Autonomous Replication In Yeast Research Articles

21] Expression and screening in yeast of genes mutagenized in vitro

Publisher Summary This chapter describes two methods that allow the expression of mutant alleles in yeast. One method produces mutations that reside in the chromosome and the other produces mutations that reside on plasmids. Yeast plasmids are grouped into several classes based on their mode of replication. All commonly used yeast plasmids are shuttle vectors; they can be propagated in both yeast and Escherichia coli. Yeast integrating plasmids (YIp) contain a yeast gene that can be used as a selectable marker for yeast transformation. These plasmids can be introduced into virtually any genetically marked yeast strain. Chromosomal alleles of several yeast genes exhibit low reversion frequencies, allowing the wild-type copies of these genes to be used as selectable markers on plasmids. The procedure for generating chromosomal mutations depends on the properties of YIp plasmids, which do not contain sequences that allow autonomous replication in yeast and can only be propagated following integration into the yeast chromosome. They integrate by the recombination between yeast sequences carried on the plasmid and the homologous sequences present in the yeast genome.

Characterization of new DNA sequences of Chlamydomonas reinhardtii that replicate autonomously in Saccharomyces cerevisiae

After cloning in pUC9 ura3, a newly constructed 3.84 kb yeast integrating vector, two Sau3A DNA fragments of 0.4 kb (ARS09) and 1.5 kb (ARS10) promoting plasmid autonomous replication in yeast have been isolated from a Chlamydomonas purified nuclear DNA fraction slightly contaminated with chloroplast DNA. Plasmids carrying these fragments were unstable in yeast but transformed it at high frequency. ARS09 and ARS10 were shown to hybridize to the chloroplast EcoRI fragments R19 and R21 for ARS09 and to the fragment R26 for ARS10. Since the amount of chloroplast DNA in the DNA fraction used was minor it can be concluded that the frequency of ARSs in nuclear DNA is low. The sequence analysis of ARS09 and ARS10 shows a high AT content, numerous direct and inverted repeats and 11 bp elements closely related to the yeast ARS consensus sequence. ARS09 and ARS10 contain in addition an 11 bp sequence differing by one or two nucleotides only from the 3′ conserved element present in many yeast ARSs and which might play a role in promoting ARS activity.

Transformation of Phycomyces with a bacterial gene for kanamycin resistance.

Phycomyces protoplasts transformed with a plasmid containing the bacterial gene for kanamycin resistance grow in the presence of G418, a kanamycin analogue. The plasmid also contains a Phycomyces DNA sequence that supports autonomous replication in yeast. We obtained about 250 transformants per microgram DNA or one per 5000 viable protoplasts. The transformant phenotype is retained under selective conditions and lost in the majority of the vegetative spores. Recovered plasmids and Southern analysis indicate that the plasmid probably replicates autonomously in Phycomyces.

Transformation of Phycomyces blakesleeanus to G-418 resistance by an autonomously replicating plasmid.

The fungus, Phycomyces blakesleeanus, shows many well-defined responses to a number of external stimuli. Genetic analysis shows that at least eight genes are involved in Phycomyces sensory transduction. As a first step toward the molecular analysis of these genes and their products, we have developed a transformation protocol for Phycomyces by using a plasmid containing the kanamycin-resistance gene from Tn903 and a Phycomyces DNA fragment capable of supporting autonomous replication in yeast (ARS). Our results demonstrate that the Tn903 gene is expressed in Phycomyces and that the ARS fragment selected in yeast supports autonomous replication in Phycomyces as well.

Yeast/E. coli shuttle vectors with multiple unique restriction sites

Two yeast/E. coli shuttle vectors have been constructed. The two vectors, YEp351 and YEp352, have the following properties: (1) they can replicate autonomously in Saccharomyces cerevisiae and in E. coli; (2) they contain the beta-lactamase gene and confer ampicillin resistance to E. coli; (3) they contain the entire sequence of pUC18; (4) all ten restriction sites of the multiple cloning region of pUC18 including EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI and HindIII are unique in YEp352; these sites are also unique in YEp351 except for EcoRI and KpnI, which occur twice; (5) recombinant plasmids with DNA inserts in the multiple cloning region of YEp351 and YEp352 can be recognised by loss of beta-galactosidase function in appropriate E. coli hosts; (6) YEp351 and YEp352 contain the yeast LEU2 and URA3 genes, respectively, allowing for selection of these auxotrophic markers in yeast and E. coli; (7) both plasmids are retained with high frequency in yeast grown under non-selective conditions indicative of high plasmid copy number. The above properties make the shuttle vectors suitable for construction of yeast genomic libraries and for cloning of DNA fragments defined by a large number of different restriction sites. The two vectors have been further modified by deletion of the sequences necessary for autonomous replication in yeast. The derivative plasmids YIp351 and YIp352 can therefore be used to integrate specific sequences into yeast chromosomal DNA.

