Cloning of the complete biosynthetic gene cluster for an aminonucleoside antibiotic, puromycin, and its regulated expression in heterologous hosts.

Puromycin, produced by Streptomyces alboniger, is a member of the large group of aminonucleoside antibiotics. The genes pac and dmpM, encoding a puromycin N-acetyl transferase and an O-demethyl puromycin O-methyltransferase, respectively, are tightly linked in the DNA of S. alboniger. The entire set of genes encoding the puromycin biosynthesis pathway was cloned by screening a gene library from S. alboniger, raised in the low copy number cosmid pKC505, with a DNA fragment containing pac and dmpM. Puromycin was identified by biochemical and physicochemical methods, including 1H NMR, in the producing transformants. This pathway was located in a single DNA fragment of 15 kb which included the resistance, structural and regulatory genes and was expressed when introduced into two heterologous hosts Streptomyces lividans and Streptomyces griseofuscus. In addition to pac and dmpM, two other genes have been identified in the pur cluster: pacHY, which determines an N-acetylpuromycin hydrolase and prg1, whose deduced amino acid sequence is significantly similar to that of degT, a Bacillus stearothermophilus pleiotropic regulatory gene.     

Recognition and cleavage of the bacteriophage P1 packaging site (pac). I. Differential processing of the cleaved ends in vivo

The packaging of bacteriophage P1 DNA into viral capsids is initiated at a specific DNA site called pac. During packaging, that site is cleaved and at least one of the resulting ends is encapsidated into a P1 virion. We show here that pac is located on a 620 base-pair fragment of P1 DNA (EcoRI-20). When that fragment is inserted into the chromosome of cells that are then infected with P1, packaging of host DNA into phage particles is initiated at pac and proceeds down the chromosome, unidirectionally, for about five to ten P1 "headfuls" (about 5 X 10(5) to 10 X 10(5) bases of DNA). Using an assay for pac cleavage that does not depend on DNA packaging, we have identified a set of five amber mutations that are mapped adjacent to pac, and that define a gene (gene 9) essential for pac cleavage. Amber mutations that are located in genes necessary for viral capsid formation (genes 4, 8 and 23), or in a gene necessary for "late" protein synthesis (gene 10), do not affect pac cleavage. The latter result suggests that the synthesis of the pac cleavage protein is not regulated co-ordinately with other phage morphogenesis proteins. The products of pac cleavage were analyzed using two different DNA substrates. In one case, a single copy of pac was placed in the chromosome of P1-sensitive cells. When those cells were infected with P1, we could detect the cleavage of as much as 70% of the pac-containing DNA. The pac end destined to be packaged in the virion was detected five to 20 times more efficiently than was the other end. Since this result is obtained whether or not the infecting P1 phage can encapsidate the cut pac site, the differential detection of pac ends is not simply a consequence of one end being packaged and the other not. In a second case, pac was located in cells on a small (5 X 10(3) bases) multicopy plasmid. When those cells were infected with P1, neither pac end was detected efficiently after P1 infection, unless the cells carried a recBCD- mutation. In recBCD- cells, the results with plasmid-pac substrates were similar to those obtained with chromosomally integrated pac substrates. We interpret these results to mean that, following pac cleavage, the end destined to be packaged is protected from cellular nucleases while the other end is degraded by the action of at least two nucleases, one of which is the product of the host recBCD gene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Permanent cell lines established from ts-COS cells that regulate by temperature the amplification and expression of cloned genes.

Temperature-sensitive COS cells have been transformed at restrictive temperature with SV40 replicons containing the neo or pac markers. Puromycin-resistant cell clones maintained at the restrictive temperature contain the pac gene integrated into the cell DNA. However, when the cells are shifted to the permissive temperature the pac gene is amplified in episomal forms up to 2-4 X 10(4) copies per cell. Concomitant with this, an induction of 35-300 fold in the levels of puromycin acetyl transferase activity is observed, leading to the accumulation of the enzyme up to 10-60 mU/mg of total cell protein. A band of apparent molecular weight 26,500 daltons is observed by polyacrylamide gel electrophoresis of induced culture extracts, that accounts for approximately 3% of the newly synthesized protein. The expression of non-selectable genes can also be regulated, as shown by the induction of influenza virus nucleoprotein synthesis in transformed cells. These results indicate that the ts-COS cells can be used as a highly efficient, regulable mammalian expression system.     

