Animal and Plant Viruses as vector,

Binary and shuttle vector

Promila SheoranPh.D. BiotechnologyGJU S&T Hisar

Animal viruses as vector

•The first eukaryotic DNA virus was SV40, for which a complete nucleotide sequence and a detailed understanding of transcription were available.

•The genome of SV40 contains very little non-essential DNA so it is necessary to insert the foreign gene in place of essential viral genes and to propagate the recombinant genome in the presence of a helper virus.

•This virus is capable of infecting several mammalian species, following a lytic cycle in some host and a lysogenic cycle in others.

The genome is 5.2kb in size and contains two sets of genes, the early genes, expressed early in the infection cycle and coding for proteins involved in viral DNA replication, and the late genes, coding for viral capsid proteins.

However, all work using SV40 virions to propagate recombinant DNA molecules is severely constrained by the facts that the viral genome is small, 5.24 kb, and that the packaging limits are strict. Such systems can not, therefore, be used for the analysis of most eukaryotic genes.

Adenoviruses, are a group of viruses which enable larger fragments of DNA to be cloned than it is possible with an SV40 vector. Adenoviruses are more difficult to handle because the genomes are bigger.

•Papillomaviruses, which also have a relatively high capacity for inserted DNA, have the important advantage of enabling a stable transformed cell line to be obtained.

•Papillomavirus transformed cells don't contain integrated viral DNA rather they contain between 50 and 300 copies of unintegrated, circular viral DNA although some proportion of these viral genomes exists as concatamers and/or catenates.

• Bovine papillomavirus (BPV), which causes warts on cattle, has an unusal infection cycle in mouse cells, taking the form of a multi copy plasmid with about 100 molecules present per cell.

•It doesn't cause the death of the mouse cell, and BPV molecules are passed to daughter cells on cell division. Shuttle vectors consisting of BPV and pBR322 sequences, and capable of replication in both mouse and bacterial cells, are therefore of great value in animal cell biotechnology.

Retroviruses

•Retroviruses, though have single-stranded RNA genomes but provides perhaps the most promising vector system of all.

• During the process of reverse transcription, sequences from the termini of viral RNA are duplicated to generate long terminal repeats(LTRs).

• These long terminal repeats contain both the promoter and the polyadenylation signal for the transcription of viral mRNAs.

•The specificity of proviral DNA integration is also determined by the long terminal repeats. Although retroviruses can integrate at many sites within the cellular genome, integrative recombination always occurs at particular sites at the ends of the LTRs.

•The sequences appropriately inserted between the two LTRs will be integrated intact. A further great advantage of retroviruses is that they are natural transducing viruses.

•Retroviral vectors are most frequently based upon the Moloney murine leukaemia virus (Mo-MLV), which is an amphotrophic virus, capable of infecting both mouse cells, enabling vector development in mouse models, & human cells, enabling human treatment.

• The viral genes (gag, pol & env) are replaced with the transgene of interest & expressed on plasmids in the packaging cell line. Because the non-essential genes lack the packaging sequence (psi) they are not included in the virion particle.

•To prevent recombination resulting in replication competent retroviruses, all regions of homology with the vector backbone should be removed & the non-essential genes should be expressed by at least two transcriptional units.

•The essential regions include the 5' & 3' LTRs & the packaging sequence lying downstream of the 5' LTR. To aid identification of transformed cells selectable markers, such as neomycin & beta galactosidase, can be included & transgenes expression can be improved with the addition of internal ribosome sites. The available carrying capacity for retroviral vectors is approximately 7.5 kb, which is too small for some genes even if the cDNA is used.

Plant viruses as vector

•The expression of foreign genes in plants has proven advantageous to the study of molecular biology. Stable gene transfer to whole plants can be achieved by a combination of DNA transformation and tissue culture techniques or byvirus transfection.

• With viral-based vectors the ease of infection avoids the time-consuming procedures, "position" effects, and "somaclonal" variation seen when foreign genesare integrated into the plant genome

• Several features of tobacco mosaic virus (TMV) suggest that it might be usefullyadapted for such a purpose:

(i) tobamoviruses have a wide host range; (ii) they can move cell-to-cell mediated by a virus-encoded peptide;

(iii) they exhibit rapid systemic spread in plants; (iv) TMV infections are maintained for the lifetime of the plant; (v) TMV RNA is replicated to high levels as autonomous sequences; (vi) this replication results in rapid and productive cytoplasmic gene expression; (vii) temperature-sensitive mutations of RNA synthesis are available tomodulate expression of foreign genes; (viii) TMV also lacks the packaging constraints found with nonhelical viruses,including existing DNA plant virus vectors; and (ix) the TMV genome can now be manipulated as a DNA copy and thentranscribed in vitro to produce infectious RNA molecules.

•The TMV genome consists of one 6395-nucleotide molecule of messenger-sense, single-stranded RNA, encoding at least four proteins.

