Breakpoint junctions of chromosome 9p deletions in two human glioma cell lines.

Interstitial deletions of the short arm of chromosome 9 are associated with glioma, acute lymphoblastic leukemia, melanoma, mesothelioma, lung cancer, and bladder cancer. The distal breakpoints of the deletions (in relation to the centromere) in 14 glioma and leukemia cell lines have been mapped within the 400 kb IFN gene cluster located at band 9p21. To obtain information about the mechanism of these deletions, we have isolated and analyzed the nucleotide sequences at the breakpoint junctions in two glioma-derived cell lines. The A1235 cell line has a complex rearrangement of chromosome 9, including a deletion and an inversion that results in two breakpoint junctions. Both breakpoints of the distal inversion junction occurred within AT-rich regions. In the A172 cell line, a tandem heptamer repeat was found on either side of the deletion breakpoint junction. The distal breakpoint occurred 5' of IFNA2; the 256 bp sequenced from the proximal side of the breakpoint revealed 95% homology to long interspersed nuclear elements. One- and two-base-pair overlaps were observed at these junctions. The possible role of sequence overlaps, and repetitive sequences, in the rearrangement is discussed.     

Localization of the t(2;13) breakpoint of alveolar rhabdomyosarcoma on a physical map of chromosome 2.

A characteristic translocation t(2;13)(q35;q14) has been previously identified in the pediatric soft tissue tumor alveolar rhabdomyosarcoma. We have assembled a panel of lymphoblast, fibroblast, and somatic cell hybrid cell lines with deletions and unbalanced translocations involving chromosome 2 to develop a physical map of the distal 2q region. Twenty-two probes were localized on this physical map by Southern blot analysis of the mapping panel. The position of these probes with respect to the t(2;13) rhabdomyosarcoma breakpoint was then determined by quantitative Southern blot analysis of an alveolar rhabdomyosarcoma cell line with two copies of the derivative chromosome 13 and one copy of the derivative chromosome 2 and by analysis of somatic cell hybrid clones derived from an alveolar rhabdomyosarcoma cell line. We demonstrate that the t(2;13) breakpoint is situated within a map interval delimited by the distal deletion breakpoint in fibroblast line GM09892 and the t(X;2) breakpoint in somatic cell hybrid GM11022. Furthermore, from a comparison of our data with the linkage map of the syntenic region on mouse chromosome 1, we conclude that the t(2;13) breakpoint is most closely flanked by loci INHA and ALPI within this map interval.     

毛细胞白血病5q13.3断裂位点线性DNA荧光原位杂交定位

毛细胞白血病经常与５ｑ１３．３断裂位点相关联,该断裂位点区域及位于这一区域的重要基因有待研究。我们探索了ＤＮＡ纤维荧光原位杂交方法（即ＤＮＡ纤维ＦＩＳＨ）检测该断裂位点的可行性。实验选用含有断裂位点区域的两个基因组克隆及位于断裂位上是的两个ｃｏｓ质粒探针与带有结构性到位（５）（ｐ１３．１ｑ１３．３）的线性ＤＮＡ共杂交（ＤＮＡ来自ＨＣＬ患者的细胞系）。实验证明该断裂位点将探针信号一分为二。根据这些结果描绘出断裂位点区域图。研究表明,ＤＮＡ纤维ＦＩＳＨ方法是绘制高精度物理图谱和检出遗传重排的一种有效的研究手段。     

An analysis of Xq deletions

We characterized by fluorescence in situ hybridization and Southern blotting 14 partial Xq monosomies, 11 due to terminal deletions and 3 secondary to X/autosome translocations. Three cases were mosaics with a XO cell line. In view of the possible role played by telomeres in chromosome segregation, we hypothesize a relationship between the loss of telomeric sequences in terminal deletions and the presence of 45,X cells. A correlation between phenotype and extent of deletion revealed that there is no correspondence between the size of the deletion and impairment of gonadal function. Turner stigmata are absent in patients without an XO cell line, when the breakpoint is distal to Xg24. A low birthweight is present whenever the breakpoint is at q22 or more proximal.     

