Bio 313 Test 4

  1. Gene regulation. difference between 
    pro vs. euk.
    For EUKARYOTES:
    • 1. no operons: each structural gene has its own promoter, and is transcribed separately
    • 2. chromatin structure: DNA must unwind from histone before transcript.
    • 3. nuclear membrane. : transcription and translation separate steps
    • 4. RNA stability: significance of longer half-life?
  2. Histone tail modification
    • A. Activation B. RepressionC. Both
    • 1. methylation, acetylation to HISTONE: 
    • a. methylation of H: activation/repression of transcription
    • b. acetylation of H: activation of transcription
    • 2. chromatin remodeling: 
    • a. nucleosome slide along DNA
    • b. conformational change
    • 3. DNA methylation
    • a. methyl to nucleotide bases
    • b. most common to C (cytosine)
    • c. repression of transcription
  3. histone tail modification: acetylation
    • 1. + charge histone tails interact w/ - charge phosphate on DNA
    • 2. acetylation of the tails : WEAKENS interaction between w/ DNA , allows transcription factors to attach
    • 3.
  4. histone modification : chromatin modeling
    • 1. nucleosome slide along DNA 
    • 2. conformational change
    • 3. the reconfiguration process exposes binding sites for transcription
    • 4. transcription factors and RNA polymerase bind to DNA and initiate transcription
  5. DNA Methylation
    • 1. addition of methyl groups to nucleotide bases
    • 2. most common: cytosine to produce 5-methylcytosine
    • 3. repression of transcription
  6. Regulation of Initiation of transcription
    • 1. typical eukaryotic promoters consist of 2 parts
    • A. core promoter: sequence that is common to most genes 
    • B. Regulatory promoter: sequence UNIQUE to gene (the R word has a U in it!)
    • 2. transcriptional activator proteins bind to : and INCREASE rate of transcription
    • 3. BASAL Transcription apparatus: RNA polymerase II, gen. transcription factors, mediator) binds to: promoter
  7. Regulatory Promoters
    • 1. upstream from core promoter
    • 2. bind to transcriptional activator proteins to increase rate of gene transcription
    • 3. activator proteins bind to response elements (RE) that contain short consensus sequences
    • 4. RE are located in regulatory promoter and enhancer
    • 5. Each RE binds a different activator
  8. Coordinated gene expression
    • 1. one stimulus -> multiple genes
    • 2. eukaryotes: different genes can have the same response elemtent -> this means a given stimulus can activate many genes at the same time via binding the same activator protein to RE
    • EXAMPLE: heat shock proteins expressed from dif. genes in resonse to extreme heat or other stress stimulus by containing the same response element (Heat shock element) to which transcription factors bind to turn "ON" gene
  9. Regulation of Initiation transcription
    • 1. repressors : silencer
    • 2. activators/co-activators : enhancer
    • 3. insulator binding protein : insulator
    • 4. trans factors
  10. Regulation of Initiation of transcription: Transcriptional Activators and Coactivators
    • 1. bind enhancers: DNA sequences that enhance transcription ,  generally not specific to the gene they enhance , location varies
    • 2. stimulate and stabilize basal transcription apparatus at core promoter
  11. what stimulates transcription
    • 1. transcription, RNA polymerase , transcriptional activator proteins
    • 2.
  12. Regulation of Initiation of transcription
    • 1. bind to silencers or sequences in the regulatory promoter
    • 2. binding repress transcription
    • a. compete w/activators for same DNA binding site
    • B. bind to a sequence near the activator binding site and prevent activator binding
    • C. interfere w/ assembly of basal transcription apparatus
  13. Different Regulation roles:
    • 1. repressors= silencers
    • 2. activators /coactivators= enhancers
    • 3. insulator binding protein=
  14. the regulation transcriptor role of activator/coactivator:
    • 1. bind enhancers
    • a. DNA seq. that enhance transcription,
  15. repressors in reg. of transcription:
    • 1. bind to silencers or sequences in regulatory promoter
    • 2. binding repress transcription
  16. insulator binding proteins:
    • 1. (a typre of transcription regulator)
    • 2. enhancer: DNA sequence stimulating transcription from a distance away from promoter, have RE and bind transcriptional activator proteins
    • 3. insulator: DNA sequence that binds insulator protein , which blocks or insulates the effect of an enhancer
  17. posttransciptional regulation
    • 1. degradation of RNA
    • a. degradation is controlled by ribonucleases (RNAses)
    • b. degradation off 5' UTR , coding sequence , and 3' UTR
  18. eukaryotes regulation happens when?
