Involvement of telomeric sequences in chromosomal aberrations

The genomes of higher eukaryotes are not homogeneous in terms of structure or function. Many examples of chromosomal regions particularly prone to involvement in aberrations have been reported. The molecular structures of some of these regions have now been determined, most notably the folate-sensitive fragile sites and FRA16B-a distamycin A-sensitive fragile site. In addition, a number of cytological studies suggest that telomeric sequences can in some circumstances be involved in chromosomal aberrations more frequently than expected. Here, the roles of telomeric DNA sequences, both terminal and interstitial, and telomerase in chromosomal aberration formation are reviewed.     

Amplification of telomeric DNA and the extent of karyotypic evolution

The distribution of telomeric DNA in the genomes of the antelope ground squirrel, Ammospermophilus harrisii (family Sciuridae; 2n = 32) and the African black-footed cat, Felis nigripes (family Felidae; 2n = 38) were compared by fluorescence in situ hybridization (FISH) technique. These two mammalian species have the highest and the lowest amount of C-banded regions, respectively. FISH preparations with the human telomeric DNA probe showed that all C-banded segments in the A. harrisii chromosomes, except a few intercalary segments, were hybridizing with this DNA. F. nigripes showed hybridization only on the termini of each chromosome, and the C-banded regions did not hybridize with telomeric DNA on FISH analysis. The C-banded chromosomal arms in another rodent species, Peromyscus eremicus (family Cricetidae; 2n = 48), when hybridized with human telomeric DNA showed signals only in the termini of chromosomes but not in the heterochromatic arms. These observations indicate that not all C-banded regions in rodent species are telomeric DNA. The amplification of telomeric DNA in relation to speciation is discussed.     

Citrinin-induced chromosomal abnormalities in the bone marrow cells of Mus musculus

Oral administration of various doses of citrinin (5 to 20 ppm) to young weanling mice for 8 weeks induced both gross as well as individual types of abnormalities and breakages in the chromosomes of bone marrow cells. The rate of such abnormalities was 5.5% in the controls and 36.0% in the treated variants. Hypoploidy, stickiness and clumping of chromosomes were some of the gross types of abnormalities. Structural chromosomal changes observed were chromosome/chromatid breaks, gaps, acentric fragments, metacentric chromosomes and ring formations.     

Molecular cloning and chromosomal localization of Chinese hamster telomeric protein chTRF1. Its potential role in chromosomal instability

Chinese hamster cells frequently have altered karyotypes. To investigate the basis of recent observations that karyotypic alterations are related to telomeric fusions, we asked whether these alterations are due to lack of telomere repeat binding factor/s. Further, Chinese hamster chromosomes contain large blocks of interstitial telomeric repeats, which are preferentially involved in chromosome breakage and exchange, rendering it an interesting model for such studies. Here, we report on the cloning and the chromosomal localization of the Chinese hamster telomere repeat binding factor, chTRF1. The sequence analysis revealed, similar to human TRF1 (hTRF1), an N-terminal acidic domain, a TRF1 specific DNA binding motif and a C-terminal Myb type domain. Unlike mouse TRF1 (mTRF1), chTRF1 shows 97.5% identity to hTRF1. chTRF1 gene was localized on the long arm of chromosome 5. In vitro translation of chTRF1 resulted in protein product similar in molecular weight to hTRF1. Immunostaining of Chinese hamster ovary cells (CHO) with anti-TRF1 antibody revealed punctate nuclear staining. At metaphase, antibodies failed to detect TRF1 on most of the chromosome ends and the interstitial telomeric repeat bands. These studies suggest that chTRF1 does not bind the interstitial telomeric repeats, and its presence at the metaphase chromosome ends is limited. The later could be a factor contributing to frequent karyotypic alterations observed in Chinese hamster cells.     

