Identification of an amplified gene cluster in glioma including two novel amplified genes isolated by exon trapping

Gene amplification, which occurs in more than 50% of malignant gliomas, is considered to play a pivotal role in tumorigenesis. There are, however, few studies aimed toward the isolation of novel genes from amplified sequences. Previously, we reported amplification of the protooncogene MET (hepatocyte growth factor receptor; 7q31) in more than 20% of glioblastomas. For an approximate size estimation of the amplification unit we analyzed three glioblastomas all of which carried an amplified MET gene, by Southern blot analysis and/or competitive polymerase chain reaction using eight DNA markers. Although the extent of the amplified domain varied, the close vicinity of the MET gene was the only region consistently amplified in these glioblastomas. A yeast artificial chromosome (YAC) contig of 900 kb was refined spanning the amplified region flanking the MET gene. The YAC inserts were subcloned into 59 cosmids, which were used for exon trapping. Eight sequences were identical to parts of the genes MET and CAPZA2 (human actin capping protein α-subunit). Two newly identified exons and the CAPZA2 exons were amplified in tumor TX3095, which retains an amplified MET gene. The new exons were localized close to MET and CAPZA2. Characterization of the clones, which were termed glioma-amplified sequence (GAS)7-1 and GAS7-2, showed an open reading frame and a different expression pattern in multiple human tissues. This study reports the identification of a cluster of amplified genes including two novel genes in a region amplified in more than 20% of glioblastomas. Received: 27 June 1997 / Accepted: 18 July 1997     

Heterogeneity of the glutathione transferase genes encoding enzymes responsible for insecticide degradation in the housefly

One of the four glutathione-S-transferases (GST) that is overproduced in the insecticide-resistant Cornell-R strain of the housefly (Musca domestica) produces an activity that degrades the insecticide dimethyl parathion and conjugates glutathione to lindane. In earlier work, it was shown that the resistant Cornell-R carries an amplification, probably a duplication, of one or more of its GST loci and that this amplification is directly related to resistance. Using polymerase chain reaction (PCR) amplification with genomic DNA, multiple copies of the gene encoding the parathion-degrading activity (called MdGst-3) were subcloned from both the ancestral, insecticide-susceptible strain BPM and from the insecticide-resistant Cornell-R. In BPM, three different MdGst-3 genes were identified while in Cornell-R, 12 different MdGst-3 sequences were found that, though closely related to ancestral genes, had diverged by a few nucleotides. This diversity in MdGst-3 genomic sequences in Cornell-R is reflected in the expressed sequences, as sampled through a cDNA bank. Population heterozygosity cannot account for these multiple GST genes. We suggest that selection for resistance to insecticides has resulted in not only amplification of the MdGst-3 genes but also in the divergence of sequence between the amplified copies. Received: 22 November 1995 / Accepted: 23 February 1996     

A different view on DNA amplifications indicates frequent, highly complex, and stable amplicons on 12q13-21 in glioma

To further understand the biological significance of amplifications for glioma development and recurrencies, we characterized amplicon frequency and size in low-grade glioma and amplicon stability in vivo in recurring glioblastoma. We developed a 12q13-21 amplicon-specific genomic microarray and a bioinformatics amplification prediction tool to analyze amplicon frequency, size, and maintenance in 40 glioma samples including 16 glioblastoma, 10 anaplastic astrocytoma, 7 astrocytoma WHO grade 2, and 7 pilocytic astrocytoma. Whereas previous studies reported two amplified subregions, we found a more complex situation with many amplified subregions. Analyzing 40 glioma, we found that all analyzed glioblastoma and the majority of pilocytic astrocytoma, grade 2 astrocytoma, and anaplastic astrocytoma showed at least one amplified subregion, indicating a much higher amplification frequency than previously suggested. Amplifications in low-grade glioma were smaller in size and displayed clearly different distribution patterns than amplifications in glioblastoma. One glioblastoma and its recurrencies revealed an amplified subregion of 5 Mb that was stable for 6 years. Expression analysis of the amplified region revealed 10 overexpressed genes (i.e., KUB3, CTDSP2, CDK4, OS-9, DCTN2, RAB3IP, FRS2, GAS41, MDM2, and RAP1B) that were consistently overexpressed in all cases that carried this amplification. Our data indicate that amplifications on 12q13-21 (a) are more frequent than previously thought and present in low-grade tumors and (b) are maintained as extended regions over long periods of time.     

