c-myc and immunoglobulin kappa light chain constant genes are on the 8q+ chromosome of three Burkitt lymphoma lines with t(2;8) translocations.

We have determined the localization of c-myc and the immunoglobulin kappa light chain genes on the 8q+/2p- chromosomes of the three Burkitt lymphoma lines BL21, LY66 and LY91 with t(2;8) translocation by in situ hybridization. BL21 is characterized by a complex translocation in which a piece of chromosome 9 appears to be located between the fragments of chromosome 8 and 2 on the 8q+ chromosome. Our data indicate that in all three cell lines the c-myc gene is located on the 8q+ chromosome proximal to the breakpoint in band 8q24. In all cell lines examined the cluster of kappa variable genes has remained on the 2p- chromosome. In LY91 cells the major part of the joining region remained on 2p-, while the joining region has moved to 8q+ in the cell lines BL21 and LY66. In all three cell lines the constant kappa light chain gene was found on the 8q+ chromosome. The fact that an essentially identical pattern was found in the cell line BL21, with the complex translocation, suggests that the insertion of the piece of chromosome 9 into the 8q+ chromosome might be a secondary event. Our present data fit into the concept that in all Burkitt lymphoma lines investigated so far, including cases with t(8;14) and the variant translocations t(2;8) and t(8;22), the c-myc gene becomes situated at the 5' side of an immunoglobulin constant gene. This may have implications for the generation of somatic mutations in the coding and non-coding part of the c-myc gene.     

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.     

Clustered somatic mutations in and around first exon of non-rearranged c-myc in Burkitt lymphoma with t(8;22) translocation.

We have examined the restriction map of the c-myc gene in 15 BL cell lines carrying the variant t(8;22) translocation in which c-myc is known to remain on chromosome 8. Using 3 restriction enzymes cutting outside the c-myc domain (EcoRI, BamHI, HindIII), we found no evidence for a c-myc/Ig lambda rearrangement in 14 BL cell lines. In the last one, BL 37, the 3' flanking region was rearranged corresponding to the already identified breakpoint located 400 pb downstream from the c-myc gene (9). Using 4 restriction enzymes cutting inside the c-myc gene (PvuII, PstI, SacI, HincII) we looked for discrete abnormalities within the gene limits, and we found in 9 BL cell lines several abolished and created sites, compatible with multiple independent somatic mutations. They are significantly clustered in the 5' non coding region, with a striking prevalence at the end of exon 1. The role of mutations in the non-coding first exon region for the deregulation of c-myc expression is discussed.     

Episomal and integrated copies of Epstein-Barr virus coexist in Burkitt lymphoma cell lines.

The Epstein-Barr virus genome is present in more than 95% of the African cases of Burkitt lymphoma. In this tumor, the viral genome is usually maintained in multiple episomal copies. Viral integration has been described only for Namalwa, a cell line lacking episomes. In this study, we have addressed the question of whether integrated and episomal copies can coexist in Burkitt lymphoma cells. Gel electrophoresis was used to demonstrate the presence of episomal as well as free linear DNA in three Burkitt lymphoma cell lines. The numbers of episomal copies per cell were estimated to be 5 to 10 in BL36 and BL137 cells and below 1 in BL60 cells, indicating that BL60 does not represent a homogeneous cell population. Fluorescence in situ hybridization was combined with chromosomal banding to study the association of the viral DNA with metaphase chromosomes. A symmetrical pattern of signals at both chromatids located at the same chromosomal sites in many if not all metaphases was taken as evidence for viral integration. In each of the three cell lines, one site of integration was identified: at chromosome 11p15 in BL36 cells, at chromosome 1p34 in BL137 cells, and at the site of a reciprocal t(11;19) translocation in BL60 cells. Integrated, episomal and linear copies of Epstein-Barr virus DNA thus coexist in Burkitt lymphoma cells. The biological significance of viral integration in Burkitt lymphoma cells remains to be elucidated.     

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.     

Structural analysis of both products of a reciprocal translocation between c-myc and immunoglobulin loci in Burkitt lymphoma.

