The FLP protein of the yeast 2-microns plasmid: expression of a eukaryotic genetic recombination system in Escherichia coli.

The FLP gene of the yeast 2-microns plasmid is involved in a site-specific recombination event that results in the inversion of a set of sequences within the plasmid. This gene has been cloned and expressed in Escherichia coli. Expression of the FLP gene results in efficient recombination within the bacterial cell, which is specific for plasmids containing at least one 2-microns plasmid recombination site. This work demonstrates that (i) FLP protein is actively involved in 2-microns plasmid recombination; (ii) no other factors specific to yeast are required for the reaction; (iii) FLP protein acts efficiently in trans; (iv) FLP protein will promote site-specific insertion and deletion reactions in addition to the inversion reaction; and (v) FLP-promoted recombination is not dependent upon any DNA structural features unique to yeast chromatin.     

Yeast plasmid 2-micron circle promotes recombination within bacterial transposon Tn5.

The site-specific recombination system (FLP) encoded by the yeast plasmid 2-micron circle can also act in yeast on the inverted repeats of the bacterial transposon Tn5. The efficiency of this recombination is dependent on the location of Tn5 within the 2-micron circle genome but can be as high as that observed for 2-micron circle itself. Comparison of the DNA sequences between the Tn5 repeat and the 2-micron circle recombination region reveals certain strikingly similar structural features that might be important in the recombination reaction.     

Holliday junctions in FLP recombination: resolution by step-arrest mutants of FLP protein.

The FLP "recombinase" of the 2-micron circle yeast plasmid can resolve synthetic FLP site-Holliday junctions. Mutants of the FLP protein that are blocked in recombination but are normal in substrate cleavage can also mediate resolution. The products of resolution by these mutants are almost exclusively nicked molecules with a protein-bound 3' end. There is no significant asymmetry in strand cleavage (top versus bottom) by the mutants in linear or in circular FLP substrates; nor is there a bias in resolution (toward parentals or toward recombinants) of Holliday junctions (corresponding to top- or to bottom-strand exchange) by wild-type FLP. During normal FLP recombination, a small amount of the expected Holliday intermediate can be detected.     

FLP recombinase is an enzyme.

The FLP protein of the yeast 2-microns plasmid catalyzes intermolecular site-specific recombination with a turnover number of approximately equal to 0.12 min-1 (per FLP monomer) for relaxed DNA substrates. Under conditions that enhance its stability, the protein can be used in catalytic rather than stoichiometric amounts. The reaction rate exhibits a strong dependence on FLP protein concentration even when the protein is present in excess relative to available recombination sites.     

Mutations in the 2-microns circle site-specific recombinase that abolish recombination without affecting substrate recognition.

The site-specific recombinase encoded by the yeast plasmid 2-microns circle (FLP) forms a transient covalent linkage with its substrate DNA via a tyrosine residue, which appears to be located near its COOH terminus. The homology of the COOH terminus of FLP with that of the Int family of recombinases suggests that tyrosine-343 of FLP could be involved in forming the DNA-protein bridge. We have mutated tyrosine-343 to a phenylalanine or serine. We demonstrate that the binding of each of the two mutant proteins to its substrate is indistinguishable from that of wild-type FLP. However, both mutant proteins are incapable of catalyzing strand cleavage and recombination.     

Two-micrometer circle site-specific recombination: the minimal substrate and the possible role of flanking sequences.

The 2-mum circle DNA of yeast encodes a site-specific recombination system (FLP recombination). The recombination region had been mapped earlier to a 65-base-pair (bp) segment within the 599-bp-long inverted repeats of the molecule. I have shown that the "minimal" FLP substrate resides in a 13-bp dyad symmetry plus an 8-bp core located within the 65-bp recombination region. Further, as determined by different in vivo assays, sequences extraneous to the minimal FLP site and the 65-bp recombination region can affect the efficiency of the recombination reaction.     

Functional expression in yeast of the Escherichia coli plasmid gene coding for chloramphenicol acetyltransferase.

The Escherichia coli R factor-derived chloramphenicol resistance (camr) gene is functionally expressed in the yeast Saccharomyces cerevisiae. the gene was introduced by transformation into yeast cells as part of a chimeric plasmid, pYT11-LEU2, constructed in vitro. The plasmide vector consists of the E. coli plasmid pBR325 (carrying the camr gene), the yeast 2-micron DNA plasmid, and the yeast LEU2 structural gene. Yeast cells harboring pYT11-LEU2 acquire resistance to chloramphenicol and cell-free extracts prepared from such cells contain chloramphenicol acetyltransferase (acetyl-CoA: chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28), the enzyme specified by the camr gene in E. coli. Resistance to chloramphenicol and the presence of chloramphenicol acetyltransferase activity segregate with the yeast marker LEU2, carried by the transforming plasmid, during both mitotic growth and meiotic division.     

