Relationship of the human protooncogene CBL2 on 11q23 to the t(4;11), t(11;22), and t(11;14) breakpoints

A probe identifying CBL2, the human cellular homolog of the murine oncogene v-cbl and murine cellular protooncogene Cbl-2, and panels of rodent X human somatic cell hybrids were used to study the relationship of this protooncogene to translocations associated with acute leukemia, lymphoma, and Ewing sarcoma. CBL2 was mapped to 11q23 and found to translocate from chromosome 11 to 4 in an acute leukemia cell line possessing a t(4;11)(q21;q23) and from chromosome 11 to 14 in a B-cell lymphoma with a t(11;14)(q23;q32). In an Ewing sarcoma cell line with a t(11;22)(q23;q12), however, CBL2 remained on chromosome 11. Additional studies of other genes in the region of 11q23 allowed the following ordering of these genes and breakpoints: 11cen--q23--NCAM--CD3(E-D-G)--[t(11;14), t(4;11)]--(THY1, CBL2, ETS1)--t(11;22)--11qter. The gross structure of the CBL2 sequences examined was not altered by either of the flanking breakpoints. Given that the 5' and 3' ends of the CBL2 gene are not known and are probably not evaluated by the v-cbl probe, these results do not rule out the possibility of CBL2 involvement in the pathogenesis of a subset of acute leukemias possessing a t(4;11), B-cell lymphomas possessing a t(11;14), or Ewing sarcomas possessing a t(11;22).     

Comparative mapping of the constitutional and tumor-associated 11;22 translocations.

The reciprocal t(11;22)(q23;q11) is the most common non-Robertsonian constitutional translocation in humans. The tumor-associated 11;22 rearrangement of Ewing sarcoma (ES) and peripheral neuroepithelioma (NE) is cytologically very similar to the 11;22 constitutional rearrangement. Using immunoglobulin light-chain constant region, ETS1 probes, and the technique of in situ hybridization, we previously were able to show that the constitutional and ES/NE breakpoints are different. As a first step toward isolating these translocation junctions and to further distinguish between them, we have made somatic cell hybrids. Cells from a constitutional 46,XX,inv(9),t(11;22) carrier and from an ES cell line with a t(11;22) were separately fused to a hypoxanthine-guanine phosphoribosyltransferase-deficient Chinese hamster cell line (RJK88). Resulting clones were screened with G-banding and Southern hybridization. Hybrid clones derived from the constitutional t(11;22) were established which contained the der(22) and both the der(22) and the der(11). Hybrid clones derived from the ES cell line containing the der(11) were isolated. Using the technique of Southern hybridization we have sublocalized the loci; ApoA1/C3, CD3D, ETS1, PBGD, THY1, D11S29, D11S34, and D11S147 to the region between the two breakpoints on chromosome 11 and V lambda I, V lambda VI, V lambda VII, and D22S10 to the region between the breakpoints on chromosome 22. Using anonymous DNA probes, we found that D22S9 and D22S24 map proximal to the constitutional breakpoint and that D22S15 and D22S32 map distal to the ES breakpoint on chromosome 22.     

The ataxia-telangiectasia gene (ATA) on chromosome II is distinct from the ETS-1 gene.

We have studied the segregation of an RFLP detected with a human ETS-1 genomic probe in 25 families containing members affected with ataxia-telangiectasia (AT) and in 27 families from the Centre d'Etude du Polymorphisme Humain (CEPH) panel. We have recently mapped a gene for AT to 11q22-23 by linkage to the markers THY1 and D11S144. Multipoint linkage analysis of the CEPH families indicated that ETS-1 is located on chromosome 11q approximately 19.2 centimorgans telomeric to THY1. Analysis of the segregation of ETS-1 alleles in AT families yields strongly negative LOD scores, excluding an AT gene from a region extending 15 cM to either side of ETS-1. Multipoint mapping of ETS-1, D11S144, THY1, and AT also excludes the possibility that an AT gene is telomeric to ETS-1.     