The balanced translocations that occur between the c-myc and immunoglobulin loci in Burkitt lymphoma provide an unusual opportunity to analyze both products of a reciprocal recombination. Accordingly, we have determined the structure of the two reciprocal products of a translocation that joins the 5' portion of the c-myc gene on chromosome 8 to the immunoglobulin mu switch recombination signal on chromosome 14. By determining the nucleotide sequences at the translocation crossover points of both product chromosomes, we precisely locate these points with respect to nearby genes. This determination allows us to conclude that translocation involves nonhomologous recombination, is highly conservative of c-myc sequences (deleting only 16 bp at the crossover point), but deletes over 2 Kb of immunoglobulin sequences from the mu switch signal. The mu constant and c-myc genes are joined head-to-head about 3 Kb apart, while the IgH enhancer and an aberrantly rearranged D/J region are linked to sequences 5' of c-myc on the reciprocal product.     

Radiation-induced nonhomoeologous wheat-Agropyron intermedium chromosomal translocations conferring resistance to leaf rust

Summary The Agropyron intermedium chromosome 7Ai #2 is the source of the leaf rust resistance gene Lr38 which was transferred to wheat by irradiation. The chromosomal constitutions of eight radiation-induced rust-resistant wheat-Agropyron intermedium derivatives were analyzed by C-banding and genomic in-situ hybridization (GISH). Five lines were identified as wheat Ag. intermedium chromosome translocation lines with the translocation chromosomes T2AS·2AL-7Ai#2L, T5AL · 5AS-7Ai # 2L, T1DS · 1DL-7Ai # 2L, T3DL · 3DS-7Ai#2L, and T6DS · 6DL-7Ai#2L. The sizes of the 7Ai#2L segments in mitotic metaphases of these translocations are 2.42 m, 4.20 m, 2.55 m, 2.78 m, and 4.19 m, respectively. One line was identified as a wheat-Ag. intermedium chromosome addition line. The added Ag. intermedium chromosome in this line is different from 7Ai # 2. This line has resistance to leaf rust and stem rust. Based on the rust reactions, and the C-banding and GISH results, the remaining two lines do not contain any Ag. intermedium-derived chromatin.     

小麦遗传背景对黑麦抗叶锈基因Lr26的抗性表达的影响

利用1套从小麦纯系和黑麦自交系培育出的1R附加系、代换系和易位系,研究了1RS上的抗叶锈基因Lr26在小麦中的表达。结果发现,1R二体附加系和纯合1RS/1BL易位系高抗小麦叶锈病;而其小麦亲本、1R(1B)代换系和1BS/1RL易位系重感叶锈病。这一结果指出了黑麦染色体臂1RS上的抗小麦叶锈病基因Lr26在小麦中的表达受小麦染色体臂1BL上的基因的强烈影响,指出了外源基因在小麦中的表达可受染色体臂或基因水平上的相互作用的制约。文中讨论了外源基因与小麦遗传背景相互作用在小麦育种中的意义。     

Mapping by chromosome sorting of several gene probes, including c-myc, to the derivative chromosomes of a 3;8 translocation associated with familial renal cancer

In eight members of a single family a constitutional translocation t(3;8) (p14.2;q24.1) is associated with the development of renal cancer. Chromosomes isolated from a cell line established from a subject with this translocation were analysed in flow with a fluorescence-activated cell sorter (FACS II). Nearly six million chromosomes from the flow karyotype region containing the der(8) and 5.5 million from the region containing the der(3) were sorted, the DNA extracted, digested with EcoRI, size fractionated by electrophoresis, and transferred to nitrocellulose. Hybridization with gene probes for c-mos, which has been localized to 8q11-q22 and somatostatin, which has been mapped to 3q28, confirmed that the sorted fractions contained, respectively, the der(8) and der(3) chromosomes. The cellular oncogenes c-raf-1 (3p25) and c-myc (8q24) were found to be translocated to the der(8) and der(3) chromosomes, respectively. The possible role that the relocation of c-myc might have on the development of renal carcinoma in carriers of this 3;8 translocation was further studied by analysis of the region surrounding the c-myc gene. By the use of cosmid cloning, no rearrangement 31 Kb 5'(or 19 Kb 3') of the translocated gene was found, indicating that the break-point is not immediately adjacent to c-myc. In an associated study, the DNA fragment D3S2 from chromosome 3 was found to map to 3p14.2-pter. This assignment in conjunction with published somatic cell hybrid data enabled D3S2 to be mapped more precisely to the interval 3p14.2-3p21.     

