Transferable Streptomyces DNA amplification and coamplification of foreign DNA sequences.

The 8.8-kb amplifiable unit of DNA of Streptomyces achromogenes subsp. rubradiris, AUD-Sar 1, which carries 0.8-kb terminal direct repeats and a spectinomycin resistance determinant, can mediate high-level amplification of an AUD-Sar 1-derived 8.0-kb DNA sequence not only in S. achromogenes but also in the heterologous host Streptomyces lividans. This was seen upon introduction of AUD-Sar 1 into chloramphenicol-sensitive strains of S. lividans via the temperature-sensitive (39 degrees C) plasmid pMT660, which contains the thiostrepton resistance gene tsr. Following the cultivation of transformants at 39 degrees C on media containing spectinomycin, a number of strains which were unable to grow on thiostrepton and which carried the amplified 8.0-kb DNA sequence as arrays of 200 to 300 copies of tandem 8.0-kb repeats were found. Chloramphenicol-resistant strains of S. lividans did not yield amplified sequences under similar conditions. Studies with plasmids carrying inserted antibiotic resistance genes at two sites of AUD-Sar 1 yielded coamplified sequences which contain the inserted DNA. Transformation with a plasmid carrying a 1.0-kb deletion in AUD-Sar 1 followed by growth under similar conditions yielded a 7.0-kb repeated DNA sequence. Southern analysis revealed the absence of vector sequences located on the right side of AUD-Sar 1 in the input plasmids in all examined DNA samples of amplified strains. In contrast, a majority of the samples revealed the presence at unit copy level of AUD-Sar 1 left-adjacent sequences which are part of the input plasmids and in several samples the presence of certain vector sequences located near them. The results suggest input plasmid integration into the S. lividans chromosome prior to the generation of the amplified sequences and the deletion of AUD-Sar 1 adjacent sequences.     

Spectinomycin resistance and associated DNA amplification in Streptomyces achromogenes subsp. rubradiris.

Streptomyces achromogenes subsp. rubradiris plated at low density on 1,000 micrograms of spectinomycin per ml initially produces slow-growing, bald colonies from which arise, in a spatially and temporally random fashion, foci of rapidly growing aerial mycelium-forming cells whose DNA contains an approximately 200- to 300-fold amplification of an 8-kilobase (kb) sequence. This sequence was cloned in Escherichia coli on pBR322 and physically characterized. It was separately cloned also in Streptomyces lividans as a BglII fragment and shown to impart high-level resistance to spectinomycin in an orientation-independent manner when present in either the high-copy-number vector pIJ702 or the unit-copy-number vector pIJ943. A spectinomycin resistance determinant was shown to reside on a 1.7-kb SphI-BglII subfragment. Analysis of Southern blots of restriction enzyme digests of wild-type S. achromogenes DNA probed with the labeled 8-kb DNA sequence resulted in the identification and subsequent cloning in S. lividans of a 10.4-kb BamHI fragment which probably includes the complete 8.8-kb amplifiable unit of DNA. This unit is present in wild-type S. achromogenes and in the initially slow-growing, bald colonies arising on 1,000 micrograms of spectinomycin per ml as a single copy. It carries two 0.8-kb direct repeats at its termini as well as the spectinomycin resistance determinant close to one of these termini. About 5% of protoplast regenerants from wild-type S. achromogenes and 77% of protoplast regenerants from the rapidly growing strains lost both the ability to grow on spectinomycin at 10 micrograms/ml and the sequences that hybridize with the 8-kb probe DNA. The 1.7-kb Bg/II-SphI resistance fragment, when introduced via the vector pIJ702 into an S. achromogenes strain sensitive to 10 microgram of spectinomycin per ml, permitted its vigorous growth on 1,000 micrograms of the antibiotic per ml.     

Deamplification and deletion of amplified DNA in Streptomyces lividans and S. fradiae

