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PubMed1991

A system, using neural cell lines, to characterize HSV-1 vectors containing genes which affect neuronal physiology, or neuronal promoters.

Among the potential uses of defective herpes simplex virus (HSV-1) vectors are to study neuronal physiology, neuronal gene regulation, and to perform gene therapy of neuronal diseases. The prototype HSV-1 vector, pHSVlac, stably expresses Escherichia coli beta-galactosidase from the HSV-1 immediate early (IE) 4/5 promoter in cultured rat peripheral and CNS neurons, and in neurons in the adult rat brain. The LacZ gene and the IE 4/5 promoter in pHSVlac can be replaced with genes which affect neuronal physiology or cellular promoters, respectively. A system is required to characterize these HSV-1 vectors; cultured neurons, a mixture of different kinds of neurons and glia, cannot be used. In contrast, neural cell lines represent a homogenous population of neural cells available in virtually unlimited quantities. A system, using neural cell lines, to characterize HSV-1 vectors carrying other genes or promoters is now reported: First, 4 assays are described to detect HSV-1 vector DNA, RNA transcribed from the vector, and to quantitate beta-galactosidase expression. Second, 8 cell lines derived from rodents, primates, and humans were infected with pHSVlac virus and shown to express beta-galactosidase. The cell lines tested included adrenergic and cholinergic mouse neuroblastoma cells, rat pheochromocytoma cells, rodent pituicytes, and human neuroblastoma cells. Infection of these cell lines should prove useful for characterizing HSV-1 vectors with molecular and biochemical assays. Third, differentiated rat pheochromocytoma and mouse neuroblastoma cells, which resemble neurons, were infected with pHSVlac virus and shown to stably express beta-galactosidase. Infection of these cells should be useful for determining the effect of various HSV-1 vectors on neuronal physiology. Thus, HSV-1 vectors containing various genes or promoters can be characterized using the system described in this study.

Animals↗
PubMedDate not supplied

The use of integrating DNA vectors to analyse the molecular defects in ionising radiation-sensitive mutants of mammalian cells including ataxia telangiectasia.

Integrating DNA vectors, encoding selectable recombinant genes, were used to assess rejoining and recombination in wild-type mammalian cells and their ionising radiation-sensitive mutants. To provide a simple model of an important radiation-induced lesion - the DNA double-strand break - the vectors were cut with restriction endonucleases at specific single sites. If these breaks were made in the coding sequence of a selectable gene, the fidelity of the rejoin/recombination process could be measured by survival of vector-transformed cells in selective medium. Rejoining was assessed using vectors without internal homologies, while recombination was measured using pairs of fragments or deletion vectors carrying homologous regions. Initial experiments were made with vectors carrying a single selectable gene but, to overcome potential artefacts, 2-gene vectors were then constructed where one gene acts as a linked marker and (unbroken) control for the other (broken) gene. Available data are reviewed to show that, compared to their respective wild-type counterparts: (1) an ataxia telangiectasia (A-T) cell line and the hamster irs1 mutant show a consistent reduction in the fidelity of rejoining double-strand breaks (while the hamster mutants irs2, irs3, xrs series, and EM9 show wild-type fidelity); (2) the hamster EM9 mutant shows a reduction in ability to recombine homologous vector fragments (while the A-T line and probably the xrs mutants show show wild-type abilities); and (3) the xrs mutants show a reduction in overall transformation frequency with vector DNA, whether broken or not, while the other mutants tested show approximately wild-type frequencies. A critical account of the techniques and data is given, together with speculations on the molecular nature of the processes which are defective in these mutants, leading to radiosensitivity.

Ataxia Telangiectasia↗
PubMed1988

Bunyavirus-vector interactions.

