Unusual chromosomal organization of telomeric sequences and expeditious karyotypic differentiation in the recently evolved Mus terricolor complex

The Mus terricolor complex displays a stable homozygous arrangement of autosomal heterochromatin variations in the form of accretion of definitive autosomal short arms among three nonoverlapping populations, in concert with an expeditious evolutionary differentiation into three chromosomal species: M. terricolor I, II, and III. In contrast to the highly conservative M. musculus-like chromosomes in the coexisting sibling species, M. booduga, reshuffling and differentiation of centric heterochromatin has occurred in harmony with a revision of centric configurations, resulting in acrocentric and submetacentric autosomes. The chromosomal distribution of the prevalent vertebrate telomeric sequence (TTAGGG)n was examined by fluorescence in situ hybridization to metaphase cells of M. terricolor I, II, and III. An unusual centric organization of internal telomeric sequences was detected in all the submetacentric and acrocentric autosomes. An auxiliary role of these presumably fragile, recombinogenic telomeric sequences in the evolutionary revision of centric configurations in the terricolor complex is hypothesized.     

Processing of telomeric DNA ends requires the passage of a replication fork

During telomere replication in yeast, chromosome ends acquire a long single-stranded extension of the strand making the 3' end. Previous work showed that these 3' tails are generated late in S-phase, when conventional replication is virtually complete. In addition, the extensions were also observed in cells that lacked telomerase. Therefore, a model was proposed that predicted an activity that recessed the 5' ends at yeast telomeres after conventional replication was complete. Here, we demonstrate that this processing activity is dependent on the passage of a replication fork through yeast telomeres. A non-replicating linear plasmid with telomeres at each end does not acquire single-stranded extensions, while an identical construct containing an origin of replication does. Thus, the processing activity could be associated with the enzymes at the replication fork itself, or the passage of the fork through the telomeric sequences allows a transient access for the activity to the telomeres. We therefore propose that there is a mechanistic link between the conventional replication machinery and telomere maintenance.     

Amplification of closed circular DNA in vitro

The polymerase chain reaction is a powerful technique used to amplify nucleic acids in vitro . The reaction produces linear products, and as of yet, closed circular products have not been possible. Since the replicatively competent form of many DNA molecules is the closed circular form, it would be adventitious to amplify closed circular DNA as closed circular molecules. Until now, these molecules could only be amplified in vivo in appropriate host cells. Here, we describe an in vitro procedure, ligation-during-amplification (LDA), for selective amplification of closed circular DNA using sequence-specific primers. LDA is useful for site-directed mutagenesis, mutation detection, DNA modification, DNA library screening and circular DNA production.     

Amplification of closed circular DNA in vitro

The polymerase chain reaction is a powerful technique used to amplify nucleic acids in vitro. The reaction produces linear products, and as of yet, closed circular products have not been possible. Since the replicatively competent form of many DNA molecules is the closed circular form, it would be adventitious to amplify closed circular DNA as closed circular molecules. Until now, these molecules could only be amplified in vivo in appropriate host cells. Here, we describe an in vito procedure, ligation-during-amplification (LDA), for selective amplification of closed circular DNA using sequence-specific primers. LDA is useful for site-directed mutagenesis, mutation detection, DNA modification, DNA library screening and circular DNA production.     

Clonal karyotypic evolution in an embryonal rhabdomyosarcoma with trisomy 8 as the primary chromosomal abnormality

An embryonal rhabdomyosarcoma was analyzed cytogenetically. In primary cultures fed a serum-containing medium, 11 clones with karyotypic abnormalities were found. One had trisomy 8 only. The other 10 clones had trisomy 8 as well as additional evolutionary changes that included trisomy for part or all of chromosome 2, isochromosomes for the short and long arms of chromosome 11, isochromosomes for the long arm of chromosome 8, and extra copies of chromosome 8, some of which had an interstitial deletion in 8q. In those primary cultures that had grown in a chemically defined, serum-free medium and in all passaged cultures, trisomy 8 was the only aberration. Our findings and a survey of published information point to gain of one chromosome 8 as a frequent primary karyotypic abnormality in embryonal rhabdomyosarcomas. Trisomy for part or all of chromosomes 2 and 11 and additional gains of chromosome 8 material seem to be common secondary changes.     