Summary Streptomyces lividans arginine auxotrophs which show amplification of a 5.7-kb DNA sequence, arose at a very high frequency, varying from 10% to 25% of Cml^s spores. The amplifiable DNA sequence was shown to be stable over many generations. However, treatment of Cml^s arg mutants with subinhibitory concentrations of antibiotics such as spectinomycin, streptomycin, chloramphenicol, thiostrepton and kanamycin, either during sporulation or during vegetative growth of mycelia, led to the deletion of the entire amplified DNA sequence, including the left and right junction sequences. Depending upon the method of antibiotic treatment a reduction in the copy number of the amplified DNA was also observed. This reduction in copy number apparently occurred without drastically affecting the basic structure of the amplifiable unit of DNA. This phenomenon appears to be universal since deamplification and deletion were observed also in S. fradiae. Further, spontaneous arg mutants arose at much lower frequency from spectinomycin-pretreated Cml^s cells compared to untreated cells. These arg mutants isolated in the presence of spectinomycin did not show amplification of the 5.7-kb sequence. Southern blot analysis using the 5.7-kb probe showed that the entire DNA sequence homologous to the amplifiable DNA sequence had been deleted. Offprint requests to: K. Dharmalingam     

Characterization of endogenous and exogenous mouse mammary tumor virus proviral DNA with site-specific molecular clones.

Restriction fragments of the mouse mammary tumor virus (MMTV) proviral DNA were obtained by molecular cloning procedures. A 4-kilobase fragment delimited by two PstI sites was isolated from unintegrated, linear MMTV DNA and amplified in the pBr322 plasmid vector. EcoRI fragments of proviral DNA, integrated into the genome of a GR mammary tumor cell line, were isolated as lambda recombinant molecules. Five different recombinant phages which contained the 3' region of the MMTV proviral DNA and adjacent host DNA sequences were isolated. Heteroduplex analysis and S1 nuclease digestion suggested that there is no extensive sequence homology in the host DNA flanking the different proviral genes. The cloned DNA was fractionated into site-specific restriction fragments which served as molecular probes in the analysis of the endogenous MMTV proviral copies of C3H, GR, BALB/c, and feral mice. This allowed the correlation of MMTV-specific EcoRI fragments obtained from genomic DNA of these strains with the 5' and 3' ends of the proviral gene. Restriction fragments of two clones which contained the proviral sequences adjacent to the flanking host DNA as well as 1 to 2 kilobases of host DNA were used as hybridization probes, and the results allow the following conclusions: the proviral DNA of both clones contains nucleotide sequences complementary to the 5' and 3' ends of proviral DNA; and the host DNA flanking one clone belongs to the unique class of genomic DNA, whereas the DNA flanking the second clone is reiterated at least 15 times within the mouse genome.     

Concerted deletions and inversions are caused by mitotic recombination between delta sequences in Saccharomyces cerevisiae.

Deletions of a tyrosine tRNA suppressor gene, SUP4-o, are mediated by recombination between short repeated delta sequences in Saccharomyces cerevisiae. The arrangement of the five solo delta sequences that surround the SUP4 locus was established by DNA sequence analysis. Seven deletion classes were identified by genomic blotting. DNA sequence analysis also showed that the delta sequences within a 6.5-kilobase region of the SUP4 locus were the endpoints of these events. In three of these classes, an adjacent interval surrounded by delta sequences was inverted in concert with the deletion. The frequency of all deletion classes decreased in strains that contained mutations in the recombination and repair gene RAD52. We present two gene conversion mechanisms by which these rearrangements could have been generated. These models may also explain deletions between repeated sequences in other systems.     

Transformation and transfection of anthracycline-producing streptomycetes.

Streptomyces peucetius and Streptomyces strain C5, producers or anthracycline antibiotics, were converted to protoplasts from vegetatively growing mycelia. Conditions are described for maximal protoplast formation (greater than 99%) and for regeneration frequencies of up to 13%. Streptomycete plasmids pIJ61, pIJ702, and pIJ922, from the replicons SLP1, pIJ101, and SCP2, respectively, were isolated from Streptomyces lividans 66 and successfully introduced into S. peucetius and Streptomyces strain C5 by polyethylene glycol-mediated protoplast transformation. Frequencies of up to 10(6) transformations X microgram of plasmid DNA-1 were achieved by these procedures. Analyses showed that the two anthracycline-producing strains can stably harbor the plasmids without deletion of plasmid sequences or loss of the plasmids for several transfers through selective media. Fragments of DNA from S. peucetius ligated into pIJ702 and introduced into Streptomyces strain C5 were stable after several transfers through selective media. Both anthracycline producers also were sensitive to infection and transfection by actinophages KC401 and KC515, clear plaque derivatives of bacteriophage phi C31. Optimal conditions were determined for the transfection of S. peucetius and Streptomyces strain C5 protoplasts with phi C31 KC401 and KC515 DNA with liposome-assisted, polyethylene glycol-mediated protoplast transfection.     

Aerobactin genes in clinical isolates of Escherichia coli.

