TT1 notes part 2.pdf

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Department
Cell and Systems Biology
Course
CSB351Y1
Professor
Mounir Abou Haidar
Semester
Fall

Description
41 The 35S RNA is particularly complex, containing highly structured 600 nt long leader sequence with 6-8 short ORF (encode proteins 2-35 aa in length) This leader is followed by 7 tightly arranged longer ORFs that encode all the viral proteins The mechanism of expression of these proteins is very special ORF VI protein (TAV, encoded by the 19S RNA) controls translation reinitiation of major ORF on the polycistronic 35S RNA Ribosomal shunt translational strategy 42 Replication cycle of CaMV 1. Entry of the virus into the host cell 2. The virion is transported to the nuclear pore 3. Genome imported into the nucleus 4. Repair of DNA sequence discontinuities and association of the genome with histones to form a minichromosome o The viral DNA is transcribed by cellular RNA polymerase II into the pregenomic 35S and subgenomic 19S RNA 5. Translation of the 19S RNA results in production of P6 6. P6 condenses into cytoplasmic virus factories and transactivates the translation of all other viral proteins from 35S RNA 7. Among these, P5 replicates the genome by reverse transcriptase using 35S RNA as a template 8. The newly transcribed viral DNA is encapsidated, resulting in generation of virus particles in the virus factories 9. With this last step, the minimal time required for one round of replication is over, symbolised by the clock The replication is similar to that of other retroviruses The 35S RNA contains at the 5 end region 13 nt complementary to the 3 end of tRNA Met and 190 nt of repeated sequences at the two ends of the RNA The 3 OH end of the tRNA Met is used as a primer for the reverse transcriptase to start the synthesis of a complementary DNA of the virus The ribonuclease H activity of the RT degrades the RNA hybridised to the cDNA However, small pieces of RNA are resistant to the RNase H activity They serve for synthesis of a 2dstrand DNA These small RNA pieces are located at 3 different positions on the viral DNA and at their position the DNA is interrupted Assembly A specific interaction takes place between the CP of CaMV and the conserved purine-rich sequence in the central part of the CaMV pgRNA leader (35S RNA) 35S RNAs are RT into dsDNAs in the form of immature virions which are likely assembled in inclusion bodies in the cytoplasm Summary of CaMV Replication cycle Once introduced within a host cell, virions migrate to the nuclear envelope, where they presumably decapsidate (uncoating) The viral genomes then enter the nucleus where they form minichromosomes that are transcribed by the host RNA polymerase II to generate 2 mRNAs, the polycistronic 35S RNA comprising the entire genome encoding a single protein, P6 (TAV or transactivator protein) In the cytoplasm, P6 is translated from the 19S RNA and aggregates in small inclusion bodies, where it transactivates translation of all other viral proteins from the 35S RNA The 35S RNA is also the template for the production of viral genomic dsDNA, which is probably encapsidated into virions during or shortly after its synthesis The progeny virions are sequestered mostly into the P6 inclusions that grow over time and form viral factories The 35S promoter of CaMV is widely used in biotechnology The promoter of the 35S RNA is a very strong constitutive promoter responsible for the transcription of the whole CaMV genome It is well known for its use in plant transformation It causes high levels of gene expression in dicot plants However, it is less effective in monocots, especially in cereals The differences in behavior are probably due to differences in quality and/or quantity of regulatory factors43 The promoter was named CaMV 35S promoter because the coefficient of sedimentation of the viral transcript, whose expression is naturally driven by this promoter, is 35S One of the most widely used, general-purpose constitutive promoters The CaMV promoter is used in most transgenic crops to activate foreign genes which have been artificially inserted into the host plant It is inserted into transgenic plants in a form which is different to its naturally occurring state arising in its natural Brassica plant hosts This enables it to operate in a wide range of host-organism environments which would otherwise not be possible 35S promoter is active in all plant tissues: leaves, roots Recombination Cloned CaMV DNA is infectious 2 overlapping pieces of the genome are also infectious, suggesting that recombination of CaMV DNA exists 2 pieces of CaMV DNA from 2 different strains of the virus were used to infect plants The results indicate that a portion of the virus particles obtained have the characteristics of the 2 strains Can Caulimoviruses integrate into the host genome like retroviruses? Do not integrate obligatorily into the host genome But illegitimately integrated sequences have been found for several genera to date These have been named commonly endogenour plant pararetroviruses region of chromosomes, are passively replicated together with the host DNA, and are inherited from generation to generation Lecture 14 Viroids, virusoids and ribozymes Identification of viroids like a virus Eg. Po
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