Download Dr. Becker`s Review – Exam 4 Notes provided by Kadie Keen

Dr. Becker’s Review – Exam 4
Notes provided by Kadie Keen
Disclaimer: I tried to scan Kadie’s notes for all of us. My technological abilities this evening
were VERY lacking. Hence, I am typing up Kadie’s notes for you guys. Sorry you couldn’t have
her originals. She put a lot of work into them for everyone. THANKS, Kadie!
DNA Structure and Replication
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Phage: Protein and DNA
Protein has sulfur (sulfide bonds)
DNA has phosphorus
DNA is a double helix (discovered by Watson and Crick)
Pictures taken by x-ray defraction (Rosalind Franklin)
Sugar and phosphorous backbone of DNA held together by covalent bonds (phosphodiester
bonds)
hydrogen bonds between nitrogen bases (A-T; CG)
Covalent bonds (phosphodiester bonds between
deoxyribose sugars and phosphate groups)
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Strands are antiparallel
A=T and C ≡ G
Rules of Chargaff :
o A = T%; If A is 30%, T is approximately 30% of total nitrogen bases
o C = G%; If C is 20%, G is approximately 20% of total nitrogen bases
o NOTE: All four (% of A, % of T, % of C, and % of G should add up to 100%)
One old strand of DNA (one strand from the parent, original DNA) and one newly formed
strand: Called semiconservative replication
DNA replication occurs in interphase (S phase)
Semi-conservative replication
RED = parental DNA
BLUE = newly formed DNA
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Okazaki fragments make lagging strand
DNA polymerase I replaces RNA
Spaces are closed by ligase
Helicase unzips
Topisomerase prevents strands from supercoiling as well as SSB (single-strand binding
protein)
DNA polymerase III builds new DNA strand and binds to RNA
Primase puts in an RNA primer and some DNA polymerase connect
Always builds 5’ to 3’
Always reads 3’ to 5’
Proof Reading
 DNA polymerase III checks DNA to make sure A didn’t get matched with C etc…
 DNA polymerase II fixes it
 Before the S phase, it checks to see if the strand is worth replicating
 If strand is damaged DNA polymerase cuts and fixes it
 Prokaryotes have 1 circular chromosome
 The origin of replication (ORI) is where it starts
 A and T have 2 hydrogen bonds
 C and G have 3 hydrogen bonds
 Prokaryotes have 1 ORI
 Eukaryotes have multiple ORIs
 A telomere is the end of a chromosome (telomere cap)
 Telomerase is built in the RNA primer
 Chromosomes shorten as you age which causes shortened telomeres; the shorter the
telomere, the shorter the life span remaining
 Proteins are made in DNA
 Central Dogma:
transcription
DNA
mRNA
 3 Steps to Transcription and Translation
o Initiation
o Elongation
o Termination
Initiation
 Transcription
 Eukaryotes: the DNA is in the nucleus
 Prokaryotes: the DNA is in the cytoplasm
 RNA polymerase II builds RNA
translation
protein
Elongation
 Promoter sequence is the DNA sequence where RNA polymerase binds (also called
initiation sequence or TATA box)
 Initiation factors help bind
 RNA polymerase builds by 5’ to 3’ and reads 3’ to 5’ and A binds with U instead of T
Termination
 Occurs when polymerase separates, mRNA separates, and etc.
