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MCDB 310 > Chapters 8 and 9 > Flashcards

Chapters 8 and 9 Flashcards

1
Q
  1. What are the 3 components of nucleotides?
  2. What are the 2 components of nucleosides?
  3. Which bases are Purines and which bases are Pyrimidines?
A
  1. Nitrogenous base + pentose + phosphate
  2. Nitrogenous base + pentose
  3. Purines: Adenine and Guanine, Pyrimidines: Cytosine, Thymine, and Uracil. Complementary bases pairing helps w/ stability, folding, and structure.
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2
Q
  1. What separates DNA form RNA in terms of their base structure?
  2. Is the ring puckered or planar? What the most important positions in the rings?
  3. What does this puckered or planar structure help with?
A
  1. The two pentoses are closed ring forms (furanoses)
    1. 2’-deoxy-D-ribose is DNA
    2. D-ribose is RNA
  2. The ring is puckered. Significant in complex structures. The most important positions in the rings are the the 2’, 3’ and 5’ positions.
  3. Helps with flexibility and stacking.
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3
Q
  1. What is different about minor bases?
  2. What nucleotides have additional rings?
A
  1. Most are methylated forms of the major bases.
  2. cAMP and cGMP
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4
Q
  1. What type of bonds are nucleotides joined together by?
  2. What kind of linkages does the backbone have?
  3. What are oligonucleotides?
A
  1. Phosphodiester bonds 5’ phosphate + 3’ hydroxyl
    1. Creates a backbone
  2. pentose-phosphate-pentose-phosphate: Ester-linkages at the 3’ and 5’ positions.
  3. Up to 50 bases. 3’-5’ allows for the double turn of DNA.
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5
Q
  1. What part of the molecule is hydrophilic? What group forms the h-bonds?
  2. What charge does phosphate have at 7.0 pH?
  3. At what maxima is UV light absorption for nucleotides?
A
  1. The backbone is hydrophilic, bases are hydrophobic. Pentose hydroxyls form hydrogen bonds with water.
  2. Negative, because phosphates are completely ionized at pH 7.0.
  3. 260 nm
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6
Q
  1. Are bases hyrdophobic or hydrophilic?
  2. What is the reason for h-bonding between the bases of two different strands?
  3. Why do bases stack?
A
  1. Bases are hydrophobic, relatively insoluble in water.
  2. The reason for h-bonding and the bases being hydrophobic is to stabilize binding between two or more strands. (A:T 2 H-bonds) (G:C 3 H-bonds)
  3. Bases stack to minimize contact with water.
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7
Q
  1. What method did Rosalind Franklin play in helping with the identification of the structure of DNA?
  2. What was the major observation?
  3. What were the other observations?
  4. What are some important features of DNA?
  5. Structure leads to function: what two grooves are present on the DNA helix?
A
  1. x-ray diffraction of DNA crystals.
  2. DNA is a helix with about two periodic sequences.
  3. Phosphate is on the outside of the helix and DNA has 1-3 strands.
  4. DNA is a double right-handed helix. Base-pairing is complementary. Backbones are on the outside (phosphate and sugar). Bases face inward. Strands are H-bonded together. Crick deduced that strands are anti-parallel.
  5. Major and Minor grooves
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8
Q
  1. What kind of mutations does Ethidium bromide cause?
  2. How can we make this molecule safer?
  3. What molecule can we use instead and why doesn’t it cross the membrane?
A
  1. Frameshift mutations
  2. Ensure it cannot contact DNA, binds to the DNA and causes a frameshift.
  3. We could use gel red, it does not cross the membrane because it is big and insoluble.
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9
Q
  1. What bonds does DNA rotate about?
  2. What does thermal fluctuations and the presence/absence of water produce?
  3. What does this DNA flexibility result in?
A
  1. Rotation about sugar-phosphate and glycosyl bonds.
  2. Bending, stretching and melting (broken H-bonds)
  3. Results in different DNA structures. Natural changes in bonds result in different structures.
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10
Q
  1. What is A-form DNA favored in?