PCR analysis of x- and y-type genes present at the complex Glu-A1 locus in durum and bread wheat

Genes (x-type) corresponding to different high-molecular-weight glutenin subunits encoded at the Glu-A1 locus present in bread- and durum-wheat cultivars have been selectively amplified by the polymerase chain reaction (PCR). DNA fragments corresponding to an unexpressed x-type gene were also amplified. As unexpressed y-type genes may or may not contain an 8-kb transposon-like insertion, two different sets of primers were designed to obtain amplification of DNA fragments corresponding to these genes. Amplified DNA fragments were also digested with restriction enzymes. The digestion patterns of amplified fragments corresponding to unusual x-type subunits showed similarities with genes encoding the most common subunits 2^* and 1. The unexpressed amplified x-type gene showed a restriction pattern similar to the one obtained with the allelic gene encoding high-molecular-weight glutenin subunit 1; homologies were also found within the repetitive region of the linked y-type genes. On the basis of these observations it is postulated that an ancestral active x-type gene, most likely corresponding to subunit 1, was silenced following the insertion of the 8-kb transposon-like fragment into the linked y-type gene. Received: 8 April 1996 / Accepted: 30 August 1996     

Superantigen-like gene(s) in human pathogenic Streptococcus dysgalactiae,subsp equisimilis: genomic localisation of the gene encoding streptococcal pyrogenic exotoxin G (speG(dys))

Streptococcus pyogenes (GAS) causes about 90% of streptococcal human infections while group C (GCS) and G (GGS) streptococci can be pathogenic for different mammalians. Especially the human pathogenic GCS and GGS, Streptococcus dysgalactiae, subsp. equisimilis, account for 5-8% of the human streptococcal diseases like wound infections, otitis media, purulent pharyngitis and also streptococcal toxic shock syndrome. A defined superantigen so far was not identified in GCS and GGS strains. In the present investigation we screened DNA of GCS and GGS human isolates for the presence of genes for streptococcal pyrogenic exotoxins (spe) by hybridisation with probes that stand for the GAS genes speA, speC, speZ (smeZ), speH, speG, speI, speJ and ssa. In many GCS and GGS strains we found positive reactions with the probes speG, speJ and ssa, but not with the probes for the remaining genes under investigation. PCR amplification with subsequent sequence analysis of the PCR fragments revealed only the presence of the gene speG in GCS and GGS strains, while no DNA fragments specific for speJ and ssa could be amplified. Additionally, the upstream and downstream regions flanking speG in GGS strain 39072 were sequenced. Remarkable differences were found in the neighbourhood of speG between GAS and GGS sequences. Downstream of speG we identified in strain GGS 39072 two new open reading frames encoding proteins with no similarity to protein sequences accessible in the databases so far. In the compared GAS strains SF370 and MGAS8232, this segment, apart from some small fragments, had been deleted. Our analysis suggests that a gene transfer from GGS to GAS has preceded following deletion of the two genes orf1 and orf2 in GAS.     

Identification of novel candidate oncogenes in chromosome region 17p11.2-p12 in human osteosarcoma

