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    Gene, gene expression, gene silencing and RNAi
    By Ashwani Kumar | November 8th 2012 02:30 AM | 6 comments | Print | E-mail | Track Comments
    About Ashwani

    Professor Emeritus ,Former Head of the Department of Botany, and Director Life Sciences, University of Rajasthan, Jaipur. 302004, India At present...

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    Gene Expression?–What is a Gene?

    A gene codes for a homogeneous ‘functional unit’ – classically a protein, or rRNA, tRNA (other RNAs are emerging). The DNA sequence defines the gene, along with processing steps that determine the product. Generally gene product is a protein which is synthesised by a process of transcription i.e. DNA to mRNA and translation i.e. mRNA to Protein synthesis. 

    In a given organism every cell has the same genomic DNA, the different characteristics of tissues are the result of differential expression.         Which genes are expressed at what stage ?  What is the relationship between external or internal signal perception ?  All this  has important role in gene expression systems.    Once activated it is important to know    how much of each of those gene products are synthesised. Genome interacts with environment and final products are   reproducible, and represent  diagnostic expression differences between individuals, between healthy and sick or stressed tissues and as the result of many environmental interactions.     What is the function of a gene (or set of genes) in a cell and how does this affect the total biology of the cell (tissue,organism, etc.)

    The 3 major strategies for determining the sequence of a gene which could be expressed.

    (1) Reverse engineer from the protein, ie sequence the protein and  convert the amino acids to appropriate nucleotides. - There is ambiguity because of the redundancy of the genetic code-more than  one nucleotide triplet can give the same amino acid. Only when a full-length (m)RNA or protein has been sequenced is there definitive evidence for the existence of a particular gene.

    Types of Gene  Expression Data Measurement

    1. Tag- id (sequence part and record # occurrences)

            ESTs, SAGE and MAGE, andMPSS (LYNX).

    2. Chromatographic (separation on physical characteristics   eg. length, mass, enzyme recognition site).

            cDNA-AFLP,GC/HPLC/MS-MALDI-TOF

            Differential Display

    3. Sequence recognition/amplification (e.g. PCR and     internal sequence recognition)

            RT-PCR and TaqMan, Beacons

    4. Array and chip (use a “known” sequence and intensity)

            Nylon, glass and liquidarrays of cDNAs/ESTs and

            silicon “chips” ofoligomers (also Northern blots).

     

     

    The Gene Expression Analysis in the Genomic era depends on : 

    •       High throughput, parallel assays have been developed, in which many genes or gene products are studied simultaneously. This involves: 

         DNA:High throughput shotgun sequencing or sequencing by hybridization chips

         RNA:Microarrays and GeneChips, SAGE, cDNA-AFLP (Functional Genomics,Trancriptomics)

         Proteins:Protein arrays, yeast 2-hybrids, MALDI-TOF MSMS (Proteomics,        Expressionmonitoring)

    Gene silencing: (a) Silencing of transgenes

    Transgene silencing falls in the field of epigenetics: 

    Epigenetics is the  study of mitotically/ meiotically heritable changes in the function of a gene that cannot be explained by changes in its DNA sequence.

    Modes of silencing:

    (a) Transcriptional silencing (TGS)i.e.  silending of gene and inhibition of  mRNA synthesis . It can be of two types:                    a. cis TGS                            b. trans TGS

    (b) Post-transcriptional silencing (PTGS):  Here post transcriptional regulation is carried out between mRNA and protein synthesis: It can also be of two types: a. cis PTGS                         b. trans PTGS

      Post-Transcriptional Gene Silencing (PTGS) was discovered by R. Jorgensen in (1990) in plants.        When Jorgensen introduced a re-engineered gene into Petunia that had a lot of homology with an endogenous Petunia gene, both genes became suppressed. This is also called as –  Co-suppression. This –       suppression was mostly due to increased degradation of the mRNAs (from the endogenous and introduced genes). The regulation was to manipulate pigment               synthesis genes in petunia.       Genes produced  proteins which were  enzymes of the flavonoid/ anthocyanin pathway:        CHS:chalcone synthase;        DFR:dihydroflavonol reductase

    Introduction  of these genes in  Petunia   using a strong viral promoter,  resulted  in drop of mRNA levels                and so did pigment levels in many  transgenics i.e. genetically modified plants.

    RNAi interference is further advanced technique discovered recently 

      The discovery of this technique resulted in was discovered  by  Dr. Craig Mello    and        Dr.  Andrew Fire   who were awarded 2006 Nobel Prize in    Physiology& Medicine, ). 

     C. elegans (first animal : a nematode) where   antisense RNA in vivo was employed. 

     Control “sense” RNAs also produced suppression of        target gene!

    Sense RNAs were contaminated with dsRNA and it came out that      dsRNA was the suppressing agent. e.g. –        Double-stranded RNA (dsRNA) induced interference of the Mex-3 mRNA in the nematode C. elegans.

    –       Antisense RNA  or dsRNA  for the mex-3 (mRNA) was injected into C. elegans ovaries, and then mex-3 mRNA was detected in embryos by in situ hybridization with a mex-3 probe.

