BIOL 112

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BIOL 112
2012-04-23 22:47:17

Biology Notes
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  1. Experiment to prove that some genetic traits can be induced
    • Using streptococcus pneumoniae, Griffith used two strains to infect mice with the virus.
    • - S (smooth) was the lethal strain
    • - R (rough) was the non-lethal strain
    • By combining dead S-bugs and live R-bugs, mice died.
    • Conclusion: A chemical component from one cell is capable of genetically transforming another cell.
  2. Hershey Chase Experiment
    • Used virus where:
    • - Coat labelled with radioactive S
    • - DNA labelled with radioactive P
    • Conclusion: DNA, not protein, enters bacterial cells and directs the assembly of new virus particles.
  3. Watson and Crick
    • Developed the structure of DNA to further comprehend replication and phenotypes.
    • - Used Rosalind Franklin's and Maurice Wilkins interpreted an x-ray so establish shape
  4. DNA Model
    • - Nitrogenous bases in the middle and phosphate backbone runs along the outside
    • - Charged backbone exposed to water
    • - Purines always paired with pyramidines (which are planar and hydrophobic)
    • - Strands run antiparallel
  5. DNA Replication Hypotheses
    • 1. Conservative Replication: each strand makes new DNA. Old bind to old, new binds to new.
    • 2. Semiconservative Replication: each strand makes a new strand and binds to it.
    • 3. Dispersive replication: DNA breaks apart and rejoins to produce 4 strands mixing old and new DNA
  6. Meselson and Stahl
    • Proved semiconservative DNA replication
    • - tagged with heavier N isotope
    • - after one replication all DNA was half as heavy
    • - after two replications half the DNA was half as heavy
  7. DNA Replication
    • 1. Helicase breaks the hydrogen bonds between nucleotides (TATA box)
    • 2. RNA primase primes the lagging strand
    • 3. DNA Polymerase 3 begins to elongate both the leading and lagging strands from the RNA primer locations
    • 4. DNA Polymerase 1 chews up RNA primers and fills in gaps
    • 5. DNA Ligase joins ends of the newly synthesized DNA
  8. Mistakes in Replication
    • - proofreading immediately removed bases hat are not complementary to the template strand.
    • - mismatch repair removal of one nucleotide
    • - excision repair removal of a group of nucleotides
  9. Garrod
    • Proves Alkaptonurea is a recessive hereditary trait
    • Results form the absence of an enzyme
    • Deduces gene gives rise to a specifid enzyme
  10. Beadle and Tatum
    • Using Neospora Colony proved that one gene gives rise to one protein
    • Studied the pathway of the organism to produce arginine
    • Removing the gene that allowed for the production of the enzyme that allowed the step to occur destroys pathway
    • If arganine is provided, none of the genes are necessary
  11. mRNA
    • Transfers the genetic information from the nucleus to the cytoplasm where proteins are made
    • Same as sequence on mRNA is called sense strand and complement is called template strand
    • 1. RNA Polymerase binds to the promoter (TATA) and starts to unwind DNA strands
    • 2. Reads DNA template 3' to 5' and produces RNA transcript by adding nucleotides to the 3' end.
    • 3. Reaches termination site, RNA transcript is set free from the template.
