Cloning and Expression in Escherichia coli of the Polysaccharide Depolymerase Associated with Bacteriophage-Infected Erwinia amylovora

The bacteriophage-encoded polysaccharide depolymerase produced in Erwinia amylovora has been cloned and expressed in Escherichia coli. The bacteriophage ERA103 genome was observed to consist of five EcoRI fragments, labeled as follows: A, 7.5 kilobases (kb); B, 5.0 kb; C, 2.7 kb; D, 2.1 kb; and E, 1.8 kb. A restriction map for ERA103 was also prepared. Each of the fragments were cloned into the positive-selection vector pOP203(A₂⁺) and pBR322.     

Mitochondrial DNA analysis ofExophiala moniliae

Mitochondrial DNA(mtDNA) analysis with restriction enzymes, Hae III, Hind III and Msp I was performed in 17Exophiala moniliae strains. The results were as follows: (1)E. moniliae could be classified into 10 types based on restriction patterns, (2)E. moniliae is suggested to be a complex organism because of extensive mtDNA polymorphism among strains likeE. jeanselmei and (3) two types ofE. moniliae are identical with two types ofE. jeanselmei. These results suggest thatE. moniliae is not genetically defined fromE. jeanselmei and the taxonomical status ofE. moniliae requires reevaluation     

Cleavage map of the simian-virus-40 genome by the restriction endonuclease III of Haemopholus aegyptius.

Enzymic digestion of Simian virus 40 (SV40) DNA with Haemophilus aegyptius restriction endonuclease Hae III results in 10 major and eight minor fragments. These were resolved by electrophoresis on graduated polyacrylamide slab gels. All fragments have been characterized with respect to the size relative to the Haemophilus influenzae Rd fragments (Hind). They were ordered on the SV40 DNA map by means of overlap analysis of the double cleavage products derived from sequential digestion of Hind fragments with Hae III endonuclease and Hae fragments with Hind II + III enzyme, as well as by other reciprocal cleavage experiments, including those involving Haemophilus para-influenzae fragments. In this way the 18 Hae III cleavage sites and the 13 Hind sites have been localized on the circular SV40 DNA map.     

Release of Free DNA by Membrane-Impaired Bacterial Aerosols Due to Aerosolization and Air Sampling

We report here that stress experienced by bacteria due to aerosolization and air sampling can result in severe membrane impairment, leading to the release of DNA as free molecules. Escherichia coli and Bacillus atrophaeus bacteria were aerosolized and then either collected directly into liquid or collected using other collection media and then transferred into liquid. The amount of DNA released was quantified as the cell membrane damage index (I_D), i.e., the number of 16S rRNA gene copies in the supernatant liquid relative to the total number in the bioaerosol sample. During aerosolization by a Collison nebulizer, the I_D of E. coli and B. atrophaeus in the nebulizer suspension gradually increased during 60 min of continuous aerosolization. We found that the I_D of bacteria during aerosolization was statistically significantly affected by the material of the Collison jar (glass > polycarbonate; P < 0.001) and by the bacterial species (E. coli > B. atrophaeus; P < 0.001). When E. coli was collected for 5 min by filtration, impaction, and impingement, its I_D values were within the following ranges: 0.051 to 0.085, 0.16 to 0.37, and 0.068 to 0.23, respectively; when it was collected by electrostatic precipitation, the I_D values (0.011 to 0.034) were significantly lower (P < 0.05) than those with other sampling methods. Air samples collected inside an equine facility for 2 h by filtration and impingement exhibited I_D values in the range of 0.30 to 0.54. The data indicate that the amount of cell damage during bioaerosol sampling and the resulting release of DNA can be substantial and that this should be taken into account when analyzing bioaerosol samples.     

Location of the T4 gene 32 protein-binding site on polyoma virus DNA

Three easily denatured regions can be demonstrated in polyoma virus DNA. T4 gene 32 protein which binds to single stranded DNA, but not to duplex DNA, will specifically bind to any of these sites when viral DNA is in its superhelical configuration. These sites were mapped relative to a unique E. coli R_I endonuclease cleavage site by electron microscopy.     

Long range periodicities in mouse satellite DNA.