Amplification of mitochondrial DNA in acute myeloid leukaemia

There is a long-standing interest in the possible role of mitochondria in malignancy. We sought to discover whether amplification of mitochondrial DNA (mtDNA) occurred in leukaemia, and found it was often remarkably amplified in the blast cells of acute myeloid leukaemia (AML). We used gene dosage experiments to quantify the amount of mtDNA relative to nuclear DNA. DNA extracted from peripheral blood leucocytes or bone marrow of healthy individuals or patients was simultaneously hybridized with a probe for the mitochondrial genome and a control probe for the renin gene on human chromosome 1. Comparative densitometric ratios of approximately 1 were obtained between the two signals in 20 normal control peripheral blood samples. In contrast, comparative ratios in the range of 2-50 were observed in 25 AML samples and 13 of these showed 8-fold or greater amplification of mtDNA relative to normal peripheral blood controls. An additional four cases of AML were investigated at both presentation and remission and showed 3-10-fold amplification of mtDNA at presentation, but no amplification when in clinical remission. 18 cases of chronic granulocytic leukaemia (CGL) were also studied in chronic phase and showed mtDNA dosage levels equivalent to normal peripheral blood controls. However, 8/9 CGL patients showed mtDNA amplification during transformation from chronic phase. We conclude that amplification of mtDNA is an invariable feature of acute myeloid leukaemia and that it may be a useful marker for detecting transformation of CGL.     

Sequence-specific binding protein of single-stranded and unimolecular quadruplex telomeric DNA from rat hepatocytes

A rat liver nuclear protein, unimolecular quadruplex telomere-binding protein 25, (uqTBP25) is described that binds tightly and specifically single-stranded and unimolecular tetraplex forms of the vertebrate telomeric DNA sequence 5'-d(TTAGGG)n-3'. A near homogeneous uqTBP25 was purified by ammonium sulfate precipitation, chromatographic separation from other DNA binding proteins, and three steps of column chromatography. SDS-polyacrylamide gel electrophoresis and Superdex copyright 200 gel filtration disclosed for uqTBP25 subunit and native Mr values of 25.4 +/- 0.5 and 25.0 kDa, respectively. Sequences of uqTBP25 tryptic peptides were closely homologous, but not identical, to heterogeneous nuclear ribonucleoprotein A1, heterogeneous nuclear ribonucleoprotein A2/B1, and single-stranded DNA-binding proteins UP1 and HDP-1. Complexes of uqTBP25 with single-stranded or unimolecular quadruplex 5'-d(TTAGGG)4-3', respectively, had dissociation constants, Kd, of 2.2 or 13.4 nM. Relative to d(TTAGGG)4, complexes with 5'-r(UUAGGG)4-3', blunt-ended duplex telomeric DNA, or quadruplex telomeric DNA had >10 to >250-fold higher Kd values. Single base alterations within the d(TTAGGG) repeat increased the Kd of complexes with uqTBP25 by 9-215-fold. Association with uqTBP25 protected d(TTAGGG)4 against nuclease digestion, suggesting a potential role for the protein in telomeric DNA transactions.     

In vivo linearization and autonomous replication of plasmids containing human telomeric DNA in Aspergillus nidulans

We have previously shown the existence of a novel peripheral reflex inhibitorily modulating the vas deferens sympathetic activity. An interaction between noradrenergic and histamine-containing neurons is involved in this reflex. As an overall mechanism of sympathetic autoregulation, we found that enhanced sympathetic activity in the rat during the stress induced by brief inescapable footshocks caused a marked rise of blood histamine that was seemingly dependent upon sympathetic activity. This rise was prevented by either previous ganglionic blockade with hexamethonium or chronic guanethidine-induced sympathectomy. Previous adrenal demedullation did not impair this rise. Thus, it appears that only the sympathetic postganglionic neuron, interacting with a histamine-containing neuron, is involved in the rise of blood histamine induced by footshocks.     