  12. tRNA
    • At least one for every amino acid, often more than one
    • Contains an anticodon sequence that is the reverse complement of the codon for an amino acid
    • Unique sequence causes it to have an overall unique shape
  13. Origins of Genetic Code
    • Code is arbitrary
    • All organisms share a genetic code
    • the code is fixed by convention
    • we must all share a common ancestor
  14. Translation
    • Begins with the indentification of AUG on the mRNA (start codon)
    • mRNA and tRNA recruit the large ribosomal subunits
    • the ribosome has a A site and P site (where first tRNA-amino acid complex sit first in the A site then move to the P site)
    • Amino acids for peptide bonds and let go of tRNA
    • A relelase factor causes the ribosome to let go of the mRNA
  15. Wobble
    • Does not always have to match up perfectly with the 3' residue of the codon
    • tRNA anticodon does not strictly obey base-pairing rules
    • wobble pairing and the degeneracy of the genetic code
  16. Steps of protein synthesis
    • not always sequential
    • in prokaryotes, translation can begin before translation is over (lack of nuclear membrane)
    • many ribosomes can bind to the same transript, starting at AUG, one after the other - polysome
  17. Linus Pauling and Vernan Ingram
    • Relates a genetic change in a protein to a phenotype
    • Sickle Cell Anemia - Deduced that hemoglobin must be defective in sickle cell anemia
    • Hemoglobin was digested and purified (normal and sickle) then found that they differed by only one or a few amino acids
    • Conclusion: the specific change in DNA that resulted in that amino acid was identified thus proving the final link in the chain of logic that connects genotype and phenotype
  18. Point Mutations
    • Point mutations: small changes in a single gene
    • - silent mutations: no effect due to the degeneracy of the genetic code
    • - missense mutation: changes one amino acid to another (sickle cell anemia)
    • - nonsense mutation: change an amino acid to a stop codon thus truncating the protein
    • - Frame shift mutation: results from an insertion or deletion and changes the reading frame from that point forward
  19. Chromosomal Mutations
    • Chromosomal: changes that affect a large portion of a chromosome - affects many genes
    • - deletion: remove a large piece of the chromosome
    • - duplication: duplicate large chunks of the chromosome
    • - inversion: when a piece of DNA flips around re-entering the chromosome in the reverse orientation
    • - translocation: when a piece of DNA jumps from one chromosome to another (reciprocal - trade)
  20. Mutagens
    • anything extraneous to the organism
    • effects of DNA change depends on whether the change occurs in somatic tissue or germ line tissue
    • Somatic: change could kill the cell, make it sick, or cancerous
    • change will not be transmitted to the progeny of the organism simply inherited by mitotic progeny of the cell
    • Germ line: mutation inherited to the progeny of the organism - leads to natural selection and acts to produce new species
  21. Virions
    • virus particles
    • consist of simple protein coat/capsid and nucleic acid
    • animal viruses often have in addition a lipid bilayer
    • instructions for subverting the host and making it do something not necessarily in the host's best interest
    • cell stops doing cell stuff and starts making more virus
  22. Bacteriophages
    • 2 phases:
    • - virulent : undergo a lytic reproductive cycle
    • - temperate : undergo lysogenic cycle
  23. Virus Classification
    • Host specificity and pathology
    • Genetic material (RNA/DNA)
    • Size/shape (electron microscopy)
  24. Lysogenic cycle
    • Wait and see approach
    • inserts DNA (prophage) into the bacterial chromosome and waits for bacteria to divide
    • prophage pops out of chromosome and resumes the lytic reproductive cycle
  25. Lytic cycle
    • Cell take over
    • Cell forced to make parts of the virus
    • Virus kills cell and releases virus parts and more viruses into the environment
  26. Virus Genes
    • Phenotypes change the way they are able to infect and lyse bacteria
    • high multiplicity of infection leads to more than one phage entering the same bacterium
    • DNA of both phage may recombine allowing for mapping of phage genes
  27. Viruses that infect eukaryotes
    • envelope glycoprotein
    • lipid bilayer
    • nucleocapsid
    • viral genome
  28. Retroviruses
    • do something similar to the lysogenic phage
    • bend rules of central dogma making a DNA copy from an RNA genome
    • DNA copy of the retrovirus can be integrated into the host chromosome and remain there, inactive, before it starts producing viral particles again
  29. Auxotrophic vs. Prototrophic
    bacteria that can only grow when certain substances are added to their media - auxotrophic

    the wild type is called prototrophic
  30. Specialized Transduction
    • prophage excises itself from the chromosome it could be sloppy and take some of the bacterial genome
    • then inserted into the chromosome of another bacteria when infected by that phage