Escherichia coli restriction enzyme II breaks mouse satellite DNA into fragments which form a series of bands on gel electrophoresis. The DNA in the strongest band has a length of 220 to 260 nucleotide pairs and the other bands are multiples of this length. It is shown that these fragments are linked together in long arrays in the satellite sequence. The reassociation register of the DNA is about half the length of the 220 to 260 nucleotide pair fragment. In the electrophoresis pattern of the Eco RII² fragments other weaker bands can be seen. The stronger bands of the minor patterns fall half-way between the bands of the main pattern and the smallest is 120 to 130 nucleotide pairs long. The properties of the minor fragments suggest short spacings of the restriction site which have been produced by unequal crossing-over. The extents of divergence and unequal crossing-over are estimated. From this analysis and the sequence analysis described in the accompanying paper (Biro et al., 1975) it is proposed that mouse satellite DNA consists of an hierarchy of four periodicities which reflect stages in the evolution of the sequence.Digestion of mouse satellite DNA with Hae III produces fragments with the same sizes as those produced by Eco RII, but the yields are much lower. It is suggested that Hae III sites have been introduced by divergence and subsequently spread by unequal crossing-over.     

The selective digestion of polytene and mitotic chromosomes of Drosophila melanogaster by the Alu I and Hae III restriction endonucleases

Polytene and mitotic chromosomes of Drosophila melanogaster were treated with either Alu I or Hae III restriction endonucleases. Subsequent staining with the DNA-specific fluorochrome ethidium bromide showed that these enzymes are capable of selectively digesting chromosomal DNA in fixed cytological preparations, as previously shown in mammalian metaphase chromosomes. Alu I or Hae III digestion made possible the localization in situ of some highly repetitive DNAs in both polytene and mitotic chromosomes, while only Alu I permitted the localization of the 5S RNA genes on the polytene chromosomes of D. melanogaster.     

Plasmid Conjugation from Proteobacteria as Evidence for the Origin of Xenologous Genes in Cyanobacteria

Comparative genomics have shown that 5% of Synechococcus elongatus PCC 7942 genes are of probable proteobacterial origin. To investigate the role of interphylum conjugation in cyanobacterial gene acquisition, we tested the ability of a set of prototype proteobacterial conjugative plasmids (RP4, pKM101, R388, R64, and F) to transfer DNA from Escherichia coli to S. elongatus. A series of BioBrick-compatible, mobilizable shuttle vectors was developed. These vectors were based on the putative origin of replication of the Synechococcus resident plasmid pANL. Not only broad-host-range plasmids, such as RP4 and R388, but also narrower-host-range plasmids, such as pKM101, all encoding MPF_T-type IV secretion systems, were able to transfer plasmid DNA from E. coli to S. elongatus by conjugation. Neither MPF_F nor MPF_I could be used as interphylum DNA delivery agents. Reciprocally, pANL-derived cointegrates could be introduced in E. coli by electroporation, where they conferred a functional phenotype. These results suggest the existence of potentially ample channels of gene flow between proteobacteria and cyanobacteria and point to MPF_T-based interphylum conjugation as a potential mechanism to explain the proteobacterial origin of a majority of S. elongatus xenologous genes.     

Sequences encoding identical peptides for the analysis and manipulation of coding DNA

The use of sequences encoding identical peptides (SEIP) for the in silico analysis of coding DNA from different species has not been reported; the study of such sequences could directly reveal properties of coding DNA that are independent of peptide sequences. For practical purposes SEIP might also be manipulated for e.g. heterologous protein expression. We extracted 1,551 SEIP from human and E. coli and 2,631 SEIP from human and D. melanogaster. We then analyzed codon usage and intercodon dinucleotide tendencies and found differences in both, with more conspicuous disparities between human and E. coli than between human and D. melanogaster. We also briefly manipulated SEIP to find out if they could be used to create new coding sequences. We hence attempted replacement of human by E. coli codons via dicodon exchange but found that full replacement was not possible, this indicated robust species-specific dicodon tendencies. To test another form of codon replacement we isolated SEIP from human and the jellyfish green fluorescent protein (GFP) and we then re-constructed the GFP coding DNA with human tetra-peptide-coding sequences. Results provide proof-of-principle that SEIP may be used to reveal differences in the properties of coding DNA and to reconstruct in pieces a protein coding DNA with sequences from a different organism, the latter might be exploited in heterologous protein expression.     

Organization of ribosomal protein genes in Escherichia coli: I. Physical structure of DNA from transducing λ phages carrying genes from the aroE-str region

The physical structures of the genomes of five transducing bacteriophages (λaroE, λtrkA, λspc1, λspc2, and λfus2) carrying various portions of the aroE-trkA-spc-str segment of the Escherichia coli chromosome have been determined. Two methods were used: (a) heteroduplex analysis of DNA molecules from these phages, and (b) analysis of fragments obtained from digestion of the DNA by restriction endonucleases EcoRI and HindIII. In λaroE, λtrkA, λspc1 and λspc2, whose genome lengths vary from about 75% to about 104% of the λpapa genome, the right arm of λ DNA is present, whereas various portions of the left arm have been replaced by E. coli DNA. In λfus2, however, about 93% of the λ DNA molecule is replaced by E. coli DNA, the resultant genome being 103.5 %λ units long (Figs 1 and 2). All five phages contain an identical λ-E. coli junction at 1.9 %λ units from the left λ terminus, and there is complete homology between the common portions of the inserted E. coli DNA. Since these phages were independently isolated, we believe that the genetic organization of the E. coli DNA carried by these phages probably reflects the organization of the relevant segments of the E. coli chromosome. Comparison of the physical and genetic maps of these transducing phages has allowed us to assign a physical position to the ribosomal and neighbouring genes, including those coding for the α subunit of RNA polymerase and the elongation factors G and Tu, on the bacterial DNA.     