Chromosome assignment of the gene loci ETS1 and THY1 in the owl monkey

Our previous assignment of the gene loci HBB, HRAS1, INS, PTH, LDHA, and CAT to owl monkey chromosome 19 of karyotype VI (K-VI) indicated a putative homology of this owl monkey chromosome with the short arm of human chromosome 11 (HSA 11p). To investigate further the extent of shared homology, we localized in the owl monkey complement two genes known to be on HSA 11q. Segregation analysis of ETS1 and THY1 homologous DNA in three karyotypically different panels of rodent x owl monkey somatic cell hybrids provided evidence for the syntenic assignment of these loci to homologous chromosomes of three owl monkey karyotypes, namely, chromosomes 4 (K-VI), 3 (K-II), and 5 (K-V). The results indicate a disruption of syntenic gene loci on the distal portion of HSA 11q from 11p during primate evolution.     

Further mapping of an ataxia-telangiectasia locus to the chromosome 11q23 region.

We recently mapped the gene for ataxia-telangiectasia group A (ATA) to chromosome 11q22-23 by linkage analysis, using the genetic markers THY1 and pYNB3.12 (D11S144). The most likely order was cent-AT-S144-THY1. The present paper describes further mapping of the AT locus by means of a panel of 10 markers that span approximately 60 cM in the 11q22-23 region centered around S144 and THY1. Location scores indicate that three contiguous subsegments within the [S144-THY1] segment, as well as three contiguous segments telomeric to THY1, are each unlikely to contain the AT locus, while the more centromeric [STMY-S144] segment is most likely to contain the AT locus. These data, together with recent refinements in the linkage and physical maps of 11q22-23, place the AT locus at 11q23.     

DNase I-defined chromatin configuration of the human CD3 gene cluster.

The three CD3 genes on human chromosome 11q23 encode proteins (gamma, delta and epsilon) which form part of the antigen receptor on T lymphocytes. All three genes are clustered within 50 kb and are activated approximately contemporaneously during the early stages of T cell ontogeny. In order to pinpoint potential regulatory sequences important for locus activation and tissue-specific gene expression, the chromatin structure of almost 90 kb of this region has been probed in five cell lines using the endonuclease pancreatic DNase I. A set of DNase I hypersensitive (HS) sites has been defined in T cell chromatin, five of which were strong and not found in non-T cells, with the exception of the erythroleukaemia cell line K562, in which three sites were weakly expressed, correlating with a low level of delta mRNA. The subset of five HS sites map close to the CD3 genes and lie in regions which contain elements of defined function: the gamma promoter; the delta promoter and its 3' enhancer; and the epsilon promoter and its 3' enhancer. Since no further major T cell-restricted HS sites lie within the 90kb of the CD3 locus analysed, these five regions may contain all the sequences important for CD3 gene expression.     

Gene order, amplification, and rearrangement of chromosome band 11q23 in hematologic malignancies

Eleven genes were found to be amplified in a patient with acute myelogenous leukemia and a homogeneous staining region 11q23qter. The gene order of such region was determined by using transverse alternating field electrophoresis of normal cell DNA and Southern blots of DNA from somatic cell hybrids, each containing a single human derivative chromosome 11 from six different chromosomal defects. This in turn allowed us to uncover a breakpoint in band 11q23.3 between the CD3 gamma and the ets-1 genes in genomic rearrangements found in acute myelogenous leukemia, acute lymphocytic leukemia, and B-cell diffuse lymphoma. The breakpoint of a constitutional deletion from a patient whose mother and brother have a heritable 11q23.3 fragile site occurs in the same region.     

Precise in situ localization of NCAM, ETS1, and D11S29 on human meiotic chromosomes

In order to sublocalize NCAM, ETS1, and the anonymous DNA fragment D11S29 within 11q23, in situ hybridization was performed on pachytene bivalents. Analysis of the grain distribution within the band 11q23 indicated that the chromosomal sublocalization of both NCAM and D11S29 was in 11q23.1, whereas ETS1 was found to be localized in 11q23.3. These results clearly demonstrate the usefulness of in situ hybridization applied to pachytene bivalents to obtain accurate gene sublocalization.     

