What affects protein expression and function
Sequence and regulation of DNA
How does DNA get to being a protein
DNA transcribe to mRNA and translated to protein
Define gene
Segment of DNA encoding a polypeptide chain, includes regulatory sequences, introns and extrons.
Gives information on proteins present in cell, sequence, structure and function
Define genomics
Study of all the genes in an organism. Gives information on how genes/proteins work together, regulate each other, interact, function together
Define genetics
Study of inheritance of genes. Gives information on mutations and inherited diseases
What are the inheritance patterns inherited genes show?
Autosomal: dominant and recessive
X-linked: dominant (rare), recessive
What are diseases caused by autosomal dominant allele?
Huntingtons disease, hypercholesteroleamia
What are diseases caused by autosomal recessive allele?
Sickle cell anemia, cystic fibrosis, phenylketonuria
What are diseases caused by x-linked recessive alleles?
Haemophilia, colour blindness, duchenne muscular dystrophy
Describe the process of genetic linkage
During meiosis 1 chromatids crossover and homologous recombination appears
Mixing of maternal and paternal DNA
Regions of DNA close together will be swapped together and hence inherited together
Identification of molecular markers which are inherited with disease can help locate region of disease gene
Describe molecular markers
Molecular markers used to map disease loci are based on polymorphisms
Polymorphisms are common differences in DNA sequences that vary between individuals
Occur approx once every 1000-2000 bases
Many found in non coding regions
Inherited with nearby genes
Currently location of 10^4 polymorphisms have been mapped
Identification of different markers in normal and affected individuals can aid mapping of disease loci
Differences may be in a single base pair (snp, RFLP) or may be in the number of copies of a repeated sequence (ssr)
What are RFLPs?
Restriction fragment length polymorphisms. Digestion of DNA results in different length fragments due to loss of restriction enzyme sites
What are SSRs?
Simple sequence repeats.
Tandem repeats of 2-8 bases (e.g. TGTGTGTGTG)
Due to recombination or slippage of template or new strand
Different individuals inherit different number of respects at each loci
If varying repeat numbers occur within a family can be used to map disease gene
How do we map genes causing a disease?
Requires large database of family groups.
Individuals mapped at several different loci
Linkage made between inheritance of particular polymorphisms and presence or absence of disease
Observe trend of inheriting particular polymorphisms and disease through different generations
Look for mutations in genes near to inherited markers
What are the problems with mapping genes?
Linkage mapping can narrow region containing mutated gene to 10^5 bp- still 10 genes!
Isolate mRNA and analyse by Northern blotting and in-situ hybridisation
Most monogenetic disease now identified
Polygenetic and heterogenetic diseases more complicate e.g. Diabetes, obesity, predisposition to cancer/autoimmunity
Define inherited disease
Mutations in genes encoding for essential proteins
Define cancer
Mutations in genes encoding for proteins involved in the regulation of growth and survival
Define polymorphisms
Different gene sequences- can cause altered phenotype but not necessarily disease
Define alleles
Genes encoding for different polymorphic forms of proteins
How is cell growth and survival mainly controlled
Mainly under transcriptional regulation
What are the 4 normal control mechanisms?
Heterozygosity- 2 copies of genes to minimise loss of function due to mutations
Apoptosis- damaged cells killed to prevent transmission of mutated genes
Cell cycle control- checkpoints during cell division to ensure cell is healthy, no damaged DNA
Regulation of gene transcription- requirement for appropriate activation signals to induce gene transcription
What are the loss of control mechanisms?
Dominant mutations
Inherited mutation followed by mutation of remaining copy
Increased/constitutively active pathways
Gene translocations
Reduced apoptotic pathways
Loss of checkpoint proteins
Loss of repair mechanisms
What are the hallmarks of cancer?
Sustained angiogenesis Tissue invasion and metastasis Limitless replicative potential Evasion of apoptosis Self sufficiency in growth signals Insensitive to anti growth signals
How could sequence mutations change normal genes to aberrant genes?
Deletions or insertions might scramble the encoded mRNA. Complete loss of functional protein
Single base mutation could lead to a single amino acid difference in the altered protein which could lead to a change in functional activity. (+ or -)
Why is cancer not much more common?
Many mutations are silent
Multi step process that requires 3-7 hits on relevant genes. Those regulating cell growth and division, DNA repair, cell death
DNA repair is very efficient
What are oncogenes?
Mutated forms of normal cellular genes which control cell growth
What happens when Ras is mutated?
Makes it constitutively active, not longer requires signals from receptors
Activates Ras-MAPK signalling pathway. Increase gene transcription
What increases growth signals?
Receptors on cell surface receives growth signals
Increased receptor number- HER2 breast cancer
Gene amplification
Translocation to region of high transcription rate
Conformational changes in receptor leading to ligand I depends constitutive activation
Constitutive dimerisation
Describe steroids
Lipophilic hormones
Bind intracellular receptors
Execute long term effects
Regulate gene transcription and mRNA stabilisation
Give examples of steroid hormones, receptors they bind and their functions
Glucocorticoids. GR. Increase blood sugar, anti inflammatory
Mineralcorticoids (aldosterone) MR. Maintain water/salt balance
Testosterone. AR. Masculinisation
Beta-estradiol. ER. Feminisation
Vitamin D. VDR. Bone development, calcium metabolism
What are the steroid response elements?
Often located in enhancers- activate nearby promoters
Consist of 2 half sites
Half sites (repeats) separated by 0-4bp
Suggests receptors bind as multimers
How do steroid receptors interact with DNA
Homodimers and heterodimers
What are homodimers?
GR, MR, AR, ER and PR are all form homodimers
Cytoplasmic and nuclear localisation
Each subunit of dimer binds one repeat
Half sites are inverted repeats (palindromes)
What are heterodimers?
Nuclear location- activated when ligand binds
RXR forms a dimer with either vitamin d receptor, retinoic acid receptor, triiodothyronine receptor
Bind direct half sites
Recognition determined by spacing. 3bp VDR, 4bp t3r, 5bp RAR
Give examples of genes controlled be steroids
Many and varied
Specific to receptors
E.g. vitamin d receptors. Calcium binding proteins, osteocalcin (bone development)
E.g. Steroid activated glucocorticoid receptor. Inhibit IL-1. Collagenase genes
Explains some of anti-inflammatory properties of steroids
What is NFkB?
Nuclear factor kappa b (nuclear factor of kappa chain transcription in B cells)
Family of dimeric transcription factors
5 members: p65(relA), p52, p50, c-rel, rely
Cytoplasmic proteins
all contain conserved 300 amino acid region
Rel-homology domain
What is RHD involved in?
DNA binding, dimerisation, interaction with IkB proteins
What does the carboxyl terminus if NFkB contains?
Transactivation domain
What is southern blotting?
Used to detect a specific sequence of DNA in a large sample e.g. Genomic DNA or cDNA library
Can detect ~1pg of the DNA of interest but ~1microgram of sample DNA needed
DNA-DNA hybridization
Hybridization will occur if temperature < Tm
Influenced by:
Percentage of g and c
Lengths of fragments to be hybridized
The number of mismatches
Buffer composition
How does southern blotting work?
Allows the visualisation of any portion of genomic DNA by means of specific probe complementary to the sequence of the gene to be analysed
Uses restriction enzymes to digest DNA into smaller fragments
Probe: ssDNA, specific for gene of interest, labelled (32P-dNTPs, biotin, digoxigenin, fuorescein)
What can Southern blotting be used for?
Gene mapping
Assessing relationships between organisms
Determining incorporation and copy number of transgenes
Detecting sequence differences at specific restriction sites