  2. What is B-form DNA favored in?
  3. Describe Z-form: Where is it found and what does it play a role in?
A
  1. Favored in anhydrous solutions. Favored by DNA-RNA hybrids and RNA alone.
  2. Most stable in biological systems for randome sequence DNA. Called “Watson and Crick” DNA.
  3. Certain base sequences and high salt favor this form. A left-handed helix, a more elongated than A or B, almost no minor grooves, flat major groove.
    1. Short stretches found in eukaryotes and prokaryotes.
    2. Plays a role in gene regulation.
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11
Q
  1. What do four or more Adenosine residues lead to?
  2. What is a palindromic sequence?
  3. What are inverted repeats? What type of structure can they lead to?
  4. What are mirror repeats?
A
  1. A tight bend in the helix.
  2. The same sequence forward and backward.
  3. Occur over 2 strands of DNA.
    1. Self Complementary: Hairpins and cruciforms. Proteins recognize these loops and turn genes on.
  4. Occur often on the same strand.
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12
Q
  1. What is Triplex DNA?
  2. What is Tetraplex DNA?
  3. What do they play a role in?
A
  1. B-form of DNA makes additional H-bonds in Major Groove.
    1. Special H-bonds: Hoogsteen Positions
    2. Non-Watson-Crick pairing: Hoogsteen Pairing
  2. 4-strand DNA where there is a high proportion of Guanosine residues, very stable
  3. DNA replication, recombination, and transcription.
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13
Q
  1. What are the three RNAs?
  2. What is siRNA (iRNA or RNAi)? What does it do, and how can it be used?
A
  1. rRNA, mRNA (carries genetic information, DNA to ribosome), tRNA (translates information coded in mRNA into protein.
  2. Silencing RNA
    1. Long dsRNA “diced” into siRNA
    2. siRNA unwinds and becomes ssRNA.
    3. Binds to mRNA to inhibit translation via endogenous endoribonuclease activity.
    4. Can be used as a tool to manipulate gene expression.
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14
Q
  1. What is always the product of transcription?
  2. What kind of helix does it form?
  3. After transcription, what can complementation produce?
  4. What form of helix does this product produce?
  5. What does the final structure of RNA form? What interactions play a role in stabilizing RNA?
A
  1. A single stranded RNA molecule.
  2. Right-handed helix.
  3. Double stranded RNA.
  4. Complementary strands tend to produce the A-form helix, B-form is not observed.
  5. Its final structure is complex with many forms. Weak interactions play a role in stabilizing the RNA.
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15
Q
  1. What is DNA at room temperature in aqueous solution?
  2. What temperature does DNA melt at?
  3. What kinds of bonds are broken?
  4. What is disrupted?
  5. What happens when DNA is unwound and strands separate?
  6. Does each species have a unique DNA denaturation temperature?
  7. What would drive the denaturation temperature up?
A
  1. Viscous
  2. 80°C and pH extremes
  3. H-bonds are broken
  4. Base stacking is disrupted.
  5. Can be partial or complete, only change is in conformation. Function is not lost.
  6. Yes, each species has a characteristic denaturation temperature.
  7. The higher the GC content, the higher the denaturation temperature, due to H-bonding.
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16
Q
  1. Sometimes during Renaturation (Annealing) it happens in one, rapid step. What could this be due to?
  2. What are the two steps of renaturation if strands are completely separated?
A
  1. If some small parts of the two DNA strands are still attached, annealing occurs in one step.
  2. 1st step is slow, strands randomly collide. 2nd step is fast, strands zipper.
17
Q
  1. What can Denaturation-Renaturation reveal?
  2. Give an example
  3. What does the relative number of Hybrid Duplexes reveal?
A
  1. Evolutionary History
  2. Melt human and mouse DNA, some sequence anneal together over a few hours.
  3. Sequence Homology
18
Q
  1. What is a Southern Blot used for?
  2. What is the process?
  3. What is PCR used for?
  4. What is the process?
A
  1. Used to show a DNA sequence is present. Others indicate a gene is transcribed and translated.
  2. Process
