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BIOB11 Lecture Notes (1-11)
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- University Of Toronto (U Of T )
BIOB11 Lecture Notes (1-11)
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BIOB11 NotesLECTURE 01
● Topoisomerases - regulates supercoiling
● Catenate - link or unlink circular DNA
● Linkage group - inherited together
● Recombination / Crossing over occurs at chaismata
LECTURE 02
● Area of nucleation - reannelling area
● Thermal Denaturation - heat denatures DNA
● Melting Temperature (Tm) - temp to shift absorbance readings to 50% ssDNA and 50% dsDNA
● G-C pairing is stronger than A-T pairings because of number of hydrogen bonds and twist stacking (van der wall forces)
● Smaller and unique genomes renature quicker as they have less to match compared to larger and unique genomes
○ Greater time to renaturation = greater complementary pieces to find each other
● Rate of renaturation of genomes (C0t plots)
○ C0 = initial DNA concentration
○ T = time
● Copy numbers = repeated DNA = repeated nucleotide sequences
● Prokaryotic DNA is a simple progression of one gene after another.
● Tandem repeats = short repeat sequences
● SLIDE 18
○ Satellite DNAs (generally found at centromeres and telomeres) - 5-500 bp in tandem repeats of up to 100 kb that can
form very large clusters base composition is distinct from bulk DNA. Ex. Packing structure.
○ Minisatellite DNAs - 10-100 bp with up to 3000 repeats highly variable (polymorphic), therefore differ between
individuals/generations – basis for DNA fingerprinting in criminal and paternity tests.
○ Microsatellite DNAs - 1-5 bp in clusters of 10-40 bp scattered quite evenly throughout the genome highly variable
(lots mutations), used to compare populations.
● FISH - biotin binds to avidin, avidin has GFP. Satellite DNA found on centromere of chromosome
○ High repeats = in centromere of chromosome
● Moderate repeats
○ Coding = histones and ribosomal RNA = identical tandems
○ Non-coding = SINEs and LINEs (short/long interspersed elements), sequences scattered
● Non-repeated
○ Sequences code for all proteins but histones
○ Single copy per haploid set of chromosomes
○ Highly localized
● VNTR = variable numbers of tandem repeats
○ Minisatellite DNA and fingerprinting
● Restriction enzymes are endonucleases that cut DNA into fragments
○ Cut at restriction site
● Genetic Mapping
○ Centimorgan (cM) = location of gene in genetic map
■ Closer the genes, lower recombination chances
■ Takes generations
● Physical Mapping
○ Restriction enzyme sites
● Isoform = similar proteins
○ Pseudogenes = similar to protein family however have too many mutations = non-functional
● exons (protein coding region) and introns (non-coding region)
● Mobile elements = DNA sequences that can move in genome (changes in quantity and expression)
○ Mobile DNA = dna that moves in the genome = transposition with the mobile genetic elements called transposable
elements
○ Bacterial transposable elements = transposons (jumping genes)
■ Not tandem arrays
■ Transposases = proteins
, ● Cuts in overhangs for DNA polymerase to fill the rest
● Transposons inserted into DNA:
○ Inverted repeats on ends of transposons for recognition by transposase
○ Direct repeats to recognize insertion on other DNA
● Two methods of transposon movements
○ Cut and paste
■ Donor DNA has transposon, transposase cuts it out, inserted into recipient DNA. Donor DNA joins back
together without transposon.
○ Copy and paste
■ Donor DNA has transposon, RNA polymerase makes ssRNA, retrotransposon reverses it into complementary
DNA (cDNA), make dsDNA = transposon created, insert into recipient DNA
○ Shows that genome is not stationary / stable / static repository
● Two types of Interspersed elements
○ LINEs
○ SINEs
● Non coding DNA = junk DNA
○ Functional non coding DNA = not junk DNA
● Histones highly conserved
● SNPs = single nucleotide polymorphism
○ Change in one nucleotide
○ Coding or non-coding region
● CNPs = copy number polymorphisms
○ Deletion, insertion (3 insertions restores reading frame), inversion, frameshift, missense (one change in nucleotide =
different amino acid), nonsense (stops early).
