Bio115 Exam 3 flashcards

  1. Four steps of DNA Replication
    • Initiation
    • Unwinding
    • Elongation
    • Termination
  2. DNA Helicase binds to...
    The lagging-strand in the 5' to 3' direction and breaks hydrogen bonds while moving along the replication fork
  3. DNA Gyrase ...
    relieves torsional strain that builds up ahead of the replication fork due to unwinding
  4. Primase ...
    sysnthesizes short strands of RNA including a 3'OH group for replication to begin
  5. How many primers are required on the leading strand?
    Just one
  6. DNA polymerase III ...
    • 5' to 3': polymerase activity: adds nucleotides (primary)
    • 3' to 5': exonuclease activity for error correction
  7. DNA polymerase I ...
    • 5' to 3': polymerase activity: adds nucleotides
    • 3' to 5': exonuclease activity
    • 5' to 3': exonuclease activity - remove RNA primers
    • Not as efficient as DNA polymerase III
  8. DNA ligase ...
    links together DNA at nicks/Okazaki fragments by forming a phosphodiester bond between adjacent nucleotides
  9. Initiator protein
    Binds to origin and separates strands of DNA to initiate replication
  10. Single-strand-binding proteins
    Attach to single-stranded DNA and prevent secondary structures from forming (stabilizes)
  11. Termination can occur when...
    • Replication forks meet
    • A termination protein (Tus in E. coli) binds to specific sequences to block helicase
  12. What is an Autonomously Replicating Sequence?
    Origin of replication found in yeast
  13. Why do origins of replication typically have numerous A-T base pairs?
    Easier to break - only 2 H-bonds compared to 3 for C-G base pairs
  14. What is an Origin-Replication Complex?
    In eukaryotes, an ORC binds to origins and unwinds the DNA in this region
  15. What is licensing?
    In eukaryotes, it is the regulation of precise replication once per cell cycle from a large number of origins
  16. What is telomerase?
    Telomerase is a ribonucleoprotein which maintains telomeres by extending the DNA and filling in the gap due to removal of the RNA primer after replication
  17. When does homologous recombination/crossing over take place?
    Prophase I, after DNA replication
  18. What are the two models of recombination?
    • Holliday
    • Double-strand break(*)
  19. What are three DNA binding motifs?
    • Helix-turn-helix
    • zinc fingers
    • leucine zipper
  20. lac operon is ...
    negative inducible
  21. trp operon is ...
    negative repressible
  22. trp operon is also regulated by attenuation - when tryptophan level is high:
    region 3 and 4 pair resulting in termination of transcription
  23. Antisense RNA regulates transcription by ...
    binding to mRNA to create double-stranded RNA blocking the ribosome-binding site
  24. Riboswitches with a regulatory protein...
    blocks the ribosome binding site by conformation change
  25. Ribozymes with a regulatory molecule ...
    induce cleavage
  26. Histone modification includes:
    Methylation, acetylation, and phosphylation
  27. Acetyl groups are added by:
    acetyl transferase
  28. Chromatin remodeling complexes...
    bind to sites in DNA and reposition nucleosomes allowing transcription factors to bind to promoters and initiate transcription
  29. Heavily methylated DNA ...
    is associated with the repression of transcription in vertebrates and plants
  30. Transcriptional activator proteins...
    bind to sites on DNA and stimulate transcription specific to a gene or subset of genes
  31. In eukaryotes, repressors ...
    do not directly block RNA polymerase, but instead compete with activators or interfere with the basal transcription apparatus
  32. Enhancers ...
    can operate at distant promoters by way of DNA looping out
  33. Insulators ...
    block the action of enhancers
  34. An example of coordinated gene regulation is:
    Several eukaryotic genes respond via consensus sequences to extreme heat producing heat-shock proteins
  35. Alternative splicing allows pre-mRNA to...
    be spliced in multiple ways generating different proteins in different tissue or at different times in development
  36. A female fruit fly has a ratio of:
  37. A male fruit fly has a ratio of:
  38. A ratio of 1.0 in fruitfly embryos activates the Sxl gene to produce a protein that causes ...
    tra pre-mRNA to be spliced at a downstream 3' site resulting in tra protein which leads to female fruit flies