Recent advances in the genetics and molecular biology of bunyaviruses have been applied to understanding bunyavirus-vector interactions. Such approaches have revealed which virus gene and gene products are important in establishing infections in vectors and in transmission of viruses. However, much more information is required to understand the molecular mechanisms of persistent infections of vectors which are lifelong but apparently exert no untoward effect. In fact, it seems remarkable that LAC viral antigen can be detected in almost every cell in an ovarian follicle, yet no untoward effect on fecundity and no teratology is seen. Similarly the lifelong infection of the vector would seem to provide ample opportunity for bunyavirus evolution by genetic drift and, under the appropriate circumstances, by segment reassortment. The potential for bunyavirus evolution by segment reassortment in vectors certainly exists. For example the Group C viruses in a small forest in Brazil seem to constitute a gene pool, with the 6 viruses related alternately by HI/NT and CF reactions, which assay respectively M RNA and S RNA gene products (Casals and Whitman, 1960; Shope and Causey, 1962). Direct evidence for naturally occurring reassortant bunyaviruses has also been obtained. Oligonucleotide fingerprint analyses of field isolates of LAC virus and members of the Patois serogroup of bunyaviruses have demonstrated that reassortment does occur in nature (El Said et al., 1979; Klimas et al., 1981; Ushijima et al., 1981). Determination of the genotypic frequencies of viruses selected by the biological interactions of viruses and vectors after dual infection and segment reassortment is an important issue. Should a virus result that efficiently interacts with alternate vector species, the virus could be expressed in different circumstances with serious epidemiologic consequences. Dual infection of vectors with different viruses is not unlikely, because many bunyaviruses are sympatric in nature. For example, the Ae. trivittatus-cottontail rabbit and the Ae. triseriatus-squirrel arbovirus cycles are sympatric in the ecotone between their respective grassland and forest ecosystems (LeDuc, 1979). Should a LaCrosse virus variant or reassortant evolve that was efficiently vectored by Ae. trivittatus mosquitoes, significantly more human infections with La Crosse virus would likely occur. Unlike Ae. triseriatus, Ae. trivittatus mosquitoes are not restricted to forested areas and consequently are more likely to encounter and to feed upon humans.(ABSTRACT TRUNCATED AT 400 WORDS)

Aedes↗
PubMed1988

A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers.

Several families of positive-selection cloning vectors were constructed, based on the principle of palindrome nonviability first used by Hagan and Warren [Gene 19 (1982) 147-151]. Each vector, derived from either pBR322 or RSF1010 (a broad-host-range plasmid), contains a long inverted repeat (2 x 366 to 2 x 1008 bp) ending in a symmetrical polylinker. Plasmids with long palindromes are not viable in most strains of Escherichia coli and in at least one Gram-positive bacterium. These palindrome-containing vectors therefore transform such strains at a very low frequency unless a DNA fragment is cloned within the polylinker at the center of the palindrome. Transformation by plasmids lacking an insert is reduced by two to four orders of magnitude. Such vectors can be propagated in a palindrome-tolerant strain; however, long symmetrical deletions then occur within the palindrome. To suppress the resulting deletion derivatives, vectors have been constructed so that an extensive deletion would remove the selectable marker. Alternatively, the vectors can be propagated in any strain of E. coli so long as the palindrome is interrupted by a nonpalindromic DNA fragment. We also present several symmetrical polylinkers and drug-resistance cassettes within the vectors. These components can be interchanged to make new positive-selection vectors as needed, and the cassettes are useful in insertional mutagenesis as well. A general method is described to convert virtually any small or medium-sized plasmid into a positive-selection vector.

Base Sequence↗
PubMed1989

Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells.

A cDNA expression vector containing the element oriP and the sequence encoding the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) as well as the hygromycin B-resistance dominant marker gene has been constructed. Its characteristics have been compared to a similar vector lacking the EBV sequences. (a) The EBV+ vector is maintained as an episome with a copy number of approx. 50 per cell, whereas the number of the integrated EBV- copies is in general smaller than 10, when simian virus 40-transformed xeroderma pigmentosum fibroblasts (XP20S-SV) constitute the recipient cell line. (b) The presence of the EBV sequences in the vector resulted in a five- to ten-fold higher transfection efficiency with the Ca.phosphate precipitation technique. (c) cDNA inserts in the EBV+ vector are shown to be efficiently and properly expressed in the recipient cell. (d) If transfection is performed with a mixture of EBV+ vectors with different inserts, transfectants are shown to harbour different plasmids within one cell. (e) The ratio between these plasmids in one cell can be shifted in favour of a vector with a particular insert, when selection for this insert is performed. (f) Reconstruction experiments indicated that isolation of a low-abundance sequence from a mixture of vectors is at least 100-fold more efficient with the EBV+ system, than with the EBV- system. (g) Rescue of the episomal vector from transfected cells can be readily achieved.