Comparative mapping of the human 22q11 chromosomal region and the orthologous region in mice reveals complex changes in gene organization

The region of human chromosome 22q11 is prone to rearrangements. The resulting chromosomal abnormalities are involved in Velo-cardio-facial and DiGeorge syndromes (VCFS and DGS) (deletions), "cat eye" syndrome (duplications), and certain types of tumors (translocations). As a prelude to the development of mouse models for VCFS/DGS by generating targeted deletions in the mouse genome, we examined the organization of genes from human chromosome 22q11 in the mouse. Using genetic linkage analysis and detailed physical mapping, we show that genes from a relatively small region of human 22q11 are distributed on three mouse chromosomes (MMU6, MMU10, and MMU16). Furthermore, although the region corresponding to about 2.5 megabases of the VCFS/DGS critical region is located on mouse chromosome 16, the relative organization of the region is quite different from that in humans. Our results show that the instability of the 22q11 region is not restricted to humans but may have been present throughout evolution. The results also underscore the importance of detailed comparative mapping of genes in mice and humans as a prerequisite for the development of mouse models of human diseases involving chromosomal rearrangements.     

Effect of thymine tract length on the structure and stability of model telomeric sequences

DNA from the telomeres at the ends of eukaryotic chromosomes contains a stretch of simple tandemly repeated sequences in which clusters of G residues alternate with clusters of T/A sequences along one DNA strand. Model telomeric G-clusters form four-stranded structures in Na+ or K+, stabilized by Hoogsteen pairing between G bases. DNA containing a single copy of the G-cluster can self-associate to form tetramers, with a parallel-stranded, right-handed helical structure. Two copies of the 3'-terminal G strand form a folded-back hairpin that dimerizes to create an antiparallel quadruplex structure. We show here that the tetrameric structure is strongly influenced by the T residue flanking either side of the G-cluster. The parallel tetraplex formed by single copies of the sequences dTnG4 is most stable for n = 1 and least stable for n = 8, the longest tract we have studied. At least two thymine residues are required to allow formation of antiparallel folded-back hairpin dimers from two-copy oligomers of sequence d(TnG4)2 in Na+; additional T's destabilize this structure. In K+, the predominant structure formed is the four-stranded parallel tetramer in all cases. Kinetic analysis indicates that the quadruplex structure formed by Oxytricha telomeric DNA overhangs in the presence of Na+ arises by dimerization of two Hoogsteen base-paired hairpins, with a relatively low energy barrier.     

The organization of DNA sequences in the mouse genome

Analysis of the organization of nucleotide sequences in mouse genome is carried out on total DNA at different fragment size, reannealed to intermediate value of Cot, by Ag+--Cs2SO4 density gradient centrifugation.--According to nuclease S-1 resistance and kinetic renaturation curves mouse genome appears to be made up of non-repetitive DNA (76% of total DNA), middle repetitive DNA (average repetition frequency 2X10(4) copies, 15% of total DNA), highly repetitive DNA (8% of total DNA) and fold-back DNA (renatured density 1.701 g/ml, 1% of total DNA).--Non-repetitive sequences are intercalated with short middle repetitive sequences. One third of non-repetitive sequences is longer than 4500 nucleotides, another third is long between 1800 and 4500 nucleotides, and the remainder is shorter than 1800 nucleotides.--Middle repetitive sequences are transcribed in vivo. The majority of the transcribed repeated sequences appears to be not linked to the bulk of non-repeated sequences at a DNA size of 1800 nucleotides.--The organization of mouse genome analyzed by Ag+--Cs2SO4 density gradient of reannealed DNA appears to be substantially different than that previously observed in human genome using the same technique.     

Genomic organization and chromosomal localization of the Itm2a gene

BACKGROUND: Pemphigus vulgaris is a potentially life-threatening autoimmune disease. Although combination therapies with prednisone and azathioprine are usually effective in controlling the disease, some patients either do not respond to this treatment or show early relapses. OBJECTIVE: To find out whether mycophenolate mofetil would be an effective drug in controlling pemphigus vulgaris in patients who failed initial treatment with azathioprine and prednisone. RESULTS: Twelve patients who were initially diagnosed as having pemphigus vulgaris and had relapsed while undergoing treatment with azathioprine (1.5-2 mg/kg of body weight) and prednisolone (2 mg/kg of body weight) subsequently received combination therapy with mycophenolate mofetil (2 x 1 g/d) and prednisolone (2 mg/kg of body weight per day). Eleven of the 12 patients responded to therapy and showed no relapse of their disease even after tapering of the steroid dose. One patient did not respond. Toxic effects were low with only mild gastrointestinal symptoms in 5 patients and mild lymphopenia (World Health Organization grade I) in 9 patients. During the 9- to 12-month follow-up, none of the 11 patients showed reappearance of pemphigus lesions. CONCLUSION: Treatment of pemphigus vulgaris with mycophenolate is a safe and effective treatment.     