Hypomagensemia with secondary hypocalcemia in a female with balanced X;9 translocation: mapping of the Xp22 chromosome breakpoint

Magnesium-dependent hypocalcaemia (HSH), a rare inherited disease, is caused by selective disorders of magnesium absorption. Both X-linked and autosomal recessive modes of inheritance have been reported for HSH; this suggests a genetically heterogeneous condition. A balanced de novo t(X;9)(p22;q12) translocation has been reported in a female manifesting hypomagnesemia with secondary hypocalcemia. In a lymphoblastoid cell line, derived from this patient, the normal X chromosome is preferentially inactivated, suggesting that the patient's phenotype is caused by disruption of an HSH gene in Xp22. In an attempt to define more precisely the position of the X breakpoint, we have constructed a hybrid cell line retaining the der(X)(Xqter-Xp22.2::9q12-9qter) in the absence of the der(9) and the normal X chromosome. Southern blot analysis of this hybrid and in situ hybridization on metaphase chromosomes have localized the breakpoint between DXS16 and the cluster (DXS207, DXS43), in Xp22.2. Thus, if a gene involved in HSH resides at or near the translocation breakpoint, our findings should greatly facilitate its isolation.     

Molecular characterisation of the t(1;15)(p22;q22) translocation in the prostate cancer cell line LNCaP

Although chromosome translocations are well-documented recurrent events in hematological malignancies and soft tissue sarcomas, their significance in carcinomas is less clear. We report here the molecular characterization of the reciprocal translocation t(1;15)(p22;q22) in the prostate carcinoma cell line, LNCaP. The chromosome 1 breakpoint was localized to a single BAC clone, RP11-290M5, by sequential FISH analysis of clones selected from the NCBI chromosome 1 map. This was further refined to a 580-bp region by Southern blot analysis. A 2.85-kb fragment spanning the der(1) breakpoint was amplified by long-range inverse PCR. The breakpoint on chromosome 1 was shown to lie between the CYR61 and the DDAH1 genes with the der(1) junctional sequence linking the CYR61 gene to the TSPAN3 (TM4SF8) gene on chromosome 15. Confirmatory PCR and FISH mapping of the der(15) showed loss of chromosome material proximal to the breakpoint on chromosome 15, containing the PSTPIP1 and RCN2 genes. On the available evidence we conclude that this translocation does not result in an in-frame gene fusion. Comparative expressed sequence hybridization (CESH) and comparative genomic hybridization (CGH) analysis, showed relative down-regulation of gene expression surrounding the breakpoint, but no gross change in genomic copy number. Real-time quantitative RT-PCR for genes around the breakpoint supported the CESH data. Therefore, here we may have revealed a gene down-regulation mechanism associated with a chromosome translocation, either through small deletion at the breakpoint or through another means of chromosome domain related gene regulation.     

双色FISH和DNA纤维FISH方法的建立与应用

在构建了含毛细胞白血病相关的结构性倒位inv (5) (p13.1q13.3)的细胞系后,为了确定该新建细胞系在建株过程中其倒位断裂点关键区遗传物质是否发生改变,以生物素或地高辛标记的cCI5-216 和cCI5-267黏粒DNA为探针,进行染色体中期、间期和DNA纤维3种双色荧光原位杂交的分析。结果表明：该新建细胞系的3种双色荧光原位杂交结果,均与该细胞系的原代细胞的完全相同,证实了该细胞系倒位断裂点关键区的遗传物质结构未发生改变。该细胞系是揭示毛细胞白血病发病的分子机理的重要研究材料。     

Molecular characterization of ataxia telangiectasia T cell clones

Summary We compared inversions of chromosome 14 in an ataxia telangiectasia clone and in a malignant T cell line (SUPT1). The R-banding chromosome analysis showed a clear difference between the distal breakpoint of the two inversions. Fine mapping of the distal breakpoint in the ataxia telangiectasia inv(14) was performed by in situ hybridization. We conclude that this breakpoint is centromeric to the immunoglobulin heavy chain locus and to the D14S1 anonymous locus. Our results favor the existence of an unknown oncogene in band 14q32.1.     

Localization of the translocation breakpoint in a female with Menkes syndrome to Xq13.2-q13.3 proximal to PGK-1.

Menkes syndrome is a rare X-linked recessive disorder characterized by an inability to metabolize copper. A female patient with both this disease and an X; autosome translocation with karyotype 46,X,t(X;2)(q13;q32.2) has previously been described. The translocation breakpoint in Xq13 coincides with a previous assignment of the Menkes gene at Xq13 by linkage data in humans and by analogy to the mottled mutations which are models for Menkes disease in the mouse. Therefore, this translocation probably interrupts the gene for Menkes syndrome in band Xq13. We describe here experiments to precisely map the translocation breakpoint within this chromosomal band. We have established a lymphoblastoid cell line from this patient and have used it to isolate the der(2) translocation chromosome (2pter----2q32::Xq13----Xqter) in human/hamster somatic cell hybrids. Southern blot analyses using a number of probes specific for chromosomes X and 2 have been studied to define precisely the location of the translocation breakpoint. Our results show that the breakpoint in this patient--and, therefore, likely the Menkes gene--maps to a small subregion of band Xq13.2-q13.3 proximal to the PGK1 locus and distal to all other Xq13 loci tested.     

