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Recombinant DNA Technology
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Recombinant DNA Technology

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@WellRoundedArtePovera

Questions and Answers

What is the function of the σ factor in RNA polymerase?

  • To synthesise the enzyme
  • To transcribe the DNA into RNA
  • To recognise specific promoter sequences (correct)
  • To bind to the activator-binding site

    • What is the purpose of genetic control of enzyme activity?

  • To synthesise enzymes directly
  • To block enzyme activity
  • To control transcription of the mRNA needed for an enzyme's synthesis (correct)
  • To degrade mRNA

    • What is the function of repressors in genetic control?

  • To block transcription of mRNA (correct)
  • To degrade RNA polymerase
  • To promote transcription of mRNA
  • To synthesise proteins

    • What is the difference between an operon and a regulon?

    <p>Operon is a set of genes transcribed as polycistronic message, while regulon is a set of genes transcribed as monocistronic units</p> Signup and view all the answers

    What is the function of activators in genetic control?

    <p>To promote transcription of mRNA</p> Signup and view all the answers

    What is the sequence of the promoter recognised by the classical 'vegetative' E. coli sigma factor?

    <p>5'- TTGACA--17 bp spacing—TATAAT-5</p> Signup and view all the answers

    What is the function of translational control in genetic control?

    <p>To control translation of mRNA</p> Signup and view all the answers

    What is the purpose of the activator-binding site in genetic control?

    <p>To bind regulatory proteins</p> Signup and view all the answers

    What is the role of lactose in the lactose operon?

    <p>To induce the synthesis of the enzymes needed for its utilization</p> Signup and view all the answers

    What happens to the repressor protein when lactose is present?

    <p>It changes shape and can no longer bind to the operator region</p> Signup and view all the answers

    What is the result of the repressor protein binding to the operator region?

    <p>RNA polymerase is unable to bind to the promoter region</p> Signup and view all the answers

    What is the function of the regulator gene in the lactose operon?

    <p>To code for the repressor protein</p> Signup and view all the answers

    What happens to the three enzyme genes in the absence of lactose?

    <p>They are not transcribed into mRNA</p> Signup and view all the answers

    What is the role of the operator region in the lactose operon?

    <p>It binds to the repressor protein</p> Signup and view all the answers

    What is the outcome when the inactivated repressor protein is unable to bind to the operator region?

    <p>RNA polymerase can bind to the promoter region and transcribe the three enzyme genes</p> Signup and view all the answers

    What type of protein is the repressor protein?

    <p>Allosteric protein</p> Signup and view all the answers

    What is the result of competitive inhibition of enzyme activity?

    <p>The enzyme can no longer bind to the starting substrate</p> Signup and view all the answers

    What is the purpose of DNA ligase in gene cloning?

    <p>To join the DNA to be cloned to the vector</p> Signup and view all the answers

    What is the advantage of using plasmids as vectors?

    <p>They allow for easier manipulation, copying, and purification of recombinant DNA</p> Signup and view all the answers

    What is the function of restriction endonucleases?

    <p>To cut DNA into smaller pieces</p> Signup and view all the answers

    What is the objective of creating a DNA library?

    <p>To isolate genes encoding a specific protein</p> Signup and view all the answers

    What is the purpose of PCR?

    <p>To amplify a specific genome sequence</p> Signup and view all the answers

    What is a characteristic of plasmids?

    <p>They are circular extrachromosomal DNA molecules</p> Signup and view all the answers

    What is the purpose of using a four-cutter restriction enzyme in creating a DNA library?

    <p>To cut the DNA into smaller fragments</p> Signup and view all the answers

    What is the result of the fourth step in gene cloning?

    <p>The transformed colony is selected and amplified</p> Signup and view all the answers

    What is the purpose of partial restriction endonuclease digestion?

    <p>To collect fragments of a specific target size</p> Signup and view all the answers

    What is the purpose of size fractionation in DNA library creation?

    <p>To separate the fragments by size</p> Signup and view all the answers

    What is the type of end produced by restriction endonucleases?

    <p>Either sticky or blunt ends</p> Signup and view all the answers

    What is used to identify the presence of the target DNA?

    <p>A DNA probe</p> Signup and view all the answers

    What is the purpose of cloning the cells that contain the target fragments?

    <p>To isolate the target fragments</p> Signup and view all the answers

    What is the result of a successful transformation reaction?

    <p>A plasmid with an insert</p> Signup and view all the answers

    What is the purpose of antibiotic selection in transformation?

    <p>To select for cells that contain the plasmid</p> Signup and view all the answers

    Why is it difficult to clone eukaryotes gene into prokaryote?

