Hairpin plasmid--a novel linear DNA of perfect hairpin structure.

The terminal structures of deletion derivatives of linear DNA killer plasmid from yeast were analyzed. The yeast Kluyveromyces lactis harbors two unique double-stranded linear DNA killer plasmids, pGKL1 of 8.9 kb and pGKL2 of 13.4 kb. The killer toxin and the resistance to the killer are coded by pGKL1, while pGKL2 is required for the maintenance of pGKL1 in the cell. When the pGKL plasmids from K. lactis were transferred into Saccharomyces cerevisiae by transformation, non-killer transformants harboring pGKL2 and new plasmids, F1 of 7.8 kb and F2 of 3.9 kb, were obtained. F2 was shown to be a linear DNA arising from a 5-kb deletion of the right part of pGKL1. F1 was an inverted dimer of F2. Here we show that F2 has two different terminal structures: one end has a protein attached at the 5' terminus whereas the two strands of duplex are linked together at the other end, thus forming a hairpin structure. This is a novel type of autonomously replicating DNA molecule.     

The yeast linear DNA killer plasmids, pGKL1 and pGKL2, possess terminally attached proteins.

The terminal structures of linear DNA killer plasmids from yeast, pGKL1 and pGKL2, were analyzed. Results obtained by exonuclease treatments of these plasmids show that both pGKL plasmids have free hydroxyl 3'-ends and blocked 5'-ends. Electrophoretic analysis of the terminal restriction fragments treated with proteases revealed that pGKL1 and pGKL2 have proteins bound at 5'termini and that the terminal protein of pGKL1 is distinct from that of pGKL2. This is the first linear DNA-terminal protein association found in yeast.     

Efficient isolation of the linear DNA killer plasmid of Kluyveromyces lactis: evidence for location and expression in the cytoplasm and characterization of their terminally bound proteins.

Differential centrifugation of an osmotic lysate of K. lactis protoplasts showed that the linear DNA killer plasmids of K. lactis, pGKL1 and pGKL2, are almost exclusively present in the cytoplasmic fraction. This fractionation procedure allows the rapid isolation of large amounts of plasmid DNA without contamination by chromosomal and mitochondrial DNA. With these DNA preparations the size of the terminally bound proteins was estimated to be 28 and 36 kDal for pGKL1 and pGKL2, respectively. The entire pGKL1 sequence (except for 21 base pairs at the right terminus) was cloned in a shuttle vector that permits autonomous replication in the nucleus of K. lactis. However, killer gene expression could not be established in transformants. In connection with the observed cytoplasmic localization, this result suggests that gene expression of the killer DNA plasmids is entirely cytoplasmic.     

A linear DNA plasmid from Streptomyces rochei with an inverted terminal repetition of 614 base pairs.

The terminal structure of a linear plasmid pSLA2 , which was isolated from Streptomyces rochei , was analysed. The 5' ends of pSLA2 DNA were blocked by the association of a protein probably covalently bonded with the DNA. This block is removed by alkali treatment and blunt ends with 5'-phosphate and 3'-hydroxy termini were released. The two terminal fragments of pSLA2 were cloned and the nucleotide sequence was determined. An inverted terminal repetition of 614 bp was found along with the presence of further interrupted homologous sequences beyond this area up to 800 bp. These are the first inverted terminal repeat sequences found in microbial linear plasmids.     

DNA sequence homology between the terminal inverted repeats of Shope fibroma virus and an endogenous cellular plasmid species.

DNA hybridization experiments indicate that the genome of a tumorigenic poxvirus. Shope fibroma virus (SFV), possesses sequence homology with DNA isolated from uninfected rabbit cells. Southern blotting experiments, either with high-complexity rabbit DNA as probe and SFV restriction fragments as targets or with high-specific activity, 32P-labeled, cloned SFV sequences as probes and rabbit DNA as target, indicate that the homologous sequences map at two locations within the viral genome, one in each copy of the terminal inverted repeat sequences. Unexpectedly, Southern blots revealed that the homologous host sequences reside in a rabbit extrachromosomal DNA element. This autonomous low-molecular-weight DNA species could be specifically amplified by cycloheximide treatment and was shown by isopycnic centrifugation in cesium chloride-ethidium bromide to consist predominantly of covalently closed circular DNA molecules. DNA sequencing of pSIC-9, a cloned 1.9-kilobase fragment of the rabbit plasmid species, indicated extensive homology at the nucleotide level over a 1.5-kilobase stretch of the viral terminal inverted repeat. Analysis of open reading frames in both the plasmid and SFV DNA revealed that (i) the N-terminal 157-amino acid sequence of a potential 514-amino acid SFV polypeptide is identical to the N-terminal 157 amino acids of one pSIC-9 open reading frame, and (ii) a second long pSIC-9 open reading frame of 361 amino acids, although significantly diverged from the comparable nucleotide sequence in the virus, possessed considerable homology to a family of cellular protease inhibitors, including alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III. The potential role of such cellular plasmid-like DNA species as a mediator in the exchange of genetic information between the host cell and a cytoplasmically replicating poxvirus is discussed.     

