Structure of amplified DNA in different Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

Syrian hamster cell lines selected in multiple steps for resistance to high levels of N-(phosphonacetyl)-L-aspartate (PALA) contain many copies of the gene coding for the pyrimidine pathway enzyme CAD. Approximately 500 kilobases of additional DNA was coamplified with each copy of the CAD gene in several cell lines. To investigate its structure and organization, we cloned ca. 162 kilobases of coamplified DNA from cell line 165-28 and ca. 68 kilobases from cell line B5-4, using a screening method based solely on the greater abundance of amplified sequences in the resistant cells. Individual cloned fragments were then used to probe Southern transfers of genomic DNA from 12 different PALA-resistant mutants and the wild-type parents. A contiguous region of DNA ca. 44 kilobases long which included the CAD gene was amplified in all 12 mutants. However, the fragments cloned from 165-28 which were external to this region were not amplified in any other mutant, and the external fragments cloned from B5-4 were not amplified in two of the mutants. These results suggest that movement or major rearrangement of DNA may have accompanied some of the amplification events. We also found that different fragments were amplified to different degrees within a single mutant cell line. We conclude that the amplified DNA was not comprised of identical, tandemly arranged units. Its structure was much more complex and was different in different mutants. Several restriction fragments containing amplified sequences were found only in the DNA of the mutant cell line from which they were isolated and were not detected in DNA from wild-type cells or from any other mutant cells. These fragments contained novel joints created by rearrangement of the DNA during amplification. The cloned novel fragments hybridized only to normal fragments in every cell line examined, except for the line from which each novel fragment was isolated or the parental population for that line. This result argues that "hot spots" for forming novel joints are rare or nonexistent.     

Properties of single-step mutants of Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate.

Eleven independent lines of Syrian hamster cells were selected by using very low levels of N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of aspartate transcarbamylase. The protocol employed insured that each resistant cell arose during one of the last divisions before selection was applied. Cells of each mutant line contained an amplification of the structural gene for CAD, a trifunctional protein which includes aspartate transcarbamylase and two other enzymes of UMP biosynthesis. Strikingly, despite the minimal selection employed, the degree of amplification of the CAD gene was 6 to 10 times the normal diploid number in all 11 cases. In situ hybridization indicated that the amplified CAD genes were almost always present at a single chromosomal site in each line. Therefore, one of the two alleles was amplified 11- to 19-fold. The rates at which cells became resistant to PALA, determined by fluctuation analysis, were 100 times less dependent on drug concentration than were the frequencies of resistant cells in steady-state populations. The relatively shallow dependence of this rate upon PALA concentration is consistent with our independent observation that most events gave rise to a similar degree of amplification. In six of six cell lines examined, the levels of CAD mRNA and aspartate transcarbamylase activity were elevated two- to fourfold. These lines were resistant to PALA concentrations 20- to 80-fold higher than the ones used for selection. The organization of amplified DNA was examined by hybridizing Southern blots with cloned DNA fragments containing amplified sequences, previously isolated from two cell lines resistant to high levels of PALA. A contiguous region of DNA approximately 44 kilobases long which included the CAD gene was amplified in five of five single-step mutants examined. Outside this region, these mutants shared amplified sequences with only one of the two highly resistant lines.     

Chromosome aberrations associated with CAD gene amplification in Chinese hamster cultured cells

Eleven sublines with increasing resistance to N-phosphonacetyl-L-aspartate (PALA) were isolated from the V79,B7 Chinese hamster cell line. Aspartate transcarbamylase activity and CAD gene copy number increased with increasing resistance of sublines. In situ hybridization with a DNA probe for the CAD gene showed that the amplified sequences resided in the terminal region of a marker chromosome with elongated q arms. This region stained homogeneously after G-banding. A high incidence of both numerical and structural chromosome aberrations was found in PALA-resistant cells. In hyperdiploid and polyploid cells, containing 2 copies of the marker chromosome, dicentrics were found at a very high frequency. As indicated by in situ hybridization and G-banding, they originated from a rearrangement involving 2 homologous marker chromosomes.     

Physical map of the Salmonella typhimurium histidine transport operon: correlation with the genetic map.

A detailed restriction map of a 12.4-kilobase EcoRI fragment of Salmonella typhimurium deoxyribonucleic acid (DNA) containing the entire histidine transport operon and the argT gene is presented. Subclones of specific regions of the transport operon of S. typhimurium were constructed in plasmid vectors. An accurate correlation between the restriction map and the location of genetically defined deletions was obtained by hybridizing restriction digests of chromosomal DNA from strains carrying each deletion with cloned transport operon DNA as a probe. These data were used to position the histidine transport genes on the cloned 12.4-kilobase fragment of DNA.     

