Mitosis and meiosis

1
Lecture 21

• Mitosis and meiosis
• What are the differences and similarities?
• Diploid vs. haploid
• Recombination
• Meselson-Weigle experiment showed recombination
  by density of DNA fragments
• Homologous Recombination
• Crossing over between different strands of DNA

2

• Mechanism of homologous recombination
• 2 pieces of ds DNA can pair through homologous
  sequences
• Crossing over occurs between a single strand of
  each ds molecule
• The Holliday junction is formed
• Breaking of the DNA strands can resolve the
  Holliday structure in 2 ways

3
parental recombinant
4
Lecture 22

• Slide 4- typo- should read represents 2 ds DNA
  molecules
• Proteins associated with recombination
• recA- strand displacement, binds ss DNA
• Also involves recBCD complex
• RuvA, B and C- involved in resolution of Holliday
  structure

5
Other processes involving recombination

• Transposable elements
• Require very little homology at target site
• Requires transposase enzyme
• Generates a duplication of the target site upon
  integration
• Can get homologous recombination between
  different transposable elements on the same
  chromosome- depending on their orientation
  relative to one another get either inversion of
  intervening sequences or deletion

6
Lecture 22

• Integration of viruses into host genomes
• Example in notes ? integration into E. coli (p.
  7)
• DNA repair
• Antibody diversity
• Recombination in each cell generates a different
  arrangement of DNA sequences encoding antibody
  chains

7
Lecture 23

• Cell cycle
• Phases, what happens during each to the cell,
  nucleus and chromosomes
• Monitoring cell division
• Use various techniques to mark cell stages, then
  calculate what percentage of total cells that is-
  get relative length of a particular cycle
• Control of cell cycle
• Proteins make sure cell does not proceed if its
  not ready- too small, replication errors, DNA
  damage
• Analysis of cell cycle through mutants

8
Lecture 24

• Transcription
• Prokaryotes- concurrent transcription and
  translation no modification
• Eukaryotes- transcription in nucleus, translation
  in cytoplasm capping, splicing, poly A tail
  addition
• Control of gene expression in prokaryotes
• at level of transcription
• mRNA stability very important

9
Lecture 24

• Terminology-
• sense, antisense, coding strands
• RNA polymerase
• Initiation
• RNA pol scans DNA for promoter sequences (-35
  and -10 regions)
• Once it finds them, it unwinds DNA and initiates
  transcription
• The sequence of promoter determines how much
  transcription will occur- how closely does it
  match consensus? What effect do mutations have?

10
Lecture 24

• Elongation
• Transcription bubble of unwound DNA moves with
  RNA pol along the length of the chromosome
• Termination
• Rho dependent
• Rho protein complex binds to RNA and disrupts
  pairing between nascent strand and template
• Rho independent
• Secondary structure within RNA due to specific
  sequences (G-C rich, followed by run of Us) leads
  to dissociation of RNA from template

11
Lecture 25

• Operons
• One promoter serves a number of genes
• One mRNA is made and each cistron (gene unit) is
  translated independently
• Examples- lac, trp
• Control of transcription
• Repressors
• Bind to operator sequences (at or near promoter)
• Their activity can be affected by effectors
• Can increase activity (trp trp repressor)? more
  active repressor
• Or decrease activity (lac lac repressor)-
  blocks activity of repressor, leading to
  transcription

12
Lecture 25

• Activators
• Bind at or near promoter and increase RNA
  polymerase binding (eg. CRP at lac operon)

Lac operon no lactose- no transcription
13
Lecture 25

• Lac operon
• No Lactose around
• Operon switched off, no mRNA regardless of
  glucose
• Lactose present glucose also present
• The presence of lactose inactivates the repressor
  
• ? Transcription occurs
• Glucose present ? cAMP is low ? CRP does not
  help transcription
• Lactose present no glucose
• The presence of lactose inactivates the repressor
• ? Transcription occurs
• NO Glucose ? cAMP is high ? cAMP binds CRP
  (becomes activated) ? CRP binds Helps
  Transcription
• High Level of transcription

14
Lecture 26

• Trp operon
• Regulated by 2 mechanisms

15
Lecture 26

• Control of gene expression by protein with
  specific motifs
• HLH proteins bind and bend DNA
• Summary
• Repressors and activators
• When is a repressor active vs. inactive?
• When is an activator active vs. inactive?

16
Lecture 27

• Eukaryotic transcription
• 3 kinds of polymerases responsible for making
  different classes of RNA
• Pol I rRNA
• Pol II mRNA
• Pol III tRNA, 5s rRNA
• Each has a different level of sensitivity to
  certain drugs- can be used to distinguish which
  RNA is made by which polymerase
• Each cannot bind to a promoter without an
  accessory protein

17
Lecture 27

• Pol I make rRNAs
• Uses 2 TF for initiation
• TFIB binds promoter
• TFIS stabilizes TFIB and allows RNA pol I to bind
• Gene for 45S rRNA encodes 3 mature rRNAs that
  emerge after RNA processing
• Pol III makes tRNAs and 5s rRNA
• Unusual regulation in which TFs nind to DNA
  sequences that will be transcribed

18
Lecture 28

• RNA pol II and mRNA
• Regulation by 2 control elemens
• Promoter
• TATA box bound by TFIID -composed of TBP and TAFs
  (TATA-binding associated factors)
• Enhancers
• DNA sequences that can bind multiple regulatory
  proteins
• Can be far away orientation doesnt matter
• Ways of studying enhancer function

19
Lecture 28

• Motifs found in DNA-binding proteins
• Zinc fingers
• Protein structural domain that binds a Zn2 ion
  though interaction with 2 alpha helices and two
  beta sheets
• Leucine zippers
• Protein structure motif in which leucine residues
  lie n one face of an alpha helix, allowing it to
  interact with a similar protein forming either a
  homo- or hetero-dimer
• HLH motif-
• Alpha helices that er spaced so as to fit into
  the DNA grooves

20
Lecture 28

• Modifications of mRNAs
• 5 capping
• Only added onto pol II transcripts
• GTP added in reverse orientation, methyl groups
  are also added
• Serves to position the ribosome and to stabilize
  the transcript
• Poly A tailing
• Polyadenylation site (AAUAAA) in sequence signals
  the end of transcription, nuclease cuts
  downstream from that
• Poly A polymerase adds on A residues
• Useful for isolating mRNAs