  31. Generalized transduction
    • phage does a poor job of chopping up the bacterial DNA
    • instead of packing phage DNA into the virions it packs chucks of bacterial genome
    • when virions infect other bacteria, the chunk of DNA is injected into the infected bacterium and can be incorporated into its chromosome
    • can be used to map genes
  32. Transformation
    • take up floating DNA from dead bacteria
    • shown in Griffith experiment
    • DNA integrated by recombination
    • non-genomic DNA called plasmids are small DNA circles that also can be used to transform bacteria - do not integrate but are able to be duplicated in the cell - express few genes
    • r-factors: plasmids that carry antibiotic resistance genes
  33. Conjugation
    • Sex-like way that bacteria exchange DNA
    • E-coli - F plus and minus distiguishes types
    • F plus are male-like and they can conjugate with f minus bacteria - donate one strand of their F plasmid to the F minus cell making it F plus
    • Integrated F-strains are called Hfr (high frequency of recombination)
  34. Inducible Enzymes
    • turned on and off as needed
    • opposite of constituitive (always on) enzymes
  35. Lac Operon
    • using B-galactosidase, B-galactoside permease and B-galactoside transacetylase E-coli breakdown lactose (genes that code for these proteins together is called operator)
    • transcribe genes on RNA for the proteins only in the presence of lactose
    • repressor protein (lac-repressor) binds to DNA stopping transcription
    • when lactose becomes present, acts as inducer to release repressor from the operator
  36. Trp Operon
    • tryptophan synthesis
    • presence of tryptophan blocks transcription
    • protein that binds near the promoter can facilitate transcription, acting as an enhancer
  37. Exons/Introns
    • exon: part of DNA that constitutes protein-coding
    • intron: non-coding sequences
  38. RNA Processing
    • Introns removed (splicing) and exons joined together by snRNPs enzymes (one snRNP finds one exon and other finds the exon beside it and join them together)
    • methylated guanosine cap added to the 5' end (translation/stability)
    • poly A tail (~100 adenosine nucleotides) added to 3' end
  39. Telomeres
    • each DNA replication leads to shortening of DNA
    • cell prevents gene loss by adding short repetative stretches of DNA called telomeres (added by telomerase)
    • in somatic cells - no telomerase - thus only 20 divisions will occur before cell senses the telomeres shortening (reason we age)
  40. Centromeres
    • non-coding DNA
    • associated with centromeric regions of chromosomes
    • involved in binding kinetichore proteins
  41. Transposable Elements
    • DNA elements that can hop around the genome
    • Retrotransposons
    • DNA transposons
    • Pseudogenes
  42. Retrotransposons
    • make copies of themselves by being transcribed and then using reverse transcriptase to make DNA copy of the transcript that then re-inserts into the genome
    • some look like retroviruses: encode retroviral proteins but can't leave cell b/c they can't make functional coat proteins (LTR retroviruses)
    • LINES (non-LTR) encode reverse transcriptase and an endonuclease that helps them insert into the genome
    • Alu elements - rely on reverse transcriptase produced by other elements to allow them to jump
    • mutated versions of functional genome (300,000 copies in human genome)
  43. DNA Transposons
    • splice themselves out of the genome and jump back in by encoding a special enzyme called transposase
    • DNA parasites that serve no useful purpose
  44. Pseudogenes
    • ordinary mRNA's get copied into DNA and inserted into genome producing processed pseudogenes
    • OR
    • chromosomal duplication can produce multiple copies of a gene - inactivated by mutation - pseudogenes
  45. Ribozymes
    • Tom Cech discovered that some organisms have introns that splice themselves out (self-splicing introns)
    • RNA used to act as both genetic material and enzymes
    • still present: ribosomes which are made almost entirely of RNA - ribozymes - RNA portion catalyzes peptide bond formation
  46. Gene Regulation
    • all cells have the same alleles
    • cell appear different because they express different genes (transcriptional regulation)
  47. Housekeeping Genes
    • all cells must express them
    • they are necessary for basic functions of metabolism, transcription, structure, etc
  48. Transcriptional Regulation
    • ability of the RNA polymerase to begin transcription is controlled
    • promoter element required
    • accessory proteins that recognize regulatory DNA sequences that are involved
  49. Promoter
    • TATA box
    • CAAT box
    • GC box
  50. 3 Eukaryotic RNA Polymerases
    • Pol I - rRNA
    • Pol II - mRNA
    • Pol III - tRNA
  51. Enhancers/Silencers
    • Act at great distance (up to 20,000 bp from promoter)
    • can be upstream or downstream of the promoter
    • work in either orientation
    • fold DNA to bind to the enhancer associated transcription factors
    • they bind specifid sequences by having protein domains that probe the major groove of the DNA
