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At least 631 records · Page 35Linked to original sources
PubMed1992

A self-transmissible, narrow-host-range endogenous plasmid of Rhodobacter sphaeroides 2.4.1: physical structure, incompatibility determinants, origin of replication, and transfer functions.

Rhodobacter sphaeroides 2.4.1 naturally harbors five cryptic endogenous plasmids (C. S. Fornari, M. Watkins, and S. Kaplan, Plasmid 11:39-47, 1984). The smallest plasmid (pRS241e), with a molecular size of 42 kb, was observed to be a self-transmissible plasmid which can transfer only to certain strains of R. sphaeroides. Transfer frequencies can be as high as 10(-2) to 10(-3) per donor under optimal mating conditions in liquid media in the absence of oxygen. pRS241e, designated the S factor, was also shown to possess a narrow host range, failing either to replicate or to be maintained in Escherichia coli, Agrobacterium tumefaciens, and Rhizobium meliloti. It was further revealed that one of the remaining four endogenous plasmids, pRS241d, was also transmissible at a frequency similar to that of the S. factor. As a cointegrate with pSUP203, S was maintained in E. coli, providing sufficient DNA from which a physical map of S could be constructed. Progressive subcloning of S-factor DNA, in conjunction with assays of plasmid transfer, led to the localization and identification of oriV (IncA), IncB, and the putative oriT locus. The DNA sequence of the 427 bp containing oriTs revealed topological similarity to other described oriT sequences, consisting of an A-T-rich DNA region, several direct and inverted repeats, and putative integration host factor (IHF)-binding sites, and was shown to be functional in promoting plasmid transfer.

Anaerobiosis↗
PubMed2001

Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria.

Full exploitation of the information available in bacterial genome sequences requires the availability of facile tools for rapid genetic manipulation. One bacterium for which new genetic tools are needed is the methylotroph Methylobacterium extorquens AM1. IncQ and small IncP vectors were shown to be unsuitable for use in this bacterium, but a spontaneous mutant of a small IncP plasmid was isolated that functioned efficiently in M. extorquens AM1. This plasmid was sequenced and used as a base for developing improved broad-host-range cloning vectors. These vectors were found to replicate in a wide variety of bacterial species and have the following advantages: (1) high copy number in Escherichia coli; (2) small size (7.2 and 8.0 kb); (3) complete sequences; (4) variety of unique restriction sites; (5) blue-white screening via lacZalpha; (6) conjugative mobilization between bacterial species; and (7) readily adaptable into species-specific promoter-probe and expression vectors. Two low-background promoter-probe vectors were constructed based on these cloning vectors with either lacZ or xylE as reporter genes; these were shown to report gene expression effectively in M. extorquens AM1. Specific expression vectors were developed for use in M. extorquens AM1, which were shown to express foreign genes at significant levels, and a simple strategy is outlined to develop specific expression vectors for other bacteria. The strong mxaF promoter was used for expression, since E. coli lac-derived promoters were expressed at very low levels. This suite of genetic tools will enable a more sophisticated analysis of the physiology of M. extorquens AM1, and these vectors should also be valuable tools in the study of a variety of bacterial species.

Alcohol Oxidoreductases↗
PubMed2006

Infection of human cancer cells with myxoma virus requires Akt activation via interaction with a viral ankyrin-repeat host range factor.

We demonstrate that the susceptibility of human cancer cells to be infected and killed by an oncolytic poxvirus, myxoma virus (MV), is related to the basal level of endogenous phosphorylated Akt. We further demonstrate that nonpermissive tumor cells will switch from resistant to susceptible for MV infection after expression of ectopically active Akt (Myr-Akt) and that permissive cancer cells can be rendered nonpermissive by blocking Akt activation with a dominant-negative inhibitor of Akt. Finally, the activation of Akt by MV involves the formation of a complex between the viral host range ankyrin-repeat protein, M-T5, and Akt. We conclude that the Akt pathway is a key restriction determinant for permissiveness of human cancer cells by MV.