    • in euks, 
    • 1. transcr. in nucleus,
    • 2. then pre-RNA travel to cytoplasm, and are slightly altered via more REGULATION called
    • A. RNA Processing
    • B. Degradation
    • 3. translation
  19. RNA processing (a posttransciptional euk regulation)
    • 1. leads to alternative proteins in different tissues (considering that this is a more isolated event)
    • a. a gene can promote a protein, and if on different chromosome, maybe same gene may NOT create that protein,
    • b. DOMINO effects as well
  20. 2. DEGRADATION Of DNA
    • a. amount of mRNA: based on mRNA synthesis and degradation rates
    • b. how long mRNA lasts depends on the amount of mRNA present!
  21. Transcriptional Activators and Coactivators
    • how activation occurs:
    • 1. stimulate and stabilize basal transcription apparatus at core promoter
    • who they are:
    • 2. transcription factors, RNA polymerase, and transcriptional activator proteins bind DNA and stimulate transcription
  22. repressors
    • 1. bind to silencers or sequences in the regulatory promoter 
    • 2. binding repress transcription: compete w/a ctivators for same, near site
    • b. can interfere w/ basal trancription apparatus
  23. RNA interference
    • 1. other names: RNA silencing and post transcriptional gene silencing
    • 2. triggered by:
    • A. microRNA's
    • i. transcriptional silencing
    • ii. inhibition of translation
    • B. small interfering RNA's :siRNA's 
    • i. RNA cleavage
    • C. Dicer enzyme either siRNA or miRNA
  24. benign
    tumor remains localized
  25. malignant
    tumor cells invade other tissue
  26. metastatic
    tumor cells induce secondary tumors
  27. stage 0 cancer
    • 1. carcinoma in situ (cancer in place") 
    • 2. not YET invaded surrounding tissue
    • 3. cure rate is 100% when no invasion
  28. stage 1 cancer
    • 1. small tumor 
    • 2. invasive into other tissue but has not spread
  29. stage 2 cancer
    • 1. primary tumor is larger
    • 2. still no clinical evidence of spread
  30. step 3 cancer
    1. tumor has apread to lynmph glands in that region of the body
  31. stage 4 cancer
    spread beyond region where it initiated to a distant tissue or organ
  32. cancer
    • 1. genetic disease
    • 2. role of carcinogens
    • 3. some cancer have specified gene mutations
    • a. DNA repair enzyme mutations
    • b. tumor suppression
    • c. proto-oncogenes
    • 4. some families are prone to cancer
  33. 1. 2 -hit mutation model for retinoblastoma
    • Germ line and single cell mutation 
    • 1. germ line mutation: "first hit". found in all somatic cells of offspring
    • 2. single cell mutation: " during mitotic cell cycle
    • 3. if this mutation occurs in a retinal cell, retinoblastoma will occur
  34. Knudson's multistep model of cancer
    • 1. multiple mutations are required to produce cancerous cells
    • 2. a person predisposed with a mutation have a likelier chance of cancer and easier chance to have more cancerous cells
  35. how many mutations to get malignant cell
    • 4,
    • allows tumor cels to become aggressive and proliferative
  36. cancer, more inherited or environmentally affected
    environmentally affected
  37. role of environmental factors in cancer
    • 1. tobacco use
    • 2. obesity
    • 3. alcohol
    • 4. UV radiation
    • 5. environmental related to 42.7% of all cancer related
  38. related to genes
    • 1. proto-oncogenes and tumor suppressor genes
    • 2. genes that control the cycle of cell division
    • 3. DNA repair genes
    • 4. genes that regulate telomerase
    • 5. genes that promote vascularization and the spread of tumors
  39. oncogenes
    • 1. proto-oncogenes: responsible for basic cellular functions in normal cells;
    • A. conversion into an oncogene (dominant -acting stimulatory gene that  cause cancer) occurs by ->
    • B. "gain of function mutation
    • 2. MECHANISMS OF CONVERSION:
    • a. mutations in a proto-oncogene 
    • b. reduplication (gene amplification) 
    • c. chromosomal translocation that brings proto-oncogene under the control
  40. tumor suppressor genes
    • 1. tumor- suppressor genes generally encode proteins that inhibit cell proliferation . loss of these "brakes" contributes to development of many cancers
    • 2. mutated tumor suppressor gene are recessive-acting "loss of function" 
  41. are heterozygotes for tumor suppressors safe?
