B-cell tumorigenesis in mice carrying a yeast artificial chromosome-based immunoglobulin heavy/c-myc translocus is independent of the heavy chain intron enhancer (Emu).

We have used YAC (yeast artificial chromosome) technology to create large translocation regions where the c-myc proto-oncogene is coupled to the core region of the human immunoglobulin heavy chain (IgH) locus (from VH2-5 through to Cdelta). Chimeric mice were obtained from embryonic stem cells carrying a single copy of the 240-kb IgH/c-myc translocation region. B-cell tumorigenesis occurs in the translocus mice, even when the entire Emu intron enhancer region between the joining segments and switch mu is deleted. This demonstrates that as yet unidentified regulatory elements in the IgH locus, independent from the known enhancers, are sufficient to cause B-cell specific activation of c-myc after translocation. The phenotype of tumors from IgH/c-myc YAC transgenic mice with or without Emu (B220+, IgM+/IgD+) is reminiscent of Burkitt's lymphoma. A rapidly expanding abnormal B-cell population is present at birth and accumulates in bone marrow, periphery, and spleen, well before discrete tumor establishment. Molecular analysis identified a clonal origin, with rearrangement of one mouse heavy chain allele retained in tumor cells from different sites, whereas subsequent rearrangements of heavy or light chain loci can be diverse. These mice routinely develop mature B-cell tumors early in life and may provide an invaluable resource of a B-cell lymphoma model.

Transplacental uptake of glucose is decreased in embryonic lethal connexin26-deficient mice.

Mice that harbor a targeted homozygous defect in the gene coding for the gap junctional protein connexin26 died in utero during the transient phase from early to midgestation. From day 10 post coitum onwards, development of homozygous embryos was retarded, which led to death around day 11 post coitum. Except for growth retardation, no gross morphological alterations were detected between homozygous connexin26-defective embryos and wild-type littermates. At day 9 postcoitum, when chorioallantoic placenta started to function, connexin26 was weakly expressed in the yolk sac epithelium, between syncytiotrophoblasts I and II in the labyrinth region of the placenta, and in the skin of the embryo. At day 10 post coitum, expression of connexin26 in the placenta was much stronger than at the other locations. To analyze involvement of connexin26 in the placental transfer of nutrients, we have measured embryonic uptake of the nonmetabolizable glucose analogue 3-O-[14C]methylglucose, injected into the maternal tail vein. At day 10 post coitum, viable, homozygous connexin26-defective embryos accumulated only approximately 40% of the radioactivity measured in wild-type and heterozygous littermates of the same size. We conclude that the uptake of glucose, and presumably other nutrients as well, from maternal blood into connexin26-deficient mouse embryos was severely impaired and apparently not sufficient to support the rapid organogenesis during midgestation. Our results suggest that connexin26 gap junction channels likely fulfill an essential role in the transfer of maternal nutrients and embryonic waste products between syncytiotrophoblast I and II in the labyrinth layer of the mouse placenta.

Yeast colony size reflects YAC copy number.

A novel strategy for separation of co-cloned YACs was developed. For this, yeast cells were grown under non-selective conditions to allow the mitotic loss of multiple YACs. Yeast colonies of different size appear on 'drop-out' selection plates with small clones consistently containing a single-copy YAC. Different auxotrophic marker genes can be used to separate co-cloned YACs or reduce their copy number, which is essential for most YAC-modification procedures.

Rapid induction of B-cell lymphomas in mice carrying a human IgH/c-mycYAC.

Activation of the c-myc proto-oncogene by one of the immunoglobulin (Ig) loci after chromosomal translocation is a consistent feature of Burkitt's lymphoma. Different subtypes of this tumor vary in the molecular architecture of the translocation region. In most cases there are no known regulatory elements of the Ig locus neighboring the oncogene and this considerably obscures the mechanism of its deregulation. In order to assess possible oncogene activation signals, we produced an experimental translocation region by insertion of a c-myc gene about 50 kb from the IgH intron enhancer in a yeast artificial chromosome (YAC) containing a 220 kb region of the human Ig heavy chain (IgH) locus. Single copy integration of this YAC into the genome of mouse embryonic stem (ES) cells was achieved by spheroplast fusion. Chimeric mice derived from these ES cells developed monoclonal B-cell lymphomas expressing surface IgM by 8-16 weeks of age. The IgH/c-myc translocus showed different V(h)DJ(H) rearrangement in almost all tumors without any alterations of the distance between c-myc and the IgH intron enhancer. This mouse model can be used for the in vivo analysis of c-myc deregulation and the tumor formation capacity of the IgH locus in aberrant rearrangements.