Amplification and cloning of the Chinese hamster glutamine synthetase gene.

A Chinese hamster ovary cell line, KG1MS which is resistant to 5 mM methionine sulphoximine overproduces glutamine synthetase. Overproduction of this 42 000 mol. wt. polypeptide is not seen in either parental KG1 or revertant KG1MSC4-0 cell lines which are resistant to 3 microM and 8 microM methionine sulphoximine, respectively. Restriction endonuclease analysis of DNA from KG1MS cells produces a pattern of amplified DNA fragments not seen in parental KG1 or revertant KG1MSC4-0 digests. Hybridization of cDNA probes complementary to KG1MS poly(A) mRNA against Southern blots of KG1MS restriction digests identifies a specific subset within these amplified sequences which is not detected by cDNA probes made from parental KG1 poly(A) mRNA. One 8.2-kb BglII fragment of KG1MS DNA identified by cDNA hybridization has been cloned to produce recombinant pGS-1. mRNA hybrid selected by pGS-1 translates to a 42 000 mol. wt. polypeptide which co-migrates in polyacrylamide gels with the over-produced protein in KG1MS cells and purified glutamine synthetase. pGS-1 also hybridizes to several mRNA species abundant in KG1MSC4-M, but not KG1, poly(A) mRNA extracts. The high level of resistance to methionine sulphoximine shown by KG1MS cells is due to amplification of a DNA sequence of at least 50 kb which contains the coding region for the enzyme glutamine synthetase.     

Unstable genetic determinant of A-factor biosynthesis in streptomycin-producing organisms: cloning and characterization.

We cloned a DNA fragment directing synthesis of A-factor from the total cellular DNA of streptomycin-producing Streptomyces bikiniensis on the plasmid vector pIJ385 . Introduction of the recombinant plasmid ( pAFB1 ) into A-factor-deficient S. bikiniensis and Streptomyces griseus mutants led to A-factor production in the host cells, as a result of which streptomycin production, streptomycin resistance, and spore formation of these mutants were simultaneously restored. The plasmid pAFB1 also complemented both afsA and afsB mutations of Streptomyces coelicolor A3(2). These results indicated that the cloned DNA fragment contained the genetic determinant of A-factor biosynthesis. The cloned fragment, when carried on a multicopy vector plasmid, induced production of a large amount of A-factor in several Streptomyces hosts. In Southern blot DNA/DNA hybridization analyses with a trimmed 5-kilobase fragment containing the intact A-factor determinant as probe, total cellular DNA from A-factor-deficient mutants gave no positive hybridization. The DNA blot experiment also showed a wide distribution of sequences homologous to the S. bikiniensis A-factor determinant among most, but not all, A-factor-producing actinomycetes with a varying extent of homology and the absence of these sequences from most A-factor nonproducers .     

DNA amplification in antifolate-resistant Leishmania. The thymidylate synthase-dihydrofolate reductase gene and abundant mRNAs

Leishmania tropica promastigotes selected for resistance to the dihydrofolate reductase inhibitor, methotrexate, or the thymidylate synthase inhibitor, 5,8-dideaza-10-propargyl folate, overproduce a bifunctional thymidylate synthase-dihydrofolate reductase and possess a 30-kilobase region of amplified DNA. Five fragments, resulting from BglII digestion of this amplified DNA, were cloned into vectors and utilized as probes to examine mRNA in these organisms. Four mRNA species which hybridize to the amplified DNA sequences were found in both resistant and wild-type Leishmania, but were about 40-fold more abundant in the drug-resistant cells. Three of the four mRNAs are transcribed from the same strand of DNA, are clustered, and appear to have partial overlapping sequences. The thymidylate synthase-dihydrofolate reductase gene was localized to a specific region of the amplified unit of DNA by hybridization with mouse cDNA containing thymidylate synthase sequences and with a synthetic oligonucleotide 41 nucleotides in length, prepared on the basis of the partial amino acid sequence of the Leishmania enzyme. Furthermore, mRNA hybrid-selected using a plasmid containing sequences of the putative gene was shown to direct in vitro synthesis of the bifunctional protein.     

Co-amplification of rRNA genes with CAD genes in N-(phosphonacetyl)-L-aspartate-resistant Syrian hamster cells.