    • 1. no. in fact more of a problem
    • 2. if dominant chromosome deleted, then lose that suppression job and causes possible cancer
    • 3. EX/ BRCA1/BRCA2: control of other genes
    • a. both are responsible for dna repair, cell cycle arrest, cell death
  42. genes that control cell cycle
    • 1. control of cell cycle 
    • a. cyclin -dependent kinases (CDK's)
    • b. p.53- tumor supressor protein that stops cell cycle
    • 2. G1-to-S transition: retinoblastoma protein (RB)
    • a. RB binds to E2F and keeps it inactive
    • b. increasing concentrations of cyclin-D-CDK and cyclin-E-CDK phosphorylate RB
    • c. which inactivates RB and it releases E2F
    • d. E2F then binds to DNA and stimulates the transcription of genes required for DNA replication

    • 3. g2-to-M transition:
    • a. mitosis -promoting factor (MPF)
    • 4. spindle assembly checkpoint
  43. genes related to cancer
    • 1. tumor-supressor genes
    • 2. cell regulation genes
    • 3. DNA repair genes
    • 4. telomerase
    • 5. genes that promote vascularization and the spread of tumors
  44. DNA repair genes
    directly (xeroderma ) or indirectly BRCA1/2
  45. telomerase
    adult cells lack this but immortal cancer cells have this
  46. genes that promote vascularization
    • 1. formation of blood vessels: feeding tumor
    • 2. extracellular matrix and cytoskeleton: spread of cancers; 
    • a. ex/ palladin gene and familial pancreatic cancer
    • b.
  47. characteristics of cancer
    • 1. chromosomal instability is a general feature of cancer cells
    • a. deletions, inversions, and translocations
    • b. ex/ a reciprocal translocation between chrom. 9 and 22 causes chronic myelogenous leukemia
    • c. aneuploidy
  48. viruses that cause cancer
    • DNA Viruses
    • 1. HPV: virus-> cervical cancer
    • 2. epstein -barr virus->  Burkitt's lymphoma
    • 3. HV8 -> Kaposi's sarcoma
    • 4. Hepatitis B -> liver cancer

    • RNA viruses:
    • 1. hepatitis C: -> liver cancer
    • 2. human T -cell leukemia virus (HTLV-1)

    • Retroviruses:
    • 1. mutating and rearranging proto-oncogenes
    • 2. inserting strong promoters near proto-oncogenes
  49. germline mutations
    • 1. occur in cells that produce gametes (sperms/eggs)
    • 2. can be passed on to offspring
    • 3. affected daughter cell will carry that mutation in all her somatic and germline cells
  50. somatic mutation
    • 1. occurs in body cell
    • 2. when somatic mutation divides, its thru mitosis
    • 3. cannot be passed on to offspring
  51. mutation types
    • 1. base substitutions
    • 2. insertions and deletions
    • 3. expanding nucleotide repeats
  52. base substitution mutations
    • 1. A and G are purines, and they each pair up with opposites , pyrimidines, T and C.
    • 2. Transition: purine to purine or pyr. to pyr.  exchange. so
    • a. G->A, A->G
    • b. T->C, C-> T
    • 3. Transversion -> G-> C, T NOT a
    • same thing goes with other bases
  53. Phenotypic effects
    • missense, nonsense, silent
    • 1. missense: changes amino acid
    • 2. nonsense: creates stop codon
    • 3.silent: no affect on identity
  54. insertions or deletions
    • 1. type of frameshift mutation (if one codon)
    • can alter the entire pattern
    • 2. if insertion of a multiple of 3, then non-frameshift
  55. intragenic suppressor mutation
    2 mutations that return a codons back to original state. more than one representation of it
  56. intergenic suppressor mutation
    • 1. occurs in the gene other than the the one bearing the original mutation.
    • 2. works like intragenic except returns to original state by working with different codon.
  57. mutation rate
    • frequency a wild type allele changes to mutant allele
    • 2.
  58. spontaneous mutations
    1. under normal conditions
  59. induced mutations
    result from changes caused by environmental chemicals or radiation
  60. expanding nucleotide repeats
    • Def: when a set of nucleotides repeats abnormally
    • 1. possible how this happens: template strand forms hair pins, which is excess copied dna
  61. in frame insertion affects
    • 1. number of repeats proportional to age
    • 2. genetic anticipation: more strongly expressed or earlier anticipation as inherited down
  62. phenotypic effects
    • 1. Forward/reverse motion: (basic)
    • a. forward: causes a mutation that changes
    • b. reverse: mutation that reverts to original, so apparent mutations
    • 2. missense/nonsense/silent: based off of changing one base that changes the amino acid
    • 3. neutral mutation: a missense mutation that changes amino acid but that change isnt such a big of a deal
    • 4. loss-of-function mutation: complete or partial absence of normal protein function
    • a. opposite of neutral mutation
    • (frequently recessive)
    • 5. lethal mutation: one that causes premature death
    • 6. supressor mutation: a genetic change that supresses or hides the effect of another mutation. (intragenic vs. intergenic)
    • 7. gain of function: causes the cell to produce a protein or gene product function is not normally present
  63. silent mutation
    • may change 1 synonymous codon for another.