Secretion of killer toxin encoded on the linear DNA plasmid pGKL1 from Saccharomyces cerevisiae

By the kar1-mediated cytoduction, linear double-stranded DNA plasmids pGKL1 and pGKL2, encoding killer toxin complex, have been successfully transferred to the recipient strains with about 30% frequency. The killer toxin was found to be secreted through the normal yeast secretory pathway by introducing pGKL plasmids into the several Saccharomyces cerevisiae sec mutants and examining the secretion of killer toxin. S. cerevisiae cells, harboring newly isolated deletion plasmid pGKL1D, expressed only the 28K protein among three killer subunits, and secreted the 28K subunit at a level of zero to 20% efficiency of the cells containing intact pGKL1 plasmid. These data indicated that subunit interaction (cosecretion) of killer proteins is required for the efficient secretion of 28K subunit. The 28K precursor protein was found to translocate across the canine pancreatic endoplasmic reticulum membrane under the direction of its own signal peptide in vitro without any other subunits. From kex2 mutant cells harboring pGKL1 plasmid, the 97K subunit, and its precursor 128K protein were not secreted, however, the 28K subunit was secreted in the same amount as that secreted from KEX2 cells. These lines of evidence suggest that the final assembly of killer toxin complex after KEX2 site of Golgi apparatus is not essential for the secretion of 28K subunit, and therefore, that putative interaction between 128K protein and 28K subunit for the transport between endoplasmic reticulum and Golgi apparatus may be required for the efficient secretion of 28K subunit.     

Expression of pGKL killer 28K subunit in Saccharomyces cerevisiae: identification of 28K subunit as a killer protein.

Saccharomyces cerevisiae and other yeast cells harboring the linear double stranded (ds) DNA plasmids pGKL1 and pGKL2 secrete a killer toxin consisting of 97K, 31K and 28K subunits into the culture medium (EMBO J. 5, 1995-2002 (1986), Nucleic Acids Res., 15, 1031-1046 (1987]. The 28K subunit of the killer toxin was successfully expressed in S. cerevisiae when it was cloned on a circular plasmid with its putative promoter region replaced with that of S. cerevisiae chromosomal genes. The expression of the 28K subunit of the killer toxin in killer-sensitive cells resulted in the death of the host cells. This killing activity by the 28K subunit was prevented by the expression of the killer immunity, indicating that the killing activity of the killer toxin complex was carried out by the 28K subunit. Although the 28K subunit was synthesized as a intact precursor protein with its own signal sequence, it was not secreted into the culture medium but remained in the host cells. This indicated that 28K subunit killed host cells from inside of the cells rather than from outside. We further suggested that 28K killer subunit without 97K and 31K subunits did not kill the killer-sensitive cells from outside.     

酿酒酵母HAL1基因的克隆及植物表达载体的构建

HAL1基因是酵母中重要的耐盐基因。以酿酒酵母AS2.375菌株的DNA为模板,根据已发表的序列设计引物,经PCR扩增得到约900 bp的HAL1基因片段,连接到pMD18-T载体上,转化大肠杆菌JM109,筛选重组质粒进行酶切分析和序列测定,结果显示已克隆到完整的可读框,该基因的序列与已知序列同源性达99%。将HAL1基因从T-载体上切下连接到pAM194载体上构建了HAL 1基因的植物表达载体,用于烟草的转化获得了耐盐性提高的转化植株。     

The complete nucleotide sequence of the gene coding for botulinum type C1 toxin in the C-ST phage genome

Two DNA fragments, 3 kbp and 7.8kbp, which encode the type C1 botulinum neurotoxin gene, were obtained from toxigenic bacteriophage DNA by treatment with a restriction enzyme. They were cloned into the plasmid vectors for nucleotide sequence determination. The nucleotide sequence contained a single open reading frame coding for 1,291 amino acids corresponding to a polypeptide with a molecular weight of 149,000. The amino acid sequence of the C1 toxin has a few regions highly homologous with tetanus toxin.     

