Transformation of Kluyveromyces fragilis

For the transformation of the yeast species Kluyveromyces fragilis, we have constructed a vector containing a bacterial kanamycin resistance (Kmr) gene, the TRP1 gene of Saccharomyces cerevisiae, and an autonomously replicating sequence of Kluyveromyces lactis called KARS2 . By utilizing the method based on treatment by alkali cations and with the Kmr gene as the selective marker, a wild-type strain of K. fragilis was transformed to resistance against the antibiotic G418 . In the transformed cell the plasmid replicates autonomously. The same plasmid could also be used to transform S. cerevisiae trp1 mutant to Trp+. Thus, KARS2 of K. lactis enables the vector to replicate in K. fragilis, K. lactis, and S. cerevisiae, whereas ARS1 of S. cerevisiae allows autonomous replication only in S. cerevisiae.     

Intergeneric transfer of deoxyribonucleic acid killer plasmids, pGKl1 and pGKl2, from Kluyveromyces lactis into Saccharomyces cerevisiae by cell fusion

Two novel linear deoxyribonucleic acid plasmids, pGKl1 and pGKl2, were isolated from the yeast Kluyveromyces lactis. K. lactis strains harboring the pGK1 plasmids killed a certain group of yeasts, including Saccharomyces cerevisiae, Saccharomyces italicus, Saccharomyces rouxii, K. lactis, Kluyveromyces thermotolerans, Kluyvermyces vanudenii, Torulopsis glabrata, Candida utilis, and Candida intermedia. In this experiment, the pGKl1 and pGKl2 plasmids were intergenerically transferred from a K. lactis killer strain into a non-killer (killer-sensitive) strain of S. cerevisiae by the use of a protoplast fusion technique. Both of the pGKl plasmids replicated autonomously and stably in the new host cells of S. cerevisiae and could coexist with the resident 2-micrometers deoxyribonucleic acid plasmid. The S. cerevisiae cells which accepted the pGKl plasmids expressed the same killer phenotype as that of the donor K. lactis killer and became resistant to the K. lactis killer. The pGKl plasmids existing in the S. cerevisiae cells were cured by treatment with ethidium bromide, and the killer and resistance characters were simultaneously lost. From there results, it was concluded that both the killer and the resistance genes are located on the pGKl plasmids.     

Random AT library: autonomously replicating sequence (ARS) activity of chemically synthesized random sequences for transformation of nonconventional yeast species

In a search for sequences that confer on bacterial plasmids the capacity of autonomous replication in yeast cells, we chemically synthesized polynucleotides 80 bp in length from an equimolar mixture of A and T. The random AT-polymer population, W80, was inserted into the plasmid YIp5-Kan1 (which carries the markers URA3 and G418(R), but does not replicate in yeast) and amplified in Escherichia coli. This library, representing 10 000 different AT sequences, was transformed into three species of yeast: Saccharomyces cerevisiae, Kluyveromyces lactis and Torulaspora delbrueckii. The aim was to evaluate the frequency, if any, of autonomously replicating sequences (ARSs) in the random sequences. A large number of transformants were obtained from each species. Many of them showed a stable transformed phenotype. Several W80 sequences were found many times for a given species, suggesting that each species preferred particular sequences for ARS function, although they are diverse in their primary sequence. In view of the high frequency and stability of the replicative plasmids found in the different hosts, this small random AT library may be conveniently used as a source of replicative gene vectors for genetic manipulation of many nonconventional yeast species, in place of searching for species-specific chromosomal ARSs.     

Mapping autonomously replicating sequence elements in a 73-kb region of chromosome II of the fission yeast, Schizosaccharomyces pombe

Autonomously replicating sequence (ARS) elements are the genetic determinants of replication origin function in yeasts. They can be easily identified as the plasmids containing them transform yeast cells at a high frequency. As the first step towards identifying all potential replication origins in a 73-kb region of the long arm of fission yeast chromosome II, we have mapped five new ARS elements using systematic subcloning and transformation assay. 2D analysis of one of the ARS plasmids that showed highest transformation frequency localized the replication origin activity within the cloned genomic DNA. All the new ARS elements are localized in two clusters in centromere proximal 40 kb of the region. The presence of at least six ARS elements, including the previously reported ars727, is suggestive of a higher origin density in this region than that predicted earlier using a computer based search.     

hEPO cDNA重组逆转录病毒的构建

通过DNA重组技术,将不含非编码区的hEPO cDNA片段重组到逆转录病毒质粒pLXSN, pLNCX中重组质粒转染PA317细胞后,经G418筛选,抗性克隆细胞培养上清能成功地感染NIH3T3细胞,使之在筛选培养基中形成典型的G418抗性克隆,该克隆细胞染色体中成功地整合了EPOcDNA,并且表达出有生物学活性的红细胞生成素(EPO)产物。     

