The Human Genome

Chromatin

• DNA is compacted by forming complexes with histone proteins

• Present in metaphase

Nucleoside Structure

• octamer that has 2x H2A, H2B, H3, H4

• DNA wraps around twice

• Can be pulled into 30nm fibre

• Regulated by H1

Euchromatin

• contains all genes

• When H1 associates - chromatin tightens and genes shut off

• Gene expression regulated by opening and closing

Heterochromatin

• permanently condensed

• Forms by recruitment of condenser proteins (HP1) in a cascade like fashion

• Circle barrier element separates euchromatin and heterochromatin to stop it from spilling over

• Chromosomal translocation can cause Barrier elements to be lost

  • Chromosomal translocation (portions of chromosomes can switch locations benign)

  • Can. Cause heterochromatin formation to be misregulation

  • Barrier elements mark the locations where heterochromatin formation should stop

  • Translocation leads to barrier elements being dislocated - position effect variegation

Structural Heterochromatin

• Contain repetitive non coding DNA sequences (satellite DNA) and has no genes

• Found in centromeres and telomeres (cap to prevent prevent fusion between chromosomes)

• Fusion leads to missegregation in mitosis

• Telomeres are composed of STRs (TTA GG and AATCCC)

  • Overhang of 30 repeat

• T loop structure gives the overhang and the telosomal shelters complex gives cap

• Telosomal sheltering complex that stabilises the T loop

• T loop structure gives cap to chromosomes

Cytogenetics

Study of chromosomes

Method of Cytogentics

1. Cells are growing in culture - metaphase chromosomes have defined structure

2. Treat cells with colcemid to get them into metaphase - disrupts mitotic spindle, halted at metaphase since there is no mitotic spindle

3. Harvest and suspend cells in hypotonic solution - swells cells to separate chromosomes for analysis

4. Apply a fixative to stabilise the chromosomes and fix onto glass slide

5. Apply DNA stain and proceed to microscopy

Karyograms

• sort by size and banding pattern

• Chromosomes in prometaphase show more detail as they are less compacted but more difficult to do

• centrosomes are not always in the centre of the chromosome

• Divide chromosomes into coordinates

  • Short arm: p

  • Long arm: q

  • the higher the number the further from the centromere

Molecular Cytogenetics

• design a oligonucleotide probe that has sequence complementarity to the sequence of interest

• more targeted means of analysis

Hybridization Oligonucleotide Probes in Experiments

• apply heat to separate DNA strands

• apply the probe against the target of interest

• probes are labelled with fluorophore

• labelled probe are allowed to hybridize with to those heat treated chromosomes and then can form an interaction with the target of interest

• can tell you if a particular sequence is present or not

Fluoresence in situ hybridisation

• colcemid treatment collects cells in metaphase

• treat with methanol and formaldehyde which fixes - helps cells attach to slide and stabilise cellular strucutre

• apply hypotonic solution

• apply it to a slide

Chromosome painting

• get an entire chromosome to light up a single colour

• a cocktall of probes that bind intermittently along the chromosome

• sequence complimentarity

• whole chromosome will then light up

Spectral karyotyping

• cocktail for each chromosome

• labelled each one a distinct colour

• important for analysing chromosomal rearrangements

• in tumour samples there can be complex arrangements of chromosomes

The Human Genome Project

• performed in 1990s

• first project used to determine the entire sequence of the human genome

• sanger sequencing used

Sanger Sequencing Outline

• DNA clones to be sequenced are generated by PCR reaction

• the clones are then subject to a polymerase mediated synthesis step

• criticial is the random termination of extnesion at each nucleotide position

• the random termination results in DNA fragments which can be used to determine the sequence

Deoxynucleotides vs dideoxynucleotides

• hydroxyl group at 3 prime position

• enables next nucleotide to be added

• ddNTPs don’t have the 3 prime group so polymerase cannot add next nucleotide and results in termination

Role of dNTPs and ddNTPs in Sanger sequencing

• add dNTPs in excess over ddNTPs in reaction mixture

• because their in small amounts - random terminations

• overall there will be terminations at every single position many times

Fluorescent Labelling of ddNTPs

• each type is labelled a different colour

• dNTPs are unlabelled

• each strand will fluoresence a colour depending on which ddNTP stopped the reaction

Post Sanger Sequencing - Poly Acrylamide Gel

• run sample on a polyacrylamide gel

• these are gels that have high resolving power - separation at low molecular weight ranges

