Construction of a Bacillus subtilis cloning vehicle with heterologous DNA sequence

A cloning vehicle, pFTB91, for the Bacillus subtilis host was constructed with DNA fragments heterologous to the host chromosome. It consists of three DNA fragments: (i) chromosomal DNA of Bacillus amyloliquefaciens which complements the leuA and ilvC mutations in B. subtilis; (ii) a B. amyloliquefaciens plasmid DNA that supplies an autonomously replicating function; and (iii) a HindIII fragment of Staphylococcus aureus plasmid pTP5 that carries gene tetr, conferring the TetR phenotype. It has sufficiently low DNA homology to prevent its integration into the host chromosome in recombination-competent cells of B. subtilis. It is 9.3 kb, and approx. 10 copies are present per chromosome. The SalI and KpnI sites in the ilvC+ and tetr genes, respectively, could be used for selection of recombinant plasmids by insertional inactivation. The plasmid has unique sites for EcoRI, PstI, and XbaI.     

Dihydrofolate reductase gene as a versatile expression marker.

The Escherichia coli dihydrofolate reductase (DHFR) gene has been used as a genetic marker specifying trimethoprim resistance (TmpR). In order to use the DHFR gene as a versatile expression marker, we have constructed three types of plasmids: promoter cloning vector, terminator cloning vector, and the plasmid containing the DHFR gene cassette. In these systems, the selection of recombinant plasmids was carried out just by examining the TmpR phenotype of the transformed cells. Then, levels of the enzymatic activity of DHFR were measured to evaluate the efficiency of promoters and terminators in the fused DNA fragment. An expression plasmid which resulted in the E. coli host cells being able to produce DHFR up to 20% of total cellular proteins was also constructed by changing the promoter and Shine-Dalgarno sequences of the DHFR gene.     

Sequential cloning by a vector walking along the chromosome.

A procedure was devised for sequential cloning of chromosomal DNA by cyclical integration and excision of a plasmid vector so that slightly overlapping chromosomal segments are successively cloned. The method depends on circular integration of the vector into the chromosome of a host nonpermissive for its replication, and on excision and reduction of a recombinant plasmid by use of an appropriately designed set of restriction enzyme sites in the vector. A vector suitable for cloning in Escherichia coli was constructed by combining a segment of pBR322 with a gene encoding chloramphenicol resistance expressible in many species. Sequential cloning was demonstrated in Streptococcus pneumoniae by extending a previously cloned segment of the region of the chromosome encoding maltosaccharide utilization by 8 kb in three cycles of cloning. Accuracy of the method was confirmed by hybridization of cloned DNA with chromosomal restriction fragments. It is pointed out that the similarity of the requisite genetic processes in bacteria and yeasts should allow use of the method for sequential cloning of yeast chromosomal DNA and of human or other mammalian DNA in artificial chromosomes of yeast.     

Plasmid vector for cloning in Streptococcus pneumoniae and strategies for enrichment for recombinant plasmids

A new plasmid, pLS101, was constructed for use as a vector for cloning in Streptococcus pneumoniae. This plasmid carries two selectable genes, tet and malM, each of which contains two or more restriction sites for cloning. Insertional inactivation of the malM gene allowed direct selection of TcRMal- clones containing recombinant plasmids. Other means of enriching a recipient population for cells containing recombinant plasmids were examined. The effect of removing vector terminal phosphate in attempts to clone heterogeneous DNA fragments, such as those from chromosomal DNA, was to abolish recombinant plasmid establishment altogether, presumably because donor DNA processing during entry into the cell prevented establishment of the hemiligated molecule. However, with homogeneous DNA fragments, such as those from plasmid or viral DNA, vector phosphate removal allowed enrichment for recombinant plasmids. In the cloning of heterogeneous DNA that was homologous to the recipient chromosome (i.e. chromosomal DNA from S. pneumoniae), recovery of recombinant plasmids could be enriched tenfold (relative to the regenerated vector) by the process of chromosomal facilitation of plasmid establishment. This involved an additional passage of the mixed plasmids in which interaction with the chromosome of plasmids containing chromosomal DNA inserts (i.e. recombinant plasmids) increased their frequency of establishment relative to the vector plasmid. An overall strategy for cloning in S. pneumoniae, depending on the nature of the fragment to be cloned, is proposed.     

