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36 Cards in this Set
- Front
- Back
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Progressive sequencing (4 points) |
-DNA larger than 1Kb -Ends of clones sequenced with primers -Sequencing occurs until sequences meet in the middle -For genome, use Bacterial artificial chromosome (BAC) - can be localised to region of genome = identify whole region of DNA/genome |
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Shotgun sequencing (4 points) |
-Sequence ends from each clone using primers from vectors -Assembled by computer programe -Advantage - no primer design needed -Disadvantage - if sequence more than 6x size of genome there will be gaps present
= sequence long DNA strands |
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"Data mining" genomic sequencing (2 points) |
-Use gene prediction software which scans sequences -Computer translates DNA into all 6 reading frames then searches for similarity to known proteins = finds genes in nucleotide sequence |
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BLAST protein alignment |
-Input amino acid sequence -Database is searched for other proteins with similar sequence - find alignment -Similarities suggest proteins evolved from same common ancestor - have similar = identify species, locate domains, DNA mapping etc. |
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Microarray |
-Small scale, fast and automated - can look at 10,000 genes at once -Take 2 genes. Purify mRNA and tag -mRNA put into array and excess is rinsed off -Reader detects which genes are "ON" -Compare two samples e.g. one from liver and one from liver tumour = identify differences |
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If a gene is lost in tumour tissue it is a... If a gene is activated in tumour tissue it is a... |
... potential tumour suppressor ... potential oncogene |
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Three ways to identify genes |
1. Make library of cDNA clones from mRNA 2. Make library of genomic clones then make predictions based on genomic sequence 3. Identify sets of genes using microarray |
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Genetic engineering (in mice) - types |
Gene replacement - small changes to endogenous gene. Gene knockout - completely remove gene to determine it's function |
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Knockout of single gene in mice (5 points) |
-Acquire genomic clone and insert NEO directly into exon - destroys gene activity -Target gene sequences are homologous arms -Introduce construct to mouse ES cells -DNA repair machinery so goes into mouse genome -If homologous recombination occurs - TK gene is lost |
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Double selection will... |
...identify the knockout |
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How does double selection work? (4 points) |
-NEO and TK - selective markers which identify colonies resulting from homologous recombination -Cells that integrate both genes are used to create clonal line -First generation mice is mosaic - bred to generate non-mosaic carries of transgene -Carriers interbred to create homozygous mutants =study role of genes which have been sequenced but unknown function |
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Forward genetics (1 point) |
Randomly mutate gene and look for interesting phenotype - identify gene causing defect |
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Forward genetics in flies |
Mutagenize males e.g. heterozygous for mutation carried by PO sperm Outcross males to wildtype females Incross family to see homozygous embryos |
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How many generations does it take to make mutations homozygous? |
3 generations |
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How can you tell if they are different alleles on the same gene? (two main points) |
-> if 1/4 is mutant offspring - mutations fail to complement - alleles on same gene -> no offspring with mutation - mutations complement - mutation in different genes |
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What does complementation analysis allow? |
Allows mutations to be sorted into distinct genes that correspond to individual genes |
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Linkage Analysis (3 marks) |
-Used to identify genes -Analyse recombination between allel and known marker on same chromosome. = Can determine whether gene and marker are linked |
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Recombination frequency |
R/T x 100 = centimorgan (cM) |
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Single nucleotide polymorphisms (SNP) |
-Markers to be analysed and vary between inviduals -Markers taken from mother (with dominant disease) and children -If SNPs are present in diseased children but not healthy, we know the gene is linked to SNP |
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Mutations can affect gene function in 3 ways |
1. Changes in regulatory sequence 2. Changes in non-coding sequence 3. Changes in coding sequence |
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Amorphic/ Non-functioning (2 points) What are +/- and -/-? |
-Missense -Mutation which inactivates DNA binding protein +/- = Haplosufficient -/- = Strong phenotype due to no transcriptional activators |
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Hypomorphic/ Weakened (2 points) What are +/- and -/-? |
-Missense -Weakens DNA binding protein +/- = Still activate transcription -/- = mild phenotype due to poor transcriptional activation |
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Antimorphic (2 points) What are +/- and -/-? |
-Missense -Destruction of dimerization domain +/- = binds to DNA but isn't active -/- = completely inactive |
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Hypermorphic (2 points)
What are +/- and -/-? |
-Missense -Activation that is independent of dimerization +/- = mutant binds and is constitutively active -/- = constitutively active |
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Three types of loss of function - what does their phenotype look like? |
Amorphic - complete loss of function - recessive Hypomorphic - reduction in wildtype function Antimorphic - competitive inhibitor - poison wildtype |
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What does gain of function phenotype look like? |
Overexpression of transcription unit/gene product |
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What creates isoforms? |
Alternate splicing |
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What is an example of the regulation of alternative splicing? Describe it. |
Sex determination in Drosophila -3 genes = Sxl, tra and dsx -Males = 1 x chromosome, the transcripts for sxl and tra are spliced which gives rise to inactive proteins. dsx gives rise to male specific repressor protein = represses transcription for female development -Females = 2 x chromosomes so sxl protein is made sxl represses splicing by blocking binding more sxl is made. Tra is bound and results is dsx isoform |
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What is polyadenylation regulated by? How does this occur? (3 points) |
B-lymphocytes -Cell produces long transcript and first codon is spliced out. This results in translation of transmembrane domain -Cell switches to short transcript, splice acceptor is lost - first stop codon is not lost |
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Alternative START sites can also be regulated |
-Kozak sequence is optimal =ACCAUGG -If sequence isn't perfect then ribosome starts at the 2nd AUG - leads to 3 isoforms from one gene |
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Where can regulated nuclear transport occur? |
In HIV After integration, entire genome is transcribed in one place |
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What can signals in untranslated region of mRNA target? |
target a specific part of the cell |
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What is the role of ferritin? What is the role of transferrin? |
= protein storing iron, reduces available Fe = imports iron into cell, increases available Fe |
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What happens if there is low Fe in cytoplasm? |
Acconitase binds to 5' UTR of ferritin mRNA This blocks translation Also binds to 3' UTR transferritin mRNA - blocks degradation |
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What happens if there is high Fe in cytoplasm? |
Acconitase binds to Fe - goes through conformational change Ferritin is made Acconitase releases mRNA - provides rapid and strong regulation |
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IRES (internal ribosome entry sites) |
These allow more than one gene to be present on a mRNA They are stem loops in RNA which can initiate formation of ribosome regardless of polyA initiation complex |
