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INTRODUCTION
All life/ biological processes require for its regulation, PROTEINS or more vital of the proteins called ENZYMES. .This is the vital difference between any physical or chemical Process or phenomenon, which may nor may not require them. . Any thing coming inside the realm of life has to be associated with the afore-said molecule. .Till date no life-form has been see unassociated with proteins or rather to say regulative ENZYMES. Even take for an example the VIRUS which is often seen as a controversial entity; you shall not find it sans proteins. The smaller forms of viruses, such as VIRIODS or VIRUSOIDS even are not free of a protein-regulation. The newly discovered infectious agent, PRIONS are even PROTEIN molecules. APTAMERS which are closely related small protein-arrangements, are even a pre-requisite for signal-transduction; if signal-transduction were to be called a pure physical or chemical process induced in the living system.
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Contd
.If we take into molecular evolution of life then RNA or Ribonucleic Acid has been acclaimed as the first to have evolved ; and today we cannot see this molecule without its self-catalytic nature or ignore its RIBOZYME form. . The simple fact is biological processes are more complex than physical or chemical processes and hence cannot take place with out a decrease in its activation energy or a controlled change in the free energy available for the physical or chemical process; so what life ultimately succumbs to what are called the ENZYMES. .But what regulates the enzyme; especially a protein / structural or functional to be synthesized or function? It occurs through a centralized nucleic acid -mediated cycle and often is called the CENTRAL DOGMA OF LIFE. It involves 3 major processes, REPLICATION of the genetic matter, TRANSCRIPTION or forming a form or code for the protein and lastly decoding and formation of peptides or proteins, i.e. TRANSLATION. EXCEPTION- PRIONS Contd
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REPLICATION
DNA RNA
TRANSCRIPTION
CHANGED TO
GENOME TRANSCRIPTOME
TRANSLATION
protein PROTEOME The basic difference or modification has not come due to the the fact that RNA formed the genome for few organisms; this fact was known from quite a time. . But the vital difference has come by the study of gene-expression i.e. TRANSCRIPTION and the related functional proteins, that form the PROTEOME for an organism; also an identifying factor for the organism, and even more vital than the GENOME, sometimes for the 2/13/2013 4 organisms characterization.
. However there is one requirement in terms of Enzyme without which REVERSE-TRANSCRIPTION can never occur, in-vivo or in-vitro. . This enzyme is .
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ENZYME- REVERSETRANSCRIPTASE
.The enzyme was (+) RNA discovered in 1970; independent work by Howard Temin and David ( - ) DNA RT Ds DNA/ Baltimore for which both RNaseH Proviral DNA shared the prestigious nobel prize in 1975. RNA-DNA HYBRID .Also known as the RNA( - ) DNA DEPENDENT dnapolymerase. .The enzyme defies the Pro-viral DNA central dogma of biology by transcribing DNA from a single stranded RNA HOST GENOME host mRNA sequence.
( +) I VIRAL RNA
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RT
Inside host-cell
Dna_hybrid formation
DNA
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ENZYME - Properties
Physical
.MOLECULAR MASS- 84 TO 170 Kd
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Enzyme SUPERSCRIPT II, marketed by Life Technologies ( Kotewicz et al. 1988), can carry out the process at temp. 9 up to 50 degree Celsius.
ENZYME ACTIONS
1) 5 to 3 DNA polymerase activity 2)lacks exo-nuclease activity i.e. of editing or proof reading both 3 to 5 and the reverse. 3)intrinsic RNase H activity 4)no nick translation or horizontal gene transfer or exchange activity Highly dependent on Mg ++ conc. and divalent cation as well as sulfahydryl reagents. Enzyme action is inhibited by drugs like AZT ( Zidovudine); it causes chain- termination, during replication. The enzyme is also sensitive to virus-specific proteases as well as insertion of anti-sense oligo-nucleotide.
Contd
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In the above all cases , RNsae H activity is important along with DNA polymerase activity . In general , but the enzyme uses a RNA strand as template Which has a 3- hydroxyl group as free for its polymerase Activity. Hence acclaimed as RNA-dependent DNA POLYMERASE.. Already also mentioned, it uses 2/13/2013 11 RNA-primed DNA strands, very effectively.
Native sources
RETRO VIRUSES 1)avian myeloblastosis virus( AMV) 2) Moloney murine leukemia virus ( Mo-MLV) Other retro-viruses like Human immunodeficiency virus (HIV) not used, being high in infection rate and difficult to isolate and culture in-vitro; tumor viruses easily form cell-lines that can be maintained in-vitro.
The enzyme is coced by the viral pol gene
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Contd
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modified form of HEPATITIS B virus and the strains are renamed as HEPADNA VIRUSES.
