Melanotic tumors of the nervous system are characterized by distinct mutational, chromosomal and epigenomic profiles.

Melanotic tumors of the nervous system show overlapping histological characteristics but differ substantially in their biological behavior. In order to achieve a better delineation of such tumors, we performed an in-depth molecular characterization. Eighteen melanocytomas, 12 melanomas, and 14 melanotic and 14 conventional schwannomas (control group) were investigated for methylome patterns (450k array), gene mutations associated with melanotic tumors and copy number variants (CNVs). The methylome fingerprints assigned tumors to entity-specific groups. Methylation groups also showed a substantial overlap with histology-based diagnosis suggesting that they represent true biological entities. On the molecular level, melanotic schwannomas were characterized by a complex karyotype with recurrent monosomy of chromosome 22q and variable whole chromosomal gains and recurrent losses commonly involving chromosomes 1, 17p and 21. Melanocytomas carried GNAQ/11 mutations and presented with CNV involving chromosomes 3 and 6. Melanomas were frequently mutated in the TERT promoter, harbored additional oncogene mutations and showed recurrent chromosomal losses involving chromosomes 9, 10 and 6q, as well as gains of 22q. Together, melanotic nervous system tumors have several distinct mutational and chromosomal alterations and can reliably be distinguished by methylome profiling.

Chromosomal assignment of the canine melanophilin gene (MLPH): a candidate gene for coat color dilution in Pinschers

Pinschers affected by coat color dilution show a specific pigmentation phenotype. The dilute pigmentation phenotype leads to a silver-blue appearance of the eumelanin-containing fur and a pale sandy color of pheomelanin-containing fur. In Pinscher breeding, dilute black-and-tan dogs are called "blue," and dilute red or brown animals are termed "fawn" or "Isabella fawn." Coat color dilution in Pinschers is sometimes accompanied by hair loss and a recurrent infection of the hair follicles. In human and mice, several well-characterized genes are responsible for similar pigment variations. To investigate the genetic cause of the coat color dilution in Pinschers, we isolated BAC clones containing the canine ortholog of the known murine color dilution gene Mlph. RH mapping of the canine MLPH gene was performed using an STS marker derived from BAC sequences. Additionally, one MLPH BAC clone was used as probe for FISH mapping, and the canine MLPH gene was assigned to CFA25q24.

Molecular characterization and chromosomal assignment of the bovine glycinamide ribonucleotide formyltransferase (GART) gene on cattle chromosome 1q12.1-q12.2

The mammalian glycinamide ribonucleotide formyltransferase (GART) genes encode a trifunctional polypeptide involved in the de novo purine biosynthesis. We isolated a bacterial artificial chromosome (BAC) clone containing the bovine GART gene and determined the complete DNA sequence of the BAC clone. Cloning and characterization of the bovine GART gene revealed that the bovine gene consists of 23 exons spanning approximately 27 kb. RT-PCR amplification of bovine GART in different organs showed the expression of two GART transcripts in cattle similar to human and mouse. The GART transcripts encode two proteins of 1010 and 433 amino acids, respectively. Eleven single nucleotide polymorphisms (SNPs) were detected in a mutation scan of 24 unrelated animals of three different cattle breeds, including one SNP that affects the amino acid sequence of GART. The chromosomal localization of the gene was determined by fluorescence in situ hybridization. Comparative genome analysis between cattle, human and mouse indicates that the chromosomal location of the bovine GART gene is in agreement with a previously published mapping report.