Osteosarcoma is the most common primary malignancy of bone. The tumours are characterized by high genomic instability, including the occurrence of multiple regions of amplifications and deletions. Chromosome region 17p11.2-p12 is amplified in about 25% of cases. In previous studies, COPS3 and PMP22 have been identified as candidate oncogenes in this region. Considering the complexity and variation of the amplification profiles for this segment, the involvement of additional causative oncogenes is to be expected. The aim of the present investigation is to identify novel candidate oncogenes in 17p11.2-p12. We selected 26 of in total 85 osteosarcoma samples (31%) with amplification events in 17p11.2-p12, using quantitative PCR for 8 marker genes. These were subjected to high-resolution SNP array analysis and subsequent GISTIC analysis to identify the most significantly amplified regions. Two major amplification peaks were found in the 17p11.2-p12 region. Overexpression as a consequence of gene amplification is a major mechanism for oncogene activation in tumours. Therefore, to identify the causative oncogenes, we next determined expression levels of all genes within the two segments using expression array data that could be generated for 20 of the selected samples. We identified 11 genes that were overexpressed through amplification in at least 50% of cases. Nine of these, c17orf39, RICH2, c17orf45, TOP3A, COPS3, SHMT1, PRPSAP2, PMP22, and RASD1, demonstrated a significant association between copy number and expression level. We conclude that these genes, including COPS3 and PMP22, are candidate oncogenes in 17p11.2-p12 of importance in osteosarcoma tumourigenesis.     

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     

DNA sequences amplified in cancer cells: an interface between tumor biology and human genome analysis.

There is growing evidence that amplification of specific genes is associated with tumor progression. While several proto-oncogenes are known to be activated by amplification, it is clear that not all the genes involved in DNA amplification in human tumors have been discovered. Our approach to the identification of such genes is based on the 'reverse genetics' methodology. Anonymous amplified DNA fragments are cloned by virtue of their amplification in a given tumor. These sequences are mapped in the normal genome and hence define a new genetic locus. The amplified domain is isolated by long-range cloning and analyzed along three lines of investigation: new genes are sought that can explain the biological significance of the amplification; the structure of the domain is studied in normal cells and in the amplification unit in the cancer cell; attempts are made to identify molecular probes of diagnostic value within the amplified domain. This application of genome technology to cancer biology is demonstrated in our study of a new genomic domain at chromosome 10q26 which is amplified specifically in human gastric carcinomas.     

Regularities of karyotypic evolution during stepwise amplification of genes determining drug resistance.

Analysis of chromosomal alterations during stepwise development of mdr1, dhfr, or CAD gene amplifications in a large number of independently selected Djungarian hamster DM-15 and murine P388 sublines revealed typical patterns of karyotypic evolution, specific for multiplication of each of these genes in each cell type. Some principal similarities of karyotypic evolution were noted in at least two different systems. They include: (i) appearance at the first selection step of a new chromosomal arm bearing the resident gene copy followed at the next selection steps by the formation in these specific chromosomal arms of amplified DNA tandem arrays; (ii) translocations of amplified DNA from its initial site to other, also non-random, chromosomal sites; and (iii) emergence in the cell variants with high degrees of gene amplification of multiple extra-chromosomal elements. The most prominent distinctions among the systems were as follows: (i) different structures, evidently containing amplified DNAs, appeared at the initial steps of amplification of different genes--additional heterogeneously staining regions in specific chromosomal segments in the case of amplification of dhfr or CAD genes in DM-15 cells, and mini-chromosomes in the case of mdr1 gene amplification in both DM-15 and P380 cells; (ii) distinct patterns of location of the amplified mdr1 gene copies are characteristic of Djungarian hamster DM-15 and murine P388 cell derivatives after subsequent steps of selection--at the site of resident gene localization or in some other, also non-random, chromosomal sites in DM-15 sublines, and predominantly extra-chromosomal in P388 sublines. We propose that different mechanisms are responsible for the initial steps of amplification of dhfr and CAD genes on the one hand and the mdr1 gene on the other: non-equal sister-chromatid exchanges and autonomous replication of the extra-chromosomal elements. It seems, however, that both mechanisms may be involved in further rounds of amplification of each of these three genes.     