The location of the aerobactin gene complex on either the chromosome or plasmid was determined in eight aerobactin-positive clinical isolates of Escherichia coli by Southern hybridization analysis, using as probes the cloned aerobactin genes from the ColV-K30 plasmid. The aerobactin genes were in two cases detected on large plasmids, whereas in the other strains the aerobactin genes are most likely located on the chromosome. Restriction mapping revealed only slight variations in the structural genes and an at least 3.4-kilobase-long upstream region conserved in all three plasmid-coded systems. A 7.7-kilobase HindIII fragment upstream and adjacent to the 16.3-kilobase HindIII fragment carrying the complete aerobactin system was cloned from the ColV-K30 plasmid. Fine-structure restriction mapping identified the left insertion sequence in the upstream region as IS1, in inverted orientation to the IS1 element downstream from the aerobactin operon. The upstream and downstream sequences of IS1 appear to have perfect homology, as indicated by S1 nuclease resistance of a 760-base-pair DNA duplex formed by both IS1 elements.     

Occurence of reiterated DNA sequences in strains of Streptomyces produced by an interspecific protoplast fusion

Summary DNA isolated from a Streptomyces strain produced by interspecific protoplast fusion of Streptomyces jumonjinensis and Streptomyces lipmanii contained a tandemly repeated six kilobase pair sequence which constituted 15%–18% of the total DNA. Examination of other strains produced by similar fusions showed that they also contained reiterated DNA sequences.     

Amplification and deletion of the amyE+-tmrB+ gene region in a Bacillus subtilis recombinant-phage genome by the tmrA7 mutation.

A 22.4-kilobase DNA fragment containing the tmrA7-amyR2-amyE+-tmrB+-aroI+ region of the Bacillus subtilis N7 chromosomal DNA was cloned into a recombinant B. subtilis bacteriophage, p11-AA248. The amyE+-tmrB+ gene region, approximately 12.6 kilobases, in the phage genome was amplified in a tunicamycin-resistant (Tmr) Amy+ AroI+ transductant of B. subtilis by p11-AA248. On the other hand, the amyE+-tmrB+ region in the genomes of 80 to 90% of the phage particles was deleted when the phages were induced from the Tmr Amy+ AroI+ transductants by treatment with 1.0 micrograms of mitomycin C per ml. From analyses of the physical maps and DNA nucleotide sequences in the junction region of the deleted phage genome and the parental DNA fragments, it is suggested that the deletion occurred within a direct repeat sequence composed of 18 base pairs. The endpoints of the amplified gene region seemed to be closely related to both terminal regions of the deleted DNA.     

Organisation of nodulation and nitrogen fixation genes on a Rhizobium trifolii symbiotic plasmid

A Rhizobium trifolii symbiotic plasmid specific gene library was constructed and the physical organisation of regions homologous to nifHDK, nifA and nod genes was determined. These symbiotic gene regions were localised to u 25 kb region on the sym-plasmid, pPN1. In addition four copies of a reiterated sequence were identified on this plasmid, with one copy adjacent to nifH. No rearrangement of these reiterated sequences was observed between R. trifolii bacterial and bacteroid DNA. Analysis of a deletion derivative of pPN1 showed that these sequences were spread over a 110 kb region to the left of nifA.     

Effect of a recA mutation on cholera toxin gene amplification and deletion events.

The cholera toxin operon (ctxAB) is located on a 7-kilobase pair variable genetic element which undergoes genetic duplication and amplification events in Vibrio cholerae. Amplification of the ctx genetic element was investigated by substituting the resident ctx loci of two V. cholerae strains with a DNA fragment encoding resistance to kanamycin. Although these strains were not normally resistant to greater than 150 micrograms of kanamycin per ml, spontaneous derivatives could be obtained that grew well on 3 mg of kanamycin per ml. Southern blot analysis of these highly resistant isolates demonstrated that the ctx element was amplified approximately 20-fold. This amplification process was completely inhibited in the absence of a functional recA gene. The V. cholerae RecA protein, therefore, is essential for cholera toxin gene amplification. Spontaneous deletions of the ctx structural genes were observed in both recA+ and recA- V. cholerae strains, although such deletions occurred at a 21-fold-lower frequency in the latter case. Structural analysis of these ctx amplification and deletion events supports a model for their formation that involves unequal crossing over between repetitive sequences located upstream and downstream of the ctx operon.     

Cloning and characterization of LOS1, a Saccharomyces cerevisiae gene that affects tRNA splicing.

