Analysis of the Formation and Autonomous Replication of an Extrachromosomal Mouse Transgene

A stable autonomously replicating shuttle transgene, pr8a was, previously isolated from Bombyx mori and characterized. Autonomous replication of pr8a and its derivatives was observed in yeast cells, B. mori embryos, and transgenic mice. To continue the previous studies, transgenic mice of several generations were examined. DNA analysis revealed a course of pr8a rearrangements resulting in extrachromosomal transgene p8-2 of F₂ transgenic mice. Consecutive directional elimination of vector DNA fragments was characteristic of rearrangements affecting autonomous transgenes in mouse cells. Analysis of the data obtained with transgenic mice and other organisms made it possible to identify the pr8a fragment that acts as minimal ARS in transformed yeast cells and transgenic mice. Another pr8a region was assumed to be a component of a pr8a ARS module involved in regulating autonomous replication. Inserted in two different integrative vectors, minimal ARS ensured their autonomous state in transformed yeast cells and transgenic mice. Transgenes pr8a and p8-2, along with some other well-known constructs, were considered as prototype autonomously replicating vectors suitable for studying the mechanism of autonomous replication and solving some problems of gene therapy.     

ARS-activity of a DNA fragment from Bombyx mori in Saccharomyces cerevisiae cells]

A previously cloned autonomous transgene (pr8a) of silkworm Bombyx mori inherited without changes in the structure was used to clarify the activity of its ARS in yeast cells. ARS of pr8a was also shown to maintain autonomous replication of hybrid plasmids in yeast cells. The same was true for its central 2.4-kb fragment devoid of flanking sequences.     

Analysis of the formation and autonomous replication of an extrachromosomal mouse transgene

A stable autonomously replicating shuttle transgene, pr8a was, previously isolated from Bombix mori and characterized. Autonomous replication of pr8a and its derivatives was observed in yeast cells, B. mori embryos, and transgenic mice. To continue the previous studies, transgenic mice of several generations were examined. DNA analysis revealed a course of pr8a rearrangements resulting in extrachromosomal transgene p8-2 of F2 transgenic mice. Consecutive directional elimination of vector DNA fragments was characteristic of rearrangements affecting autonomous transgenes in mouse cells. Analysis of the data obtained with transgenic mice and other organisms made it possible to identify the pr8a fragment that acts as minimal ARS in transformed yeast cells and transgenic mice. Another pr8a region was assumed to be a component of a pr8a ARS module involved in regulating autonomous replication. Inserted in two different integrative vectors, minimal ARS ensured their autonomous state in transformed yeast cells and transgenic mice. Transgenes pr8a and p8-2, along with some other well-known constructs, were considered as prototype autonomously replicating vectors suitable for studying the mechanism of autonomous replication and solving some problems of gene therapy.     

A Petunia hybrida chloroplast DNA region, close to one of the inverted repeats, shows sequence homology with the Euglena gracilis chloroplast DNA region that carries the putative replication origin

Summary Three distinct chloroplast (cp) DNA fragments from Petunia hybrida, which promote autonomous replication in yeast, were mapped on the chloroplast genome. Sequence analysis revealed that these fragments (called ARS A, B and C) have a high AT content, numerous short direct and inverted repeats and at least one yeast ARS consensus sequence 5A/TTTTATPuTTTA/T, essential for yeast ARS activity. ARS A and B also showed the presence of (semi-)conserved sequences, present in all Chlamydomanas reinhardii cpDNA regions that promote autonomous replication in yeast (ARS sequences) or in C. reinhardii (ARC sequences). A 431 bp BamHI/EcoRI fragment, close to one of the inverted repeats and adjacent to the ARS B subfragment contains an AT-rich stretch of about 100 nucleotides that show extensive homology with an Euglena gracilis cpDNA fragment which is part of the replication origin region. This conserved region contains direct and inverted repeats, stem-and-loop structures can be folded and it contains an ARS consensus sequence. In the near vicinity a GC-rich block is present. All these features make this cpDNA region the best candidate for being the origin of replication of P. hybrida cpDNA.     

