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Archive for the ‘Plant Transformation’ Category

The Ri (root-inducing) plasmid of Agrobacterium rhizogenes carries agropine genes. When A. rhizogenes infects a plant, a portion of the Ri plasmid DNA enters the host plant cell and causes the production of hairy roots at the site of action. A foreign gene could be inserted into modified Ri plasmid and the recombinant DNA (plasmid) could be introduced into plants in much the same way as with the Ti plasmid of A. tumefaciens. The recombinant Ri plasmid would induce the production of hairy roots after the infection of the host plant. Scientists are now trying to use these hairy roots as potential drug factories. In a new study, scientists have successfully maintained a transgenic hairy root culture alive for 4-and-a-half years, and they hope that this could be a great source of continuous drug production. Read the full story here.

via: ScientificBlogging


Digg!

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This post is about two recent developments in the field of biotechnology and environmental science.

1) Agrobacterium-mediated transformation is the method of choice for inserting foreign genes into the plant genome. Through this method one gene could be introduced at a time in a single experiment. However, to introduce two foreign genes, one needs to develop two separate plant lines each carrying one gene. The plants of the two lines are then cross bred to get both the genes in one plant. The process is quite laborious and takes a lot of time to complete. Besides, integration of the foreign gene into the plant genome can disrupt any native gene; or the foreign gene may get silenced and not expressed. To overcome these problems a team of scientists from the University of Chicago, the University of North Carolina and Chromatin, Inc., has developed a new method for constructing artificial plant mini-chromosomes. These are the rings of plant DNA that can be used to carry multiple genes in plant cells in one go. The work has been published in the October 19, 2007, issue of PLoS-Genetics. The team has developed maize mini-chromosomes (MMCs) that “…can introduce an entire “cassette” of novel genes into a plant in a way that is structurally stable and functional.” These mini-chromosomes could be suitably used as vectors for the transfer of two or more than two genes simultaneously, and would thereby save the time and effort required for lengthy and laborious hybridization experiments needed to transfer two genes in a plant genome. This would also cut down expenses involved in such experiments. The MMCs created also have maize centromere sequences in them. These sequences help them maintain independently of the maize genome and  newly introduced genes remain separate from the genome. This also helps their efficient transfer to the next generation in the Mendelian fashion. Thus these MMCs behave much like ordinary chromosomes.

You can read the full story here.

2) The second story deals with the use of algae as biofuel. Several companies are already working on the use of algae as biofuels. But LiveFuels Alliance, funded by LiveFuels Inc based in Menlo Park, CA, is trying to tap the oil producing potential of algae and hopes to replace gallons of fossil fuels with algae-based biocrude by 2010. Algae synthesize oil naturally. The raw algae is processed to “…make biocrude, the renewable equivalent of petroleum, and refined to make gasoline, diesel, jet fuel, and chemical feedstocks for plastics and drugs.” To quote from the story, “Theoretically, algae can yield between 1,000 to 20,000 gallons of oil per acre, depending on the specific strain.” What is important is that LiveFuels Alliance is a national initiative led by Sandia National Laboratories, a U.S. Department of Energy National Laboratory, and in the next 3 years it would sponsor several labs and hundreds of scientists, which makes it the largest endeavor focused on commercial biofuel production from algae. Read the whole story here. via: Inhabitat