Restriction endonucleases R.EcoKI and R.EcoR124I are probably located in different environments within the bacterial cell

We describe the phenomenon of a transient state of R124I restriction deficiency after long-term storage of theE. coli[pCP1005] strain at 4°C, or after growth of the culture in synthetic M9 medium with the nonmutagenic solvent dimethyl sulfoxide. The unusual high reversion from the R⁺ ₁₂₄ to the R^? ₁₂₄ phenotype was observed only inE. coli strain transformed with the high-copy number plasmid pCP1005 carryingECoR124IhsdR, M and S genes cloned, but not with strains carrying the natural conjugative plasmid R124. The effect of both treatments on the expression ofEcoR124I phenotype in relation to the possible location of R.EcoR124I restriction endonuclease inE. coli is discussed.     

Physical map of Aspergillus nidulans mitochondrial genes coding for ribosomal RNA: an intervening sequence in the large rRNA cistron

Summary A detailed map of the 32 kb mitochondrial genome of Aspergillus nidulans has been obtained by locating the cleavage sites for restriction endonucleases Pst I, Bam H I, Hha I, Pvu II, Hpa II and Hae III relative to the previously determined sites for Eco R I, Hind II and Hind III. The genes for the small and large ribosomal subunit RNAs were mapped by gel transfer hybridization of in vitro labelled rRNA to restriction fragments of mitochondrial DNA and its cloned Eco R I fragment E3, and by electron microscopy of RNA/DNA hybrids.The gene for the large rRNA (2.9 kb) is interrupted by a 1.8 kb insert, and the main segment of this gene (2.4 kb) is separated from the small rRNA gene (1.4 kb) by a spacer sequence of 2.8 kb length.This rRNA gene organization is very similar to that of the two-times larger mitochondrial genome of Neurospora crassa, except that in A. nidulans the spacer and intervening sequences are considerably shorter.     

Repeating units of xenopus laevis oocyte-type 5S DNA are heterogeneous in length

The restriction enzymes Hind III and Hae III cleave Xenopus laevis 5S DNA at one and three sites, respectively, in each repeating unit of approximately 700 base pairs. The cleavage sites for both enzymes have been located within the repeating unit by denaturation mapping of the restriction fragments. The Hind III products and one of the Hae III fragments are variable in length, indicating heterogeneity in the length of the repeating unit in 5S DNA. This length heterogeneity is confined to the major A + T-rich spacer region. Repeating units differ from each other by discrete quanta of approximately 15 base pairs. The A + T-rich spacer has been shown to consist largely of tandem subrepeats of just this size (Brownlee, Cartwright, and Brown, 1974). We suggest that the repeat-length heterogeneity is due to variable numbers of these subrepeats in the spacer regions of the major repeating units.     

tRNA2Lys gene clusters in Drosophila

We have isolated segments of Drosophila melanogaster DNA that contain two clusters of tRNA₂^Lys genes. In one segment, pPW511, there is a cluster of three of these genes surrounded by other tRNA genes. Two other segments, pPW516 and pPW541. share a 3 × 10³ base-pair region that has a cluster of four tRNA₂^Lys genes. This cluster is flanked by 20 × 10³ base-pairs of DNA that does not appear to have other tRNA genes. The tRNA genes in both clusters are irregularly spaced and are intermingled with moderately repetitive DNA. Each cluster is present once or perhaps twice in the haploid genome and has the same arrangement of restriction endonuclease sites in the genomic DNA as in the isolated, cloned DNA. In situ hybridization to polytene chromosomes localized the pPW511 cluster to the 42A region and the pPW516/541 cluster to the 42E region. Another region, 50B, also contains tRNA₂^Lys genes. In sum, these cloned tRNA₂^Lys genes account for most of this gene family and are irregularly spaced in two clusters.     

