Development of autonomously replicating plasmids for Candida albicans.

A pool of Candida albicans RsaI fragments cloned onto a vector containing pBR322 sequences and the Candida ADE2 gene was used to transform a Candida ade2 mutant to adenine protrophy. A potential autonomously replicating sequence (ARS) in Candida DNA was identified by two criteria: instability of the selectable marker in the absence of selection and the presence of free plasmid in total DNA preparations. Plasmids carrying the ARS transformed C. albicans at a high frequency (200 to 1,000 ADE+ transformants per microgram of DNA), and Southern hybridization analysis of these transformants indicated that multiple copies of the plasmid sequences were present and that, although they were present in high-molecular-weight molecules, these sequences had not undergone rearrangement. Orthogonal field alternation gel electrophoresis indicated that the high-molecular-weight transforming sequences were not associated with any chromosome. The simplest interpretation to account for these data is that the transforming sequences are present as oligomers consisting of head-to-tail tandem repeats. The transformed strains occasionally yield stable segregants in which the transforming sequences are integrated into the chromosome as repeats. The Candida sequence responsible for the ARS phenotype was limited to a single 0.35-kilobase RsaI fragment which is present in one copy per haploid genome.     

Replication control of autonomously replicating human sequences.

Three autonomously replicating plasmids carrying human genomic DNA and a vector derived from Epstein-Barr virus were studied by density labelling to determine the number of times per cell cycle these plasmids replicate in human cells. Each of the plasmids replicated semi-conservatively once per cell cycle. The results suggest that these human autonomously replicating sequences undergo replication following the same controls as chromosomal DNA and represent a good model system for studying chromosomal replication. We also determined the time within the S phase of the cell cycle that three of the plasmids replicate. Centromeric alpha sequences, which normally replicate late in S phase when in their chromosomal context, were found to replicate earlier when they mediate replication on an extrachromosomal vector. Reproducible patterns of replication within S phase were found for the plasmids, suggesting that the mechanism specifying time of replication may be subject to experimental analysis with this system.     

Transformation of Aspergillus parasiticus using autonomously replicating plasmids from Aspergillus nidulans

Abstract A genetic transformation system for the aflatoxin-producing fungus Aspergillus parasiticus using two autonomously replicating plasmids from A. nidulans (ARp1 and pDHG25) is reported. Transformation frequencies using the plasmid pDHG25 were from 5 × 10² to 2.5 × 10⁴ transformants per 10⁶ viable protoplasts and μg DNA. The stability of the plasmids in the transformants was also studied. This transformation system offers a new opportunity to clone genes related to aflatoxin production using appropriate aflatoxin-defective mutants.     

Construction and characterization of autonomously replicating plasmids in the green unicellular alga Chlamydomonas reinhardii

Plasmids that replicate autonomously in Chlamydomonas reinhardii were constructed by inserting random DNA fragments from this alga into a plasmid containing the yeast ARG4 locus. Arginine prototrophy was used as a selective marker. The presence of free plasmids in the DNA of the transformants was demonstrated by hybridization with a specific plasmid probe and by recovering these plasmids in E. coli after transformation. Four of them were characterized. Their inserts of 415, 257, 153, and 102 by all hybridize to chloroplast DNA and were localized on the physical map of the chloroplast genome. One of these plasmids also promotes autonomous replication in yeast. Sequence analysis of the inserts of the plasmids reveals several short direct and inverted repeats and two semiconserved AT-rich elements of 19 and 12 by that may play a role in promoting autonomous replication in C. reinhardii.     

Replication initiates at multiple locations on an autonomously replicating plasmid in human cells.

We have used a two-dimensional gel electrophoresis mapping technique to determine where DNA replication initiates on a plasmid which utilizes a fragment of human DNA to replicate autonomously in human cells. Replication was found to initiate at multiple locations on the plasmid carrying the human sequence, in contrast to the pattern seen for an Epstein-Barr virus vector which served as a control with a fixed origin. The family of repeats, a portion of the Epstein-Barr virus origin of replication which is present our plasmid, was shown to function as a replication fork barrier. The nature of the stalled replicative intermediates on the human DNA-based plasmid further indicated that replication did not initiate at a single fixed position each time the plasmid replicated. The results suggest that the replication apparatus used to duplicate DNA in human cells may not have precise sequence requirements which target initiation to specific locations.     

Cloning and sequencing of the replication origin (oriC) of theSpiroplasma citri chromosome and construction of autonomously replicating artificial plasmids

A 5.6-kbp fragment ofSpiroplasma citri DNA containing thednaA gene has been cloned and sequenced. Nucleotide sequence analysis shows that this fragment harbors the genes for the replication initiator protein (dnaA), the beta subunit of DNA polymerase III (dnaN), and the DNA gyrase subunits A and B (gyrA andgyrB). The arrangement of these genes,dnaA-dnaN-gyrB-gyrA, is similar to that found in all Gram-positive bacterial genomes studied so far, except that norecF gene was found betweendnaN andgyrB. Several DnaA-box consensus sequences were found upstream ofdnaA and in thednaA-dnaN intergenic region. ThednaA region with the flanking DnaA-boxes and the tetracycline resistance determinant,tetM, were linked into a circular recombinant DNA. This DNA was able to replicate autonomously when introduced by electroporation intoS. citri cells. These experiments show that thednaA region with the DnaA-boxes is the origin of replication ofS. citri and can be used to construct gene vectors.     