● Structural variation = change in segments of genome/DNA = change in chromosome structure
○ Coding region
● Essential Protein = needed to live, otherwise cell dies
● Haplotypes = genes inherited together as they are close on the chromosome
○ Eg. SNPs for diseases
LECTURE 03
● Central Dogma = DNA to RNA (transcription) to Protein (translation)
● Beadle Tatum experiment = one gene one enzyme hypothesis
○ Mutations in a single gene leads to protein change and function
● mRNA = intermediate of gene and polypeptide = messenger RNA
○ Template for translation to polypeptide
○ Half life of DNA = gene expression only when needed
● DNA = information storage
● Transcription
○ Initiation - RNA polymerase binds to promoter with help from transcription factors
○ Elongation - RNA polymerase advances 3’ to 5’, where new strands are made 5’ to 3’.
○ Termination - RNA polymerase releases DNA by a stop site signal, RNA strand is completed.
● Template strand = anti-sense strand = transcription unit
○ mRNA is complementary to this
● Coding Strand = sense strand
○ mRNA is identical except T is switched with U
● RNTPs = ribonucleotide triphosphates = building blocks
● Topoisomerase = help unwind or wind strands
● RNA made by phosphodiester bond 5’ to 3’
● DNA sugars
○ Ribose - OH group on second carbon, two OH
○ Deoxyribose - H group on second carbon, only one OH
● Downstream = positive = 3’
● Upstream = negative = 5’
● Prokaryote Transcription
○ Initiation (sigma factor)
, ■
Regulatory sequences
● consensus sequence
● Pribnow box
○ Elongation
■ RNA polymerase elongates RNA strand
○ Termination (rho factor)
■ Sequence or signal
● Types of RNAs in Prokaryotes
○ mRNA = intermediate of DNA and protein = non-complex
○ rRNA = structural support and catalyze reactions = complex
○ tRNA = adds amino acids = complex
● Primary transcript = pre-mRNA
○ RNPs also work on it while be transcribed
● S-value (svedberg unit)
○ Larger S values = greater weight = sinks faster to bottom on centrifuge
● rRNA made from rDNA in moderate repeat sequences
○ Synthesized in the nucleolus (nucleoli is plural)
■ except 5S which is made by polymerase III
● Internal promoter rather on the end
○ RNA is mostly rRNA to make proteins
● SLIDE 37???
● RNA polymerase makes snoRNAs (small nucleolar RNA) that makes snoRNPs (small nucleolar ribonucleoproteins)
LECTURE 04
● hnRNA (heterogeneous nuclear RNA) came before mature mRNA = primary transcript = PreRNA
○ Processed into small RNA and exported to cytoplasm
● General transcription factors (GTF) = binds many places for promoter
○ RNA polymerase recognizes it by the TATA box
● Transcription INITIATION can only occur when three things are present
○ TATA box = site of assembly
○ Preinitiation Complex (PIC)
○ TATA Binding Protein (TBP) = RNA polymerase can initiate transcription at correct site, significantly changes
conformation and apart of complex protein TFIID (transcription factor for polymerase II, fraction D) which consists of
TAFs (TATA Binding Protein Associated Factors)
● TFIID ➝ TFIIA and TFIIB ➝ RNA Pol II and TFIIF ➝ TFIIE and TFIIH
○ TFIIH = Transcription factor polymerase II, fraction H. = Transcription initiation
■ Assembles onto complex when RNA polymerase is bound
■ Contains:
● Helicase = Unwinds DNA
● Kinase = phosphorylation of RNA polymerase, lets it move forward
○ Transcription initiation occurs when RNA polymerase is phosphorylated by TFIIH on the serine residues at c-terminal
domain (CTD)
■ Once released, TFIID can then initiate assembly of preinitiation complex for another RNA polymerase so that
multiple transcription phases can occur
● ELONGATION FACTORS
○ TFIIS - helps RNA Polymerase move after a pause
○ ELL - suppresses transient pausing
○ P - TEFb - another kinase that phosphorylates CTD on serine #2 which recruits other factors
● Basal level transcription = RNA polymerase II and GTF
● mRNA
○ 5’ cap = methylated guanosine cap
■ In nucleus with primary transcript, guanosine is methylated at position 7, making a 7-methylguanosine added
to 5’ of RNA. Prevents exonucleases from degrading it and helps with transport out of the nucleus and
initiation of mRNA translation
● Process, Capping Enzyme (two active sites and two enzymes)
○ RNA Triphosphatase - removes from triphosphate to diphosphate
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