一种新的克隆基因方法——重组工程应用研究初报

重组工程(recombineering)是近几年来兴起的一种基于体内同源重组的、新型的遗传工程技术。作为重组工程应用方式之一的空隙修复(gap-repair),是一种捕捉和克隆目的DNA的方法,具有操作简单、步骤少,没有突变、保真度高,不受酶切位点限制等等优点。以pACYC184为模板,PCR扩增含p15A复制子、氯霉素抗性基因和对S.cerevisiaeALD4基因同源臂的线性片段,与酵母染色体DNA共同电击转化诱导型表达了λ噬菌体重组酶活性的大肠杆菌BW25113(pKD46)感受态细胞,通过空隙修复方式,成功地从酵母染色体DNA直接捕捉到大小为1 016bp的ALD4基因部分区段,得到3188bp的重组质粒pACYC184-ALD4。为进一步掌握和充分利用该技术直接捕捉更大片段基因打下了基础。     

Integration of an insertion-type transferred DNA vector from Agrobacterium tumefaciens into the Saccharomyces cerevisiae genome by gap repair.

Recently, it was shown that Agrobacterium tumefaciens can transfer transferred DNA (T-DNA) to Saccharomyces cerevisiae and that this T-DNA, when used as a replacement vector, is integrated via homologous recombination into the yeast genome. To test whether T-DNA can be a suitable substrate for integration via the gap repair mechanism as well, a model system developed for detection of homologous recombination events in plants was transferred to S. cerevisiae. Analysis of the yeast transformants revealed that an insertion type T-DNA vector can indeed be integrated via gap repair. Interestingly, the transformation frequency and the type of recombination events turned out to depend strongly on the orientation of the insert between the borders in such an insertion type T-DNA vector.     

Positive and negative roles of homologous recombination in the maintenance of genome stability in Saccharomyces cerevisiae

In previous studies of the loss of heterozygosity (LOH), we analyzed a hemizygous URA3 marker on chromosome III in S. cerevisiae and showed that homologous recombination is involved in processes that lead to LOH in multiple ways, including allelic recombination, chromosome size alterations, and chromosome loss. To investigate the role of homologous recombination more precisely, we examined LOH events in rad50 Delta, rad51 Delta, rad52 Delta, rad50 Delta rad52 Delta, and rad51 Delta rad52 Delta mutants. As compared to Rad(+) cells, the frequency of LOH was significantly increased in all mutants, and most events were chromosome loss. Other LOH events were differentially affected in each mutant: the frequencies of all types of recombination were decreased in rad52 mutants and enhanced in rad50 mutants. The rad51 mutation increased the frequency of ectopic but not allelic recombination. Both the rad52 and rad51 mutations increased the frequency of intragenic point mutations approximately 25-fold, suggesting that alternative mutagenic pathways partially substitute for homologous recombination. Overall, these results indicate that all of the genes are required for chromosome maintenance and that they most likely function in homologous recombination between sister chromatids. In contrast, other recombination pathways can occur at a substantial level even in the absence of one of the genes and contribute to generating various chromosome rearrangements.     

High-throughput construction of expression system using yeast Pichia pastoris, and its application to membrane proteins

The well-established method for high-throughput construction of an expression system of the yeast Saccharomyces cerevisiae uses homologous recombination between an expression plasmid and a target gene (with homologous regions of the plasmid on both ends added by PCR). This method has been widely used for membrane proteins using plasmids containing GFP, and has been successfully used to investigate the cellular localization and solubilization conditions of the proteins. Although the methanol-utilizing yeast Pichia pastoris is known as an excellent expression host, a method for high-throughput construction of an expression system like that in S. cerevisiae has not been reported. In this study, we have attempted to construct expression systems via homologous recombination in P. pastoris. The insertion of genes into a plasmid could be easily checked by colony-PCR. Expression systems for seven membrane proteins of medaka fish (Oryzias latipes) and yeast (S. cerevisiae) were constructed, and the expression of proteins was analyzed by fluorescence spectra, fluorescence microscopy, and SDS-PAGE (in-gel fluorescence detection).     

