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Fragment of plasminogen cleaved in tumours, prevents the development of micro-metastases
On chr 21, so overproduced in Down's: prevents development of micro-metastases
Example of aneuploidy (not down's)
Loss of chr 10: glioblastomas
K-Ras missense mutations cause
Example of gene amplification problem
Over expression of myc causes advanced neuroblastomas
Example of translocation causing disease
Chronic myeloid leukaemia: Abl Chr 9 -> Abl binds to BCR gene on Chr 22: fusion gene called Bcr-ABL. Bcr-abl tyrosine kinase signalling blocked by glivec!
Tn3: carries antibiotic resistance
Problems with mismatch repair cause
hereditary non-polyposis colorectal cancer syndrome
Proteins to recognise mismatches
- Mut S and Mut L in bacteria
- Eukaryotes: MSH (Mut S Homologue) identifies site, MLH (Mut L Homologue) (and MHL) cleave
Problems with NHEJ
Mutations in DNA ligase IV: some leukaemias
RecA, RAD51, RuvAB (used in restarting broken replication forks)
Methylation of DNA
- Bacteria: A in GATC by Dam methyltransferase
- Eukaryotes: CpG islands - genomic imprinting (Prader-Willi 15q)
- (low muscle tone, short, incomplete sexual development, hyperphagia)
- (happy puppet)
Rho factor needs
50-90nt region with high C content
3 drugs to stop RNA transcription
Actinomycin D (intercalates), Rifamycin (binds B subunit), a-amanatin (interferes with translocation)
Label with 32P. Add binding protein. Limited nuclease digestion with DNAse 1. Visualise using autoradiography.
How do we know most promoters are nucleosome free?
DNAse1 digestion occurs fastest in actively transcribed DNA
3 ways to alter histones
- Methylation: 3 to lysine - more bulky: REPRESSES TRANSCRIPTION
- Acetylation to lysine: less positive charge
- Phosphorylation of serine: 2 negative charges
5'cap, 3 tail
- polyadenate polymerase
how is eukaryotic transcription stopped
Pol II carries on for ages. pre-mRNA is cleaved between AAUAAA and GU/U rich sequence.
- TF. Recognises GC/TC rich regions.
- 2 domains: N: Pro + Glu rich activation domain
- C: DNA binding, 4 Zn fingers
- Normal cells: 4 alternatively spliced variants. truncated forms lack domains.
hydrolytic deaminations (C->U or A->I)
Inosin can bind to U, C or A
When does large subunit bind?
Initiating AUG codon
Distinguishing between AUG codons
- Eukaryote: first one along from 5'cap
- Bactera: Shine Dalgarno sequence (GGAGG) recognised by AACAA in 16S rRNA of 30S subunit
Non-cap dependent translation initiation
Internal initiation of translation using IRES (internal ribosomal entry site). Found using dicistronic assays: assayable enzymes. In picornaviruses (polio, hep c). Long 5' UTR. Hep C IRES major drug target.
- Met-tRNA to P site, via eIF2 (eukaryotic initiation factor) (interferon response, kinase)
- All AA-tRNA to A site, via GTP and EFTu (EEF1)
- EEFtu-GDP dissociate
- Peptide bond by transfer of peptide from P to A site, via peptidyl transferase activity of large ribosomal subunit (ribozyme) - no energy use.
- Translocation, via GTP and EFG (EEF2)
DNA sequences, bind small molecules. eg thiamine.
KFERQ: lots from liver, kidney during fasting.
E1, E2 (to SH) (activating, conjugating). UBIQUITIN LIGASE: to lysine, isopeptide bond. D box in cyclins required?