Molecular cloning and mapping of SphI restriction fragments of plasmid RP4

A combined physical and functional map of plasmid RP4 is presented including the sites for 18 restriction endonucleases. Several cleavage sites of SphI, BalI, and ApaI are suitable for the dissection of the transfer gene regions. Recombinant plasmids containing RP4 SphI fragments were constructed to assist in localizing sites relative to each other and to assign functions conferred by RP4 to the host.     

Role and specificity of plasmid RP4-encoded DNA primase in bacterial conjugation.

The role of the DNA primase of IncP plasmids was examined with a derivative of RP4 containing Tn7 in the primase gene (pri). The mutant was defective in mediating bacterial conjugation, with the deficiency varying according to the bacterial strains used as donors and recipients. Complementation tests involving recombinant plasmids carrying cloned fragments of RP4 indicated that the primase acts to promote some event in the recipient cell after DNA transfer and that this requirement can be satisfied by plasmid primase made in the donor cell. It is proposed that the enzyme or its products or both are transmitted to the recipient cell during conjugation, and the role of the enzyme in the conjugative processing of RP4 is discussed. Specificity of plasmid primases was assessed with derivatives of RP4 and the IncI1 plasmid ColIb-P9, which is known to encode a DNA primase active in conjugation. When supplied in the donor cell, neither of the primases encoded by these plasmids substituted effectively in the nonhomologous conjugation system. Since ColIb primase provided in the recipient cell acted weakly on transferred RP4 DNA, it is suggested that the specificity of these enzymes reflects their inability to be transmitted via the conjugation apparatus of the nonhomologous plasmid.     

Molecular cloning and functional analysis of DNA regions of plasmid RP4 determining the incompatibility properties

Sau3A-generated DNA fragments determining incompatibility functions of the plasmid RP4 were cloned on the vectors pTK16 and pBR322. Inc+ recombinant plasmids were divided into two types: 1) expressing incompatibility only towards the homologous RP4 replicon, 2) expressing incompatibility - both towards the homologous RP4 replicon and towards the heterologous replicons of plasmids R906 and R751. For one member of the first type plasmids it was shown that the cloned Inc+-specific insertion derived from the region of location of the EcoRI restriction site. The majority of the Inc+ recombinant plasmids showed asymmetric expression of incompatibility, predominantly eliminating the resident IncP plasmid.     

Mapping and cloning of the par-region of broad-host-range plasmid RP4

Various deletion mutants of the identical broad-host-range plasmids RP4 and RK2, obtained after conjugative transfer of these plasmids from Escherichia coli to Alcaligenes eutrophus H16, were tested with respect to their segregation behaviour. Although the parent plasmids and some of the deletion mutants were completely stable in both A. eutrophus and E. coli, other derivatives were lost under non-selective conditions. The analysis of these deletion mutants allowed the identification and mapping of a region encoding a partitioning system (par) between the tra2 region and the kanamycin resistance gene of RP4 (RK2). This area corresponds to the PstI-C restriction fragment of RP4 (RK2). Cloning of this fragment into several unstable vector plasmids including pBR322 and pACYC177 resulted in all cases in an increase of segregational stability. By insertion of the par-region into an unstable broad-host-range mobilizable plasmid and transfer to a series of gram-negative bacteria, it could be shown that the cloned par-region of RP4 is functional in a broad-host-range.     

Molecular cloning of the hamster papovavirus genome in Escherichia coli plasmid vector pBR322

The complete genome of the hamster papovavirus (HaPV) which was isolated from virions found in multiple skin tumors of Syrian hamsters was cloned in Escherichia coli using the plasmid vector pBR322. The cloned viral DNAs were identified by digestion of the recombinant DNAs with various restriction enzymes followed by comparison of their electrophoretic mobilities in agarose gels with that of similarly digested uncloned DNAs. The cloned HaPV DNAs showed the same migration pattern as the corresponding fragments from the restricted uncloned DNAs, indicating that no major insertions or deletions occurred during cloning and plasmid propagation. The electrophoretic data were confirmed by Southern blot hybridization.     

