Finding the genes in a genome

what is the function of genes

region that is transcribed (includes 5’ & 3’ UTRs)

• codes for proteins or RNAs

what is the function of cis-regulatory elements

enhancers, silencers promotors just upstream of the gene

• regulates when transcription is initiated or not

what is the function of trans-regulatory elements

located far way from the gene of interest

• non-coding RNAs that influence gene expression

what are repetitive regions

• transposons

• retrotransposon

• single sequence repeats

what are transposons

a repetitive gene region capable of moving from one region to another

• may require specific sequences of nucleotides to move

• movement is called “transposing”

what is the functional importance of transposons

• cause mutations (via insertion)

• alter gene expression

• induce chromosome rearrangment

what are retrotransposons

a repetitive gene region which must be reverse transcribed into DNA before inserting into a new region

• moves similar to transposons

what is the functional importance of retrotransposons

• causes mutations (via insertions)

• alter gene expression

• induce chromosome rearrangements

what are single sequence repeats

regions of genome composed of tandem repeats of bases

• may have structural/functional properties

• high rate of mutations allow number of repeats to contract/expand

what does each nucleotide contain

• sugar phosphate backbone

• a deoxyribose sugar

• nitrogen containing base

what is the template strand

the DNA strand transcribed by RNA polymerase to make RNA

• we read DNA 5’ - 3’

what is the coding strand

the DNA strand that has the same sequence as the RNA coded by the template strand (T rather than U)

what are some ways to analyze a genome sequence

identify genes from:

• open reading frames

• transcriptional & translational control regions (promoters, terminators, ribosome binding sites)

after identification:

• determine aa sequence

• identify intron/exon boundaries

• identify gene variation (mutation)

how many reading frames are in DNA

six reading frames

• 3 for “top strand”

• 3 for “bottom strand”

how can genes be found in the genome sequence

can be found from:

• open reading frame (start & STOP codons)

• promotor regions

• enhancer & silencer regions

• translational control elements in 5’ UTR

how does a microarray work

an RNA sample is converted into cDNA

• cDNA is exposed to transcript probes which are complementary to specific sequences

• they bind and reveal the identity of the cDNA

what are some disadvantages to microarray

• low sensitivity

• low dynamic range

• known transcript only

how does RNA sequencing work

an RNA sample is converted into cDNA

• samples are aligned and a sequence is revealed

what are some advantages to RNA sequencing

• high sensitivity

• high dynamic range

• novel transcripts sequences identified

what is the difference between a reading frame & open reading frame

reading frame - one of the 6 different ways to read codons

open reading frame - a DNA sequence that occurs between a start codon and STOP codon

• must be >=30 nucleotides

what is the difference between an open reading frame & “real” open reading frame

open reading frame - may/may not code for a protein

“real” open reading frame - region between start & stop codon, that produces a real protein

• sometimes RNA

what is defined as the “real” open reading frame in eukaryotics

the removal of all intron and splicing of the exon together to produce a sequence of codon that will code of a protein

what is a difference between searching for open reading frames in prokaryotes & eukaryotes

prokaryotes - no introns, hence we look for long sequences of codons unbroken by STOP codons

eukaryotes - introns are present hence we need to find the boundaries between them and exons, then splice them out and then search for the open reading frames.

what is a splice site

the region between an intron & exon, removed by splicosome

• 3’ splice sites: CAG|G

• 5’ splice sites: MAG|GTRAGT

NOTE: M = A or C & R = A or G

what are transcript variants

alternate use of intron/exon boundaries within genes, this means multiple mRNA transcripts can be produced from a single gene

• each variant is a different open reading frame

what is alternative splicing

refers to the different ways in which a spliceosome can remove introns (even exons) during mRNA modification

what is homology searching

if a paralogue (same species) or orthologue (different specie) has been identified for the gene you’re searching

• existing info can help identify intron/exon boundaries

how can reverse transcriptase (PCR) find RNA

1. design primer based on predicted sequences of gene regions

2. if gene exists/expressed, PCR primers will bind to RNA transcript & create cDNA copy

3. detection of cDNA copy validates that predicted gene is “real”

how can transcriptomics find RNA

1. extract RNAs from a cell sample

2. run transcriptomics experiment to detect all different RNAs present

3. detection of cDNAs or RNAs that correspond to the sequence of the predicted gene validates them as “real”

how can proteomics find proteins

1. extract proteins from a cell sample, denature & unravel & treat with protease

2. run a mass spectrometry to confirm identity

3. detection of proteins through proteomics with the same sequence as predicted proteins validates that predicted gene as “real”

how can homology modelling be used to find proteins

1. based on predicted protein sequences from predicted genes, search against a database of all known & validated proteins

2. when a high match is found, this provides strong evidence that the predicted protein is a real protein

how can a western blot find proteins

1. design an antibody that recognises a part of the protein sequence your searching for

2. extract proteins from cell sample, then denature and add antibody (second antibody as indicator)

3. run a western blot to determine if protein has been bound to by antibody

4. detection to protein validates that protein isoform exists