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Molecular Genetics (Viruses (Eukaryotic Viruses - DNA or RNA genomes,…

- - - - RNA PROCESSING
        Before mRNA can exit the nucleus and bind to a ribosome in the cytoplasm, it must be modified. First a 5’ cap and a poly-A tail are added to its 5’ and 3’ ends respectively, to protect the mature mRNA from degradation by hydrolytic enzymes in the cytoplasm. Next, noncoding segments of the primary transcript, called introns, are removed by spliceosomes. The exons, untranslated regions (UTR), 5’ cap and poly-A tail are then spliced together to produce the final mRNA, which can direct the synthesis of a particular polypeptide. Since different segments can be considered introns or exons, a single gene may be able to code for different polypeptides by alternative RNA splicing.
        
        TRANSLATION
        During translation, the genetic message carried by mRNA is translated into the appropriate polypeptide or RNA molecule. After the mature mRNA exits the nucleus through a nuclear pore, it binds to a small ribosomal subunit, initiator tRNA and large ribosomal subunit, forming the translation initiation complex. tRNA plays a vital role in the elongation of the polypeptide by transferring cytoplasmic amino acids to the growing polypeptide chain. The anticodon at one end of the tRNA binds to the complementary codon of the mRNA and the corresponding amino acid is covalently bonded to its other end by aminoacyl-tRNA synthetases. As the tRNA moves from the ribosomes A site to P site, its amino acid is added to the polypeptide chain. The tRNA then moves to the E site and exits the ribosome. This process continues until a stop codon is reached and a release factor binds to the mRNA and hydrolyzes the bond between the polypeptide and the tRNA.
  - - - base pair substitution
        
        Silent mutation
        
        No amino acid change because of redundancy in code
        
        Missense mutation
        
        1 amino acid changed
        
        Nonsense mutation
        
        Amino acid changed to stop codon
      - Lead to sickle cell anemia
        
        missense mutation --> change from hydrophilic amino acid to hydrophobic amino acid
    - - Insertions - adding bases
      - Deletions - losing bases
      - Cystic Fibrosis - primarily whites of European descent, normal allele codes for a membrane protein that transports Cl- across membrane, defective or absent channels limit transport of Cl- across membranes --> thicker and stickier mucus coats around cells, mucus builds up in pancreas, lungs, digestive tract --> causes bacterial infections
        
        Deletion of amino acid leads to cystic fibrosis
- - - - If bacteria has enough tryptophan then it doesn't need to make enzymes used to build tryptophan
    - - If bacteria encounters new sugar like lactose then it needs to start making enzymes to digest lactose
    - - Repressible
        
        Usually functions in anabolic pathways, when end product is in excess, cell allocates resources to other uses
      - Inducible
        
        Digestive pathway model - when lactose is present, binds to lac repressor protein and triggers repressor to release DNA --> induces transcription --> conformational change in repressor protein --> needs to make lactose-digesting enzymes --> lactose is allosteric regulator of repressor protein
        
        Usually functions in catabolic pathways, produce enzymes only when nutrient is available, cell avoids making proteins that have nothing to do, cell allocates resources to other uses
        
        Inducer binds and inactivates repressor protein
    - - Synthesis pathway model - when excess tryptophan is present it binds to tryp repressor protein and triggers repressor to bind to DNA --> blocks transcription ---> Tryptophan is an allosteric regulator of repressor protein
      - Corepressor binds to repressor protein, activating it
- - - - From double helix --> nucleosomes --> chromatin fiber --> looped domains --> chromosomes
        
        Nucleosomes --> beads on a string, 1st level of DNA packing, histone proteins
        
        Positively charged amino acids bind tightly to negatively charged DNA, degree of packing of DNA regulates transcription
        
        Heterochromatin - highly packed --> no transcription --> genes turned off
        
        DNA methylation - blocks transcription factors --> no transcription --> genes turned off --> attachment of methyl groups to cytosines nearly permanent inactivation of genes
        
        Euchromatin - loosely packed --> genes turned on
        
        Histone acetylation --> acetylation of histones unwinds DNA, loosely wrapped around histones, enables transcription --> genes turned ON --> attachment to histones conformational change in histone proteins --> transcription factors have easier access to genes
    - - Control regions of DNA
        
        Promoter: nearby control sequence of DNA, binding of RNA polymerase and transcription factors
        
        Enhancer: Distant control sequences on DNA, binding of activator proteins enhances rate of transcription
        
        Activator proteins: bind to enhancer sequence and block gene transcriptions
    - - Alternative RNA processing --> variable processing of exons creates a family of proteins
    - - Life span of mRNA determins amount of protein synthesis
      - RNA interference
        
        Small interfering RNAs, short segments of RNA bind to mRNA create sections of double stranded DNA --> death tag for mRNA --> triggers degradation of mRNA --> causes gene silencing --> turns off gene --> no protein produced
    - - Block initiation of translation stage, regulatory proteins attach to 5' end of RNA, prevent attachment of ribosomal subunits and initiator tRNA blocks translation of mRNA to protein
    - - Folding, cleaving, adding sugar groups, targeting for transport
    - - Ubiquitin death tag --> marks unwanted proteins with a label, labeled proteins are broken down by proteasomes
        
        Proteasomes --> protein degrading machine, cell's waste disposer, recycling
- - - - RNA viruses lack replication error checking mechanisms and thus have higher rates of mutations --> leads to rapid mutation of viruses and different types
    - - genome is RNA but virus has code for reverse transcriptase which makes a DNA copy of the genome which is then spliced into the host cell chromosome, RNA-DNA
- - - - Identify and isolate the gene of interest and the cloning vector
      - Cut both the gene of interest and the vector with the same restriction enzyme
      - Join the two pieces of DNA with ligase
      - Get the vector carrying the gene of interest into a host cell
      - Select for cells that haven been transformed