Biology 1002B Lecture Notes - Molecular-Weight Size Marker, Vaucheria, Agarose Gel Electrophoresis

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19 Jun 2013
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Lecture 18: DNA Replication
components necessary for DNA synthesis: substrates, template, primer and
enzymes.
Substrates: A,T, G, C base pairs
Template: the original strand of DNA
Primers: RNA primers base paired with the template to allow DNA polymerase to
work
Enzymes: all enzymes and proteins required to make the replisome. (to synthesize
DNA)
structure of DNA with respect to polarity and complementarity: DNA has a sugar-
phosphate backbone. It has 4 nitrogenous bases: adenine, guanine, thymine and
cytosine. Adenine and guanine are purines (double-ring structures). Thymine and
cytosine are pyrimidines (single-ring structures). A base pairs with T, C base pairs
with G. DNA is polar because it has a 5’ end and a 3’ end.
5’ and 3’ end of a DNA strand: The 5’ end of DNA has a phosphate group. The 3’ end
has a hydroxyl group. DNA synthesis occurs in the 5’ to 3’ direction.
relationship between DNA synthesis and chromosome duplication during S phase:
DNA is synthesized during S phase, and therefore all the chromosomes (bundles of
DNA) get duplicated.
structure of replication fork and replication bubble:
problem that arises during replication of the ends of linear chromosomes: DNA
polymerase can only extend DNA from a free 3’ OH group. Because the lagging
strand ends on a 3’ end, there is no way to finish the DNA even if a primer were
there, as we would not be able to replace the primer because there would be no free
OH group. This means that the chromosome would get progressively shorter and
shorter if there were no mechanism to fix this.
how telomerase addresses the above problem: the ends of chromosomes contain a G
rich series of repeats, called a telomere. Telomerase recognizes the tip of an existing
repeat series. Using an RNA template, telomerase elongates the parental strand in
the 5’ to 3’ direction and adds additional repeats as it moves down the parental
strand. The lagging strand is then completed by DNA polymerase alpha, which
carries a DNA primase as one of its subunits. In this way the original information at
the ends of linear chromosomes is completely copied into the new DNA, and the
only thing lost is a bit of the telomere.
Lecture 19: Gene Mutation
characteristics of the Ames Test as a screen for chemical mutagens: the bacterium
Salmonella typhimurium is used because there is a mutant that is unable to
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synthesize the amino acid histidine. Therefore when this mutant is grown on
minimal medium, it will not grow. The Ames test takes possible chemical mutagens
and puts them on minimal medium with the mutant bacteria. If the bacteria start to
grow, it is assumed that the chemical caused a back mutation, allowing the bacteria
to start to synthesize histidine. This means that the chemical causes mutations in
bacteria and possibly other organisms and is a positive Ames Test.
characteristics of mutation vs. DNA damage: a mutation is a heritable change in
double-stranded DNA sequence such as base substitutions,
insertions/deletions/reorganizations. DNA damage is a physical abnormality in the
DNA. Damage can be recognized by enzymes and be correctly repaired. If it cannot
be repaired then transcription, translation and replication will be blocked and/or
the cell with go through apoptosis.
mechanisms of spontaneous substitution mutation arising from errors in
replication: Sometimes a base pair can undergo a tautomeric shift, which means that
the electrons within the base pair shift and then it pairs with a different base pair.
An example is thymine, which normally has 2 hydrogen bonds with adenine.
Sometimes it undergoes a tautomeric shift and will base pair with guanine and have
3 hydrogen bonds.
mechanisms of spontaneous indel mutation arising from errors in replication:
Because of the sequence (same base pairs all in a row), one or the other of the
strands can loop out. Therefore when the DNA is replicated, one of the sides is too
short.
mechanism by which base analogues, such as 5-bromouracil, increase mutation
frequency: 5-bromouracil is indistinguishable from thymine and will be put in DNA
instead of thymine. The bromine atom leads to a molecule that is tautomerically
unstable, which is bad as it will always change which base it wants to pair with.
likely effects of silent, missense and nonsense mutations on protein function: (all are
caused by a change of a single base pair)
Silent: the changed codon will specify for the same amino acid, due to the
degeneracy of the genetic code.
Missence: the changed codon will specify for a different amino acid. This may or may
not alter the protein function in a big or small way.
Nonsense: the changed codon will specify for a stop. This causes a premature
termination of the protein during translation. This will almost certainly affect the
protein in a big way, it will likely be partially functional at best.
mechanism of frameshift mutation and likely effect on protein function: A frameshift
is cause by an insertion or deletion of a base pair. This alters the whole reading
frame from there on. The resulting polypeptide is usually non-functional because of
the significantly altered amino acid sequence.
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likely effects of mutations outside of coding regions: Some of the non-coding regions
are used for transcriptional, translational, post-transcriptional regulations. These
mutations would then affect the regulations. It really depends on which part is being
affected.
Lecture 20: Chromosome Mutation
mechanism of action of DNA damage by non-ionizing radiation (UV): pyrimidines, in
particular thymines, that are side by side can absorb photons of light and this causes
a reorganization of electrons which causes covalent bonds and creates a thymine
dimer. This distorts the helix and blocks replication and transcription.
mechanism of action of DNA repair enzymes:
Proofreading: if a newly added nucleotide is mismatched, DNA polymerase will
reverse and use a a built-in deoxyribonuclease to remove the incorrect nucleotide.
The enzyme then resumes working forward. (RNA polymerases cannot proofread).
Reverse transcriptases can’t really proofread either.
Excision repair: the part where the bad base pair is is cut out and then it is filled in.
Photoreactivation: In most organisms thymine dimers can be repaired by
photoreactivation. Photoreactivation is a repair process in which photolyase
enzymes directly reverse thymine dimers via photochemical reactions. Lesions on
the DNA strand are recognized by these enzymes, followed by the absorption of light
wavelengths (particularly blue light). This absorption enables the photochemical
reactions to occur, which results in the elimination of the pyrimidine dimer,
returning it to its original state.
mechanism of action of DNA damage by ionizing mutagenesis: When ionizing
radiation passes through your cells, it creates reactive oxygen. This then damages
everything, from proteins to DNA.
mechanism of production of aneuploid (abnormal number of chromosomes)
gametes during meiosis: This happens by nondisjunction, which is the failure of
homologous pairs to separate during the first meiotic division or of chromatids to
separate during the second meiotic division.
difficulties of studying effects of radiation exposure on large populations: its hard to
isolate large populations, and there are infinite amounts of other factors affecting
the population, because it is so large and therefore more diverse.
Lecture 21: Immunogenetics
role of phagocytosis in immune response: This is the non-specific part of the
immune system. It goes after bacteria and other antigens and engulfs them and
breaks them down.
characteristics of innate vs. adaptive immune response: The innate (non-specific)
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