Localization in the P-element of Drosophila melanogaster of sequences ensuring the autonomic replication of plasmids in yeasts

Two sequences (ARS) capable of maintaining the autonomous replication of plasmids in yeast cells were localized in the right part of Drosophila melanogaster P-element subcloned from the pi 25.1 plasmid. An ARS was found in the DNA region of genome adjacent to P-element. ARS sequences contain imperfect (10 out of 11) consensus typical of yeast ARS and have a complicated domain structure.     

DNA structures associated with autonomously replicating sequences from plants

DNA fragments capable of conferring autonomous replicating ability to plasmids inSaccharomyces cerevisiae were isolated from four different plant genomes and from the Ti plasmid ofAgrobacterium tumefaciens. The DNA structure of these autonomously replicating sequences (ARSs) as well as two from yeast were studied using retardation during polyacrylamide gel electrophoresis and computer analysis as measures of sequence-dependent DNA structures. Bent DNA was found to be associated with the ARS elements. An 11 bp ARS consensus sequence required for ARS function was also identified in the elements examined and was flanked by unusually straight structures which were rich in A+T content. These results show that the ARS elements from genomes of higher plants have structural and sequence features in common with ARS elements from yeast and higher animals.Supported by Grant 1RO1-GM41708-O1 from the National Institute of Health.     

ARS replication during the yeast S phase

A 1.45 kb circular plasmid derived from yeast chromosome IV contains the autonomous replication element called ARS1. Isotope density transfer experiments show that each plasmid molecule replicates once each S phase, with initiation depending on two genetically defined steps required for nuclear DNA replication. A density transfer experiment with synchronized cells demonstrates that the ARS1 plasmid population replicates early in the S phase. The sequences adjacent to ARS1 on chromosome IV also initiate replication early, suggesting that the ARS1 plasmid contains information which determines its time of replication. The times of replication for two other yeast chromosome sequences, ARS2 and a sequence referred to as 1OZ, indicate that the temporal order of replication is ARS1 leads to ARS2 leads to 1OZ. These experiments show directly that specific chromosome regions replicate at specific times during the yeast S phase. If ARS elements are origins of chromosome replication, then the experiment reveals times of activation for two origins.     

Characterization of a chloroplast DNA sequence from Chlorella ellipsoidea that promotes autonomous replication in yeast

Summary An EcoRI 2.7 kbp fragment from Chlorella ellipsoidea chloroplast DNA (cpDNA) cloned in YIp5 was shown to promote autonomous replication in Saccharomyces cerevisiae. The fragment was localized in the small single copy region close to the inverted repeat. The ARS activity (autonomously replicating sequences in yeast) was found to be confined within a subclone of a ca. 300 bp HindIII fragment. Sequence analysis of this fragment revealed its high AT content and the presence of several direct and inverted repeats and a few elements that were related to the yeast ARS consensus sequence. Electron microscopic studies revealed that this sequence did not coincide with the primary replication origin of chloroplast DNA. The functioning of this sequence as a possible origin of plasmid replication in vivo is discussed. This is the first report on Chlorella cpDNA sequence. re]19850821 rv]19851211 ac]19851216     

Development of an autonomously replicating linear vector of the yeast Cryptococcus humicola by using telomere-like sequence repeats

The yeast Cryptococcus humicola has several attractive properties for practical applications such as in bioremediation and as a source of industrially useful enzymes and compounds. We have developed an autonomously replicating vector of C. humicola to improve its properties. We initially tried to isolate an autonomously replicating sequence (ARS) from genomic DNA by transformation using a genomic DNA library. We obtained a candidate plasmid vector harboring an ARS that gave high transformation efficiency. Southern blot analysis of transformants revealed the autonomous replication of the introduced vector in some transformants. However, the vector was not only variously altered in length but also linearized. PCR analysis indicated that a telomere-like sequence repeat (TTAGGGGG) _n was added to the termini of linearized vector. Thus, we constructed an autonomously replicating linear vector having ten repeats of the telomere-like sequence at both ends. The vector transformed the yeast cells with high transformation efficiency (3230 CFU/μg of DNA), which was approximately 25-fold higher than that of a control vector lacking the repeats, and was autonomously replicated at a roughly constant size. The copy number was estimated to be less than one copy, and Ura⁺ mitotic stability varied widely among the transformants and was related to plasmid segregation efficiency.     