A satellite DNA of the Sparidae family (pisces, perciformes) associated with telomeric sequences

This paper gives an overview of a lecture scheduled for the opening of the 10th European Bioenergetics Congress. In this lecture I plan to first reflect on the accomplishments of some of the individuals who were involved in research on the ATP synthase during the past 50 years. Then I will give a brief view of the present information about rotational catalysis by the ATP synthase. This will be followed by a discussion of some results from my laboratory that call for additional experimentation. Finally I will direct attention to other questions about the ATP synthase that should be addressed in future studies.     

Sensitive method for measuring telomere lengths by quantifying telomeric DNA content of whole cells

Recently, a new method for measuring telomere lengths based on telomere DNA content was developed. The method, which is based on the ratio of telomere to centromere DNA content (TC ratio), is highly sensitive, allowing the analysis of small quantities of DNA. However, the method required the isolation of DNA, which can be difficult or impossible for small numbers of cells. Here, we suggest an improvement of this method that can directly estimate telomere lengths from whole cells. We optimized the method for whole cells and purified DNA and found that accurate TC ratios can be obtained from as little as 9 ng of DNA or 800 whole cells. There was no statistically significant difference between the ratios obtained with purified DNA or with whole cells, indicating that the isolation of DNA is not necessary for small samples.     

Asymmetrical DNA replication promotes evolution: disparity theory of evolution

Heredity is guaranteed by faithful DNA replication whereas evolution depends upon errors accompanying DNA replication. This contradiction existing between heredity and evolution cannot be resolved in an individual organism, but only in terms of a population, in that a delicate balance exists between wild type and variants in a population which is necessary for the survival of the species. Namely, there seems to be a key in the mechanism of DNA replication to solve some problems of evolution. DNA is replicated semiconservatively using the leading and discontinuous lagging strands. According to our 'disparity theory of evolution', the existence of a sufficient fidelity difference between the leading and lagging strands is advantageous in terms of evolution, because the diversity of genotypes is enlarged but genotypes that have occurred in the past are guaranteed. In theory, by artificially increasing the fidelity difference between the leading and lagging strand ('disparity mutator'), evolution is accelerated while avoiding the extinction of the population. Using a disparity mutator, we should be able to improve living things, including multicellular organisms, within constrained conditions. A double-stranded algorithm, which mimics the structure and replication manner of DNA, is promising for solving optimization problems.     

Construction and characterization of a quadruplex DNA selective single-chain autoantibody from a viable motheaten mouse hybridoma with homology to telomeric DNA binding proteins

An autoantibody produced by a hybridoma derived from a viable motheaten mouse was isolated and found to have moderately high binding affinity for nucleic acids and specific preference for quadruplex DNAs. Polymerase chain reaction primers were designed to link the cloned parental antibody variable region fragments together in a subcloning vector. This single-chain variable fragment construct was then subcloned into the T7 promoter-driven expression vector pET22b(+). The construct contains (N- to C-terminal) a pelB leader sequence, variable heavy chain, glycine-serine polylinker, variable light chain, and biotin mimic peptide "strep-tag" sequence (pelB-VH-linker-VL-strep-tag). The ca. 29 kDa protein was expressed, exported to the periplasmic space of NovaBlue (DE) Escherichia coli, and purified by streptavidin affinity chromatography by binding the fused strep-tag peptide. The specificity of the purified single-chain variable fragment (scFv) for quadruplex and duplex DNAs was evaluated by a radioimmunofilterbinding assay. It retained about 10-fold higher affinity for quadruplexes relative to duplex DNA, a reduction of ca. 4-fold from the relative preferences of the parent IgG. The complementary-determining regions contain sequences that are homologous to or conservatively divergent from the key DNA-binding helix-turn-helix-forming motifs of Myb/RAP1 family telomeric DNA-binding proteins (1-3). The presence of this antibody in the autoimmune repertoire suggests a possible linkage between autoimmunity, telomeric DNA binding proteins, and aging.     