Gene conversion associated with site-specific recombination in yeast plasmid pSR1.

A circular DNA plasmid, pSR1, isolated from Zygosaccharomyces rouxii has a pair of inverted repeats consisting of completely homologous 959-base pair (bp) sequences. Intramolecular recombination occurs frequently at the inverted repeats in cells of Saccharomyces cerevisiae, as well as in Z. rouxii, and is catalyzed by a protein encoded by the R gene of its own genome. The recombination is, however, independent of the RAD52 gene of the host genome. A site for initiation of the intramolecular recombination in the S. cerevisiae host was delimited into, at most, a 58-bp region in the inverted repeats by using mutant plasmids created by linker insertion. The 58-bp region contains a pair with 14-bp dyad symmetry separated by a 3-bp spacer sequence. The recombination initiated at this site was accompanied by a high frequency of gene conversion (3 to 50% of the plasmid clones examined). Heterogeneity created by the linker insertion or by a deletion (at most 153 bp so far tested) at any place on the inverted repeats was converted to a homologous combination by the gene conversion, even in the rad52-1 mutant host. A mechanism implying branch migration coupled with DNA replication is discussed.     

木酮糖激酶基因整合表达载体构建及在酿酒酵母中的过表达

【目的】以载体p406ADH1为构建骨架,构建一个酿酒酵母(Saccharomyces cerevisiae)工业菌株的整合表达载体。【方法】通过酶切连接的方式,将4个元件片段:作为筛选标记的G418抗性基因KanR,用于基因表达的ADH1终止子片段,酿酒酵母W5自身木酮糖激酶基因,18S rDNA介导的同源整合区,插入到骨架质粒p406ADH1中,得到多拷贝整合表达载体pCXS-RKTr。将该载体线性转化酿酒酵母后,对转化子中木酮糖激酶酶活进行测定,检测其表达情况。【结果】重组质粒在酿酒酵母体内实现了木酮糖激酶的高水平稳定表达,其酶活力是初始菌株的2.87倍。【结论】本实验构建了一个酿酒酵母工业菌株整合表达载体,并用此载体过表达了其自身的木酮糖激酶基因。该重组质粒载体的构建可以有效解决酿酒酵母中自身木酮糖激酶酶活较低的情况,这为利用木糖高产乙醇酿酒酵母基因工程菌株的构建和其它酵母重组质粒载体的构建奠定基础。     

Intrachromosomal movement of genetically marked Saccharomyces cerevisiae transposons by gene conversion.

In this paper, we describe the movement of a genetically marked Saccharomyces cerevisiae transposon. Ty912(URA3), to new sites in the S. cerevisiae genome. Ty912 is an element present at the HIS4 locus in the his4-912 mutant. To detect movement of Ty912, this element has been genetically marked with the S. cerevisiae URA3 gene. Movement of Ty912(URA3) occurs by recombination between the marked element and homologous Ty elements elsewhere in the S. cerevisiae genome. Ty912(URA3) recombines most often with elements near the HIS4 locus on chromosome III, less often with Ty elements elsewhere on chromosome III, and least often with Ty elements on other chromosomes. These recombination events result in changes in the number of Ty elements present in the cell and in duplications and deletions of unique sequence DNA.     

The Mre11-Rad50-Xrs2 Protein Complex Facilitates Homologous Recombination-Based Double-Strand Break Repair in Saccharomyces cerevisiae

Saccharomyces cerevisiae mre11Delta mutants are profoundly deficient in double-strand break (DSB) repair, indicating that the Mre11-Rad50-Xrs2 protein complex plays a central role in the cellular response to DNA DSBs. In this study, we examined the role of the complex in homologous recombination, the primary mode of DSB repair in yeast. We measured survival in synchronous cultures following irradiation and scored sister chromatid and interhomologue recombination genetically. mre11Delta strains were profoundly sensitive to ionizing radiation (IR) throughout the cell cycle. Mutant strains exhibited decreased frequencies of IR-induced sister chromatid and interhomologue recombination, indicating a general deficiency in homologous recombination-based DSB repair. Since a nuclease-deficient mre11 mutant was not impaired in these assays, it appears that the role of the S. cerevisiae Mre11-Rad50-Xrs2 protein complex in facilitating homologous recombination is independent of its nuclease activities.     

