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.     

Cloning of a 1.1-kb Fragment Including srnB+ Gene in the F Plasmid and Isolation of an srnB Mutant

The srnB⁺ gene, promoting stable RNA degradation at 42 C in the presence of rifampin, was cloned by using pBR322 as a vector; it was located on a 1.1-kilobase (kb) EcoRI/BamHI fragment between 1.4 and 2.5 kb of the F plasmid. The region between 93.3 and 4.0 kb of the F plasmid was physically mapped by using restriction endonucleases EcoRI, HindIII, BamHI, PstI, and SmaI, with reference to a standard HindIII site in IS3. An srnB1 mutant was isolated from a chimeric plasmid, pOY54, after treatment of its DNA with hydroxylamine. The srnB1 allele on the F fragment of the mutant plasmid was recessive to the wild-type allele. Thermal elevation of cell cultures to 39 C was high enough to promote RNA degradation in strain YS12 carrying plasmid pOY54.     

Isolation and characterization of faithful and altered clones of the genomes of cauliflower mosaic virus isolates Cabb B-JI, CM4-184, and Bari I

Full-length genomes of cauliflower mosaic virus (CaMV) isolates Cabb B-JI, CM4-184, and Bari I have been cloned in the SalGI site of plasmid pAT 153. The cloned DNAs were characterized by restriction mapping and infectivity assays. All the sites present in the virion DNAs were found in the cloned DNAs. Comparison of restriction maps with those of DNA from two other isolates which have been recently completely sequenced revealed a close relationship among the different isolates. Some of the clones appear to be faithful copies of the viral genomes and these viral inserts are infectious when inoculated into turnip plants. Various clones with deletions in the CaMV DNA have been isolated and characterized. Some of them may correspond to deletions naturally occurring in a subpopulation of the virus whereas others occurred during cloning. None of the deleted fragments are infectious when inoculated into plants. Strikingly, all the deletions overlap one or two of the specific single-stranded breaks characteristic of caulimoviruses, suggesting that sequences surrounding the breaks are not dispensable.     

The avirulence domain of Cauliflower mosaic virus transactivator/viroplasmin is a determinant of viral virulence in susceptible hosts

Cauliflower mosaic virus (CaMV) transactivator/viroplasmin (Tav) is a multifunctional protein essential for basic replication of CaMV. It also plays a role in viral pathogenesis in crucifer and solanaceous host plants. Deletion mutagenesis revealed that N- and C-terminal parts of Tav are not essential for CaMV replication in transfected protoplasts. Two deletion mutants having only minimal defects in basic replication were infectious in turnips but only with highly attenuated virulence. This was shown to be due to delayed virus spread within the inoculated leaves and to the upper leaves. Unlike the wild-type virus, the mutant viruses successfully spread locally without inducing a host defense response in inoculated Datura stramonium leaves, but did not spread systemically. These results provide the first evidence that a Tav domain required for avirulence function in solanaceous plants is not essential for CaMV infectivity but has a role in viral virulence in susceptible hosts.     

Identification of a Genetic Locus in Pseudomonas aureofaciens Involved in Fungal Inhibition

In iron-rich conditions, Pseudomonas aureofaciens PA147-2 produces an antibiotic-like compound that inhibits the growth of a plant fungal pathogen, Aphanomyces euteiches. To contribute to the potential use of PA147-2 as a biocontrol organism, we report the identification of a genetic locus important for antibiotic biosynthesis. Mutants defective for fungal inhibition (Af^-) were generated by Tn5 mutagenesis. Southern hybridization of total DNAs from three Af^- mutants indicated that loss of fungal inhibition was due to a single Tn5 insertion in each mutant. Restriction mapping of the mutation points showed that in two mutants the Tn5 insertions were in the same 16.0-kb EcoRI fragment and were separated by 2.1 kb. A genomic library of PA147-2 was constructed and screened by using a region of DNA flanking the Tn5 insertion in one mutant (PA109) as a probe to recover complementing cosmids. Three cosmids containing a 16.0-kb EcoRI fragment complementary to the two mutants were recovered. Allele replacement by homologous recombination with putative complementing cosmids restored one mutant to antifungal activity against A. euteiches. Southern analysis of the complemented mutants confirmed that allele replacement had occurred between cosmid DNA and Tn5. The wild-type 16.0-kb EcoRI fragment was cloned from the cosmid and complemented the two mutants to antifungal activity. An antifungal compound was isolated from PA147-2 grown on solid medium. Antifungal activity correlated to a peak on high-pressure liquid chromatography analysis. Under the same growth and extraction conditions, the antifungal activity seen in PA147-2 was absent in two Af^- mutants. Furthermore, absence of an antifungal compound in each mutant correlated to the absence of the wild-type “antifungal” peak on high-pressure liquid chromatography analysis.     