A versatile plasmid system for the study of prokaryotic transcription signals in Escherichia coli

For comparing the relative efficiencies of Escherichia coli promoters, a modified plasmid system, pKO-2 and pKM-2, has been constructed using short synthetic DNA fragments. The new vectors were derived from the plasmids pKO-1 and pKM-1. The plasmids contain seven clustered unique restriction sites which can be used for promoter insertions. Also, three adjacent stop codons were introduced to abort any undesired translational initiation from various upstream origins. The DNA sequence of any insert in pKO-2 and pKM-2 can be determined rapidly by the supercoiled plasmid DNA sequencing method using a single oligonucleotide primer. The plasmid pKM-2 is especially suitable for the cloning and sequence determination of strong promoters.     

New cloning vectors for integration in the lambda attachment site attB of the Escherichia coli chromosome.

A set of plasmid cloning vectors has been constructed, allowing the integration of any DNA fragment into the bacteriophage lambda attachment site attB of the Escherichia coli chromosome. The system is based upon two components: (i) a number of cloning vectors containing the lambda attachment site attP and (ii) a helper plasmid, bearing the lambda int gene, transcribed from the lambda PR promoter under the control of the temperature-sensitive repressor cI857. The DNA fragment of interest is cloned into the multicloning site of one of the attP-harboring plasmids. Subsequently, the origin of the plasmid, located on a cloning cassette, is cut out and the DNA becomes newly ligated, resulting in a circular DNA molecule without replication ability. The strain of choice, containing the int gene carrying helper plasmid, is transformed with this DNA molecule and incubated at 42 degrees C to induce int gene expression. Additionally, the temperature shift leads to the loss of the helper plasmid after a few cell generations, because the replication ability of its replicon is blocked at 42 degrees C. These vectors have been successfully used for integration of several promoter-lacZ fusions into the chromosome. The ratio between integration due to homologous recombination and Int protein-mediated integration has been determined.     

Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.     

铜绿假单胞菌中快速基因操作工具的开发和应用

针对基因的操作,包括缺失和插入基因、替换基因元件(如启动子)、融合荧光蛋白基因、构建原位的基因报告系统,是大多数生物技术实验室的必备技术。目前广泛使用的基于2次同源重组的基因操作方法,在构建质粒、转化和筛选方面较为繁琐。另外使用该方法进行长片段敲除的效率较低。为了简化基因操作的流程,本研究构建了一个最小化的铜绿假单胞菌(Pseudomonas aeruginosa)整合型质粒pln2,只需将目标基因内部一段序列克隆到pln2质粒并导入细菌,由于质粒不能在细菌内自我复制而只能通过单次等位基因交换整合到基因组上,从而使目的基因断裂为两部分而失去活性。在pln2的基础上开发了一整套工具质粒适用于基因组的不同操作,包括融合荧光蛋白基因、替换基因元件(如启动子)、构建原位的转录型荧光报告系统。此外,本研究借助该系统成功实现超长片段基因簇的敲除,单次可以敲除长达270 kb的片段。     

Plasmid vectors for positive galactose-resistance selection of cloned DNA in Escherichia coli

Insertion of a HindIII-EcoRI fragment carrying part of the gal operon from lambda gal+ into pBR322 yields a plasmid (pAA3) which confers strong galactose sensitivity on E. coli strains deleted for the gal operon. Sensitivity to galactose is caused by the expression of kinase and transferase (but not epimerase) genes from a promoter located in the tet gene of pBR322. Insertion of a DNA fragment carrying Tn9 at the HindIII junction blocks gal expression and produces a galactose-resistant phenotype. Hence, galactose resistance can be used to select DNA fragments cloned at the HindIII site. The system was used efficiently for cloning lambda, yeast, and human DNA. The cloned fragments can be screened directly for the presence of promoters by testing for tetracycline resistance. Alternatively, these plasmids can be used as cosmids for cloning large fragments of DNA at a number of sites. Construction of several related vectors is described.     

Transformation in fungi.