Animals↗
PubMed2000

[Receptor sialylsugar chains as determinants of host range of influenza viruses].

All types of the hemagglutinin(HA) of human, pig, horse and aq. bird influenza A viruses, recognize sialyl lacto-series type I and II sugar chains(Sialic acid(SA) alpha 2-3(6)Gal beta 1-3(4) GlcNAc beta 1-) in glycoproteins and glycolipids in the target cells as common receptor molecules. Avian and equine influenza viruses preferentially binds the terminal sialic acid alpha 2-3Gal(SA2-3Gal) linkage, while human influenza viruses preferentially bind the SA2-6Gal linkage. SA distribution in animal species influence influenza virus host range. Swine trachea has both receptors for avian influenza viruses (SA2-3Gal specific) and for human influenza viruses(SA2-6Gal specific). In the case of horses, a virus with an HA recognizing Neu5Ac2-6Gal, but not Neu5Ac2-3Gal, failed to replicate in horses, while one with an HA recognizing the Neu5Gc2-3Gal moiety replicated in horses. The abundance of the Neu5Gc2-3Gal moiety in epithelial cells of horse trachea supports that recognition of Neu5Gc2-3Gal moiety is critical for viral replication in horses. The Neu5Gc2-3Gal is also associated with viral replication in duck intestine, primarily in the crypt epitherial cells. Such recognition, together with biochemical evidence of Neu5Gc in crypt cells, correlated exactly with the ability of the virus to replicate in duck colon. These results indicate the evidence of biologic effect of different sialic acid species in different animals.

Animals↗
PubMed1987

Unique carcinogen enhancement of transformation phenotype displayed by cloned rat embryo fibroblast (CREF) cells treated with methyl methanesulfonate and infected with a specific host-range mutant of type 5 adenovirus.

Pretreatment of cloned rat embryo fibroblast (CREF) cells with methyl methanesulfonate (MMS) prior to infection with wild-type 5 adenovirus (H5wt) or a temperature-sensitive mutant of Ad5 (H5ts125) results in an MMS dose-dependent enhancement of viral transformation. With both viral isolates, MMS enhanced the transformation frequency when normalized for cell toxicity but did not induce a carcinogen dose-dependent increase in the absolute number of foci above solvent-treated controls. In contrast, pretreatment of CREF cells with MMS prior to infection with a host-range mutant of Ad5 (H5hr1) which contains a single basepair deletion in the E1a-transforming region of Ad5 and displays a cold-sensitive transformation phenotype, results in an MMS dose-dependent increase in the absolute number of transformed foci in comparison with solvent-treated controls as well as an increase in transformation frequency when normalized for cell toxicity. To explore the possible mechanism by which H5hr1 displays its unique carcinogen-enhancement of transformation (CET) phenotype we have examined the effect of MMS pretreatment on the frequency of transformation of CREF cells infected with mutants of Ad5 which were engineered to contain either a deletion (H5dl101) or an insertion (H5in106) mutation in the E1a gene region resulting in a cold-sensitive transformation phenotype similar to H5hr1. MMS-pretreated CREF cells infected with H5dl101 or H5in106 did not demonstrate a dose-dependent increase in the absolute number of transformed foci as was observed with carcinogen-pretreated H5hr1-infected CREF cells. These findings suggest that the unique CET phenotype displayed by H5hr1 may result from a second site mutation in a region of H5hr1 other than the E1a-transforming region and/or a novel interaction between gene products resulting from the specific mutation in E1a and other region(s) of the H5hr1 genome. Our investigations also indicate that the CREF/H5hr1 system should prove useful in analyzing chemical-viral interactions in cell transformation.

