Showing papers on "Vector (molecular biology) published in 1983"

A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria

Abstract: We have developed a new vector strategy for the insertion of foreign genes into the genomes of gram negative bacteria not closely related to Escherichia coli. The system consists of two components: special E. coli donor strains and derivatives of E. coli vector plasmids. The donor strains (called mobilizing strains) carry the transfer genes of the broad host range IncP–type plasmid RP4 integrated in their chromosomes. They can utilize any gram negative bacterium as a recipient for conjugative DNA transfer. The vector plasmids contain the P–type specific recognition site for mobilization (Mob site) and can be mobilized with high frequency from the donor strains. The mobilizable vectors are derived from the commonly used E. coli vectors pACYC184, pACYC177, and pBR325, and are unable to replicate in strains outside the enteric bacterial group. Therefore, they are widely applicable as transposon carrier replicons for random transposon insertion mutagenesis in any strain into which they can be mobilized but not stably maintained. The vectors are especially useful for site–directed transposon mutagenesis and for site–specific gene transfer in a wide variety of gram negative organisms.

pEMBL: a new family of single stranded plasmids.

Abstract: We have constructed a series of plasmids, the pEMBL family, characterized by the presence of 1) the bla gene as selectable marker, 2) a short segment coding for the alpha-peptide of beta-galactosidase and containing a multiple cloning sites polylinker, 3) the intragenic region of phage F1. pEMBL plasmids have the property of being encapsidated as single stranded DNA, upon superinfection with phage F1. These vectors have been used successfully for DNA sequencing with the dideoxy-method, and can be used for any other purpose for which M13 derivatives are used. However, the pEMBL plasmids have the advantage of being smaller than M13 vectors, and the purification of the DNA is simpler. In addition, and most importantly, long inserts have a higher stability in pEMBL plasmids than M13 vectors.

Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors

Abstract: We wished to construct cell lines that supply the gene products of gag, pol, and env for the growth of replication-defective reticuloendotheliosis retrovirus vectors without production of the helper virus. To do this, first we located by S1 mapping the donor and acceptor splice sites of reticuloendotheliosis virus strain A. The donor splice site is ca. 850 base pairs from the 5' end of proviral DNA. It is close to or overlaps the encapsidation sequences for viral RNA. The splice acceptor site is ca. 5.6 kilobase pairs from the 5' end of proviral DNA. Therefore, the encapsidation sequences and the donor splice site were removed from viral DNA to give expression of the gag and pol genes without virus production. The promoter in the long terminal repeat was fused to a site near the first ATG codon of the env gene, thereby deleting the encapsidation sequences and the gag and pol genes to give expression of the env gene without virus production. The permissive canine cell line D17 was transfected with the two modified viral DNAs. Two cell clones that contain both modified viral DNAs support the production of replication-defective spleen necrosis virus-thymidine kinase recombinant retrovirus vectors without the production of helper virus. To prevent recombination, the vector contains deletions that overlap with deletions in the integrated helper virus DNAs. This helper cell-vector system will be useful to derive infectious recombinant virus stocks of high titer (over 10(5) thymidine kinase transforming units per ml) which are able to infect avian, rat, and dog cells without the aid of helper virus.

Versatile cosmid vectors for the isolation, expression, and rescue of gene sequences: studies with the human alpha-globin gene cluster.

Abstract: We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers--SV2-gpt, SV2-DHFR, and SV2-neo--in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human alpha-globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of alpha-globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult alpha-globin genes were more actively expressed than the embryonic zeta-globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.

Multipurpose Expression Cloning Vehicles in Escherichia coli

Abstract: Publisher Summary This chapter reviews the construction of several high-level expression vectors using the Escherichia coli lipoprotein promoter and part of the protein. These vectors are designed to produce a large amount of an inserted gene product and to localize it to a specific compartment of the E. coli cell—cytoplasm, cytoplasmic membrane, periplasm, or outer membrane. These vectors have three restriction enzyme sites—EcoRI, HindIII, and BamHI—in each of the three reading frames at various positions along the lipoprotein gene. The pIN-I vectors constitutively produce a large amount of the inserted gene product. The pIN-II and pIN-III vectors produce the foreign gene product only in the presence of an inducer. Once a gene has been inserted properly into one of these vectors it can easily be moved to the other vectors, like a cassette, with the use of several possible unique restriction-enzyme sites on either side of the gene. The chapter also describes two promoter cloning vectors that produce a protein—tetracycline resistance or β-galactosidase—when a promoter is inserted.