Antigens, Viral↗
PubMed1991

A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing.

A new family of vectors including cloning vectors (pEK0; pEC5), an expression vector (pEKEx1), and promoter probe vectors (pEKpllacZ; pEKplCm), has been constructed. All these shuttle vectors are based on the replication origins of the corynebacterial pBL1 and the Escherichia coli ColE1 plasmids, and thus are able to replicate in Corynebacterium glutamicum and E. coli. Plasmids pEK0 and pEC5 carry multiple restriction sites useful for gene cloning and the kanamycin- or chloramphenicol-resistance-encoding gene from Tn903 or from Tn9, respectively. In C. glutamicum, both vectors are compatible with vectors containing the corynebacterial pHM1519 replicon. Based on plasmid pEK0, the expression vector pEKEx1 was developed to allow for isopropyl-beta-D-thiogalactopyranoside-inducible expression of inserted genes in C. glutamicum and E. coli. Also based on pEK0, the promoter probe vectors pEKpllacZ and pEKplCm were constructed to carry the promoterless lacZ or cat reporter genes downstream from useful cloning sites, for assaying the transcriptional activity of cloned fragments.

Cloning, Molecular↗
PubMed1989

Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector.

This study describes a type of retroviral vector called double-copy (DC) vector that was designed to improve the expression of transduced genes. The unique feature of DC vectors is that the transduced gene is inserted within the U3 region of the 3' long terminal repeat (LTR). Consequently, in the infected cell the gene is duplicated and transferred to the 5' LTR. The important result is that in its new position the gene is placed outside the retroviral transcriptional unit, eliminating or at least reducing the negative effects of the retroviral transcriptional unit. The utility of the DC vector design was tested by using a 2.1-kilobase-pair (kbp)-long adenosine deaminase (ADA; EC 3.5.4.4) minigene that was inserted into the 3' LTR of the N2 retroviral vector, generating a 2.7-kbp-long chimeric LTR. DNA blot analysis was used to show that the chimeric LTR was faithfully duplicated in cells infected with the corresponding virus, generating two copies of the ADA minigene, one copy in each LTR. Insertion of the ADA minigene into the 3' LTR of the N2 vector led to a 10- to 20-fold increase in ADA transcripts and human ADA isozyme synthesized in NIH 3T3 cells as compared to cells harboring the same vector in which the ADA minigene was inserted between the two LTRs. A similar increase in ADA expression was observed in two human lymphoid cell lines tested, HUT 78 and Raji. These results are consistent with previous observations that upstream promoters exert an inhibitory effect on promoters placed downstream and bear out the predictions used in the design of DC vectors. The use of DC vectors may contribute to the solution of the problems encountered in expressing retrovirally transduced genes in cultured cells and, in particular, when introduced into the live animal.

Adenosine Deaminase↗
PubMed1992

Transduction of primary human hepatocytes with amphotropic and xenotropic retroviral vectors.

Experiments in animal models suggest that it is feasible to consider hepatic gene therapy using a strategy in which hepatocytes would be isolated by partial hepatectomy, transduced with recombinant retroviral vectors containing genes of therapeutic importance, and then transplanted back into the patient by autologous hepatocellular transplantation. The application of this strategy in clinical trials will require adapting these methods to human cells. We describe the transduction of primary human hepatocytes with two forms of retroviral vectors: amphotropic vectors, which have been used previously in clinical trials, and xenotropic vectors, which have a different host range. Human hepatocytes were harvested from organs preserved in Belzer's solution and were cultivated in a serum-free, tyrosine-free, hormonally defined medium. These cells proliferated for 3-5 days in culture, exhibited characteristic hepatocyte morphology, and expressed liver-specific functions, including phenylalanine hydroxylase, alpha 1-antitrypsin, and glutamine synthase. Transduction with an amphotropic LNL6 retroviral vector resulted in stable incorporation of the provirus into 1% of the cells as estimated by semiquantitative PCR. Consistently higher transduction efficiencies (as much as 10% of the cells) were observed with a xenotropic N2 vector. These data support the feasibility of using LNL6 as a marker gene in clinical trials of hepatocellular transplantation. These data also suggest that the efficiency of transducing hepatocytes with amphotropic vectors in animal models may not accurately reflect the utility of these vectors for human applications. Consideration should be given to the use of xenotropic vectors for optimizing the efficiency of transduction for human applications.

