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Query: UNIPROT:P06889 (Mol)
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The autonomously replicating sequence ARS121 was cloned as a 480-base-pair (bp) long DNA fragment that confers on plasmids autonomous replication in Saccharomyces cerevisiae. This fragment contains two OBF1-binding sites (sites I and II) of different affinities, as identified by a gel mobility shift assay and footprint analysis. Nucleotide substitutions (16 to 18 bp) within either of the two sites obliterated detectable in vitro OBF1 binding to the mutagenized site. Linker substitution (6 bp) mutations within the high-affinity site I showed effects similar to those of the complete substitution, whereas DNA mutagenized outside the binding site bound OBF1 normally. We also tested the mitotic stability of centromeric plasmids bearing wild-type and mutagenized copies of ARS121. Both deletion of the sites and the extensive base alterations within either of the two OBF1-binding sites reduced the percentage of plasmid-containing cells in the population from about 88% to 50 to 63% under selective growth and from about 46% to 15 to 20% after 10 to 12 generations of nonselective growth. Furthermore, linker (6 bp) substitutions within site I, the high-affinity binding site, showed similar deficiencies in plasmid stability. In contrast, plasmids containing linker substitutions in sequences contiguous to site I displayed wild-type stability. In addition, plasmid copy number analysis indicated that the instability probably resulted not from nondisjunction during mitosis but rather from inefficient plasmid replication. The results strongly support the notion that the OBF1-binding sites and the OBF1 protein are important for normal ARS function as an origin of replication.
Mol Cell Biol 1989 Jul
PMID:The OBF1 protein and its DNA-binding site are important for the function of an autonomously replicating sequence in Saccharomyces cerevisiae. 267 74

Regulation of the DNA damage-inducible RAD2 gene was investigated in yeast cells transformed with centromeric plasmids containing RAD2-lacZ fusion constructs. Deletion analysis defined several regions in the 350bp region upstream of the translational start codon which are required for induction of beta-galactosidase activity. No deletions resulted in constitutively enhanced expression. We therefore conclude that induction of RAD2 by DNA-damaging agents is positively regulated. Two domains required for induction have a similar sequence and are located approximately 70 and approximately 140bp upstream of the major transcriptional start site. Four other sequence domains required for induction contain uninterrupted poly(dA) poly(dT) stretches 9-13bp long. Deletion of some of these AT-rich domains also affects constitutive expression of RAD2. Expression of RAD2 is not cell-cycle-regulated in mitotic cells. However, meiosis is accompanied by increased steady-state levels of RAD2 mRNA in the absence of DNA damage. This enhanced transcription is not dependent on the presence of upstream sequences required for regulation of induction by DNA damage. Increased steady-state levels of RAD2 mRNA are induced by cycloheximide in asynchronously dividing populations of cells, but not in non-replicating cells arrested in G1 phase of the cell cycle. Following exposure to u.v. irradiation induction is also dramatically reduced in non-replicating cells.
Mol Microbiol 1989 Dec
PMID:Regulation of the RAD2 gene of Saccharomyces cerevisiae. 269 43

We have isolated and cloned a tandemly repeating element from trypanosoma vivax for use as a species-specific DNA probe. The repeat hybridises only with DNA from T. vivax, not with DNA from other Salivarian trypanosome species (T. brucei spp., T. congolense, T. simiae). The monomer of the repeat is approximately 180 bp long and is 64% GC rich. Hybridisation of the cloned fragment with size-fractionated large DNA molecules of 3 T. vivax stocks revealed a band in the position expected for minichromosomes, although these were believed absent in T. vivax. This band migrated to the 100-250 kb area of the gel at 4 different pulse frequencies and also hybridised with a telomeric repeat probe from T. brucei. The band is unlikely to be simply degraded material, since it failed to hybridise with another highly repetitive sequence from T. vivax and was consistently present in different trypanosome preparations. We conclude that T. vivax does possess mini-chromosomes, although possibly only 1 or 2 per cell.
Mol Biochem Parasitol 1989 Mar 01
PMID:Hybridisation with a repetitive DNA probe reveals the presence of small chromosomes in Trypanosoma vivax. 272 82