Construction and Characterization of a Versatile Broad Host Range DNA Cloning System for Gram–Negative Bacteria

Abstract: A set of broad host range cloning vectors has been constructed from the IncW plasmid pSa These vectors have been constructed from the transfer defective deletion derivative pSa151, which encodes resistance to kanamycin and spectinomycin–streptomycin Two of the vectors also contain the chloramphenicol resistance gene of Tn9, and a third contains the chloramphenicol resistance gene of pSa One of the vectors contains the cos sequence of bacteriophage λ and can be used with in vitro packaging systems in the construction of large recombinant plasmids Two of these vectors can be mobilized and transferred in the presence of a pBR322 derivative containing the transfer genes of pSa Together these vectors contain cloning sites for SstII, HindIII, EcoRI, KpnI, PvuII, BamHI, SmaI, and Bg1II, and recombinants at certain of these sites can be detected by insertional inactivation of a drug resistance phenotype The broad host range properties of the origin of replication of pSa allow the use of these vectors in a variety of gram–negative bacteria

Shuttle cloning vectors for the cyanobacterium Anacystis nidulans.

Abstract: Hybrid plasmids capable of acting as shuttle cloning vectors in Escherichia coli and the cyanobacterium Anacystis nidulans R2 were constructed by in vitro ligation. DNA from the small endogenous plasmid of A. nidulans was combined with two E. coli vectors, pBR325 and pDPL13, to create vectors containing either two selectable antibiotic resistance markers or a single marker linked to a flexible multisite polylinker. Nonessential DNA was deleted from the polylinker containing plasmid pPLAN B2 to produce a small shuttle vector carrying part of the polylinker (pCB4). The two polylinker-containing shuttle vectors, pPLAN B2 and pCB4, transform both E. coli and A. nidulans efficiently and provide seven and five unique restriction enzyme sites, respectively, for the insertion of a variety of DNA fragments. The hybrid plasmid derived from pBR325 (pECAN1) also transforms both E. coli and A. nidulans, although at a lower frequency, and contains two unique restriction enzyme sites.

Cloning system for Kluyveromyces species

Abstract: A new cloning system is described capable of expressing genetic material derived from recombinant DNA material, which comprises a yeast of the genus Kluyveromyces as a host. Suitable vectors are e.g. vectors containing autonomously replicating sequences (ARS) and vectors containing homologous Kluyveromyces DNA acting as a site for recombination with the host chromosome. New and preferred vectors are those containing ARS sequences originating from Kluyveromyces (KARS vectors). The genetically engineered new strains of Kluyveromyces produce, inter alia, lactase and chymosin.

Cloning Vectors Derived from Animal Viruses

Abstract: Conclusions The examples discussed above show clearly that the development of vectors for use in eukaryotic cells is presently proceeding very rapidly. Knowledge gained from the use of one type of vector defines the design criteria for the next type and thus many systems become obsolete before they are fully developed. Some future work can be predicted with reasonable certainty. The vast majority of future vectors will incorporate dominant selectable markers and regulatable promoters and we can expect to see vectors containing several different origins of DNA replication so that their replication can be controlled in several ways. In parallel with these refinements will occur the development of eukaryotic vectors suitable for the primary cloning of genomic DNA or cDNA. This will be of the greatest importance as it will allow the immunological or phenotypic recognition of genes by virtue of their expression in eukaryotic cells, thus facilitating the isolation of eukaryotic genes which can not be detected by the techniques available for screening prokaryotic clones. The use of such vector systems will surely be a central feature of all future work in eukaryotic molecular biology.