• read the colours to get sequence

• Excite fluorophores with laser to get electropherogram

Improvement in Gels for Sanger sequencing

• use capillary electrophoresis that can be commercially purchased

• Gels are a major bottleneck so this method allows good separation

• Works in the exact same way, you buy a gel polymer and inject into capillary

• Allows many samples in parallel - up to 834

Sequencing the human genome for the first time

• 1987 global effort

3 key limitations of Sanger Sequencing

1. The necessity to have a clone of the DNA template (so that the levels of fluorescence emitted is adequate for detection)

2. The requirement that at least some sequence information is known beforehand (so that primers can bind to the template)

3. The short sequencing read length

Cloning vector strategy

1. Fragment human genome

2. Clone those fragments into cloning vector

3. Design primer to be at boundary between cloning vector and human genome insert - complimentary

Overall plan of sequencing the human genome

• fragment genome into large pieces

• Clone those into certain vectors

• Order this clones

• Fragment these clones further

• Clone these into bacterial vectors

• Then sequence

• Certain amount of mapping is needed as well to keep things organised and remember info on where those fragments came from

Constructing the initial framework

• each lab was given a single chromosome sequence

• Fragmented chromosome sequence into larger pieces

• Clone them into yeast artificial chromosomes - bacterial cannot handle large pieces

• Map the fragments location back onto the original chromosome - reordering

How to map the original location of a fragment?

• after fragmentation you don’t know where they belong in the chromosome

• Using fluorescence in situ hybridisation experiments

• Extract some DNA from a single clone

• Label it a certain colour

• Using that DNA as a probe you can perform a FISH experiment

• One part of the chromosome lights up the colour which says the location of the fragment

• Using STS based PCR screening - sequence tagged sites (sequencing in the human hen genome where people had already worked out a bit of the sequence)

• Design a primer based on that sequence information

• Screen those clones in a PCR reaction

• Positive PCR reaction allows mapping because of sequence complimentarity

Clone Contigs

• Once all the locations of the overlapping clones have been mapped

Sequencing and Final Assembly of sequencing a genome

• fragment each clone in a compartmentalized way - means reassembly is more reliable

How do we reorder fragmented sequence?

• add restriction nuclease at small concentration

• because small concentration you wont get a cut in every single position

• you get random cutting here and there

• this means you get overlapping fragments

• sequence those and then you can have matching sequences

• you can reorder by finding matches

Whole genome shotgun sequencing

• initial construction wasn’t necessary

• do a fragmentation of the whole genome - no fragmentation

• do reassembly based on overlapping regions of homology

• if you sequence a genome many times, the degree of overlap is going to be very high meaning there is still a reliable assembly in the end

• reduce both time and money

Non coding proteins

• genes that encode an RNA that is expressed but not translated

Gene annotation

• the process of identifying and describing the biological features of genes with a genom

• we know that protein coding genes should be devoid from stop codons in the open reading frame (stretch of sequence that has no stop codons)

• clusters on exons instead of long stretches

What could interspecies sequence comparison be used for

• further suggest that the detected ORF represented a true coding gene

• they are important between species

• there should be a good degree of conservation

• if peaks are similar to ORFs then that proves its an exon

How was more definitive confirmation obtained about gene annotation across species?

use of expressed sequence tags

• obtain the mRNA from a tissue type

• make cDNA via reverse transcription

• clone them into a vector - each clone corresponds to a sequence for an mRNA

• use primers complimentary to the vector insert boundary

• sequence, read into that cDNA, and therefore determine what gene is expressed

• if it matches - it is more definitive proof you have a protein coding gene

Conclusions from Gene annotations

• 22,000 protein coding genes

• good amount of non protein coding genes - highly repetitive

• heterochromatin associated sequences (microsatellite sequences)

• half of our genome is made of transposable repeats

• half of genome is junk DNA

What is relevance of junk DNA

• 90% of genome is transcribed into RNA

Long non coding RNAs

• 200 nucleotides

• play a role in regulating gene transcription (some)

• quite poorly conserved between species

Protein vs lncRNA

Protein

• highly conserved

• low flexibility to sequence change - easily to become non function

LncRNA

• low conservation

• high flexibility - changes do not affect function (means why it is poorly conserved)

• larger area for mediate evolutionary advances

microRNAs

• 20 nucleotides

• highly conserved

Transposable elements

• highly repetitive

• autonomous units that can multiply and spread

• not much of a biological role

• mediate advances in genome complexity

Four classes of Transposable Elements

• long interspersed nuclear elements

• short interspersed nuclear elements

• long terminal repeats

• DNA transposons - a copy will cut out from a chromosome and inserted into another location

