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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; youshall 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.

Contd…

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.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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DNA

RNA

protein

TRANSCRIPTION

TRANSLATION

REPLICATION

CHANGED TO GENOME

TRANSCRIPTOME

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 organism’s characterization.

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TRANSCRIPTION and ITS REVERSE THAT OCCURS…

.The RNA copies of the active protein- coding gene( essentially DNA being a more stable form) is called TRANSCRIPTOME. Generally the non-coding part of the gene is the template and its complementary code is transcribed i.e. RNA synthesis occurs, which is direct sequence for amino-acids to be arranged and poly-peptide to be formed..But some RNA genomes( viral) take an alternate path. Inside the host, they form DNA from the RNA and the viral RNA forms the template; the DNA formed by the transcription is complementary to the viral RNA often also and often also called c-DNA and is integrated in to host genome (eukaryotic mostly) in double-stranded form with respect to the host genome..This reverse process of transcription can also take place without being virus-mediated i.e. eukaryotic mRNA forming the template for c-DNA synthesis; but this has to bean in-vitro process.

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. 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- REVERSEENZYME- REVERSETRANSCRIPTASETRANSCRIPTASE

.The enzyme was discovered .The enzyme was discovered in 1970; independent work in 1970; independent work by Howard Temin and by Howard Temin and David Baltimore for which David Baltimore for which both shared the both shared the prestigious nobel prize in prestigious nobel prize in 1975.1975.

.Also known as the .Also known as the RNA-RNA-DEPENDENT dna-DEPENDENT dna-polymerase.polymerase.

.The enzyme defies the .The enzyme defies the central dogma of biology central dogma of biology by transcribing DNA from a by transcribing DNA from a single stranded RNA single stranded RNA sequence.sequence.

(+) RNA

( - ) DNA

RT

RNA-DNA HYBRID

RTRNaseH

( - ) DNA

Ds DNA/Proviral DNA

I

Pro-viral DNA

host mRNA

( +) VIRAL RNA

HOST GENOME

Inside host-cell

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DNA

RNAGenome

+ ENZYME

Structure of the enzyme

Ribonuclease H component of the enzyme

Dna_hybrid formation

ENZYME

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ENZYME - Properties

.MOLECULAR MASS- 84 TO 170 Kd

.consist of 1 or 2 visible sub-units ( NMR spectrometry used).

. pH 7.6, 8.3 ( slight alkaline is optimum)

.temperature 37 to 40 degree Celsius is optimum

Physical

Enzyme SUPERSCRIPT II, marketed by Life Technologies ( Kotewicz et al. 1988), can carry out the process at temp. up to 50 degree Celsius.

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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 activity4)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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SUBSTRATES AND THE RESPECTIVE ACTIONS…

.the templates can be- 1)DNA duplexes with gaps / ss DNA with protruding 5’ termini ( acted upon by restriction -endo nucleases or native segmented genome) 2)ss DNA with a primer essentially RNA or mRNA 3) Unprimed single-stranded DNA but with hair-pin loop structure.

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 RNA-primed DNA strands, very effectively.

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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-linesthat can be maintained in-vitro.

Contd…

The enzyme is coced by the viral “ pol ” gene

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.One of the native source for the virus, is also a 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 mRNA along side host -protein, produces a viral-protein with 3 activities-

DNA POLYMERASE, REVERSE-TRANSCRIPTASE and RNase H activity. But it cannot be a source for the enzyme as 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 Reverse transcriptase.

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Genetically Engineered sources…

As also mentioned , many enzymes more thermo-stable and with same activity have been designed.

More so, T7 and T4 phage-derived; more Importantly the T7 DNA Polymerase, have great functional resemblance with the reverse-transcriptase and can be used in its place.

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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.

RT – PCR ( reverse transcription based polymerase reaction) i.e. in-vitro cloning using Reverse Transcriptase Has become a quite popular tool. It uses eukaryotic mRNA as template.

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 endCan be used to amplify RNA sequences into DNA duplexes.