Proteins encoded by the human c-myc oncogene: differential expression in neoplastic cells.

To examine myc protein products in the wide variety of human tumor cells having alterations of the c-myc locus, we have prepared an antiserum against a synthetic peptide corresponding to the predicted C-terminal sequence of the human c-myc protein. This antiserum (anti-hu-myc 12C) specifically precipitated two proteins of 64 and 67 kilodaltons in quantities ranging from low levels in normal fibroblasts to 10-fold-higher levels in Epstein-Barr virus-immortalized and Burkitt's lymphoma cell lines, to 20- to 60-fold-higher levels in cell lines having amplified c-myc. The p64 and p67 proteins were found to be highly related by partial V8 proteolytic mapping, and both were demonstrated to be encoded by the c-myc oncogene, using hybrid-selected translation of myc-specific RNA. In addition, the p64 protein was specifically precipitated from cells transfected with a translocated c-myc gene. Both p64 and p67 were found to be nuclear phosphoproteins with extremely short half-lives. In tumor cell lines having alterations at the c-myc locus due to amplification or translocation, we observed a significant change in the expression of p64 relative to p67 when compared with normal or Epstein-Bar virus-immortalized cells.     

Three breakpoints of variant t(2;8) translocations in Burkitt''s lymphoma cells fall within a region 140 kilobases distal from c-myc.

The variant translocations t(2;8) in Burkitt's lymphoma cells join band q24 of chromosome 8, distal from c-myc, to the Igkappa locus, with considerable variation in the location of the breakpoints on chromosome 8. We report the cloning and molecular characterization of a chromosome 8 region, distal from the c-myc locus, which encompasses the breakpoints of the Burkitt's lymphoma cell lines BL64, BL21, and LY91 within 11 kilobase pairs, termed provisionally bvr-1 (Burkitt's variants' rearranging region 1). Using probes from the c-myc, the bvr-1, and the human pvt-1 loci obtained by chromosome walking coupled with pulsed-field gel electrophoresis, we have constructed a physical map of the region 3' of c-myc. We map bvr-1 and pvt-1 about 140 and 260 kilobase pairs, respectively, distal from c-myc.     

Genetic recombination in a chromosomal translocation t(2;8)(p11;q24) of a Burkitt's lymphoma cell line, KOBK101

We analyzed a chromosomal translocation, t(2;8)(p11;q24), in a Burkitt's lymphoma cell line, KOBK101. The translocation reciprocally occurred between a site about 150 bp upstream from the J5 segment in the Ig kappa-encoding gene on chromosome 2 and the A-rich end of an Alu repetitive element located far downstream from the c-myc gene on chromosome 8. Short segments of both parental chromosomes were deleted at the rearrangement site. A sequence related to the heptamer recognition signal for the V-J recombination of Ig genes and a topoisomerase I-recognition sequence were detected at the breakpoints. The V-J recombination occurred on both chromosome 2 and the translocated chromosome 2p- at the J3 and J4 segments, respectively. The J region on the translocated chromosomes was mutated, as compared with that on the untranslocated chromosome, while the Alu element and its upstream sequence were conserved. These results suggest the following aspects to the chromosomal translocation of this cell line. A V-J recombination seems to have occurred at the proximal end of the J4 segment first, and then the translocation took place in the region between the J4 and J5 segments. The translocation may have been mediated by the functions of topoisomerase I and the Alu repetitive sequence located at the breakpoint, although the possibility cannot be ruled out that the recombination machinery for Ig gene rearrangements functioned irregularly.     

Human lens γ-crystallin sequences are located in the p12-qter region of chromosome 2

Summary The human -crystallin genes constitute a multigene family whose members are only expressed in the eye lens. The chromosomal location of these sequences has been determined by screening a panel of human/rodent hybrid cell lines containing overlapping subsets of human chromosomes for the presence of human -crystallin sequences. By correlating these genomic hybridization data with the chromosomal constitution of the somatic cell hybrids, all human -crystallin sequences could be assigned to chromosome 2. The use of human/hamster cell hybrids derived from human Burkitt lymphoma cells carrying a reciprocal translocation between human chromosomes 2 and 8, allowed a further localization of the sequences to the region 2p12-qter.