Two autonomously replicating sequences near oli-1 gene of yeast mitochondrial DNA

We cloned two autonomously replicating sequences from a short segment of mtDNA of an oligomycin-resistant petite yeast, O-111, into a vector pYleu 12 constructed from yeast LEU 2 gene and pBR 322. These plasmids, pYmit 4 and pYmit 1, had frequencies of transformation of yeast as high as that of YEp 13, having a replicator of 2 mu DNA. They were maintained as plasmids in yeast under selective conditions and shuttled from yeast to E. coli. No evidence was obtained that these plasmids were incompatible with the wild-type mitochondrial genome. These sequences were located in intergenic regions.     

The localization of (3H) thymidine incorporation in the DNA of replicating spinach chloroplasts by electron-microscope autoradiography.

Electron-microscope autoradiography has been used to obtain information on the localization of DNA labelled with [3H]thymidine in chloroplasts known to be replicating and concomitantly synthesizing and segregating DNA, in cultured leaf disks. The studies were made using both Microdol-X developer and a 'compact' developer which gave a smaller grain size. About 80% of the grains were associated with the granal membranes and with presumptive DNA regions (3-nm fibril material in clear areas). Few grains occurred in association with the chloroplast envelope. We suggest that the DNA of chloroplasts is associated with the grana lamellae and extends into the stroma. Some light-microscope autoradiographs of whole chloroplasts show spiral or helical-like labelling patterns. We interpret these patterns as demonstration of the possibility that DNA occurs along the length of a continuous lamellar membrane system. Chloroplast fractionation experiments provided data consistent with the electron-microscope autoradiographic studies as most of the label was associated with chlorophyll-containing lamellae. We consider an association of chloroplast DNA molecules along the length of a continuous lamellar system would ensure an orderly segregation of DNA to daughter chloroplasts, during the binary fission of spinach chloroplasts by constriction division.     

Structure of replicating herpes simplex virus DNA.

We have investigated the molecular anatomy of the herpes simplex virus replicative intermediates by cleavage with the restriction endonuclease BglII. We find that in populations of multiply infected cells, pulse-labeled replicating herpes simplex virus DNA contains at least two and probably all four sequence isomers. Also, it contains no detectable termini. In pulse-chase experiments, we show that endless replicative intermediates are the precursors to virion DNA and that maturation is a relatively slow process. The results are discussed in terms of their significance to possible models of herpes simplex virus DNA replication.     

Genetic analysis of replicating instabilities in yeast

Strains showing ethyl methanesulfonate (EMS)-induced replicating instability were genetically analysed to test whether within a given line, mosaics from different plating generations carry a mutation at the same site within the locus. A forward mutation system involving five loci controlling adenine biosynthesis in Schizosaccharomyces pombe was used. Genetic analysis was carried out by interallelic complementation and intragenic recombination tests. The data showed that EMS-induced instabilities are site-specific in being confined to the same recombination unit. This finding is discussed in relation to the possible mechanism(s) of replicating instabilities after different mutagenic treatments in a variety of biological systems.     

Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha.

Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene were shown to replicate autonomously in the yeast at an approximate copy number of 6 (copies per genome equivalent). This autonomous behaviour is probably due to an H. polymorpha replicon-like sequence present on the S. cerevisiae LEU2 gene fragment. Plasmids replicated as multimers consisting of monomers connected in a head-to-tail configuration. Two out of nine transformants analysed appeared to contain plasmid multimers in which one of the monomers contained a deletion. Plasmids containing internal or flanking regions of the genomic alcohol oxidase gene were shown to integrate by homologous single or double cross-over recombination. Both single- and multi-copy (two or three) tandem integrations were observed. Targeted integration occurred in 1-22% of the cases and was only observed with plasmids linearized within the genomic sequences, indicating that homologous linear ends are recombinogenic in H. polymorpha. In the cases in which no targeted integration occurred, double-strand breaks were efficiently repaired in a homology-independent way. Repair of double-strand breaks was precise in 50-68% of the cases. Linearization within homologous as well as nonhomologous plasmid regions stimulated transformation frequencies up to 15-fold.     

Chromatin assembly on replicating DNA in vitro.

Replicating single-stranded DNA is preferentially assembled into chromatin in Xenopus egg extracts relative to non-replicating double-stranded DNA. We have examined the molecular basis of this phenomenon. Single-stranded DNA itself is not a favored template for nucleosome assembly in comparison to double-stranded DNA. Complementary strand synthesis is required for the rapid assembly of nucleosomes. We present evidence that the assembly of chromatin on replicating DNA is a two step phenomenon. The first step involves the replication of DNA and the assembly of an intermediate structure, the second step involves the sequestration of histones H2A/H2B onto DNA. Histones H2A/H2B are preferentially sequestered onto replicated DNA in comparison to non-replicated DNA incubated in the extract.     

Nature of the single-stranded DNA in replicating adenovirus type 5 DNA.

Single-stranded regions in replicating adenovirus type 5 DNA were isolated and hybridized in solution to the separated strands of adenovirus 2 or 5 DNA. The results showed that the two strands of adenovirus 5 DNA are exposed to almost the same extent during replication, suggesting that displacement synthesis may start from either end of the viral DNA.     