Temporal comparison of recombination and synaptonemal complex formation during meiosis in S. cerevisiae

In synchronous cultures of S. cerevisiae undergoing meiosis, an early event in the meiotic recombination pathway, site-specific double strand breaks (DSBs), occurs early in prophase, in some instances well before tripartite synaptonemal complex (SC) begins to form. This observation, together with previous results, supports the view that events involving DSBs are required for SC formation. We discuss the possibility that the mitotic pathway for recombinational repair of DSBs served as the primordial mechanism for connecting homologous chromosomes during the evolution of meiosis. DSBs disappear during the period when tripartite SC structure is forming and elongating (zygotene); presumably, they are converted to another type of recombination intermediate. Neither DSBs nor mature recombinant molecules are present when SCs are full length (pachytene). Mature reciprocally recombinant molecules arise at the end of or just after pachytene. We suggest that the SC might coordinate recombinant maturation with other events of meiosis.     

Versatile use of Schizosaccharomyces pombe plasmids in Saccharomyces cerevisiae

The two model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe appear to have diverged 1000 million years ago. Here, we describe that S.?pombe vectors can be propagated efficiently in S.?cerevisiae as pUR19 derivatives, and the pREP and pJR vector series carrying the S.?cerevisiae LEU2 or the S.?pombe ura4(+) selection marker are maintained in S.?cerevisiae cells. In addition, genes transcribed from the S.?pombe nmt1(+) promoter and derivatives are expressed in budding yeast. Thus, S.?pombe vectors can be used as shuttle vectors in S.?cerevisiae and S.?pombe. Our finding greatly facilitates the testing for functional orthologs of protein families and simplifies the cloning of new S.?pombe plasmids by using the highly efficient in vivo homologous recombination activity of S.?cerevisiae.     

Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae.

Restriction enzyme-mediated events (REM events; integration of transforming DNA catalyzed by in vivo action of a restriction enzyme) and illegitimate recombination events (IR events; integration of transforming DNA that shares no homology with the host genomic sequences) have been previously characterized in Saccharomyces cerevisiae. This study determines the effect of mutations in genes that are involved in homologous recombination and/or in the repair of double-stranded DNA breaks on these recombination events. Surprisingly, REM events are completely independent of the double-strand-break repair functions encoded by the RAD51, RAD52, and RAD57 genes but require the RAD50 gene product. IR events are under different genetic control than homologous integration events. In the rad50 mutant, homologous integration occurred at wild-type frequency, whereas the frequency of IR events was 20- to 100-fold reduced. Conversely, the rad52 mutant was grossly deficient in homologous integration (at least 1,000-fold reduced) but showed only a 2- to 8-fold reduction in IR frequency.     

Sgs1, a Homologue of the Bloom''s and Werner''s Syndrome Genes, Is Required for Maintenance of Genome Stability in Saccharomyces Cerevisiae

The Saccharomyces cerevisiae SGS1 gene is homologous to Escherichia coli RecQ and the human BLM and WRN proteins that are defective in the cancer-prone disorder Bloom's syndrome and the premature aging disorder Werner's syndrome, respectively. While recQ mutants are deficient in conjugational recombination and DNA repair, Bloom's syndrome cell lines show hyperrecombination. Bloom's and Werner's syndrome cell lines both exhibit chromosomal instability. sgs1Δ strains show mitotic hyperrecombination, as do Bloom's cells. This was manifested as an increase in the frequency of interchromosomal homologous recombination, intrachromosomal excision recombination, and ectopic recombination. Hyperrecombination was partially independent of both RAD52 and RAD1. Meiotic recombination was not increased in sgs1Δ mutants, although meiosis I chromosome missegregation has been shown to be elevated. sgs1Δ suppresses the slow growth of a top3Δ strain lacking topoisomerase III. Although there was an increase in subtelomeric Y' instability in sgs1Δ strains due to hyperrecombination, no evidence was found for an increase in the instability of terminal telomeric sequences in a top3Δ or a sgs1Δ strain. This contrasts with the telomere maintenance defects of Werner's patients. We conclude that the SGS1 gene product is involved in the maintenance of genome stability in S. cerevisiae.     