Perceptual organization of complex auditory sequences: Effect of number of simultaneous subsequences and frequency separation.

Previous findings on streaming are generalized to sequences composed of more than 2 subsequences. A new paradigm identified whether listeners perceive complex sequences as a single unit (integrative listening) or segregate them into 2 (or more) perceptual units (stream segregation). Listeners heard 2 complex sequences, each composed of 1, 2, 3, or 4 subsequences. Their task was to detect a temporal irregularity within 1 subsequence. In Experiment 1, the smallest frequency separation under which listeners were able to focus on 1 subsequence was unaffected by the number of co-occurring subsequences; nonfocused sounds were not perceptually organized into streams. In Experiment 2, detection improved progressively, not abruptly, as the frequency separation between subsequences increased from 0.25 to 6 auditory filters. The authors propose a model of perceptual organization of complex auditory sequences. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Isolation and chromosomal distribution of natural Z-DNA-forming sequences in Halobacterium halobium

Conditions favoring left-handed Z-DNA such as high salinity (> 4 ), high negative DNA supercoiling, and GC-rich DNA [statistically favoring d(CG)n repeat sequences], are all found in the extremely halophilic archaeum (archaebacterium) Halobacterium halobium. In order to identify and study Z-DNA regions of the H. halobium genome, an affinity chromatography method with high Z-DNA selection efficiency was developed. Supercoiled plasmids were incubated with a Z-DNA-specific antibody (Z22) and passed over a protein A-agarose column, and the bound plasmids were eluted using an ethidium bromide gradient. In control experiments using mixtures of pUC12 (Z-negative) and a d(CG)5-containing (Z-positive) pUC12 derivative, up to 4,000-fold enrichment of the Z-DNA-containing plasmid was demonstrated per cycle of the Z-DNA selection procedure. The selection efficiency was determined by transformation of Escherichia coli DH5alpha with eluted plasmids and blue-white screening on X-gal plates. Twenty recombinant plasmids containing Z-DNA-forming sequences of H. halobium were isolated from a genomic library using affinity chromatography. Z-DNA-forming sequences in selected plasmids were identified by bandshift and antibody footprinting assays using Z22 monoclonal antibody. Alternating purine-pyrimidine sequences ranging from 8 base pairs (bp) to 13 bp with at least a 6-bp alternating d(GC) stretch were found in the Z22 antibody binding regions of isolated plasmids. The distribution of Z-DNA-forming sequences in the Halobacterium salinarum GRB chromosome was analyzed by dot-blot hybridization of an ordered cosmid library using the cloned H. halobium Z-DNA segments as probe. Among the 11 Z-DNA segments tested, five were found to be clustered in a 100-kilobase pair region of the genome, whereas six others were distributed throughout the rest of the genome.     

The chromosomal organization of simple sequence repeats in wheat and rye genomes

The physical distribution of ten simple-sequence repeated DNA motifs (SSRs) was studied on chromosomes of bread wheat, rye and hexaploid triticale. Oligomers with repeated di-, tri- or tetra-nucleotide motifs were used as probes for fluorescence in situ hybridization to root-tip metaphase and anther pachytene chromosomes. All motifs showed dispersed hybridization signals of varying strengths on all chromosomes. In addition, the motifs (AG)12, (CAT)5, (AAG)5, (GCC)5 and, in particular, (GACA)4 hybridized strongly to pericentromeric and multiple intercalary sites on the B genome chromosomes and on chromosome 4A of wheat, giving diagnostic patterns that resembled N-banding. In rye, all chromosomes showed strong hybridization of (GACA)4 at many intercalary sites that did not correspond to any other known banding pattern, but allowed identification of all R genome chromosome arms. Overall, SSR hybridization signals were found in related chromosome positions independently of the motif used and showed remarkably similar distribution patterns in wheat and rye, indicating the special role of SSRs in chromosome organization as a possible ancient genomic component of the tribe Triticeae (Gramineae).     