Sequence specificity of the human autonomously replicating sequence binding protein

We have previously found that the protein which bound to the essential region of human autonomously replicating sequences (ARS) could be the c-myc proto-oncogene product. Here we examined the binding specificity of human ARS binding protein purified from Burkitt's lymphoma Raji cells. Among several oligodeoxynucleotides, this protein bound to the fragments containing the sequence of 5'-CAPyCTCTNA-3'. Competition analysis revealed that the ARS binding protein could recognize not only the nucleotide sequences but also the high ordered structure.     

Murine genomic DNA sequences replicating autonomously in mouse L cells

Plasmids that replicate autonomously in mouse L cells were constructed by inserting random genomic DNA fragments from Ltk- cells into a plasmid containing the HSV-1 thymidine kinase gene with a truncated low-efficiency promoter. HAT resistance was used as a selective marker. The presence of free plasmids in the DNA of transformants was demonstrated by hybridization with a specific plasmid probe, by electron microscopic visualization of circular DNA, and by recovering these plasmids by E. coli transformation. Nineteen different DNA fragments were isolated. They were characterized as murine autonomously replicating sequences by Mbol restriction endonuclease sensitivity, by bromodeoxyuridine substitution, by copy number determination, and by segregation analysis. Sequence analysis of the inserts of nine plasmids revealed a conserved element of 12 bp (CTCATGAGAGGCCAA) in five out of nine autonomously replicating sequences.     

Plasmid R6K DNA replication: I. Complete nucleotide sequence of an autonomously replicating segment

A 1565-base segment of the antibiotic resistance plasmid R6K carries sufficient information to replicate as a plasmid in Escherichia coli. This segment contains a functional origin of replication and the structural gene pir for a protein, designated π, that is required for the initiation of R6K DNA replication. The nucleotide sequence of this 1565 base-pair replicon was determined. From the sequence and the analysis of proteins produced by minicells of E. coli strains carrying the wild-type pir gene and a deletion of the pir gene, it can be concluded that the π structural gene encodes for a protein of a molecular weight of approximately 35,000.On the basis of the nucleotide sequence, the π protein is the only protein containing more than 50 amino acid residues that is encoded by this 1583 base-pair replicon. The nucleotide sequence also contains eight 22 base-pair direct repeats. Seven of the direct repeats are in tandem and located in the origin region, while the eighth repeat is near or part of the promoter for the π structural gene. This eighth repeat may play a role in the autoregulated expression of the π structural gene.     

Phospholipid modulates in vitro replication of autonomous replicating sequence from human cells

A cloned plasmid, pmyc(H-K), containing sequences derived from human c-myc gene replicated in vitro in Raji nuclear extract in a semiconservative manner. Using this system, it was found that phosphatidylinositol and cardiolipin strongly inhibited the replication of pmyc(H-K) in vitro, whereas other phospholipids, i.e., phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, and sphingomyelin, had no appreciable effect. The concentrations of phosphatidylinositol and cardiolipin producing 50% inhibition of the replication were 4.6 and 5.4 microM, respectively. Phosphatidylinositol and cardiolipin inhibited the relaxation of pmyc(H-K) supercoiled DNA, but showed little or weaker effects on DNA polymerase alpha and topoisomerase II in Raji nuclear extract. These results suggest that phosphatidylinositol and cardiolipin antagonize the replication of pmyc(H-K) in vitro, through, at least in part, the interaction with topoisomerase I.     

Chromosomal mapping and nucleotide sequence of a human DNA autonomously replicating sequence

A 1.1-kb human DNA fragment (ARSH1) capable of functioning as a putative origin of replication in yeast cells has been characterized both by in situ hybridization to human metaphase chromosomes and by DNA sequencing. Our hybridization studies show a preferential localization of ARSH1 in chromosome regions 1p34-36 and 2q34-37. DNA sequence analysis indicates that in addition to the consensus sequence required for ARS function in yeast cells, nuclear matrix-associated DNA motifs are also present in the 1.1-kb fragment. These results suggest that ARSH1 sequences may serve as points of anchorage to the nuclear matrix for chromosomes 1 and 2.     

High-frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces cerevisiae.

We have developed a transformation system which uses autonomous replicating plasmids for a methylotrophic yeast, Candida boidinii. Two autonomous replication sequences, CARS1 and CARS2, were newly cloned from the genome of C. boidinii. Plasmids having both a CARS fragment and the C. boidinii URA3 gene transformed C. boidinii ura3 cells to Ura+ phenotype at frequencies of up to 10(4) CFU/micrograms of DNA. From Southern blot analysis, CARS plasmids seemed to exist in polymeric forms as well as in monomeric forms in C. boidinii cells. The C. boidinii URA3 gene was overexpressed in C. boidinii on these CARS vectors. CARS1 and CARS2 were found to function as an autonomous replicating element in Saccharomyces cerevisiae as well. Different portions of the CARS1 sequence were needed for autonomous replicating activity in C. boidinii and S. cerevisiae. C. boidinii could also be transformed with vectors harboring a CARS fragment and the S. cerevisiae URA3 gene.