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After its entry into the plant cell the ssT-DNA complex crosses the nuclear membrane to enter the nucleus and subsequently integrates into the plant genome. VirD2 and VirE2 are the two most important proteins required for this process. VirF also plays a minor role in the same (Hooykaas, P.J.J. and Shilperoort, R.A. (1992). Agrobacterium and plant genetic engineering. Plant Molecular Biology 19: 15-38). The ss-T-DNA complex is coated by a single molecule of VirD2, attached covalently to the 5′ end of T-DNA; however, a large number of VirE2 molecules (approximately 600 per a 20 kb T-DNA) coat the ssT-DNA complex. The nuclear location signals (NLS) of VirD2 and VirE2 play an important role in nuclear targeting of the ss-T-DNA complex. The two NLS of each VirE2 are important for the continuos nuclear import of ss-T-DNA complex. They keep both sides of nuclear pores simultaneously open. The process of nuclear import is also probably mediated by certain NLS-binding proteins, which are present in plant cytoplasm.
The final step is the incorporation of T-DNA into the plant genome. The integration occurs by illegitimate recombination (Gheysen, G., Villarroel, R. and Van Montagu, M. (1989). Illegitimate recombination in plants: a model for T-DNA integration. Genes Development 5: 287-297), which requires pairing of a few bases, known as micro-homologies. “These homologies are very low and provide just a minimum specificity for the recombination process by positioning VirD2 for the ligation” (de la Riva et al., 1998. Agrobacterium tumefaciens: a natural tool for plant transformation. Elec. J. Biotech. Vol. 1). The 3´-end or adjacent sequences of T-DNA find some homologies with plant DNA and form a gap in 3′-5′ strand of plant DNA. The displaced plant DNA is cut at the 3′-end position of the gap by endonucleases. The first nucleotide of the 5′ attaches to VirD2, and pairs with a nucleotide in the top (5′-3′) plant DNA strand. The 3′ overhanging part of T-DNA and the displaced plant DNA are digested. “Then, the 5′ attached to VirD2 end and other 3′-end of T-strand (paired with plant DNA during since the first step of integration process) joins the nicks in the bottom plant DNA strand.” The repair mechanism of the plant cell is also activated during the process to make the complementary strand using T-DNA strand as a template. VirD2 plays an active role in the integration process (Jayaram, M. (1994). Phosphoryl transfer if FLP recombination: a template for strand transfer mechanisms. Trends Biotechnology. 19: 78-82).

Here is the figure detailing the complete T-DNA transfer process.

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The T-DNA transfer process starts with the formation of a nick at the right 25 bp repeat. This provides the priming end for synthesis of a DNA single strand. Synthesis of new strand displaces the old strand. The old strand is used in the transfer process: The old strand is transferred to the nucleus. The old strand is transferred coated with the VirE2 single-strand binding protein. As mentioned in the earlier post the VirE2 has a nuclear localization signal (NLS) and is responsible for transporting T-DNA into the plant cell nucleus. VirD2 is covalently bound to 5′ end of ssT-DNA.
Many more proteins are required for the transfer of T-DNA. They are:
1) virB operon: The 9.5 kb operon is required for the generation of cell surface structure for the ssT-DNA complex transfer (Finberg , K.E., Muth, T.R. Young, S.P., Maken, J.B., Heitritter, S.M., Binns, A.N. and Banta, L.M. (1995). Interactions of VirB9, -10 and -11 with the membrane fraction of Agrobacterium tumefaciens: solubility studies provide evidence of tight associations. Journal of Bacteriology 177: 4881-4889). virB operon codes for 11 products. The proteins of virB operon are “homologous to the Tra proteins of certain bacterial plasmids that are involved in conjugation” (Lewin, B. Genes VIII, pp. 529). VirB proteins present hydrophathy characteristics similar to other membrane-associated proteins (Kuldau, G.A., DeVos, G., Owen, J., McGaffrey, G. and Zambryski, P. (1990). The virB operon of Agrobacterium tumefaciens pTiC58 encodes 11 open reading frames. Molecular General Genetics 221: 256-266). Most of the VirB proteins are assembled as membrane-spanning protein channel involving both membranes (Shirasu, K., and Kado, C.I. (1993a). The virB operon of the Agrobacterium tumefaciens virulence regulon has sequence similarities to B,C, and D open reading frames downstream of the Pertussis toxin-operon and to the DNA transfer-operons of broad-host-range conjugative plasmids. Nucleic Acid Research 21: 353-354). All the VirB proteins, except VirB11, have multiple periplasmic domains.
2) VirB1: It is found in the extracellular milieu.
3) VirB2: It performs some extracellular functions. It is translated as a 12 kDa proprotein. The mature functional form of the VirB2 is 7 kDa.
4) VirB4 and VirB11: These are hydrophilic ATPases and are necessary for active DNA transfer. VirB4 is tightly associated with the cytoplasmic membrane. The functional forms of VirB4 and VirB11 are homo- and heterodimers (Dang, T.A.T. and Christie, P.J. (1997). The VirB4 ATPase of Agrobacterium tumefaciens is a cytoplasmic membrane protein exposed at the periplasmic surface. Journal of Bacteriology 179: 453-462).
5) VirB7 and VirB9: VirB7 is important for the conformation of the transfer apparatus. VirB7 and VirB9 interact to form heterodimers. The VirB7-VirB9 heterodimer stabilizes other Vir proteins during the assembly of functional transmembrane channel.
6) VirB1: It functions as a transglycosidase. It doesn’t contribute significantly to the transfer process.
During the biogenesis of ssT-DNA complex apparatus, VirB7 and VirB9 are exported to the membrane and processed and interact to form heterodimers and homodimers. However, only the VirB7-VirB9 heterodimer is essential for the process. The heterodimer is sorted to the outer membrane and interacts with other Vir proteins leading to the formation of the transfer channel.