Plasmid library of rapidly renaturating fraction of human DNA was constructed. The library was used for isolation of primate-specific DNA repeats. A clone (1hsp-4) which was intensively hybridizable with human DNA exclusively and produced no signals when hybridized with animal DNAs including the ones from orangoutan and chimpanzee was isolated. The cloned sequences 1hsp-4 have been found to be highly specific to centromeric heterochromatin of the 18 chromosome. Primary structure of a short 1hsp-4 fragment has been determined. The obtained data suggest the emergence of a DNA family homologous to the 1hsp-4 probe to be due to the thousandfold leapwise amplification occuring less than 5-8 millions years ago.
Mol Gen Mikrobiol Virusol 1989 Apr
PMID:Species specific variant of human centromeric DNA repeats: localization on chromosome 18 and recent amplification in human ancestral line. 274 96

Gene conversion is one mechanism of antigenic variation in Trypanosoma brucei. Variant surface glycoprotein (VSG) genes are duplicated by this process to telomeric locations from which they may be expressed. We examined four independent antigenic switches in which the IsTaR 1.1 minichromosomal VSG gene is duplicated to a large chromosome where it is expressed. An unusual feature of three of these telomeric gene conversions is that the distance between the VSG gene and the end of the chromosome is identical for both the basic and duplicated copies following the antigenic switch. This suggests that the gene conversion is initiated 5' to the VSG gene and extends to the end of the telomere. The data also suggest that events other than simple nucleotide addition account for telomeric growth.
Mol Biochem Parasitol 1989 Jun 01
PMID:A novel telomeric gene conversion in Trypanosoma brucei. 276 71

Pigeon genome long sequences containing clusters of moderately repeating elements have been cloned. Molecular analysis has shown a dispersed distribution of the repeats in both pigeon and chicken genomes. Within a single cluster, a scrambled distribution of elements belonging to different families of repeats has been shown. Similar repeated sequences have been revealed within clusters. The analysed clusters of repeats are characterized by a limited structural variability in the genomes. In situ hybridization revealed the localization of sequences complementary to the cloned clusters in pigeon and chicken macrochromosomes. Preferential localization has been demonstrated in telomeric and centromeric chromosome regions as well as in the region of R-bands.
Mol Biol (Mosk)
PMID:[Molecular and cytologic analysis of clusters of moderate repeats of bird genomes]. 277 Jul 28

In this study we examine the amounts of four different human satellite DNA sequences in a series of human-hamster hybrid cells, which contain a human minichromosome including the centromere of human chromosome 1. Comparisons with the corresponding amounts in an intact human chromosome 1 suggest that the minichromosomes have lost satellite DNA sequences, and in one case a substantial fraction of several satellite DNAs is lost, without affecting the stability and normal mitotic segregation of the minichromosome. The smallest minichromosome appears to have lost all of the long arm and a significant portion of centromeric heterochromatin, while retaining 1000-2000 kb of the short arm of human chromosome 1. The satellite sequences examined include: a chromosome 1-specific satellite III probe, a chromosome 1-specific alpha satellite DNA, another alpha satellite DNA originally derived from the X chromosome, and an alphoid EcoRI dimer whose isolation from one of the minichromosomes and characterization is also described in this paper. One interpretation of these data indicates that an interspersion of blocks of satellite sequences occurs in the centromere region of chromosome 1. If these satellite sequences have functional significance, then there may be redundancy in the system that allows for a variation in the size of the kinetochore and the number of attachment sites for microtubules.
Somat Cell Mol Genet 1989 Sep
PMID:Molecular characterization of human minichromosomes with centromere from chromosome 1 in human-hamster hybrid cells. 278 15