Isolation of 1001 new markers from human chromosome 11, excluding the region of 11p13-p15.5, and their sublocalization by a new series of radiation-reduced somatic cell hybrids.

The determination of the physical map of human chromosome 11 will require more clones than are currently available. We have isolated an additional 1001 new markers in a bacteriophage vector from a somatic cell hybrid cell line that contains most of chromosome 11, except the middle of the short arm. These markers were localized to five different regions, 11p15-pter, 11p12-cen, 11q11-q14, 11q14-q23, and 11q23-qter, by a panel of previously characterized somatic cell hybrids. The region 11q11-14 harbors genes that have been shown to be important in breast cancer, B-cell lymphomas, centrocytic lymphomas, asthma, and multiple endocrine neoplasia, type 1 (MEN1). To determine the positions of the recombinant clones located there, we developed a new series of radiation-reduced somatic cell hybrids. These hybrids, together with those previously characterized, allowed us to map the 11q11-q14 markers into 11 separate segregation groups.     

Localization of multiple melanoma tumor-suppressor genes on chromosome 11 by use of homozygosity mapping-of-deletions analysis

Loss-of-heterozygosity (LOH) studies have implicated one or more chromosome 11 tumor-suppressor gene(s) in the development of cutaneous melanoma as well as a variety of other forms of human cancer. In the present study, we have identified multiple independent critical regions on this chromosome by use of homozygosity mapping of deletions (HOMOD) analysis. This method of analysis involved the use of highly polymorphic microsatellite markers and statistics to identify regions of hemizygous deletion in unmatched melanoma cell line DNAs. Regions of loss were defined by the presence of an extended region of homozygosity (ERH) at > or =5 adjacent markers and having a statistical probability of < or =.001. Significant ERHs were similar in nature to deletions identified by LOH analyses performed on uncultured melanomas, although a higher frequency of loss (24 [60%] of 40 vs. 16 [34%] of 47) was observed in the cell lines. Overall, six small regions of overlapping deletions (SROs) were identified on chromosome 11 flanked by the markers D11S1338/D11S907 (11p13-15.5 [SRO1]), D11S1344/D11S11385 (11p11.2 [SRO2]), D11S917/D11S1886 (11q21-22.3 [SRO3]), D11S927/D11S4094 (11q23 [SRO4]), AFM210ve3/D11S990 (11q24 [SRO5]), and D11S1351/D11S4123 (11q24-25 [SRO6]). We propose that HOMOD analysis can be used as an adjunct to LOH analysis in the localization of tumor-suppressor genes.     

Localization of Human Cadherin Genes to Chromosome Regions Exhibiting Cancer-Related Loss of Heterozygosity

This report presents the chromosomal localization of cadherin genes. Cadherins are cellular adhesion molecules. Since disturbance of intracellular adhesion is important for invasion and metastasis of tumor cells, cadherins are considered prime candidates for tumor suppressor genes. A variety of solid tumors show loss of heterozygosity of the long arm of chromosome 16, which is indicative of the potential location of tumor suppressor genes. Refined and new localizations of six cadherin genes (CDH3, 5, 8, 11, 13, and 15) to the long arm of chromosome 16 are shown. CDH15 was localized to 16q24.3, in a region that exhibits loss of heterozygosity in a number of sporadic breast cancer tumors. Previous localization of CDH13 (H-cadherin) to 16q24 suggested this gene as a tumor suppressor candidate in the 16q24.3 loss of heterozygosity region; however, refined mapping presented in this report localizes CDH13 proximal to this region. A human EST homologous to the chicken cadherin-7 was partially sequenced and found to represent a new human cadherin. This cadherin mapped to chromosome 18q22–q23, a region that exhibits loss of heterozygosity in head and neck squamous cell carcinomas. CDH16 was localized to 8q22.1, a region exhibiting loss of heterozygosity in adult acute myeloid leukemia.     