Unmethylated DNA in mouse L cells. I. DNA fibre autoradiography.

Enzymatic methylation of DNA in mouse L cells has been studied using DNA fibre autoradiography to analyse the distribution of 5-methylcytosine in chromosomal DNA. The autoradiographic pattern of DNA labelled in the 5-methylcytosine is in several respects similar to the pattern of DNA replication. Two mean features are apparent: (1) the silver grains appear in well defined sections, and (2) the labelled sections are arranged in tandem along each DNA double helix. After a short pulse of radioactivity in the rate of growth of labeled sections in the pattern of DNA replication and the enzymatic methylation of DNA are identical. Unlike the replication pattern, DNA labeled during the S phase with L-[Me-3H] methionine is not completely labeled. There are distinct, 8-20 mum intervals in the autoradiographic pattern of this DNA. The length of these intervals may correspond to unmethylated sections of chromosomal DNA of about 23 to 58 kilo base pairs. These unmethylated sections of chromosomal DNA represent about 10% of the total genome.     

Nucleotide sequences and stability of a Nicotiana nuclear DNA segment possessing autonomously replicating ability in yeast

The nucleotide sequence of a tobacco (Nicotiana tabacum) chromosomal DNA segment(t3-ars) capable of replication in yeast (ars: autonomously replicating sequences) is presented. The subcloned region (618 bp) contained 11 bp consensus (5 A/TTTTATP_uTTTA/T 3) essential for several yeast ars, and 73% A and T. Unique 70 bp repetitive sequences resided next to this sequence. Thirty-two bp AT repeats were also seen in the neighbourhood of the repetitive sequence. The hybrid plasmid containing t3-ars was mitotically stabilized by the help of yeast centromere (CEN4).     

Hybridization method for quantitation of replicating polyoma DNA sequences.

Polyoma DNA was cleaved with restriction endonuclease HpaII, the fragments were separated by gel electrophoresis and transferred in good yield to separate nitrocellulose filters by a modification of the procedure of E. M. Southern (1975, J. Mol. Biol.98, 503–517). The filters were then used in hybridization experiments to localize the isotope in different parts of the polyoma genome after in vitro incorporation of labeled deoxyribonucleoside triphosphates into the DNA.     

Topoisomerase II can unlink replicating DNA by precatenane removal.

We have analysed the role of topoisomerase II (topo II) in plasmid DNA replication in Xenopus egg extracts, using specific inhibitors and two-dimensional gel electrophoresis of replication products. Topo II is dispensable for nuclear assembly and complete replication of plasmid DNA but is required for plasmid unlinking. Extensive unlinking can occur in the absence of mitosis. Replication intermediates generated in the absence of topo II activity have an increased positive superhelical stress (+DeltaLk), suggesting a deficiency in precatenane removal. The geometry of replication intermediates cut by poisoning topo II with etoposide and purified by virtue of their covalent attachment to topo II subunits demonstrates that topo II acts behind the forks at all stages of elongation. These results provide direct evidence for unlinking replicating DNA by precatenane removal and reveal a division of labour between topo I and topo II in this eukaryotic system. We discuss the role of chromatin structure in driving DNA unlinking during S phase.     

四种DNA复制起始方式的比较

近年来有关DNA复制的机制研究取得一些重要进展,但现有高等生物学相关专业的《生物化学》、《分子生物学》和《基因工程》等教材中DNA复制机理尤其是复制起始相关章节的内容更新不够。结合最新的研究进展,本文综述了四种DNA复制方式:双向复制(原核大肠杆菌和真核生物)、单起点单向(质粒ColE1)、哺乳动物线粒体DNA(取代环)以及最近发现的不需要引物的DNA聚合酶起始的DNA复制方式,丰富了教材相关的内容,强调复制的不同方式是不同的复制起始复合物装备的结果。文末对四种不同的复制方式异同进行了比较,根据复制原点在复制复合物装备中的重要作用,对教材中复制原点的概念进行了进一步的剖析。本文内容将有利于学生跳出课程的框架,将相关的知识点融会贯通,夯实理论基础并在将来能有所应用。     

The structure of replicating kinetoplast DNA networks

Kinetoplast DNA (kDNA), the mitochondrial DNA of Crithidia fasciculata and related trypanosomatids, is a network containing approximately 5,000 covalently closed minicircles which are topologically interlocked. kDNA synthesis involves release of covalently closed minicircles from the network, and, after replication of the free minicircles, reattachment of the nicked or gapped progeny minicircles to the network periphery. We have investigated this process by electron microscopy of networks at different stages of replication. The distribution of nicked and closed minicircles is easily detectable either by autoradiography of networks radiolabeled at endogenous nicks by nick translation or by twisting the covalently closed minicircles with intercalating dye. The location of newly synthesized minicircles within the network is determined by autoradiography of network is determined by autoradiography of networks labeled in vivo with a pulse of [3H]thymidine. These studies have clarified structural changes in the network during replication, the timing of repair of nicked minicircles after replication, and the mechanism of division of the network.