 Eukaryotes aren’t as important as prokaryotes
 Prokaryotes can create a hairpin loop
 Eukaryotes it will just produce a stop codon
 RNA polymerase III makes tRNA
 RNA polymerase II makes rRNA
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pre-mRNA processed in spliceosome within the nucleus to form mature mRNA
Requires 3 things
1. Remove introns (splice)
2. Apply 5’ cap (guanine cap – represented by a backwards “G”)
3. Apply 3’ tail (polyA – tail …AAAAAAAAA)
 Mature mRNA goes to translation (ribosomes are attaching at the same time)
NOTE: Proteins that are released from the cell into the body are processed by the
ribosomes attached to the ER (rough ER). Proteins that will be used within the cell
are processed by the ribosomes that are free in the cytosol (cytoplasm)
Translation
 Anticodon pairs with codon on mRNA
 There are 3 nucleotides in each
 64 codons total
 20 amino acids
 Amino acids have multiple codons
 First 2 likely to be the same and 3rd is most likely different
 rRNA builds ribosomes and proteins
 translation occurs in the cytoplasm for both prokaryotes and eukaryotes
 ribosome attaches to mRNA by small subunit and 1st tRNA
 start codon is always AUG (methionine, MET)
 there are 2 parts of a ribosome:
o small ribosomal subunit (attaches first)
o large ribosomal subunit (attaches second)
 large subunit attaches to mRNA to catalyze the reaction and synthesize the protein
(polypeptide chain)
 Methanine is always the 1st amino acid (start codon)
 During translation, shifts 3 nucleotides down every phase
 1st codon is in the A site where tRNA binds
 Then ribosome shifts to next codon in P site
 When it shifts again as it’s shifting in P site it forms peptide bond
 The empty tRNA exits at the E site
 Translation, 5’ to 3’ direction, continues until reaches the STOP codon
 3 stop codons are UAA, UAG, and UGA (no amino acid name, just STOP codon)
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Termination factor binds to stop codon
No tRNA matches stop codon
If mRNA begins with SRP it stays inside the cell
SRP (signal recognition particle) sends to rough ER then ends up outside the cell.
Mutations
 Base pair or point: will only change 1
 Missense: changes proteins which make different amino acids
 Nonsense: codes for stop codon
 Silent: no change, probably 3rd position (wobble)
 Frameshift: reading frame shifts, caused by insertion or deletion; NOTE: worst mutation
that could happen)
Gene regulation
 Transcriptional: promoter sequence, enhancer sequence; some factors are activators
 Posttranscriptional: RNA interferences; lncRNA, miRNA, siRNA; distracts mRNA
 Translation: translation factors help
 Posttranslational: manipulates proteins because it is breaking down; tagged with ubiquitin
signals and starts breakdown process; regulation of gene expression
 Activators help, repressors block
Differentiation:
 the ability for a cell to become a different type of cell
 Cells able to be different: STEM CELLS
 Totipotent: any cell in embryo or supporting tissue (placenta, chorion, amnion) of embryo
 Pluripotent: inside the developing early-stage embryo
 Multipotent: more than one
 Unipotent: only develops into one cell type
Animal Cloning
 Needs DNA technology/engineering
 Animal donates an egg
 Chromosome number in egg is 23 (haploid)
 Somatic cell nuclear transfer is required for cloning
 Diploid: somatic cell nucleus in clone (46 in humans)
 Clone is like the nucleus donor
 Clone life is shorter (difference in length of in adult chromosome telomeres)
 Gene cloning is used to make a good copy to replace a bad copy
 Used to make human insulin
 Can modify bacteria (cloning vector)
 If cloning gene you need two different DNA strands (recombinant DNA ) – 2 sources for
the DNA
 A cloning vector is needed for the plasmid (part of the bacteria) to reproduce the
eukaryotic gene sequence such as human insulin
 The enzyme restriction endonuclease (restriction enzyme) looks for palindrome and cuts
 Pallindrome sequence ex: ABC… CBA;
 Cutting ex: AAC….GUU
UUG…CAA
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Ligase is needed to close it
PCR (polymerase chain reaction)
 Used to create millions of copies of a specific gene sequence or to produce millions of
copies of DNA evidence from crime scene in order to analyze
 Thermocycler repeats hot and cold cycles to create the copies
 Has to be mature mRNA since it only contains exons
 Used to make specific DNA
 Wants to make cDNA
 Single to double strand requires reverse transcriptase
 RNase gets rid of RNA strand
 PCR is used to make amny copies in a test tube
 You put DNA of interest into test tube, put in DNA polymerase taq (needed because of hot
temperatures)
 DNA polymerase binds to DNA primers
 Gel electrophoresis is the technique used to visualize the DNA
 The charge of DNA is negative
 Smaller DNA segments will migrate farther to the positive end during electrophoresis;
 Larger particles move more slowly and therefore , the largest segments of DNA will
remain closest to the negative end of the electrophoresis tray
 Good ones: small repeating, noncoding DNA, RFCP, SNP
o SNP used to determine relationships – can analyze different base pair mutations –
different alleles, different lengths, will be different for different people.
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 DNA fingerprinting
 Parternity testing
 Crime scene testing
 Certain genetic trait isolations (genetic disorders)