ETIOLOGIC AND CLINICAL FACTORS WHICH DIFFERENTIATE PATIENTS WITH CHROMOSOMAL ABNORMALITIES FROM DIPLOID PATIENTS IN ACUTE NONLYMPHOCYTIC LEUKEMIA

Tumor-specific chromosomal abnormalities have been demonstrated in bone marrow of approximately 50% of newly diagnosed acute nonlymphocytic (ANLL) patients. This study examined two hypotheses: (1) Aneuploid (AA) patients are diagnosed later in the course of their disease than diploid (NN) patients; and (2) AA patients are more likely to have been exposed to environmental agents. Of 324 patients eligible for study, environmental exposure data were obtained for 236 (73%) of them. No evidence was found to suggest that AA patients had more advanced disease than NN patients. Aneuploid patients were more likely than NN patients to: (a) report treatment with cytotoxic drugs for a prior medical condition (odds ratio, adjusted for age, sex and other exposures (OR) = 4.25, 95% confidence intervals, 1.38 to 13.17); (b) smoke cigarettes, OR = 1.82 (1.02, 3.26) and (c) drink alcoholic beverages, OR = 1.91 (1.05, 3.48). No statistically significant associations between aneuploidy and occupational exposures were present, OR = 3.59 (0.76, 17.13). Problems in interpreting these ORs are discussed. ^

Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers

We report the cloning and characterization of a tumor-associated carbonic anhydrase (CA) that was identified in a human renal cell carcinoma (RCC) by serological expression screening with autologous antibodies. The cDNA sequence predicts a 354-amino acid polypeptide with a molecular mass of 39,448 Da that has features of a type I membrane protein. The predicted sequence includes a 29-amino acid signal sequence, a 261-amino acid CA domain, an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a hydrophilic C-terminal cytoplasmic tail of 29 amino acids that contains two potential phosphorylation sites. The extracellular CA domain shows 30–42% homology with known human CAs, contains all three Zn-binding histidine residues found in active CAs, and contains two potential sites for asparagine glycosylation. When expressed in COS cells, the cDNA produced a 43- to 44-kDa protein in membranes that had around one-sixth the CA activity of membranes from COS cells transfected with the same vector expressing bovine CA IV. We have designated this human protein CA XII. Northern blot analysis of normal tissues demonstrated a 4.5-kb transcript only in kidney and intestine. However, in 10% of patients with RCC, the CA XII transcript was expressed at much higher levels in the RCC than in surrounding normal kidney tissue. The CA XII gene was mapped by using fluorescence in situ hybridization to 15q22. CA XII is the second catalytically active membrane CA reported to be overexpressed in certain cancers. Its relationship to oncogenesis and its potential as a clinically useful tumor marker clearly merit further investigation.

Natural genetic exchange between Haemophilus and Neisseria: Intergeneric transfer of chromosomal genes between major human pathogens

Members of the bacterial families Haemophilus and Neisseria, important human pathogens that commonly colonize the nasopharynx, are naturally competent for DNA uptake from their environment. In each genus this process is discriminant in favor of its own and against foreign DNA through sequence specificity of DNA receptors. The Haemophilus DNA uptake apparatus binds a 29-bp oligonucleotide domain containing a highly conserved 9-bp core sequence, whereas the neisserial apparatus binds a 10-bp motif. Each motif (“uptake sequence”, US) is highly over-represented in the chromosome of the corresponding genus, particularly concentrated with core sequences in inverted pairs forming gene terminators. Two Haemophilus core USs were unexpectedly found forming the terminator of sodC in Neisseria meningitidis (meningococcus), and sequence analysis strongly suggests that this virulence gene, located next to IS1106, arose through horizontal transfer from Haemophilus. By using USs as search strings in a computer-based analysis of genome sequence, it was established that while USs of the “wrong” genus do not occur commonly in Neisseria or Haemophilus, where they do they are highly likely to flag domains of chromosomal DNA that have been transferred from Haemophilus. Three independent domains of Haemophilus-like DNA were found in the meningococcal chromosome, associated respectively with the virulence gene sodC, the bio gene cluster, and an unidentified orf. This report identifies intergenerically transferred DNA and its source in bacteria, and further identifies transformation with heterologous chromosomal DNA as a way of establishing potentially important chromosomal mosaicism in these pathogenic bacteria.