  39. RNA is degraded by ...
  40. P bodies are:
    specialized complexes in which RNA molecules are degraded or sequestered for later release
  41. RNAi inhibits gene expression through:
    • Cleavage of mRNA leading to degradation using Dicer, siRNAs and RISC
    • Inhibition of translation using Dicer, miRNAs and RISC
    • Transcriptional silencing using RITS, siRNA and methylation
    • Degradation of mRNA (not via cleavage) using Dicer and RISC
  42. Posttranslational modifications of proteins includes
    • Selective cleavage and trimming of amino acids from the ends
    • Acetylation
    • Addition of phosphate groups
    • Addition of carboxyl groups
    • Addition of methyl groups
    • Addition of carbohydrates
  43. When the tinman gene has a mutation, ...
    the transcription factor is not produced and the heart doesn't develop
  44. What are the three major types of gene mutations?
    • Base substitution
    • Base insertion
    • Base deletion
  45. Purine to purine mutations are
    transition mutations
  46. The phenotypic effects of base mutations are:
    • Missense
    • Nonsense
    • Silent mutations
  47. A neutral mutation is:
    a missense mutation that alters the amino acid, but does not change its function
  48. Examples of spontaneous mutations are
    • Wobble
    • Unequal crossing over
    • Deamination
    • Depurination
  49. A base analog is
    a chemical with a structure similar to any of the four standard bases
  50. Chemical mutagens include
    • EMS: alkylation
    • Nitrous acid: deamination
    • Hydroxylamine: hydroxylation
  51. intercalating agents
    molecules which insert into DNA in place of nitrogenous bases causing insertions and deletions, e.g. proflavin and acridine orange
  52. Hermann Muller
    1927: showed mutations in fruit flies inducible by radiation
  53. x-rays, gamma rays, cosmic rays
    alter base structure, break phosphodiester bonds, and even cause double-strand breaks
  54. UV light
    less energy, but still mutagenic
  55. pyrimidine dimers
    can be caused by UV light; create covalent bonds between bases which block replication
  56. SOS system
    eukaryotic system of eta polymerase that can bypass pyrimidine dimers
  57. Four mechanisms of DNA repair are:
    • Mismatch repair
    • Direct repair
    • Base excision
    • Nucleotide excision
  58. Mismatch repair
    Replication errors, including mispaired bases and strand slippage
  59. Direct repair
    Pyrimidine dimers; other specific types of alterations
  60. Base excision
    Abnormal bases, modified bases, and pyrimidine dimers
  61. Nucleotide excision
    DNA damage that distors the double helix, including abnormal bases, modified bases, and pyrimidine dimers
  62. Mismatch repair Mechanism
    • Detects 3D distortion
    • cuts out distored part with exonuclease
    • fill in using original strand as template with DNA polymerase
    • repairs nicks with DNA ligase
    • Methylation at GATC sequence on old strand for differentiation
  63. exonuclease
    removes nucleotides usually at end of DNA strand
  64. DNA polymerase
    replaces nucleotides
  65. DNA ligase
    seals nick in sugar-phosphate backbone
  66. Direct Repair Mechanism
    • Converts modified nucleotides to original form
    • e.g. O6-Methyltransferase removes methyl group restoring base to guanine
    • e.g. photolyase uses light to break covalent bonds that link pyrimidine dimers
  67. photolyase
    an enzyme that uses light energy to break covalent bonds in pyrimidine dimers
  68. Base-excision repair mechanism
    • Excises modified bases and then replaces entire nucleotide
    • DNA glycosylase recognizes and removes damaged base
    • AP endonuclease cleaves phosphodiester bond on 5' site and removes sugar
    • DNA polymerase adds new nucleotide to 3'
    • DNA ligase fixes nick in sugar-phosphate backbone
  69. AP endonuclease
    removes nucleotide usually in middle of DNA strand
  70. Nucleotide-excision repair mechanism
    • Enzyme complex recognizes 3D distortion
    • DNA strand is separated and stabilized with binding proteins
    • An enzyme cleaves the strand on both sides of the damage
    • Part of damage strand is removed
    • Gap filled by DNA polymerase
    • Sealed by DNA ligase
  71. Homologous recombination
    A type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks.
