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.     

Novel DNA sequences at chromosome 10q26 are amplified in human gastric carcinoma cell lines: molecular cloning by competitive DNA reassociation.

Molecular cloning of genomic sequences altered in cancer cells is believed to lead to the identification of new genes involved in the initiation and progression of the malignant phenotype. DNA amplification is a frequent molecular alteration in tumor cells, and is a mode of proto-oncogene activation. The cytologic manifestation of this phenomenon is the appearance of chromosomal homogeneously staining regions (HSRs) or double minute bodies (DMs). The gastric carcinoma cell line KATO III is characterized by a large HSR on chromosome 11. In-gel renaturation analysis confirmed the amplification of DNA sequences in this cell line, yet none of 42 proto-oncogenes that we tested is amplified in KATO III DNA. We employed the phenol-enhanced reassociation technique (PERT) to isolate 21 random DNA fragments from the amplified domain, and used 6 of them to further clone some 150 kb from that genomic region. While in situ hybridization performed with some of these sequences indicated that in KATO III they are indeed amplified within the HSR on chromosome 11, somatic cell hybrid analysis and in situ hybridization to normal lymphocyte chromosomes showed that they are derived from chromosome 10, band q26. The same sequences were found to be amplified in another gastric carcinoma cell line, SNU-16, which contains DMs, but were not amplified in other 70 cell lines representing a wide variety of human neoplasms. One of these sequences was highly expressed in both KATO III and SNU-16. Thus, the cloned sequences supply a starting point for identification of novel genes which might be involved in the pathogenesis of gastric cancers, and are located in a relatively unexplored domain of the human genome.     

The dihydrofolate reductase amplicons in different methotrexate-resistant Chinese hamster cell lines share at least a 273-kilobase core sequence, but the amplicons in some cell lines are much larger and are remarkably uniform in structure.

We have previously cloned and characterized two different dihydrofolate reductase amplicon types from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). The largest of these (the type I amplicon) is 273 kilobases (kb) in length. In the present study, we utilized clones from the type I amplicon as probes to analyze the size and variability of the amplified DNA sequences in five other independently isolated methotrexate-resistant Chinese hamster cell lines. Our data indicated that the predominant amplicon types in all but one of these cell lines are larger than the 273-kb type I sequence. In-gel renaturation experiments as well as hybridization analysis of large SfiI fragments separated by pulse-field gradient gel electrophoresis showed that two highly resistant cell lines (A3 and MK42) have amplified very homogeneous core sequences that are estimated to be at least 583 and 653 kb in length, respectively. Thus, the sizes of the major amplicon types can be different in different drug-resistant Chinese hamster cell lines. However, there appears to be less heterogeneity in size and sequence arrangement within a given methotrexate-resistant Chinese hamster cell line than has been reported for several other examples of DNA sequence amplification in mammalian systems.     

Viral DNA in transformed cells: I. A study of the sequences of adenovirus 2 DNA in a line of transformed rat cells using specific fragments of the viral genome

³²P-labeled adenovirus 2 DNA was treated with restricting endonuclease isolated from Escherichia coli strain RY-13 (Yoshimori, 1971) and the resulting six fragments were separated by electrophoresis through polyacrylamide-agarose gels. The kinetics of renaturation of each of the fragments and of complete adenovirus 2 DNA were measured in the presence of DNA extracted from a line of transformed rat cells and from control cells. The entire sequences of two of the fragments and part of the sequence of a third fragment were not detectable in the transformed cell DNA. Thus the line of adenovirus 2-transformed rat cells contains sequences homologous to only about 46% of the viral DNA. From the order of the fragments, formed by this restricting endonuclease on the adenovirus 2 map, it seems that the viral sequences that are absent from transformed cells form one continuous segment located in the center of the viral genome.     

Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells

The structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension-cultured cell lines selected for glyphosate resistance was analyzed to determine the mechanism of gene amplification in this plant system. Southern hybridization of the amplified DNA digested with several restriction enzymes probed with a petunia EPSPS cDNA clone showed that there were differences in fragment sizes in the amplified DNA from one highly resistant cell line in comparison with the parental line. Cloning of the EPSPS gene and 5 flanking sequences was carried out and two different DNA structures were revealed. A 13 kb clone contained only one copy of the EPSPS gene while a 16 kb clone contained an inverted duplication of the gene. Southern blot analysis with a carrot DNA probe showed that only the uninverted repeated DNA structure was present in all of the cell lines during the selection process and the inverted repeat (IR) was present only in highly amplified DNA. The two structures were present in about equal amounts in the highly amplified line, TC 35G, where the EPSPS gene was amplified about 25-fold. The presence of the inverted repeat (IR) was further verified by resistance to S1 nuclease hydrolysis after denaturation and rapid renaturation, showing foldback DNA with the IR length being 9.5 kb. The junction was also sequenced. Mapping of the clones showed that the size of the amplified carrot EPSPS gene itself is about 3.5 kb. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells.     

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.     

Characterization of N-myc amplification in a human neuroblastoma cell line by clones isolated following the phenol emulsion reassociation technique and by hexagonal field gel electrophoresis.

The N-myc amplification of human neuroblastomas was characterized by the amplified DNA cloned from the cell line MC-NB-1 using the phenol emulsion reassociation technique (PERT). A number of PERT clones exhibiting amplification in this cell line were tested for amplification in other neuroblastoma cell lines. In almost all cell lines examined, only a few clones were co-amplified with N-myc and most of the others were exclusively amplified in a subset of the cell lines. The total aggregate size of the Hind III fragment identified by the PERT clones was approximately 350 kb. Most of the PERT clones were mapped to human chromosome (chr) 2p23-2pter, where the N-myc gene is located. Four types of amplicons, the 100, 420, 480 and 520 kb fragments, shown to be Not I fragments, were identified by hexagonal field gel electrophoresis. Three fragments are ordered in a head-to-tail array, and the remaining fragment is either ordered in a tail-to-head array or something else. Despite the extremely unusual construction of the amplified sequences in this cell line as compared with others, there was a low degree of sequence heterogeneity among the amplicons within this cell line. These observations lead to the idea that the complex rearrangements that give rise to the heterogeneous organization of the amplified sequences among the different cell lines precede the amplification of these sequences.     

Concatameric replication of Epstein-Barr virus: structure of the termini in virus-producer and newly transformed cell lines.

The linear form of Epstein-Barr virus (EBV) DNA has homologous direct tandem repeats of approximately 500 bp at each terminus (TR). After infection, EBV DNA circularizes via the TR to form the intracellular episomal DNA. To analyze the mechanism of the synthesis of linear DNA through possible replicative intermediates, the terminal fragments were identified in the total intracellular DNA and the covalently closed circular DNA from a productively infected cell line after induction of replication or after treatment with an inhibitor of viral DNA synthesis. These studies indicate that some of the fused terminal fragments detected in the total intracellular DNA are replication-dependent forms which are selectively excluded from the covalently closed circular fraction and are eliminated after treatment with acyclovir. The EBV terminal restriction enzyme fragments were identified in three producer cell lines, each with a characteristic number of TR in the intracellular episomal DNA. Identification of the termini in cell lines established with the three virus strains revealed that the newly transformed cell lines had a greater number of TR than did the template DNA in the producer cell line. The increase in the number of TR in progeny episomes indicates that linear DNA is produced from concatameric replicative intermediates rather than from amplified catenated circular intermediates.     

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.     

Replication origins are attached to the nuclear skeleton.

DNA fragments containing replication origins (oriDNA) were isolated from a chicken erythroblast cell line by a modified procedure of Zannis-Hadjopoulos et al. and studied in the renaturation reaction driven by either total or nuclear matrix DNA (nmDNA) from the same cells or from mature erythrocytes. We found that the unique sequences of nmDNA from erythroblasts (5 kb long) represented a specific subset of sequences constituting about a quarter of total DNA unique sequences, while the erythrocyte nmDNA 5 kb fragments constitute only about one tenth of total unique DNA and all are recovered among erythroblast nmDNA. Virtually all oriDNA sequences are present in the fraction of erythrocyte nmDNA. Thereafter, the putative positions of replication origins within the alpha-globine gene domain have been mapped by hybridization experiments. They were found to coincide with the previously established positions of permanent sites of DNA attachment to the nuclear matrix.     

