Gene Expression

  1. Gene expression
    the multistep process that ultimately results in the production of a functional gene product, either ribonucleic acid (RNA) or protein.
  2. Where regulation of gene ene expression happen and how it is mediated?
    - Regulation of gene expression occurs primarily at the level of transcription in both prokaryotes and eukaryotes

    • - Mediated through the binding of trans-acting proteins to
    • cis-acting regulatory elements on the DNA

    • - In Eukaryotes, regulation also occurs through
    • 1- modifications to the DNA
    • 2- Posttranscriptional and
    • 3- posttranslational events
  3. Function of lactose (lac) operon in prokaryotic
    The lactose (lac) operon contains the genes that code for three proteins involved in the breakdown of the disaccharide, lactose
  4. What are the three genes that code for 3 proteins in lactose
    (lac) operon in prokaryotic?
    • - lac Z gene codes for B-galactosidase, 
    •           which hydrolyzes lactose to galactose and glucose
    • - lac Y gene, which codes for a permease
    •           facilitates the movement of lactose into the cell
    • - lac A gene that codes for thiogalactoside transacetylase
    •           function is unknown
  5. Function of trp operon in prokaryotic
    The trp operon contains genes needed for the synthesis of tryptophan
  6. How trp operon is regulated in prokaryotic ?
    • It is regulated by 
    • Positive and negative control .
    • Attenuation
  7. Attenuation
    mRNA synthesis that escaped repression by trp is terminated before completion.
  8. Translation is also a site of prokaryotic gene regulation in prokaryotic
    When ribosomal proteins are in excess, they bind the Shine-Dalgarno sequence on their own polycistronic mRNA, preventing ribosomes from binding.
  9. Differences between gene regulation of Eukaryotic Gene Expression and Prokaryotic Gene Expression 
    • Gene regulation is more complex in eukaryotes.
    • Operons are not present
    • Coordinate regulation of the transcription of genes located on different chromosomes can be achieved through the Binding of trans-acting proteins to cis-acting elements
  10. mRNA stability
    • How long an mRNA remains in the cytosol before it
    • is degraded influences
    • how much protein product can be produced from it
  11. mRNA stability is important for
    • Regulation of iron metabolism
    • Gene-silencing process of RNA interference
  12. Gene expression in eukaryotes is also influenced by
    • Availability of DNA to the transcriptional apparatus
    • Amount of DNA
    • Arrangement of the DNA
  13. Plasmids are
    Small DNA molecules capable of self-replication
  14. rDNA
    Engineered DNA molecules generated
  15. Restriction Endonucleases Definition and the function
    • - Recognize short stretches of DNA (4-8 bp) that contain specific nucleotide sequences
    • - These sequences are palindromes

    - Biological function is to protect cells from foreign DNA.
  16. A Palindrome
    • Specific nucleotide sequences when read in the 5-3 direction, the sequence on the top strand is identical to the bottom strand.
    • Restriction endonnuclease recognize plaindrome
    • Image Upload 1
  17. Sticky or cohesive ends
    Image Upload 2

    • TaqI form staggered cuts that  produce sticky or cohesive ends
    • - DNA ligase, join the sticky ends of a DNA fragment 
  18. Blunt ends
    • Image Upload 3
    • HaeIII: produce fragments that have "blunt"ends that are double stranded
  19. Restriction sites
    DNA sequence cut by restriction enzyme/endonulcease is called a restriction site
  20. Difference between enzyme that recognizes a specific four-base-pair and enzyme that recognizes six base pairs
    Enzyme that recognizes a specific

    - Four-base-pair sequence produces many cuts in the DNA molecule, one every 44 bp.