Retention of autonomous replicating plasmids in cultured Drosophila cells

Summary Six kinds of autonomously replicating sequences (ARSs) derived from Drosophila or tobacco were inserted into the vector pDSV, constructed with pSV2-gpt and the copia long terminal repeat (LTR). The resulting ARS-containing plasmids, pDSV-ARSs, were transfected into the cultured Drosophila cells of GM1 S1cl1. Most of the plasmids remained for about 2 weeks and some for about 1 month in these cells. The retention time of the plasmid was not directly correlated with autonomously replicating activity of ARSs detected in the yeast. Two plasmids, one carrying ARS of Drosophila nuclear DNA and the other carrying tobacco DNA, showed the longest retention time in transformed cells and replication was confirmed in these cells. Some of these long lived plasmids were recovered, however, as modified forms. Other plasmids had disappeared 1 month after transfection. Two months following transfection, none of plasmids were recovered but they were detected in nuclear DNA as the integrated form. The integration patterns in all the cells transformed by different kinds of ARS-containing plasmids were similar to each other, and to the distribution pattern of copia LTR in the genome. These results suggest that copia LTR sequences contained in the pDSV-ARSs may participate in the integration process of these plasmids into Drosophila DNA.     

Extrachromosomal replication of shuttle vectors in Dictyostelium discoideum.

We cloned a 12.3-kilobase (kb) endogenous plasmid, Ddp1, found in several wild-type and laboratory strains of Dictyostelium discoideum into pBR322. The cloned plasmids have been used to cotransform D. discoideum cells with B10S, a transformation vector carrying a gene fusion conferring resistance to G418. Whereas B10S DNA alone appears to integrate in a tandem array, the cloned Ddp1 plasmids replicate extrachromosomally and are stably maintained in the absence of selection with an average copy number of 50 to 100 copies per cell. The Ddp1-derived plasmids can be directly recovered by transforming Escherichia coli with bulk nuclear DNA from these cells. Preliminary deletion analysis indicates that not all regions of Ddp1 are necessary for stable replication in D. discoideum. Several recombinant vectors which replicate extrachromosomally in D. discoideum were also isolated. One contains the Act6-neor gene fusion from B10S recombined into one of the cloned derivatives of Ddp1 and can be used to directly transform D. discoideum amoebae, selecting for G418 resistance. Another recombinant is only 5.6 kb and resulted from a deletion of a 16.6-kb cloned Ddp1 hybrid plasmid. An analysis of the vector DNAs present in clones derived from single D. discoideum transformants is also described.     

黑曲霉木聚糖酶在工业酒精酵母中的分泌表达

用重叠延伸PCR方法从黑曲霉 (Aspergillusniger)UV 11的基因组DNA中克隆出木聚糖酶的cDNA基因 ,构建了由酵母乙醇脱氢酶 (ADH1)启动子和终止子引导表达、木聚糖酶自身信号肽引导分泌、rDNA序列介导的酵母整合型分泌表达质粒pAX2。用pAX2与酵母YEp型G4 18抗性质粒共转化野生型工业酒精酵母S .cerevisiae 2 346 ,获得了整合型分泌表达木聚糖酶的酵母重组菌株XY2。发酵分析表明该工程菌能够明显提高酒精生产率     

Introduction of the plasmid pKM101-associated muc genes into Saccharomyces cerevisiae

Bacteria-yeast shuttle plasmids containing the pKM101-associated muc genes were constructed by cloning an ARS TRP fragment into the plasmid pGW270 in both possible orientations. The insertion of Saccharomyces cerevisiae DNA into pGW270 had no effect on the mutator and protective phenotypes associated with the plasmid in Escherichia coli. Two such recombinant plasmids, pAA90 and pAA91 , were capable of efficient transformation of S. cerevisiae and were stably maintained in this organism. Hybridization experiments suggest that muc-specific mRNA was present in transformed yeast cells and a small amount was polyadenylated. The RNAs were not of a discrete size, all being smaller than the muc genes. The presence of the plasmid pAA91 , and to a lesser extent, pAA90 , in yeast resulted in a detectable increase in the reversion frequencies of three markers and in ultraviolet protection. These results are discussed in terms of studying the relationship of error-prone repair in bacteria and yeast and of developing improved yeast tester strains.     