  52. Gene Regulation Transcription
    • 1. TFII - TF for Pol II family form advanced team to prepare the way
    • 2. Main TFIID complex (including TBP) binds to TATA box
    • 3. TFIID recruits other TFs that recruit more TFs and eventually recruit RNA Pol II
  53. Silencer/Enhancer 2
    • enhancer-silencer TFs determine what proteins are synthesized
    • eukaryotic genes that are expressed in the same cell under control of the same enhancer elements are often widely distributed amongst chromosomes
    • each gene has own promoter/enhancer/silencer combination
  54. Heat Shock Genes
    • 20 genes respond to high temperature
    • promoters called HRE (heat shock response element)
    • HSF (a TF) binds to HRE and transcribes genes
  55. Problem of Histones
    • nucleosomes may inhibit transcription
    • particularly tightly wound DNA - heterochromatin - stains dark and is not transcribed
    • lightly stained - euchromatin - more transcription
  56. X-chromosome: Calico Cat
    • example of use of heterochromatin
    • preventing transcription of one X chromosome
    • inactivated chromosome - barr body - tightly wound
    • ex: calico coat in cats where different X chromosome is expressed in different parts of the body thus resulting in fur patches
  57. Gene Amplification
    • having multiple codes of the same gene to control rate of transcription
    • cancer cells do this to make themselves resistant to anti-cancer drugs
  58. Alternative splicing
    • post transcriptional control
    • when intron spliced, exon spliced as well
    • protein missing a chunk
    • the presence/absence of the chunk determines its function
    • ex: fruit flies double sex genome
  59. RNA Stability
    • post transcriptional control
    • RNA must be unstable or there is little point in regulating transcription
    • RNA once made would always be present and turning off transcription doesn't change that
    • 3' and 5' untranslated regions of a transcript affect stability and therefore accumulation of a particular transcript
  60. Control of Translation
    • changing the amount of capping
    • factors binding to the RNA to prevent ribosome attachment
    • RNA interference - small intefering RNA (do not encode) complement to mRNA. using Dicer siRNAs bind to complementary RNA and block translation
  61. Post-translational controls
    • enzymes that phosphylate other proteins - kinase
    • selective protein degradation
    • one way proteins destined for degradation are marked is by ubiquitination
  62. Expression/ Function of a Protein
    • regulating transcription is energetically favourable but slow to actually make proteins
    • post-transcriptional regulation is faster but often metabolically wasteful
  63. Environmental Contributions to Phenotype
    Penetrance - refers to the % of individuals of a given phenotype that show the phenotype at all

    Expressivity - the degree to which a phenotype is expressed (ex. shades of pink in a flower)
  64. Cis/Trans (Gene Linkage)
    • If genes are linked;
    • Dominant alleles are cis (alleles on the same homologous chromosome)
    • or trans (alleles on different homologous chromosomes)
  65. Types of alleles
    • Wild type - the predominant allele (~99% of the population)
    • Mutant allele - a change from the wild type allele, typically the result of a recent mutation. Also can refer to alleles that cause disease
    • Polymorphic - present in >1% of population
  66. Variation
    • continuous - seemingly infinite number of traits for a given character "blended inheritance"
    • discrete - there are only two or a few traits for a given character
  67. Polygenetic Traits (multigenetic)
    • multiple genes contribute to a trait
    • ex: heart attack risk - 6 genes affect, if each gene has 2 semidominant alleles, 12 genetic risk levels
  68. Meiosis I
    • 1. Prophase I:
    • -DNA compacts
    • -synapsis; pairing of homologous chromosomes
    • -chiasmata form; crossing over
    • 2. Prometaphase I:
    • -nuclear envelope b/d
    • -spindles form
    • 3. Metaphase I:
    • -line up at metaphase plate held together by chiasmata
    • -Microtubules attach to kinetichores (1 per chromosome not chromatid)
    • 4. Anaphase I:
    • -separation of homologous chromosomes into separate cells
    • 5. Telophase: optional
  69. Meiosis II
    • 1. Prophase II: shorten and thicken
    • 2. Metaphase II: chromosomes line up on plate
    • 3. Anaphase II: chromatids pulled apart (all distinct)
    • 4. Telophase II: chromosomes gather into nuclei and cells divide
  70. Down Syndrome
    Caused by non-disjunction during Anaphase I
  71. Chromosomes
    • circular (bacterial), linear (most other organisms)
    • duplicates are called chromatids (held by centromere)
    • X is a typical shape for a mitotic chromosome
    • chromosomes come in nearly identical pairs called homologs ( X X )
  72. Chromosome organization
    • 1. DNA condenses around histones (proteins)
    • 2. Protein/DNA mix is called chromatin
    • 3. Bundle of 8 histones is called nucleosome
  73. Mitosis
    • 1. Prophase: Chromatin coils into short, thick chromosomes (cohesin - holds sister chromatids together)