Class II mobilizable gram-negative plasmid

Abstract: Vectors capable of replication in a broad host range of gram-negative bacteria are provided. The vectors are composite plasmids comprising a segment derived from an E. coli vector plasmid and a segment derived from a broad host range plasmid. The broad host range plasmid segment includes Mob-functions and DNA coding for replication functions with regions non-essential for broad host range replication or mobilizablity having been deleted. Bacterial strains containing the above plasmids as well as methods of cloning DNA employing such plasmids are also provided.

Conditionally replicating plasmid vectors that can integrate into the Klebsiella pneumoniae chromosome via bacteriophage P4 site-specific recombination.

Abstract: P4 is a satellite phage of P2 and is dependent on phage P2 gene products for virion assembly and cell lysis. Previously, we showed that a virulent mutant of phage P4 (P4 vir1) could be used as a multicopy, autonomously replicating plasmid vector in Escherichia coli and Klebsiella pneumoniae in the absence of the P2 helper. In addition to establishing lysogeny as a self-replicating plasmid, it has been shown that P4 can also lysogenize E. coli via site-specific integration into the host chromosome. In this study, we show that P4 also integrates into the K. pneumoniae chromosome at a specific site. In contrast to that in E. coli, however, site-specific integration in K. pneumoniae does not require the int gene of P4. We utilized the alternative modes of P4 lysogenization (plasmid replication or integration) to construct cloning vectors derived from P4 vir1 that could exist in either lysogenic mode, depending on the host strain used. These vectors carry an amber mutation in the DNA primase gene alpha, which blocks DNA replication in an Su- host and allows the selection of lysogenic strains with integrated prophages. In contrast, these vectors can be propagated as plasmids in an Su+ host where replication is allowed. To demonstrate the utility of this type of vector, we show that certain nitrogen fixation (nif) genes of K. pneumoniae, which otherwise inhibit nif gene expression when present on multicopy plasmids, do not exhibit inhibitory effects when introduced as merodiploids via P4 site-specific integration. Images

An Insect Virus for Genetic Engineering: Developing Baculovirus Polyhedrin Substitution Vectors

Abstract: Baculoviruses are exceptionally attractive candidates as vectors for propagating and expressing exogenous DNAs in a eukaryotic (invertebrate) environment (Miller, 1981a). Among the features which make baculoviruses highly advantageous as recombinant DNA vector systems are (1) a covalently-closed, circular, nuclear-replicating DNA genome, (2) an extendable rod-shaped capsid, (3) a group of genes, involved in occlusion, that are nonessential for infectious virus production and thus deletable, and (4) a strong promoter which is turned on after infectious virus production and controls the synthesis of the major occlusion body protein (poly-hedrin), constituting approximately ten percent of the protein of infected cells. The replacement of the polyhedrin gene with passenger DNA was previously suggested as an approach to using baculoviruses as recombinant DNA vectors (Miller, 1981a). The initial experimental advances our laboratory has made in developing the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector in insect cells are described herein.

Plant Viral Vectors: CaMV as an Experimental Tool

Abstract: Viruses provide natural examples of genetic engineering, since viral infection of a cell results in the addition of new genetic material which is expressed in the host. In both microbial and mammalian systems, viruses have played important roles in vector development, so it is not unexpected that plant viruses are considered as candidates for plant gene vectors. Additional genetic material incorporated into the genome of a plant virus might be replicated and expressed in the plant cell along with the other viral genes.

Promoter probe vectors

Abstract: Promoter probe vectors for determining the presence of efficient promoter regions on DNA segments, which segments permit microbiological expression of genetic information. The promoter probe vector comprises replicons active in E. coli and B. subtilis, a structural gene for B-galactosidase and at least one endonuclease restriction site suitable for insertion of promoter-containing DNA fragments.

Plasmidic vectors for expressing in bacillus subtilis and a method of preparing them

Abstract: A special description is given of plasmids pSM15, pSM16 and pSM17, which are hybrid plasmids derived by coupling pUB110 and modified pE194. The described plasmids are cloning vectors in Bacillus subtilis and are characterised by a marker of resistance to Kanamycin and an EcoRI site located near the end of the ribosome recognition sequence; they can also be used to induce the coded heterologous protein from cloned DNA with sub-inhibiting doses of erythromycin. These plasmids are prepared via the formation of plasmids pSM4, pSM5 or pSM6, which can be expressed either in B. subtilis or in E. coli.