Animals↗
PubMed1989

A simple and versatile method for the preparation of vector-primers by adapter-end-primer ligation.

A group of efficient cDNA cloning strategies employs vector-primers where cDNA synthesis starts from the oligo(dT)-primer tail, which is conventionally attached to cloning vectors by use of terminal deoxynucleotidyl transferase. An alternative, efficient and more versatile method of vector-primer preparation is to directly ligate, by use of T4 DNA ligase, a double-digested vector, e.g., pTZ18R/Pst I/Bam HI, to a synthetic (Bam HI)-adapter-end-primer, 5'-pGATCC-Tn or 5'-pGATCC-site-specific sequence. The use of a utility-vector containing a sizable spacer between the two selected restriction sites enables unambiguous separation on agarose gels of the double-digested vector precursors from single-digested ones, further simplifying the vector preparation. The adapter-end-primer ligation method can be applied to any suitable vectors with multiple cloning sites for the preparation of not only oligo(dT)-tailed, but also site-specific sequence-tailed vectors. Thus, the method enables the cDNA cloning of total poly (A+)-mRNAs, as well as specific RNA or mRNA species with or without poly(A)-tail.

Biotechnology↗
PubMedDate not supplied

[Vectors--derivatives of phage lambda for construction and analysis of genome libraries].

Basic features of lambda phage derived, cosmid and plasmid vectors are described. Plasmid vectors combine the most useful features of phage and plasmid vectors. Plasmids can exist in vivo as a plasmid or as a phage. Plasmid vectors are similar to large capacity phage vectors, but can be maintained in vivo without a stuffer fragment. That is why plasmids are easier in preparation for cloning than phage vectors. The yield of recombinants is higher with plasmid vectors (up to 3.10(6)) and the background of non-recombinants in the library is lower. Analysis of recombinant plasmids is more simple and effective than analysis of recombinant phages and plasmids. Probably plasmid vectors will soon be widely used instead of phage or cosmid vectors for genomic libraries construction and analysis.

Bacteriophage lambda↗
PubMed1991

Effects of vector cutting on its recombination with the chromosomal immunoglobulin gene in hybridoma cells.

We have analyzed the effects of linearizing vector DNA on the frequency and pathway of its recombination with the homologous chromosomal gene. The pSV2neo vector bearing a 4.3-kb fragment encoding the mouse immunoglobulin mu heavy chain constant (C mu) region was cut either at sites within the C mu segment or outside C mu and then transferred to hybridoma cells bearing a mutant mu gene. The frequency of recombinant cells producing normal mu was then measured. For most cut sites, whether in regions of homology or of nonhomology, linearization of the transferred DNA enhanced the recombination frequency between the vector and chromosomal mu genes. When the vector was either uncut or cut at SacI in the region of homology, G418-resistant mu m+ recombinants were found to have integrated the vector by a single reciprocal homologous crossover; the enzyme site (SacI) used for cutting was present in the recombinants. By contrast, when the vector had been linearized at PvuI or SfiI in the region of nonhomology, vector integration involved nonhomologous crossovers, either between transferred DNA molecules or between transferred and chromosomal DNA, and the vector cut sites were absent in these recombinants. Some recombinants were found to have an unaltered as well as recombinant mu gene, suggesting that the nonhomologous recombination process might have involved sister chromatids.

Animals↗
PubMed1992

Improved gene expression by a U3-based retroviral vector.

To improve the expression of the genes transduced by retroviral vectors, we have constructed a U3-based retroviral vector and evaluated its effect on the expression of an insert from the internal promoter. The unique feature of the vector is that the transduced gene is inserted at the U3 region of the 3' long terminal repeats (LTR). Consequently, in the infected cells the gene is duplicated and transferred to the 5'-LTR. When compared with the conventional retroviral vectors which insert the gene within the retroviral transcriptional unit, the U3-based vectors greatly enhanced the expression of the transduced gene under all three promoters tested, viz. the cytomegalovirus immediately early gene promoter (CMV), the SV40 early gene promoter (SV), and the herpes simplex virus thymidine kinase gene promoter (TK). The SV and TK promoters which were previously shown suppressed by the retroviral promoter in the conventional construction restored their potencies in the U3-based vectors. Our results therefore suggested that the U3-based vectors are more advantageous than the conventional vectors for gene expression.

