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.     

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     

The growth-inhibitory Ndrg1 gene is a Myc negative target in human neuroblastomas and other cell types with overexpressed N- or c-myc

A major prognostic marker for neuroblastoma (Nb) is N-myc gene amplification, which predicts a poor clinical outcome. We sought genes differentially expressed on a consistent basis between multiple human Nb cell lines bearing normal versus amplified N-myc, in hopes of finding target genes that might clarify how N-myc overexpression translates into poor clinical prognosis. Using differential display, we find the previously described growth-inhibitory gene Ndrg1 is strongly repressed in all tested Nb cell lines bearing N-myc amplification, as well as in a neuroepithelioma line with amplified c-myc. Overexpression of N-myc in non-amplified Nb cells leads to repression of Ndrg1, as does activation of an inducible c-myc transgene in fibroblasts. Conversely, N-myc downregulation in N-myc-amplified Nb cells results in re-expression of the Ndrg1, and stimuli known to induce Ndrg1 do so in Nb cells while simultaneously down-regulating N-myc. Relevant to these results, we demonstrate an in vitro interaction of Myc protein with the Ndrg1 core promoter. We also find that Ndrg1 levels increase dramatically during in vitro differentiation of two cell lines modeling neural and glial development, while c- and N-myc levels decline. Our results combined with previous information on the Ndrg1 gene product suggest that downregulation of this gene is an important component of N-Myc effects in neuroblastomas with poor clinical outcome. In support of this notion, we find that re-expression of Ndrg1 in high-Myc Nb cells results in smaller cells with reduced colony size in soft-agar assays, further underscoring the functional significance of this gene in human neuroblastoma cells.     

Cloning and physical mapping of DNA sequences encompassing a region in N-myc amplicons of a human neuroblastoma cell line.

Cloning and physical mapping of DNA sequences encompassing N-myc amplicons of a human neuroblastoma cell line were done. A number of lambda phage clones within this region were isolated using the probes prepared by the phenol emulsion reassociation technique. Based on the restriction mapping, they were integrated into 8 contigs with sizes of 25-60 kb which, in total, encompassed a 330 kb region. Several amplicons, 100, 420, 480 and 520 kb in size as a Notl fragment, were identified using hexagonal field gel electrophoresis, and the contigs were assigned in these Notl fragments. The region encompassed by the contigs was equivalent to some 60-80% of the amplicons identified as a Notl fragment. In order to compare the amplified regions flanking the N-myc gene among the cell lines, the phage clones to cover the whole contigs were used for hybridization as a probe. The results showed that the portions of the whole contigs ranging 18-45% were also amplified in the cell lines examined. These results allowed us to identified the 'rearranged sites' which were rather evenly distributed, one at every 40 kb, through the contigs. These observations lead to the idea that an amplified DNA domain is constructed after the multiple rearrangements and then increases in number, finally resulting in the formation of subsets of amplicons with sequence homogeneity.     

Cloning and characteristics of DNA sequences amplified in Djungarian hamster cells with multidrug resistance

A number of DNA clones containing the amplified DNA sequences were isolated from the genomic library of multidrug-resistant (MDR) Djungarian hamster cells using the DNAC0t 10-250 hybridization probe. Five independent nonoverlapping clones were obtained that covered more than 100 kb of the amplified genomic region. These clones were used as hybridization probes in blot-hybridization with DNA from 7 independently derived MDR Djungarian hamster cell lines selected for the resistance to colchicine or actinomycin D. Some clones contained the DNA sequences amplified in all of the cell lines tested while the others contained the cell line specific amplified sequences. Hybridization in situ was used to localize the amplified DNA in metaphase chromosomes of a MDR cell line that contained about 140 copies of these sequences. The approximate size of an amplicon calculated on the basis of the obtained data is about 1-2 X 10(3) kb.     