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RT PCR

Contd…

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2) c-DNA libraries/ genomic libraries

A cDNA library is a population of bacterial -transformants or phage -lysates in which each mRNA isolated from an organism or tissue is represented as its cDNA in such a library would ordinarily depend on the frequency of the concerned mRNA in the tissue/organism in question.

cDNA is the copy or complementary DNA produced by using mRNA (usually) as a template.DNA copy of an RNA molecule is produced by the enzyme “ reverse transcriptase” or RNA dependent DNA polymerase.

The enzyme has an absolute requirement for a primer with a free 3’-OH end.

Contd..

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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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Primer construct using RT -PCR

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*PRIMER AND PROBE DESIGNING & MANUFACTURE/ SYNTHESIS

.Creation of DNA /RNA primers for PCR reactions and also for in-vivo amplification or gene-constructs/vector designing made easier by having a c-DNA library.

.Probes for DNA analysis is also made easier by having a DNA library or instantaneous creation of one by the using atemplate 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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Probe-design using RT-PCR

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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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•Activator and Reverse activator systems

Repressor-based systems are required very essentially in transgenic technology but require a very high level of constitutive expression of repressor molecules required to suppress background trans-gene activity; however keeping in mind cytotoxicity and feed-back inhibition for the enzymes or proteins.. A model for the drug resistance study against Tetracycline (Bohl et al.) shows that the MYOBLASTS were transformed with a reverse tetracycline trans-activator or rtTA system. The myoblasts were transformed with the system using a retro-viral vector in which the erythropoietin cDNA which was placed under the control of a tetracycline-inducible promoter. These cells were implanted into mice, and erythropoietin secretion could be controlled by feeding with a derivative of the drug called doxycycline with a shorter life period. Hence drug-effect was reduced.

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4) STABLE CELL- LINE PRODUCTION

The insertion of foreign or homologous ( or both integrated) is supposedly to form a stable cell-line.Retro-virus based cell-line have immense applications in research-based therapeutics.They are more stable in terms of expressing a gene product which could be a potent drug or vaccine.More-over toxicity testing and inhibiting a drug action or else enhancement is easier with a stable cell-line production.

However this is also not a direct application of the enzyme.

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5) GENE- SILENCING AND RNA INTERFERENCE

Following can effect in-activation of a trans-gene expressionor cause instability of a gene-product .Sequence homology of an a gene-insert with host .Endogenous expression of host –gene , by addition of more number of cDNA copies. .RNA interference i.e. insertion of mRNA or transcriptome or even small RNA fragments derived from viral/bacterial or else eukaryotic/prokaryotic cell organelle -based RNA also causes transient and unstable gene -expression ; sometimes stable gene-silencing. .Protein interference or introducing gene-product into the cell in large or effective small quantities .

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6) PROTEIN -CHIP PRODUCTION/ INTRABODIES…DIAGNOSTIC TOOL

In-vivo selected proteinaceous or protein derived /peptides that play a role as signal –transducers and are expressed on the surface are called APTAMERS. Such aptamers can be used a diagnostic tool and are also called INTRABODIES and like DNA-CHIPS used for rapid detection of genetic simliarity , these can function as PROTEIN-CHIPS and also serve as inert libraries. (Being more stable in general than DNA or RNA). But production on a large level requires the gene-library. Here cDNA libraries are of high applicability.

The importance of the protein-chips is that they form a tool for very rapid detection of diseases which are very difficult to be diagnosed in their early stages. E.g. Cancer

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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

cDNA

RNA extracted

rRNA sequencing Probe design

Data base of the sample

Reverse transcriptase

Procedures like DGGE* and TGGE* for sample analysis and purification

*Denaturing gradient gel electrophoresis*Temperature gradient gel electrophoresis

CREATION OF A DATABASE FOR AN ENVIRONMENTAL SAMPLE

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*Characterization OF ENVIRONMENTAL MICROBIAL COMMUNITIES using the enzyme-

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 arbitrarilychosen 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 assay-

Probe* + Target RNA

Hybridization in a solution

The mixture is diluted

Treatment with RNase

Hybridized portion free of single-stranded RNA

(Visualixed by electrophorsis and auto-radiography)

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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

SWATI MOHANTY 12MB/O9

Submitted to- Dr. Pratima Ray