The CDK regulates repair of double-strand breaks by homologous recombination during the cell cycle

DNA double-strand breaks (DSBs) are dangerous lesions that can lead to genomic instability and cell death. Eukaryotic cells repair DSBs either by nonhomologous end-joining (NHEJ) or by homologous recombination. We investigated the ability of yeast cells (Saccharomyces cerevisiae) to repair a single, chromosomal DSB by recombination at different stages of the cell cycle. We show that cells arrested at the G1 phase of the cell cycle restrict homologous recombination, but are able to repair the DSB by NHEJ. Furthermore, we demonstrate that recombination ability does not require duplicated chromatids or passage through S phase, and is controlled at the resection step by Clb-CDK activity.     

Schizosaccharomyces pombe Mms1 channels repair of perturbed replication into Rhp51 independent homologous recombination

In both Schizosaccharomyces pombe and Saccharomyces cerevisiae, Mms22 and Mms1 form a complex with important functions in the response to DNA damage, loss of which leads to perturbations during replication. Furthermore, in S. cerevisiae, Mms1 has been suggested to function in concert with a Cullin-like protein, Rtt101/Cul8, a potential paralog of Cullin 4. We performed epistasis analysis between Δmms1 and mutants of pathways with known functions in genome integrity, and measured the recruitment of homologous recombination proteins to blocked replication forks and recombination frequencies. We show that, in S. pombe, the functions of Mms1 and the conserved components of the Cullin 4 ubiquitin ligase, Pcu4 and Ddb1, do not significantly overlap. Furthermore, unlike in S. cerevisiae, the function of the H3K56 acetylase Rtt109 is not essential for Mms1 function. We provide evidence that Mms1 function is particularly important when a single strand break is converted into a double strand break during replication. Genetic data connect Mms1 to a Mus81 and Rad22(Rad52) dependent, but Rhp51 independent, branch of homologous recombination. This is supported by results demonstrating that Mms1 is recruited to a site-specific replication fork barrier and that, in a Δmms1 strain, Rad22(Rad52) and RPA recruitment to blocked forks are reduced, whereas Rhp51 recruitment is unaffected. In addition, Mms1 appears to specifically promote chromosomal rearrangements in a recombination assay. These observations suggest that Mms1 acts to channel repair of perturbed replication into a particular sub-pathway of homologous recombination.     

Mismatch Correction Acts as a Barrier to Homeologous Recombination in Saccharomyces Cerevisiae

A homeologous mitotic recombination assay was used to test the role of Saccharomyces cerevisiae mismatch repair genes PMS1, MSH2 and MSH3 on recombination fidelity. A homeologous gene pair consisting of S. cerevisiae SPT15 and its S. pombe homolog were present as a direct repeat on chromosome V, with the exogenous S. pombe sequences inserted either upstream or downstream of the endogenous S. cerevisiae gene. Each gene carried a different inactivating mutation, rendering the starting strain Spt15(-). Recombinants that regenerated SPT15 function were scored after nonselective growth of the cells. In strains wild type for mismatch repair, homeologous recombination was depressed 150- to 180-fold relative to homologous controls, indicating that recombination between diverged sequences is inhibited. In one orientation of the homeologous gene pair, msh2 or msh3 mutations resulted in 17- and 9.6-fold elevations in recombination and the msh2 msh3 double mutant exhibited an 43-fold increase, implying that each MSH gene can function independently in trans to prevent homeologous recombination. Homologous recombination was not significantly affected by the msh mutations. In the other orientation, only msh2 strains were elevated (12-fold) for homeologous recombination. A mutation in MSH3 did not affect the rate of recombination in this orientation. Surprisingly, a pms1 deletion mutant did not exhibit elevated homeologous recombination.     