Animals↗
PubMedDate not supplied

Studies on direct and indirect effects of DNA damage on mutagenesis in monkey cells using an SV40-based shuttle vector.

We are using an SV40-based shuttle vector, pZ189, to study mechanisms of mutagenesis in mammalian cells. The vector can be treated with mutagens in vitro and replicated in animal cells; resulting mutants can be selected and amplified in bacteria for DNA sequencing. This versatile vector system has allowed us to explore several different questions relating to the mutagenic process. We have studied the direct effects of template damage caused by UV or benzo[a]pyrene diolepoxide by treating vector DNA with these agents and then replicating the damaged DNA in monkey cells. Mutational mechanisms were deduced from the spectrum of mutations induced in the supF target gene of the vector DNA. To study the role of indirect effects of DNA damage on mutagenesis in mammalian cells, we have treated the cells and the vector DNA separately with DNA-damaging agents. We find that pretreatment of cells with DNA-damaging agents, or with conditioned medium from damaged cells, causes an enhancement of mutagenesis of a UV-damaged vector. Thus, DNA damage can act indirectly to enhance the mutagenic process. We also have preliminary evidence that pZ189 can be used in an in vitro DNA replication system to study the process of mutation fixation on the biochemical level. We believe that the pZ189 vector will prove to be as useful for in vitro studies of mutational mechanisms as it has been for in vivo studies.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1991

Amplification of Epstein-Barr virus-based shuttle vectors by ultraviolet light in human cells.

In order to approach the mechanism of gene amplification, we have developed a model system in human cells based on the use of episomally-replicating shuttle vectors. Shuttle vectors carrying the replication origin of the Epstein-Barr virus can be stably maintained in human cells. These vectors replicate as an episome with a low copy number. We also constructed hybrid plasmids containing both the EBV and the SV40 replication origins. These molecules are able to replicate episomally either like an EBV vector or like SV40 if the SV40 large T antigen is provided at the same time. UV irradiation of both human adenovirus transformed 293 or SV40-transformed MRC5 host cells leads to vector amplification whatever the type of replication origin used for the episomal maintenance. Our result clearly shows that the EBV latent replication origin (OriP), in the presence of the Epstein-Barr nuclear antigen-1 (EBNA-1) and the SV40 large T antigen, is sensitive to over-replication in UV-irradiated human cells. Since the UV doses were small enough to induce very little damage, if any, on the plasmid sequences, this amplification should be mediated through a cellular factor acting in trans. The interest in using shuttle vectors for this kind of study lays in the easy analysis of the amplified vectors in rescued bacterial colonies. The accuracy of the amplification process can be monitored by studying restriction maps of individual plasmid molecules or more precisely the integrity of a target gene, such as the lacZ' sequence, carried by our vectors.

Cells, Cultured↗
PubMed1988

High-level transient expression of influenza virus proteins from a series of SV40 late and early replacement vectors.

We have constructed a collection of simian virus 40 (SV40) plasmid vectors useful for transient or constitutive expression of cDNA or genomic DNA in animal cells. Most vectors contain several unique restriction sites downstream from the SV40 late or early promoter, and are available with or without the virus-specific splicing signals. The use of these vectors for transient expression in monkey cells of X47 (H3N2) influenza hemagglutinin (HA) and matrix protein (M1) was demonstrated. Membrane-bound (HAm) as well as secreted forms of the HA glycoprotein lacking the sequence of the C-terminal anchor (HA-) have been obtained. Depending on the insert, the type of vector and the amount of transfected DNA, HA levels in COS cells [Gething and Sambrook, Nature 293 (1981) 620-625] transfected with late replacement SV40 vectors vary from 10(9) (HAm) to 10(8) (HA-) molecules per transfected cell. The maximum expression levels with early replacement vectors in COS cells are at least 50 times lower. In addition to the optimalization and the characterization of the expression of each vector-coded influenza protein, cotransfections, including vectors expressing HAm, neuraminidase (NA) and M1, were undertaken. The latter experiments did not result in a measureable amount of HAm or NA in the cell culture medium, suggesting that expression of these three structural viral proteins does not result in budding of (empty) influenza particles from the cell surface.