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 amplification of oligonucleotide themes in the evolution of themyc protooncogene family

The evolutionary past of intragenic repeats in protein-coding exons of c-, N-, L-, and s-myc-protooncogene subfamilies was elucidated. Apparently these genes evolved by succession of distinct unit events rather than by a steady flow of random point mutations. An evolutionary event probably involved a duplication of the whole gene, which was followed by amplification of progressively shorter oligonucleotide themes and motifs. The repeats were either joined in tandem or one of the copies was transposed and integrated elsewhere within the same exon. In some instances multiple fragments of an amplified theme were integrated at several sites. Direct repeats were found to prevail over inverted ones. By reconstructing the fate of repeats in the course of evolution of vertebrates, the origins of some functional domains could be traced to the initial amplification event. For example, an N-myc-specific domain was created by tandem duplication of a single-copy theme of L-myc exon; at the time of divergence of the c-myc and N-myc, the tandem duplex underwent a new round of duplication followed by transposition of the new copy, thus accounting for the formation of a new domain specific for c-myc. The model proposed here may be regarded as a molecular-level equivalent of the theory of punctuated equilibria. This is the author's last paper; it was submitted shortly before his death. The proofs were corrected by Michal Dvorak Correspondence to: M. Dvorak     

Diversity of rat strains and tumor lines of DNA fragments homologous to an amplified 5.8 kilobase ECO R1 fragment of Novikoff hepatoma cells

The nuclear DNA of several different rat strains and rat tumor lines have been analyzed with respect to Eco R1 fragments homologous to the amplified 5.8 kb Eco R1 fragment (fragment A) of Novikoff hepatoma cells. Two Eco R1 fragments, 4.8 and 4.4 kb, which hybridized to the 5.8 kb Eco R1 fragment, were found in all the genomes investigated. Although none of the examined genomes exhibited evidence of the same degree of amplification of fragment A related sequences as that of Novikoff hepatoma, several had Eco R1 fragments of various other sizes which were homologous to fragment A. These results indicate that the family of fragment A homologous sequences consists of two populations, the constant 4.8 and 4.4 kb fragments, and a second group of sequences which varies with respect to size.     

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.     

Registration of S alleles in Brassica campestris L by the restriction fragment sizes of SLGs

Polymorphism of SLG (the S-locus glycoprotein gene) in Brassica campestris was analyzed by PCR-RFLP using SLG-specific primers. Nucleotide sequences of PCR products from 15 S genotypes were determined in order to characterise the exact DNA fragment sizes detected in the PCR-RFLP analysis. Forty-seven lines homozygous for 27 S-alleles were used as plant material. One combination of primers, PS5 + PS 15, which had a nucleotide sequence specific to a class-I SLG, gave amplification of a single DNA fragment of approximately 1.3kb from the genomic DNA of 15 S genotypes. All the DNA fragments showed different electrophroetic profiles from each other after digestion with MboI or MspI. Different lines having the same S genotype had an identical electrophoretic profile even between the lines collected in Turkey and in Japan. Another class-I SLG-specific primer, PS 18, gave amplification of a 1.3-kb DNA fragment from three other S genotypes in combination with PS 15, and the PCR product also showed polymorphism after cleavage with the restriction endonucleases. Genetic analysis, Southern-hybridization analysis, and determination of the nucleotide sequences of the PCR products suggested that the DNA fragments amplified with these combinations of primers are class-I SLGs. Expected DNA fragment sizes in the present PCR-RFLP condition were calculated from the determined nucleotide sequence of SLG PCR products. A single DNA fragment was also amplified from six S genotypes by PCR with a combination of primers, PS3 + PS21, having a nucleotide sequence specific to a class-II SLG. The amplified DNA showed polymorphisnm after cleavage with restriction endonucleases. The cleaved fragments were detected by Southern-hybridization analysis using a probe of S ⁵ SLG cDNA, a class-IISLG. Partial sequencing revealed a marked similarity of these amplified DNA fragments to a class-II SLG, demonstrating the presence of class-I and class-II S alleles also in B. campestris. The high SLG polymorphism detected by the present investigation suggests the usefulness of the PCR-RFLP method for the identification of S alleles in breeding lines and for listing S alleles in B. campestris.     