    <p>Due to post translation modification and the presence of introns</p> Signup and view all the answers

    How is mRNA extracted from other ribosomal RNA?

    <p>Using a chromatography method with beads attached to poly TTTTT tail</p> Signup and view all the answers

    What is the first step in converting mRNA into dsDNA?

    <p>Reverse transcription of the mRNA</p> Signup and view all the answers

    What is the function of the klenow fragment of E. coli DNA polymerase I?

    <p>To synthesize dsDNA from ssDNA</p> Signup and view all the answers

    What is formed after reverse transcription of the mRNA?

    <p>ssDNA with a hairpin loop</p> Signup and view all the answers

    What is the purpose of treating the mRNA-bead complex with a buffer?

    <p>To break A:T hydrogen bonds and release the mRNA</p> Signup and view all the answers

    Study Notes

    Recombinant DNA Technology

    Promoter Reminder

    • Promoter sequences are recognized by RNA polymerase holoenzyme
    • Promoter lies adjacent to an activator-binding site
    • RNA polymerase is composed of 5 polypeptides (α2ββσ) when it binds to promoters
    • σ factor is the protein that recognizes specific promoter sequences
    • Most important nucleotides are centered at –35 and –10 relative to the transcription start-site for promoters recognized by the classical "vegetative" E. coli sigma factor, σ70
    • Promoter sequence: 5'- TTGACA—17 bp spacing—TATAAT-5 to 9 bp- START Codon

    Genetic Control

    • Genetic control of enzyme activity refers to controlling transcription of mRNA needed for an enzyme's synthesis
    • In prokaryotic cells, this involves induction or repression of enzyme synthesis by regulatory proteins that can bind to DNA and either block or enhance the function of RNA polymerase
    • Regulatory proteins are part of either an operon or a regulon
    • Operon: a set of genes transcribed as a polycistronic message that is collectively controlled by a regulatory protein
    • Regulon: a set of related genes controlled by the same regulatory protein but transcribed as monocistronic units
    • Regulatory proteins may function either as repressors or activators

    Genetic Control Groups

    • Repressors: Regulatory proteins that block transcription of mRNA
    • Activators: Regulatory proteins that promote transcription of mRNA
    • Translational control (antisense RNA): A strand of RNA complementary to a mRNA coding for some polypeptide or protein

    Lactose Operon

    • E. coli will only synthesize the three enzymes it requires to utilize lactose if that sugar is present in the surrounding environment
    • Lactose functions as an inducer
    • In the absence of lactose, the repressor protein binds to the operator and RNA polymerase is unable to transcribe the genes for utilization of lactose
    • When lactose is present, it binds to the allosteric repressor protein and causes it to change shape, allowing RNA polymerase to transcribe the genes required for lactose degradation

    Gene Cloning

    • Gene cloning involves four steps:
      • The vector DNA is cleaved with one or more restriction enzymes
      • The DNA to be cloned is joined to the vector by ligase enzyme, generating a recombinant molecule
      • The recombinant DNA is introduced into a host cell (e.g., bacteria)
      • The transformed colony is selected to amplify

    Vectors

    • Vectors: DNA molecules used to transfer foreign DNA fragments between cells
    • Types of vectors:
      • Plasmid
      • Phage (virus)
      • Cosmid
      • Yeast Artificial Chromosome (YAC)
      • Bacterial Artificial Chromosome (BAC)

    Creating and Screening a Library

    • Isolation of genes encoding proteins is a fundamental objective of molecular biotechnology
    • A DNA library for the selected organism can be generated by:
      • Using restriction enzyme to cut the DNA into smaller fragments
      • Inserting each fragment into a vector
      • Cloning and identifying the cells containing the target fragments
    • The presence of the target DNA can be identified with a DNA probe

    Cloning DNA Sequence That Encode Eukaryotes Protein

    • Due to post-translational modification and the presence of introns, it is difficult to clone eukaryotic genes into prokaryotic ones
    • To solve this problem, mRNA can be extracted and used as all introns regions are already removed
    • mRNA can be isolated using a special isolation technique based on devising a chromatography method where the beads are attached to poly TTTTT tail, which will bind to the poly AAAAA tail present at the end of the mRNA
    • Cloning DNA sequence that encodes eukaryotic protein involves:
      • Converting mRNA into dsDNA using reverse transcription and DNA synthesis
      • Cloning the cDNA
      • Identifying the transformed cell

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    Description

    This quiz covers the basics of recombinant DNA technology, including promoter sequences, RNA polymerase, and activator-binding sites. It's based on Lecture 5 of Microbial Biotechnology.

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