Localization of the rhabdomyosarcoma t(2;13) breakpoint on a physical map of chromosome 13.

Previous investigations of the pediatric soft tissue tumor alveolar rhabdomyosarcoma have identified a characteristic translocation t(2;13)(q35;q14). We have employed a physical mapping strategy to localize the site of this translocation breakpoint on chromosome 13. Using a panel of somatic cell hybrid and lymphoblast cell lines with deletions and unbalanced translocations involving chromosome 13, we have mapped numerous probes from the 13q12-q14 region and demonstrate that this region is divisible into five physical intervals. These probes were then mapped with respect to the t(2;13) rhabdomyosarcoma breakpoint by quantitative Southern blot analysis of an alveolar rhabdomyosarcoma cell line with two copies of the derivative chromosome 13 and one copy of the derivative chromosome 2. Our findings demonstrate that the t(2;13) breakpoint is localized within a map interval delimited by the proximal deletion breakpoints in lymphoblast lines GM01484 and GM07312. Furthermore, the breakpoint is most closely flanked by loci D13S29 and TUBBP2 within this map interval. These findings will facilitate chromosomal walking strategies for cloning the regions disrupted by the alveolar rhabdomyosarcoma translocation. In addition, this physical map will permit rapid determination of the proximity of new cloned sequences to the translocation breakpoint.     

Equivalent circuit models for theI–V relationship in epithelial tissues

In many epithelia, a plot of the voltage resulting from a short (1 sec) current pulse vs. current (theI–V relationship) has been found to consist of a number of straight line regions (constant resistance) which intersect at breakpoints. If the tissue is modeled by an equivalent circuit consisting of a single emf and resistances, it is shown that replacement of a resistance with a non-ideal diode (with finite forward and back resistances) produces a single breakpoint at a voltage calculable from the circuit parameters. One breakpoint is produced per diode, up to a maximum of three. It is shown that no single-emf circuit can produce more than 3 breakpoints, or produce a breakpoint at a voltage greater than the open circuit potential. Such observations imply that more complex circuits containing more than one emf must be used for these cases.     

Molecular studies in three patients with isodicentric Y chromosome

Three patients carrying an isodicentric (idic) Y chromosome associated with a mosaic 45,X cell line were studied using molecular techniques. Genotype-phenotype correlations suggested an effect of the 45,X cell line on sexual differentiation. A relationship was established between instability of the idic(Y) chromosome and localization of the breakpoint on Yq, and between azoospermia and deletion of interval 6 on Yq. Received: 31 May 1996 / Revised: 12 July 1996     

N segment insertion and region-directed somatic hypermutation in a kappa gene of a t(2;8) chromosomal translocation.

A detailed molecular analysis of both reciprocal recombination products of the variant t(2;8) chromosomal translocation of the Burkitt lymphoma derived cell line JI and their germline counterparts was carried out. The breakpoint on chromosome 8 is localized 28 kb to the 3' side of the c-myc protooncogene, the breakpoint on chromosome 2 was found to be within an aberrantly rearranged VK gene (abbreviations ref. 1). Novel features of the immunoglobulin moiety involved in this process include insertion of extra nucleotides in the V-J junction which have the characteristics of a N segment as it has been found up to now only in heavy chain and T cell receptor genes; the occurrence of somatic mutations in 8q+ and not in 2p-. These data allow a reconstruction of the course of events in the cell line JI; remarkable sequence regularities at the chromosomal breakpoints consisting of symmetrically placed dinucleotides and elements related to the hepta- and nonanucleotide recombinase recognition sequences are discussed in the context of the translocation mechanism.     

Burkitt lymphoma cell line carrying a variant translocation creates new DNA at the breakpoint and violates the hierarchy of immunoglobulin gene rearrangement.