Kinetics of T cell receptor β, γ, and δ rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44+CD25+ Pro-T thymocytes

We performed a comprehensive analysis of T cell receptor (TCR) γ rearrangements in T cell precursors of the mouse adult thymus. Using a sensitive quantitative PCR method, we show that TCRγ rearrangements are present in CD44+CD25+ Pro-T thymocytes much earlier than expected. TCRγ rearrangements increase significantly from the Pro-T to the CD44−CD25+ Pre-T cell transition, and follow different patterns depending on each Vγ gene segment, suggesting that ordered waves of TCRγ rearrangement exist in the adult mouse thymus as has been described in the fetal mouse thymus. Recombinations of TCRγ genes occur concurrently with TCRδ and D-Jβ rearrangements, but before Vβ gene assembly. Productive TCRγ rearrangements do not increase significantly before the Pre-T cell stage and are depleted in CD4+CD8+ double-positive cells from normal mice. In contrast, double-positive thymocytes from TCRδ−/− mice display random proportions of TCRγ rearranged alleles, supporting a role for functional TCRγ/δ rearrangements in the γδ divergence process.

Loss of heterozygosity induced by a chromosomal double-strand break

The repair of chromosomal double-strand breaks (DSBs) is necessary for genomic integrity in all organisms. Genetic consequences of misrepair include chromosomal loss, deletion, and duplication resulting in loss of heterozygosity (LOH), a common finding in human solid tumors. Although work with radiation-sensitive cell lines suggests that mammalian cells primarily rejoin DSBs by nonhomologous mechanisms, alternative mechanisms that are implicated in chromosomal LOH, such as allelic recombination, may also occur. We have examined chromosomal DSB repair between homologs in a gene targeted mammalian cell line at the retinoblastoma (Rb) locus. We have found that allelic recombinational repair occurs in mammalian cells and is increased at least two orders of magnitude by the induction of a chromosomal DSB. One consequence of allelic recombination is LOH at the Rb locus. Some of the repair events also resulted in other types of genetic instability, including deletions and duplications. We speculate that mammalian cells may have developed efficient nonhomologous DSB repair processes to bypass allelic recombination and the potential for reduction to homozygosity.

The human XRCC9 gene corrects chromosomal instability and mutagen sensitivities in CHO UV40 cells

The Chinese hamster ovary (CHO) mutant UV40 cell line is hypersensitive to UV and ionizing radiation, simple alkylating agents, and DNA cross-linking agents. The mutant cells also have a high level of spontaneous chromosomal aberrations and 3-fold elevated sister chromatid exchange. We cloned and sequenced a human cDNA, designated XRCC9, that partially corrected the hypersensitivity of UV40 to mitomycin C, cisplatin, ethyl methanesulfonate, UV, and γ-radiation. The spontaneous chromosomal aberrations in XRCC9 cDNA transformants were almost fully corrected whereas sister chromatid exchanges were unchanged. The XRCC9 genomic sequence was cloned and mapped to chromosome 9p13. The translated XRCC9 sequence of 622 amino acids has no similarity with known proteins. The 2.5-kb XRCC9 mRNA seen in the parental cells was undetectable in UV40 cells. The mRNA levels in testis were up to 10-fold higher compared with other human tissues and up to 100-fold higher compared with other baboon tissues. XRCC9 is a candidate tumor suppressor gene that might operate in a postreplication repair or a cell cycle checkpoint function.

Macrophage inflammatory protein-2: Chromosomal regulation in rat small intestinal epithelial cells