The amplified CAD genes in N-(phosphonacetyl)-L-aspartate (PALA)-resistant Syrian hamster cells are located in an expanded chromosomal region emanating from the site of the wild-type gene at the tip of the short arm of chromosome B-9. The terminus of B-9 in PALA-sensitive cells contains a cluster of rRNA genes (i.e., a nucleolus organizer, rDNA). We have used a molecular clone containing sequences complementary to Syrian hamster 28S rRNA to investigate whether rDNA is coamplified with CAD genes in the PALA-resistant mutants. In situ hybridization of this probe to metaphase chromosomes demonstrates that rDNA and CAD genes do coamplify in two independently isolated PALA-resistant mutants. The tight linkage of CAD and rDNA genes was demonstrated by their coordinate translocation from B-9 to the end of the long arm of chromosome C-11 in one mutant. Blot hybridization studies substantiate the in situ hybridization results. Both types of analysis indicate that only one or two rDNA genes, on the average, are coamplified per CAD gene. The data are consistent with the model that unequal exchanges between rDNA genes mediate the amplification of CAD genes in the Syrian hamster mutants that were analyzed.     

General method for cloning amplified DNA by differential screening with genomic probes.

Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite.This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or more can be cloned, even if no function for the region is known. The method for removing highly repetitive sequences from genomic DNA probes should also be of general use.     

Large inverted duplications are associated with gene amplification

Amplified DNA can be found in arrays of large repeated units, with each repeat unit containing a marker gene and surrounding DNA sequences. Amplified DNA sequences from established cell lines were assessed for the presence of repeat units in the form of inverted duplications. Inverted duplicated DNA was detected by virtue of its concentration-independent resistance to S1 hydrolysis after denaturation and rapid renaturation. Using this assay, inverted duplications were detected in amplified DNA (both DM and HSR configurations) containing the myc gene (16-50 copies/cell) in four human tumor cell lines and in amplified DNA containing the CAD gene (30-200 copies/cell) in three PALA-resistant BHK cell lines. The widespread association of inverted duplications with amplified DNA must bear on the amplification mechanism.     

Unstable and stable CAD gene amplification: importance of flanking sequences and nuclear environment in gene amplification.

We analyzed the amplification of the CAD gene in independently isolated N-(phosphonacetyl)-L-aspartate-resistant clones derived from single parental clones in two mouse cell lines. We report for the first time that the CAD gene is amplified unstably in mouse cells, that the degree of instability varies greatly between clones, and that minute chromosomes and highly unstable chromosomelike structures contain the amplified sequences. These data are most consistent with the idea that the amplified unit in each clone consists of different flanking DNA and that such differences engender amplified sequences with unequal stability. We also introduced the mouse chromosome containing the CAD gene into hamster cells by microcell-mediated chromosome transfer to determine whether the propensity for unstable extrachromosomal amplification of the mouse CAD gene would prevail in the hamster cell nuclear environment. We report that the mouse CAD gene was amplified stably in expanded chromosomal regions in each of seven hybrids that were analyzed. This observation is consistent with the idea that the nuclear environment influences whether mutants containing intra- or extrachromosomally amplified sequences will be isolated.     

Cloning and expression of the transhydrogenase gene of Escherichia coli.

Based on the rationale that Escherichia coli cells harboring plasmids containing the pnt gene would contain elevated levels of enzyme, we have isolated three clones bearing the transhydrogenase gene from the Clarke and Carbon colony bank. The three plasmids were subjected to restriction endonuclease analysis. A 10.4-kilobase restriction fragment which overlapped all three plasmids was cloned into the PstI site of plasmid pUC13. Examination of several deletion derivatives of the resulting plasmid and subsequent treatment with exonuclease BAL 31 revealed that enhanced transhydrogenase expression was localized within a 3.05-kilobase segment. This segment was located at 35.4 min in the E. coli genome. Plasmid pDC21 conferred on its host 70-fold overproduction of transhydrogenase. The protein products of plasmids carrying the pnt gene were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes from cells containing the plasmids. Two polypeptides of molecular weights 50,000 and 47,000 were coded by the 3.05-kilobase fragment of pDC11. Both polypeptides were required for expression of transhydrogenase activity.     

Cloning of DNA involved in sporulation of Streptomyces griseus

Twenty-two bald mutants of Streptomyces griseus were isolated and classified into four phenotypic groups, two of which showed conditional sporulation. A 3-kilobase fragment of DNA was cloned in a high-copy-number vector and detected by its ability to restore sporulation to one class of conditionally bald mutants. Analysis of subclones demonstrated that the sporulation property was contained within a 2.5-kilobase fragment. Hybridization studies and restriction analysis indicated that this DNA fragment was present in several Streptomyces species and was distinct from DNA that has been shown to complement afsA mutants of S. bikiniensis and bldA mutants of S. coelicolor.     