The replication behavior of Saccharomyces cerevisiae DNA in human cells

We studied the replication of random genomic DNA fragments from Saccharomyces cerevisiae in a long-term assay in human cells. Plasmids carrying large yeast DNA fragments were able to replicate autonomously in human cells. Efficiency of replication of yeast DNA fragments was comparable to that of similarly sized human DNA fragments and better than that of bacterial DNA. This result suggests that yeast genomic DNA contains sequence information needed for replication in human cells. To examine whether DNA replication in human cells would initiate specifically at a yeast origin of replication, we monitored initiation on a plasmid containing the yeast 2-micron autonomously replicating sequence (ARS) in yeast and human cells. We found that while replication initiates at the 2-micron ARS in yeast, it does not preferentially initiate at the ARS in human cells. This result suggests that the sequences that direct site specific replication initiation in yeast do not function in the same way in human cells, which initiate replication at a broader range of sequences.by J.A. Huberman     

Cloning of DNA sequences from Methanococcus vannielii capable of autonomous replication in yeast

Total DNA of the archaebacterium Methanococcus vannielii was digested with BamHI or BamHI/HindIII, cloned with plasmid Yip5 and analyzed for sequences capable of autonomous replication (ARSs) in the eukaryote Saccharomyces cerevisiae. Two recombinant plasmids were isolated which contained 3.3 kb and 8 kb fragments of methanogen derived DNA with ARS activity. They exhibited low transformation efficiencies for yeast and promoted slow growth of yeast transformants.Abbreviations Ap ampicillin - ARS autonomously replicating sequence - EtBr ethidium bromide - kb kilobase(s) - Mc. Methanococcus - R resistance - RE replication enhancer - RS replication sequence - Tc tetracycline     

Genomic organization of two families of highly repeated nuclear DNA sequences of maize selected for autonomous replicating activity in yeast

Maize nuclear DNA sequences capable of promoting the autonomous replication of plasmids in yeast were isolated by ligating Eco RI-digested fragments into yeast vectors unable to replicate autonomously. Three such autonomously replicating sequences (ARS), representing two families of highly repeated sequences within the maize genome, were isolated and characterized. Each repetitive family shows hybridization patterns on a Southern blot characteristic of a dispersed sequence. Unlike most repetitive sequences in maize, both ARS families have a constant copy number and characteristic genomic hybridization pattern in the inbred lines examined. Larger genome clones with sequence homology to the ARS-containing elements were selected from a lambda library of maize genomic DNA. There was typically only one copy of an ARS-homologous sequence on each 12–15 kb genomic fragment.     

Yeast DNA replication in vitro: initiation and elongation events mimic in vivo processes

An enzyme system prepared from Saccharomyces cerevisiae carries out the replication of exogenous yeast plasmid DNA. Replication in vitro mimics that in vivo in that DNA synthesis in extracts of strain cdc8, a temperature-sensitive DNA replication mutant, is thermolabile relative to the wild-type, and in that aphidicolin inhibits replication in vitro. Furthermore, only plasmids containing a functional yeast replicator, ARS, initiate replication at a specific site in vitro. Analysis of replicative intermediates shows that plasmid YRp7, which contains the chromosomal replicator ARS1, initiates bidirectional replication in a 100 bp region within the sequence required for autonomous replication in vivo. Plasmids containing ARS2, another chromosomal replicator, and the ARS region of the endogenous yeast plasmid 2 microns circle give similar results, suggesting that ARS sequences are specific origins of chromosomal replication. Used in conjunction with deletion mapping, the in vitro system allows definition of the minimal sequences required for the initiation of replication.     