Gene amplification profiling of esophageal squamous cell carcinomas by DNA array CGH

Gene amplification is one of the basic mechanisms that lead to overexpression of oncogenes. DNA array comparative genomic hybridization (CGH) has great potential for comprehensive analysis of both a relative gene-copy number and altered chromosomal regions in cancers, which enables us to identify new amplified genes and unstable chromosomal loci. We examined the amplification status in 32 esophageal squamous cell carcinomas (ESCCs) and 13 ESCC cell lines on 51 frequently amplified loci in a variety of cancers by both DNA array CGH and Southern blot analyses. The 1p34 locus containing MYCL1, 2p24 (MYCN), 7p12 (EGFR), and 12q14 (MDM2) were amplified in one of the 32 cases (3%), and the 17q12 locus (ERBB2) and 8p11 (FGFR1) in two of the 32 cases (6%), while only the 11q13 locus (Cyclin D1, FGF4, and EMS1) was frequently amplified (28%, 9/32), demonstrating this locus to be a major target in ESCCs. One locus, 8q24 (c-MYC) was found to be amplified only in the cell lines. Eight out of 51 loci (15.7%) were found to be amplified in at least one of the 32 primary ESCCs or the 13 ESCC cell lines, suggesting that chromosomal loci frequently amplified in a type of human cancer may also be amplified in other types of cancers. This paper is the first report of an application of DNA array CGH to ESCCs.     

Construction and analysis of an HN-cDNA library derived from the p-arm of pig Chromosome 12

Our aim is to find unidentified genes on specific pig chromosomes or chromosome fragments. Our approach has involved the construction of a heterogeneous nuclear complementary (hn-c) DNA library of the p-arm of pig Chromosome (Chr) 12, the only pig chromosome present in the pig × hamster hybrid cell line 8990. Total RNA was extracted from the cells and first-strand synthesis of hn-cDNA carried out with random and oligo dT primers. Pig hn-cDNA was isolated by amplification of first-strand synthesized hn-cDNA with primers specific for Short Interspersed Repeat Elements (SINEs) via the polymerase chain reaction (PCR). Hn-cDNAs were size selected and cloned in E. coli XL-1 blue cells with PCR-Script as the vector. The library consisted of 6000 clones. Clone inserts were amplified by PCR with vector-specific primers, and randomly picked inserts greater than 600 bp were sequenced. Homology searches were carried out with the FASTA search program on the GenEmbl database. Thirty clones were sequenced, and of these three showed strong homologies to GenEmbl sequences: (1) to sheep, mouse, human, and rat mammary gland factor (MGF); (2) to MLN-50, a gene that is amplified in human familial breast cancer and is present on human Chr 17; the latter is homologous to pig chromosome 12; (3) to a family of unassigned overlapping human ESTs. Of the other sequenced clones, seven were over 80% homologous with pig SINE sequences; three were over 75% homologous to human LINE sequences; six displayed open reading frames over a mean distance equivalent to 50 amino acids, although these showed no significant similarities with sequences in the databases. Using this approach, we have been able to identify several new genes on the p-arm of pig Chr 12. This is the first report of gene isolation from a library derived from a pig chromosome fragment. Received: 9 February 1996 / Accepted: 14 May 1996     

Coamplification on chromosomes 7p12-13 and 9q12-13 identified by reverse chromosome painting in a glioblastoma multiforme

DNA amplification is known to occur in approximately 50% of glioblastomas, with the epidermal growth factor receptor (EGFR) gene being the most frequently amplified. Whereas previous amplification studies have largely been limited to the analysis of known tumor-related genes, reverse chromosome painting allows us to search for as yet unidentified amplified domains. Here, we report the analysis of a glioblastoma multiforme by reverse chromosome painting. Hybridization signals were found on chromosome 7p12-13 and chromosome 9q12-13. Standard Southern blot analysis revealed amplification of the EGFR gene, which is localized on band 7p13. These findings corroborate previous reports on coamplification of sequences on different chromosomes in glioblastoma.     