  72. Interstrand cross-link
    Two DNA strands are connected thru covalent bonds. Not much know about this.
  73. Common Mechanisms for Nucleotide removal
    • Detection
    • Excision
    • Polymerization
    • Ligation
  74. Detection
    Damaged section of DNA is recognized
  75. Excision
    DNA repair endonucleases nick phosphodiester backbone on one or both sides of the DNA damage and one or more nucleotides are removed
  76. Polymerization
    DNA polymerase addes nucleotides to the newly exposed 3'-OH group by using the other strand as a template and replacing the damaged nucleotides
  77. Ligation
    DNA ligase seals the nices in the sugar-phoshate backbone
  78. Differences in Mechanisms for Nucleotide removal
    How detection and excision are accomplished
  79. xeroderma pigmentosum
    autosomal recessive condition caused by nonfunctional repair mechanism for pyrimidine dimers
  80. 1973, Cohen and Boyer at UCSF
    Created first recombinant DNA molecule
  81. Recombinant DNA technology
    Set of molecular techniques for locating, isolating, altering, and studying DNA segments
  82. Stemps requiring Recombinant DNA techniques
    • Find gene
    • Separate gene
    • Make copies of gene
    • Insert gene into plasmid without degredation
    • Induce bacteria to take up plasmid
    • Select bacteria that take up plasmid
  83. Restriction enzymes/endonucleses
    • Enzymes that recognize and make double-strand cuts in DNA at specific nucleotide sequences
    • Produced naturally by bacteria to defend against viruses
  84. Type I and III Restriction enzymes
    Cut outside recognition sequence
  85. Type II restriction enzyme
    • Cuts within recognition sequence
    • Used in molecular genetic work
    • Names indicate original bacteria
    • More than 800 isolated
  86. Characteristics of restriction enzymes
    • Palindromic recognition sequence
    • Fragment end either cohesive or blunt
  87. Cohesive end restriction enzyme
    Staggered cut -> sticky ends
  88. Restriction Digest
    Reaction of mixture of DNA, buffer, restriction enzyme, water heated at about 37 C
  89. Electrophoresis
    Standard technique for separating molecules on basis of size/electrical charge
  90. agarose
    polysaccharide isolated from seaweed
  91. Viewing DNA fragments using electrophoresis
    DNA fragments move to positive pole with smaller fragments moving faster
  92. probe
    fluorescent or radioactive DNA or RNA fragment complementary to sequence of interest
  93. Southern blotting
    Transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by radioactive probe
  94. Northern blotting
    • A technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample.