Isolation and characterization of Saccharomyces cerevisiae mutants with a different degree of resistance to killer toxins K1 and K2

Killer toxin K1 of Saccharomyces cerevisiae kills sensitive cells of the same species by disturbing the ion gradient across the plasma membrane after binding to the receptor at cell wall beta-1,6-glucan. Killer protein K2 is assumed to act by a similar mechanism. To identify the putative plasma membrane receptors for both toxins we mutagenized three sensitive S. cerevisiae strains and searched for clones with killer-resistant spheroplasts. The well diffusion assay identified three phenotypically different groups of clones: clones resistant simultaneously to both toxins, clones with lowered sensitivity to only K1 toxin and those with strongly lowered sensitivity to K2 and partially lowered sensitivity to K1 toxin. These phenotypes are controlled by recessive mutations that belong to at least four different complementation groups. This indicates certain differences at the level of interaction of K1 and K2 toxin with sensitive cells.     

Incompatibility of linear DNA killer plasmids pGKL1 and pGKL2 from Kluyveromyces lactis with mitochondrial DNA from Saccharomyces cerevisiae.

Two linear killer plasmids (pGKL1 and pGKL2) from Kluyveromyces lactis stably replicated and expressed the killer phenotype in a neutral petite mutant [( rho0]) of Saccharomyces cerevisiae. However, when cytoplasmic components were introduced by cytoduction from a wild-type [( rho+]) strain of S. cerevisiae, the linear plasmids became unstable and were frequently lost from the cytoductant cells during mitosis, giving rise to nonkiller clones. The phenomenon was ascribed to the incompatibility with the introduced S. cerevisiae mitochondrial DNA (mtDNA), because the plasmid stability was restored by [rho0] mutations in the cytoductant cells. Incompatibility with mtDNA was also apparent for the transmission of plasmids into diploid progeny in crosses between killer cells carrying the pGKL plasmids and [rho+] nonkiller cells lacking the plasmids. High-frequency transmission of the plasmids was observed in crosses lacking mtDNA [( rho0] by [rho0] crosses) and in crosses involving mutated mtDNA with large deletions of various regions of mitochondrial genome. In contrast, mutated mtDNA from various mit- mutations also exerted the incompatibility effect on the transmission of plasmids. Double-stranded RNA killer plasmids were stably maintained and transmitted in the presence of wild-type mtDNA and stably coexisted with pGKL killer plasmids in [rho0] cells of S. cerevisiae.     

Copy number control by a yeast centromere

Plasmids containing a cloned yeast (Saccharomyces cerevisiae) centromere (CEN3) in combination with a suitable DNA replication system are maintained in yeast at the low copy number typical of a chromosome. In composite plasmids containing CEN3 plus the yeast 2 mu plasmid, the CEN3 copy number control is dominant over the amplification system that normally drives the 2 mu plasmids to high copy number. The CEN3-2 mu composite plasmids are relatively stably maintained in yeast at a copy number of about one per haploid genome, and segregate through meiosis in a typical Mendelian pattern. Some of the CEN3-2 mu composite plasmids isolated from yeast contain deletions of variable size that remove the functional centromere, resulting in loss of the CEN3 control and reversion to high copy number. Formation of the CEN3 deletions requires the specialized recombination system (inverted repeat sequences and FLP gene) of the yeast 2 mu plasmid.     

Characterisation of an autonomously replicating sequence from the fission yeast Schizosaccharomyces pombe

Summary A DNA sequence has been isolated from Schizosaccharomyces pombe which promotes high frequency transformation of plasmids in the same organism. It is closely linked to the DNA ligase gene CDC17 and has therefore been named ARS17 although in structure it differs substantially from ARS elements in Saccharomyces cerevisiae. ARS17 spans some 1.8 kb of DNA and deletion of any part of this region affects activity. Moreover, there does not appear to be any short sequence which is, by itself, sufficient for high frequency transformation. ARS17 lies between and partly overlaps two divergently transcribed genes and it is extremely AT rich. It lacks the consensus sequence found in S. cerevisiae ARSs and it has no ARS activity in S. cerevisiae.     

Plasmidial maintenance in rodent fibroblasts of a BPV1-pBR322 shuttle vector without immediately apparent oncogenic transformation of the recipient cells.

A recombinant plasmid was constructed (pV69) which comprises a subgenomic fragment of bovine papilloma virus type 1 (BPV1) DNA, part of plasmid pBR322 DNA and a drug resistance gene expressed in both mammalian fibroblasts and Escherichia coli. This gene (vv2) is a modified form of the bacterial neomycin resistance gene (neo) linked to the herpes simplex virus thymidine kinase (tk) promoter (plasmid pAG60), to which the original bacterial neo promoter from transposon Tn5 was added back, upstream of the eukaryotic promoter. It induced kanamycin resistance in E. coli, as well as resistance to the drug G418 in rat and mouse fibroblasts. Its expression in FR3T3 rat cells was enhanced as compared with the original tk-neo construction. After transfer of plasmid pV69 into C127 mouse cells or FR3T3 rat cells, the number of resistant colonies selected in medium containing G418 was one to two orders of magnitude higher than that of transformed foci in normal medium. In eight independent cell lines selected by drug resistance, pV69 DNA was found to be maintained in a plasmidial state, without any detectable rearrangement or deletion and could be transferred back in E. coli. In contrast, cell lines selected by focus formation in normal medium maintained deleted forms of the original plasmid DNA, and only part of them were resistant to G418. Most of the drug-resistant clones had kept the morphology and growth control of the normal fibroblasts. However, with further passages in culture, these cells spontaneously produced transformed foci with increasing frequencies.     