Maize DNA enrichment by representational difference analysis in a maize chromosome addition line of oat

 The recent recovery of maize (Zea mays L.) single-chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses has provided novel source materials for the potential isolation of maize chromosome-specific sequences for use in genetic mapping and gene cloning. We report here the application of a technique, known as representational difference analysis (RDA), to selectively isolate maize sequences from a maize chromosome-3 addition line of oat. DNA fragments from the addition line and from the oat parent were prepared using BamHI digestion and primer ligation followed by PCR amplification. A subtractive hybridization technique using an excess of the oat parental DNA was employed to reduce the availability for amplification of DNA fragments from the addition lines that were in common with the ones from the oat parental line. After three rounds of hybridization and amplification, the resulting DNA fragments were cloned into a plasmid vector. A DNA library containing 400 clones was constructed by this method. In a test of 18 clones selected at random from this library, four (22%) detected maize-specific repetitive DNA sequences and nine (50%) showed strong hybridization to genomic DNA of maize but weak hybridization to genomic DNA of oat. Among these latter nine clones, three detected low-copy DNA sequences and two of them detected DNA sequences specific to chromosome 3 of maize, the chromosome retained in the source maize addition line of oat. The other eight out of the 13 clones that had strong hybridization to maize DNA detected repetitive DNA sequences or high-copy number sequences present on most or all maize chromosomes. We estimate that the maize DNA sequences were enriched from about 1.8% to over 72% of the total DNA by this procedure. Most of the isolated DNA fragments detected multiple or repeated DNA sequences in maize, indicating that the major part of the maize genome consists of repetitive DNA sequences that do not cross-hybridize to oat genomic sequences. Received: 18 November 1997 / Accepted: 12 March 1998     

Construction of two BAC libraries from cucumber (Cucumis sativus L.) and identification of clones linked to yield component quantitative trait loci

Two bacterial artificial chromosome (BAC) libraries were constructed from an inbred line derived from a cultivar of cucumber (Cucumis sativus L.). Intact nuclei were isolated and embedded in agarose plugs, and high-molecular-weight DNA was subsequently partially digested with BamHI or EcoRI. Ligation of double size-selected DNA fragments with the pECBAC1 vector yielded two libraries containing 23,040 BamHI and 18,432 EcoRI clones. The average BamHI and EcoRI insert sizes were estimated to be 107.0 kb and 100.8 kb, respectively, and BAC clones lacking inserts were 1.3% and 14.5% in the BamHI and EcoRI libraries, respectively. The two libraries together represent approximately 10.8 haploid cucumber genomes. Hybridization with a C₀t-1 DNA probe revealed that approximately 36% of BAC clones likely carried repetitive sequence-enriched DNA. The frequencies of BAC clones that carry chloroplast or mitochondrial DNA range from 0.20% to 0.47%. Four sequence-characterized amplified region (SCAR), four simple sequence repeat, and an randomly amplified polymorphic DNA marker linked with yield component quantitative trait loci were used either as probes to hybridize high-density colony filters prepared from both libraries or as primers to screen an ordered array of pooled BAC DNA prepared from the BamHI library. Positive BAC clones were identified in predicted numbers, as screening by polymerase chain reaction amplification effectively overcame the problems associated with an overabundance of positives from hybridization with two SCAR markers. The BAC clones identified herein that are linked to the de (determinate habit) and F (gynoecy) locus will be useful for positional cloning of these economically important genes. These BAC libraries will also facilitate physical mapping of the cucumber genome and comparative genome analyses with other plant species.     