Organization of delta-crystallin genes in the chicken.

Double-stranded DNA was synthesized from delta-crystallin mRNA prepared from lens fibers of 15-day-old chick embryos and cloned at the Pst I site of the plasmid pBR322. Using the cloned cDNA and single-stranded cDNA as hybridization probes, a number of genomic DNA fragments containing delta-crystallin gene sequences have been cloned from the partial and complete EcoRI digests of chick brain DNA. One of the clones from the partial digests contains a DNA fragment that consists of four EcoRI fragments of 7.6 kb, 4.0 kb, 2.6 kb, and 0.8 kb. The gene sequences reside in the (5')7.6 kb - 0.8 kb - 4.0 kb (3') fragments. Electron microscopy has provided evidence that the cloned DNA fragment includes the entire gene sequences complementary to delta-crystallin mRNA except for the 3' terminal poly(A) tail, and that the delta-crystallin gene is interrupted by at least 13 intervening sequences. Another clone contains a genomic fragment that consists of two EcoRI fragments of 3.0 kb and 11 kb. The DNA fragment in the latter clone represents a different delta-crystallin gene, as judged by restriction endonuclease mapping and by electron microscopy.     

2-micrometers DNA-like plasmids in the osmophilic haploid yeast Saccharomyces rouxii.

DNA plasmids were detected in two independent strains of Saccharomyces rouxii among 100 yeast strains other than Saccharomyces cerevisiae tested. The plasmids, pSR1 and pSR2, had almost the same mass (approximately 4 X 10(6) daltons) as 2-micrometers DNA of S. cerevisiae. pSR1 and pSR2 gave identical restriction maps with restriction endonucleases BamHI, EcoRI, HincII, HindIII, and XhoI, and both lacked restriction sites for PstI, SalI, and SmaI. These maps, however, differed significantly from that of S. cerevisiae 2-micrometers DNA. Restriction analysis also revealed two isomeric forms of each plasmid and suggested the presence of a pair of inverted repeat sequences in the molecules where intramolecular recombination took place. DNA-DNA hybridization between the pSR1 and pSR2 DNAs indicated significant homology between their base sequences, whereas no homology was detected between pSR1 and pJDB219, a chimeric plasmid constructed from a whole molecule of 2-micrometers DNA, plasmid pMB9, and a 1.2-kilobase DNA fragment of S. cerevisiae bearing the LEU2 gene. A chimeric plasmid constructed with pSR1 and YIp1, the larger EcoRI-SalI fragment of pBR322 ligated with a 6.1-kilobase DNA fragment of S. cerevisiae bearing the HIS3 gene, could replicate autonomously in an S. cerevisiae host and produced isomers, presumably by intramolecular recombination at the inverted repeats.     

Clustering of mutations affecting alginic acid biosynthesis in mucoid Pseudomonas aeruginosa.

A 10-kilobase DNA fragment previously shown to contain the phosphomannose isomerase gene (pmi) of Pseudomonas aeruginosa was used to construct a pBR325-based hybrid that can be propagated in P. aeruginosa only by the formation of a chromosomal-plasmid cointegrate. This plasmid, designated pAD4008, was inserted into the P. aeruginosa chromosome by recombination at a site of homology between the cloned P. aeruginosa DNA and the chromosome. Mobilization of pAD4008 into P. aeruginosa PAO and 8830 and selection for the stable acquisition of tetracycline resistance resulted in specific and predictable changes in the pattern of endonuclease restriction sites in the phosphomannose isomerase gene region of the chromosomes. Chromosomal DNA from the tetracycline-resistant transformants was used to clone the drug resistance determinant with Bg/II or XbaI, thereby allowing the "walking" of the P. aeruginosa chromosome in the vicinity of the pmi gene. Analysis of overlapping tetracycline-resistant clones indicated the presence of sequences homologous to the DNA insert of plasmid pAD2, a recombinant clone of P. aeruginosa origin previously shown to complement several alginate-negative mutants. Restriction mapping, subcloning, and complementation analysis of a 30-kilobase DNA region demonstrated the tight clustering of several genetic loci involved in alginate biosynthesis. Furthermore, the tetracycline resistance determinant in PAO strain transformed by pAD4008 was mapped on the chromosome by plasmid FP2-mediated conjugation and was found to be located near 45 min.