    • may change one similar amino acid for another
  64. neutral mutation
    –May change an amino acid in a non-critical area of the protein à it will still function
  65. spontaneous replication errors
    • 1. strand slippage: small loop is formed, insertion if on new strand, deletion if on template(old) strand
    • 2. deamination: C turns to U ,due to the loss of an amino group from a base.
    • 3. unequal crossing over: one pair with deletion and other with insertion
    • 4. depurination: loss of apurine base from a nucleotide.
  66. mutagens
    • 1. DEF: type of induced mutation , not natural like spontaneous, an unnatural exposure to chemical or radiatino
    • 2. extracellular physical or chemical factors that cause or increase frequency of mutations
    • EX/ alkylating , changes the original nucleotide to a different one thru methylation (or at least enough for a diffrent reciprocal to attach than what would before the mutation)
    • 2nd EX/ intercalating: induces frameshift
    • 3rd EX/ RADIATION
    • a. pyrimidine dimers: since pyrimidine ones (C and T) readily absorb UV light, they attach to each other covalently to become a dimer, no longer ready for transcription and die off .
  67. how to test for mutagens
    • AMES test!
    • 1. has a salmonella bacteria that lacks a the coat that protects it from environment
    • 2. also dna repair mechanisms deactivated
    • 3. also lack histidine (really most important part of all 3)
    • -ones that do reverse , or his- to his +, are on plate.
    • ---higher number of colonies means higher number of mutagens OBVI
  68. how to repair dna"
    • 1. mismatch pair
    • 2. nucleotide excision repair
  69. rules of thumb for DNA repair:
    • 1. 2 nucleotides repaired at a time
    • 2. redundancy, important to catch these errors
  70. types of dna repair
    • 1. mismatch repair: corrects mismatched dna after it escapes the grasp of dna polymerase. 
    • a. detected by enzymes
    • how it works:
    • i. wrong nucleotide put in repsonse to template. random methyl on the old dna later down the road 
    • ii. the mismatch repair complex brings mismatched pair close to methylated pair., so loop of old on inside track while new dna on outside track
    • iii. exonucleases remove new outside track in between these two checkpoints taking out of 1 of 2 of those endpoints (obviously mismatched new end )
    • iv. dna polymerase then replaces the nucleotides, correcting the mismatch. adn DNA ligase seals the nick.
    • 2. nucleotide excision: removes bulky dna lesions (such as pyrimidine dimers)
    • a. can repair all sorts of dna damages
  71. restriction enzymes
    • 1. make dsDNA cuts (so through BOTH!)
    • 2. reads 5' to 3' (palindromic)
    • 3. cohesive, staggered ends
    • blunt: equal length ends
  72. how do wee see DNA fragments?
    • Gel electrophoresis!
    • shorter fragments move farther along than long ones (density?)
  73. how to increase amount of same dna:
    gene cloning: amplifying a specific piece of DNA via a bacteria cell
  74. gene cloning:
  75. 1.insert DNA into plasmid vectors:
    • a. plasmid : circular dna present in bacteria
    • b. 3 important elements:
    •     i. origin of replication
    •     ii. selectable marker
    •     iii. unique restriction enzyme sites
    • 2. transformation of host cells with plasmids
    • 3. selection and screening cell for recombinant plasmids
    • a. selectable markers used
    • 3 characteristics:
    • 1. oriT
    • 2. selectable markers within vector so these parts can be easily identified
    • 3. 1 or more restriction site into which a DNA fragment can be inserted in
  76. test of recombination
    1. white colony is recombinant plasmid. no longer performs the blue color gene bc dna inserted in the middle of that blue gene making it + to -.
  77. requirements for PCR
    • 1. Template DNA
    • 2. dnTps (A,T,G,C)
    • 3. Primers (are short pieces of single stranded nucleotides that are made in a laboratory)
    • 4. thermal stable polymerase:
  78. PCR
    • Def: allows replication of dna to a billionfold in a few hours
    • 1. primers: short fragments of dna, 17-25 nuc's. long, complementary to template.
