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Ch 9 DNA-Based Information Technologies, 9.2 Exploring Protein Function on…

- - - - paralogs: genes similarly related to each other within a single species
      - orthologs: genes that occur in different species but have a clear sequence and functional relationship to each other
      - analogs: another gene, but with a similar function
    - - provides additional evidence for an orthologous relationship between genes at identical locations within the related segments
      - for example, seen between human and mouse genomes
- - - - RNA-Seq: method that determines the RNAs that are transcribed from a genome under a given set of conditions
        
        single cell RNA-seq (scRNA-Seq): application of RNA-Seq to a single cell, possible due to the high sensitivity of the method
        
        process:
        
        Fragment and prime isolated RNA
        
        Create RNA-DNA hybrid
        
        Create dsDNA
        
        Ligate to duplex adapters for seqencing
        
        Use PCR to amplify for sequencing
    - - mass spectrometry can accurately catalog and quantify the thousands of proteins present in a typical cell
        
        complementary approach to RNA-Seq
        
        provides information about how proteins are modified
    - - green fluorescent protein (GFP): jellyfish protein that serves as a useful location marker
        
        a target gene fused to the GFP gene generates a highly fluorescent protein
        
        variants of other colors and characteristics also exist
      - immunofluorescence: alternative approach for visualizing the endogenous protein that involves fixation (and death) of the cell
        
        epitope tag: short protein sequence that is bound tightly by an antibody, with the tag being on the protein of interst
- - - - immunoprecipitation: process of precipitating a fusion protein containing the gene of interest and a gene for an epitope tag by antibodies to the epitope
        
        proteins that bind to the tagged protein will also precipitate
      - Tandem Affinity Purification (TAP) tags: two consecutive tags that are fused to a target protein to enhance the selectivity of immunoprecipitation
        
        first tag (protein A) binds mammalian immunoglobulin G (IgG)
        
        second tag binds calmodulin
    - - the two domains of Gal4p must be brought together to function correctly
      - probes molecular interactions in vivo
- - - - components of the CRISPR/Cas complex:
        
        guide RNAs: transcribed viral spaced sequences that are cleaved
        
        trans-activating: CRISPR RNA (tracrRNA)
        
        1+ Cas proteins
      - the complex binds and destroys invading bacteriophage DNA by the Cas protein nuclease activities
    - - single Cas protein (Cas9)
        
        has two separate nuclease domains, which each cleaving one strand of DNA
      - single guide RNA (sgRNA): consists of gRNA and tracrRNA fused into a single RNA
    - - Plasmid and fragment introduced into cell by electroporation
      - Plasmid directs synthesis of CRISPR/Cas9 complex
      - CRISPR/Cas9 complex scans genome for target sequence
      - In a double-strand break, nonhomologous end joining leads to gene mutation. In single-strand break, recombination with added fragment leads to homology-directed repair.
    - - high-throughput genetic screening can be used to identify genes required for a specific stress or selection response
      - cells in which genes required to survive the treatment are inactivated or activated by the CRISPR/Cas9 variant will die or thrive
- - - - generated by PCR; including restriction endonuclease cleavage sites facilitates the subsequent cloning of amplified DNA
        
        cleavage of PCR-amplified DNA creates sticky ends used to ligate the amplified DNA to a cloning vector
    - - Restriction Endonucleases and DNA ligases yield recombinant DNA
        
        Restriction endonucleases (restriction enzymes): recognize and cleave DNA at specific sequences
        
        Type II restriction endonucleases: catalyze the hydrolytic cleavage of DNA phosphodiester bonds within the recognition sequence; require no ATP
        
        restriction-modification system: the restriction endonuclease and the corresponding methylase
        
        methylases: catalyze methylation of host DNA to protect it from digestion by the host cell's restriction endonucleases
        
        leave either sticky ends or blunt ends
        
        sticky ends: unpaired bases on the ends
        
        due to endonucleases making staggered cuts
        
        can base pair with each other or complementary sticky ends
        
        blunt ends: no unpaired bases on the ends due to endonucleases making straight cuts
        
        DNA Ligases: join the DNA fragment to be cloned to a suitable cloning vector
        
        DNA polymerase: fills gaps in duplexes by stepwise addition of nucleotides to 3' ends
        
        DNA ligase: joins two DNA molecules or fragments
        
        reverse transcriptase: makes a DNA copy of an RNA molecule
  - - - allow amplification of inserted DNA segments
      - popular examples:
        
        plasmid: circular DNA molecule that replicates separately from the host chromosome
        
        5,000-400,000 bp
        
        usually have a symbiotic role in the cell
        
        key features of constructed E. coli plasmid:
        
        origin of replication (ori): sequence where replication is initiated
        
        resistance genes
        
        recognition sequences for restriction endonucleases
        
        bacterial transformation and electroporation:
        
        transformation: laboratory process by which small plasmids are introduced into bacterial cells through heat shock treatment
        
        reason why is not well understood
        
        becomes less successful as plasmid size increases
        
        bacterial artificial chromosomes
        
        BACs: composed of the plasmid vector and large segments of cloned DNA
        
        have stable origins of replications that maintain low copy numbers
        
        contain par genes from an F plasmid that direct the reliable distribution of the recombinant chromosomes at cell division
        