    1. Separate DNA segments by size in a gel.
    2. Transfer separated segments to membrane.
    3. Identify segment(s).
  3. A specific sequence of DNA can be replicated many times using a simple set of reactions
    1. Heat to denature
    2. Bind oligonucleotide primers to the specific sequence.
    3. Add heat-stable DNA poly
    4. Duplicate the DNA sequence between the primers.
    5. Repeat the steps above 20-30 times.
19
Q
  1. What is DNA microarrays used for?
  2. What is the process?
A
  1. Identifies gene sequences in a whole genome or plasmid.
    1. Quantitative
    2. May identify several sequences on the same array at the same time.
      3.
20
Q
  1. What is Deamination?
  2. What does deamination of Cytosine result in? How often does it occur? How is it repaired?
  3. What does deamination of A and G result in? What is it caused by? How often does it occur? How can it be prevented?
A
  1. The loss of an amine group.
  2. Results in Uracil
    1. 1 in 107 per cell per 24 hours.
    2. Repaired by excision of mutated DNA.
  3. A to hypoxanthine and G to Xanthine
    1. Caused by nitrous acid and bisulfite.
    2. 1 in 105 per cell per 24 hours.
    3. Prevent bacterial growth in the food industry.
21
Q
  1. What is Depurination?
  2. What is the rate of loss?
A
  1. Hydrolysis of the N-β-glycosidic linkage between the sugar and base.
  2. Rate of loss is 105 purines per cell per 24 hours.
22
Q
  1. What does Ultraviolet light result in?
  2. What does Ionizing radiation and X-rays result in?
  3. What does oxidative damage drom hydrogen peroxide, superoxide radicals and hydroxyl radicals result in?
A
  1. Thymidine dimers on the same DNA strand.
  2. Open ring structures and caused covalent bond breaks in DNA backbone.
  3. Many alterations in DNA.
23
Q
  1. What are some other functions of nucleotides?
  2. How do they operate as cofactors? What happens if you remove adenosine?
  3. What is a Nucleotide-binding fold? What is it specific for?
  4. What else may they help with?
  5. What is The Rossman Fold? What does that area bind? What alternates in this structure?
A
  1. Chemical energy sources (Phosphate covalently linked to the 5’ hydroxyl of ribonucleotide.
  2. Adenosine is present in all of the important ones.
    1. Adenosine does no participate in the chemical reaction.
    2. Removal of adenosine reduces cofactor function.
  3. A domain present in many enzymes. Specific for adenosine nucleotides over others.
  4. May assist in substrate/active site induced fit.
  5. A binding motif or super-secondary structure. Area of a protein that binds nucleotides with specificity (As a Motif, they are a pattern of secondary structure found in many proteins. Alternating beta strands (6) and alpha helices (4).
24
Q
  1. Can nucleotides act as electron carriers?
  2. What do the Nitrogens in Riboflavin do?
  3. Nucleotides as Regulatory molecules?
A
  1. Yes!
  2. N picks up an electron and a proton, and another does the same, for a total of 2 e- and 2 protons.
  3. Can act as signal transducers and second messengers.
25
Q
  1. What is the overall goal of the Sangar method?
  2. What do we prepare the 4 tubes with?
  3. Why can’t ddNTPs covalently bind another NTP at 5’ position?
  4. How do we separate labeled fragments?
  5. What is the result?
A
  1. To obtain 4 sets of complements of original DNA. Each set is labeled at a different position of 5’ end.
  2. DNA as a template, All 4 dNTPs, DNA Poly, DNA primer. After add a small amount of ddNTPs
  3. There is no 3’ hydroxyl, as a result DNA polymerization stops at positions with that complementary base. Fragment sizes correspond to relative position of 1 base in sequence.
  4. Separate with by electrophoresis.
  5. As as result, a pattern forms in each lane for each of 4 bases.
26
Q
  1. What does restriction endonucleases types 1 and 3 do?
  2. What does restriction endonucleases type 2 do?
  3. What does DNA ligase do?
A
  1. Random or cut at 25 bp from recog. sequence. ATP dependent
  2. Cleave phosphodiester bond at very specific sequences. Produce sticky or blunt ends. Use recombinant DNA tech.
  3. re-form the phosphodiester bonds.
27
Q

How do we get the DNA into the cloning vector? How does it make many copies?

A

MCDB 310 (9 decks)

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