End structure and mechanism of packaging of bacteriophage T4 DNA.

We analyzed by restriction enzyme digestion the end structure of T4 phage DNA by comparing mature, concatemeric, first-packaged, and incompletely packaged DNAs. The structure of mature DNA was also studied using 3' end labeling with terminal transferase. Our data support the hypothesis that T4 DNA packaging is not initiated at specific packaging initiation sequences on the concatemeric precursor (cos or pac site mechanisms) but by a different packaging mechanism.     

Expression in mammalian cells of a gene from Streptomyces alboniger conferring puromycin resistance.

The gene encoding a puromycin N-acetyl transferase from Streptomyces alboniger has been cloned next to the SV40 early promoter in a mammalian cells-Escherichia coli shuttle vector. When this construction was introduced into VERO cells it expressed the relevant enzymic activity. Moreover, the puromycin N-acetyl transferase gene has been used as a dominant marker for the selection of transformed mammalian cells able to grow in the presence of the antibiotic.     

A novel shuttle vector for Streptomyces spp. and Escherichia coli as a tool in site-directed mutagenesis.

This paper describes the construction and utilization of a novel shuttle vector for Streptomyces spp. and Escherichia coli as a useful vector in site-directed mutagenesis. The shuttle vector pIAFS20 (6.7 kb) has the following features: a replicon for Streptomyces spp., isolated from plasmid pIJ702; the thiostrepton-resistance gene as a selective marker in Streptomyces; the ColE1 origin, allowing replication in E. coli; and the ampicillin-resistance gene as a selective marker in E. coli. Vector pIAFS20 also contains the phage f1 intergenic region, which permits production of single-stranded DNA in E. coli after superinfection with helper phage M13K07. Moreover, the lac promoter is located in front of the multiple cloning sites cassette, allowing eventual expression of the cloned genes in E. coli. After mutagenesis and screening of the mutants in E. coli, the plasmids can be readily used to transform Streptomyces spp. As a demonstration, a 3.2-kb DNA fragment containing the gene encoding the xylanase A from Streptomyces lividans 1326 was inserted into pIAFS20, and the promoter region of this gene served as a target for site-directed mutagenesis. The two deletions reported here confirm the efficiency of this new vector as a tool in mutagenesis.     

适用于链霉菌大片段基因组DNA 克隆和异源表达的细菌人工染色体(BAC)载体的构建及应用

【目的】很多链霉菌来源的天然产物的生物合成基因簇往往很大,用传统的cosmid载体很难完整的克隆和异源表达。本研究通过载体改造,成功构建出一个新的细菌人工染色体(BAC)载体,用于链霉菌来源的天然产物生物合成基因簇的克隆及异源表达实验。【方法】从复合型载体pCUGIBAC1出发,通过λRED介导的PCR-targeting方法,用链霉素抗性基因替换掉原有的氯霉素抗性基因标记,同时插入链霉菌中常用的安普拉霉素抗性标记、转移起始位点oriT、φC31整合酶基因int、整合位点attP等元件。【结果】成功构建出可装载链霉菌大片段DNA的BAC载体pMSBBACs。使用pMSBBACs构建出链霉菌U27的基因组BAC文库,平均插入片段大小为100 kb。选取其中一个大小为140 kb的BAC质粒进行功能验证,实验证明通过接合转移和原生质体转化的方法都能够将这个大型BAC质粒导入链霉菌模式菌株,并通过位点特异性重组整合到染色体中进行异源表达。【结论】BAC载体pMSBBACs可成功用于放线菌大片段基因组DNA的克隆和异源表达实验。     

Construction of a PAC vector system for the propagation of genomic DNA in bacterial and mammalian cells and subsequent generation of nested deletions in individual library members