Improved expression vectors

Abstract: Improved expression vectors comprising an expression control sequence characterized by at least one promoter selected from the group consisting of the promoter of RNA I, the promoter of the primer RNA for initiation of DNA replication and derivatives thereof; a DNA sequence within the replicon of the vector encoding RNA I and the primer RNA for initiation of DNA replication and their regulatory regions, the DNA sequence being characterized by at least one modification increasing the copy number of the vector in an appropriate host as compared to a vector without that modification and further amplifying the expression of genes and DNA sequences under the control of the expression control sequence of the vector; and at least one restriction site wherein a DNA sequence encoding a desired polypeptide may be inserted into the vector and operatively-linked therein to the expression control sequence. Using these vectors, DNA sequences encoding useful prokaryotic and eukaryotic polypeptides may be expressed to produce those polypeptides in high yield in appropriate hosts.

Bacterial-Plant Gene Cloning Shuttle Vectors for Genetic Modification of Plants

Abstract: Directed genetic transformation of plant cells is one of the fundamental requirements for tailoring plants’ cells to harbor desirable characteristics. This requirement can be achieved by the development of systems for the delivery and integration of foreign genes into the plant genome. Two potential gene delivery systems have been the focus of considerable attention: the Ti plasmid of Agrobacterium tumefaciens and the DNA plant virion cauliflower mosaic virus (CaMV)1–8. Although these are the most thoroughly characterized systems, reports on the successful application of these vectors in the manipulation of the plant genome have been premature. The inherent limitations of these vectors have been previously pointed out3,4,6,7, yet the design of a cloning vector capable of replication in bacteria and plants continues to center around the Ti plasmid and DNA plant viruses.

Plasmid vehicle for cloning in Agrobacterium tumefaciens

Abstract: A cloning vector comprises a replication system derived from the pTAR plasmid and capable of stable maintenance in Agrobacterium tumefaciens. By combining the pTAR replication system with a second replication system from a host other than A. tumefaciens, shuttle vectors are obtained which allow manipulation in more than one host. The cloning vectors will usually include selectable markers having restriction enzyme sites which allow identification of recombinant molecules by insertional inactivation. By providing at least a fragment of the T-DNA region from the Ti plasmid, the subject vectors can be used to clone desired DNA fragments and transfer these fragments to the genome of a higher plant. The strain E. coli HB101/puc0400 was deposited on June 7, 1983 at the A.T.C.C. for patent purposes and granted accession no. 39377.

Novel cloning vectors

Abstract: Vector plasmids are constructed to provide sites for insertion of a structural gene in phase with three reading frames downstream from the promoter of alkaline phosphatase gene of Escherichia coli.

Cloning vectors for cyanobacterium

Abstract: OF THE DISCLOSURE Shuttle cloning vectors, for the cloning of DNA in both E. coli bacteria and cyanobacteria (blue-green algae) are constructed using a bacterial plasmid with a polylinker containing many different restriction enzyme sites. These shuttle vectors contain an antibiotic resistance gene, an origin of replication for E. coli, an origin of replication for cyanobacteria and a polylinker with many restriction enzyme sites for the insertion of DNA fragments. Plasmid vectors generated by this procedure can be used for genetic engineering of cyanobacteria and to clone and manipulate plant genes, for the genetic engineering of plants.

Plasmid vectors including Tn904

Abstract: Cloning vectors are described which include the streptomycin resistance (Sm r ) determinant derived from Tn904. A single site for the restriction endonuclease, AvaI, is present within the Tn904 determinant for Sm r . A method is described for preparing the Tn904 containing cloning vectors through transposition of Tn904 to a parent cloning vector and then cloning of the Sm r gene into another vector segment. The cloning vector is important for inserting deoxyribonucleic acid segments, which encode for various characteristics such as chemical production, antibiotic resistance or bacterial cell properties, in the Sm r gene AvaI cleaved site and which normally provides a marker for identification of transformed strains of bacteria.