cleavage sites. Alignment of the restriction endonuclease maps with the heteroduplex maps showed that differences in cleavage sites occurred most frequently in regions of nonhomology. However, differences in cleavage sites in regions of apparent homology were also detected, indicating that point mutations may have occurred in addition to block recombination events.     

Recombination Between Endogenous and Exogenous Simian Virus 40 Genes. II. Biochemical Evidence for Genetic Exchange

The genome of the simian virus 40 (SV40) temperature-sensitive (ts) mutant tsD202 rescued by passage on transformed permissive monkey lines (see accompanying paper [Y. Gluzman et al., J. Virol. 24:534-540, 1977]) was analyzed by restriction endonuclease cleavage mapping to obtain biochemical evidence that the rescue of the ts phenotype results from recombination with the resident SV40 genome of the transformed cell. It was demonstrated that the endonuclease R. HaeIII cleavage site, which is located at 0.9 map unit in the standard viral genome (and which is in the proximity of the known map position of the tsD lesion), is missing in the DNAs of the parental tsD202 virus and of three independent revertants of tsD202. In contrast, this cleavage site was shown to be present in the DNAs of four out of five independently derived rescued D202 populations and in the DNA of the SV40 strain, 777, used to transform the monkey cells. Comparison of the endonuclease R. Hin(II + III) cleavage patterns of SV40 strain 777 DNA and tsD202 DNA revealed differences in the electrophoretic mobilities of Hin fragments A, B, and F. However, the corresponding Hin fragments from all four rescued D202 genomes were identical in their mobilities to those of tsD202 DNA, indicating that these regions of the rescued D202 genome are characteristic of the tsD202 parent. We conclude, therefore, that the genome of the rescued D202 virus is a true recombinant, since it contains restriction endonuclease cleavage sites characteristic of both parents, the endogenous resident SV40 genome of the transformed monkey cells and the exogenous tsD202 mutant.     

Physical mapping of beta-converting and gamma-nonconverting corynebacteriophage genomes

Deoxyribonucleic acids (DNAs) from wild-type and mutant strains of beta-converting and gamma-nonconverting corynebacteriophages were isolated and physically characterized. The data obtained from DNA heteroduplexes, restriction enzyme banding profiles, and restriction maps reinforce the conclusion that beta and gamma phages are very closely related. The major physical differences seen in the DNA heteroduplexes are a small substitution bubble and one or two insertions which are present on the gamma phage genome. The insertions account for the differences in the genome sizes of beta and gamma phages, and with the substitution they are responsible for most of the differences in the restriction endonuclease profiles and maps of the corynebactriophage genomes, two special sites and the DNA fragments carrying them were identified. These were the cohesive (cos) sites and the specific attachment (attP) site of the vegetative phage genome. The behavior of these sites indicated that the transition of phage DNA from the vegetative to the prophage state involves the circularization of vegetative DNA through the cos sites and its integration into the bacterial chromosome via the attP site. The mechanism of corynebacteriophage integration was similar to that employed by Escherichia coli phage gamma. From the data assembled the physical and genetic maps of beta and gamma phage were oriented with respect to one another. The extensive similarity in their maps provides additional confirmation of a close evolutionary relationship.     

Evidence for the Asiatic origin of endogenous AKR-type murine leukemia proviruses.

A restriction endonuclease cleavage map of the genome of AKV, the endogenous, ecotropic leukemia virus of AKR mice, has been derived. By using this map and analyzing DNA from congenic mice, we have defined four DNA fragments diagnostic for AKV proviruses. Analysis of DNAs from 10 strains of American laboratory mice revealed that all strains carrying inducible, ecotropic murine leukemia viruses yielded DNAs which contained the four DNA fragments diagnostic for AKV. Virus-negative strains lacked these fragments in their DNA. Screening DNA from 23 additional mice revealed that, among these mice, only mice from Asia gave rise to the DNA fragments diagnostic of an AKV provirus. We conclude that all of the endogenous ecotropic murine leukemia proviruses in American laboratory mice are closely related since they share a common set of restriction endonuclease cleavage sites. These proviruses appear to derive from the East Asian ancestors of these mouse strains. Analysis of DNA from six selected mice with an additional restriction endonuclease showed that greater than 97% of the nucleotide sequences in each provirus are contigous and that these endogenous proviruses are indistinguishable from proviruses introduced by exogenous infection.     

Physical map of the BK virus genome.

Two new human papovavirus isolates (JMV and MMV) from the urines of patients with Wiskott-Aldrich syndrome were morphologically and serologically identical to BK virus (BKV). The genomes of these two new isolates were found to be indistinguishable from prototype BKV DNA in a variety of nucleic acid hybridization experiments. Like BKV DNA, JMV and MMV DNAs share approximately 20% of their polynucleotide sequences with simian virus 40 DNA. The genome of JMV was indistinguishable from that of BKV by restriction endonuclease analysis; MMV DNA contained three instead of four R-Hind cleavage sites and one rather than no R-HpaII cleavage sites. Physical maps of the BKV and MMV genomes were constructed using restriction endonucleases, and these maps were oriented to the map of simian virus 40 DNA.     