Analysis of chromosomal aberrations involving chromosome 1q31-->q53 in a DMBA-induced rat fibrosarcoma cell line: amplification and overexpression of Jak2

In a study of DMBA-induced rat fibrosarcomas we repeatedly found deletions and/or amplifications in the long arm of rat chromosome 1 (RNO1). Comparative genome hybridization showed that there was amplification involving RNO1q31-->q53 in one of the DMBA-induced rat fibrosarcoma tumors (LB31) and a cell culture derived from it. To identify the amplified genes we physically mapped rat genes implicated in cancer and analyzed them for signs of amplification. The genes were selected based on their locations in comparative maps between rat and man. The rat proto-oncogenes Ccnd1, Fgf4, and Fgf3 (HSA11q13.3), were mapped to RNO1q43 by fluorescence in situ hybridization (FISH). The Ems1 gene was mapped by radiation hybrid (RH) mapping to the same rat chromosome region and shown to be situated centromeric to Ccnd1 and Fgf4. In addition, the proto-oncogenes Hras (HSA11p15.5) and Igf1r (HSA15q25-->q26) were mapped to RNO1q43 and RNO1q32 by FISH and Omp (HSA11q13.5) was assigned to RNO1q34. PCR probes for the above genes together with PCR probes for the previously mapped rat genes Bax (RNO1q31) and Jak2 (RNO1q51-->q53) were analyzed for signs of amplification by Southern blot hybridization. Low copy number increases of the Omp and Jak2 genes were detected in the LB31 cell culture. Dual color FISH analysis of tumor cells confirmed that chromosome regions containing Omp and Jak2 were amplified and were situated in long marker chromosomes showing an aberrant banding pattern. The configuration of the signals in the marker chromosomes suggested that they had arisen by a break-fusion-bridge (BFB) mechanism.     

Analysis of thymidylate synthase gene amplification and of mRNA levels in the cell cycle

We report that the gene for thymidylate synthase (TS) is amplified in the mouse cell line L1210:C15 that was selectively grown in increasing concentrations of the competitive inhibitor of thymidylate synthase, CB3717. The gene is amplified 50-fold compared to the parental cell line. Amplification has not been accompanied by any major rearrangements, and the increase in gene copy number is reflected in elevation of thymidylate synthase mRNA levels. The amplification is relatively stable as there was only a 2- to 3-fold decrease in the number of amplified TS genes when cells were grown in the absence of selection for 375 generations. We also observe a 30- to 40-fold increase in number of copies of the dihydrofolate reductase gene with 7-fold elevation of the RNA product, and we suggest that this may be due to cross-inhibition of dihydrofolate reductase by CB3717. Thymidylate synthase mRNA levels in L1210 and L1210:C15 show no variation within the different phases of the cell cycle but are significantly reduced during quiescence.     

DNA copy number amplifications in sarcomas with homogeneously staining regions and double minutes

To identify DNA amplifications in sarcomas, comparative genomic hybridization was performed on 27 cases that were likely to display high-level DNA copy number gains. In all cases, chromosome banding analysis had revealed homogeneously staining regions or double minutes, i.e., cytogenetic signs of gene amplification. In most cases, gains predominated over losses. Low-level amplifications (ratio 1.3:1.5) were seen in 20 cases. High-level amplifications (ratio >1.5) exceeded the frequencies seen in published, unselected sarcomas of similar histotypes and were detected in 16 tumors: 4/4 osteosarcomas, 5/8 malignant fibrous histiocytomas, 3/7 leiomyosarcomas, 1/2 myosarcomas, 0/1 liposarcoma, 0/1 rhabdomyosarcoma, 1/1 pleomorphic sarcoma, 0/1 myxofibrosarcoma, 1/1 malignant mesenchymona, and 1/1 malignant schwannoma, with two to four chromosomal regions involved in nine tumors. Recurrent amplifications involved 1p33-p32, 5p15-p14, 7pter-p12, 7q21-qter, 8q21.3-qter, 11q22-q23, 16p13.2-p12, 19q12-q13.1, 20q11.2-qter, and 22q12-q13. Most of the recurrent gains/amplifications we detected have been reported in sarcomas previously. A novel gain/amplification was seen at 2q14.3-q21 in five cases of four sarcoma types. The disparate pattern of amplified sequences, the poor correspondence between the localization of low- and high-level amplifications, and the chromosomal position of homogeneously staining regions suggest the involvement of many genes in the amplifications and that the genes rarely maintain their native position in these tumors.     