Association of the 2-micron DNA plasmid with yeast folded chromosomes.

The 2-micron DNA plasmid cosedimented in sucrose gradients with the folded chromosome on centrifugation of lysates from logarithmically growing yeast cells. It banded with both the slow-sedimenting g1 form derived from prereplicative cells and the fast-sedimenting g2 form from postreplicative cells. When cells were induced to withdraw from the cell cycle by nitrogen starvation, the folded chromosome was converted to the g0 form, which sedimented more slowly than the g1 form, and only 1/10th the amount of 2-micron DNA cosedimented with this g0 form as compared to the logarithmic-phase forms, g1 and g2. Expression of the temperature-sensitive cell division cycle (cdc) 28 mutation, which defines the "start" of the cell cycle, resulted in a rapid alteration in the folded chromosome sedimentation pattern, and less than 20% of the 2-micron DNA cosedimented with the folded chromosome. These results raise the possibility that association of the 2-micron DNA plasmid with structures in the cell corresponding to the folded chromosome is subject to cell division cycle control.     

High-frequency transformation of yeast by plasmids containing the cloned yeast ARG4 gene

Hybrid ColE1 plasmids, containing cloned DNA from the yeast ARG4 region [e.g., pYe(arg4)1], transform yeast arg4 mutants to ARG4(+) with a frequency of 10(-4) (about 10(3) transformants per mug of plasmid DNA) and can replicate autonomously without integrating into the yeast genome. The yeast transformants are genetically unstable when grown on nonselective medium, but can be readily grown and maintained on minimal medium lacking arginine. The existence of unintegrated replicating plasmid DNA in the yeast transformants was demonstrated by Southern gel hybridization and by transformation of Escherichia coli argH mutants with DNA preparations from yeast transformants and subsequent recovery of intact plasmid DNA from the bacterial transformants. Plasmid DNAs recovered from the E. coli-yeast-E. coli "shuttle" remain essentially unchanged, as judged by DNA restriction fragment patterns. Some plasmid mutations leading to increased efficiency of expression of the ARG4 gene in E. coli do not appear to affect expression of the cloned ARG4 gene in yeast. Appropriate derivatives of these ARG4 plasmids are of potential usefulness as vectors for cloning genes in yeast and for studying the mechanism of yeast DNA replication.     

乙型肝炎病毒前S2基因酵母表达载体的构建及表达

目的：探讨乙型肝炎病毒（HBV）前S2蛋白的功能。方法：以质粒pCP10 (含有HBV ayw亚型全长序列)为模板，多聚酶链反应（PCR）扩增HBV前S2基因，克隆到pGEM-T载休整 ，测序鉴定、酶切后回收，与酵母表达质粒pGBKT7连接。将重组载体转化酵母细胞AH109，提取酵母蛋白质，进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western免疫印迹分析。结果：成功构建HBV前S2基因酵母表达载体，Western免疫印迹显示HBV前S2蛋白在酵母细胞中表达，表达产物在胞内存在，分子量24kD左右。结论：HBV前S2蛋白在酵母细胞中表达成功。     

Mitotic and meiotic stability of linear plasmids in yeast.

Circular recombinant DNA plasmids that contain autonomously replicating sequences (ARSs) are maintained in extrachromosomal form in transformed yeast cells. However, these plasmids are unstable, being rapidly lost from cells growing without selection. Although the stability of such a plasmid can be increased by the presence of yeast centromere DNA (CEN), even CEN plasmids are lost at a high rate compared to a bona fide yeast chromosome. Natural yeast chromosomes are linear molecules; therefore, we have asked if linearization can improve the stability of recombinant DNA plasmids. Linear plasmids with and without yeast CENs were constructed in vitro by using termini from the extrachromosomal ribosomal DNA (rDNA) of the ciliated protozoan Tetrahymena thermophila as "telomeres." These linear plasmids transformed yeast at high frequency and were maintained as linear extrachromosomal molecules during mitotic growth. Moreover, linear plasmids containing CENs were also transmitted through meiosis: these plasmids segregate predominantly 2+:2- at the first meiotic division, indicating that Tetrahymena rDNA termini can provide telomere function during yeast meiosis. Linear plasmids without CENs were about as stable in mitosis as the comparable circular plasmid. Thus, the Tetrahymena rDNA termini have no marked positive or negative effect on the mitotic stability of ARS plasmids. However, linear plasmids containing CENs are three to four times less stable in mitotic cells than circular CEN plasmids. This decrease in stability is not due to a functional change in the centromere itself; rather, linearization of a CEN plasmid has a direct detrimental effect on its mitotic stability. These results may reflect the existence of spatial constraints on the positions of centromeres and telomeres, constraints which must be satisfied to achieve stable segregation of chromosomes during mitosis.     