Transformation of yeast and Podospora: innocuity of senescence-specific DNAs

Two senscence-specific DNAs (sen-DNAs α and β) were tested for their ability to drive autonomous replication in yeast and Podospora. The β but not the α sequence has autoreplicative (ARS) properties in yeast; the ARS sequences of β are not included in the region common to all the β sen-DNAs. Neither the α nor the β sequences can confer autoreplicative properties in Podospora. These sequences inserted into a hybrid vector carrying the suppressor tRNA, su4-1, do not change the mode of transformation of a suppressible leu-1-1 strain of Podospora: the transformation is by integration whether or not the plasmid carries a sen-DNA sequence. The su4-1 gene integrates at its homologous site in a minority of cases. It is possible to reisolate free plasmids at a low frequency from some transformants. The presence of a sen-DNA on the transforming vector has no effect upon the longevity of the transformants.

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

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 5′A/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.

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

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.

Expression of human salivary α-amylase gene in Saccharomyces cerevisiae and its secretion using the mammalian signal sequence

A cDNA fragment coding for human salivary α-amylase precursor was joined to the promoter of the Saccharomyces cerevisiae PHO5 gene, and the recombinant gene was inserted into a vector plasmid capable of autonomous replication in yeast. Yeast cells transformed with this recombinant plasmid synthesized about 5 × 10 5 molecules of the enzyme per cell when synthesis was induced by deprivation of inorganic phosphate and released about half of the synthesized enzyme into the medium. The enzyme is stable, and exhibited the same specific activity as α-amylase in human saliva. The amylase-producing yeast grew on starch and produced alcohol.

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.

Isolation of Cyanidium caldarium and Porphyridium purpureum DNA Fragments Capable of Autonomous Replication in Yeast

Summary: DNA fragments from the unicellular red algae Cyanidium caldarium and Porphyridium purpureum were inserted at various sites of plasmid pLG4 (pBR325 + a HindIII fragment bearing the yeast arg4 gene) with the purpose of isolating sequences supporting autonomous replication of plasmids in Saccharomyces cerevisiae. Plasmid pools were prepared in Escherichia coli then used to transform the arg4 yeast strain X3656-7 D to prototrophy. The presence of free plasmids in the yeast transformants was demonstrated by Southern blotting hybridization between yeast DNA and 32P-labelled pBR325 and by the transformation of E. coli argH with DNA from yeast transformants. Hybrid plasmids recovered from Arg+bacterial transformants transformed yeast at high frequency. They contained Aval fragments of C. caldarium total DNA, EcoRI fragments of P. purpureum satellite DNA and one BamHI fragment of P. purpureum main DNA. These new plasmids have unique restriction sites which make them convenient vectors for cloning in yeast and possibly in algae and other plants.

Chloroplast origins of DNA replication are distinct from chloroplast ARS sequences in two green algae

A 5.3 kb chloroplast restriction fragment of Chlamydomonas reinhardii containing an origin of DNA replication and a sequence capable of promoting autonomous replication in C. reinhardii (ARC sequence) also carries an ARS sequence (autonomous replication in yeast). The ARC and ARS elements have been physically mapped and shown to be distinct from the origin of DNA replication. Similarly, restriction fragments containing the origin of chloroplast DNA replication from Euglena gracilis are unable to promote autonomous replication in yeast.