The BAC and PAC cloning systems allow investigators to propagate large genomic DNA fragments up to 300 kb in size in E. colicells.We describe a new PAC shuttle vector that can be propagated in both bacterial and human cells. Specifically, the P1 cloning vector pAd10sacBII was modified by the insertion of a puromycin-resistance gene (pac), the Epstein-Barr Virus (EBV) latent replication origin oriP,and the EBV EBNA1 gene. Transfection studies in HEK 293 cells demonstrated that the modified vector was stably maintained as an episome for at least 30 generations. And since pJCPAC-Mam1 contains a loxP site, genomic DNA cloned into this vector can be subjected to loxP-Cre -mediated deletion events. The transposon vector pTnPGKpuro/loxP was modified to make this system amenable to propagation in human cells by inserting pac, oriP, and EBNA1 elements into the vector (Chatterjee, P.K., Coren, J.C., 1997. Isolating large nested deletions in PACs and BACs by in vivo selection of P1 headful-packaged products of Cre-catalyzed recombination between the loxP site in PAC and BAC and one introduced in transposition. NAR 25, 2205-2212.). pTnPGKpuro/loxP-EBV was then used to generate deletions in an individual library member to demonstrate that all of the deletions still contain the required eukaryotic elements and that they were nested. All library members constructed in pJCPAC-Mam1 can be directly transformed into human cells to assess function. And the deletion technology can be used to aid in delineating the boundaries of genes and other cis-acting elements.     

Properties of the streptomycete temperate bacteriophage FP43.

FP43 is a temperate bacteriophage for Streptomyces griseofuscus that forms plaques on many Streptomyces species. FP43 virions contain 56 kb of double-strand DNA that is circularly permuted and terminally redundant, and contains 65% G + C. A physical map of the FP43 genome was constructed, and the origin for headful packaging (pac) was localized to an 8.8-kb region of the genome (hft) that mediates high-frequency transduction by FP43 of plasmid pRHB101. The phage attachment site (attP), a replication origin (rep), a region that inhibits plaque formation (pin), and a 3-kb deletion (rpt) that caused a 100-fold reduction in plasmid transduction were mapped.     

Transposition of Tn5096 from a temperature-sensitive transducible plasmid in Streptomyces spp.

Transposon Tn5096 was inserted into a derivative of the temperature-sensitive plasmid pMT660 containing the bacteriophage FP43 pac site. The resulting plasmid, pRHB126, was transduced by FP43 into several Streptomyces species. Tn5096 transposed from pRHB126 into different sites in the genomes of Streptomyces ambofaciens, Streptomyces cinnamonensis, Streptomyces coelicolor A3(2), Streptomyces fradiae, Streptomyces griseofuscus, and Streptomyces thermotolerans.     

Cloning and expression of an alpha-amylase gene from Streptomyces thermoviolaceus CUB74 in Escherichia coli JM107 and S. lividans TK24

A gene coding for a thermostable extracellular alpha-amylase, carried by a 5.7 kb BamHI chromosomal DNA fragment isolated from Streptomyces thermoviolaceus strain CUB74, was cloned into Escherichia coli JM107 using, as a cloning vector, the high-copy-number plasmid pUC8. E. coli containing a recombinant plasmid pQR300 expressed the amylase gene and exported the enzyme into the periplasmic space and the culture medium. The amylase protein expressed by E. coli had the same molecular mass (50 kDa) as that expressed by the Streptomyces parent strain, which suggests that the enzyme is processed similarly by both strains. The amylase gene was also cloned into Streptomyces lividans TK24 using pIJ702 as vector. The enzyme was stable at 70 degrees C when CaCl2 was present.     

Cloning, expression in Escherichia coli and nucleotide sequence of a tetracycline-resistance gene from Streptomyces rimosus

Determinants of tetracycline resistance have been cloned from two different tetracycline-producing industrial strains of Streptomyces into Streptomyces lividans using the plasmid vector pUT206. Three plasmids, pUT250 and pUT260 with a 9.5 and a 7.5 kb insert respectively of Streptomyces rimosus DNA, and pUT270 with a 14.0 kb insert of Streptomyces aureofaciens DNA, conferring resistance to tetracycline, have been isolated. By in vitro sub-cloning, a similar fragment of 2.45 kb containing the tetracycline resistance gene (tet347) was further localized on these plasmids. The S. rimosus gene has been cloned into Escherichia coli and expressed under the control of lambda pL or Lpp promoters. Differential protein extraction of E. coli cells revealed the presence of an additional membrane-embedded protein in tetracycline-resistant cells. On the basis of available restriction endonuclease maps, the tet347 gene is probably identical to the tetB gene from S. rimosus recently identified by T. Ohnuki and co-workers as responsible for the reduced accumulation of tetracycline. The nucleotide sequence of a 2052 bp DNA fragment containing the TcR structural gene from S. rimosus has been determined. The amino acid sequence of the tet347 protein (Mr35818) deduced from the nucleotide sequence shows a limited but significant homology to other characterized tetracycline transport acting determinants from pathogenic bacteria.