    • Size of mRNA molecule
    • Relative abundance of mRNA
    • Tissue in which MRNA is transcribed
  95. Western Blotting
    • Transfer of proteins from gel to a membrane
    • Probe is usually an antibody
    • Determine size of protein
    • Pattern of protein's expression
  96. Gene cloning
    create identical copies of a piece of DNA
  97. Cloning vector
    DNA molecule into which a foreign DNA fragment can be inserted for introduction into and replication in a cell
  98. Characteristics of an effective cloning vector
    • Origin of replication
    • selectable marker
    • one or more unique restriction sites where DNA can be inserted
  99. Types of cloning vectors
    • Plasmid, e.g. pUC19
    • Bacteriophage
    • Cosmid
    • BAC
    • YAC
    • Retroviral vectors
    • Transposons
    • Expression
  100. Transformation
    The capacity of bacterial cells to take up DNA from the environment
  101. Cosmid
    Plasmids that are packaged into empty viral protein coats and transferred to bacteria by viral infection
  102. BAC
    Originally contructed from F plasmids
  103. YAC
    DNA molecule that has a yeast ORI, pair of telomeres, and a centromere
  104. Ti plasmid
    Plasmid that can be used to introduce DNA into plants
  105. Expression vector
    Vector that allows the production of protein (i.e. transcription and translation)
  106. PCR Steps
    • 1) Heat to 90-100 C for denaturation
    • 2) Cool to 30-65 C for primers to anneal
    • 3) Heat to 60-70 C for DNA synthesis
    • Repeat
  107. PCR Limitations
    • Requires knowledge of at least part of sequence for primers
    • Taq polymerase is poor at proofreading
    • Fragments larger than 50Kb cannot be isolated
  108. PCR as a diagnostic tool
    Detects presence of a particular sequence, e.g. HIV
  109. Shotgun cloning
    Clone all sequences in an organism into vectors
  110. DNA library
    Collection of clones containing all the DNA fragments from one source
  111. Genomic library
    Set of bacterial colonies or phages containing fragments in a DNA library
  112. cDNA library: isolating mRNA
    isolation of mRNA using oligo(dT) chains
  113. cDNA library: making cDNA from mRNA
    • oligo(dT) act as primers
    • reverse transcriptase for DNA strand
    • Rnase digests most of RNA strand
    • Remaining RNA act as primers for second DNA strand
  114. In situ hybridization
    A type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue
  115. Restriction Fragment Length Polymorphisms (RFLPs)
    Variations in the patterns of fragments produced when DNA is cut with a restriction enzyme typically caused by mutation
  116. Dideoxyribonucleoside triphosphate(ddNTP)
    No OH groups
  117. Sanger Method of DNA sequencing
    Uses ddNTP to terminate synthesis of strands of different length which can be read by electophoresis to sequence
  118. Pyrosequencing
    allows sequencing of entire genomes in a couple months
  119. DNA fingerprinting
    • PCR used to amplify STR loci each with large numbers of alleles which assort independently
    • Difference in number of tandem repeats have no phenotypic consequence
    • Probability of two randomly selected people having the same DNA profile is less than 1 in 10 billion
  120. DNA fingerprinting procedure
    • DNA extracted from tissue samples
    • PCR primers for specific STR loci used to amplify fragments
    • DNA from sample is compared with reference DNA
    • Usually use genomic DNA, but mitochondrial DNA can be used as well
  121. Forward Genetic Approach
    • Function -> gene
    • Frequently used in less complex organisms to discover new genes
  122. Forward Genetic Procedure
    • 1. Isolate mutants that have phenotypic mutation.
    • 2. Map the mutations.
    • 3. Sequence the gene to find the mutation.
    • 4. Clone the gene using molecular techniques.
    • 5. Further genetic, molecular genetic, and biochemical experiments can further define a gene's function in that process.
  123. Reverse Genetic Approach
    • gene -> function
    • Frequently used in mice to see if genes discovered in simpler organisms
    • have a similar phenotype in mammals
  124. Reverse Genetic Procedure
    • 1. Begin with a gene with known sequence.
    • 2. Induce a mutation in that gene.