    • 2. Prometaphase: Nuclear envelope b/d, kinetichore microtubules appear and connect kinetichores to the poles
    • 3. Metaphase: Centromeres become aligned at equator (checkpoint)
    • 4. Anaphase: sister chromatids separate/move to poles
    • 5. Telophase: daughter chromatids reach poles, nuclear envelope reforms
  74. Cytokinesis
    • Animals: actin/myosin contractile ring
    • Plants: vescicles fuse to make cell membrane/plate
  75. Syncytial
    • multiple nuclei
    • telophase does not occur at the end of mitosis
  76. Cell cycle
    • M - mitosis
    • G1 - Gap 1
    • S - DNA synthesis
    • G2 - Gap 2

    • G1 to S commitment to cell division is made
    • G1 to S/G2 to M: depend on the activation of protein - cyclin dependent kinase (cdk) which bind to cyclin and phosphorylate protein substrate
  77. Messing with checkpoint between S and G2
    • Hydroxyurea - prevents DNA replication
    • Caffeine - deactivated checkpoint
  78. Epistasis
    • complex gene interactions
    • 1st locus determines something
    • 2nd locus determines something else
    • one will hide or mask the effect of the other
    • often results from genes involved in different steps of the same process/ pathway
    • ex: albino and agouti loci in mice
  79. Recombination (linkage group genes)
    • linkage of alleles with crossing over
    • gene is defined by a locus - or its location on chromosome
    • rates: measure of distance
    • occurs at random points on chromosome
    • depends on how far apart genes are on the chromosome
    • measure of physical distance along the chromosome
    • the more recombination the father apart the genes are
  80. Hemizygous
    gene is missing from 1 chromosome
  81. Genetic Engineering
    • taking a single gene from one organism and putting it into another organism
    • reasons:
    • 1. make organism express new phenotype
    • 2. understand how gene itself works
  82. Insulin
    • made by beta islet cells in pancreas
    • triggers glucose absorption out of blood stream
    • glucagon - does the opposite
  83. Banting and Best
    • purified insulin from a dog and used it to treat diabetes
    • many years after, pig insulin was used (differs from the human insulin by one nucleotide)
  84. 3 Obstacles of making your own insulin
    • 1. where to get the human genome
    • 2. how to put it into another organism so that it could be inherited
    • 3. how to get the host organism to recognize gene and express it

    make gene, put it into plasmid, tranform it into bacteria
  85. Making Insulin 1
    • Bacteria don't know how to splice thus need gene without introns
    • make DNA copy of mRNA of insulin using reverse transcriptase enzyme from a retrovirus
    • call this DNA copy of the mRNA - cDNA
  86. Making insulin 2
    • purify all RNA in a cell
    • add oglio-dT primers and reverse transcriptase
    • make cDNA double stranded with DNA polymerase
    • mix with linear plasmid and ligase
  87. Vector
    the plasmid that you use to carry the gene you are cloning
  88. Restriction endonucleases or restriction enzymes
    • proteins that identify a stretch of 6 base pairs (restriction site) of a specific sequence and cut both strands of DNA at that point
    • leave ragged end also known as overhanging or sticky end characteristic to each enzyme
  89. Making insulin 3
    using restriction enzyme cut plasmid and ligate your cDNA using DNA ligase (forms phosphodiester bonds between cDNA and plasmid vector)
  90. Transformation Marker
    • phenotype that allows you to indentify organisms that have been transformed
    • in case of insulin procedure antibiotic resistance to ampicillin is used
  91. cDNA library
    • spread bacteria on plates with ampicillin, each transformed bacterium will grown and divide creating colonies
    • every bacterium in that colony will have identical plasmids - creating a cDNA library
  92. DNA probe
    • piece of DNA that is used to find complementary DNA
    • melt DNA (pull strands apart) and allow DNA to cool - the complementary sequences will find each other (anneal or hybridize)
    • synthesize a short piece of radioactive DNA(the probe) and expose bacterial colonies of the cDNA library to the probe, the complement to insulin gene should stick to DNA or the correct colony (use a mixture of all possibilities b/c code is degenerate)
  93. Expression Vector
    • a vector that is "good for expressing proteins"
    • this means it will include a promoter and a terminator
  94. Making insulin 4
    • Repeat the process of cutting the DNA and making the cDNA attach to this new expression vector
    • add ligase to make sure that the cDNA stays stuck to the vector
    • transform bacteria with mixture and look for ampicillin resistant bacteria (expression vectore has antibiotic resistance too)
  95. Making insulin 5
    • plasmids dont always find the ends of the insulin DNA
    • ligation reaction has many different combinations
    • gel electrophoresis use it to find the right size of DNA
    • once correct bacterium is found (used lac promoter and operon) you have everything you need to make insulin!