Nonpathogenic, resident bacteria participate in the pathogenesis of inflammation in the small intestine, but the molecular messages produced by such bacteria are unknown. Inflammatory responses involve the recruitment of specific leukocyte subsets. We, therefore, hypothesized that butyrate, a normal bacterial metabolite, may modulate chemokine secretion by epithelial cells, by amplifying their response to proinflammatory signals. We studied the expression of the chemokine, macrophage inflammatory protein-2 (MIP-2) by the rat small intestinal epithelial cell line, IEC-6. Cells were stimulated with lipopolysaccharide or with interleukin 1β (IL-1β) and incubated with sodium butyrate. Acetylation of histones was examined in Triton X acetic acid–urea gels by PAGE. Unstimulated IEC-6 cells did not secrete MIP-2. However, lipopolysaccharide and IL-1β induced MIP-2 expression. Butyrate enhanced MIP-2 secretion both in lipopolysaccharide-stimulated and IL-1β-stimulated enterocytes; but butyrate alone did not induce MIP-2 expression. Butyrate increased the acetylation of histones extracted from the nuclei of IEC-6 cells. Furthermore, acetylation of histones (induced by trichostatin A, a specific inhibitor of histone deacetylase) enhanced MIP-2 expression by cells stimulated with IL-1β. In conclusion, trichostatin A reproduced the effects of butyrate on MIP-2 secretion. Butyrate, therefore, increases MIP-2 secretion in stimulated cells by increasing histone acetylation. We speculate that butyrate carries information from bacteria to epithelial cells. Epithelial cells transduce this signal through histone deacetylase, modulating the secretion of chemokines.

Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea

Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5′ upstream region of the grisea ORF. One mutation is a G → A transition at the 5′ splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.

The ARS309 chromosomal replicator of Saccharomyces cerevisiae depends on an exceptional ARS consensus sequence

Autonomously replicating sequence (ARS) elements, which function as the cis-acting chromosomal replicators in the yeast Saccharomyces cerevisiae, depend upon an essential copy of the 11-bp ARS consensus sequence (ACS) for activity. Analysis of the chromosome III replicator ARS309 unexpectedly revealed that its essential ACS differs from the canonical ACS at two positions. One of the changes observed in ARS309 inactivates other ARS elements. This atypical ACS binds the origin recognition complex efficiently and is required for chromosomal replication origin activity. Comparison of the essential ACS of ARS309 with the essential regions of other ARS elements revealed an expanded 17-bp conserved sequence that efficiently predicts the essential core of ARS elements.

Highly conservative reciprocal translocations formed by apparent joining of exchanged DNA double-strand break ends

Chromosomal translocations induced by ionizing radiation and radiomimetic drugs are thought to arise by incorrect joining of DNA double-strand breaks. To dissect such misrepair events at a molecular level, large-scale, bleomycin-induced rearrangements in the aprt gene of Chinese hamster ovary D422 cells were mapped, the breakpoints were sequenced, and the original non-aprt parental sequences involved in each rearrangement were recovered from nonmutant cells. Of seven rearrangements characterized, six were reciprocal exchanges between aprt and unrelated sequences. Consistent with a mechanism involving joining of exchanged double-strand break ends, there was, in most cases, no homology between the two parental sequences, no overlap in sequences retained at the two newly formed junctions, and little or no loss of parental sequences (usually ≤2 bp) at the breakpoints. The breakpoints were strongly correlated (P < 0.0001) with expected sites of bleomycin-induced, double-strand breaks. Fluorescence in situ hybridization indicated that, in six of the mutants, the rearrangement was accompanied by a chromosomal translocation at the aprt locus, because upstream and downstream flanking sequences were detected on separate chromosomes. The results suggest that repair of free radical-mediated, double-strand breaks in confluence-arrested cells is effected by a conservative, homology-independent, end-joining pathway that does not involve single-strand intermediate and that misjoining of exchanged ends by this pathway can directly result in chromosomal translocations.

Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats

The ultra-long telomeres that have been observed in mice are not in accordance with the concept that critical telomere shortening is related to aging and immortalization. Here, we have used quantitative fluorescence in situ hybridization to estimate (T2AG3)n lengths of individual telomeres in various mouse strains. Telomere lengths were very heterogeneous, but specific chromosomes of bone marrow cells and skin fibroblasts from individual mice had similar telomere lengths. We estimate that the shortest telomeres are around 10 kb in length, indicating that each mouse cell has a few telomeres with (T2AG3)n lengths within the range of human telomeres. These short telomeres may be critical in limiting the replicative potential of murine cells.