Triplication of a unique genetic segment in a simian virus 40-like virus of human origin and evolution of new viral genomes

The non-defective (heavy) virions from a simian virus 40-like virus (DAR virus) isolated from human brain have been serially passaged at high input multi-plicities in primary monkey kidney cells. The ³²P-labeled, progeny DAR-viral genomes have been purified and tested for sensitivity to the R_I restriction endouclease from Escherichia coli (Eco RI3 restriction nuclease). The parental DAR-viral genomes share many physical properties with “standard” simian virus 40 DNA and are cleaved once by the Eco RI restriction nuclease. After the fourth serial passage, three populations of genomes could be distinguished: Eco RI resistant, Eco RI sensitive (one cleavage site) and Eco RI “supersensitive” (three, symmetrically-located, cleavage sites). The Eco RI cleavage product of the “supersensitive” form is one-third the physical size (10.4 S) of simian virus 40 DNA and reassociates about three times more rapidly than sheared, denatured simian virus 40 DNA. From the fourth to the eighth serial passages, the genomes containing this specific triplication of viral DNA sequences were selected for and became the predominant viral DNA species.     

A biochemical method for increasing the size of deletion mutations in simian virus 40 DNA.

A biochemical procedure has been developed for increasing the size of deletion mutations in closed-circular, double-stranded DNA. Specifically, the deletion in a simian virus 40 (SV40) mutant (dl892), a viable deletion mutant lacking about 35 base-pairs at 0.675 to 0.68 SV40 map units, has been enlarged to produce a series of new mutants lacking from 45 to 90 base-pairs. To enlarge the deletion, the following steps were involved: mutant and wild-type SV40 DNAs were cleaved with the EcoRI restriction endonuclease to form full-length linear molecules, and then they were mixed, denatured and annealed to reform duplex structures. The linear heteroduplex DNAs were re-circularized by treatment with DNA ligase. These closed-circular molecules, half of which contain a small deletion loop at 0.675 to 0.68 map units, were treated with S1 endonuclease, which cleaves at the site of the deletion loops to produce linear molecules with ends at 0.675 to 0.68 map units. Mutants containing enlarged deletions were obtained by infecting permissive monkey kidney cells with the linear DNA. The location of the enlarged deletion in each mutant was compared to that of the parental mutant, dl892. One end of the parental deletion (at about 0.675 map units) remained essentially unmoved; the deletions were enlarged almost entirely in the opposite direction. Since these mutants were all selected for viability, 0.675 map units very likely marks the boundary between a region of the genome previously shown to contain non-essential sequences (from 0.675 to about 0.74 map units) and a portion of the genome required for lytic growth.     

Two different satellite DNAs in Beta vulgaris L.: evolution,quantification and distribution in the genus

Summary Two highly repeated EcoRI (0.45 × 10⁶) and BamHI (0.17 × 10⁶) fragments per haploid genome were found in sugar beet genomic DNA. Both fragments were located by 6% acrylamide-gel electrophoresis, purified and cloned in pUC18. Four of the inserts corresponding to each family were chosen for further study. Both fragment families display the main characteristics of the satellite DNA of animals and plants. The EcoRI and BamHI fragment families are arranged in long tandem arrays. Fragments of the EcoRI family (pBVE) were analyzed. They vary both in sequence and in length (158–160 nt) in comparison with the consensus sequence of 159nt. Both families are A-T rich; pBVE is 59% rich while pBVB is 69% rich. The BVESAT family is present in all the members of the section Vulgares. It is conserved in the section Procumbentes with 80% homology and the same length, but is not detectable in the Corollinae. The sequence variation rate and the variation in length (330±5 nt) are of the same order in comparison with those of the BVESAT family. However, the BVBSAT family is present in species of the section Vulgares only. As regards other plant satellite DNAs, the BVESAT family shares homology with Allium cepa satellite DNA, with three of the yeast centromeric sequences, and with three Arabidopsis thaliana sequences. The BVBSAT family is unique to the Vulgares and does not share any homology with other plant or animal satellite DNAs sequences so far.     

EcoRI analysis of bacteriophage P22 DNA packaging.