Isolation of a condensed, intracellular form of the 2-micrometer DNA plasmid of Saccharomyces cerevisiae.

We have initiated an investigation of the proteins bound in vivo to the 2-micrometer DNA plasmid found in the yeast Saccharomyces cerevisiae by examining its intracellular form. To isolate 2-micrometer DNA without disturbing proteins bound to the plasmid, an extract was prepared from a strain lacking mitochondrial DNA and the nuclear chromatin was removed from the extract by centrifugation. When the DNA in this extract was sedimented through a sucrose gradient containing 0.15 M NaCl, plasmid DNA had a sedimentation coefficient of approximately 70. This S value is greater than the S value of 25 for naked, superhelical 2-micrometer DNA. Cosedimenting with the DNA were proteins of the same size as the histone proteins associated with yeast nuclear chromatin. Digestion of the plasmid DNA with micrococcal nuclease and electrophoresis of the resulting DNA fragments revealed that segments of discrete sizes are protected from degradation. Examination of the plasmid DNA protein complex by electron microscopy showed nucleosome structures. We conclude that 2-micron DNA exists as a condensed chromosome body within the cell.     

Inversions of specific DNA segments in flagellar phase variation of Salmonella and inversion systems of bacteriophages P1 and Mu.

Prophages P1 and Mu produces a trans-acting factor possessing the din+ activity which catalyzes the inversion of the specific DNA segment responsible for flagellar phase variation of Salmonella, din mutants were isolated from PICMclr100 phage by selecting phages that did not suppress the yh2 mutation of Salmonella in prophage state. No inversion loop structure was detected among DNA forms arising after denaturation and rehybridization of DNAs extracted from the din mutants. The DNA fragment containing C region of P1 was cloned on a plasmid vector, pCR1. The resulting hybrid plasmid, pKK2, was shown to possess the din+ activity: the vh2 mutant of Salmonella harboring the plasmid changed the flagellar phase. From analysis of the plasmid by use of BamHI and Bgl II, the din gene specifying the din+ activity was located near or within the C region of P1. It is highly plausible that the din gene of P1 was also involved in the inversion of the C region. Similarly, the DNA fragment containing the G and beta segments of Mu was cloned on pCR1. The resulting hybrid plasmid, pII101, also possessed the din+ activity.     

Occurrence of crossed strand-exchange forms in yeast DNA during meiosis.

The crossed strand-exchange form (Holliday structure, half chiasma) has been predicted as an intermediate in the genetic recombination of eukaryotes. We report here the detection of this form in the yeast plasmid, 2-micron DNA, isolated during meiosis. Physical mapping has previously suggested that two forms of 2-micron DNA arise because of recombination between inverted repeat regions. After appropriate digestion with restriction endonuclease, a crossed strand-exchange form intermediate in this recombination would yield an X-shaped form resistant to loss by branch migration because of nonhomology in sequences flanking the region of homology. We first generated this X-shaped form artificially by reannealing melted restriction fragments of 2-micron DNA. This enabled us to develop a procedure for the physical separation of the X-shaped form by agarose gel electrophoresis. We then used this electrophoretic procedure to isolate a naturally occurring form of identical structure from the 2-micron DNA of meiotic cells. Electron microscopy demonstrated that the exchange junction had the expected configuration of strands and indicated that the junction occurred within the region of homology.     

Functional expression of cloned yeast DNA in Escherichia coli.