Construction and analysis of recombinant lambda phages containing mitochondrial DNA fragments.

Rat mtDNA has a molecular length of about 16 kilobase (kb) pairs and is cleaved into seven fragments by restriction endonuclease EcoRI. These fragments were cloned in Escherichia coli K-12 host using lambda gtWES.lambda B' (lambda gtWES.lambda B, for short, in this paper) as a vector. Recombinant DNAs containing one or a few fragments of the mtDNA were transfected to CaCl2-treated E. coli, and the plaques containing specific recombinant phages were selected. DNA amplified in the recombinanat phage lambda gt.mt was shown to contain the same restriction endonuclease cleavage sites as those found in the mtDNA. Present results permitted the DNA sequencing of any portion of the mitochondrial genome.     

A new method for specific cleavage of megabase-size chromosomal DNA by lambda-terminase.

The development of methods for cleavage of DNA at specific site(s) that are widely spaced would facilitate physical mapping of large genomes. Several methods for rare and specific cleavage of chromosomal DNAs require a nearly complete methylation of a given type of restriction site except the one that is specifically protected. It is expected that as the target DNA increases in length, it will become less likely to achieve nearly complete methylation. The intron-encoded endonucleases may also provide a capability to cleave megabase-sized DNA segments due to their very large recognition sequences. However, there are endogenous cleavage sites in the chromosomes of most organisms. We present here a new method to specifically cleave intact chromosomal DNA using lambda-terminase. A plasmid containing two specific cleavage sites (cohesive-end sites) for lambda-terminase was specifically introduced into the E.coli genome and into chromosome V of S.cerevisiae. Chromosomal DNA was prepared from the resulting strains, and then cleaved with lambda-terminase. The results showed that the 4.7-megabase pair (Mb) circular E.coli chromosome and the 0.58-Mb linear yeast chromosome V were specifically cleaved at the desired sites with very high efficiencies. The approach of using the lambda-terminase cleavage reaction is a simple one-step procedure with a high specificity which is particularly suitable for mapping very large genomes of eucaryotes.     

Analysis of a restriction endonuclease map for a rabbit papillomavirus DNA

A restriction endonuclease map was constructed for rabbit papillomavirus DNA. Analysis of the viral genome by cleavage with specific endonucleases, when compared with bovine papillomavirus types 1 and 2 genome maps, revealed similarities among the three genomes. A number of cleavage sites and their relative order on each genome were found to be conserved.     

Cloning of human papilloma virus genomic DNAs and analysis of homologous polynucleotide sequences.

The complete DNA genomes of four distinct human papilloma viruses (human papilloma virus subtype 1a [HPV-1a], HPV-1b, HPV-2a, and HPV-4) were molecularly cloned in Escherichia coli, using the certified plasmid vector pBR322. The restriction endonuclease patterns of the cloned HPV-1a and HPV-1b DNAs were similar to those already published for uncloned DNAs. Physical maps were constructed for HPV-2a DNA and HPV-4 DNA, since these viral DNAs had not been previously mapped. By using the cloned DNAs, the genomes of HPV-1a, HPV-2a, and HPV-4 were analyzed for nucleotide sequence homology. Under standard hybridization conditions (Tm = --28 degrees C), no homology was detectable among the genomes of these papilloma viruses, in agreement with previous reports. However, under less stringent conditions (i.e., Tm = --50 degrees C), stable DNA hybrids could be detected between these viral DNAs, indicating homologous segments in the genomes with approximately 30% base mismatch. By using specific DNA fragments immobilized on nitrocellulose filters, these regions of homology were mapped. Hybridization experiments between radiolabeled bovine papilloma virus type 1 (BPV-1) DNA and the unlabeled HPV-1a, HPV-2a, or HPV-4 DNA restriction fragments under low-stringency conditions indicated that the regions of homology among the HPV DNAs are also conserved in the BPV-1 genome with approximately the same degree of base mismatch.     

Restriction of two-replicon shuttle Escherichia coli-Streptomyces plasmids in Streptomyces lividans strain 66

The shuttle Escherichia coli - Streptomyces plasmids were used to transform S. lividans 66. Plasmid DNAs isolated from this strain transform it 10-1000-fold more efficiently than DNAs from E. coli. Rare transformant cured from most restricted plasmid is more efficient recipient of plasmid DNA from E. coli and has the property of R +/- M+ mutant. Restriction in S. lividans 66 correlates with the appearance in DNA from E. coli of the sites susceptible to Scg2I restriction endonuclease. The latter was isolated earlier from recombinant strain Rcg2, a hybrid between S. griseus Kr. 15 and S. coelicolor A3(2). Scg2I possesses the recognition sequence CCTAGG, like EcoRII, MvaI and Eco dcm methylase. The DNA resistant to Scg2I cleavage retained this ability after in vitro modification by EcoRII methylase. So, the resistance of DNA to Scg2I cleavage is not connected with methylation at 4th and 5th position of second cytosine in the recognition sequence. Neither restriction of plasmid DNA in S. lividans 66 is dependent on dcm modification in E. coli, though its dependence on dam modification is not excluded. It is assumed that the restriction in S. lividans 66 is specified by endonuclease analogous to Scg2I.     