        Camr: positive selection marker
        
        IacZ: screenable marker
        
        yeast artificial chromosomes
        
        YACs: composed of the plasmid vector and large segments of cloned DNA
        
        DNA cloned in a YAC can be altered to study:
        
        the function of specialized sequences in chromosome metabolism
        
        mechanisms of gene regulation and expression
        
        includes an origin of replication (ori), a centromere (CEN), two telomeres (TEL), and selectable markers (x and Y)
        
        Construction of a YAC Process:
        
        Digestion with appropriate restriction enzymes generates two separate DNA arms, each with a telomeric end and one selectable marker
        
        A large segment of DNA is ligated to the two arms to create a YAC.
        
        The YAC transforms yeast cells, and the cells are selected for X and Y; the surviving cells propagate the DNA insert.
        
        stability:
        
        increases with the length of the cloned DNA segment
        
        inserts more than 150,000 bp are very stable
        
        inserts less than 100,000 bp are gradually lost during mitosis
        
        YACs that lack a telomere at either end are rapidly degraded
  - - - DNA metabolism is well understood
      - many naturally occurring cloning vectors (plasmids and bacteriophages)
      - techniques for moving DNA from one bacterial cell to another
  - - - useful for inserting additional DNA at a later point
  - - - elucidating protein function
      - studying reaction mechanisms
      - generating antibodies to the proteins
      - reconstituting complex cellular activities in the test tube with purified components
      - examining protein binding partners
    - - bacteria
        
        expressing a eukaryotic protein in a bacterium
        
        eukaryotic genes have surrounding sequences needed for their transcription and regulation
        
        expression vectors: cloning vectors with transcription and translation signals needed for the regulated expression of a cloned gene
        
        sequences do not function in bacteria
        
        transcription from the Lac Promoter
        
        gene of interest fused to lactose operon promotor and regulatory sequences
        
        transcription occurs when lactose is added to the medium
        
        regulation is "leaky"
        
        transcription from the bacteriophage T7 Promoter and RNA polymerase
        
        cloned gene is fused to a T7 promoter and transcribed by T7 RNA polymerase
        
        affords tight regulation
        
        most common hosts for protein expression
        
        advantages:
        
        regulatory sequences are well-understood
        
        can express high levels of cloned protiens
        
        easy to store and grow
        
        efficient methods for transforming and extracting DNA
        
        can be grown in huge amounts
        
        disadvantages:
        
        some heterologous proteins do not fold correctly
        
        proteins may not undergo necessary posttranslational modifications or proteolytic cleavage
        
        some gene sequences can be difficult to express
        
        many eukaryotic proteins aggregate into insoluble cellular precipitates (inclusion bodies)
      - yeast
        
        advantages:
        
        probably the best understood eukaryotic organism
        
        expression of eukaryotic genes can be more efficient
        
        proteins may be folded and modified more accurately
        
        underlying principles:
        
        cloned genes must be linked to appropriate promoters
        
        gene expression can be controlled by choosing an appropriate medium
        
        disadvantages:
        
        heterologous proteins may not fold properly
        
        yeast may lack the enzymes needed to modify the proteins to their active forms
        
        certain features of the gene sequence may hinder expression of a protein
      - insects and insect viruses
        
        baculoviruses: insect viruses with double-stranded DNA genomes
        
        p10 and polyhedrin genes can be replaced with the gene for a heterologous protein
        
        hard to work with, so replaced with bacmids
        
        bacmids: large circular DNAs that include the entire baculovirus gene and sequences that allow replication of the bacmid in E. coli
        
        recombinant bacmids are transfected into insect cells
        
        transfection: term used when the DNA used for transformation includes viral sequences and leads to viral replication
        
        advantages:
        
        may successfully replicate protein-modification patterns and produce active, correctly modified proteins
        
        wide range commercially
        
        disadvantages:
        
        not successful with all proteins
      - mammalian cells in culture
        
        DNA introduced into mammalian cells using engineered mammalian viruses as vectors
        
        advantages:
        
        proteins can be expressed either transiently or permanently
        
        proper posttranslational modification can be ensured
        
        disadvantage:
        
        very expensive
- - - - Add synthetic DNA oligonucleotide primer to target RNA to be amplified
      - Add reverse transcriptase to catalyze 5'->3' DNA synthesis
      - Heat to separate strands; reverse transcriptase inactivated; RNA no longer used as a template.
      - Add synthetic oligonucleotide primers.
      - Thermostable Taq DNA polymerase catalyzes 5'->3' DNA synthesis.
      - Steps three and four are repeated; the DNA polymerase from step five remains present and active. After 20 cycles, the target sequence has been amplified about 10^6-fold.