Eukaryotic DNA segments capable of autonomous replication in yeast.

Abstract

A selective scheme is presented for isolating sequences capable of replicating autonomously in the yeast Saccharomyces cerevisiae. YIp5, a vector Table 1. YNN27 is a ura3-52 strain that is transformed by YRp12 (see Fig. 2) at a particularly high frequency (2000-10,000 colonies per gg of DNA). It was obtained by crossing YNN6 and YNN34 and assessing the transformation ability of strains grown from individual spores. Growth and storage conditions used for all strains have been described (27).DNA. Bacterial plasmid DNA was purified by repeated isopycnic centrifugation in CsCl (27). Chromosomal yeast DNA was prepared by the method of Cameron (28). N. crassa DNA was purified from conidia (unpublished method). E. coil, D. melanogaster, D. discoideum, C. elegans, and Z. mays DNAs were generous gifts of Lee Rowan, Louise Prestidge, Alan Jacobsen, David Hirsh, and Irwin Rubenstein, respectively. pSY317, a kanamycin-resistant plasmid carrying the E. coli origin of replication, was provided by Seiichi Yasuda.Enzymes and Reagents. EcoRI endonuclease was purified by the published procedure (29). T4 DNA ligase and DNA polymerase I were generously provided by Stewart Scherer. All other enzymes and reagents were purchased from commercial suppliers and were used as described (27).Construction of Hybrid DNA Molecules. Random DNA fragments were inserted into YIp5 to produce pools of hybrid molecules. After digestion with the appropriate restriction endonuclease(s) (EcoRI, HindIII, BamHI, or codigestion with EcoRI and HindIII), the YIp5 and chromosomal DNAs (each at 15-20 ,ug of DNA per ml) were mixed and ligated with 0.1 ,g of T4 DNA ligase in 100 mM NaCl/50 mM Tris-HCl, pH 7.4/10 mM MgSO4/1 mM ATP/10 mM dithiothreitol at 40C for hr. This ligation mixture was directly used to transform yeast cells.Hybrids were constructed between YIp5 and the E. coil origin of replication, oriC, by mixing and ligating EcoRI-digested pSY317 and YIp5 DNAs (as described above). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Targeting sequences of the two major peroxisomal proteins in the methylotrophic yeast Hansenula polymorpha

“…Amplification of plasmids was done in Escherichia coli MB 1000 (hsrK hsmK lac trp pyrF) (Stinchcomb et al 1980), either grown in LB supplemented with 100 ~tg/ml ampicillin or 50 lag/ml neomycin or in M9 medium with 20 gg/ml tryptophan (Maniatis etal. 1982).…”

“…Structure of pME4b. The plasmid is a derivative of the yeast integration vector YIp5 (Stinchcomb et al 1980) To construct plasmid pDH258 (d258), the internal EcoRI-BglII fragment of the DAS gene was excised from the vector pDH131 (H. Hansen, Dissertation, University of Dfisseldorf 1990) and the vector was religated after the ends had been blunted with Klenow polymerase. The vector pDH131 carries the whole DAS gene as a BamHI fragment between the PvuI and the EcoRV sites of plasmid pHARS1.…”

Functional complementation of catalase‐defective peroxisomes in a methylotrophic yeast by import of the catalase A from Saccharomyces cerevisiae

“…Escherichia coli MB 1000 (hsrK hsmK lac trp pyrF) was used as bacterial host for plasmid amplification [12]. It was grown either in LB or M9 medium [13] supplemented with 100 kg ampicillin/ml and 20 pg tryptophan/ml, respectively.…”

Nuclear scaffold/matrix attached region modules linked to a transcription unit are sufficient for replication and maintenance of a mammalian episome

“…Although the activation of individual replication origins may depend on epigenetic factors (1,2), all of these autonomously replicating sequences (ARSs) allow extrachromosomal replication when inserted into plasmids (3). In the fission yeast Saccharomyces pombe, replication origins are more extended than in budding yeast, and, although they show no consensus sequence in the strict sense, they contain AT-rich elements that serve as binding sites for the origin recognition complex (4).…”