PCR-RFLP of S locus for identification of breeding lines in cruciferous vegetables

A simple method of detecting polymorphism of S locus glycoprotein gene, SLG, in Chinese cabbage and cabbage was developed, and used for identification of breeding lines. DNA was amplified by the polymerase chain reaction (PCR) with a pair of primers having S ₆ SLG sequences from inbred lines, and digested with restriction endonucleases which recognize tetranucleotide sequences. The cleaved DNA fragments were size-fractionated by polyacrylamide gel electrophoresis and detected by silver staining. PCR with S ₆ SLG primers amplified a fragment of ca. 1.3kb in more than half of the inbred lines tested. After digestion, polyacrylamide gel electrophoresis revealed polymorphism between the amplified 1.3kb DNA fragments. These polymorphic bands were detected by Southern hybridization using a probe of S ₆ SLG cDNA, suggesting that the amplified DNA was SLG. Primers having the SLG sequences of S ₂ , a representative of recessive S alleles, were used for amplification of SLG in the lines which did not give the 1.3kb DNA fragment by the PCR with S ₆ SLG primers. Polymorphism of amplified DNA was found in these lines. However these primers also appeared to amplify an invariant SLR-2 sequence of 1.3kb in addition to the polymorphic S ₂ SLG related sequences. Although the used primer sequences still need improvement for the analysis of recessive S alleles, PCR-RFLP of SLG was considered to be useful for identification of breeding lines as well as for S allele identification in cruciferous vegetables. F₁ hybrids exhibited the sum of the bands of both parents, and, therefore, this method is expected to be used for a purity test of F₁ seeds.     

A p53 mutation is required for stable transformation of REF52 cells by themyc andras oncogenes

It is known that neoplastic transformation of rodent primary embryonic fibroblasts culturedin vitro requires coexpression at least of two cooperating oncogenes. In the case of transduction into cells of oncogenesras andmyc, the cell transformation is poorly effective. To study some additional factors necessary for such transformation, c-myc and N-ras ^Asp12 were consecutively introduced into REF52 cells by retroviral infection, and the cell cultures obtained were analyzed. Expression ofmyc broke the regulation of the cell cycle, in particular, canceled the G1 phase arrest for cells with damaged DNA, despite the normal function of protein p53 and induction of the p53-responsive genep21 ^Waf1 in these cells. The subsequent transduction ofras led to morphological transformation of cells and an increase of p53 level. However, reversion of the transformed phenotype to normal morphology took place after less than five passages. On this background, rare clones generated the stable transformed cell lines characterized by accelerated proliferation and having a mutation in thep53 gene. Attempts to obtain stable transformed cell lines by transduction ofras into REF52 cells not expressing exogenousmyc were unsuccessful. Analysis of the stable transformed clones revealed a mutation at codon 271 of thep53 gene, a hot spot of mutations, which led to the replacement of arginine by cysteine. In these clones, p53 is accumulated owing to the increased life time, and has a flexible conformation, being able to interact with monoclonal PAb1620 and PAb240 antibodies recognizing alternative protein conformations. The results obtained suggest that p53 participates in negative regulation of the cell cycle under conditions of oncogenic stimulation, and its inactivation is necessary for full transformation of cells by cooperating oncogenesmyc andras.     

Selection of cells with different chromosomal localizations of the amplified c-myc gene during in vivo and in vitro growth of the breast carcinoma cell line SW 613-S