Saccharomyces cerevisiae strains carrying los1-1 mutations are defective in tRNA processing; at 37 degrees C, such strains accumulate tRNA precursors which have mature 5' and 3' ends but contain intervening sequences. Strains bearing los1-1 and an intron-containing ochre-suppressing tRNA gene, SUP4(0), also fail to suppress the ochre mutations ade2-1(0) and can1-100(0) at 34 degrees C. To understand the role of the LOS1 product in tRNA splicing, we initiated a molecular study of the LOS1 gene. Two plasmids, YEpLOS1 and YCpLOS1, that complement the los1-1 phenotype were isolated from the YEp24 and YCp50 libraries, respectively. YEpLOS1 and YCpLOS1 had overlapping restriction maps, indicating that the DNA in the overlapping segment could complement los1-1 when present in multiple or single copy. Integration of plasmid DNA at the LOS1 locus confirmed that these clones contained authentic LOS1 sequences. Southern analyses showed that LOS1 is a single copy gene. The locations of the LOS1 gene within YEpLOS1 and YCpLOS1 were determined by deletion and gamma-delta mapping. Two genomic disruptions of the LOS1 gene were constructed, i.e., an insertion of a 1.2-kilobase fragment carrying the yeast URA3 gene, los1::URA3, and a 2.4-kilobase deletion from the LOS1 gene, los1-delta V. Disruption or deletion of most of the LOS1 gene was not lethal; cells carrying the disrupted los1 alleles were viable and had phenotypes similar to those of cells carrying the los1-1 allele. Thus, it appears that the los1 gene product expedites tRNA splicing at elevated temperatures but is not essential for this process.     

Certain chromosomal regions in Streptomyces glaucescens tend to carry amplifications and deletions

Summary Streptomycetes are subject to a high degree of genetic instability. One manifestation of this phenomenon is the occurrence of tandemly reiterated DNA stretches within the chromosome. We describe the analysis of ten reiterated sequences observed in various ethidium bromide-treated streptomycin-sensitive and melanin-negative mutant strains of Streptomyces glaucescens. The repeated DNA units were 2.9 to 35 kb in lenght. No two sequences were identical. The amplified sequences occupied up to 45% of the total genomic DNA. Structural analysis of the cloned repeated DNA stretches by means of restriction enzymes and by cross hybridization revealed the presence of two chromosomal areas rich in DNA reiterations. In some cases reiterated regions were accompanied by nearby rearrangements.     

Cloning of the gene for the surface array protein of Aeromonas salmonicida and evidence linking loss of expression with genetic deletion.

A gene bank of DNA from the fish pathogenic bacterium Aeromonas salmonicida was constructed in the bacteriophage lambda gt11. Phage lambda gt11/10G, a recombinant carrying a 4.0-kilobase fragment of A. salmonicida DNA, was found to express the surface array protein (A protein) in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the protein expressed from the cloned gene had a subunit molecular weight of 49,000, which was identical to that of subunits in the native assembled A layer. Genomic Southern analysis showed that the gene coding for this predominant cellular protein was in a single copy on the chromosome and was conserved among a wide range of A. salmonicida strains with different phenotypic characteristics and isolated from diverse geographic locations, fish species, and means of pathogenesis. Results of genomic blotting experiments also showed that loss of expression of the A layer resulting from growth at 30 degrees C was accompanied by genetic rearrangement in which N-terminal sequences of the gene for A protein were lost by deletion.     

Transformation and transfection of anthracycline-producing streptomycetes

Streptomyces peucetius and Streptomyces strain C5, producers or anthracycline antibiotics, were converted to protoplasts from vegetatively growing mycelia. Conditions are described for maximal protoplast formation (greater than 99%) and for regeneration frequencies of up to 13%. Streptomycete plasmids pIJ61, pIJ702, and pIJ922, from the replicons SLP1, pIJ101, and SCP2, respectively, were isolated from Streptomyces lividans 66 and successfully introduced into S. peucetius and Streptomyces strain C5 by polyethylene glycol-mediated protoplast transformation. Frequencies of up to 10(6) transformations X microgram of plasmid DNA-1 were achieved by these procedures. Analyses showed that the two anthracycline-producing strains can stably harbor the plasmids without deletion of plasmid sequences or loss of the plasmids for several transfers through selective media. Fragments of DNA from S. peucetius ligated into pIJ702 and introduced into Streptomyces strain C5 were stable after several transfers through selective media. Both anthracycline producers also were sensitive to infection and transfection by actinophages KC401 and KC515, clear plaque derivatives of bacteriophage phi C31. Optimal conditions were determined for the transfection of S. peucetius and Streptomyces strain C5 protoplasts with phi C31 KC401 and KC515 DNA with liposome-assisted, polyethylene glycol-mediated protoplast transfection.     

Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA

Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA.     

Isolation and characterization of a human locus homologous to the transforming gene (v-fes) of feline sarcoma virus

A single locus (designated c-fes) in the human genome which exhibits homology to the transformation-specific onc gene (v-fes) of Snyder-Theilen feline sarcoma virus was identified by the Southern blot technique. Recombinant clones containing 16- to 18-kilobase inserts of human DNA including the c-fes locus were constructed. Restriction endonuclease mapping of these clones verified their identity with native human c-fes and demonstrated the presence of at least two sequences in human c-fes interrupting v-fes-homologous regions. The v-fes-homologous locus in the human genome spans about 4 kilobases. The 5'-3' orientation of the c-fes clones with respect to feline sarcoma virus proviral DNA was determined. The region of the human genome that is homologous to v-fes is proximal to the highly reiterated human Alu sequence but not to the highly reiterated human alphoid sequence.     

A new symbiotic cluster on the pSym megaplasmid of Rhizobium meliloti 2011 carries a functional fix gene repeat and a nod locus.

A 290-kilobase (kb) region of the Rhizobium meliloti 2011 pSym megaplasmid, which contains nodulation genes (nod) as well as genes involved in nitrogen fixation (nif and fix), was shown to carry at least six sequences repeated elsewhere in the genome. One of these reiterated sequences, about 5 kb in size, had previously been identified as part of a cluster of fix genes located 220 kb downstream of the nifHDK promoter. Deletion of the reiterated part of this fix cluster does not alter the symbiotic phenotype. Deletion of the second copy of this reiterated sequence, which maps on pSym 40 kb upstream of the nifHDK promoter, also has no effect. Deletion of both of these copies however leads to a Fix- phenotype, indicating that both sequences carry functionally reiterated fix gene(s). The fix copy 40 kb upstream of nifHDK is part of a symbiotic cluster which also carries a nod locus, the deletion of which produces a marked delay in nodulation.     

DNA sequence homology between the terminal inverted repeats of Shope fibroma virus and an endogenous cellular plasmid species.

DNA hybridization experiments indicate that the genome of a tumorigenic poxvirus. Shope fibroma virus (SFV), possesses sequence homology with DNA isolated from uninfected rabbit cells. Southern blotting experiments, either with high-complexity rabbit DNA as probe and SFV restriction fragments as targets or with high-specific activity, 32P-labeled, cloned SFV sequences as probes and rabbit DNA as target, indicate that the homologous sequences map at two locations within the viral genome, one in each copy of the terminal inverted repeat sequences. Unexpectedly, Southern blots revealed that the homologous host sequences reside in a rabbit extrachromosomal DNA element. This autonomous low-molecular-weight DNA species could be specifically amplified by cycloheximide treatment and was shown by isopycnic centrifugation in cesium chloride-ethidium bromide to consist predominantly of covalently closed circular DNA molecules. DNA sequencing of pSIC-9, a cloned 1.9-kilobase fragment of the rabbit plasmid species, indicated extensive homology at the nucleotide level over a 1.5-kilobase stretch of the viral terminal inverted repeat. Analysis of open reading frames in both the plasmid and SFV DNA revealed that (i) the N-terminal 157-amino acid sequence of a potential 514-amino acid SFV polypeptide is identical to the N-terminal 157 amino acids of one pSIC-9 open reading frame, and (ii) a second long pSIC-9 open reading frame of 361 amino acids, although significantly diverged from the comparable nucleotide sequence in the virus, possessed considerable homology to a family of cellular protease inhibitors, including alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III. The potential role of such cellular plasmid-like DNA species as a mediator in the exchange of genetic information between the host cell and a cytoplasmically replicating poxvirus is discussed.     

A male-specific DNA probe detects heterochromatin sequences in a familial Yq- chromosome.

Using a recombinant DNA probe, we have demonstrated the presence of residual 3.4-kilobase (kb) repeat sequences in a family with a Yq- chromosome. The heterochromatin of this Y variant was not readily detectable with conventional chromosome-banding techniques. These data suggest that the breakpoint of the deletion occurs at the heterochromatin region proximal to the euchromatin/heterochromatin junction.