Adenoviridae↗
PubMed1989

The host range of the pKD1-derived plasmids in yeast.

pKD1 is a 2 mu-like circular plasmid found in the yeast Kluyveromyces drosophilarum that can also stably replicate in Kluyveromyces lactis. We have found a short intergenic region in this genome that appears to be functionally neutral; that is, the introduction of foreign sequences into the single EcoRI restriction site located near one of the inverted repeats did not affect the high stability of the natural plasmid. By introducing a G418 resistance gene at this site, we constructed an autonomous recombinant plasmid. Since this vector did not require cir+ hosts for its stable maintenance, it could be used to examine the transformation host range of pKD1 among all the species belonging to the genus Kluyveromyces. Both species closely related to K. drosophilarum as well as a few other species that are very different in chromosomal GC% could be transformed to yield highly stable transformant clones.

DNA Transposable Elements↗
PubMed2000

Transient host range selection for genetic engineering of modified vaccinia virus Ankara.

Recombinant vaccinia viruses are extremely valuable tools for research in molecular biology and immunology. The extension of vaccinia vector technology to replication-deficient and safety-tested virus strains such as modified vaccinia virus Ankara (MVA) have made this versatile eukaryotic expression system even more attractive for basic and clinical research. Here, we report on easily obtaining recombinant MVA using stringent growth selection on rabbit kidney RK-13 cells. We describe the construction and use of new MVA vector plasmids that carry an expression cassette of the vaccinia virus host range gene, K1L, as a transient selectable marker. These plasmids allow either stable insertion of additional recombinant genes into the MVA genome or precisely targeted mutagenesis of MVA genomic sequences. Repetitive DNA sequences flanking the K1L gene were designed to remove the marker gene from the viral genome by homologous recombination under nonselective growth conditions. The convenience of this new selection technique is demonstrated by isolating MVA recombinants that produce green fluorescent protein and by generating MVA deletion mutants.

Animals↗
PubMed1989

Broad-host-range vectors for delivery of TnphoA: use in genetic analysis of secreted virulence determinants of Vibrio cholerae.

Gene fusions between the cholera toxin structural genes and phoA, which encodes bacterial alkaline phosphatase, were identified after TnphoA mutagenesis of the cloned genes in Escherichia coli and were then mobilized into Vibrio cholerae. The activities of the hybrid proteins were detectable in V. cholerae and suggested that, like cholera toxin, they were secreted beyond the cytoplasm. To extend the utility of TnphoA to identify additional genetic export signals in V. cholerae and other gram-negative bacteria, TnphoA delivery vectors utilizing broad-host-range plasmids were developed. By using V. cholerae as a model system, insertion mutants carrying active phoA gene fusions were identified as colonies expressing alkaline phosphatase, which appeared blue on agar containing the indicator 5-bromo-4-chloro-3-indolyl phosphate. Since alkaline phosphatase is active only upon export from the cytoplasm, PhoA+ colonies resulting from the mutagenesis procedure were enriched for insertions in genes that encode secreted proteins. Insertion mutations were identified in the gene encoding a major outer membrane protein, OmpV, and in tcpA, which encodes a pilus (fimbrial) subunit. Mutant strains harboring chromosomal insertions isolated in this manner can be used to assess the role of the corresponding inactivated gene products on survival of V. cholerae in vivo. The expression of the hybrid proteins as determined by measuring alkaline phosphatase activity also allowed the convenient study of virulence gene expression.

Alkaline Phosphatase↗
PubMed1988

Isolation and characterization of herpes simplex virus type 1 host range mutants defective in viral DNA synthesis.

