protein synthesis/genetic mutations/ genetic engineering

proteins: macromolecules

• building block: amino acids (bonded w polypeptide bonds)   * coded by a sequence of 3 nucleotides (codons)
• ex. transport/membrane proteins, enzymes, muscle tissue

genetic code: DNA sequence that gives instructions to make proteins

RNA: ribonucleic acid

• single stranded
• nucleotide w ribose sugar, phosphate, and 4 bases (a, u, c, g)
• found in nucleus, cytoplasm (ribosomes)
• 3 types   * mRNA (messenger)     * takes info from DNA in the nucleus and brings it to ribosome     * found in nucleus, cytoplasm, ribosome   * tRNA (transport)     * takes amino acids to ribosomes and assembles into proteins     * found in cytoplasm, ribosome   * rRNA (ribosomal)     * loading dock for mRNA/tRNA     * found in ribosome (makes up ribosomes?)

scientific dogma: an established principle believed by scientists

• central dogma of biology: information is transferred from DNA → RNA → proteins (all DNA codes for proteins)

\ protein synthesis

• 2 parts   * transcription: making mRNA using DNA as a template → like DNA replication but simpler     * DNA = recipe book   * translation: taking RNA and changing it to proteins
• codons: set of 3 mRNA bases that code for an amino acid   * 64 possible codons
• anticodons: set of 3 tRNA bases (carry amino acid and match up w mRNA codons [complementary bases])

part 1: transcription ==[diagram]==

• occurs in the nucleus (eukaryotes) and nucleoid region (prokaryotes)
• makes mRNA as a messenger for DNA’s genetic info/instructions as it never leaves the nucleus
• RNA polymerase does all jobs: unzips/unwinds, adds new nucleotides, etc.   * builds on leading strand from 5’ to 3’
• ~~steps~~   * ~~DNA unzips~~   * ~~mRNA nucleotides pair up with 1 DNA strand only (leading strand)~~     * ~~complementary base pairs ( A → U, T → A, C → G, G → C)~~   * ~~when completed copying DNA information, mRNA floats off and travels to ribosome / DNA strands come back together (temporarily split to be used as template)~~
• 3 stages   * initiation: start transcription as RNA polymerase binds to a promoter region and starts to unwind DNA and add RNA bases   * elongation: RNA polymerase continues to add complementary bases   * termination: ends transcription as RNA polymerase lands at the stop/termination region and mRNA molecule floats off

post transcriptional modification

• DNA has different sections of nucleotides: “junk” introns that don’t code for proteins and “good” exons that do
• before leaving the nucleus, the introns are spliced out of the mRNA sequence of the exons are put together

part 2: translation ==[diagram]==

• occurs at the ribosome
• information carried by mRNA assembles amino acids carried by tRNA to make proteins (amino acid chain/polypeptide)   * start codon: starts translation (green light / capital letter) → AUG (met)   * stop codon: ends translation (red light / period) → UGA, UAA, UAG
• ribosome ==[diagram]==   * A: attachment site (for tRNA)   * P: peptide bond site where peptide bonds form   * E: exit site (tRNA leaves ribosome after unloading amino acid)
• steps   * mRNA attaches to ribosome at start codon (travels to ribosome through nuclear membrane and cytoplasm w microtubules)   * start codon (AUG) matches with tRNA anticodon (with amino acid methionine) at P site     * complementary bases   * next codon matches with tRNA anticodon (with an amino acid) at A site   * the 2 amino acids join together w peptide bonds     * amino acid at P “jumps ship” to A site     * tRNA at P moves to E and exits the ribosome after unloading its amino acid       * can be reused → goes to find new amino acid     * tRNA at A moves to P with attached amino acids   * new tRNA comes at A site and attaches more amino acids to growing chain w ^   * repeats until ribosome reaches a stop codon → protein is completed and floats away to be used

\ codon chart → use to find what amino acid is coded by a certain codon/DNA sequence/etc.

• if an mRNA codon chart → reference mRNA codon sequence to find amino acid
• if mRNA codon is AUG → find A on left side, U on top side, G on right side

base pairing practice

• DNA: TAT / GGA / CAG / TAG
• mRNA: AUA / CCU / GUC / AUC
• tRNA: UAU / GGA / CAG / UAG
• amino acids: ile / pro / val / ile

