Study of the structure of amplifying sequence of Streptomyces antibioticus

A low productive laboratory strain of S. antibioticus and a strain with an increased productivity of oleandomycin derived from it were studied comparatively with using restriction analysis and blotting hybridization. Amplification, site specific integration and segregation of the DNA sequence 32.0 kb in size were detected in the strains. The chromosomes of the laboratory strain contained one copy of the amplifying sequence AUD. After uniting of the end sequences AUD appeared to be capable of segregating from the chromosomes and its one copy per five genomes was present in the form of an extrachromosomal genetic element eSA1. The genome of the strain with increased productivity of oleandomycin contained in its chromosomes sequence ADS-Sa1 amplified to 150 copies and the eSA1 extrachromosomal genetic element in the form of mono-, di- and trimeric structures in the quantity of approximately one copy per genome. The BamHIB fragment of the eSA1 DNA 4 kb in size was identified. The fragment was able to participate in segregation or integration of eSA1 from or into the chromosomes since its subfragments were flanking AUD and ADS-SA1 in the chromosomes. The BamHIB fragment was hybridizing with a number of fragments of the chromosomal DNA of S. antibioticus, S. erythraeus. S. lividans and other strains of streptomycetes. It probably contained an IS-like element or a dispersed genetic element of another class. The DNA sequence of the eSA1 genetic element contained regions homologous to the sequence of the Erm E gene in S. erythraeus NRRL 2338.     

Amplified DNA in Streptomyces fradiae.

A spontaneous mutant of Streptomyces fradiae contained an amplifiable unit of DNA with a sequence length of approximately 10.5 kilobases that was amplified to approximately 500 copies per chromosome. The amplified DNA appears to be cryptic. SalI fragments of the amplified DNA were cloned into Escherichia coli to construct a restriction map and characterize the amplified DNA. The amplified DNA contained tandem repeats of the amplifiable unit of DNA. The unit had an average base composition of 71% guanine plus cytosine, similar to the chromosomal DNA of Streptomyces species. At least a portion of the amplifiable unit of DNA was present at a low copy number in the wild-type strain. The phenotype of amplified DNA was designated Ads1SF for amplified DNA sequence 1 in S. fradiae.     

Isolation of a repeated DNA sequence from Bordetella pertussis

A repeated DNA sequence in the genome of Bordetella pertussis has been demonstrated. At least 20 copies of this sequence could be observed in either BamHI or EcoRI restriction enzyme digests of chromosomal DNA; fragments carrying the repeated DNA sequence ranged in size from about 1.5 to 20 kbp. The repeated DNA sequence was cloned from two separate regions of the B. pertussis genome, as shown by restriction enzyme site maps of the two clones and by hybridization studies. A small number of differences in the pattern of hybridization of the repeated DNA sequence to chromosomal DNA from several strains of B. pertussis was observed. No repeated DNA sequences were observed in one strain each of B. parapertussis and B. bronchiseptica, and there was no hybridization of B. pertussis DNA to Escherichia coli chromosomal DNA. The repeated DNA sequence was subcloned on a 2.54 kbp BamHI fragment from one of the two original clones. Restriction enzyme digests and hybridization studies showed that the repeated DNA sequence was about 1 kbp in size and had a single, internal ClaI site.     

The very large amplifiable element AUD2 from Streptomyces lividans 66 has insertion sequence-like repeats at its ends.

In a spontaneous, chloramphenicol-sensitive (Cms), arginine-auxotrophic (Arg-) mutant of Streptomyces lividans 1326, two amplified DNA sequences were found. One of them was the well-characterized 5.7-kb ADS1 sequence, amplified to about 300 copies per chromosome. The second one was a 92-kb sequence called ADS2. ADS2 encoding the previously isolated mercury resistance genes of S. lividans was amplified to around 20 copies per chromosome. The complete ADS2 sequence was isolated from a genomic library of the mutant S. lividans 1326.32, constructed in the phage vector lambda EMBL4. In addition, the DNA sequences flanking the corresponding amplifiable element called AUD2 in the wild-type strain were isolated by using another genomic library prepared from S. lividans 1326 DNA. Analysis of the ends of AUD2 revealed the presence of an 846-bp sequence on both sides repeated in the same orientation. Each of the direct repeats ended with 18-bp inverted repeated sequences. This insertion sequence-like structure was confirmed by the DNA sequence determined from the amplified copy of the direct repeats which demonstrated a high degree of similarity of 65% identity in nucleic acid sequence to IS112 from Streptomyces albus. The recombination event leading to the amplification of AUD2 occurred within these direct repeats, as shown by DNA sequence analysis. The amplification of AUD2 was correlated with a deletion on one side of the flanking chromosomal region beginning very near or in the amplified DNA. Strains of S. lividans like TK20 and TK21 which are mercury sensitive have completely lost AUD2 together with flanking chromosomal DNA on one or both sides.     