Antibodies, Viral↗
PubMed1988

Entrapment of recombinant plasmids in SeaPlaque agarose plugs and their rapid purification from recircularised vectors.

A simple method is described which permits both the separation and concentration of circular recombinant plasmids from smaller plasmid vectors that are an undesirable by-product of a ligation reaction. SeaPlaque agarose plugs are used to entrap open-circular forms of recombinant plasmids during electrophoresis. In the example described over 98% of supercoiled, open-circular and linear forms of the 2.9-kb Bluescript plasmid vector, as well as the equivalent dimer forms, pass through the 1.4% SeaPlaque plug. Circular recombinant plasmids greater in length than the vector dimer are entrapped within the plug. By increasing the concentration of SeaPlaque, recombinants smaller than the vector dimer are retained in the trap, but with a concomitant increase in contamination by open-circular vector dimers. For most library constructions the high ratio of insert to vector used during the ligation reaction reduces the formation of vector dimers and makes this level of contamination inconsequential. The recombinant plasmids can be extracted readily from the SeaPlaque plug by excising it, melting the agarose and extracting with phenol. Alternatively, the excised plug can be melted and the recombinant plasmids used to transform bacteria, or mammalian cells, directly in the agarose. The procedure should be valuable for cloning large inserts for 'jumping' and 'linking' libraries, for large inserts in general where recircularisation is a low-frequency event, e.g., minichromosomes, for pulsed-field gel electrophoresis applications, and for hosts and vectors where genetic selection of the recombinant is not possible.

Cloning, Molecular↗
PubMed1991

Plasmid cloning vectors that integrate site-specifically in Streptomyces spp.

Cloning vectors based on the Streptomyces ambofaciens plasmid pSAM2 and the streptomycete phage phi C31 were developed for use in Streptomyces spp. These vectors replicate in Escherichia coli but integrate by site-specific recombination in Streptomyces spp. Both pSAM2-based and phi C31-based vectors transformed a number of different Streptomyces spp; however, the phi C31-based vectors consistently transformed at higher frequencies than pSAM2-based vectors. Southern analysis indicated that the phi C31-based vectors integrated at a unique site in the S. ambofaciens chromosome, while the pSAM2-based vectors gave complex patterns which could indicate structural instability or use of multiple loci. Both types of vectors utilize the apramycin (Am)-resistance gene which can be selected in E. coli and Streptomyces spp. with either Am or the commercially available antibiotic Geneticin (G418).

Blotting, Southern↗
PubMed1992

Characterization and properties of a novel plasmid vector for Bacillus thuringiensis displaying compatibility with host plasmids.

A novel plasmid vector, composed of a 1.7-kb Bacillus thuringiensis (B.t.) replicon, a multiple cloning site, and an erythromycin-resistance marker gene from Bacillus subtilis, was constructed for use in B.t. Unlike other vectors which have been reported to be acceptable for B.t., this new B.t. vector was stably maintained in the absence of Er and did not displace host plasmids, some of which carry crystal protein-encoding genes (cry genes). The compatibility of this B.t. vector with native plasmids is highly desirable when introducing new cry genes into a wild-type B.t. strain. When a cryIIIA gene of B.t. tenebrionis was cloned in this vector and introduced into B.t. kurstaki (kur) HD119, cryIIIA was highly expressed without affecting the level of expression of native cry genes. The stability of this vector and its compatibility with native B.t. plasmids were achieved by subcloning only nucleotide sequences required for the vector to replicate in B.t. The origin of replication was first cloned on a 9.6-kb Bg/II fragment from a 75-kb plasmid of B.t. kur HD73 and then localized to a 2.4-kb region within the 9.6-kb fragment. Sequencing of the 2.4-kb region revealed the presence of an open reading frame (ORF), encoding a putative 312-amino acid (aa) protein. The deduced aa sequence of the ORF showed no homology to any published aa sequences. Deletion analysis indicated that the B.t. vector required at least the ORF and up to 300 bp surrounding the ORF, in order to replicate.

Amino Acid Sequence↗