    • STEPS of pcr:
    • 1. heat to high temp, breaks H bonds of original dna paired strands
    • 2. cooled so primers attach
    • 3. heated to 72 C, for Dna polymerase to synthesize new strands,
    • 4. repeated over and over again
  79. limitations to pCR
    • 1. accuracy
    • 2. contamination
    • 3. known info on target DNA
    • 4. amplified fragments are less than 2 kb
  80. dna sequencing
    • Def.: allows Dna information to be read
    • methods:
    • 1. Sanger/dideoxy : fragment read is used as a template to make more dna.
    • a. use dideooxyribonucleases instead of the famous deoxyribo.. chemical difference is no OH and H in place for dideo
    • b. 4 separate tubes each with ddA,T,G,C... but they have the deoxyribo... availble too.
    • c. all make different cuts , using d_ first since there is more of this
    • d. can read new template by reading down to up
    • ANOTHER SANGER WAY (only one tube)
    • 1. ddNTP marked by dye
    • rESULTS: longest to shortest fragment, showcase an equal but opposite representation of the 5' to 3' strand.
  81. pyrosequencing
    • addition of nucleotide is paired with a flash of light, which is generated as  the nucleotide is added.
    • 1. broken fragments of blunt dsDNA
    • 2. primers put on 5' ends of both strands, now separate strands
    • 3. attached to bead, amplified by pcr
    • 4. put in plates.
    • 5. the dA,T,C, G, flow over the plate, and if the next one should be added to growing template, a light will go off
  82. Microsatellites:
    • type of way to determine dna fingerprinting
    • 1. use very short DNA sequences repeated in tandem
    • 2. these repeats found in many loci throughout the human genome
    • 3. uses PCR. tags these primers fluorescently . size determined from laser and gel. (shorter fragments are faster)
  83. genotype-> phenotype
    Begins with a known phenotype --> find and sequence the gene that encodes the phenotype
  84. reverse genetics (pheno-> genotype)
    Begins with a scientist inducing mutations in a gene of interest--> checking the effect of the mutation on the phenotype to learn about gene’s function
  85. structural genomics
    -determines the DNA sequences of entire genomes
  86. functional genomics
    studies the functions of genes by using genomic-based approaches
  87. comperative
    -studies how genomes evolve
  88. map based genome sequencing
    • 1. Generate random genomic DNA libraries of large clones (~200000 bp)
    • 2. Assemble large clones into long contigs on the basis of genetic and physical maps
    • 3. Then select large clones (break into small pieces) and sequence each individually
  89. whole genome shotgun sequencing
    • 1. Break the genome into short sequences
    • 2. Generate random genomic DNA libraries of small clones (~2000bp)
    • 3. Sequence each small, random clone
    • 4. Assembles them into contigs on the basis of sequence overlap.
  90. genetic map
    • 1. approximate locations of genes, relative to the location of other genes, measured in centiMorgans (cM)
    • 2. based on the rates of recombination
    • 3. low resolution
    • 4. do not correspond to physical distances between genes
  91. physical map
    • 1. based on the direct analysis of DNA
    • 2. places genes in relation to distances measured in bp, kbp, and mbp
    • 3. higher resolution and more accurate than genetic maps
  92. difference between a map-based approach to sequencing a whole genome and whole -genome shotgun approach?
    • whole genome:
    • 1. categorize long contigs based off of physical and genetic maps
    • Genome shotgun:
    • 1. break into small pieces and categorize those based off of sequence overlap
  93. SNP
    • 1. single-nucleotide polymorphism
    • 2. a site at which individual members of a species differ
    • 3. are valuable markers in linkage studies
  94. important findings of metagenomics studies:
    • 1. Determine the genome sequences of an entire group of organisms that inhabit an environment
    • 2. Typically microbes: the organisms in a cup of seawater;the organisms at a toxic mining waste dump; the human microbiome
    • 3. essential to understanding the human microbiome
  95. microarray:
    • rely on nucleic acid hybridization (DNA fragment is used to probe to find complementary sequences)
    • b. allows thousands of genes to be monitored simultaneously
  96. proteomes and genomes list discovered for liver cell. bigger difference in genomes or proteomes?
    genomes will always be the same. however the proteins expressed will vary cell to cell.
  97. transcriptome
    1.identification of all the RNA molecules transcribed from a genome in a certain tissue and stage of development
Author
haleygreenbean
ID
325639
Card Set
Bio 313 Test 4
Description
gene expression, etc
Updated