Expression of prokaryotic genes for hygromycin B and G418 resistance as dominant-selection markers in mouse L cells

Novel bacterial resistance genes were cloned and expressed as dominant selection markers in mammalian cells. Escherichia coli genes coding for resistance to the aminocyclitol antibiotics hygromycin B (Hm) and G418 were cloned into the eukaryotic expression plasmid pSV5GPT [Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78 (1981) 2072–2076]. Mouse cells normally sensitive to 100 μg/ml Hm were transformed with these plasmids and selected in 200 μg/ml Hm. Transformants resistant to as much as l mg/ml Hm and 500 μg/ml G418 were isolated. Cell extracts contained an acetyltransferase activity capable of acetylating G418 and an Hm amino-cyclitol phosphotransferase activity. Plasmid DNA sequences were identified by Southern blot analysis of high M_r DNA isolated from transformed cells.     

小鼠DLL1真核表达载体的构建及其在肿瘤细胞中的表达

目的：构建带绿色荧光蛋白的小鼠DLL1全长基因真核表达载体,并在肿瘤细胞中表达。方法：利用PCR特异性引物扩增出DLL1基因全长,将克隆的基因片段插入带绿色荧光蛋白的真核表达载体pIRES2-EGFP质粒中。然后利用脂质体将重组质粒pIRES2-EGFP-DLL1转染进小鼠B16黑色素瘤细胞中,并通过G418筛选后选取生长良好、荧光强度高的三株单克隆进行mRNA水平DLL1表达的鉴定。结果：成功扩增小鼠DLL1的全长基因。克隆入质粒载体后,通过DNA序列测定证实其序列正确。将构建的pIRES2-EGFP-DLL1质粒转染小鼠B16黑色素瘤细胞,经过G418筛选和荧光显微镜观察后,挑选得到GFP阳性率90%以上的稳定转染细胞株。RT-PCR检测稳定转染细胞的mDLL1的表达显著增加,进一步证实了pIRES2-EGFP-DLL1的表达效能。结论：成功构建了小鼠DLL1基因的真核表达质粒,证实其在真核细胞B16中可以表达。     

Construction of a Genetic Transformation System for a Freeze-tolerant Yeast Kluyveromyces thermotolerans

We have developed a genetic transformation system for a freeze-tolerant yeast Kluyveromyces thermotolerans. K. thermotolerans spheroplasts could be transformed with a YRp-type vector containing an autonomously replicating sequence (ARS) of Saccharomyces cerevisiae. However, transformation with a YEp-type vector containing a replication origin of S. cerevisiae 2 μM DNA was not successful. The cycloheximide resistance gene (RIM-C) of Candida maltosa and the URA3 gene of S. cerevisiae were successfully used to transform a prototrophic strain of K. thermotolerans and an Ura^? mutant of this yeast isolated in this study, respectively. Transformation was also possible by using intact cells treated with lithium salts or thiol compounds. The YRp-type vectors were maintained as plasmids in the transformants under selective conditions. This is the first report of successful transformation of K. thermotolerans.     

Expression and Antigenic Characterization of the Epitope-G1 of the Bovine Ephemeral Fever Virus Glycoprotein in Pichia pastoris

The epitope-G1 gene of Bovine ephemeral fever virus(BEFV) glycoprotein was synthesised by PCR and cloned into expression vector pPIC9K to construct recombinant plasmid pPIC9K-G1.Then the pPIC9K-G1 was linearized and transformed into Pichia pastoris GS115.The recombinant P.pastoris strains were selected by a G418 transformation screen and confirmed by PCR.After being induced with methanol,an expressed protein with 26 kDa molecular weight was obtained,which was much bigger than the predicted size(15.54 kDa).Deglycosylation analysis indicated the recombinant G1 was glycosylated.Western blot and ELISA tests,as well as rabbit immunization and specificity experiments indicated that the target protein had both higher reaction activity and higher immunocompetence and specificity.The recombinant G1 protein could be used as a coating antigen to develop an ELISA kit for bovine ephemeral fever diagnosis.     

Interaction between phosphatidylserine vesicles and rat brain synaptosomes

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 extra-chromosomally, 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.