Morphological and chromosomal descriptions of new species in the Anopheles subpictus complex

In the present work, the role of lipid peroxidation in cellular lethal injury induced by various types of oxidative stress has been studied in both normal and tumor thymocytes. The prooxidants included either a xanthine/xanthine oxidase system, which is an exogenous source of oxyradicals, or tert-butyl hydroperoxide (t-BOOH), which enters the cell and endogenously produces free radicals. Our data demonstrate that: (A) Using xanthine/xanthine oxidase system as a prooxidant, normal thymocytes are more sensitive than thymoma cells to oxidative damage, as their lactate dehydrogenase (LDH) and malondialdehyde (MDA) release is higher than that of tumor cells. By varying Fe3+/ADP ratios, a positive correlation can be established between LDH and MDA release only in normal thymocytes. While thymoma cells still show a very high level of vitamin E (80%) after 15 min of incubation with this prooxidant, normal thymocytes lose it after the same incubation time. (B) Using t-BOOH as a prooxidant, normal thymocytes release a higher amount of MDA but a lower amount of LDH than thymoma cells. In agreement with the results obtained with the xanthine/xanthine oxidase system, by varying the concentrations of the prooxidant, a correlation between LDH and MDA release can be established only in normal thymocytes. Although high levels of the antioxidant are still present in both kinds of cells after 15 min of incubation with t-BOOH, normal thymocytes consume vitamin E faster than thymoma cells. These data suggest that the role of lipid peroxidation in cell lethal injury is influenced by the source and the site of radical production as well as by the cell type. With t-BOOH as a prooxidant in normal thymocytes, lipid peroxidation is only partially involved in the induction of irreversible cell injury, but it plays a crucial role when the xanthine/xanthine oxidase system is used as a prooxidant. Moreover, whatever the prooxidant used in tumor thymocytes, membranes are more resistant to lipid peroxidation, suggesting that this mechanism is not causally related to cell death.     

Structural organization and chromosomal localization of the human ribosomal protein L9 gene

Monoclonal antibodies (MoAbs) raised against Trypanosoma cruzi microsomal fraction (Mc) and cross-reactive with mammalian tissues were used to evaluate the ability of cross-reactive T. cruzi antigens to induce an immune response in Chagas' disease. Thus, we studied the ability of sera from Chagas' disease patients (CDP) with different degrees of cardiac dysfunction to block the immune recognition of these MoAb to the target antigen determining for each serum an inhibition index (II). By means of this approach we inferred that blocking of monoclonal antibody binding to T. cruzi microsomes by subjects' serum represents antibodies with the same reactivity. After serological and medical examinations, individuals were separated into the following groups: Chagas' disease patients without manifest cardiac involvement (CDP-0), CDP with suspected or borderline cardiac disease (CDP-1), CDP with moderate myocardial dysfunction (CDP-2), CDP with overt cardiac dysfunction (CDP-3) and controls including healthy subjects (HS) and patients with idiopathic myocarditis (IMP). The reactivity between MoAb 5F2 and its target antigen was significantly (p < 0.05) inhibited by sera from CDP irrespective of the clinical stage [CDP: n = 46, 50 +/- 20, mean II +/- SD: control: n = 16, 18 +/- 8]. Moreover, 5F2 was able to distinguish (p < 0.05) sera from CDP with mild disease (CDP clinical grade 0/1: n = 26, 34 +/- 18) from that of CDP with severe disease (CDP clinical grade 2/3: n = 20, 67 +/- 7). Moreover, the inhibitory capacity of sera from asymptomatic CDP (CDP-0) correlated with patients age (r = 0.66, p < 0.05). CDP-0 below or equal 40 years of age had results (n = 15, 25 +/- 13) comparable (p > 0.05) to that of controls while mean inhibition of CDP-0 over 40 years of age (n = 5, 60 +/- 5) was indistinguishable (p > 0.05) from that of patients with severe disease. Competitive assay with MoAb 5A9B11 also showed significant differences (p < 0.05) between sera from CDP (n = 46, 46 +/- 24) and controls (n = 13, 5 +/- 5). On the contrary, the differences observed between CDP with different cardiac involvement was not significant (mild: n = 26, 31 +/- 22; severe: n = 20, 66 +/- 11). However a thorough study of data from asymptomatic sera revealed the existence of two levels of reactivity, with low and high capacity to inhibit the reaction of 5A9B11 against Mc. On the contrary, CDP sera showed a blocking activity for 1A10C11 comparable to that of controls (CDP: n = 25, 19 +/- 9; control: n = 12, 14 +/- 6). Some cross-reactive MoAbs recognized epitopes partially composed of carbohydrates. Interestingly, 5F2 and 5A9B11 epitopes did not appear to have carbohydrates moieties. In summary, immunoinhibition assays revealed differences in the immune response of chronic chagasic patients against parasite epitopes. These results have opened the possibility to identify a prognosis marker of the disease suggesting the clinical utility of monitoring levels of these anti-Mc antibodies in patients with chronic Chagas' disease.     