•The 126- and 183-kDa replicase proteins are translated directly from the genomic RNA, whereas the 30-kDa cell-to-cell movement protein and 17.5-kDa coat protein are translated from two 3'-coterminal subgenomic RNAs produced during replication.

•Because the 126- and 183-kDa proteins are required for TMV RNA replication, it was decided to express the foreign sequences by means of a subgenomic RNA 3' of these genes. For brome mosaic virus, subgenomic RNA synthesis has been shown to be internally initiated on minus-sense RNA.

•Functionally similar sequences occur upstream of the highly expressed TMV coat protein ORF and are within the 30-kDa protein-coding region. The 203 bases upstream of the respective coat protein initiation codons of TMV-U1 and odontoglossum ringspot virus (ORSV), a tobamovirus isolated from Cymbidium spp., exhibit a remarkably low sequence similarity [45% compared with 65% for the 3' 2.4 kilobases (kb) of the respective genomes].

•The expression vector pTB2 was designed to avoid deletion of foreign inserts due to flanking repeated sequences by using these two heterologous promoters from TMV-U1 and ORSV to synthesize subgenomic RNAs for the foreign gene and ORSV coat protein gene, respectively.

•To determine whether foreign sequences inserted into such a TMV-based expression vector could be replicated stably and whether they could be propagated systemically, two bacterial sequences were tested in the vector.

•The DHFR sequence from plasmid R67 chosen for its small size of 238 base pairs (bp)] and the larger (832-bp) NPTII ORF from transposon TnS (24) were clonedinto the Xho I site of pTB2, downstream of the TMV-U1 coat protein subgenomic promoter, to produce pTBD4 and pTBN62, respectively

FIG. 1. Construction of expression vector pTB2 and derivatives containing dihydrofolate reductase (DHFR; pTBD4) and neomycin phosphotransferase II (NPTII; pTBN62) genes.

Thin lines and open boxes (ORFs) are sequences transcribed in vitro by E. coli RNA polymerase from its promoter (hatched box in top diagram).

ORFs for the 126-, 183-, 30-kDa and coat proteins (126K, 183K, 30K, and cp, respectively) are TMV-U1 sequences, except where noted as being from ORSV (o).

Thick lines represent bacterial cloning vector sequences. Narrow open box (o) delineates 203 ORSV bases upstream of coat protein initiation codon.

Vertical lines in pTBD4 and pTBN62 indicate extent of foreignsequences. Selected restriction sites are shown (B, BamHI; K, Kpn I; N, Nsi I; Nc, Nco I; P, Pst I; S, Spi I; and X, Xho I).

•Two bacterial sequences inserted independently into TB2 moved systemically in Nicotiana benthamna, although they differed in their stability on serial passage.

•Systemic expression of the bacterial protein neomycin phosphotransferase was demonstrated. Hybrid RNAs containing both TMV-U1 and the inserted bacterial gene sequences were encapsidated by the odontoglossum ringspot virus coat protein,facilitating their transmission and amplification on passaging to subsequent plants.

•The vector TB2 provides a rapid means of expressing genes and gene variants in plants.

Shuttle Vector•These vectors have been designed to replicate in cells of two different species; therefore, they contain two origins of replication, one specific for each host species, as well as those genes necessary for their replication and not provided by the host cells.

•These vectors are created by recombinant techniques.

•Some of them can be grown in two different prokaryotic species, usually E. coli and a eukaryotic one, e.g. yeast, plants, animals.

•Since these vectors can be grown in one host and then moved into another without any extra manipulation, they are called shuttle vectors.

•YEp13 is an example of shuttle vector.

•The yeast Saccharomyces cerevisiae is one of the most important organisms inbiotechnology. Development of cloning vectors for yeast was initially stimulated by the discovery of a plasmid that is present in most strains of S. cerevisiae. The2 µm plasmid, as it is called, is one of only a very limited number of plasmids found in eukaryotic cells.

•Vectors derived from the 2 µm plasmid are called yeast episomal plasmids (YEps). Some YEps contain the entire 2 µm plasmid, others include just the 2 µm origin of replication. An example of the latter type is YEp13.

•The 2 µm plasmid is an excellent basis for a cloning vector. It is 6 kb in size, which isideal for a vector, and exists in the yeast cell at a copy number of between 70 and 200. Replication makes use of a plasmid origin, several enzymes provided by the host cell, and the proteins coded by the REP1 and REP2 genes carried by the plasmid.

•However, all is not perfectly straightforward in using the 2 µm plasmid as a cloning vector. First, there is the question of a selectable marker.

•Here gene LEU2, which codes for b-isopropyl-malate dehydrogenase, one of the enzymes involved in the conversion of pyruvic acid to leucine is used as marker.

•In order to use LEU2 as a selectable marker, a special kind of host organism isneeded. The host must be an auxotrophic mutant that has a non-functional LEU2 gene.