Cell lines were generated by cotransfection of Vero cells with pSV2neo and a plasmid containing the herpes simplex virus type 1 (HSV-1) EcoRI D fragment (coordinates 0.086 to 0.194). One such cell line (S22) contained the genes for alkaline exonuclease and several uncharacterized functions. Three mutant isolates of HSV-1 strain KOS which grew on S22 cells but not on normal Vero cells were isolated and characterized. All three mutants (hr27, hr48, and hr156) were defective in the synthesis of viral DNA and late proteins when grown in nonpermissive Vero cells. Early gene expression in cells infected with these host range mutants appeared to be normal at the nonpermissive condition. The mutations were mapped by marker rescue to a 1.5-kilobase fragment (coordinates 0.145 to 0.155). The mutation of one of these mutants, hr27, was more finely mapped to an 800-base-pair region (coordinates 0.145 to 0.151). This position of these mutations is consistent with the map location of a putative 94-kilodalton polypeptide as determined by sequence analysis (D. McGeoch, personal communication). Complementation studies demonstrated that these mutants formed a new complementation group, designated 1-36. The results presented in this report indicate that the 94-kilodalton gene product affected by these mutations may have a direct role in viral DNA synthesis.

Animals↗
PubMed2001

A variable region on the chlorovirus CVK2 genome contains genes possibly involved in the host range determination.

A 22.6-kbp variable region near the left end of the chlorovirus CVK2 genome which was characterized. This region contained a tandem array of 5 gene copies for Vp260-like protein, a viral surface glycoprotein. The authentic 104-kDa Vp260 was encoded at another site on the genome and contained 13 internally located, tandem repeats of 61-65 amino acids like the prominent Rickettsia surface antigen. By Northern and Western blot analyses, these genes were demonstrated to be expressed late in infection and the proteins were incorporated into virions. These results implied that the extra copies of Vp260-like proteins may be involved in host range in the natural environment.

Gene Dosage↗
PubMed1994

Determinants of receptor specificity of coliphages of the T4 family. A chaperone alters the host range.

E. coli phages of the T4 family (T4, TuIa, TuIb) recognize their cellular receptors with a C-terminal region of protein 37. This protein, common to all three phages, is present as a dimer located at the distal part of the long tail fibers and possesses a C-terminal domain consisting of 40 to 70 highly conserved C-terminal residues, followed by a variable region of 50 to 80 residues which is again followed by a highly conserved area. Protein 38, not being a component of the mature virion, is required for dimerization of protein 37; this represents a non-covalent association of a structural protein. Seven host range mutants of TuIa or TuIb were analyzed which were able to use proteinaceous receptors other than those recognized by their parents. All had suffered amino acid substitutions within the variable region. It is concluded that in all probability it is this region which interacts directly with the cellular receptors. Conditional mutants of T4 are known which, when propagated at the non-permissive temperature (42 degrees C), yield phage of normal morphology but these are more or less unable to adsorb to cells. The causative amino acid substitutions were found both downstream and upstream from the variable area. Distortion of it in the mutants could suggest a "snap-back" conformation of the tail fiber; the conserved C-terminal region may fold back and expose the variable region as a loop at the tip of the fiber. One of the phage mutants (L93), when grown at the permissive temperature, had lost the ability to use the OmpC porin (a receptor for T4) as a receptor. A secondary mutant, able to do so, was isolated. An additional mutation, leading to one amino acid substitution, had occurred in gene 38. This mutant gene acted in trans and caused a much enhanced temperature-sensitivity of infectivity without conferring temperature-sensitivity per se, i.e. the mutant protein 38 apparently altered the conformation of the receptor-recognizing area of the dimer of protein 37. A gene from phage lambda, about 40% identical to gene 38 of T4, complements gene 38 amber mutants. The corresponding protein also restored the ability of L93 to recognize OmpC but did not cause any such temperature-sensitivity. Hence, protein 38, classifying as a chaperone, appears to act instructively in conveying steric information to the target polypeptide.

Adsorption↗
PubMed1997

From the natural evolution to the genetic manipulation of the host-range of retroviruses.