Chromosomal and genomic organization of Ty1-copia-like retrotransposon sequences in the genus Avena

Regulation of ligand-mediated signal transduction through transmembrane tyrosine kinase growth factor receptors involves phosphorylation of tyrosine residues in the intracellular domain of the receptor. The insulin-like growth factor-I (IGF-I) receptor contains three tyrosine residues in the carboxy-terminal domain at positions 1250, 1251, and 1316. Of these, only the tyrosine at position 1316 is conserved in the homologous position of the insulin receptor. Mutational analysis was used to study the role of these tyrosines in specific outcomes of IGF-I-mediated signal transduction. Mutations in the human IGF-I receptor were either replacement of tyrosines 1250 and 1251 with phenylalanine and histidine (yyFH), respectively, or replacement of the conserved distal tyrosine (position 1316) with phenylalanine (yCF). The yyFH mutation results in an IGF-I receptor with the amino acids found in the homologous position of the human insulin receptor. Cells overexpressing mutated IGF-I receptors were compared with cells expressing only endogenous IGF-I receptors or overexpressing wild-type IGF-I receptors. The ability of yyFH mutant IGF-I receptors to autophosphorylate the beta-subunit or phosphorylate insulin receptor substrate-1 was not significantly different from wild-type type IGF-I receptors. However, one or both of the proximal tyrosine residues (positions 1250 and 1251) in the carboxy-terminus of the IGF-I receptor are essential for IGF-I-stimulation of mitogenic and tumorigenic pathways. IGF-I-induced mitogenesis, measured as thymidine incorporation and cellular proliferation, was abrogated in cells overexpressing mutant IGF-I receptors with replacement of the proximal double tyrosines (positions 1250 and 1251). Fibroblasts expressing this mutant IGF-I receptor formed fewer tumors than the negative control cells, whereas cells expressing wild-type IGF-I receptors formed large tumors in all recipient mice injected. Conversely, cells expressing mutant IGF-I receptors with only the conserved distal tyrosine (position 1316) replaced had slightly reduced IGF-I-stimulated beta-subunit autophosphorylation, thymidine incorporation, and cellular proliferation when compared with cells expressing wild-type receptors. Phosphorylation of insulin receptor substrate-1 by the yCF mutant receptors was not impaired. Despite the ability of these mutant receptors to stimulate mitogenic growth, fibroblasts expressing this mutant receptor were also incapable of forming tumors in recipient nude mice. The distal tyrosine (position 1316) of the IGF-I receptor is crucial for tumor formation but is not essential for IGF-I stimulated mitogenesis. Thus, the tyrosine moieties in the carboxy-terminus of the IGF-I receptor participate in the signal transduction pathways that affect the mitogenic and tumorigenic potentials of cells expressing mutant IGF-I receptors.     