Molecular cloning of heterologous chromosomal DNA by recombination between a plasmid vector and a homologous resident plasmid in Bacillus subtilis

Summary The difficulty experienced in the shotgun cloning of chromosomal DNA on plasmid vectors in Bacillus subtilis is analyzed and an explanation for this difficulty is offered based on an inherent property of competent cells which imposes a requirement of plasmid multimers in transformation of plasmidfree recipients (Canosi et al., 1978). A stratagem which uses cloning by recombination between the vector and a resident homologous plasmid is tested and shown to be successful. Several recombinant plasmids are obtained containing Bacillus licheniformis DNA fragments which complement aromatic amino acid mutants of Bacillus subtilis. The yield of recombinant clones ranges from 6.7 to 210 per g of chromosomal DNA, depending on the selection and the restriction endonuclease. The various trp clones obtained after cutting chromosomal DNA with BglII and BclI do not complement trpE and exhibit both orientations with respect to the vector. The location of several restriction endonuclease cleavage sites in the cloned trp fragments is presented, and their relationship to the genetic map of Bacillus licheniformis is described.Abbreviations Km kanamycin - Cm chloramphenicol - Em erythromycin - CCC covalently closed circular - OC open circular - resistant - MDal megadalton In partial fulfillment of the requirements for the doctoral degree in the Department of Microbiology at the New York University School of Medicine, for S.C.     

Method for cloning single-stranded oligonucleotides in a plasmid vector

A method for cloning single-stranded oligonucleotides in a plasmid vector has been developed. The method relies on ligation of the oligonucleotide into suitable restriction enzyme sites of the cloning vector such that the site at the 5' end has a 5' overhang [for example, a Bgl II site (A decreases GATCT)], and the site at the 3' end has a 3' overhang [for example, a Sac I site (GAGCT decreases C)]. This arrangement allows the oligonucleotide to anneal to the single-stranded ends of the vector and to be covalently joined by T4 DNA ligase. The complementary strand can be synthesized in vitro to generate a double-stranded plasmid, or the partially single-stranded molecule can be used as a target for site-directed mutagenesis. The subsequent transfer of the oligonucleotide to test plasmids or excision for other manipulations, such as band shift experiments to identify protein binding sites, is facilitated by cloning of the oligonucleotide into a polylinker containing multiple restriction enzyme sites. For this purpose, the plasmid vector, pKP59, which is a 2.0 kB derivative of pBR322 lacking "poison sequences" and containing 16 cloning sites, has been the most satisfactory.     

SOS-induction of the RP4 plasmid tet-determinant

Induction of the tetracycline-resistance genes function by the inducer of the DNA-repair and mutability SOS-system, UV-light, has been tested. Activity of the tet-genes residing on the plasmid RP4 in Escherichia coli cells has been shown to be inducible by the low doses of tetracycline as well as by the mutagenic doses of UV-light. The induction was quantitatively registered by measuring the activity of beta-galactosidase of bacteriophage Mud1 (Ap, Lac) inserted into the tet-genes of the plasmid RP4. The bacteriophage integration inactivates the tet-genes function of the constructed plasmid fusing the lac-operon to a promoter of inactivated genes. Precise excision of bacteriophage restores the activity of the tet-genes proving together with the plasmid DNA-restriction analysis the fusion of tet-promoter with Iac-operon. The tet-genes of RP4 are concluded to be a part of the SOS-regulon, a set of genes inducible by the conditions harming the bacterial cell. Preliminary data on the mutagenic activity of tetracycline obtained in the bacterial test-system of mutagens are discussed.     

Molecular cloning of complementary DNA: preparation of a plasmid vector with low transformation background

A simple method that allows the rapid preparation of oligo dG-tailed plasmid vectors is presented. The procedure involves purification of the tailed molecules by hybridization to oligo dC-cellulose followed by a stepwise thermal elution. The resulting plasmid is virtually devoid of transformation activity in the absence of oligo dC-tailed DNA fragments. It allows construction of cDNA libraries with as low as 1% of colonies harboring wild-type plasmids.     

Gene organization and nucleotide sequence of the primase region of IncP plasmids RP4 and R751.