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.     

Rapid Evolution of Recombinant Saccharomyces cerevisiae for Xylose Fermentation through Formation of Extra-chromosomal Circular DNA

Circular DNA elements are involved in genome plasticity, particularly of tandem repeats. However, amplifications of DNA segments in Saccharomyces cerevisiae reported so far involve pre-existing repetitive sequences such as ribosomal DNA, Ty elements and Long Terminal Repeats (LTRs). Here, we report the generation of an eccDNA, (extrachromosomal circular DNA element) in a region without any repetitive sequences during an adaptive evolution experiment. We performed whole genome sequence comparison between an efficient D-xylose fermenting yeast strain developed by metabolic and evolutionary engineering, and its parent industrial strain. We found that the heterologous gene XylA that had been inserted close to an ARS sequence in the parent strain has been amplified about 9 fold in both alleles of the chromosomal locus of the evolved strain compared to its parent. Analysis of the amplification process during the adaptive evolution revealed formation of a XylA-carrying eccDNA, pXI2-6, followed by chromosomal integration in tandem arrays over the course of the evolutionary adaptation. Formation of the eccDNA occurred in the absence of any repetitive DNA elements, probably using a micro-homology sequence of 8 nucleotides flanking the amplified sequence. We isolated the pXI2-6 eccDNA from an intermediate strain of the evolutionary adaptation process, sequenced it completely and showed that it confers high xylose fermentation capacity when it is transferred to a new strain. In this way, we have provided clear evidence that gene amplification can occur through generation of eccDNA without the presence of flanking repetitive sequences and can serve as a rapid means of adaptation to selection pressure.     

A 2.2-kilobase repeated DNA segment is associated with DNA amplification in Streptomyces fradiae.

We have previously identified a 10.5-kilobase DNA sequence which is highly amplified and tandemly repeated in the mutant Streptomyces fradiae JS85. A library of DNA was prepared from S. fradiae T776, which does not contain amplified DNA. The library was screened by plaque hybridization to identify phage clones containing the unamplified 10.5-kilobase DNA sequence. Four phage isolates were identified which contained DNA homology to the amplified DNA sequence. This sequence was designated the amplifiable unit of DNA. None of the clones carried an entire amplifiable unit of DNA, and so overlapping regions were aligned to create a map of the entire region. Detailed restriction mapping identified a 2.2-kilobase direct repeat at the ends of the amplifiable unit of DNA. Analysis by Southern hybridization confirmed that the direct repeats were homologous to each other. The DNA of S. fradiae contained at least two additional copies of DNA that was homologous to the repeat sequence.     

Gene amplification in Tetrahymena thermophila: formation of extrachromosomal palindromic genes coding for rRNA.

Tetrahymena thermophila contains in the macronucleus multiple copies of extrachromosomal palindromic genes coding for rRNA (rDNA) which are generated from a single chromosomal copy during development. In this study we isolated the chromosomal copy of rDNA and determined the structure and developmental fate of the sequence surrounding its 5' junction. The result indicates that specific chromosomal breakage occurs at or near the 5' junction of rDNA during development. The breakage event is associated with DNA elimination and telomeric sequence addition. Similar results were also found previously for the 3' junction of this gene. These results could explain how the extrachromosomal rDNA is first generated. Near both junctions of the chromosomal rDNA, a pair of 20-nucleotide repeats was found. These sequences might serve as signals for site-specific breakage. In addition, we found a pair of perfect inverted repeats at the 5' junction of this gene. The repeats are 42 nucleotides long and are separated by 28 nucleotides. The existence of this structure provides a simple explanation for the formation of the palindromic rDNA.     

PCR amplification of tandemly repeated DNA: analysis of intra- and interchromosomal sequence variation and homologous unequal crossing-over in human alpha satellite DNA.

Tandemly repeated DNA can comprise several percent of total genomic DNA in complex organisms and, in some instances, may play a role in chromosome structure or function. Alpha satellite DNA is the major family of tandemly repeated DNA found at the centromeres of all human and primate chromosomes. Each centromere is characterized by a large contiguous array of up to several thousand kb which can contain several thousand highly homogeneous repeat units. By using a novel application of the polymerase chain reaction (repPCR), we are able to amplify a representative sampling of multiple repetitive units simultaneously, allowing rapid analysis of chromosomal subsets. Direct sequence analysis of repPCR amplified alpha satellite from chromosomes 17 and X reveals positions of sequence heterogeneity as two bands at a single nucleotide position on a sequencing ladder. The use of TdT in the sequencing reactions greatly reduces the background associated with polymerase pauses and stops, allowing visualization of heterogeneous bases found in as little as 10% of the repeat units. Confirmation of these heterogeneous positions was obtained by comparison to the sequence of multiple individual cloned copies obtained both by PCR and non-PCR based methods. PCR amplification of alpha satellite can also reveal multiple repeat units which differ in size. Analysis of repPCR products from chromosome 17 and X allows rapid determination of the molecular basis of these repeat unit length variants, which appear to be a result of unequal crossing-over. The application of repPCR to the study of tandemly repeated DNA should allow in-depth analysis of intra- and interchromosomal variation and unequal crossing-over, thus providing insight into the biology and genetics of these large families of DNA.     