The yeast telomere length regulator TEL2 encodes a protein that binds to telomeric DNA

TEL2 is required for telomere length regulation and viability in Saccharomyces cerevisiae. To investigate the mechanism by which Tel2p regulates telomere length, the majority (65%) of the TEL2 ORF was fused to the 3'-end of the gene for maltose binding protein, expressed in bacteria and the purified protein used in DNA binding studies. Rap1p, the major yeast telomere binding protein, recognizes a 13 bp duplex site 5'-GGTGTGTGGGTGT-3' in yeast telomeric DNA with high affinity. Gel shift experiments revealed that the MBP-Tel2p fusion binds the double-stranded yeast telomeric Rap1p site in a sequence-specific manner. Analysis of mutated sites showed that MBP-Tel2p could bind 5'-GTGTGTGG-3' within this 13 bp site. Methylation interference analysis revealed that Tel2p contacts the 5'-terminal guanine in the major groove. MBP-Tel2p did not bind duplex telomeric DNA repeats from vertebrates, Tetrahymena or Oxytricha. These results suggest that Tel2p is a DNA binding protein that recognizes yeast telomeric DNA.     

The telobox, a Myb-related telomeric DNA binding motif found in proteins from yeast, plants and human

The yeast TTAGGG binding factor 1 (Tbf1) was identified and cloned through its ability to interact with vertebrate telomeric repeats in vitro. We show here that a sequence of 60 amino acids located in its C-terminus is critical for DNA binding. This sequence exhibits homologies with Myb repeats and is conserved among five proteins from plants, two of which are known to bind telomeric-related sequences, and two proteins from human, including the telomeric repeat binding factor (TRF) and the predicted C-terminal polypeptide, called orf2, from a yet unknown protein. We demonstrate that the 111 C-terminal residues of TRF and the 64 orf2 residues are able to bind the human telomeric repeats specifically. We propose to call the particular Myb-related motif found in these proteins the 'telobox'. Antibodies directed against the Tbf1 telobox detect two proteins in nuclear and mitotic chromosome extracts from human cell lines. Moreover, both proteins bind specifically to telomeric repeats in vitro. TRF is likely to correspond to one of them. Based on their high affinity for the telomeric repeat, we predict that TRF and orf2 play an important role at human telomeres.     

SRY and karyotypic status of one abnormal and two intersexual marsupials

An intersexual agile wallaby (Macropus agilis) with a penis, a pouch and four teats had a sex-chromosome constitution of XXY in lymphocytes and cultured fibroblasts; the sex-determining region Y (SRY) gene was present, consistent with the presence of a testis. An intersexual eastern grey kangaroo (Macropus giganteus) with a small empty scrotum and no penis, and an abnormal red kangaroo (Macropus rufus) with no penis, pouch or teats, both had XX sex-chromosome complements; the SRY gene was not present, consistent with testis absence. The agile wallaby and grey kangaroo described here provide further evidence that scrotal development in marsupials is independent of the Y chromosome. The cause of the abnormalities in the XX individuals cannot be determined until candidate genes are identified. These animals provide a basis for further genetic studies into marsupial intersexuality and sex differentiation.     

Molecular evolution of the family Camelidae: a mitochondrial DNA study

We report the first molecular evolutionary analysis of the family Camelidae by analysing the full DNA sequence of the mitochondrial cytochrome b gene. Estimates for the time of divergence of the Old World (Camelini) and New World (Lamini) tribes obtained from sequence data are in agreement with those derived from the fossil record. The DNA sequence data were also used to test current hypotheses concerning the ancestors of the domesticated llama and alpaca. The results show that hybridization has occurred in the ancestry of both domesticated camelids, obscuring the origin of the domestic species.