The discovery that retroviruses share many features with retrotransposons led to the proposal that retroviruses evolved from cellular movable elements and became independent genetic entities, which started a parasitic life on their own. It has been further hypothesized that in the course of this evolutionary process, retrotransposons incorporated a cellular membrane glycoprotein gene into their genome, which finally became the envelope gene of the virus. Equipped with a error-prone polymerase and a high recombination rate during replication, the resulting genetic element has been equipped with a powerful machinery to change and modify itself, in particular the envelope gene, not only to escape the immune system, but also to change the host species. The HIV-1 epidemiology may serve as the best example of this still on-going evolutionary process. Since retroviruses insert their genome into that of the host cell as part of their life cycle, they have been extensively utilized as gene transfer vectors (retroviral vectors). Recently, experiments have been initiated to modify the envelope of retroviral vectors in the laboratory to alter the host range for specific gene transfer applications. This review highlights aspects of the natural evolution and the genetic modification of the retroviral envelope protein. Its implication on the safety of retroviral vectors in human gene therapy is also discussed.

Animals↗
PubMed2003

Lysogeny and bacteriophage host range within the Burkholderia cepacia complex.

The Burkholderia cepacia complex comprises a group of nine closely related species that have emerged as life-threatening pulmonary pathogens in immunocompromised patients, particularly individuals with cystic fibrosis or chronic granulomatous disease. Attempts to explain the genomic plasticity, adaptability and virulence of the complex have paid little attention to bacteriophages, particularly the potential contribution of lysogenic conversion and transduction. In this study, lysogeny was observed in 10 of 20 representative strains of the B. cepacia complex. Three temperate phages and five lytic phages isolated from soils, river sediments or the plant rhizosphere were chosen for further study. Six phages exhibited T-even morphology and two were lambda-like. The host range of individual phages, when tested against 66 strains of the B. cepacia complex and a representative panel of other pseudomonads, was not species-specific within the B. cepacia complex and, in some phages, included Burkholderia gladioli and Pseudomonas aeruginosa. These new data indicate a potential role for phages of the B. cepacia complex in the evolution of these soil bacteria as pathogens of plants, humans and animals, and as novel therapeutic agents.

Bacteriophages↗
PubMed2002

Paired charge-to-alanine mutagenesis of dengue virus type 4 NS5 generates mutants with temperature-sensitive, host range, and mouse attenuation phenotypes.

Charge-to-alanine mutagenesis of dengue virus type 4 (DEN4) NS5 gene generated a collection of attenuating mutations for potential use in a recombinant live attenuated DEN vaccine. Codons for 80 contiguous pairs of charged amino acids in NS5 were individually mutagenized to create uncharged pairs of alanine residues, and 32 recombinant mutant viruses were recovered from the 80 full-length mutant DEN4 cDNA constructs. These mutant viruses were tested for temperature-sensitive (ts) replication in both Vero cells and HuH-7 human hepatoma cells. Of the 32 mutants, 13 were temperature sensitive (ts) in both cell lines, 11 were not ts in either cell line, and 8 exhibited a host range (tshr) phenotype. One tshr mutant was ts only in Vero cells, and seven were ts only in HuH-7 cells. Nineteen of the 32 mutants were 10-fold or more restricted in replication in the brains of suckling mice compared to that of wild-type DEN4, and three mutants were approximately 10,000-fold restricted in replication. The level of temperature sensitivity of replication in vitro did not correlate with attenuation in vivo. A virus bearing two pairs of charge-to-alanine mutations was constructed and demonstrated increased temperature sensitivity and attenuation relative to either parent virus. This large set of charge-to-alanine mutations specifying a wide range of attenuation for mouse brain should prove useful in fine-tuning recombinant live attenuated DEN vaccines.

Alanine↗
PubMed1983

Definition of the simian virus 40 early promoter region and demonstration of a host range bias in the enhancement effect of the simian virus 40 72-base-pair repeat.