苜蓿红豆草属间体细胞杂种的分子生物学鉴定

通过原生质体融合和培养获得苜蓿红豆草属间体细胞杂种植株。采用一种简便方法从杂种组织再生植株叶片、红豆草羟脯氨酸抗性系再生植株叶片和苜蓿根癌农杆菌702转化系愈伤组织提取DNA用于RAPD和Southern杂交分析。随机引物扩增结果显示两种亲本的RAPD多态具有明显差异。在所用20种随机引物中,6种产生较多的DNA片段。杂种组织具有两种亲本特有的DNA片段,但倾向于排除红豆草亲本的染色体,表明该杂种为非对称杂种,两种亲本染色体之间可能发生了重组。由于红豆草DNA的介入,杂种组织表现出较强的分化能力。分别利用RAPD扩增得到的OPA141000bp红豆草羟脯氨酸抗性系特异产物和OPA141600bp苜蓿根癌农杆菌702转化系特异产物为探针进行Southern分子杂交,证明杂种组织同时具有这两种DNA片段的同源序列。     

Characterization of N-myc amplification in a human neuroblastoma cell line by clones isolated following the phenol emulsion reassociation technique and by hexagonal field gel electrophoresis

The N-myc amplification of human neuroblastomas was characterized by the amplified DNA cloned from the cell line MC-NB-1 using the phenol emulsion reassociation technique (PERT). A number of PERT clones exhibiting amplification in this cell line were tested for amplification in other neuroblastoma cell lines. In almost all cell lines examined, only a few clones were co-amplified with N-myc and most of the others were exclusively amplified in a subset of the cell lines. The total aggregate size of theHind III fragment identified by the PERT clones was approximately 350 kb. Most of the PERT clones were mapped to human chromosome (chr) 2p23-2pter, where the N-myc gene is located. Four types of amplicons, the 100, 420, 480 and 520 kb fragments, shown to beNot I fragments, were identified by hexagonal field gel electrophoresis. Three fragments are ordered in a head-to-tail array, and the remaining fragment is either ordered in a tail-to-head array or something else. Despite the extremely unusual construction of the amplified sequences in this cell line as compared with others, there was a low degree of sequence heterogeneity among the amplicons within this cell line. These observations lead to the idea that the complex rearrangements that give rise to the heterogeneous organization of the amplified sequences among the different cell lines precede the amplification of these sequences.     

Detection of amplified genomic sequences in human small-cell lung carcinoma cells by arbitrarily primed-PCR genomic fingerprinting

The arbitrarily primed-PCR (AP-PCR) genomic fingerprinting method was applied to evaluate its effectiveness in detecting and characterizing amplified DNA fragments in two small-cell lung carcinoma (SCLC) cell lines, NCI-H69 and NCI-H82. Of the 2428 DNA fragments detected by AP-PCR using 62 arbitrary primers, 2 (0.08%) DNA fragments were amplified in NCI-H69 and 6 (0.25%) DNA fragments were amplified in NCI-H82. Based on these results, we estimate the total size of the amplified genomic regions in these cell lines to be 3000 megabase pairs (Mb) × 0.0008 = 2.4 Mb in NCI-H69 and 3000 Mb × 0.0025 = 7.5 Mb in NCI-H82. The 2 amplified fragments in NCI-H69 were mapped to chromosome 2, and all 6 amplified fragments in NCI-H82 were mapped to chromosome 8. This strongly suggests that restricted chromosomal regions are specifically amplified in these SCLC cell lines. Since the N-myc gene at 2p24 is amplified in NCI-H69 and the c-myc gene at 8q24 is amplified in NCI-H82, it is possible that these DNA fragments are co-amplified with N-myc or c-myc in these cell lines. However, since the 2 amplified fragments in NCI-H69 were not amplified in 42 other human cancer cell lines including 11 cell lines carrying amplified N-myc genes, it is also possible that there are amplified regions on chromosome 2 other than the N-myc locus at 2p24 in NCI-H69. In contrast, all 6 amplified fragments in NCI-H82 were amplified in several other human cancer cell lines carrying amplified c-myc genes. This result further indicates that these fragments were derived from an amplification unit that includes the c-myc gene. Our results show the ability of the AP-PCR method to analyze the fraction of the genome with amplification in human cancer cells. Received: 10 April 1995 / Revised: 18 December 1995, 15 April 1996     

Shotgun polymerase chain reaction: construction of clone libraries specific to a NotI fragment of flow-sorted human chromosome 22.