A novel meiosis-specific protein of fission yeast, Meu13p, promotes homologous pairing independently of homologous recombination.

Meiotic homologous pairing is crucial to proper homologous recombination, which secures subsequent reductional chromosome segregation. We have identified a novel meiosis-specific protein of fission yeast Schizosaccharomyces pombe, Meu13p, to be a molecule that is required for proper homologous pairing and recombination. Rec12p (homologue of Saccharomyces cerevisiae Spo11p), which is essential for the initiation of meiotic recombination, is also shown for the first time to participate in the pairing process of S.pombe. Meu13p, however, contributes to pairing through a recombination-independent mechanism, as disruption of the meu13(+) gene reduces pairing whether the rec12(+) gene is deleted or not. We also demonstrate a dynamic nature of homologous pairing in living meiotic cells, which is markedly affected by meu13 deletion. Meu13p is not required for telomere clustering and the nuclear movement process, which are well known requirements for efficient pairing in S.pombe. Based on these results, together with the localization of Meu13p on meiotic chromatin, we propose that Meu13p directly promotes proper homologous pairing and recombination.     

The Drosophila Meiotic Recombination Gene Mei-9 Encodes a Homologue of the Yeast Excision Repair Protein Rad1

Meiotic recombination and DNA repair are mediated by overlapping sets of genes. In the yeast Saccharomyces cerevisiae, many genes required to repair DNA double-strand breaks are also required for meiotic recombination. In contrast, mutations in genes required for nucleotide excision repair (NER) have no detectable effects on meiotic recombination in S. cerevisiae. The Drosophila melanogaster mei-9 gene is unique among known recombination genes in that it is required for both meiotic recombination and NER. We have analyzed the mei-9 gene at the molecular level and found that it encodes a homologue of the S. cerevisiae excision repair protein Rad1, the probable homologue of mammalian XPF/ERCC4. Hence, the predominant process of meiotic recombination in Drosophila proceeds through a pathway that is at least partially distinct from that of S. cerevisiae, in that it requires an NER protein. The biochemical properties of the Rad1 protein allow us to explain the observation that mei-9 mutants suppress reciprocal exchange without suppressing the frequency of gene conversion.     

Polymorphic extracellular glucoamylase genes and their evolutionary origin in the yeast Saccharomyces diastaticus.

DNA coding for extracellular glucoamylase genes STA1 and STA3 was isolated from DNA libraries of two Saccharomyces diastaticus strains, each carrying STA1 or STA3. Cells transformed with a plasmid carrying either the STA1 or STA3 gene secreted glucoamylases having the same enzymatic and immunological properties and the same electrophoretic mobilities in acrylamide gel electrophoresis as those of authentic glucoamylases. Southern blot analysis of genomic DNA from S. diastaticus and a glucoamylase-non-secreting yeast, Saccharomyces cerevisiae, revealed that the STA1 and STA3 loci of S. diastaticus showed a high degree of homology, and that both yeast species (S. diastaticus and S. cerevisiae) contained DNA segments highly homologous to those of the extracellular glucoamylase genes. Restriction maps of the homologous DNA segments suggested that the extracellular glucoamylase genes of S. diastaticus may have arisen from recombination among the resident DNA segments in S. cerevisiae.     

Ctp1/CtIP and the MRN complex collaborate in the initial steps of homologous recombination

In a recent issue of Molecular Cell, Limbo et al. (2007) identify a new homologous recombination factor, Ctp1 in S. pombe, a homolog of Sae2/Com1 in S. cerevisiae and CtIP in mammals, that operates cooperatively with the MRN complex.