Identification of sites required for repression of a silent mating type locus in yeast

There are three loci in the yeast Saccharomyces, each containing one of two possible genetic elements that can determine cell type. At one of these loci, MAT, this information is expressed to establish the mating type of the cell. At the other two loci. HML and HMR, this same information is phenotypically and transcriptionally silent, even though a large amount of identical sequence flanks MAT, HML and HMR coding regions. Transcriptional repression of HML and HMR requires the trans active gene products of four loci, designated variously as MAR or SIR, that are unlinked to each other or to MAT, HML or HMR. We have examined the phenotypic expression of a cloned, plasmid-borne copy of HML and of various deletion and insertion derivatives of this plasmid following their reintroduction into Mar +/Sir + yeast strains. From these data, we have identified two sites flanking the locus, both of which are required for MAR/SIR repression of the locus. In addition, we demonstrate that each of these sites promotes autonomous replication in yeast. Abraham et al. (1984) have presented evidence demonstrating that a similar regulatory structure exists at the other silent locus. HMR. From an analysis of the sequences of these four regulatory sites, we have identified several specific sequences that may be involved in mediating repression of these loci and in promoting replication in yeast. These results are discussed in the context of potential models for the mechanism of regulation of the silent mating type loci.

Expression of an artificial yeast TRP-gene cluster in yeast and Escherichia coli.

All five tryptophan biosynthetic genes of Saccharomyces cerevisiae were unified on plasmid pME554, which is based on 2 micrometer DNA and pBR322 sequences allowing for autonomous replication in yeast and E. coli. Homologous and heterologous expression of this artificial yeast TRP-gene cluster was studied. Plasmid pME554 allowed for nearly normal growth of a yeast strain bearing auxotrophic mutations in all five TRP-genes. The plasmid-borne genes TRP2 to TRP5 were expressed and regulated normally in the frame of the general control. Gene TRP1, carried on an EcoRI/Bg/II fragment lacking the ARS1 function, was expressed poorly and did not respond to the general control like the chromosomally-borne TRP1 gene. Plasmid pME554 allowed for poor growth of E. coli strain W3110 tna- delta trpEA2 on minimal medium. Marked stimulation was observed, however, when anthranilic acid or indole were added. Accordingly, poor expression of the first Trp-enzyme anthranilate synthase and the last enzyme tryptophan synthase was found, whereas the other three genes were moderately well expressed in E. coli.

Structural requirements for the function of a yeast chromosomal replicator

A sequence closely linked to the Saccharomyces cerevisiae HO gene confers autonomous replication in yeast. I have subjected this putative replication origin to deletion and point mutagenesis in order to identify structural features that are important requirements for autonomous replication in vivo. This analysis identifies a 14 bp core region, which is crucial for function and shows partial sequence conservation between a number of autonomously replicating sequences. Point mutations within the core region can abolish autonomous replication. The core region is flanked on one side by a sequence of about 20 bp, which is important for efficient autonomous replication. Deletion of this flanking sequence reduces, but does not necessarily eliminate, autonomous replication.

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 bp 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 bp that may play a role in promoting autonomous replication in C. reinhardii.

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.

DNA sequences from a ligninolytic filamentous fungus [formula omitted] capable of autonomous replication in yeast

Five different DNA sequences of Phanerochaete chrysosporium capable of supporting autonomous replication of yeast integration plasmid (YIp5) in Saccharomyces cerevisiae were isolated. These hybrid plasmids with the autonomous replication sequences from P . chrysosporium are maintained extrachromosomally, are mitotically unstable and transform Ura3 deletion mutant of S . cerevisiae to Ura + phenotype with high frequency. The autonomous replication sequence in pRR2, one of the recombinant plasmids, was further characterized and was shown to be homologous to P . chrysosporium genomic DNA. Restriction analyses showed that this plasmid has unique PvuII and SalI restriction sites for cloning.

Cloning of Petunia hybrida chloroplast DNA sequences capable of autonomous replication in yeast

Sequences from Petunia hybrida chloroplast DNA which have the property to promote autonomous replication in Saccharomyces cerevisiae were cloned in vector YIp5. Seven cloned chloroplast DNA fragments are localized at one of two different sites on the chloroplast genome. One site, arsA was mapped on a 1.8 Kb fragment at position 27.0-28.8 Kb on the P. hybrida chloroplast genome. The plasmids containing this arsA are stable both in yeast and E. coli. The other site, arsB, was shown to be very unstable and is located either in the small single copy region close to the inverted repeat or just in the inverted repeat. The functioning of these sequences as a possible origin of replication in vivo is discussed.

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.