The Burkitt lymphoma cell line KK124, which contains a reciprocal t(8;22) translocation, was shown to have rearranged in a region 3' to the c-myc proto-oncogene on chromosome 8 and 5' to the lambda constant region on chromosome 22. The breakpoint was cloned and sequenced, revealing that c-myc and a portion of its 3' region abutted a complete lambda variable gene that had undergone V-J recombination. Since this cell line expresses kappa light chain, this lambda rearrangement violates the previously proposed hierarchy of immunoglobulin gene rearrangement. A novel duplication of normal chromosome 8 sequences was also found at the breakpoint. The first exon of c-myc and its flanking sequence from the translocated allele was sequenced and compared with a normal counterpart. Extensive mutation was found within the first exon in contrast to its 3' and 5' flanking regions. S1 nuclease analysis revealed that it was the translocated c-myc being expressed and that there was a promoter shift from P2 to P1. The detailed structural analysis of this cell line provides clues concerning mechanisms of chromosomal translocation and c-myc deregulation in Burkitt lymphomas.     

Rearrangement of the c-myc proto-oncogene locus in a cell line of T-lymphoblastic origin

A molecular rearrangement of the proto-oncogene c-myc located downstream of the exon III 3' end has been found in a cell line derived from KE37 cell line established from an acute T-lymphoblastic leukemia. This rearrangement resulted from a chromosomal translocation t(8; 14)(q24; q11). Since the 14q11 chromosomal band has been found to be involved in several T-cell leukemias and lymphomas, the importance of the rearrangement of c-myc discovered in the KE37 cell line lies in the possibility of analyzing chromosome 14 DNA near the breakpoint involved in the translocation.     

Chromosome translocations in breast cancer with breakpoints at 8p12

Unbalanced chromosome translocations with breakpoints around 8p12, resulting in loss of distal 8p, are common in carcinomas. We have mapped the 8p12 breakpoints in three breast cancer cell lines, T-47D, MDA-MB-361, and ZR-75-1, using YACs and PACs between D8S540 and D8S255 by fluorescence in situ hybridization. All three lines had a breakpoint close to D8S505, proximal to HGL. Each breakpoint was distinct, but all were within 0.5 to 1.5 Mb of each other. The T-47D cell line had a straightforward translocation, but in MDA-MB-361 and ZR-75-1 the translocations were accompanied by local rearrangements of surprising complexity. Small regions of 8p from close to the breakpoint were duplicated or amplified as inserts in the attached chromosome fragment. ZR-75-1 also had retained a separate fragment of about 1 Mb, from the region 1 to 3 Mb telomeric to the common breakpoint, that included HGL. This line also had an interstitial deletion several megabases more centromeric. The data suggest that breakpoints on 8p12 are clustered in a small region and show that translocations breaking there may be accompanied by additional rearrangements.     

The Cloning of the Bar Region and the B Breakpoint in Drosophila Melanogaster: Evidence for a Transposon-Induced Rearrangement

We have cloned the B breakpoint in Drosophila melanogaster using DNA from a P-M-induced revertant of B, which has a P element inserted at the B breakpoint. The analysis of the B DNA reveals that there is a transposable element, B104, right at the breakpoint. This suggests that this element may have been involved in the generation of the B breakpoint and the associated tandem duplication. One possible mechanism to generate the B duplication is a recombination event between two B104 elements, one at 16A1 and the other at 16A7. DNA sequencing data of the junctions of the B104 element support this model. Four partial revertants of B are the result of insertions of transposable elements very close to the B breakpoint. This supports the hypothesis that the breakpoint is the cause of the B mutation. The clones from B were used to isolate wild-type clones from 16A1, the location of the Bar gene. Four rearrangement breakpoints associated with various Bar mutations map within a 37-kb region, suggesting that the Bar gene is very large.     

The t(8;14) breakpoint of the EW 36 undifferentiated lymphoma cell line lies 5' of MYC in a region prone to involvement in endemic Burkitt's lymphomas

One of the best analyzed tumor-specific cytogenetic abnormalities is the t(8;14) chromosomal translocation observed in cases of Burkitt's and undifferentiated lymphomas (ULs), and acute lymphoblastic leukemias (ALLs). Here we analyze the cloned (8;14) chromosomal breakpoint of the UL cell line EW 36. We show that the region of chromosome 8 involved in the translocation is situated near a site previously demonstrated to harbor a cluster of endemic Burkitt's lymphoma breakpoints, approximately 50 kb 5' of MYC. In those cases, we demonstrated that malfunction of the V-D-J recombinase generated the translocations. However, in this case the isotype switch mechanism of translocation is implicated: at the breakpoint, S mu/S gamma and C gamma sequences are found on chromosome 14. Thus, the features of the EW 36 t(8;14) breakpoint are consonant with our model for B-cell lymphomagenesis which relates the precursor cell that gives rise to malignancy, the mechanism of translocation, and the phenotype of the tumor.