The primase genes of RP4 are part of the primase operon located within the Tra1 region of this conjugative plasmid. The operon contains a total of seven transfer genes four of which (traA, B, C, D) are described here. Determination of the nucleotide sequence of the primase region confirmed the existence of an overlapping gene arrangement at the DNA primase locus (traC) with in-phase translational initiation signals. The traC gene encodes two acidic and hydrophilic polypeptide chains of 1061 (TraC1) and 746 (TraC2) amino acids corresponding to molecular masses of 116,721 and 81,647 Da. In contrast to RP4 the IncP beta plasmid R751 specifies four large primase gene products (192, 152, 135 and 83 kDa) crossreacting with anti-RP4 DNA primase serum. As shown by deletion analysis at least the 135 and 83 kDa polypeptides are two separate translational products that by analogy with the RP4 primases, arise from in-phase translational initiation sites. Even the smallest primase gene products TraC2 (RP4) and TraC4 (R751) exhibit primase activity. Nucleotide sequencing of the R751 primase region revealed the existence of three in-phase traC translational initiation signals leading to the expression of gene products with molecular masses of 158,950 Da, 134,476 Da, and 80,759 Da. The 192 kDa primase polypeptide is suggested to be a fusion protein resulting from an in frame translational readthrough of the traD UGA stopcodon. Distinct sequence similarities can be detected between the TraC proteins of RP4 and R751 gene products TraC3 and TraC4 and in addition between the TraD proteins of both plasmids. The R751 traC3 gene contains a stretch of 507 bp which is unrelated to RP4 traC or any other RP4 Tra1 gene.     

New cloning and expression vector derived from Escherichia coli plasmid pIGWZ12; a potential vector for a two-plasmid expression system

We constructed pIGPZ, a new cloning and expression vector derived from Escherichia coli plasmid pIGWZ12::Kan. pIGPZ contains a kanamycin resistance marker, a multiple-cloning-site (MCS) region, and a promoter for constitutive expression of cloned genes. pIGPZ has the same high level of stability as the original plasmid, even in the absence of antibiotic selection. Furthermore, we show that pIGPZ is compatible with ColE1-based plasmids and a pSC101-like plasmid. All the characteristic elements of theta-replicating plasmids were found in the pIGPZ putative origin of replication. Finally, we demonstrate that pIGPZ can be used in a double-plasmid expression system by co-expressing UBP1 protease from pIGPZ with ubi-interferon alpha (IFNA13; GenBank Accession No. NM_006900.3) or ubi-human growth hormone (ubi-hGH; patent No. WO 2005/066208 A2) cloned in another plasmid. In this system, both ubi-interferon alpha and ubi-human growth hormone were deubiquitinated efficiently in E. coli cells.     

Stabilization of the cloning vector pACYC184 by insertion of F plasmid leading region sequences

The leading region of the F plasmid is, by definition, the first part of the plasmid DNA to be transferred to the recipient cell during conjugation. Restriction fragments of the leading region, when cloned into the plasmid vector pACYC184, extended the maintenance of the normally unstable p15A-derived vector replicon in rec+ Escherichia coli K-12 cells. Mutations in the host's general recombination systems were found to influence the maintenance of these hybrid plasmids.     

Molecular cloning of the T4 genome; organization and expression of the frd--DNA ligase region

Summary derivatives including the thymidylate synthetase (td) gene of T4 were selected by their ability to substitute for the thyA gene of E. coli. Two HindIII fragments of T4 DNA, but only one EcoRI fragment, are required for a functional td gene; one of the HindIII fragments includes a functional frd gene. The organisation of the EcoRI and HindIII fragments in the td region and their orientation with respect to the T4 genome have been deduced from genetic, physical, and functional evidence. The T4 genes can be transcribed from phage promoters and the T4td derivatives include genes specifying five T4 polypeptides. Three of these are identified as the products of the frd, td, and nrdA genes; two, neither of which appears to be the nrdB gene product, remain to be identified. Some td phages yield lysogens of thyA bacteria which are thymine-independent and some frd phages yield trimethoprim-resistant lysogens, indicating that the td and frd genes can be transcribed from included T4 DNA sequences. EcoRI fragments of DNA from the td and lig regions, used as probes, identified a single large HindIII fragment that joins the HindIII fragment carrying the DNA ligase gene to that carrying the td gene. Since this fragment, which must include genes coding for RNA ligase and polynucleotide kinase, could not be recovered in either phage or plasmid vectors, a derivative of it was used to identify the EcoRI fragments located between the td and DNA ligase genes. The order of these fragments within the T4 genome was deduced and all but one of them cloned in a vector. As none of these recombinants rescued T4 phage having mutations within the RNA ligase gene, the missing fragment may include this gene. Three adjacent EcoRI fragments, each of which has been cloned, are missing in a mutant of T4 deleted for the polynucleotide kinase gene.     