Restriction map of the single-stranded DNA genome of Kilham rat virus strains 171, a nondefective parvovirus.

We constructed a physical map of Kilham rat virus strains 171 DNA by analyzing the sizes and locations of restriction endonuclease-generated fragments of the replicative-form viral DNA synthesized in vitro. BglI, KpnI, BamHI, SmaI, XhoI, and XorII did not appear to have any cleavage sites, whereas 11 other enzymes cleaved the genome at one to eight sites, and AluI generated more than 12 distinct fragments. The 30 restriction sites that were mapped were distributed randomly in the viral genome. A comparison of the restriction fragments of in vivo- and in vitro-replicated replicative-form DNAs showed that these DNAs were identical except in the size or configuration of the terminal fragments.     

Site-specific dissection of E. coli chromosome by lambda terminase.

We have succeeded the targeted cleavage of chromosomes by lambda terminase that introduces double-strand cleavages in DNA recognizing the lambda cos sequence. When chromosomal DNAs of various Escherichia coli K-12 strains were subjected to terminase digestion, all were found to contain two common cleavage sites. Therefore, DNAs from lambda lysogens in which lambda DNA was inserted at different chromosomal sites were specifically cleaved at one more additional site. The two sites, termed ecos1 and ecos2, were mapped at approximately 35.1' and 12.7' of E. coli genetic map. The ecos1 and ecos2 sites were included in qin and qsr' regions, respectively. Therefore, the cleavage sites were associated with cryptic prophages. Sequences at the ecos1 and ecos2 sites showed 98% homology to the lambda cos sequence, indicating high fidelity of sequence recognition by the terminase. Since the strategy for integration of a DNA segment into chromosomal DNA through homologous recombination has been established, the dissection method that uses lambda terminase should be applicable for gene mapping as well as construction of macrophysical maps of larger genomes.     

Virion DNA of ground squirrel hepatitis virus: structural analysis and molecular cloning.

The structure of the encapsidated DNA genome of ground squirrel hepatitis virus (GSHV) has been examined by restriction endonuclease cleavage, nucleic acid hybridization, and molecular cloning. GSHV virion DNA is a relaxed circular molecule of approximately 3,200 bases in length; most molecules harbor an extensive single-stranded region which is largely confined to one-half of the genome. The full-length viral DNA strand is covalently bound to protein. The single-stranded region can be repaired in vitro by the action of the endogenous virion polymerase, exogenously added DNA polymerase from avian myeloblastosis virus, or both. Restriction enzyme cleavage of viral DNA from different isolates demonstrated that multiple variants of GSHV exist in nature. The genomes of two such strains have been cloned in Escherichia coli, and their physical maps have been determined. Nucleic acid hybridization studies revealed that the strains share sequence homology with the DNA of human hepatitis B virus. Regions homologous to the coding regions for the surface and core antigens of human hepatitis B virus have been localized on the GSHV chromosome. Molecular cloning experiments have also led to the identification of a region of the viral genome which is altered in a procaryotic host.     

An electron microscopic method for studying and mapping the region of weak sequence homology between simian virus 40 and polyoma DNAs.

A procedure for investigating the possibility of small amounts of partial DNA sequence homology between two defined DNA molecules has been developed and used to test for sequence homology between simian virus 40 and polyoma DNAs. This procedure, which does not necessitate the use of separated viral DNA strands, involves the construction of hybrid DNA molecules containing a simian virus 40 DNA molecule covalently joined to a polyoma DNA molecule, using the sequential action of EcoRI restriction endonuclease and Escherichia coli DNA ligase. Denaturation of such hybrid DNA molecules then makes it possible to examine intramolecularly rather than intermolecularly renatured molecules. Visualization of these intramolecularly renatured “snapback” molecules with duplex regions of homology by electron microscopy reveals a 15% region of weak sequence homology. This region is denatured at about 35 °C below the melting temperature of simian virus 40 DNA and therefore corresponds to about 75% homology. This region was mapped on both the simian virus 40 and polyoma genomes by the use of Hemophilus parainfluenzae II restriction endonuclease cleavage of the simian virus 40 DNA prior to EcoRI cleavage and construction of the hybrid molecule. The 15% region of weak homology maps immediately to the left of the EcoRI restriction endonuclease cleavage site in the simian virus 40 genome and halfway around from the EcoRI restriction endonuclease cleavage site in the polyoma genome.