Transformation with exogenous deoxyribonucleic acid (DNA) now appears to be possible with all fungal species, or at least all that can be grown in culture. This field of research is at present dominated by Saccharomyces cerevisiae and two filamentous members of the class Ascomycetes, Aspergillus nidulans and Neurospora crassa, with substantial contributions also from fission yeast (Schizosaccharomyces pombe) and another filamentous member of the class Ascomycetes, Podospora anserina. However, transformation has been demonstrated, and will no doubt be extensively used, in representatives of most of the main fungal classes, including Phycomycetes, Basidiomycetes (the order Agaricales and Ustilago species), and a number of the Fungi Imperfecti. The list includes a number of plant pathogens, and transformation is likely to become important in the analysis of the molecular basis of pathogenicity. Transformation may be maintained either by using an autonomously replicating plasmid as a vehicle for the transforming DNA or through integration of the DNA into the chromosomes. In S. cerevisiae and other yeasts, a variety of autonomously replicating plasmids have been used successfully, some of them designed for use as shuttle vectors for Escherichia coli as well as for yeast transformation. Suitable plasmids are not yet available for use in filamentous fungi, in which stable transformation is dependent on chromosomal integration. In Saccharomyces cerevisiae, integration of transforming DNA is virtually always by homology; in filamentous fungi, in contrast, it occurs just as frequently at nonhomologous (ectopic) chromosomal sites. The main importance of transformation in fungi at present is in connection with gene cloning and the analysis of gene function. The most advanced work is being done with S. cerevisiae, in which the virtual restriction of stable DNA integration to homologous chromosome loci enables gene disruption and gene replacement to be carried out with greater precision and efficiency than is possible in other species that show a high proportion of DNA integration events at nonhomologous (ectopic) sites. With a little more trouble, however, the methodology pioneered for S. cerevisiae can be applied to other fungi too. Transformation of fungi with DNA constructs designed for high gene expression and efficient secretion of gene products appears to have great commercial potential.     

Transformation in fungi.

Transformation with exogenous deoxyribonucleic acid (DNA) now appears to be possible with all fungal species, or at least all that can be grown in culture. This field of research is at present dominated by Saccharomyces cerevisiae and two filamentous members of the class Ascomycetes, Aspergillus nidulans and Neurospora crassa, with substantial contributions also from fission yeast (Schizosaccharomyces pombe) and another filamentous member of the class Ascomycetes, Podospora anserina. However, transformation has been demonstrated, and will no doubt be extensively used, in representatives of most of the main fungal classes, including Phycomycetes, Basidiomycetes (the order Agaricales and Ustilago species), and a number of the Fungi Imperfecti. The list includes a number of plant pathogens, and transformation is likely to become important in the analysis of the molecular basis of pathogenicity. Transformation may be maintained either by using an autonomously replicating plasmid as a vehicle for the transforming DNA or through integration of the DNA into the chromosomes. In S. cerevisiae and other yeasts, a variety of autonomously replicating plasmids have been used successfully, some of them designed for use as shuttle vectors for Escherichia coli as well as for yeast transformation. Suitable plasmids are not yet available for use in filamentous fungi, in which stable transformation is dependent on chromosomal integration. In Saccharomyces cerevisiae, integration of transforming DNA is virtually always by homology; in filamentous fungi, in contrast, it occurs just as frequently at nonhomologous (ectopic) chromosomal sites. The main importance of transformation in fungi at present is in connection with gene cloning and the analysis of gene function. The most advanced work is being done with S. cerevisiae, in which the virtual restriction of stable DNA integration to homologous chromosome loci enables gene disruption and gene replacement to be carried out with greater precision and efficiency than is possible in other species that show a high proportion of DNA integration events at nonhomologous (ectopic) sites. With a little more trouble, however, the methodology pioneered for S. cerevisiae can be applied to other fungi too. Transformation of fungi with DNA constructs designed for high gene expression and efficient secretion of gene products appears to have great commercial potential.     

Cloning vehicles for the homologous Bacillus subtilis host-vector system

A series of Bacillus subtilis plasmids was constructed which carry either the leu region or both the leu and the dihydrofolate reductase (DHFR) regions of the B. subtilis chromosome. The DHFR-coding gene was derived from a trimethoprim resistant (Tmpr) B. subtilis strain, and cells harboring the DHFR plasmid showed resistance to trimethoprim (Tmp). One such leu+tmpr plasmid, pTL12, was found to be useful for cloning DNA fragments at the BamHI, EcoRI, BglII and XmaI sites. It was also shown that insertion of DNA fragments at the BamHI and XmaI sites of pTL12 inactivated the leuA gene function (insertional inactivation) but not tmpr, indicating that cells carrying recombinant plasmids can be detected easily by selecting Leu-Tmpr colonies. Combination of B. subtilis 168 and plasmid pTL12 should serve as an efficient homologous cloning system in B. subtilis.