The c-myc gene is amplified in the human breast carcinoma cell line SW 613-S. At early in vitro passages, the extra copies of the gene were mainly localized in double minute chromosomes (DMs), as shown by in situ hybridization with a biotinylated c-myc probe. However, cells without DMs were also present in which the c-myc genes were found integrated into any of several distinct chromosomes (mainly 7q+, 4 and 4q+, and 1). When this cell line was propagated in vitro, the level of c-myc amplification decreased because cells with DMs and a high amplification level were lost and replaced by cells without DMs and having a low amplification level. On the contrary, when early passage SW 613-S cells were grown in vivo, as subcutaneous tumours in nude mice, cells with numerous DMs and a high level of c-myc amplification were selected for. In one cell line (SW 613-Tu1) established from such a tumour, the DM-containing cells were substituted at late passages for cells with a high number of c-myc copies integrated within an abnormally banded region, at band 17q24 of a 17q+ chromosome. When only cells with integrated genes were present, this cell line was still highly tumorigenic indicating that the localization of the c-myc genes in DMs was not required for these cells to be tumorigenic in nude mice. Furthermore, cells of the secondary tumours induced by SW 613-Tu1 did not contain any DMs showing that in vivo growth did not promote the release of integrated c-myc copies into DMs.     

Gene mapping on human metaphase chromosomes by in situ hybridization with 3H, 35S,and 32P labeled probes and transmission electron microscopy

A method based on in situ hybridization, autoradiography and transmission electron microscopy for mapping genes on human metaphase chromosomes is presented. Successful mapping of the tandemly repeated rDNA genes and of two nucleic acid probes, N-myc and probe 3 (Kanda et al. 1983), that are amplified in a homogeneously staining region (HSR) of the neuroblastoma cell line, IMR-32 is described. By using sufficiently thin AgBr emulsions, it is possible to obtain observable grains and good resolution with probes radiolabeled with ³H, ³⁵S, or ³²P, but the former gives the best results. We observe that neither of the two probes, N-myc and probe 3, has a uniform spatial distribution along the HSR and that the distributions of the two probes differ from each other. These observations support previous studies which indicated that the formation of an HSR is a more complex process than uniform amplification of a single DNA segment to form an n-fold set of perfect tandem repeats. The present study shows that the electron microscopic method is useful for extending the results of light microscopic studies for problems where higher resolution mapping is needed.     

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.     

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.     

Pattern of evolution of amplicons during the development of multiple drug resistance in Djungarian hamster cells

Six cloned DNA fragments representing different portions of the genomic region amplified in multidrug resistant Djungarian hamster cells were used to study amplicon variations in a large number of the resistant cell lines. Expressed correlation exists between the degree of 3 cloned sequences amplification and the level of multidrug resistance. Three other cloned regions amplify coordinately with the latter ones at the initial steps of selection. Later their amplification halts and they mao even eliminate from amplicons of highly resistant cells. The rates and order of elimination of these sequences vary among different independently derived series of multidrug resistant cell lines.     

Detection of Two Fungal Biocontrol Agents against Root-knot Nematodes by RAPD Markers

The strain ZK7 of Pochonia chlamydosporia var. chlamydosporia and IPC of Paecilomyces lilacinus are highly effective in the biological control against root-knot nematodes infecting tobacco. When applied, they require a specific monitoring method to evaluate the colonization and dispersal in soil. In this work, the randomly amplified polymorphic DNA (RAPD) technique was used to differentiate between the two individual strains and 95 other isolates, including isolates of the same species and common soil fungi. This approach allowed the selection of specific fragments of 1.2 kb (Vc1200) and 2.0 kb (Vc2000) specific for ZK7, 1.4 kb (P1400) and 0.85 kb (P850) specific for IPC, using the random Primers OPL-02, OPD-05, OPD-05 and OPC-11, respectively. These fragments were cloned, sequenced, and used to design sequence-characterized amplification region (SCAR) primers specific for the two strains. In classical polymerase chain reaction (PCR), with serial dilution of ZK7 and IPC pure culture DNAs template, the detection limits of these oligonucleotide SCAR-PCR primers were found to be 10, 1000, 500, 100 pg, respectively. In the dot blotting, digoxigenin (DIG)-labeled amplicons from these four primers specifically recognized the corresponding fragments in the DNAs template of these two strains. The detection limit of these amplicons were 0.2, 0.2, 0.5, 0.5 μg, respectively.