Assembly and extension of yeast artificial chromosomes to build up a large locus.

For the assembly of a large human locus, overlapping regions on yeast artificial chromosomes (YACs) and cosmids were linked up using their regions of homology. By site-specific recombination a YAC of 410 kb was created accommodating the major part of the human lambda light chain locus in authentic configuration with 28 variable (V) genes, all joining (J) segments, all constant (C) genes and the downstream enhancer. A contiguous region was first constructed from three overlapping cosmids. Each of these was linearized at unique sites in the vectors and YAC arms were ligated to the 5' and 3' ends. After cells of Saccharomyces cerevisiae were transformed with the three cosmids, YACs of 120 kb were obtained which contained the reassembled 3' J-C region in authentic configuration. The assembled YAC was further extended by mitotic recombination with a YAC containing a 280-kb region of the C-proximal part of the V gene cluster with a 15-kb 3' overlap. For this, a simple three-way selection procedure was developed involving the integration of different selectable marker genes at specific sites in the left and right YAC arms. Rare recombination events between two overlapping YACs could be identified in yeast clones able to grow in lysine- and adenine-deficient medium in the presence of 5-fluoro-orotic acid which is toxic for yeast cells containing a YAC with a functional URA3 gene. This approach made it possible to assemble and extend large YACs from an unlimited number of smaller overlapping YACs by positive-negative selection.

Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average approximately 17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was approximately 1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.

Short DNA sequences from the cytoplasm of mouse tumor cells induce immortalization of human lymphocytes in vitro.

Cytoplasts of mouse L929 and Ehrlich ascites tumor cells harbor DNA sequences that induce unlimited proliferation ("immortalization") of human lymphocytes after transfection in vitro. By equilibrium centrifugation of cytoplasmic lysates in a neutral CsCl gradient, the immortalizing activity was recovered together with extramitochondrial fractions at high salt densities (1.85-1.87 g/cm3). Unexpectedly, these fractions contain linear DNA molecules of 50-500 bp in length. In contrast, cytoplasts of primary, senescent cells (mouse embryo fibroblasts, human lymphocytes) do not harbor DNA in the corresponding fractions. Cytoplasmic DNA isolated from high-density fractions of mouse tumor cells was cloned in subset libraries, and of 45 DNA sequences we identified 2 clones--one from L929 cytoplasts (203 bp) and another one from the cytoplasm of Ehrlich ascites cells (372 bp)--that induce unlimited proliferation of human lymphocytes in vitro. Immortalized lymphoid cells harbor 1-5 copies of transfected DNA integrated into chromosomal DNA, whereas about 100 copies were found as episomal DNA in the cytoplasmic fraction. No immortalization could be induced by transfection of nuclear DNA randomly fragmented to 200-500 bp. Although the cloned DNA sedimented at 1.70 g/cm3, after transient transfection into lymphocytes, these DNA sequences form salt-stable complexes that sediment in fractions at the same high density (1.82-1.88 g/cm3) from which they were originally cloned. The high-density banding of these cytoplasmic DNA sequences may be due to association with RNA and/or with (metallo-) proteins in vivo. Since both cloned DNA sequences with immortalizing activity have stop codons for protein translation in all possible reading frames, immortalization may be induced by insertional inactivation or functional suppression of genes that are needed to be expressed during cellular senescence or programmed cell death.

Re-organization of the immunoglobulin kappa gene on both alleles is not an obligatory prerequisite for Ig lambda gene expression in human cells.