Nuclear scaffold attachment stimulates, but is not essential for ARS activity in Saccharomyces cerevisiae: analysis of the Drosophila ftz SAR

Nuclei isolated from eukaryotic cells can be depleted of histones and most soluble nuclear proteins to isolate a structural framework called the nuclear scaffold. This structure maintains specific interactions with genomic DNA at sites known as scaffold attached regions (SARs), which are thought to be the bases of DNA loops. In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, genomic ARS elements are recovered as SARs. In addition, SARs from Drosophila melanogaster bind to yeast nuclear scaffolds in vitro and a subclass of these promotes autonomous replication of plasmids in yeast. In the present report, we present fine mapping studies of the Drosophila ftz SAR, which has both SAR and ARS activities in yeast. The data establish a close relationship between the sequences involved in ARS activity and scaffold binding: ARS elements that can bind the nuclear scaffold in vitro promote more efficient plasmid replication in vivo, but scaffold association is not a strict prerequisite for ARS function. Efficient interaction with nuclear scaffolds from both yeast and Drosophila requires a minimal length of SAR DNA that contains reiteration of a narrow minor groove structure of the double helix.     

Localization and sequence analysis of chloroplast DNA sequences of Chlamydomonas reinhardii that promote autonomous replication in yeast

Four distinct chloroplast DNA segments from Chlamydomonas reinhardii of 400, 415, 730 and 2300 bp which promote autonomous replication in yeast have been mapped on the chloroplast genome. Plasmids carrying these chloroplast DNA fragments are unstable in yeast when the cells are grown under non-selective conditions. Sequence analysis of three of these chloroplast ARS regions (autonomously replicating sequences in yeast) reveals a high AT content, numerous short direct and inverted repeats and the presence of at least one element in each region that is related to the yeast ARS consensus sequence. A/T TTTATPuTTT A/T. These three chloroplast regions share, in addition, two common elements of 10 and 11 bp which may play a role in promoting autonomous replication.     

Analysis of sequences conferring autonomous replication in baker's yeast

A method is presented for rapid sequencing and mapping of elements which support autonomous replication in yeast. The strategy relies on a novel phage M13 vector which allows detection of ARS (autonomously replicating sequence) function in cloned fragments. Deletion mapping of an ARS element linked to the HO gene of Saccharomyces cerevisiae has identified a 57-bp region 3' to the gene, which is essential for autonomous replication. This region shows sequence homology to other ARS elements.     

In addition to the ARS core, the ARS box is necessary for autonomously replicating sequences

Summary Autonomously replicating sequences (ARSs) were cloned from nuclear and mitochondrial DNA of D. melanogaster using YIp5, which is composed of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^- yeast strain as the recipient. The nucleotide sequences of six ARSs, two from nuclear bulk, two from the nuclear 1.688 satellite, and two from mitochondorial DNA, were determined. The relationship between the transformation frequency and the inclusion of the ARS core, 5 _T ^A TT-TAT _A ^G TTT _T ^A 3, of these fragments was analysed. All the ARSs contained an ARS core or a single base change of it. However, not all the fragments that contained a single base change of the ARS core were able to transform the recipient cells, suggesting that certain bases in the ARS core were not exchangeable. It is suggested by transformation experiments with subfragments that in addition to an ARS core, an ARS box which is located within 25 bp upstream of the ARS core and whose sequence is composed of 5TNT _G ^A AA 3, is necessary for autonomous replication.     

In vitro deletion analysis of ARS elements spanning the replication origin in the 5'' non-transcribed spacer of Tetrahymena thermophila ribosomal DNA.

Two adjacent but non-overlapping restriction fragments that encompass the replication origin of the macronuclear copy of rDNA from Tetrahymena thermophila allow autonomous replication of plasmids in the yeast Saccharomyces cerevisiae; i.e. they function as autonomously replicating segments (ARS). Deletions generated in vitro into these fragments yield an 82 bp segment from each as the smallest sequence specifying ARS function. These 82 bp segments are at the 5' end of a 220 bp region of homology between the two original ARS restriction fragments. A 39 bp region of almost complete sequence identity between the two 82 bp fragments is suggested to be a core sequence element necessary for ARS function. This 39 bp sequence contains a region identical or nearly identical to the 11 bp yeast ARS consensus sequence (T/ATTTATPuTTTA/T) which is suggested to be essential for ARS function. Detailed comparisons of the 82 bp segments and of the 39 bp core with other ARS sequences reveal no extensive homologies aside from the consensus.