Characterization of a rearrangement in viral DNA: mapping of the circular simian virus 40-like DNA containing a triplication of a specific one-third of the viral genome

Serial passage of the non-defective form of a simian virus 40-like virus (DAR) isolated from human brain results in the appearance of three distinct classes of supercoiled DNAs: R_I resistant, R_I sensitive (one cleavage site) and R_I “supersensitive” (three cleavage sites). The R_I cleavage product of the “super sensitive” form is one-third the physical size of simian virus 40 DNA (10.4 S) and reassociates about three times more rapidly than “standard” viral DNA. To identify the portions of the DAR genome present in the 10.4 S segment, the plus strand of each of the 11 fragments of ³²P-labeled simian virus 40 DNA, produced by cleavage with the Hemophilus influenzae restriction endonuclease, was hybridized in solution with the sheared R_I cleavage product of the “supersensitive” class of viral DNA. Reaction was observed with fragments located in two distinct regions of the simian virus 40 genome: (1) Hin-A and C; (2) Hin-G, J, F and K.Further studies indicated that simian virus 40 complementary RNA transcribed in vitro with Escherichia coli RNA polymerase from one strand of simian virus 40 DNA reacts with both strands of the denatured 10.4 S cleavage product when hybridization is monitored with hydroxyapatite. Treatment of the 10.4 S DNA-simian virus 40 cRNA hybrid with the single-strand spcific nuclease, S₁, converted approximately 50% of the radioactive counts to an acid-soluble product. These results indicate that the 10.4 S product contains a transposition of sequences originally present on one of the DAR DNA strands to the other strand. Examination of heteroduplexes formed between the 10.4 S segment and unique linear forms of DAR DNA produced with the R · Eco RI restriction endonuclease have confirmed these observations. Thus it appears that a molecular rearrangement(s) has resulted in the recombination and inversion of viral DNA sequences from two separate loci on the parental DAR genome. This 1.1 × 10⁶ dalton segment is reiterated three times in a supercoiled molecule equivalent in physical size to parental DAR DNA.     

Relative stabilities of RNA/DNA hybrids: effect of RNA chain length in competitive hybrization

Escherichia coli DNA and fragmented rRNA were used as a model system to study the effect of RNA fragment size in hybridization-competition experiments. Though no difference in hybridization rates was observed, the relative stabilities of the RNA/DNA hybrids were found to be largely affected by the fragment size of the RNA molecule. Intact rRNA was shown to replace shorter homologous rRNA sequences in their hybrids, the rate of the displacement being dependent on the molecular size of the RNA fragments. Hybridization-competition experiments between molecules of different lengths are expected to be complicated by the displacement reaction. The synthesis of tRNA^Tyr-like sequences transcribed in vitro on φ80psu₃⁺ bacteriophage DNA was measured by hybridization competition assays. Indirect competition with labelled E. coli tRNA^Tyr hybridization revealed that the in vitro-synthesized RNA contained significant amounts of tRNA^Tyr; these sequences could not, however, be detected by the direct competition method in which labelled in vitro-synthesized RNA competes with E. coli tRNA^Tyr for hybridization to φ80psu₃⁺ DNA. These contradictory results can be traced to the differences in size of the competing molecules in the hybridization-competition reaction. Indeed, in vitro-transcribed tRNA^Tyr-like sequences, longer than mature tRNA, were found to displace efficiently E. coli tRNA^Tyr from its hybrids with φ80psu₃⁺ DNA. These findings explain why such sequences could not be detected by direct competition with E. coli tRNA^Tyr.     

Analysis of Drosophila melanogaster satellite IV with restriction endonuclease MboII.

The products of digestion of Drosophila melanogaster satellite IV DNA with restriction endonuclease MboII have been analysed and found to be consistent with a repeating pentamer sequence (A-G-A-A-G)_n for satellite IV. More than 95% of the satellite DNA is digested to fragments less than 25 base-pairs in length, suggesting that the DNA sequence is highly conserved.     

Cassette-like variation of restriction enzyme genes in Escherichia coli C and relatives

A surprising result of comparative bacterial genomics has been the large amount of DNA found to be present in one strain but not in another of the same species. We examine in detail one location where gene content varies extensively, the restriction cluster in Escherichia coli. This region is designated the Immigration Control Region (ICR) for the density and variability of restriction functions found there. To better define the boundaries of this variable locus, we determined the sequence of the region from a restrictionless strain, E.coli C. Here we compare the 13.7 kb E.coli C sequence spanning the site of the ICR with corresponding sequences from five E.coli strains and Salmonella typhimurium LT2. To discuss this variation, we adopt the term ‘framework’ to refer to genes that are stable components of genomes within related lineages, while ‘migratory’ genes are transient inhabitants of the genome. Strikingly, seven different migratory DNA segments, encoding different sets of genes and gene fragments, alternatively occupy a single well-defined location in the seven strains examined. The flanking framework genes, yjiS and yjiA, display approximately normal patterns of conservation. The patterns observed are consistent with the action of a site-specific recombinase. Since no nearby gene codes for a likely recombinase of known families, such a recombinase must be of a new family or unlinked.