Re-arrangement and expression of the immunoglobulin (Ig) genes in B cells occurs under an ordered developmental control. The sequential model of Ig light chain exclusion predicts that only after non-productive re-organization or deletion of both Ig kappa alleles would re-arrangement of Ig lambda gene segments occur. To prove this model, we asked whether expression of Ig lambda light chains is always associated with rearrangement and/or deletion of both Ig kappa alleles in human cells. Therefore, we established human diploid B-cell clones in vitro that produce Ig lambda light chains. Southern blot analysis of the Ig kappa alleles revealed that three Ig lambda expressing cell lines (out of six Ig lambda+ cell lines tested) harbour one Ig kappa allele in germline configuration. Furthermore, a 1.5 kb RNA derived from the germline Ig kappa locus was detected by Northern blot hybridizations. The results implicate that the mechanism of Ig light chain exclusion is not precisely sequential and that it does not necessarily need re-arrangement or deletion of both Ig kappa alleles as a prerequisite for Ig lambda light chain expression in human cells.

Altered expression of proto-oncogenes in human lymphoid cells immortalized by transfection with extrachromosomal DNA of mouse L cells.

Human lymphocytes were induced to proliferate continuously in vitro by transfection with extrachromosomal DNA from mouse L929 cytoplasts. The immortalized cells contain 2- to 5-fold increased mRNA levels of only 3 (c-Ha-ras-1, c-Ki-ras, c-myc) out of 18 proto-oncogenes tested compared to quiescent lymphocytes. Furthermore, the immortalized cells contain decreased amounts of c-fos mRNA, 2- to 20-fold decreased amounts of lck and c-fgr mRNA and 10-fold increased levels of JD15 mRNA compared to primary lymphocytes. The immortalised cells do not harbor amplified copies or any major rearrangement of these genes. No additional copy of these genes transferred from the mouse genome could be detected in the immortalized human cell lines. Thus, the changes in the level of these proto-oncogene transcripts in the lymphoid cells are likely to be induced by the immortalizing DNA from L929 cytoplasts and appear to be associated with continuous proliferation of these cells. Since the immortalized cell lines do not form colonies in soft agar medium and do not induce tumors in nude mice, we suggest that the altered expression of these proto-oncogenes may be due to the transition from quiescent to continuous proliferation in vitro ("immortalization"), but does not correlate with a tumorigenic phenotype of the cells.

Immortalization of human lymphocytes by transfection with DNA from mouse L929 cytoplasts.

Transfection of human peripheral blood lymphocytes with DNA from mouse L929 cytoplasts induced proliferation of lymphocytes and the formation of B and T cell-derived cell lines with apparently unlimited growth potential. The cell lines could be grown in serum-containing media as well as in chemically defined serum-free media, have a nearly normal human karyotype, did not form colonies in soft-agar medium, and were not tumorigenic after injection into nude mice. For immortalization of human lymphocytes, DNA from L929 cytoplasts was 100-fold more efficient than L929 nuclear DNA. The ability of cytoplast DNA to immortalize lymphocytes could be consecutively transferred by using total cellular DNA from primary or secondary transfectants. Circular or linear mitochondrial DNA of L929 cells did not lead to immortalization of human lymphocytes. Since DNA with immortalizing activity could be isolated from cytoplasts, the Hirt supernatant, and a mitochondria-depleted cytoplasmic fraction of L929 cells, we conclude that the immortalizing DNA is located extramitochondrially in the cytoplasm of L929 cells.

Adenovirus type 5 persisting in human lymphocytes is unlikely to be involved in immortalization of lymphoid cells by fusion with cytoplasts or by transfection with DNA of mouse L cells.

Human peripheral blood lymphocytes were induced to proliferate indefinitely in vitro by fusion with cytoplasts from mouse L 929 cells or, alternatively, by transfection with DNA isolated from L929 cytoplasts. Since adenoviruses epidemically infect human lymphocytes, the newly established cell lines were assayed for the presence of adenoviral DNA sequences. In 8 of 10 lymphoid cell lines of both B and T cell origin, adenovirus type 5 (serogroup C) DNA sequences were detected. The cells harbor approximately 40 to 70 genome copies per cell. Adenovirus type 5 transcripts of the E1A and E1B early regions that are responsible for the transforming capacity of adenoviruses could not be detected in the lymphoid cell lines. Therefore we conclude that the adenovirus genome persisting in immortalized lymphoid cells is not involved in the maintenance of indefinite proliferation induced by fusion with L929 cytoplasts or by DNA transfection.