Molecular cloning in plasmid pBR322 giving altered expression of the tetracycline resistance gene

The two HindIII fragments of polyoma virus DNA were cloned in the HindIII site of plasmid pBR322, a site located in the RNA polymerase promoter involved in the expression of tetracycline resistance. Although insertion of foreign DNA into this site did not always result in the complete loss of tetracycline resistance, Escherichia coli K12 strain chi 1776 harbouring recombinant plasmids exhibited reduced growth properties in liquid culture with tetracycline and could easily be differentiated from bacteria transformed by non-recombinant plasmids. The formation of plasmid multimers increased the resistance to tetracycline at the level of the induction period, presumably as a result of a gene dosage effect.     

Molecular cloning of a thermostable neutral protease gene from Bacillus stearothermophilus in a vector plasmid and its expression in Bacillus stearothermophilus and Bacillus subtilis.

The structural gene for a thermostable protease from Bacillus stearothermophilus was cloned in plasmid pTB90. It is expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about 15-fold more protease (310 U/mg of cell dry weight) than did the wild-type strain of B. stearothermophilus. Some properties of the proteases that have been purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant difference was observed among the enzyme properties studied here despite the difference in host cells. We found that the protease, neutral in pH characteristics and with a molecular weight of 36,000, retained about 80% of its activity even after treatment of 65 degrees C for 30 min.     

High-copy-number derivatives of the plasmid cloning vector pBR322

A stable copy-number mutant of a pBR322-derived plasmid was isolated. The mutation was found to be a single G → T transversion located near the 3' end of a DNA segment coding for the regulatory RNA I. The resulting copy number for this plasmid is approx. 1000 per cell or 65 % of total cellular DNA. Several cloning vectors have been constructed from this copy-number mutant and their practical application is discussed.     

斜纹夜蛾inx1,inx4基因的克隆及表达载体构建

无脊椎动物innexin(inx)基因是生物进化上高度保守的基因,区别于脊椎动物的connexin。Inx基因可在细胞膜表面形成功能性的半通道蛋白hemichannel和间隙连接蛋白gap junction,这两种蛋白都与信号传导,胚胎发育,神经系统分化等功能相关。本研究在斜纹夜蛾Spodoptera litura血细胞转录组数据中发现特异的inx1,inx4序列,通过RT-PCR克隆出了Spli-inx1,Spli-inx4,经过比对,确定它们都属于保守的inx基因家族,具有四个保守的跨膜结构域,两个胞外环,一个胞内环,一个位于第二个跨膜结构域前端的YYQWV基序及在胞外环上各有2个半胱氨酸残基。其中,Spli-inx1开放阅读框全长1086 bp,编码361个氨基酸;Spli-inx4开放阅读框全长1116 bp,编码371个氨基酸。构建了包含ORF全长编码区的原核表达载体及真核表达载体,经Western blot证明Spli-inx1和Spli-inx4均可在大肠杆菌及鳞翅目昆虫High Five,Sf9和Spli221细胞系中的表达,初步探索了它们在细胞中的结构和功能。为深入研究Spli Inx1和Spli Inx4的功能奠定基础。     

Molecular cloning and expression of chondroitin 4-sulfotransferase

Chondroitin 4-sulfotransferase (C4ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine residue of chondroitin. The enzyme has been previously purified to apparent homogeneity from the serum-free culture medium of rat chondrosarcoma cells (Yamauchi, A., Hirahara, Y., Usui, H., Takeda, Y., Hoshino, M., Fukuta, M., Kimura, J. H., and Habuchi, O. (1999) J. Biol. Chem. 274, 2456-2463). The purified enzyme also catalyzed the sulfation of partially desulfated dermatan sulfate. We have now cloned the cDNA of the mouse C4ST on the basis of the amino acid sequences of peptides obtained from the purified enzyme by protease digestion. This cDNA contains a single open reading frame that predicts a protein composed of 352 amino acid residues. The protein predicts a Type II transmembrane topology. The predicted sequence of the protein contains all of the known amino acid sequence and four potential sites for N-glycosylation, which corresponds to the observation that the purified C4ST is an N-linked glycoprotein. The amino acid sequence of mouse C4ST showed significant sequence homology to HNK-1 sulfotransferase. Comparison of the sequence of mouse C4ST with human HNK-1 sulfotransferase revealed approximately 29% identity and approximately 48% similarity at the amino acid level. When the cDNA was introduced in a eukaryotic expression vector and transfected in COS-7 cells, the sulfotransferase activity that catalyzes the transfer of sulfate to position 4 of GalNAc residue of both chondroitin and desulfated dermatan sulfate was overexpressed. Northern blot analysis showed that, among various mouse adult tissues, 5.7-kilobase message of C4ST was mainly expressed in the brain and kidney.