•Such a leu2− yeast is unable to synthesize leucine and can survive only if this amino acid is supplied as a nutrient in the growth medium. Selection is possiblebecause transformants contain a plasmid-borne copy of the LEU2 gene, and so are able to grow in the absence of the amino acid.

• In a cloning experiment, cells are plated out onto minimal medium, which contains no added amino acids. Only transformed cells are able to survive and form colonies.

•YEp13 illustrates several general features of yeast cloning vectors. First, it is a shuttle vector.

• As well as the 2 µm origin of replication and the selectable LEU2 gene, YEp13 also includes the entire pBR322 sequence, and can therefore replicate and be selected for in both yeast and E. coli.

Uses of Shuttle Vectors

•There are several lines of reasoning behind the use of shuttle vectors. One is that it might be difficult to recover the recombinant DNA molecule from a transformed yeast colony.

•This is not such a problem with YEps, which are present in yeast cells primarily as plasmids, but with other yeast vectors, which may integrate into one of the yeast chromosomes, purification might be impossible.

•This is a disadvantage because in many cloning experiments purification of recombinant DNA is essential in order for the correct construct to be identified by, for example, DNA sequencing.

•The standard procedure when cloning in yeast is therefore to perform the initialcloning experiment with E. coli, and to select recombinants in this organism.Recombinant plasmids can then be purified, characterized, and the correct molecule introduced into yeast.

Binary vector

•The binary vector is a shuttle vector, so-called because it is able to replicate in multiple hosts (E. coli and Agrobacterium tumefaciens).

•A transfer DNA (T-DNA) binary system is a pair of plasmids consisting of a binary plasmid and a helper plasmid. The two plasmids are used together (thus binary) to produce genetically modified plants. They are artificial vectors that have both been created from the naturally occurring Ti plasmid found in Agrobacterium tumefaciens.

•Systems in which T-DNA and vir genes are located on separate replicons are called T-DNA binary systems. T-DNA is located on the binary vector (the non-T-DNA region of this vector containing origin(s) of replication that could function both in E. coli and in Agrobacterium tumefaciens, and antibiotic-resistance genes used to select for the presence of the binary vector in bacteria, became known as vector backbone sequences).

•The replicon containing the vir genes became known as the vir helper. Strains harboring this replicon and a T-DNA are considered disarmed if they do not contain oncogenes that could be transferred to a plant.

•The discovery that the vir genes do not need to be in the same plasmid with a T-DNA region to lead its transfer and insertion into the plant genome led to the construction of a system for plant transformation where the T-DNA region and the vir region are on separate plasmids.

•In the binary vector system, the two different plasmids employed are:

•First is a wide-host-range small replicon, which has an origin of replication (ori) that permits the maintenance of the plasmid in a wide range of bacteria including E. coli and Agrobacterium. This plasmid typically contains:

1. foreign DNA in place of T-DNA,2. the left and right T-DNA borders (or at least the right T-border),3. markers for selection and maintenance in both E. coli and A. tumefaciens,4. a selectable marker for plants.

•The plasmid is said to be "disarmed", since its tumor-inducing genes located in the T-DNA have been removed.

Second is a helper Ti plasmid, harbored in A. tumefaciens, which lacks the entire T-DNA region but contains an intact vir region.

In general, the transformation procedure is as follows:

•The recombinant small replicon is transferred via bacterial conjugation or direct transfer to A. tumefaciens harboring a helper Ti plasmid,

•the plant cells are co-cultivated with the Agrobacterium, to allow transfer of recombinant T-DNA into the plant genome, and

•transformed plant cells are selected under appropriate conditions.

Possible pitfalls

•A possible disadvantage may ensue from the fact that the stability of wide host range replicons in E. coli and Agrobacterium varies considerably. Depending on the orientation, plasmids with two different origins of replication may be unstable in E. coli where both origins are active.

Advantages

•Compared with co-integrated vectors, binary vectors present some advantages:No recombination process takes place between the molecules involved.Instead of a very large, recombinant, disarmed Ti plasmid, small vectors are used, which increases transfer efficiency from E. coli to Agrobacterium

•This vector system is most widely used nowadays.

• Different types of binary vectors have been devised to suit different needs in a plant transformation process. One example is pGA series vectors.

1.pGA series vectors, which contain:• an ori derived from RK2 for replication in E. coli and Agrobacterium,• a tetracycline resistance gene,• the cis-acting factor required for conjugal transfer,• the right (RB) and left (LB) T-DNA borders,• a neomycin phosphotransferase (nptII) gene, which confers resistance to • kanamycin and G418 in transformed plants, and• a polylinker site (multicloning site).

•Specific vectors in this series are designed for cloning large fragments (colE1 origin of replication and phage l cos), analyzing promoters (multiple cloning site immediately upstream of a promoterless cat gene), and expressing a gene of interest (polylinker site between a plant promoter and a terminator).

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