We have developed the "shotgun polymerase chain reaction," a method for obtaining a large number of DNA markers specific to a giant DNA fragment, which facilitates analysis of a particular chromosomal region. We applied this method to a giant NotI fragment which carries the immunoglobulin lambda constant region on chromosome 22. NotI digests of chromosome 22 flow-sorted from human B-lymphoblastoid cell line GM130B were size fractionated by pulsed-field gel electrophoresis. Preliminary Southern hybridization analysis revealed that the immunoglobulin lambda constant region was conveyed on 1.4- and 1.3-Mb NotI fragments in this cell line. The agarose gel corresponding to 1.2 to 1.5 Mb in size was excised into slices and subjected to polymerase chain reaction to identify gel slices containing NotI fragments carrying Ke-Oz+, a subtype of the immunoglobulin lambda constant region. From the NotI fragment thus identified, a large number of small DNA segments were amplified through the ligation-mediated random polymerase chain reaction method. The amplified products were cloned and analyzed for chromosomal origin and localization to particular NotI fragments. Seven of eighteen clones originated from the 1.4-Mb NotI fragment of chromosome 22 in GM130B cells, which appears to be exactly the same as detected by a probe for the immunoglobulin lambda constant region.     

Excision of amplified viral DNA at palindromic sequences from the adenovirus type 12-transformed hamster cell line T637.

In the DNA of the adenovirus type 12 (Ad12)-transformed hamster cell line T637 approximately 20-22 viral DNA molecules per cell are covalently linked to cellular DNA. Spontaneously arising morphological revertants of T637 cells have lost the bulk of the viral DNA. We have been able to mimic the excision event of viral DNA, as it occurs during reversion, by autoincubation of isolated nuclei from T637 cells. The same Ad12 DNA sequences, which had been deleted in morphological revertants, proved highly sensitive to endogenous nucleases in isolated nuclei of T637 cells. Viral DNA sequences, which persisted in the revertants, are resistant to endogenous nucleases in isolated T637 nuclei. All attempts to clone the nuclease-sensitive sites of Ad12 DNA in cell line T637 have so far failed. After denaturation and renaturation of T637 DNA followed by treatment with S1 nuclease, large fold-back structures of DNA have been found. These snap-back structures were derived from precisely those viral DNA restriction fragments which were uncloneable. The fragments containing palindromic sequences were both highly sensitive to endogenous nucleases in isolated T637 nuclei and were absent from the DNA of all revertant cell lines. Moreover, the palindromic sequences are susceptible to the phage T4-specific endonuclease VII which specifically attacks cruciform structures in DNA. The peculiar structures at the termini of integrated Ad12 DNA molecules are highly sensitive to endogenous nucleases in isolated nuclei. These nucleases may be related to the reversion event.     

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.     

Characterization of N-myc amplification units in human neuroblastoma cells.

A set of DNA clones comprising 48 independent HindIII fragments (215 kilobases of sequence) was derived from the N-myc amplification unit of the neuroblastoma cell line NGP. These clones were used to investigate N-myc amplification units in NGP cells and 12 primary neuroblastoma tumors. Three parameters were evaluated: (i) the number of rearrangements from germ line configuration that had occurred during the amplification process; (ii) the homogeneity of amplification units within individual tumors; and (iii) the conservation of amplified sequences among different tumors. The results indicated that remarkably few rearrangements had occurred during amplification, that the amplification units within any one tumor were quite homogeneous, and that although each tumor contained a unique pattern of amplified DNA fragments, there was considerable similarity between the amplification units of different tumors. In particular, the amplification units were strikingly similar over a contiguous domain of at least 140 kilobases surrounding the N-myc structural gene.     

Studies on DNA bound to histones in chromatin]

Regions of DNA protected by histones against the action of DNAse 1 in the chromatin were isolated. Such DNA fragments ("subhistones" DNA) have 80% double helix structure, their nucleotide composition is close to that of total DNA, and their sedimentation constant is within the range of 2-2.7S for completely denatured molecules. Kinetics of renaturation of "subhistone" DNA was studied: within a wide range of Cot values, renaturation curves of total and "subhistone" DNA are almost identical. According to the data on hybridization with nuclear d-RNA, "subhistone" DNA is transcribed in the cell. The data obtained witness for uniform character of distribution of histones along the DNA chain in the chromatin. DNA sites which are active in RNA synthesis seem to be bound to histones as well as the non-active ones. No significant difference was found in the hybridization of "subhistone" DNA from rat liver and thymus with ibver nuclear RNA.