Some extrachromosomal circular DNAs from Drosophila embryos are homologous to tandemly repeated genes

In Drosophila melanogaster embryos we have identified three classes of extrachromosomal circular DNA molecules homologous to the three main families of tandemly repeated genes, 5 S, rDNA and histone. 5 S genes are present in circular multimeric molecules containing up to 16 copies of the 375(±7) base-pair repeated unit. Circular molecules homologous to rDNA are also multimeric molecules, which contain up to ten copies of the 240 base-pair tandemly repeated sequence of the non-transcribed spacer. The two major genomic classes of histone units (4800 and 5000 bases) are found only as monomeric circular molecules.No circular intermediate of the I transposable element was detected in embryos laid by f₁ dysgenic females produced by the I-R system of hybrid dysgenesis.As far as we know, it is the first time that genes have been identified among extrachromosomal circular molecules independently of any specific amplification phenomenon.     

Genetic transactivation of Dissociation elements in transgenic tomato plants

Summary A DNA amplification is correlated with the dominant, unstable cob-354 cobalt resistance trait in the cellular slime mold, Dictyostelium discoideum. The amplified DNA is present as about 50 copies of an extrachromosomal element. Cells grown under nonselective conditions in the absence of cobalt ions lose both the cobalt resistance trait and all extrachromosomal copies of the amplified DNA. The amplified DNA is transferrable to new genetic backgrounds by parasexual genetic crosses. These results explain the inability to map the cob-354 trait to a linkage group. The chromosomal origin of the amplified DNA is group III or VI. Thus the resistance trait appears to be independent of the previously known cobalt resistance locus, cobA, which maps to group VII. A developmental defect involving the production of multiply-tipped aggregates that do not complete fruiting body formation also is correlated with the presence of the amplified DNA.     

DNA amplification in arsenite-resistant Leishmania

Arsenite-resistant variants of a trypanosomatid protozoan, Leishmania mexicana amazonensis, were selected in vitro by stepwise increases of sodium arsenite concentrations up to 30 microM in the culture medium. These variants were found to contain amplified DNA as extrachromosomal supercoiled molecules of about 69 kb. They originate from a single chromosome in the wild-type cells. There is evidence of chromosomal changes in these cells associated with the selection for arsenite resistance. The apparent absence of these circular molecules in the wild type and their loss from the drug-sensitive revertants suggest amplification of chromosomal DNA into these extrachromosomal circles as the mechanism of arsenite resistance.     

Isolation and characterization of sequences from mouse chromosomal DNA with ARS function in yeasts.

Fragments of chromosomal DNA from a variety of eucaryotes can act as ARSs (autonomously replicating sequence) in yeasts. ARSs enable plasmids to be maintained in extrachromosomal form, presumably because they function as initiation sites for DNA replication. We isolated eight different sequences from mouse chromosomal DNA which function as ARSs in Saccharomyces cerevisiae (bakers' yeast). Although the replication efficiency of the different mouse ARSs in yeasts appears to vary widely, about one-half of them functions as well as the yeast chromosomal sequence ARS1. Moreover, five of the ARSs also promote self replication of plasmids in Schizosaccharomyces pombe (fission yeast). Each of the ARSs was cloned into plasmids suitable for transformation of mouse tissue culture cells. Plasmids were introduced into thymidine kinase (TK)-deficient mouse L cells by the calcium phosphate precipitation technique in the absence of carrier DNA. In some experiments, the ARS plasmid contained the herpes simplex virus type 1 TK gene; in other experiments (cotransformations), the TK gene was carried on a separate plasmid used in the same transformation. In contrast to their behavior in yeasts, none of the ARS plasmids displayed a significant increase in transformation frequency in mouse cells compared with control plasmids. Moreover, only 1 of over 100 cell lines contained the original plasmid in extrachromosomal form. The majority of cell lines produced by transformation with an ARS TK plasmid contained multiple copies of plasmid integrated into chromosomal DNA. In most cases, results with plasmids used in cotransformations were similar to those for plasmids carrying TK. However, cell lines produced by cotransformations with plasmids containing any one of three of the ARSs (m24, m25, or m26) often contained extrachromosomal DNAs.     