Sequence-based differentiation of strains in the Mycobacterium avium complex

The complete 16S-23S rDNA internal transcribed spacer (ITS) was sequenced in 35 reference strains of the Mycobacterium avium complex. Twelve distinct ITS sequences were obtained, each of which defined a "sequevar"; a sequevar consists of the strain or strains which have a particular sequence. ITS sequences were identified which corresponded to M. avium (16 strains, four ITS sequevars) and Mycobacterium intracellulare (12 strains, one ITS sequevars). The other seven M. avium complex strains had ITS sequences which varied greatly from those of M. avium and M. intracellulare and from each other. The 16S-23S rDNA ITS was much more variable than 16S rDNA, which is widely used for genus and species identification. Phylogenetic trees based on the ITS were compatible with those based on 16S rDNA but were more detailed and had longer branches. The results of ITS sequencing were consistent with the results of hybridization with M. avium and M. intracellulare probes (Gen-Probe) for 30 of 31 strains tested. Serologic testing correlated poorly with ITS sequencing. Strains with the same sequence were different serovars, and those of the same serovar had different sequences. Sequencing of the 16S-23S rDNA ITS should be useful for species and strain differentiation for a wide variety of bacteria and should be applicable to studies of epidemiology, diagnosis, virulence, and taxonomy.     

Levels of personality organization and production of associative sequences in process-reactive schizophrenia.

The 1st part of this study was essentially a replication of an earlier study which reported that genetic theory helped clarify the process-reactive dimension of schizophrenia. The Rorschach and Proverbs tests of 40 schizophrenic Ss were scored according to a genetic scoring system and correlated with Elgin Prognostic Scale scores which measured process-reactive status. Results were negative. Ss were also given 2 association tasks in which they were asked to name animals and persons they had known and several measures were obtained from these associative sequences. A significant difference between process and reactive groups was found on total number of associations on both tasks. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Identification, characterization, and chromosomal organization of cell division cycle genes in Caulobacter crescentus

We report a detailed characterization of cell division cycle (cdc) genes in the differentiating gram-negative bacterium Caulobacter crescentus. A large set of temperature-sensitive cdc mutations was isolated after treatment with the chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Analysis of independently isolated mutants at the nonpermissive temperature identified a variety of well-defined terminal phenotypes, including long filamentous cells blocked at various stages of the cell division cycle and two unusual classes of mutants with defects in both cell growth and division. The latter strains are uniformly arrested as either short bagel-shaped coils or large predivisional cells. The polar morphology of these cdc mutants supports the hypothesis that normal cell cycle progression is directly responsible for developmental regulation in C. crescentus. Genetic and physical mapping of the conditional cdc mutations and the previously characterized dna and div mutations identified at least 21 genes that are required for normal cell cycle progression. Although most of these genes are widely scattered, the genetically linked divA, divB, and divE genes were shown by genetic complementation and physical mapping to be organized in one gene cluster at 3200 units on the chromosome. DNA sequence analysis and marker rescue experiments demonstrated that divE is the C. crescentus ftsA homolog and that the ftsZ gene maps immediately adjacent to ftsA. On the basis of these results, we suggest that the C. crescentus divA-divB-divE(ftsA)-ftsZ gene cluster corresponds to the 2-min fts gene cluster of Escherichia coli.     

Dihydropyrimidinase deficiency: structural organization, chromosomal localization, and mutation analysis of the human dihydropyrimidinase gene

Dihydropyrimidinase (DHP) deficiency (MIM 222748) is characterized by dihydropyrimidinuria and is associated with a variable clinical phenotype. This disease might be associated with a risk of 5-fluorouracil toxicity, although no cases have been reported. We present here both the molecular characterization of the human DHP gene and, for the first time, the mutations causing DHP deficiency. The human DHP gene spans >80 kb and consists of 10 exons. It has been assigned to 8q22, by FISH. We performed mutation analysis of genomic DNA in one symptomatic and five asymptomatic individuals presenting with dihydropyrimidinuria. We identified one frameshift mutation and five missense mutations. Two related Japanese adult subjects were homozygous for the Q334R substitution, whereas two other, unrelated Japanese infant subjects were heterozygous for the same mutation, but this mutation is not common in the Japanese population. A Caucasian pediatric patient exhibiting epileptic attacks, dysmorphic features, and severe developmental delay was homozygous for W360R. Using a eukaryotic expression system, we showed that all mutations reduced enzyme activity significantly, indicating that these are crucial DHP deficiency-causing mutations. There was no significant difference, in residual activity, between mutations observed in the symptomatic and those observed in the asymptomatic individuals.