Bacteriophage P22 linear DNA molecules are a set of circularly permuted sequences with ends located in a limited region of the physical map. This mature form of the viral chromosome is cut in headful lengths from a concatemeric precursor during DNA encapsulation. Packaging of P22 DNA begins at a specific site, which we have termed pac, and then proceeds sequentially to cut lengths of DNA slightly longer than one complete set of P22 genes (Tye et al., 1974b). The sites of DNA maturation events have been located on the physical map of EcoRI cleavage sites in P22 DNA. EcoRI digestion products of mature P22 wild-type DNA were compared with EcoRI fragments of two deletion and two insertion mutant DNAs. These mutations decrease or increase the length of the genome, but do not alter the DNA encapsulation mechanism. Thus the position of mature molecular ends relative to EcoRI restriction sites is different in each mutant, and comparison of the digests shows which fragments come from the ends of linear molecules. From the positions of the ends of molecules processed in sequential headfuls, the location of pac and the direction of encapsulation relative to the P22 map were deduced. The pac site lies in EcoRI fragment A, 4.1 × 10³ base-pairs from EcoRI cleavage site 1. Sequential packaging of the concatemer is initiated at pac and proceeds in the counterclockwise direction relative to the circular map of P22. One-third of the linears in a population are cut from the concatemer at pac, and most packaging sequences do not extend beyond four headfuls.Fragment D is produced by EcoRI cleavage at a site near the end of a linear chromosome which has been encapsulated starting at pac. The position of the pac site is therefore defined by one end of fragment D. The pac site is not located near genes 12 and 18, the only known site for initiation of P22 DNA replication, but lies among late genes at a position on the physical gene map approximately analogous to the cohesive end site (cos) of bacteriophage λ at which λ DNA is cleaved during encapsulation. Our results suggest that P22 and λ DNA maturation mechanisms have many common properties.     

Replication of cauliflower mosaic virus DNA in leaves and suspension culture protoplasts of cotton

Cauliflower mosaic virus (CaMV) replicated in protoplasts and in inoculated leaves of the non-host, cotton (Gossypium hirsutum, L.). Protoplasts prepared from suspension-cultured cotton cells were infected by incubation with liposome-encapsulated CaMV virions. During a 1-week culture period the amount of CaMV nucleic acid as detected by nucleic acid hybridization in the protoplasts increased significantly regardless of whether or not the protoplasts contained vacuoles. In leaves inoculated with CaMV virions or CaMV DNA, viral DNA sequences were found by leaf skeleton hybridization to be located in small circular areas. DNA extracted from ultracentrifugal pellets of homogenates of inoculated leaves contained circular, gapped CaMV DNA only when inocula contained CaMV virions, CaMV DNA, or partial nested dimer CaMV plasmid DNA. When plants had been heavily watered, the CaMV DNA recovered contained degraded CaMV DNA. The results suggest that the host range limitation for CaMV is not due to an inability to replicate or spread locally in inoculated leaves.     

High-fidelity PCR amplification of infectious copies of the complete simian virus 40 genome from plasmids and virus-infected cell lysates

We describe here a long-polymerase chain reaction (PCR) method that can be used to amplify complete simian virus 40 (SV40) DNA with high fidelity, and we show that authentic, viable virus can be produced from molecular clones of the PCR-amplified viral DNAs. A commercial long-PCR kit that employed a combination of Taq and GB-D polymerases was used, together with a pair of overlapping primers that recognized a unique EcoRI site in the SV40 genome. Efficient amplification required linearization of the circular SV40 genomic DNAs with EcoRI. Entire SV40 genomes were successfully PCR-amplified from an SV40 plasmid and from two different SV40-infected cell lysates and were cloned into pUC-19. Three separate segments of the cloned viral genomes were DNA sequenced, and no nucleotide changes relative to the parental virus were detected, suggesting that the viral DNAs had been amplified with high fidelity. Each PCR clone was infectious, and no differences were detected in the growth characteristics of viruses derived from these clones as compared to the original viral strain. The procedure we utilized shortens and simplifies the molecular cloning of small double-stranded DNA viruses and will be useful for viral diagnostic tests and for recovery of virus from clinical samples. The results of these experiments have broad implications, as the methodology is applicable to many systems.     

Restriction endonuclease analysis of DNA from the Streptomyces phages SH3, SH5, SH10 and SH13

Using 26 restriction endonucleases, a cleavage site survey was undertaken for DNAs of several unrelated Streptomyces phages SH3, SH5, SH10 and SH13. Only EcoRI was found to produce single cleavage in SH3 and SH10 DNA. The complete maps were prepared for the 2, 9 and 11 fragments of SH10 DNA, as generated by EcoRI, KpnI and BglII, respectively. The evidence is presented that SH10 DNA contains cohesive ends. Moreover, a clearplaque mutant of SH10 was shown to contain a deletion of 790 bp in the right part of the genome, including two KpnI sites.     

