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Angiostatin
Fragment of plasminogen cleaved in tumours, prevents the development of micro-metastases
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Endostatin
On chr 21, so overproduced in Down's: prevents development of micro-metastases
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Example of aneuploidy (not down's)
Loss of chr 10: glioblastomas
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K-Ras missense mutations cause
pancreatic tumours
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Example of gene amplification problem
Over expression of myc causes advanced neuroblastomas
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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!
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Bacterial retrotransposon
Tn3: carries antibiotic resistance
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Problems with mismatch repair cause
hereditary non-polyposis colorectal cancer syndrome
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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
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Problems with NHEJ
Mutations in DNA ligase IV: some leukaemias
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HR things
RecA, RAD51, RuvAB (used in restarting broken replication forks)
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Methylation of DNA
- Bacteria: A in GATC by Dam methyltransferase
- Eukaryotes: CpG islands - genomic imprinting (Prader-Willi 15q)
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Prader-Willi /Angelman
- 15q
- (low muscle tone, short, incomplete sexual development, hyperphagia)
- (happy puppet)
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Rho factor needs
50-90nt region with high C content
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3 drugs to stop RNA transcription
Actinomycin D (intercalates), Rifamycin (binds B subunit), a-amanatin (interferes with translocation)
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DNA footprinting
Label with 32P. Add binding protein. Limited nuclease digestion with DNAse 1. Visualise using autoradiography.
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How do we know most promoters are nucleosome free?
DNAse1 digestion occurs fastest in actively transcribed DNA
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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
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5'cap, 3 tail
- m7g5'ppp5'n
- polyadenate polymerase
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how is eukaryotic transcription stopped
Pol II carries on for ages. pre-mRNA is cleaved between AAUAAA and GU/U rich sequence.
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WT1
- 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.
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RNA editing
hydrolytic deaminations (C->U or A->I)
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Wobble example
Inosin can bind to U, C or A
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When does large subunit bind?
Initiating AUG codon
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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
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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.
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Translation
- 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)
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Riboswitches
DNA sequences, bind small molecules. eg thiamine.
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Lysosyme sequence
KFERQ: lots from liver, kidney during fasting.
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Ubiquitination
E1, E2 (to SH) (activating, conjugating). UBIQUITIN LIGASE: to lysine, isopeptide bond. D box in cyclins required?
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