Alphoid DNA is a family of tandemly repeated simple sequences found mainly at the centromeres of the chromosomes of many primates. This paper describes the structure of the alphoid DNA at the centromere of the human Y chromosome. We have used pulsedfield gradient gel electrophoresis, cosmid cloning and DNA sequencing to determine the organization of the alphoid DNA on each of the Y chromosomes present in two somatic cell hybrids. In each case there is a single major block of alphoid DNA. This is approximately 470,000 bases (475 kb) long on one chromosome and approximately 575 kb long on the other. Apart from the size difference, the structures of the two blocks and the surrounding sequences are very similar. However, one restriction enzyme, AvaII, detects two clusters of sites within one block but does not cleave the other. The alphoid DNA within each block is organized into tandemly repeating units, most of which are about 5.7 kb long. A few variant units present on one chromosome are about 6.0 kb long. These variants, like the AvaII site variants, are clustered. The 5.7 kb and 6.0 kb units themselves consist of tandemly repeating 170 base-pair subunits. The 6.0 kb unit has two more of these subunits than the 5.7 kb unit. Our results provide a basis for further structural analysis of the human Y chromosome centromeric region, and suggest that long-range structural polymorphisms of tandemly repeated sequence families may be frequent.
J Mol Biol 1987 Jun 05
PMID:Structure of the major block of alphoid satellite DNA on the human Y chromosome. 282 Dec 79

The centromeric regions of human chromosomes are characterized by diverged chromosome-specific subsets of a tandemly repeated DNA family, alpha satellite, which is based on a fundamental monomer repeat unit approximately 171 bp in length. We have compared the nucleotide sequences of 44 alphoid monomers derived from cloned representatives of the multimeric higher-order repeat units of human chromosomes 1, 11, 17, and X. The 44 monomers exhibit an average 16% divergence from a consensus alphoid sequence, and can be assigned to five distinct homology groups based on patterns of sequence substitutions and gaps relative to the consensus. Approximately half of the overall sequence divergence can be accounted for by sequence changes specific to a particular homology group; the remaining divergence appears to be independent of the five groups and is randomly distributed, both within and between chromosomal subsets. The data are consistent with the proposal that the contemporary tandem arrays on chromosomes 1, 11, 17, and X derive from a common multimeric repeat, consisting of one monomer each from the five homology groups. The sequence comparisons suggest that this pentameric repeat must have spread to these four chromosomal locations many millions of years ago, since which time evolution of the four, now chromosome-specific, alpha satellite subsets has been essentially independent.
J Mol Evol 1987
PMID:Chromosome-specific subsets of human alpha satellite DNA: analysis of sequence divergence within and between chromosomal subsets and evidence for an ancestral pentameric repeat. 282 35

The transition from lineform DNA to cruciform DNA (cruciformation) within the cloned telomere sequences of the Leporipoxvirus Shope fibroma virus (SFV) has been studied. The viral telomere sequences have been cloned in recombination-deficient Escherichia coli as a 322 base-pair, imperfect palindromic insert in pUC13. The inverted repeat configuration is equivalent to the arrangement of the telomere structures observed within viral DNA replicative intermediates. A major cruciform structure in the purified recombinant plasmid has been identified and mapped using, as probes, the enzymes AflII, nuclease S1 and bacteriophage T7 endonuclease I. It was extruded from the central axis of the cloned viral inverted repeat and, by unrestricted branch migration, attained a size commensurate with the superhelical density of the plasmid molecule at native superhelical densities. This major cruciform extrusion event was the only detectable duplex DNA perturbation, induced by negative superhelical torsion, in the insert viral sequences. No significant steady-state pool of extruded cruciform was identified in E. coli. However, the identification of a major deletion variant generated even in the recombination-deficient E. coli strain DB1256 (recA recBC sbcB) suggested that the cruciform may be extruded transiently in vivo. The lineform to cruciform transition has been further characterized in vitro using two-dimensional agarose gel electrophoresis. The transition was marked by a high energy of formation (delta Gf = 44 kcal/mol), and an apparently low activation energy that enabled facile transitions at physiological temperatures provided there was sufficient torsional energy. By comparing cruciformation in a series of related bidirectional central axis deletions of the telomeric insert, it has been concluded that the presence of extrahelical bases in the terminal hairpin structures contributes substantially to the high delta Gf value. Also, viral sequences flanking the extruded cruciform were shown to influence the measured delta Gf value. Several general features of poxvirus telomere structure that would be expected to influence the facility of cruciform extrusion are discussed along with the implications of the observed cruciform transition event on the replicative process of poxviruses in vivo.
J Mol Biol 1987 Aug 05
PMID:Cruciform extrusion in plasmids bearing the replicative intermediate configuration of a poxvirus telomere. 282 85


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