Expression of normal and translocated c-myc alleles in Burkitt''s lymphoma cells: evidence for different regulation.

In Burkitt's lymphoma (BL) cells the normal c-myc allele is usually silent or expressed at very low levels. Here we demonstrate that the normal c-myc allele can be induced in BL cells by 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA did activate the normal c-myc alleles in Raji(P207), BL36, P3HR1, Jijoye and LY91 cells, but not in Raji(DE88), BL41, BL67, LY47 and KK124 cells. C-myc RNA derived from the normal allele appeared 6 h after treatment with TPA and showed the characteristic preferential usage of the second promoter. This induction could not be inhibited by cycloheximide. Despite the differences in c-myc induction in Raji(P207) and Raji(DE88) cells, c-fos and the early Epstein-Barr virus gene DR were induced to a similar extent and with similar kinetics by TPA. Nuclear run-on experiments suggest that the normal c-myc allele in Raji cells is activated at least in part by releasing a block to RNA elongation at the end of c-myc exon 1. Expression of the translocated c-myc alleles was also affected by TPA; however, only if cycloheximide was simultaneously present. TPA plus cycloheximide induced a rapid decrease of c-myc RNA derived from the translocated allele within 6 h, whereas cycloheximide alone led to abolition of c-myc RNA after 16-24 h. This rapid decline of c-myc RNA was observed in Raji and BL41 cells, but not in three cell lines with variant t(2;8) and t(8;22) translocations.     

Chromatin structure and protein binding in the putative regulatory region of the c-myc gene in Burkitt lymphoma

A chromosomal myc gene displays one of three patterns of activity depending upon the arrangement of the gene and its allelic partner. In nonmalignant B cells both myc alleles are normally expressed. In Burkitt lymphoma cells carrying both a translocated and a nontranslocated myc allele, the translocated allele is inappropriately expressed, while the nontranslocated allele is virtually inactive. Here we examine the chromatin structure of these genes using DNAase I hypersensitivity in nonmalignant lymphoblastoid cells and in the Burkitt lymphoma, BL31 . Three hypersensitivity patterns emerge that correlate with the state of the gene and reveal sites associated with putative regulatory structures. One region is associated with the two myc promoters, one with a specific nuclear protein binding site, and one--which is markedly enhanced in the inactive germline gene in the Burkitt cell--with a putative negative control region. The perturbation of the normal pattern in this particular Burkitt cell may be due to the action of an immunoglobulin enhancer.     

NF-kappa B sites function as positive regulators of expression of the translocated c-myc allele in Burkitt''s lymphoma.

An in vivo footprint over a potential NF-kappa B site in the first exon of the c-myc gene has been identified on the translocated allele in the Ramos Burkitt's lymphoma cell line. The potential NF-kappa B site in the 5' flanking sequence of c-myc was found to be occupied on the translocated allele in the Raji Burkitt's cell line. Electrophoretic mobility shift assays with each of these sequences demonstrated complexes with mobilities identical to those of the NF-kappa B site from the kappa light-chain gene. A supershift was obtained with anti-p50 antibody with the exon site. The upstream-site shift complex disappeared with the addition of anti-p50 antibody. Binding of NF-kappa B proteins to the c-myc exon and upstream sites was demonstrated by induction of binding upon differentiation of pre-B 70Z/3 cells to B cells. UV cross-linking experiments revealed that a protein with a molecular mass of 50 kDa bound to the exon and upstream sites. Transfection experiments with Raji cells demonstrated that both sites functioned as positive regulatory regions, with a drop in activity level when either site was mutated. Access to these sites is blocked in the silent normal c-myc allele in Burkitt's lymphoma cells, while Rel family proteins bind to these sites in the translocated allele. We conclude that the two NF-kappa B sites function as positive regulatory regions for the translocated c-myc gene in Burkitt's lymphoma.     

Evidence for an elevated frequency of in vivo somatic cell mutations in ataxia telangiectasia.

Somatic cell mutation frequency in vivo was measured in individuals with high cancer risk who were from ataxia telangiectasia (A-T) families. The assay for somatic mutation measures the frequency of variant erythrocytes which are progeny of erythroid precursor cells with mutations that result in a loss of gene expression at the polymorphic glycophorin A (GPA) locus. Samples from 14 of 15 A-T homozygotes showed high frequencies of GPA gene expression-loss variant cells with normal expression of only one of the two alleles at the GPA locus (i.e., GPA hemizygous variant cells). The mean elevation of the frequency of hemizygous variant cells over those in normal controls and unaffected family members was 7-14-fold. A-T homozygotes also showed an increase in the frequency of cells in which one allele at the GPA locus had lost expression and in which the remaining allele was expressed at a homozygous level (i.e., GPA homozygous variant cells). Family members who are obligate A-T heterozygotes did not appear to have a significantly elevated frequency of GPA hemizygous or homozygous variant cells. These indications of elevated in vivo frequencies of variant erythrocytes in A-T homozygotes support a causal link between susceptibility to somatic mutation and susceptibility to cancer.     

Chromosomal elements regulate gene activity and chromatin structure of the human serpin gene cluster at 14q32.1

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 contains a number of genes that are specifically expressed in hepatic cells. Cell-specific enhancers have been identified in several of these genes, but elements involved in locus-wide gene and chromatin control have yet to be defined. To identify regulatory elements in this region, we prepared a series of mutant chromosomal alleles by homologous recombination and transferred the specifically modified human chromosomes to hepatic cells for functional tests. We report that deletion of an 8-kb DNA segment upstream of the human alpha1-antitrypsin gene yields a mutant serpin allele that fails to be activated in hepatic cells. Within this region, a 2.3-kb DNA segment between kb -8.1 and -5.8 contains a previously unrecognized control region that is required not only for serpin gene activation but also for chromatin remodeling of the entire locus.     