    • 3. Look to see what effect these mutations have on the phenotype of the organism
  125. Transgenic animal
    An organism with an added transgene
  126. Transgene
    non-innate DNA added to an organism
  127. Knockout mice
    Mouse in which known gene has been disabled via homologous recombination
  128. Knockin mouse
    wildtype gene is replaced with known mutant gene
  129. Knockout mouse procedure
    • Target "normal" gene disabled by inserting neo+ gene in the middle and a tk+ gene is added at the end
    • Disabled gene is transferred to embryonic mouse stem cells to undergo recombination with normal cells resulting in some neo+ tk- cells
    • Cells grown in antibiotic, and only recombinated ones survive
    • Surviving cells injected into early mouse embryo resulting in variegated mouse
    • Variegated progeny interbred resulting in some homozygous mice for the knocked-out gene
  130. Site-Directed Mutagenesis: Method 1
    • Short sequence of nucleotides removed and replaced by synthetic sequence containing mutated bases
    • Requires flanking restriction sites that are nowhere else in DNA
  131. Site-Directed Mutagenesis: Method 2
    • Oligonucleotide created that differs from target sequence by single nucleotide
    • Two sequences pair
    • Oligonucleotide used as primer which yields molecule with single mismatched pair
    • DNA transferred back to bacteria where about half are repaired
    • Bacteria then screened for altered sequence
  132. Oligonucleotide-directed mutagenesis (2)
    • Often used for making small changes in DNA sequences already cloned into plasmids
    • Can't be used in multicellular organism: long, noncircular DNA, multiple ori, etc.
  133. Silencing with RNAi: RNA Knockdown
    Can be delivered to cell by injecting or soaking to turn down expression without inducing mutation
  134. Short hairpin RNA (shRNA)
    Can be cloned into vectors and used to make transgenic animals
  135. RNAi for the treatment of disease
    • siRNAs could be used against RNA viruses, such as HIV
    • siRNAs could be used to treat genetic diseases, high cholesterol and cancer
  136. Stable nucleic-acid-lipid-particles (SNALPs)
    Used in delivery of siRNA to lower cholesterol thru silencing in monkeys
  137. Gene therapy targets what kinds of cells?
  138. What is genomics?
    It is the field of genetics that attempts to understand the content, organization, function, and evolution of genetic information contained in whole genomes
  139. The first living organism to be sequenced was ?
    Haemophilus influenza
  140. For the Human Genome Project, what kind of method was used for sequencing?
    A map-based method
  141. Craig Venter and Celera Genomics used what method to sequence the human genome?
    A whole-genome shotgun technique using computers
  142. What is a single nucleotide polymorphism (SNP)?
    A site in the genome at which individual members of a species differ in a single base pair
  143. Why are SNPs more commonly found in non-coding regions?
    Because there is no selective pressure to weed out the mutations.
  144. What is a haplotype?
    It is the set of SNPs and other genetic variants found on a particular part of a chromosome.
  145. Of Africans, Japanese, Chinese, and Europeans, which group has the greatest diversity of SNPs and why?
    Africans, as this is consistent with many other studies that suggest humans first evolved in Africa.
  146. What is a contig?
    A continuous stretch of DNA
  147. What is bioinformatics?
    A field that fuses molecular genetics and computer science
  148. What are two methods to identify genes?
    • ab initio approach: scans the sequences looking for characteristics such as an open reading frame
    • comparative approach: Looks for similarities between a new sequence and sequences of all known genes
  149. What is an open reading frame?
    A frame which includes a start and stop codon in the same reading frame
  150. What is BLAST?
    Basic Local Alignment Search Tool, is a program to determine whether a similar gene sequence has already been found in the same or another species
  151. What are two types of homologs?
    • Orthologs are homologous genes found in different species that evolved from the same gene in a common ancestor.
    • Paralogs are homologous genes in the same organism that arise by duplication of a single gene
  152. What is a protein domain?
    A region in a protein that has a specific function or shape
  153. What is a microarray?
    An array of numerous microscopic DNA fragments/probes used to find complementary sequences corresponding to known genes
  154. What is a reporter gene?
    A gene that researchers attach to a regulatory sequence of another gene of interest that allows for visual identification (e.g. Green Fluorescent Protein, GFP)
Card Set
Bio115 Exam 3 flashcards
Bio115 Exam 3 flashcards