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ssT-DNA-protein complex is transferred to the plant nucleus, and during the translocation it passes through three membranes, the plant cell wall and cellular spaces. The ssT-DNA-VirD2 complex formed after the cleavage is coated by the 69 kDa VirE2 protein. The VirE2 protein is a single strand DNA binding protein. It prevents the DNA from the attack of nucleases, and also helps in making the translocation through the membrane channels easier. However, this association does not stabilize T-DNA complex inside Agrobacterium (Zupan et al., 1996). VirE2 and VirD2 both contain plant nuclear location signals (NLS): VirE2 contains two and VirD2 contains one. The presence of NLS indicates that these proteins play pivotal role after the ssT-DNA-protein complex is inside the plant cell and mediate its uptake to the nucleus.

However, an alternative model of T-DNA transfer “proposes that the transfer complex is a single-strand DNA covalently bound at its 5′-end with VirD2, but uncoated by VirE2” (de la Riva et al., 1998). It is proposed that VirE2 is exported independently to the plant cell, and once the naked ssT-DNA-VirD2 complex is inside the plant cell, it is coated by VirE2.

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The T-DNA is flanked by 25 bp direct repeats. These repeats differ at only two positions between the left and right ends. Mutation or deletion studies (Hille, J., Wullems, G. and Schilperoort, R.A. (1983). Non-oncogenic T-region mutants of Agrobacterium tumefaciens do transfer T-DNA into plant cells. Plant Molecular Biology 2: 155-163) of the left and right border sequences have conclusively proved that extensive mutations of the right T-DNA border lead to an almost complete loss of T-DNA transfer capacity. However, mutations of left border only lower the transfer efficiency. These studies clearly indicate that T-strand synthesis is initiated at the right border, and proceeds in the 5′ to 3′ direction. The left border may act as initiation site of  ssT-DNA , but the efficiency is much lower. It has been shown that this difference occurs because of the presence of an enhancer sequence next to the right border. This enhancer sequence is specifically recognized by VirC1 protein (Toro, N., Datta, A., Carmi, O.A., Young, C., Prusti, R.K. and Nester, E.W. (1989) The Agrobacterium tumefaciens virC1 gene products binds to overdrive, a T-DNA transfer enhancer. Journal of Bacteriology 171: 6845-6849.).

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T-DNA transfer

virD is an operon of 4 genes. Out of these four genes two code for VirD1 and VirD2 proteins. VirD1 and VirD2 play a key role in generating T-DNA transfer complex: They recognize the T-DNA border sequences and nick the bottom strand at each border by their endonuclease activity. VirD2 remains covalently attached to to the 5′-end of the ssT-strand (single strand T-strand) after the cleavage. This association is important as it prevents the exonucleolytic attack to the 5′-end of the ssT-strand. It also distinguishes the 5′-end as the leading end of the T-DNA transfer complex. VirD1 is found to be essential for the cleavage of supercoiled DNA by VirD2.

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