DNA amplification in tunicamycin-resistant Leishmania mexicana. Multicopies of a single 63-kilobase supercoiled molecule and their expression

Tunicamycin-resistant variants of Leishmania mexicana were found to contain elevated activity of N-acetylglucosamine-1-phosphate transferase and amplified DNA (Kink, J. A., and Chang, K.-P. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1253-1257). Complete digestion of their DNA with restriction endonucleases produced discrete ethidium bromide-staining bands after agarose gel electrophoresis. All four BamHI fragments of the amplified DNA were cloned separately into pBR322 and found to share no substantial sequence homology. DNA complementary to each of the cloned fragments is 64-128-fold more abundant in the variants than in the wild type cells. The amplified DNA appears to originate from a single chromosomal region of 63 kilobases. Individual copies of the 63 kilobases are each circularized at the newly formed junction site producing multiple extrachromosomal supercoiled molecules in the drug-resistant cells. There is overproduction of RNA ranging in size from 1.9 to 6.6 kilobases complementary to the amplified DNA in these cells.     

Plasmid loss and changes within the chromosomal DNA of Streptomyces reticuli.

The sporulating wild-type strain of Streptomyces reticuli, which produces a melanin pigment and the macrolide leucomycin, contains plasmid DNA of 48 to 49 megadaltons. Plasmidless variants had an altered secondary metabolism and a changed antibiotic resistance pattern. By using a new colony hybridization technique developed for streptomycetes, it could be shown that plasmidless variants could be transformed with the wild-type plasmid DNA, which, however, is quickly lost from regenerated mycelium. In contrast to the wild-type strain, the plasmidless variants contain amplified nucleotide sequences within the chromosomal DNA. The number and size of these sequences vary with the strain tested. Hybridization studies revealed that the reiterated sequences are neither amplified ribosomal nor plasmid genes, but are present in small concentrations within the wild-type chromosome. Some of them share extensive homologies with each other and are located at different positions within the chromosome. It is assumed that alterations in secondary metabolism are due to changes within both the chromosomal and the extrachromosomal DNAs of S. reticuli.     

Distribution of oxoacyl synthase homology sequences within Streptomyces DNA

Abstract A genomic DNA sequence of Streptomyces strain ISP 5485 was cloned, sequenced and compared with corresponding information from nucleic acid data banks. The DNA sequence was unique, but showed homology to DNA coding for the condensing enzyme, 2-oxoacyl synthase, of the deoxyerythronolide B synthase complex (DEBS) from Saccharopolyspora erythraea NRRL 2338. A subfragment of the sequenced DNA was used to construct a gene-specific probe that formed part of the putative 2-oxoacyl synthase gene. The PCR-amplified and labelled probe was used in hybridization experiments involving 33 streptomycete strains that produced different classes of antibiotics. The probe showed widespread homology with DNA considered to be part of analogous genes within genomes of different polyketide producers. The implications of the probe homology to bacterial chromosomal DNA are discussed.     

Regulated Formation of Extrachromosomal Circular DNA Molecules during Development in Xenopus laevis

Extrachromosomal circular DNA molecules of chromosomal origin have been detected in many organisms and are thought to reflect genomic plasticity in eukaryotic cells. Here we report a developmentally regulated formation of extrachromosomal circular DNA that occurs de novo in preblastula Xenopus embryos. This specific DNA population is not detected in the male or female germ cells and is dramatically reduced in later developmental stages and in adult tissues. The activity responsible for the de novo production of extrachromosomal circles is maternally inherited, is stored in the unfertilized egg, and requires genomic DNA as a template. The formation of circular molecules does not require genomic DNA replication but both processes can occur simultaneously in the early development. The production of extrachromosomal circular DNA does not proceed at random since multimers of the tandemly repeated sequence satellite 1 were over-represented in the circle population, while other sequences (such as ribosomal DNA and JCC31 repeated sequence) were not detected. This phenomenon reveals an unexpected plasticity of the embryonic genome which is restricted to the early developmental stage.     

Cloning of the gene topA encoding for DNA topoisomerase I and the physical mapping of the cysB-topA-trp region of Escherichia coli.

The gene topA of Escherichia coli that encodes for DNA topoisomerase I has been cloned by a combination of genetic and radioimmunal screening. The gene has been mapped to be within a 3.4 Kb segment of the bacterial genome. The intracellular level of the enzyme in strains harboring extrachromosomal copies of topA gene increases with increasing copy number of the gene and the introduction of extrachromosomal copies of the topA gene truncated at its 3' side into a topA strain of E. coli does not significantly influence the expression of the chromosomal copy of topA. These results suggest that the expression of topA is not tightly regulated. Strains in which DNA topoisomerase I is overproduced grow significantly slower in broth and give smaller size colonies on agar plates. Physical mapping of a 20 Kb region containing cysB; topA and trp has also been carried out with a number of restriction enzymes; topA is found to be immediately adjacent to cysB and is separated from trp by a 7 Kb segment where no known gene resides.