A collection of hybrid circular DNAs was constructed in vitro using the poly(dA-dT) "connector" method: each hybrid circle contained one molecule of poly(dT)-tailed DNA of plasmid ColE1 (made linear by digestion with EcoRI endonuclease) annealed to a poly(dA)-tailed fragment of yeast (Saccharomyces cerevisiae) DNA, produced originally by shearing total yeast DNA to an average size of 8 X 10(6) daltons. This DNA preparation was used to transform E. coli cells, selecting colicin-E1-resistant clones that contain hybrid ColE1-yeast DNA plasmids. Sufficient numbers of transformant clones were obtained to ensure that the hybrid plasmid population was representative of the entire yeast genome. Various hybrid ColE1-yeast DNA plasmids capable of complementing E. coli auxotrophic mutations were selected from this population. Plasmid pYeleu 10 complements several different point or deletion mutations in the E. coli or S. typhimurium leuB gene (beta-isopropylmalate dehydrogenase); plasmids pYeleu11, pYeleu12, and pYeleu17 are specific suppressors of the leuB6 mutation in E. coli C600. Plasmid pYehis2 complements a deletion in the E. coli hisB gene (imidazole glycerol phosphate dehydratase). Complementation of bacterial mutations by yeast DNA segments does not appear to be a rare phenomenon.     

Cloning by function: an alternative approach for identifying yeast homologs of genes from other organisms.

Studies of cell physiology and structure have identified many intriguing proteins that could be analyzed for function by using the power of yeast genetics. Unfortunately, identifying the homologous yeast gene with the two most commonly used approaches--DNA hybridization and antibody cross-reaction--is often difficult. We describe a strategy to identify yeast homologs based on protein function itself. This cloning-by-function strategy involves first identifying a yeast mutant that depends on a plasmid expressing a cloned foreign gene. The corresponding yeast gene is then cloned by complementation of the mutant defect. To detect plasmid dependence, the colony color assay of Koshland et al. [Koshland, D., Kent, J. C. & Hartwell, L. H. (1985) Cell 40, 393-403] is used. In this paper, we test the feasibility of this approach using the well-characterized system of DNA topoisomerase II in yeast. We show that (i) plasmid dependence is easily recognized; (ii) the screen efficiently isolates mutations in the desired gene; and (iii) the wild-type yeast homolog of the gene can be cloned by screening for reversal of the plasmid-dependent phenotype. We conclude that cloning by function can be used to isolate the yeast homologs of essential genes identified in other organisms.     

Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene.

The structural gene for yeast DNA topoisomerase I (TOP1) has been cloned from two yeast genomic plasmid banks. Integration of a plasmid carrying the gene into the chromosome and subsequent genetic mapping shows that TOP1 is identical to the gene previously called MAK1. Seven top1 (mak1) mutants including gene disruptions are viable, demonstrating that DNA topoisomerase I is not essential for viability in yeast. A 3787-base-pair DNA fragment including the gene has been sequenced. The protein predicted from the DNA sequence has 769 amino acids and a molecular weight of 90,020.     

Autonomously replicating sequences in Saccharomyces cerevisiae.

A method is presented for isolating DNA segments capable of autonomous replication from Saccharomyces cerevisiae chromosomal DNA based on the differential transforming ability of autonomously replicating plasmids and nonreplicating plasmids. DNA plasmids that are capable of self-replication in yeast transform yeast spheroplasts at about 1000-fold higher frequency than nonreplicating plasmids. We have cloned from total yeast DNA a number of DNA segments that permit the pBR322 plasmid carrying the yeast LEU2 gene to replicate autonomously. These plasmid clones are characterized by their ability to transform Leu- spheroplasts to Leu+ at a high frequency and their ability to replicate autonomously. Analysis of the insert DNAs carried in some of these self-replicating plasmids divides them into two categories: those that are unique in the yeast genome and those that are repetitive.     

乙型肝炎病毒e抗原真核表达载体的构建及在酵母中的表达

为探讨乙型肝炎病毒(HBV)e抗原(HBeAg)的功能，用多聚酶链反应(PCR)的方法以HBV ayw亚型全序质粒pCP10为模板扩增HBeAg基因，克隆到pGEM—T载体中扩增并测序，鉴定符合GenBank报告序列。用EcoRI和Pstl双酶切后回收片段，连接到真核表达载体pGBK17中并转化酵母AH109，在色氨酸缺陷型培养基(SD／—Trp)上筛选阳性菌落。提取阳性酵母菌的蛋白质，进行十二烷基磺酸钠—聚丙烯酰胺凝胶电泳(SDS—PAGE)和Western免疫印迹分析，显示HBeAg基因在酵母细胞中表达，表达产物在胞内存在，相对分子质量(MT)为43000左右。