Construction of a recombinant plasmid composed of B. subtilis leucine genes and a B. subtilis (natto) plasmid: its use as cloning vehicle in B. subtilis 168.

Summary Recombinant plasmids composed of Bacillus subtilis 168 leucine genes and a B. subtilis (natto) plasmid have been constructed in a recombination deficient (recE4) mutant of Bacillus subtilis 168. The process involved EcoRI fragmentation and ligation of a B. subtilis (natto) plasmid and a composite plasmid RSF2124-B · leu in which B. subtilis 168 leucine genes are linked to the R-factor RSF2124. A constructed plasmid (pLS102) was found to be composed of an EcoRI fragment derived from the vector plasmid and two tandemly repeated EcoRI fragments carrying the leucine genes. A derivative plasmid (pLS101 or pLS103) consisting of one molecule each of the EcoRI fragments was obtained by in vivo intramolecular recombination between the repeated leucine gene fragments in pLS102. pLS103 was cleaved once with BamNI, SmaI and HpaI. Insertion of foreign DNA (Escherichia coli plasmid pBR322) into the BamNI site inactivated leuA but not the leuC function which thus can serve as selective marker if the plasmid is used as vector in molecular cloning. The penicillin resistance carried in pBR322 was not functionally expressed in B. subtilis cells. By partial digestion of pLS103 with HindIII followed by ligation with T4-induced ligase, pLS107 was obtained which contained only one EcoRI site. However, insertion of exogenous DNA (pBR322) into this EcoRI site inactivated both leuA and leuC functions.     

Replication intermediate of a hybrid plasmid carrying the replication terminus (ter) site of R6K as revealed by agarose gel electrophoresis

Summary A 4.32 kb DNA fragment, on which the DNA replication terminus (terR) site of plasmid R 6K was located, was inserted into the unique EcoRI site of plasmid pUC9. To detect replication intermediate molecules with a replication fork halted at the terR site, a cell DNA extract was digested with EcoRI, electrophoresed through an agarose gel and stained with ethidium bromide. In addition to two major bands, one derived from vector DNA and the other from the ter insert fragment, two extra minor bands were detected. Following DNA-DNA hybridization and electron microscopic observation we concluded that the two minor bands corresponded to the two Y-shaped molecules, produced from the -shaped intermediate molecules by EcoRI digestion.Abbreviations Ap ampicillin - kb kilobase pair(s) - EtBr ethidium bromide     

Highly transformable mutants of Streptomyces fradiae defective in several restriction systems

Summary Streptomyces fradiae JS85 is a mutant defective in tylosin production and an efficient recipient for conjugal transfer of tylosin genes. JS85 was mutagenized with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and derivatives defective in restriction were isolated by sequential selection for increased transformability by several plasmid DNAs. From the number of mutation and selection cycles required to eliminate most restriction, it was estimated that wild type S. fradiae expressed at least five restriction systems. From the patterns of restriction enzyme digestion of chromosomal DNA observed in the series of mutants that became progressively less restricting, it was suggested that wild type S. fradiae normally expresses modification (and presumably restriction) systems similar or analogous to PstI, XhoI, ScaI and EcoRI. The least restricting mutant of S. fradiae was readily transformable by many plasmids, including a bifunctional cosmid vector containing a large insert of Streptomyces DNA.     

Structural analysis of EcoRI-DNA complexes as revealed by electron microscopy

Electron microscopy of negatively stained isolated restriction enzyme EcoRI revealed particle projections with triangular or square outlines, indicating that the enzyme, in its tetrameric state, is tetrahedron-like. The two dimers making up the tetramer appear to be arranged in two planes orthogonal to each other. Complexes formed by EcoRI with the plasmids pBR322 or pGW10 were investigated by electron microscopic spreading techniques. In the presence of Mg²⁺, EcoRI was bound to the DNA molecules to form pearl necklace-like aggregates. The number of bound EcoRI particles was much higher as the sum of EcoRI-and 5..AATT..3 sites (with exceptions, the 5..AATT..3 sites may function as one type of EcoRI^* sites) along the DNAs, indicating unspecific binding. In the absence of Mg²⁺, EcoRI was bound to the DNA only at the recognition site for EcoRI and the sites where the tetranucleotide sequence 5..AATT..3 was present. A direct correlation of the local concentrations of the bases A and T within the flanking sequences of the binding sites with the frequency of EcoRI to the DNA was observed. Dimers and tetramers of the enzyme was found to bind to the DNA. Tetramers occasionally exhibited two binding sites for DNA as indicated by the observation of DNA loops originating at the sites of bound tetrameric EcoRI particles.Abbreviations BAC Benzyldimethylalkylammoniumchloride - bp base pairs - Kb kilobases - SDS sodium dodecylsulfate Enzymes (EC 3.1.23.13) Restrictionendonuclease EcoRI - (EC 3.1.23.21) Restrictionendonuclease HindIII - (EC 3.1.23.37) Restrictionendonuclease SalGI Dedicated Professor H. G. Schlegel on occasion of this 60th birthday     