Regional assignment of five genes on human chromosome 19

A human-mouse hybrid segregant HM76Dd40-6 with new characteristics was derived from the hybrid cell line HM76Dd containing human chromosome 19 as the only human chromosome. Three virus sensitivities located on human chromosome 19 (PVS, E11S and RDRC) were lost in HM76Dd40-6, while six other genes (C3, LDLR, EF2, GPI, PEPD and MANB) were retained. Cytogenetic analysis and in situ hybridization using human or mouse repeated sequences as probes showed that the region q13.1-qter of human chromosome 19 had been replaced by a fragment of mouse chromosome. Our results permit further regional assignment for the following five genes on human chromosome 19: GPI in the region cen-q12, MANB in p13.2-q12, E11S and RDRC in q13.1-qter, and EF2 in pter-q12.     

Genetic linkage analysis and homology relationships of genes located on human chromosome 11q.

We have used DNA polymorphisms detected by probes for 11q to order 16 genes and to determine the genetic distances between them. Our map includes the genes for CD20, tyrosinase, progesterone receptor, stromelysin, collagenase, N-CAM, dopamine-D2 receptor, apolipoproteins AI-CIII-AIV, CD3-epsilon, -delta, and -gamma, porphobilinogen deaminase, thy-1, and ets-1. These genes have previously been sequenced as well as placed on the 11q cytogenetic map, which now makes them anchor points between the cytogenetic, genetic, and physical maps of this region. The ordering and distances between these genes are of immediate use in testing hypotheses of candidate genes for human genetic diseases associated with chromosome 11q. A comparison between our genetic map and similar maps from other species defines regions of homologous synteny that may be useful in mapping human genetic disease genes localized to the 11q region. Analysis of such homology provides additional bases for speculation of the evolutionary histories of gene families in this region.     

Localization of the human NCAM gene to band q23 of chromosome 11: the third gene coding for a cell interaction molecule mapped to the distal portion of the long arm of chromosome 11

cDNA clones containing sequences coding for the murine neural cell adhesion molecule (N-CAM) were used in Southern hybridizations on human genomic DNA and demonstrated approximately 90% homology between human and murine NCAM genes. In situ hybridization with one of these clones was performed on human metaphase chromosomes and allowed the localization of the human NCAM gene to band q23 of chromosome 11. The genes for two other cell surface molecules believed to be involved in cell-cell interactions, Thy-1 and the delta chain of the T3-T cell receptor complex, have recently been localized to the same region of chromosome 11 in man. Moreover, this region of the human chromosome 11 appears to be syntenic to a region of murine chromosome 9 that also contains the staggerer locus: staggerer mice show abnormal neurological features which may be related to abnormalities in the conversion of the embryonic to the adult forms of the N-CAM molecule.     

An activity associated with human chromosome 21 permits nuclear colocalization of the adenovirus E1B-55K and E4orf6 proteins and promotes viral late gene expression

The adenovirus E1B-55K and E4orf6 proteins cooperate during virus infection while performing several tasks that contribute to a productive infection, including the selective nucleocytoplasmic transport of late viral mRNA. Previous studies have shown that the E4orf6 protein retains the E1B-55K protein in the nucleus of human and monkey cells, but not in those of rodents, suggesting that primate-specific cellular factors contribute to the E4orf6-mediated retention of the E1B-55K protein in the nucleus. In an effort to identify these proposed primate-specific cellular factors, the interaction of the E1B-55K and E4orf6 proteins was studied in a panel of stable human-rodent monochromosomal somatic cell hybrids. Analysis of this panel of cell lines has demonstrated the existence of an activity associated with human chromosome 21 that permits the E1B-55K and E4orf6 proteins to colocalize in the nucleus of a rodent cell. Additional hybrid cells bearing portions of human chromosome 21 were used to map this activity to a 10-megabase-pair segment of the chromosome, extending from 21q22.12 to a region near the q terminus. Strikingly, this region also facilitates the expression of adenovirus late genes in a rodent cell background while having little impact on the expression of early viral genes.