\ ~~cell organelles~~

• ~~ribosomes (free): make proteins to be used by the cell~~
• ~~ribosomes on rough ER: make proteins to be shipped out of the cell~~   * ~~golgi apparatus: quality check proteins that are being shipping out~~   * ~~proteins shipped out in vacuoles/vesicles~~

genetic mutations

• genes: controls what an organism looks like / its inherited traits by controlling protein shape and structure
• everyone has distinct DNA/genomes   * polymorphisms = variations in DNA/genes     * account for slight differences in people ex. hair color / but can also cause disease     * although all polymorphisms are the result of a gene mutation, only polymorphisms that are not within “normal variations” are called mutations
• how mutations occur → everyone accumulates changes in DNA throughout lives   * copying errors in DNA replication   * DNA damage through environmental agents ex. radiation, chemical mutagens   * inherited/hereditary     * mutations occurring in somatic (body) cells, ex. skin cells, are not hereditary     * germ line mutations are mutations in gametes (egg/sperm) and are hereditary / passed down from parent → child       * responsible for hereditary diseases
• DNA repair of mutations does not work as effectively as we age, causing accumulation of mutations and aging
• types of mutations   * any change in a gene sequence of bases can alter the gene’s meaning and change the protein that is made, or how or when a cell makes the protein   * 2 large classes: point mutation and frame shift mutation     * point mutation → substitution       * change/replacement in one or more bases of a gene sequence (none are added/deleted)     * frame shift mutation → insertion or deletions       * one or more bases are inserted or deleted → because cells read DNA in three-base codons, this changes each subsequent codon / “shifts”     * inversions       * a section of DNA is reversed     * DNA expression mutation       * do not change the protein but where and how much of a protein is made       * proteins may be made at the wrong time, wrong cell type, or wrong amount     * translocation       * DNA is transferred to another chromosome       * reciprocal (even exchange between chromosomes where there is no change in amount)       * non-reciprocal (one-way transfer resulting in change in amount)     * silent mutation: DNA base change occurs but does not change resulting amino acids     * nonsense mutation: DNA base change occurs and stop codon appears too early, stopping translation too early and resulting in shortened protein     * missense mutation: DNA base change occurs and replaces one amino acid with another, resulting in protein not formed properly

genetic engineering (synthetic biology): making changes in genome (DNA sequence) of organisms

gel electrophoresis ==[diagram]==

• agarose solution poured into glass plate casting tray, wells formed with comb
• DNA segments fragmented by restriction enzymes (ex. eco r1) are loaded into wells with a micropipette
• gel plate is immersed in a charged butter solution → power supply charges side with wells negatively and side far from the wells positively   * negatively charged DNA fragments move through the filter-like gel towards the positive side due to attraction
• this separates DNA fragments by length as smaller fragments move faster through holes in the gel
• result: DNA fingerprint ==[diagram]==   * unique pattern of bands to each organism as no one has identical DNA (except identical twins)   * length of DNA fragments are smaller as they are farther from the wells → measured by bp (base pairs)   * standard sample is used as a reference for size of samples
• purpose: identifying crime suspects, medical (transplants, etc.), parentage   * crime: a suspect’s DNA and the DNA of the perpetrator found at a crime scene would be an exact match   * parentage: all of the baby’s DNA must be a match to either the mom or dad’s DNA in a DNA fingerprint ==[diagram]==

PCR (polymerase chain reaction)

• purpose: to make copies of specific DNA fragments
• process: based on DNA replication   * denaturation (95 C): separate DNA strands by breaking hydrogen bonds at high temperature   * annealing (55 C): primers added to complementary sequences (“joining of bases”)   * extension (72 C): strand is grown as DNA polymerase attaches complementary bases
• enzyme used: taq polymerase → from archaebacteria thermophiles so it has high optimal temperature
• 2^n = amount of strands produced (n is number of PCR cycles) ==[diagram]==

genetic transformation: changing an organism’s DNA by transferring foreign DNA into host genome

• transgenic organism: an organism containing DNA from another species
• recombinant: DNA made from combining DNA from two different species
• ==[diagram]== example: inserting HGH (human growth hormone) into bacterial plasmid to make transgenic bateria   * section of human DNA (HGH gene) cut out by restriction enzyme   * same section cut out from bacterial plasmid by restriction enzyme   * plasmid and cutout HGH gene are bonded with ligase (spliced together) and inserted into bacterial cell   * human protein (HGH) is produced by bacteria
• purpose: make proteins for humans/animals (ex. insulin, HGH) more efficiently, sustainably, and ethically
• enzymes: ligase, restriction enzyme (ex. eco r1)

gene therapy: fixing DNA by replacing defective gene with normal ones

• CRISPR + CAS9 searches for and replaces bad genes with good genes   * purpose: gene therapy / process: genetic engineering   * real life examples: treating diseases like sickle cell

cloning: making genetically identical organisms

• process   * nucleus of an egg (haploid) is removed and destroyed of organism A   * empty egg is fused with a somatic/body cell of organism B   * the egg cell, with the nucleus of the body cell, starts dividing   * embryo is placed in the uterus of a foster mother where it develops to full term   * has DNA of organism B
• dolly the sheep - cloned by ian wilmut
• asexual process for plants

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