Characterization of supernumerary rings and giant marker chromosomes in well-differentiated lipomatous tumors by a combination of G-banding,CGH, M-FISH,and chromosome- and locus-specific FISH

Supernumerary ring chromosomes and/or giant marker chromosomes are often seen in soft-tissue tumors of low-grade or borderline malignancy, such as well-differentiated liposarcomas or atypical lipomas. Classic cytogenetic banding techniques have proved insufficient to identify the genomic composition and structure of such rings and markers, but fluorescent in situ hybridization (FISH) studies have shown that they consist mainly of amplified material from chromosome 12, more specifically from bands 12q13-->q15. We have used the new FISH-based screening techniques comparative genomic hybridization (CGH) and multicolor-FISH (M-FISH) in combination with G-banding and analysis by chromosome- and locus-specific fluorescent in situ probes to examine in detail the karyotypic characteristics of 22 lipomatous tumors, most of them classified histologically as well-differentiated liposarcomas, selected because they had been shown to harbor rings and/or marker chromosomes. M-FISH, in contrast to G- banding, was found to be informative with regard to the chromosomal origin of the rings and other markers present, whereas CGH and hybridizations with locus-specific probes helped identify which subchromosomal regions were involved. We found that chromosome bands 12q15-->q21 were always gained, with 12q15-->q21 being amplified (i.e., a green-to-red ratio >2 by CGH) in 14 of 22 tumors. In three tumors, two distinct but close amplicons in 12q could be identified, corresponding to bands 12q13-->q15 and 12q21. The genomic segment 1q21-->q23 was gained in 12 cases, reaching the level of amplification in seven. Bands 6q24 and 7p15, whose pathogenetic involvement in liposarcomas has not been reported previously, were gained in three cases each. In addition, the rings and giant markers often contained interspersed sequences from several other chromosomes that did not give an equally clear impression of being nonrandomly involved.     

Adaptive Amplification

ABSTRACT

Modern techniques are revealing that repetition of segments of the genome, called amplification or gene amplification, is very common. Amplification is found in all domains of life, and occurs under conditions where enhanced expression of the amplified genes is advantageous. Amplification extends the range of gene expression beyond that which is achieved by control systems. It also is reversible because it is unstable, breaking down by homologous recombination. Amplification is believed to be the driving force in the clustering of related functions, in that it allows them to be amplified together. Amplification provides the extra copies of genes that allow evolution of functions to occur while retaining the original function. Amplification can be induced in response to cellular stressors. In many cases, it has been shown that the genomic regions that are amplified include those genes that are appropriate to upregulate for a specific stressor. There is some evidence that amplification occurs as part of a broad, general stress response, suggesting that organisms have the capacity to induce structural changes in the genome. This then allows adaptation to the stressful conditions. The mechanisms by which amplification arises are now being studied at the molecular level, but much is still unknown about the mechanisms in all organisms. Recent advances in our understanding of amplification in bacteria suggests new interpretations of events leading to human copy number variation, as well as evolution in general.     

Regional mapping of two human immunoglobulin V lambda genes and analysis of the V lambda locus in chronic myeloid leukaemia.

The human immunoglobulin V lambda locus has been studied in relation to chromosomal translocations involving chromosome 22. DNA probes for two V lambda genes which belong to different subgroups and do not cross hybridize, were used to show that both V lambda genes are located on the Philadelphia chromosome in chronic myeloid leukaemia (CML). Both genes map in band 22q11 to a region that is bounded on the distal side by the breakpoints for CML 9:22 translocations and on the proximal side by the breakpoint for an X:22 translocation. We have found no evidence for rearrangements or amplification of either V lambda gene in CML, in either the chronic or acute phases of the disease. In K562 cells which are derived from the pleural effusion of a patient with Ph1-positive CML, there appears to be no rearrangement of the V lambda genes, but they are both amplified about four times. We have estimated that the minimum size for the amplification unit in K562 cells is 186 kb.