The simian virus 40 (SV40) origin region includes the viral replication origin and the early and late promoters and consists of a few palindromes, a 17-base-pair (bp) A + T-rich sequence, three copies of a G + C-rich 21-bp repeat, and two copies of a 72-bp repeat. We have made sequential deletions in the SV40 origin region and determined the early promoter efficiencies of these truncated DNA segments by connecting them in the correct orientation with the coding regions of selectable marker genes and assaying the expression of the chimeric marker genes in vivo in different host cell lines. A truncated SV40 early promoter segment containing only the TATA box and the major in vivo mRNA initiation sites has essentially no promoter efficiency. We have located the major component of the SV40 early promoter within the 21-bp repeated sequences, which consist of an alternating and mutually overlapping array of two C-rich oligonucleotides having the consensus sequences Y-Y-C-C-G-C-C-C (Y = pyrimidine nucleoside) and G-C-C-C-(C)-TA-AT-A(T)-C-T. Between one and two copies of the 21-bp repeat were adequate for gene expression under conditions in which the enhancement effect of the 72-bp repeat was minimal. We also find that the SV40 72-bp repeat exhibits a pronounced host range in its enhancement of gene expression; the enhancement is only 2-fold in the nonpermissive mouse cells but amounts to 10- or 20-fold in the permissive monkey cells or the semipermissive human cells, respectively.

Animals↗
PubMed1982

Morphological varieties and host ranges of Vibrio parahaemolyticus bacteriophages isolated from seawater.

Eighteen bacteriophages lytic for Vibrio parahaemolyticus were isolated from seawater, and their ultrastructure was examined by electron microscopy. Based on the phage morphology, they were classified in four groups. Group I phages consisted of a hexagonal head and a tail with a contractile sheath. All the phages of the other three groups had a relatively long, noncontractile tail, but there were differences in the head structure among these phages. The phages of groups II and III had a hexagonal head and an elongated polyhedral head, respectively. Group IV phages exhibited a unique hexagonal head with knoblike projections. There appeared to be no correlation between the O and K serotypes of V. parahaemolyticus strains and the host ranges of the phages. The phages had varying sensitivities to heat and organic solvents.

Bacteriophages↗
PubMed1983

Reversal of Fv-1 host range by in vitro restriction endonuclease fragment exchange between molecular clones of N-tropic and B-tropic murine leukemia virus genomes.

We molecularly cloned unintegrated viral DNA of the BALB/c endogenous N-tropic and B-tropic murine leukemia retroviruses and in vitro passaged N-tropic Gross (passage A) murine leukemia retroviruses. Recombinant genomes were constructed in vitro by exchanging homologous restriction enzyme fragments from N- or B-tropic parents and subsequent recloning. Infectious virus was recovered after transfection of these recombinant genomes into NIH-3T3 cells and cocultivation with the Fv-1 nonrestrictive SC-1 cells. XC plaque assays of recombinant virus progeny on Fv-ln and Fv-lb cells indicated that the Fv-l host range was determined by sequences located between the BamHI site in the p30 region of the gag gene (1.6 kilobase pairs from the left end of the map) and the HindIII site located in the pol gene (2.9 kilobase pairs from the left end of the map).

AKR murine leukemia virus↗
PubMed1989

Antibodies directed against a synthetic peptide enable detection of a protein encoded by a vaccinia virus host range gene that is conserved within the Orthopoxvirus genus.

A vaccinia virus gene required for multiplication in some cell lines but not in others has been previously isolated and sequenced. A synthetic peptide predicted from the nucleotide sequence and corresponding to the carboxy-terminal 18 amino acids was used to raise antibodies in rabbits. The immune serum enabled detection of a 29-kilodalton (kDa) polypeptide by either immunoprecipitation or Western immunoblot assays. Synthesis of the 29-kDa polypeptide occurred immediately after infection and lasted for about 3 h. Shutoff of its synthesis was concomitant with the appearance of a delayed early polypeptide that may be antigenically related to the 29-kDa polypeptide. Analysis of cloned segments of the genomes of other orthopoxviruses by hybridization with the vaccinia virus host range gene demonstrates that it is well conserved within this genus.

Genes, Viral↗