Retrotransposons

• a copy of a retrotransposon gene

• transcribed into RNA copy

• RNA is copied into DNA copy

• inserted into another location of the genome

• many can happen at one time

Long Interspersed Nuclear Elements

• LINE1 gene: endonuclease component, reverse transcriptase component (enzymes)

• LINE1 transcribed into RNA

• RNA is translated into a reverse transcriptase enzyme

• reverse transcriptase enzyme copies RNA into DNA

• DNA inserted by endonuclease into other part of the genome

• encodes all of proteins required for its retrotransposition

How can LINE genes enhance genomic complexity

• LINE1 gene present in an intron of a protein coding gene

• LINE1 genes have polya signal to terminate transcription

• it can happen that polya tail is skipped

• downstream exon will be included in final product

• reverse transcriptase will make that DNA

• endonuclease puts it somewhere else in the genome

Long terminal repeats

• associated with endogenous retroviral repeats

  sequences from viruses that have affected human germ line

  • lost the ability to make virus particles

SINEs

• include members of the Alu repeat family

• only found in primates

• actively transpose

• not autonomous - do not encode any of the enzymes

• hijack machinery of LINE1 genes to mediate retro transposition

The mitochondrial genome

• most of human genome is in the form of the nuclear chromosomal associated DNA

• small is in mitochondria - resemble plasmid e.g ribosomal RNA or tRNA

• functional proteins produced

• early eukaryotes engulfed bacteria

• vesicle membrane became outer and bacterial because inner

• DNA within bacteria became mitochondrial genome - very functionally important

The small interfering RNA pathway

• discovered in plants

• viral defense mechanism

PROCESS

• DNA copy of virus inserted into host genome - viral particles created to mediate infection

• Dicer will get hold of some copies and cut it into smaller pieces (siRNAs)

• some copies have already genome but some are cut up

• siRNAs enter RISK complex

• one of the strands of the siRNA molecule is ejected from the complex and one is retained (guide strand of RNA)

• RISK complex with guide strand then goes scannign the plant cell for anything with sequence complimentarity to that siRNA

• siRNA will have been derived from viral sequences - having sequence complimentarity

• form a stable interaction with mRNA

• base pairing triggers enzymatic activity of RISK complex resulting in the degradation of any bound RNA target (the viral mRNA)

• no more viral proteins are made stopping the viral infection

Dicer

• dicer generates the small double stranded RNAs

• creates two nucleotide 3 prime overhangs

• 3 prime part of the RNA is bound by the PAS domain of the dicer enzyme

• RNAs domain located upstream

• rigid helix

• maintains a corresponding distanced between the PAS and RNA domain

• RNA domain are not in perfect alignment - RNAs domains make incisions tow nucleotides apart from each other (generates overhangs)

• once incision is performed - dicer enzyme moves up along RNA and keeps on making incisions of 22 nucleotides

RISC complex

• only takes up RNA molecules in that precise configuration

• one strand ejected, guide strang retained

• searches cytoplasm for complimentarity

• once found - activates

• Argonuate has MID domain - binds to 5 prime end, PAZ domain binds to 3 prime end

• Prior to binding, N temrinal unzips and unwinds double stranded RNA

• sequence search begins and PIWI domain activates with RNAse activity and degrades the target

How are microRNAs processed?

• there is a microRNA gene sitting in our genome

• transcibed as normal - primary pmicroRNA which has several hairpin structures which has the eventual mature form

• DROSHA complex recognises this and performs incisions to release this mature form (pre-microRNA)

• Dicer cleaves off the hairpin part of the molecule with 2 nucleotide 3 prime overhang

• base pairing after RISC complex is never 100% accurate with one or two mismatches

• mismatch means no degradation - complex trigger inhibition of translation (inhibit ribosomal translocation temporarily)

piRNA pathway

• only expressed in germline

• bursts of retro transposon activity

• aim is to promote genomic complexity

• if process is uncontrolled it can lead to adverse mutagenic processes

How do we control retro transposon activity?

• piRNA pathway

• piRNA genes located on opposite strand to transposon on LINE1 gene

• line1 RNA transcribed - mediates retro transposition

• after a certain period piRNA precursor expressed

• because sequences are overlapping piRNA precursor has sequence complementarity to lineRNA

• PIWI starts recognising piRNA and degrades it into smaller pieces (piRNAs)

• that PIWI RNA complex can bind to LINE1 RNA (recruits certain enzymes that methylate the gene which turns it off)

• histone methyltransferase complex that methylates and modify histone proteins resulting in chromatin condensation and turning off the gene