They have the gapped ds DNA which on infection , circularizes and gets integrated into host genome. Host- RNA polymerases transcribe to form mRNA as well as a pre-genome ( + ) RNA. The
protein
mRNA
viral-
REVERSE-TRANSCRIPTASE and RNase H activity. But it cannot be a source for the enzyme as
DNA POLYMERASE, very little of (+) RNA remains, being digested by the RNase H and little left ,acts as primer for gapped Ds DNA synthesis. ( - ) strand DNA is the template for ds DNA synthesis, template is required for once. Host DNA POLYMERASE thus, in greater concentration than 2/13/2013 13 Reverse transcriptase.
with 3 activities-
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1) In DNA CLONING
Owing to its polymerase activity , can replace many bacterial derived DNA-Polymerases and quite apt at being more alkaline ( so resistant to hydrolytic cleavage) and also being in more thermo-stable forms.
Actually it is based upon the Anchored PCR technique where if only one end of is known then the amplification is still workable. This variation and using adapter molecule or primer to an unknown end Can be used to amplify RNA sequences into DNA duplexes.
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Contd
RT PCR
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There are but hindrances to the library creation because even if we start with a pure mRNA preparation, the end-product consists of a mixture of cDNA s which are mostly shorter than the complete RNA molecule.
There is incomplete copying of the mRNA by reverse transcriptase (5-end of mRNA is missing from the mRNA). Further more copy or synthesis of double strand from the single strand is incomlete so that 3 end wil be missing and attributes to this action goes to the nucleaes use for cleaving the hair-pin loop.
The double-stranded c-DNA preparations are always a mixture of different kinds of molecules due to the above problems in copying of the RNA and also because even highly purified mRNAs are never absolutely pure. Physical and chemical methods are incapable of resolving these mixture. Therefore, the cDNA mixture itself is used for cloning and the desired cDNA is identified and isolated in pure form from the bacterial clones or else mechanical way through RT-PCR.
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.Probes for DNA analysis is also made easier by having a DNA library or instantaneous creation of one by the using a template for gene-constructs which can be RNA or DNA . Associated PCR and usage of restriction enzymes derived from the bacterial /viral sources along with their usage in the vector-designing are additional requirements for designing an appropriate probe.
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* VECTOR DESIGNING
.Retro-viral vectors and genetically modified bacterial vectors are by retro-viral genes especially due to the LONG TERMINAL REPEATS and TRANSPOSABLE CHARACTERISTICS. . The retro-viruses or the source of reverse transcriptase can shuttle between Prokaryotes as well as eukaryotic systems have immense potential in industrial production of Drugs, therapeutic hormones and proteins. Gene therapy, though not a clinical success or else has shown much of an in-vivo success rate; still cannot be Completely discarded .
But this cannot be a direct functional application of the enzyme Reverse transcriptase. However amplified product of the enzyme i.e. a probe, primer or c-DNA can be used to make a novel vector. The c-DNA should be analogous to the host cell DNA.
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3) TRANSGENICS PRODUCTION
The very first strategy to generate a transgenic was Micro-injection of DNA and the foreign DNA inserted was of a virus, Simian virus 40. ( Mintz 1974). In plants the early example was in form of transformation experiments conducted on tobacco and petunia by simple Protoplast to plant regeneration. Meyer et al.(1987) constructed a plasmid vector containing a marker gene along with the maize cDNA.
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7) IN ENVIRONMENTAL MONITORING
.As already mentioned that degradation of antibiotics can be monitored as well as triggered by cDNA synthesis and library creation , same can be applied on a set of conditions in-vivo or ex-vivo or environmental samples. .Extraction of RNA from the environmental samples is quite effective than DNA for analysis because these are smaller fragments. In fact 16 s sub-units can be procured easily and in more quantities as well as more effectively analysed.
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Environmental sample RNA extracted Reverse transcriptase cDNA Procedures like DGGE* and TGGE* for sample analysis and purification Probe design
rRNA sequencing
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Total community RNA can be analyzed using RAP-PCR * and metabolic fingerprints were generated wit the RNA extracted from the microbial communities. The technique involves amplification using an arbitrarily chosen primer at a low annealing temperature suitable for amplification of the total RNA( both mRNA and r RNA). Later by further cloning and sequence analysis the detection is completed. This method is not biased by the selective amplification of Sequences used to contruct primers as it uses gene-specific primers. RT-PCR , RNAse protection assays and Northen blotting are an the very often used techniques used to quantify specific mRNA molecules extracted from the environmental samples. of these RNase Protection assay is an extremely sensitive technique. * RNA arbitrarily primed PCR
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RNase Protection assayProbe* + Target RNA Hybridization in a solution The mixture is diluted
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CONCLUDING REMARKS
.Recent studies on viruses and viral-enzymes have led to many new frontiers in science. Viruses have a high infection and multiplication rate; which can be applied to diverse fields such as nanotechnology based disease diagnosis, rapid DNA micro-array studies and preservation of genomes( creation of genomic libraries). .Retro-viruses and its respective polymerase enzyme REVERSE TRANSCRIPTASE have great significance in eukaryotic systems. .Further more the study of viral genetics become easier with retro-viruses which not only replicate stably in eukaryotic system but also have properties of a TRANSPOSON.( *have long terminal repeats or LTRs in the genome).
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An assignment submitted by
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