Shafter Flashcards
(1) Interphase chromatin is mainly existing of wat
Euchromatin
(1) Differences between euchromatin and heterochromatin
Euchromatin: Decondenses during interphase, most expressed sequences
Heterochromatin: Remains condensed, few expressed sequences (contains elements essential to chromosome stability and inheritance)
(1) Characteristics of histones
- Small basic and highly conserved
- Form a solenoid structure with DNA wound core of histone protein
(1) 3 main types of functional elements of chromosomes
Telomere
Replication origin
Centromere
(1) Telomere characteristics
- Vertebrate sequence is TTAGGG repeated over several kb
- Telomere gets shorter after each replication as end of lagging strand cannot be replaced as DNA can’t put a primer
- Telomerase can replace telomere sequence
(1) Example of creating new genes by duplication during evolution
Globin gene family
(1) First evidence for Eukaryotic transposable elements
MAIZE
- Change in pigmentation during development (variegation)
- Results in sector tissue with altered phenotype
- Proposed due to rearrangement of genome
- Unstable alleles
(1) 3 types of reassociation kinetics used to predict number of genes per genome
1) Highly repetitive DNA: High copy number seq, repetitive very short sequences
2) Moderately repetitive DNA: Moderate short sequences (e.g. histones, ribosomal genes, transposons)
4) Non repetitive DNA: Unique sequences, genes, gene families.
(2) 7 stages gene expression can be controlled
1) Histone modification (acetylation, methylation)
2) Translational control (RNA polymerase, TFs)
3) RNA processing control
4) RNA transport and localisation control
5) mRNA degradation
6) Translation control
7) Protein activity control
(2) WAT is histone code hypothesis
Proposes that specific combinations to histones and DNA of chromatin help determine chromatin configuration and influence transcription
(2) 3 histone modifications
1) Acetylation: Loosens chromatin and promotes initiation of trans. (Acetyl groups attached to +ve lysines in histone tails)
2) Methylation: condenses chromatin
3) Phosphorylation (next to methylated aa): Loosens chromatin
(2) 3 types of RNA Polymerase
Pol I: rRNA
Pol II: All protein-coding genes (mRNA)
Pol III: tRNA genes
(2) Eukaryotes consensus sequence
TATA box, -25 relative to transcriptional start
(2) RNA Pol II Initiation complex subunits
1) TFIID: TBP (TATA box recognised), TAF (regs DNA-binding)
2) TFIIB: Positions RNA Pol
3) TFIIF: Stabilises RNA polymerase
4) TFIIE: Attracts and regs TFIIH
5) TFIIH: Unwinds DNA, phos Ser5
(2) Role of Activators, enhancers
- Enhancers are distal control elements away from gene
- Activators: proteins that binds to enhancers and stim trans of gene.
(2) How are different genes activated in different tissues?
There are different activators in different cells to determine which specific gene is expressed
(2) Role of Leu zipper TFs
- Mediates both DNA and protein binding
- Bind to DNA as homo or deterodimers
(3) Methods for analysing expression of single gene
1) RT-PCR
2) Live cell imaging
3) Promoter studies
(3) Methods for analysing expression of all genes
1) Microarrays analysis (Tiling or Affymetrix)
2) RNA seq: using next gen sequencing
(3) Method for analysing TF binding site
1) Add reporter gene to promoter construct: encodes an easily assayed enzyme (e.g. B-galactosidase)
2) Transfect reporter DNA construct to cultured cells
3) Allow time for expression
4) Extract protein
5) Assay for reporter enzyme activity
ALSO CHROMATIN IMMUNOPRECIPITATON
(3) Method to test promoter function
TRANSFECTION
- Modify reporter construct and repeat experiement
- Find TF binding sites by PCR or expression of reporter gene/protein
(3) Modifications to make mature mRNA from pre-mRNA
1) 5’ Cap
2) Polyadenylation at 3’ end
3) RNA splicing
(3) Why do modifications to mRNA?
1) Distinguishes mRNA from other RNA
2) Helps mRNA to process and export to cytosol
3) Stabilises mRNA
4) required for efficient translation
(3) Principle of splicing
A TWO STEP TRANSESTERIFICATION 1) 5' end splice site 2) 3' end splice site 3) Branch point in intron sequence MUST BE PRECISE