    It was concluded that 

    (1) dsRNA reduced mex-3 mRNA better than antisense mRNA. 

    (2) the suppressing signal moved from cell to cell.

    Mechanism of RNAi: Role of Dicer

    1.  Cells (plants and animals) undergoing RNAi    contained small fragments (~25 nt) of the RNA being     suppressed.

    2.  A nuclease (Dicer) was purified from Drosophila    embryos that still had small RNA fragments           associated with it, both sense and antisense.

    3.  The Dicer gene is found in all organisms that exhibit    RNAi, and mutating it inhibits the RNAi effect.

    Conclusion: Dicer is the endonuclease that degrades dsRNA into 21-24 nt fragments, and in higher eukaryotes also pulls the strands apart via intrinsic  helicase activity.

     

    Comments

    From:
    http://biobabel.wordpress.com/2012/11/03/a-chimeric-fusion-of-rna-and-dn...

    Diemer, G., & Stedman, K. (2012). A novel virus genome discovered in an extreme environment suggests recombination between unrelated groups of RNA and DNA viruses Biology Direct, 7 (1) DOI: 10.1186/1745-6150-7-13

    Ashwani Kumar
    Thanks for sending this reference:Diemer, G.,&Stedman, K. (2012). A novel virus genome discovered in an extreme environment suggests recombination between unrelated groups of RNA and DNA viruses Biology Direct, 7 (1) DOI: 10.1186/1745-6150-7-13
    My pleasure.

    I'm an interested layman, so keep up the good work.

    Ashwani Kumar
    Thanks for encouragement
    Ashwani Kumar

     RNA interference or RNAi is now commercially available: 

       (a)  Process results in down-regulation of a    gene at the RNA level (i.e., after transcription)

       (b)  There is also gene silencing at the transcriptional level (TGS) –        Examples: transposons, retroviral      genes, heterochromatin

       (c) PTGS is heritable, although it can be modified in subsequent cell divisions or generations.      It is an epigenetic phenomenon.   Epigenetics- refers to heritable changes in phenotype or gene expression caused by mechanisms other than changes in the underlying DNA sequence.

     (d) Antisense Technology: •       Used from ~1980 on, to repress specific genes. It is –       alternative to gene knock-outs, which were/are very difficult to do in higher plants and animals. By introducing an antisense gene (orasRNA) into cells, the asRNA would “zipup” the complementary       mRNA intoa dsRNA that would not be translated.     The “antisense effect” was highly variable, andin light of   the discovery of RNAi, asRNA probably inhibited its target       by inducing RNAi rather than inhibiting translation.

     Morpholino antisense oligos: They have  high mRNA binding affinity.   Depending on the oligo sequence selected,they either can

    (a)   Block translation initiation in the cytosol (by targeting the 5' UTR through the first 25 bases of coding sequence), 

    (b)  Modifypre-mRNA splicing in the nucleus (by targeting splice junctions orsplice regulatory sites) 

    (c)  Inhibit miRNA maturation andactivity (by targeting mature miRNA or pri-miRNA), as well as more exotic applications such as ribozyme inhibition or translational frameshifting. They  have been shown effective in animals, protists, plants and bacteria.  Microinjection or electroporation of Morpholino oligos into the embryos of frogs, zebrafish,chicks, sea urchins and other organisms successfully and specifically shutsdown the expression of targeted genes.  

    Ashwani Kumar

    Anti-mRNA Strategies have bright future and will lead to developments of anti viral drugs also in near future once the genomic data bases are perfected for broader number of plants and animals and microarrays technique is improved. Future of biotechnology lies in improved instrumentations and faster alignment techniques and simulations for possible solutions using bioinformatics in my view.  

     Zamecnik and Stephenson in (1978) were first to use  Antisense compounds as therapeutic agents.  The revolution in the availability of viral and human genomic sequences enhanced the development of the antisense technology.

    RNA Interference (RNAi): RNAi is an innate cellular process that directs the degradation of mRNA homologous to short double stranded RNA (dsRNA).  

      Antisense Oligonucleotides are unmodified or chemically modified ssDNA, RNA or their analogs. They  are 13-25 nucleotides long made of synthetic genetic material. _They are  specifically designed to hybridize to the corresponding RNA by Watson-Crick binding. 

    Mechanism of Action of Antisense Oligonucleotides:

    1. Translational Arrest by Blocking the Ribosome.

    2. Activation of RNase H

    3. Ribozymes: Ribozymes are RNA molecules that catalyze biochemical reactions. They cleave single-stranded regions in RNA through transesterification or hydrolysis reactions that result in cleavage of phosphordiester bonds. Identification of  the minimum ribozyme structure and  enhancing stability to nucleases is an important step.

     

    Over the past decade, substantial development in antisense science and manufacturing led to the approval of the first antisense drug fomivirsen (VitraveneTM) for the treatment of AIDS-related CMV retinitis and over 50 drugs are in various stages of development or testing.