BMS2062 Lecture Notes - Lecture 7: Stem-Loop, Immunofluorescence, Peptide
30 May 2018
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Week 4. Genotype to Phenotype
AVOIDING FOOL’S GOLD: HOW DO WE PROVE THAT VRTUAL GENES ARE REAL?
• The human genome is complex
• The genes and gene product mismatch problem
• Cytosine in the dinucleotide CG (CpG)is frequently methylated in vertebrate DNA
• DNA methylation may turn off genes by altering chromatin structure (usually occurs when C is
with G)
• Vertebrates are deficient in the dinucleotide CpG:
o Does not occur at the expected frequency
o During DNA damage, deamination of unmethylated C gives rise to U which is recognised
as a fault by DNA repair machinery
o Deamination of methylated C gives rise to T which is not recognised as an error
o Oer eolutioary tie, ethylated C’s hae ee utated to so CpG is relatiely rare
in vertebrate DNA
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find more resources at oneclass.com
• CpG islads:
o Our aily at the 5’ ed of gees
o Cytosine is often methylated in inactivated genes
o Promoters of genes which are on are no methylated an mutations are repaired
efficiently
o CpG islands mark the promoters of many vertebrate genes
-GC content is not uniform along a eukaryotic chromosome
• Transcriptome:
o Consist of every RNA that can be produced from a genome
o Because only a subset of genes is active in any cell, only a subset of the transcriptome is
represented in a particular cell type
o Made up of 3 major classes of RNA in eukaryote transcriptome:
Polymerase I, II, and III
• Genes are not organised into operons
• Splicing: mRNA is derived from excision of noncoding segments (introns) from a precursor mRNA
• Splicing is sequence specific so intron/exon boundaries can sometimes be predicted using
bioinformatics genomic sequence analyses
• Alternative splicing:
o different polypeptides can be generated from the same primary transcript
o Different exons are incorporated or omitted from the final mRNA to generate transcript
isoforms
o explains why relatively few genes in genome can give rise to vastly greater number of
proteins
o mechanism for fine tuning protein function or localisation in specific tissues/cells at
specific points during development
o forces a redefinition of the gene
o New definition 1: single transcription unit (gene) encodes one set of protein isoforms
o New definition 2: a single polypeptide is the product of a single gene
• Splicing errors
o Common result of mutation and can cause disease
o Can generate new splice sequences
o Exons can be omitted, deleting sections of protein and severely affecting structure
o Use of false (cryptic) acceptor or donor sites can truncate or lengthen exons
• Why analyse RNA
o Where and when its transcribed
o How it is spliced
o How many spliceoforms
o Whether particular spliceoforms are restricted to particular cells or growth stage
o We rely on analysis of cDNA and EST (expressed sequence tags) which are derived from
RNA
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find more resources at oneclass.com
• Deducing mRNA sequence: cDNA
o Most RNA is highly susceptible to nucleases so it is very difficult to purify and maintain
an RNA molecule in an intact state
o Methods to directly sequence RNA are inefficient and cumbersome
o Solution is to make a DNA copy of the RNA molecule
-DNA is stable and easy to amplify or clone and easy to sequence
• DNA can be produced from an RNA template
• Producing cDNA:
o RNA can base pair with DNA
o mRNA has a poly A tail
o retroviral enzyme – reverse transcriptase can produce DNA from an RNA template
o no pre-existing gene sequence info is required to generate a cDNA
• producing a cDNA using PCR usually requires a pre-existing sequence information to design
primers
• Expressed Sequence Tags (ESTs)
o ESTs are copies of mRNA or mRNA fragments
o ESTs are cDNA made from mRNAs originating from a specific cell or tissue i.e. they
present a snapshot of mRNA present at that time and place
o Sequences from transcriptionally active genes should be active in EST databases
• Uses of EST and EST databases:
o Gene verification: if a DNA sequence from a genome matches exactly to a specific EST, it
can be concluded that the genomic DNA is transcribed and that it represents a gene (has
an active gene)
o Gene structure: identify intron and exon boundaries – ESTs will only match exons
therefore segments of a gene that do not match with an EST derived from that gene are
introns – copy of spliced mRNA
o Gene expression
• Gene structure and expression:
o ESTs will not match introns
o Point at which an EST to genomic DNA match ends is either an intron/exon boundary or
transcriptional start or end point
o Number of clones containing the same ESY in one library is proportional to the
transcriptional activity of the gene
• Unigene matches ESTs from various sources and organises them into transcript families
-both real and hypothetical genes are represented
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Both real and hypothetical genes are represented: deep sequencing, rna analysis, no cloning necessary but requires genome database as reference, can find: (cid:1005). Backwards and forwards: strategies for working out what genes do: total understanding of humans biology requires that we: Identify all genes and variants genomics: understand roles of heterochromatin, junk dna and mobile elements. Identify all rnas and their developmental, spatial and temporal distribution. Identify all proteins and their developmental, spatial and temporal distributions as well as their modifications: establish structure of all proteins and noncoding rnas structural biology, establish function of all proteins and rnas, transcripts can be detected by: In situ hybridisation (intact cells/tissue: northern blotting, reverse transcriptase, microarray analysis, deep sequencing. Info about whether genes are on and also whether they undergo alternative splicing and level of transcript: rna from different sources/conditions can be compared, find out where and when the gene is expressed:
