Insertion Of Foreign DNA Research Articles

Role of the TK+ phenotype in the stability of pigeonpox virus recombinant.

Insertion of foreign DNA containing the E. coli gpt marker by homologous recombination in the pigeonpox virus (PPV) thymidine kinase (TK) gene and selection for the presence of this DNA in the viral genome produced unstable recombinants after 3 plaque purifications. We highlight the persistence of duplicated TK DNA sequences arising from single crossing over, due to the growth advantage of TK+ virus. Restoration of the TK function by coinsertion of the vaccinia virus TK gene led to stable TK+ recombinants arising from double crossing over.

Deletion between direct repeats in bacteriophage T7 gene 1.2

Deletion mutagenesis in bacteriophage T7 was studied with an insertion-reversion assay involving phage containing inserts of foreign DNA that form 10-bp direct repeats. The precise deletion of the insert restores the function of the non-essential gene and is easily assayed by growth on selection strains of E. coli. Similar inserts with unique direct repeats were placed in either gene 1.2 (dGTPase Inhibitor) or gene 1.3 (DNA ligase). The deletion rates of the inserts were quantified with Luria and Delbrück fluctuation tests. Deletion rates were similar for inserts in both genes indicating that the rates of deletion were not unique to either specific site, or the sequence of the direct repeats. Deletion was independent of functional T7 ligase at 37 degrees C, while an increase in the rate of deletion was noted in some ligase-deficient phage at 43 degrees C. The effect of E. coli DNA Polymerase I on deletion rate was tested and found to decrease deletion rate 60% with the polA1 mutation and 90% with the polA546ex mutation.

Insertional inactivation of a fowlpox virus homologue of the vaccinia virus F12L gene inhibits the release of enveloped virions.

Insertion of the Escherichia coli lacZ gene into a ClaI restriction enzyme site of a 5.7 kb HindIII fragment of the fowlpox virus (FPV) genome resulted in the generation of stable recombinants. These recombinants produced plaques that were significantly smaller than those produced by parental FPV or by FPV recombinants containing the lacZ gene at other non-essential sites. Insertion of foreign DNA into the ClaI site disrupts a previously unidentified open reading frame (ORF) which potentially encodes a 74K polypeptide. The predicted amino acid sequence of this FPV ORF has 24% identity with the F12L ORF of vaccinia virus, the function of which is not currently known. Production of intracellular FPV was similar in cells infected with recombinant or parental viruses, but the number of infectious extracellular virions released into the medium by the recombinant was about 20% of that released by the parental virus. Likewise, the release of FPV particles, which were labelled in vivo with [3H]thymidine, was significantly lower in recombinant FPV-infected cells. These results suggest that the FPV homologue of the vaccinia virus F12L ORF is involved in the envelopment or release of infectious extracellular virions.

39] Generation and analysis of vaccinia virus recombinants

This chapter presents methods for the generation and analysis of recombinant vaccinia viruses. Vaccinia virus is an extremely versatile virus expression system that can be easily manipulated in the laboratory. The widespread use of vaccinia as a vector system is because of the ease with which recombinant vaccinia viruses containing large insertions of foreign DNA can be generated and because of the authenticity of expression of foreign polypeptides during viral replication. Recombinant vaccinia viruses expressing a wide variety of heterologous genes are used for a number of applications, including (1) analyzing gene regulation, (2) producing biologically active molecules in tissue culture, (3) immunizing animals for antibody production, (4) creating live recombinant vaccines, and (5) elucidating the role of specific antigens in inducing defined immune responses. As additional viral vector improvements are made, the potential of vaccinia as a tool for biological research, as a protein production system and as an immunoprophylactic continues to increase.

Construction of chimeric vaccinia viruses by molecular cloning and packaging.

Foreign DNA was inserted into unique restriction endonuclease cleavage sites (Sma I or Not I) of the 200,000-base-pair vaccinia virus genome by direct molecular cloning. The modified vaccinia virus DNA was packaged in fowlpox virus-infected avian cells, and chimeric vaccinia virus was isolated from mammalian cells not supporting the growth of the fowlpox helper virus. In contrast to the classical "in vivo" recombination technique, chimeric viruses with inserts in both possible orientations and families of chimeras with multiple inserts were obtained. The different genomic configurations of chimeric viruses provide a broader basis for screening of optimal viruses. In addition to packaging in avian cells, a second packaging procedure for vaccinia DNA, based on the abortive infection of mammalian cells with the fowlpox helper virus, was developed. This procedure permits simultaneous packaging and host-range selection for the packaged virus. The cloning/packaging procedure allows the direct insertion of foreign DNA without the need for plasmids having flanking regions homologous to viral nonessential regions and is independent of inefficient in vivo recombination events. By direct cloning and packaging, about 5-10% of the total vaccinia virus yield consisted of chimeras. The procedure is, therefore, a useful tool in molecular virology.

Trans regulation in the Ultrabithorax gene of Drosophila: alterations in the promoter enhance transvection.

We report a genetic and molecular study of UbxMX6 and Ubx195rx1, two mutations in the Ultrabithorax (Ubx) locus which appear to have a strong effect on the activity of the homologous Ubx gene. These mutations show the characteristic embryonic and adult phenotypes of Ubx null alleles, and also fail to produce any detectable Ubx product. Yet, genetic and phenotypic analyses involving a large number of trans heterozygous combinations of UbxMX6 and Ubx195rx1 with different classes of Ubx mutations, indicate that they hyperactivate the homologous gene. This effect is induced on wildtype or mutant forms of Ubx, provided that the pairing in the bithorax region is normal, i.e. these mutations have a strong positive effect on transvection. We also show that, unlike all the other known cases of transvection in Ubx, this is not zeste-dependent. Southern analyses indicate that UbxMX6 is a 3.4 kb deletion, and Ubx195rx1 is an approximately 11 kb insertion of foreign DNA, both in the promoter region. We speculate that the region altered in the mutations may have a wildtype function to ensure cis-autonomy of the regulation of Ubx transcription.

Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

The insertion of foreign DNA in plants occurs through a complex interaction between Agrobacteria and host plant cells. The marker gene β-glucuronidase of Escherichia coli and cytological methods were used to characterize competent cells for Agrobacterium-mediated transformation, to study early cellular events of transformation, and to identify the potential host-cell barriers that limit transformation in Arabidopsis thaliana L. Heynh. In cotyledon and leaf explants, competent cells were mesophyll cells that were dedifferentiating, a process induced by wounding and-or phytohormones. The cells were located either at the cut surface or within the explant after phytohormone pretreatment. In root explants, competent cells were present in dedifferentiating pericycle, and were produced only after phytohormone pretreatment. Irrespective of their origin, the competent cells were small, isodiametric with thin primary cell walls, small and multiple vacuoles, prominent nuclei and dense cytoplasm. In both cotyledon and root explants, histological enumeration and β-glucuronidase assays showed that the number of putatively competent cells was increased by preculture treatment, indicating that cell activation and cell division following wounding were insufficient for transformation without phytohormone treatment. Exposure of explants for 48 h to A. tumefaciens produced no characteristic stress response nor any gradual loss of viability nor cell death. However, in the competent cell, association between the polysaccharide of the host cell wall and that of the bacterial filament was frequently observed, indicating that transformation required polysaccharide-to-polysaccharide contact. Flow cytofluorometry and histological analysis showed that abundant transformation required not only cell activation (an early state exhibiting an increase in nuclear protein) but also cell proliferation (which in cotyledon tissue occurred at many ploidy levels). Noncompetent cells could be made competent with the appropriate phytohormone treatments before bacterial infection: this should aid analysis of critical steps in transformation procedures and should facilitate developing new strategies to transform recalcitrant plants.

A new shuttle vector forBacillus stearothermophilus andEscherichia coli

A new shuttle vector was constructed by inserting a 3.1 kbp-DNA fragment from thermophilicBacillus sp. plasmid pIH41 intoEscherichia coli plasmid pUC18. The resultant hybrid replicates in bothE. coli andB. stearothermophilus. This vector has ten unique restriction sites within a part oflacZ gene. Insertion of foreign DNA into these sites can be readily detected by a coloration method.

Vaccinia virus gene SalF5R is non-essential for virus replication in vitro and in vivo

A gene designated SalF5R from within the SalI F fragment of vaccinia virus strain WR has been characterized. The predicted primary translation product has 194 amino acids with an Mr of 22578 and has features typical of a class I membrane glycoprotein. At the amino and carboxy termini there are runs of hydrophobic residues that might function as membrane signal and anchor sequences, respectively, and after the C-terminal hydrophobic sequence there is a short charged sequence that may prevent passage of the molecule through the membrane. Between these hydrophobic regions there are two potential sites for addition of N-linked carbohydrate. Northern blotting using a probe to an internal region of the open reading frame (ORF) detected an early transcript of 750 nucleotides and late transcripts of heterogeneous length. However, accurate mapping of the 5' ends of these transcripts did not reveal a monocistronic mRNA that might allow translation of the full-length SalF5R ORF. Instead two early transcripts were found, one beginning 130 nucleotides downstream of the start of the ORF, and a second initiating in the short intergenic region between SalF5R and SalF6R. The nearest late initiation site mapped to the beginning of the upstream gene SalF4R (profilin) and not the beginning of the SalF5R ORF. A virus deletion mutant lacking an internal 554 nucleotides of the 582 bp SalF5R ORF was constructed by insertion of the Escherichia coli guanine phosphoribosyl transferase gene linked to the vaccinia virus p7.5K promoter. The rate of replication and the final titre of intracellular naked virus produced by this virus was indistinguishable from wild-type virus in CV-1 and RK13 cells. In intranasally infected mice the virus lacking SalF5R was not attenuated in comparison to wild-type virus. The SalF5R site may be useful for insertion of foreign DNA into recombinant vaccinia viruses.

Differences in the E3 regions of the canine adenovirus type 1 and type 2

Sequence analysis of a region extending between the pVIII and the fiber gene of Canine adenovirus type 1 (Cav-1, Utrecht) and type 2 (Cav-2, Manhattan) was performed. The results revealed a high level of identity between the two viruses when the pVIII gene and the N-terminal part of the fiber gene were compared. The open reading frames of region E3 in of Cav-1 and Cav-2 encoded a 22 kDa and 40.7 kDa polypeptide, respectively. The Cav-1 and Cav-2 E3 region polypeotides shared conserved amino and carboxyl domains. In Cav-2 an extra sequence of about 500 nucleotides was present, appearing like an in frame insertion of foreign DNA. It can be speculated that this insertion in the E3 region contributes to the observed biological differences between Cav-1 and Cav-2.

Adenovirus as an expression vector in muscle cells in vivo.

Attempting gene transfer in muscle raises difficult problems: the nuclei of mature muscle fibers do not undergo division, thus excluding strategies involving replicative integration of exogenous DNA. As adenovirus has been reported to be an efficient vector for the transfer of an enzyme encoding gene in mice, we decided to explore its potential for muscle cells. Advantages of adenovirus vectors are their independence of host cell replication, broad host range, and potential capacity for large foreign DNA inserts. We constructed a recombinant adenovirus containing the beta-galactosidase reporter gene under the control of muscle-specific regulatory sequences. This recombinant virus was able to direct expression of the beta-galactosidase in myotubes in vitro. We report its in vivo expression in mouse muscles up to 75 days after infection. The efficiency and stability of expression we obtained compare very favorably with other strategies proposed for gene or myoblast transfer in muscle in vivo.

Characterization of a cis element required for packaging and replication of the human hepatitis B virus

The hepatitis B virus (HBV) is a DNA virus; however, it replicates through a pregenomic RNA intermediate. Several HBV-specific mRNAs are transcribed, but only the pregenomic RNA transcript is encapsidated. The encapsidation of the HBV genome, therefore, is a highly specific and selective process. Using mutational analyses and complementation tests, we have defined a 99-nucleotide cis element located at the pre-C/C region of the HBV genome which is essential for encapsidation and the replication. Furthermore, HBV genome truncated down to 1.5 kb and HBV mutants carrying foreign DNA inserts could still replicate in our complementation system.

Insertion of a disulfide-containing neurotoxin into E. coli alkaline phosphatase: the hybrid retains both biological activities.

We have inserted a disulfide-containing snake neurotoxin into the N-terminal end of Escherichia coli alkaline phosphatase, between residues +6 and +7 of the mature enzyme. For this purpose, we have designed a cloning and expression vector which allows insertion of foreign DNA between the corresponding codons, and visual selection of the desired recombinant clones upon recovery of phosphatase activity. The hybrid protein is exported to the bacterial periplasm, the alkaline phosphatase signal peptide is correctly processed, and both domains are functionally conformed. The phosphatase domain displays catalytic activity, and the inserted toxin is able to bind to its biological target, the nicotinic acetylcholine receptor. The hybrid molecule is remarkably stable and resistant to proteolysis. Crude periplasmic extract containing the hybrid can be used as a tracer-containing reagent in competitive enzymo-immuno and enzymo-receptor assays. We propose to use the system described in this paper for fast preparation of properly folded disulfide-containing enzymatic probes.

Second-generation approach to the construction of yeast artificial-chromosome libraries

We describe an improved method for construction of yeast artificial-chromosome (YAC) libraries that contain large inserts of foreign DNA. The procedure consists of seven steps: (i) preparation of human DNA in agarose beads; (ii) partial digestion of the DNA with EcoRI; (iii) electrophoretic elimination of the smaller partial-digest fragments; (iv) ligation of the EcoRI fragments with vector arms in molten agarose; (v) hydrolysis of agarose with agarase; (vi) fractionation of the recombinant molecules by sucrose-gradient centrifugation; and (vii) transformation of yeast. More than 7000 colonies were obtained starting with 15 μg of human DNA, which was fractionated on a single sucrose gradient. The average size of these YACs was approximately 380 kb. It is estimated that the total length of human DNA present in the clones corresponds to 80% of the length of the human haploid genome. The results of screening the clones for a number of single-copy genes indicate that the clones reflect a nearly random sampling of the human genome. The efficiency of the cloning is sufficient to support the construction of multihit libraries for the human genome or for the genomes of other higher organisms.

Development of a cloning system in Mycoplasma pulmonis

A system suitable for recombinant DNA manipulations in mycoplasmas was developed using the cloned antibiotic-resistance genes of Tn 4001 and Tn 916. An integrative plasmid containing one of the resistance markers was inserted into the genome of Mycoplasma pulmonis to form a recipient strain. This was accomplished by transformation and homologous recombination between chromosomal DNA sequences cloned onto the integrative plasmid. A second vector, the cloning vector, containing the same plasmid replicon and alternate resistance marker, carried cloned foreign DNA. When transformed into mycoplasmal recipients, homologous recombination between plasmid sequences resulted in integration of the cloning vector and foreign DNA. A Brucella abortus gene coding for a 31-kDa protein and the P1 structural gene and operon from Mycoplasma pneumoniae were introduced to examine the feasibility of developing mycoplasm as cloning hosts. Recombinant plasmids as large as 20 kb were inserted into M. pulmonis, and the integrated foreign DNA was stably maintained. The maximum size of clonable DNA was not determined, but plasmids larger than 22 kb have not been transformed into mycoplasmas using polyethylene glycol. Also, the size of the genome (800–1200 kb) may affect the stability of larger inserts of foreign DNA. This system is applicable to any mycoplasma capable of transformation, homologous recombination and expression of these resistance markers. Because of their lack of a cell wall, mycoplasmas may be useful cloning hosts for membrane or excreted protein genes from other sources.

Agrobacterium‐mediated gene delivery and the biology of hostrange limitations

Insertion of foreign DNA into Ti plasmid‐derived vectors in Agrobacterium tumefaciens is currently the most frequently used strategy for generating transgenic plants in a wide variety of species. Limitations of the host range of Agrobacterium restrict its usefulness in many cases, particularly when dealing with monocotyledonous plants. The objective of this presentation is to briefly discuss the efficiency of the transformation process utilized by Agrobacterium tumefaciens, potential barriers to efficient transformation by Agrobacterium that result in limitation of its useful host range, and how an understanding of the successful Agrobacterium/plant cell interaction might lead to advances in a variety of DNA delivery methodologies.

The alpha-amylase gene as a marker for gene cloning: direct screening of recombinant clones.

We report the construction and use of a new system for the direct screening of recombinant clones after transformation. The system uses a Bacillus subtilis-Escherichia coli shuttle vector that carries the B. subtilis structural gene for alpha-amylase. Insertion of foreign DNA into this gene results in a loss of amylolytic activity in the host cells that can be assayed using a simple and inexpensive staining procedure.

Construction of fowlpox virus vectors with intergenic insertions: expression of the beta-galactosidase gene and the measles virus fusion gene

A DNA fragment from fowlpox virus cloned on a plasmid vector was modified to contain foreign DNA inserts within an intergenic region. In a first step, a 32-base-pair intergenic region from the fowlpox virus genome corresponding to the position of the thymidine kinase locus in the vaccinia virus genome was enlarged to 55 base pairs by site-directed mutagenesis. A unique restriction endonuclease site introduced upstream of the intergenic region was then used to insert various foreign DNA fragments. The lacZ gene encoding beta-galactosidase and the measles virus gene encoding the fusion protein were positioned downstream of two vaccinia virus p7.5 promoter elements in either a direct repeat or inverted repeat orientation. Foreign DNA inserts contained within the fowlpox virus sequence were transferred to the viral genome by homologous recombination occurring in cells infected with a fowlpox virus temperature-sensitive mutant and transfected with both wild-type viral DNA and plasmid DNA. Recombinant viruses were selected for the expression of beta-galactosidase activity by screening for blue plaques in the presence of a chromogenic substrate. Stable recombinants expressing both the lacZ gene and the unselected measles gene were obtained when the p7.5 promoter was present as an inverted repeat. However, when the p7.5 promoter was in the direct repeat orientation, viral recombinants which initially expressed both gene inserts readily deleted the lacZ gene flanked by the promoter repeat. The methods described enable precise insertion and deletion of foreign genes in the fowlpox virus genome and could be applied to other intergenic regions of the same virus as well as other poxviruses.

Gene replacement and expression of foreign DNA in mycobacteria

A system that permits molecular genetic manipulation of mycobacteria was developed on the basis of the yeast paradigm of gene replacement by homologous recombination. A shuttle vector that can replicate autonomously at a high copy number in Escherichia coli but must integrate into homologous DNA for survival in Mycobacterium smegmatis was constructed. The vector contains a ColE1 origin of replication, antibiotic resistance markers for ampicillin and kanamycin, a nutritional marker (pyrF) that allows both positive and negative selection in E. coli and M. smegmatis, and unique restriction sites that permit insertion of foreign DNA. Transformation of mycobacteria with this vector results in integration of its DNA into the genomic pyrF locus by either a single or a double homologous recombination event. With this system, the 65-kilodalton Mycobacterium leprae stress protein antigen was inserted into the M. smegmatis genome and expressed. This gene replacement technology, together with a uniquely useful pyrF marker, should be valuable for investigating mycobacterial pathobiology, for the development of candidate mycobacterial vaccine vehicles, and as a model for the development of molecular genetic systems in other pathogenic microorganisms.

The family of highly interrelated single-stranded deoxyribonucleic acid plasmids

Many plasmids from gram-positive bacteria replicate via a single-stranded deoxyribonucleic acid (ssDNA) intermediate, most probably by a rolling-circle mechanism (these plasmids are referred to in this paper as ssDNA plasmids). Their plus and minus origins are physically separated, and replicative initiations are not simultaneous; it is this feature that allows visualization of ssDNA replication intermediates. The insertion of foreign DNA into an ssDNA plasmid may provoke a high frequency of deletions, changes of replicative products to high-molecular-weight forms, segregational loss, and decreased plasmid copy numbers. When an ssDNA plasmid is inserted into the chromosome, both deletions and amplifications may be induced. Both the mode of replication and the copy control mechanism affect the fate of inserted foreign material, usually selecting for its loss. Thus, after having tasted various morsels of DNA, the resulting plasmid stays trim. The features of the ssDNA plasmids seem to be beneficial for their viability and propagation, but not for their use as cloning vectors. However, plasmids replicating via ssDNA intermediates are being exploited to yield insights into the mechanisms of recombination and amplification.