Promoter activity associated with the left inverted terminal repeat of the killer plasmid k1 from yeast.

The killer plasmid k1 of Kluyveromyces lactis has terminal inverted repeats of 202 base pairs (bp). The left terminal repeat is contiguous to the transcribed open reading frame, ORF1, which is supposed to code for a DNA polymerase. A 266-bp fragment (called Pk1) containing most of the terminal repeat sequence was isolated and examined for promoter activity. Pk1 was fused, in either original or inversed orientation, with a promoter-less lacZ gene of E coli and a promoter-less G418 resistance gene of Tn903. These fusions were introduced into a pKD1-derived circular vector, and transformed into a lactose-negative (lac4), and a G418-sensitive K lactis host. Lac+ and G418-resistant transformants were obtained with either orientation of Pk1. The promoter activity of Pk1 fragment was independent of the presence or absence of killer plasmids. It is not known whether Pk1 can also function bidirectionally on the natural k1 plasmid. The possible functions of Pk1 for killer plasmid gene expression and plasmid replication are discussed.     

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.     

Molecular cloning and sequencing of lysozyme gene of bacteriophage SF6 ofBacillus subtilis

Digestion of bacteriophage SF6 (ofBacillus subtilis) DNA with restriction endonuclease Pst I generates seven fragments (A-G). These fragments were cloned in pBR322 DNA, and the recombinant clones carrying the lysozyme gene were identified by measuring lysozyme activity. The complete nucleotide sequence of 996 bp fragment D, containing lysozymze gene, was determined. One open reading frame was present at 13 bp, and the termination codons were present at 961 bp. The deduced amino acid sequence of the lysozyme gene was also determined.     

Palindrome-hairpin linear plasmids possessing only a part of the ORF1 gene of the yeast killer plasmid pGKL1

Summary The yeast Kluyveromyces lactis haboring linear DNA plasmids pGKL1 and pGKL2 exhibits killer and killer-resistant phenotypes. Two new linear plasmids pK192L and pK192S were found in the weak killer mutant KUV192 induced by UV irradiation. pK192S was always accompanied by pK192L in subclones of KUV192. Both plasmids were derived from pGKL1 by deletion of the large right part of it. pK192L was 4.9 kb in size and had a palindromic structure consisting of 2.35 kb inverted terminal repetitions and a 215 base unique sequence. Analysis of denatured and renatured DNA strands suggested that pK192S was a hairpin-like form of pK192L. The pK192 plasmids were maintained only in cells haboring either pGKL1 or pGKL1S in addition to pGKL2 and competed with pGKL1 or pGKL1S for their maintenance. Since no complete ORF1 was conserved in pK192 plasmids, these results lead to the conclusion that the ORF1 gene is necessary for the replication and/or maintenance of pGKL1.     

Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus

The isopenicillin N synthetase (IPNS) gene from Streptomyces clavuligerus was isolated from an Escherichia coli plasmid library of S. clavuligerus genomic DNA fragments using a 44-mer mixed oligodeoxynucleotide probe. The nucleotide sequence of a 3-kb region of the cloned fragment from the plasmid, pBL1, was determined and analysis of the sequence showed an open reading frame that could encode a protein of 329 amino acids with an Mr of 36,917. When the S. clavuligerus DNA from pBL1 was introduced into an IPNS-deficient mutant of S. clavuligerus on the Streptomyces vector pIJ941, the recombinant plasmid was able to complement the mutation and restore IPNS activity. The protein coding region of the S. clavuligerus IPNS gene shows about 63% and 62% similarity to the Cephalosporium acremonium and Penicillium chrysogenum IPNS nucleotide sequences, respectively, and the predicted amino acid sequence of the encoded protein showed about 56% similarity to both fungal sequences.     

黄瓜线粒体DNA类质粒pC1的性质和核酸序列研究

津研四号黄瓜线粒体中除主环ＤＮＡ外,还有４种ＤＮＡ类质粒：ｐＣ１、ｐＣ２、ｐＣ３、ｐＣ４。将环形类质粒ｐＣ１ｌｐｋ ｇｃ ｐＵＣ１９的ＥｃｏＲⅠ位点上,克隆至Ｅ．ｃｏｌｉ ＪＭ１０９中。以克隆的ｐＣ１为探针,进行同源性检测,ｐＣ１与津研四号黄瓜的核基因组、叶绿体基因组、线粒体基因组和线粒体中其他类质粒不同源。对ｐＣ１进行序列测定和分析,ｐＣ１长度２ ８８９ｂｐ,含有多个正向和反向重复序列,有３个８