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Archive for August, 2007

The Ti plasmid contains genes for auxin and cytokinin formation, and for synthesis or utilization of opines. Ti plasmids range in size from 200 to 800 kbp. Of this, about 30% is common to nopaline and octopine plasmids (Genes VIII, Benjamin Lewin, pp. 525). The genes of common region are required for the interaction between Agrobacterium and plant hosts. The part of Ti plasmid which is transferred to the plant nucleus is called T-DNA. It gets incorporated into the plant genome. The T-DNA is referred to as the T-region. T-region is approximately 10 to 30 kbp in size and represents less than 10% of the Ti plasmid. Ti plasmids generally contain one T-region, but some Ti plasmids have multiple T-regions on them. T-region is flanked by T-DNA border repeats. These are 25 bp long and highly homologous in sequence. The T-DNA border sequences flank the T-region in directly repeated orientation. The T-DNA border sequences delimit the T-DNA , and are the target of the border-specific endonucleases.

Ti Plasmid

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Ti Plasmid

Ti plasmids are divided into 4 groups depending upon the types of opines that are synthesized.

1) Nopaline plasmids: These carry genes for synthesis of nopaline in tumors and for utilizing it in bacteria. The tumors carrying nopaline plasmids can differentiate into shoots with abnormal structures. They are called teratomas as they are analogous to certain mammalian tumors.

2) Octopine plasmids: The octopine tumors are undifferentiated and do not produce teratoma shoots.

3) Agropine plasmids: The tumors carrying agropine plasmids do not differentiate. They are poorly developed and die early.

4) Ri plasmids: These induce hairy root disease on some plants. They carry agropine genes. May also contain segments of both nopaline and octopine plasmids.

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Agrobacterium tumefaciens, a soil-borne bacterium, causes the crown gall disease in most dicots. The disease leads to the formation of tumor on the site of infection. The tumor-induction is dependent upon the bacterium’s ability to transfer its DNA to the plant cell: The bacterium attaches itself to the host plant cell and transfers part of its DNA to the plant cell. The plant cell is now transformed. The tumor inducing ability of A. tumefaciens resides in the Ti plasmid; the Ti plasmid is maintained as an independent replicon within the bacterium. The plasmid carries genes required for the transfer of tumor-inducing principle from bacterium to the plant cell. The Ti plasmid also carries genes for synthesis or utilization of opines (novel derivatives of arginine). Ti plasmids, and, therefore, Agrobacteria in which they (Ti plasmids) reside, are divided into four groups on the basis of the types of opines that are made:
1) Nopaline plasmids
2) Octopine plasmids
3) Agropine plasmids
4) Ri plasmids

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miRNA and siRNA

Both microRNAs (miRNA) and small interfering RNAs (siRNA) play integral roles in the RNAi. Both these molecules play similar roles but differ in their origin . siRNAs are derived from the cleavage of long dsRNA precursors, which could either be produced endogenously or introduced into the cell from outside. When produced endogenously, siRNAs are derived from the cleavage of long dsRNAs which are produced by RNA-dependent RNA polymerases or from transcription of genes or transposable elements. The cleavage of long precursor dsRNAs into siRNA is catalysed by the Dicer endonuclease. One of the two strands of siRNA is finally incorporated into the RISC.

miRNAs, in contrast, are endogenously derived from the cell. They are encoded within the host genome. The miRNA transcripts contain “near-complementary inverted repeats” which fold back on themselves to form hairpin or stem-loop structures. These are processed in the nucleus by the RNase III enzyme Drosha, and a protein called Pasha in Drosophila or DGCR8 in mammals. The processing reaction generates pre-miRNA, which is further cleaved by the cytoplasmic RNase III endonuclease Dicer complex to produce mature miRNA. This is then incorporated into the RISC. The miRNA could silence genes by two different modes: in plants, miRNAs cleave the cognate mRNAs; in animals, miRNAs predominantly inhibit translation by targeting partially complementary sequences in the 3´ UTR (untranslated region) of mRNAs.

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During the course of gene silencing by RNAi the destruction of target mRNA is catalysed by the siRNA-guided, RNA-induced silencing complex (RISC). RISC, composed of two signature components (siRNA/miRNA and Argonaute), is an endonuclease , which catalyses the cleavage of a single phosphodiester bond on the RNA target. The cleavage reaction requires “Mg2+, but not Ca2+, and the cleavage product termini suggest a role for Mg2+ in catalysis” (Current Biology, 2004, Vol 14: 787-791). Argonaute proteins, reported from RISC complexes of diverse organisms, are the key components of RISC. The Argonaute protein family is highly diverse, and the members contain two domains: a PAZ domain, which is involved in miRNA/siRNA binding, and a PIWI domain, which is related to RNaseH endonucleases and functions in slicer activity.

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RNA interference (RNAi) has emerged as one of the significant mechanisms playing a major role in gene expression regulation. The mechanism relies on diverse small noncoding RNAs, the prominent being siRNAs (small interfering RNAs) and miRNAs (microRNAs), for efficient gene silencing. The RNAi pathway is started by the enzyme Dicer, which is a ribonuclease of the RNase III family. This enzyme cleaves long double-stranded RNA (dsRNA) to short double-stranded fragments of 20–25 base pairs. One of the two strands is incorporated into the RNA-induced silencing complex (RISC). The resulting ribonucleoprotein complex (RISC-RNA) lodges itself on the target mRNA by complementary base pairing (between the mRNA and the single-stranded small RNA bound to RISC). This results into degradation of the target mRNA by the argonaute protein. Argonaute is the catalytic component of the RISC complex. The outcome of the whole process is inhibition of gene expression or gene silencing.

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A brief introduction

A desire to pen down, or key in, important happenings on diverse aspects, issues, sites, links, tutorials, protocols, research papers, journals, companies, and all things molecular biology and biotechnology gave birth to this blog.

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