An <Emphasis Type="Italic">Rhs</Emphasis>-like genetic element is involved in bacteriocin production by <Emphasis Type="Italic">Pseudomonas savastanoi</Emphasis> pv. <Emphasis Type="Italic">savastanoi</Emphasis>

The main aim of this work was the identification of genetic determinants involved in bacteriocin production by strain ITM317 of Pseudomonas savastanoi pv. savastanoi, besides bacteriocin characterization. The bacteriocin was observed to be a heat-sensitive, high molecular weight proteinaceous compound. We identified a transposon (Tn5)-induced mutant which had lost its ability to produce the bacteriocin. The Tn5 insertion’s responsibility for the above mutated phenotype was demonstrated by marker-exchange mutagenesis. An EcoRI DNA fragment, corresponding to the EcoRI Tn5-containing fragment of the mutant, was also cloned from the wild-type strain, and its introduction into the mutant complemented the mutation. Moreover, that fragment enabled bacteriocin production by P. s. pv. savastanoi ITM302, a strain not previously capable of doing so. DNA sequence analysis revealed that Tn5 insertion occurred in the mutant within a large ORF encoding a protein which showed similarity with proteins from the Rhs family. The DNA region including that ORF showed features which have been considered typical of the Rhs genetic elements previously identified in other bacteria but whose function is as yet unclear. The results of this study for the first time identify an Rhs-like element in P. s. pv. savastanoi, and for the first time indicate that an Rhs element is involved in bacteriocin production, also suggesting this possible function for Rhs genetic elements previously characterized in other bacteria.     

The cauliflower mosaic virus virion-associated protein is dispensable for viral replication in single cells

Cauliflower mosaic virus (CaMV) open reading frame III (ORF III) codes for a virion-associated protein (Vap), which is one of two viral proteins essential for aphid transmission. However, unlike the aphid transmission factor encoded by CaMV ORF II, Vap is also essential for systemic infection, suggesting that it is a multifunctional protein. To elucidate the additional function or functions of Vap, we tested the replication of noninfectious ORF III-defective mutants in transfected turnip protoplasts. PCR and Western blot analyses revealed that CaMV replication had occurred with an efficiency similar to that of wild-type virus and without leading to reversions. Electron microscopic examination revealed that an ORF III frameshift mutant formed normally structured virions. These results demonstrate that Vap is dispensable for replication in single cells and is not essential for virion morphogenesis. Analysis of inoculated turnip leaves showed that the ORF III frameshift mutant does not cause any detectable local infection. These results are strongly indicative of a role for Vap in virus movement.     

A molecular description of mutations affecting the pollen component of the Nicotiana alata S locus.

Mutations affecting the self-incompatibility response of Nicotiana alata were generated by irradiation. Mutants in the M1 generation were selected on the basis of pollen tube growth through an otherwise incompatible pistil. Twelve of the 18 M1 plants obtained from the mutagenesis screen were self-compatible. Eleven self-compatible plants had mutations affecting only the pollen function of the S locus (pollen-part mutants). The remaining self-compatible plant had a mutation affecting only the style function of the S locus (style-part mutant). Cytological examination of the pollen-part mutant plants revealed that 8 had an extra chromosome (2n + 1) and 3 did not. The pollen-part mutation in 7 M1 plants was followed in a series of crosses. DNA blot analysis using probes for S-RNase genes (encoding the style function of the S locus) indicated that the pollen-part mutation was associated with an extra S allele in 4 M1 plants. In 3 of these plants, the extra S allele was located on the additional chromosome. There was no evidence of an extra S allele in the 3 remaining M1 plants. The breakdown of self-incompatibility in plants with an extra S allele is discussed with reference to current models of the molecular basis of self-incompatibility.