No evidence for loss of genetic variation following sequential translocations in extant populations of a genetically depauperate species

Repeated population bottlenecks can lead to loss of genetic variation and normally should be avoided in threatened species to preserve evolutionary potential. We examined the effect of repeated bottlenecks, in the form of sequential translocations, on loss of genetic variation in a threatened passerine, the saddleback (Philesturnus carunculatus carunculatus), a species that has recovered from a remnant population with historically low levels of genetic variation. Although a slight but nonsignificant loss of alleles may have occurred between the first-order translocation and the extirpated source population, first-, second-, and third-order translocated populations had very similar levels of genetic variation to each other. The most obvious difference among the seven island populations appeared to lie in allele frequencies with little or no loss of alleles among extant populations. Although sequential translocations are known to cause loss of variation in genetically diverse species, our study indicates that genetically depauperate species may be less sensitive to loss of genetic variation through founder events presumably because the few remaining alleles are well represented in founding individuals. These results show that ancient bottlenecks may have a long-term effect on genetic variation, to the extent that contemporary population bottlenecks may leave no appreciable genetic signature. Our results suggest that subjecting genetically depauperate endangered species to sequential translocations could be used to rapidly establish new populations without further eroding genetic variation.     

A Somatic Origin of Homologous Robertsonian Translocations and Isochromosomes

One t(14q14q), three t(15q15q), two t(21q21q), and two t(22q22q) nonmosaic, apparently balanced, de novo Robertsonian translocation cases were investigated with polymorphic markers to establish the origin of the translocated chromosomes. Four cases had results indicative of an isochromosome: one t(14q14q) case with mild mental retardation and maternal uniparental disomy (UPD) for chromosome 14, one t(15q15q) case with the Prader-Willi syndrome and UPD(15), a phenotypically normal carrier of t(22q22q) with maternal UPD(22), and a phenotypically normal t(21q21q) case of paternal UPD(21). All UPD cases showed complete homozygosity throughout the involved chromosome, which is supportive of a postmeiotic origin. In the remaining four cases, maternal and paternal inheritance of the involved chromosome was found, which unambiguously implies a somatic origin. One t(15q15q) female had a child with a ring chromosome 15, which was also of probable postmeiotic origin as recombination between grandparental haplotypes had occurred prior to ring formation. UPD might be expected to result from de novo Robertsonian translocations of meiotic origin; however, all de novo homologous translocation cases, so far reported, with UPD of chromosomes 14, 15, 21, or 22 have been isochromosomes. These data provide the first direct evidence that nonmosaic Robertsonian translocations, as well as isochromosomes, are commonly the result of a mitotic exchange.     

Host versus in vitro signals and intrastrain allelic differences in the expression of a Candida albicans virulence gene

The yeast Candida albicans is a harmless colonizer of mucosal surfaces in healthy people but can become a serious pathogen in immunocompromised patients, causing superficial as well as systemic infections. The evolution of gene families encoding pathogenicity-related functions, like adhesins and secreted aspartic proteinases (Saps), which are differentially induced by host signals at various stages of colonization and infection, may have allowed C. albicans an optimal adaptation to many different host niches. We found that even the two alleles of a single gene can be differentially regulated in the diploid C. albicans. In the model strain SC5314, the in vitro expression of one of the two SAP2 alleles, SAP2-1, depended on the presence of a functional SAP2-2 allele. In contrast, inactivation of SAP2-1 did not in-fluence the expression of SAP2-2. The proteinase encoded by the SAP2-2 allele serves as a signal sensor and amplifier to enhance its own expression as well as to induce the SAP2-1 allele to achieve maximal proteolytic activity under appropriate conditions. Using in vivo expression technology, we could demonstrate that the SAP2-1 allele is significantly activated only in the late stages of systemic candidiasis in mice, whereas the SAP2-2 allele is induced much earlier. The differential regulation of the two SAP2 alleles was due to differences in their pro-moters, which contained a variable number of two pentameric nucleotide repeats. Mutations that reduced or increased the copy number of these repeats diminished the inducibility of the SAP2 promoter during infection but not in vitro, suggesting that the mutations affected interactions of regulatory factors that are necessary for SAP2 activation in vivo but dispensable for its induction in vitro. Therefore, the signals and signal transduction pathways that mediate SAP2 expression within certain host niches may differ from those that activate the gene in vitro. In addition to the generation of gene families whose members exhibit functional and regulatory diversification, C. albicans seems to use its diploid genome to create further variability and host adaptation by differential evolution of even the two alleles of a single gene.     

Genetic diversity and population viability in translocated North?Island saddleback (

Genetic variation in two translocated populations of North Island saddleback (Philesturnus rufusater) on Kapiti Island and at Zealandia was investigated using five microsatellite loci and compared with the source populations in the Hauraki Gulf. Although the absolute number of alleles in the two populations was low (3 alleles per locus), both populations carried all the alleles found in their immediate source populations, but lacked one rare allele found in only one individual from the original remnant population on Hen Island. Overall heterozygosity was high and inbreeding coefficients were low. Population viability analyses showed that these populations will likely reach carrying capacity by the middle of this decade, and genetic simulations predicted that they should retain between 90% (Kapiti) and 95% (Zealandia) of the heterozygosity of their sources. The difference between the two populations is most likely due to the prolonged post-translocation bottleneck on Kapiti when rats were still present on the island. While our results suggest that additional top-up translocations would be unnecessary and unwarranted at this time, further work on potentially selected loci or inbreeding depression could justify this decision to be revisited.