    - Six-base-pairs produces fewer cuts, one every46 bp, longer pieces
  21. Cloning into a Plasmid Image Upload 4
    • Plasmid
    • Occur in bacteria
    • Much smaller than chromosomal DNA
    • Carry their own origin of replication
    • Easy to manipulate and identify
    • Many copies per cell
    • Carry antibiotic resistant genes
  22. Sanger dideoxy method
    Method to confirm the DNA sequence
  23. Why do we use dideoxynucleotides in Sanger dideoxy method?
    • Image Upload 5
    • We use Dideoxynucleotides (ddNTP) in DNA chain Termination
    • Dideoxynucleotides are essentially the same as nucleotides except they contain a H group on the 3’ carbon instead of (OH).
    • When integrated into a sequence, it prevents the addition of further nucleotides.
    • This occurs because a phosphodiester bond cannot form between the dideoxynucleotide and the next incoming nucleotide, and thus the DNA chain is terminated.
  24. What are the Methods to confirm the DNA sequence? Similarities ?
    • - The Maxam–Gilbert chemical sequencing method
    • - The Sanger–Coulson enzymatic sequencing method.
    • - The Automated Sequncers non-enzymatic sequncing, sequencing by hybridization. 

    • Both involve the ultimate generation of a full set of
    • fragments of the DNA strand to be sequenced.
  25. What do you add in the 4 tubes in Sanger's method?
    • Image Upload 6"G" tube: all four dNTP's, ddGTP and DNA polymerase
    • "A" tube: all four dNTP's, ddATP and DNA polymerase
    • "T" tube: all four dNTP's, ddTTP and DNA polymerase
    •  "C" tube: all four dNTP's, ddCTP and DNA polymerase
  26. What is Polymerase chain reaction (PCR) used for?
    • Image Upload 7
    • - PCR is used to amplify a specific region of a DNA strand (the DNA target)

    • - Amplify DNA fragments of up to ~10 kp (kilo base pairs)
    • - Some techniques - fragments up to 40 kb in size
  27. Component of PCR
    • Image Upload 8
    • DNA template that contains the DNA region (target) to be
    • amplified.
    • Two primers that are complementary to the 3' (three prime) ends of the DNA target.
    • Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.
    • Deoxynucleoside triphosphates (4dNTP)(dATP- dGTP- dCTP- dTTP), the building-blocks from which the DNA polymerase synthesizes a new DNA strand.
    • Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
    • Divalent cations, magnesium or manganese ions; Mg2+ is used
  28. Advantage of PCR over Cloning
    • Speed-  amplify millions of copies in few hours- irrespective of sample size
    • Sensitive – DNA sequence present in an individual cell can be amplified and studied.
    • PCR process is less difficult than cloning
  29. Applications of PCR
    • Comparison of normal cloned gene with an uncloned mutant form of the gene.
    • Detection of low abundance nucleic acid sequences (HIV)
    • Forensic analysis of DNA samples (crimes)
    • Prenatal diagnosis and carrier detection of cystic fibrosis.
  30. Techniques to Analysis of Gene Expression
    • mRNA expression
    • 1- Northern Blots
    • 2- Microarrays

    • Protein expression
    • 1- ELISA
    • 2- Western blots
    • 3- Proteomics

    ELISA and Western blots are commonly used to detect exposure to HIV by measuring the amount of anti-HIV antibodies in patient’s blood sample.
  31. Geneomic DNA will contain..................... if the targe therapeutic protein is eukaryotic
    • • If the target therapeutic protein is eukaryotic, the genomic DNA will contain both coding (exon) and non-coding (intron) sequences,
    • whereas the cDNA will be a reflection of the exons only.
  32. Where does the Recombinant protein produced?
    • E.coli, S. cerevisiae or in
    • Animal cell lines (mainly CHO or BHK cells).
  33. Protein Engineering 
    Manipulation of a protein’s amino acid sequence.
  34. Advantages of Protein engineering?
    • Understanding of the link between a polypeptide’s amino acid sequence and its structure
    • Studying the relationship between structure and its function.
    • Tailor structural or functional attributes of therapeutic and other commercially important proteins
  35. Reasons for Primary Non-function of Islet Grafts
    • 1- Transplantation In Liver Sinusoids
    •     Transplantation Under Kidney 
    • 2- Hypoxia to the inner core of islets due to inadequate revascularization
    • 3- Inflammation and immune mediated cell death and loss of islet function
Card Set
Gene Expression
Gene Expression