BIO341 Gene Discovery Section 3 Genome Organisation

1
BIO341Gene DiscoverySection 3 - Genome
Organisation

• Jasper Rees
• Department of Biochemistry, UWC
• www.biotechnology.uwc.ac.za/teaching/BIO341

2
Eukaryote Genome organisation

• Genome sizes
• Cot analysis and genome complexity
• Repetitive and unique sequences
• mRNA expression
• How much coding sequence in a genome
• Rot analysis of mRNA expression
• Tissue specific genes

3
Genome sizes in different phyla
4
Cot analysis of genomic DNA

• Anneal DNA and measure double stranded component
• Annealing rate depends on initial concentration
  (Co) and time (t)
• Speed of annealing is dependent on sequence
  complexity
• Sequence complexity is the length of unique
  sequence

5
Complexity of Genomes

• Chemical complexity amount of DNA determined by
  chemical analysis
• Kinetic complexity amount of DNA determined by
  Cot analysis
• Generally values agree well, except of polyploid
  genomes

6
Several classes of sequence complexity in
eukaryote genomes

• Cot analysis shows three classes of sequence
  complexity
• Highly repetitive, mostly from centromeres
• Middle repetitive, mostly from longer repeat
  sequences
• Unique sequences

7
Repetitive and unique sequence in eukaryote
genomes

• As genomes get larger, proportion of repetitive
  sequence increases
• Amount of unique sequence is still high even in
  large genomes
• Distribution of three class of sequence not
  revealed by Cot analysis

8
mRNA as a product of genomic DNA

• Using mRNA as a probe for reassociation kinetics
• Shows that majority of mRNA is from unique DNA
  sequences
• Not all unique DNA sequences are transcribed

9
Estimating the amount of coding sequence in a
genome

• Saturation of reassociation experiment shows the
  total proportion of DNA transcribed in small
• The result will vary depending on the tissue
  source of mRNA
• This is with cytoplasmic (spliced) mRNA

10
Measurement of the sequence complexity of mRNA
expressed

• Rot analysis shows the abundance of mRNA
• Chick oviduct expresses large amount of ovalbumin
  (50 of mRNA)
• About 10 other abundant genes (15 of mRNA,
  including lyzosyme)
• And 35 of the mRNA represents all other
  transcribed genes in the tissue

11
Overall expression of mRNA in different tissues
12
Organisation of genes in genomes

• Overview of exon and intron size and numbers
• Differences between small genomes and large
  genomes
• Variations in gene strategy
• Implications for genome organisation

13
Overview of splicing of introns
14
Identification of introns gene and mRNA are not
contiguous
15
Globin gene structure common structure to whole
family of genes

• Position of introns in coding sequences is
  constant.
• Length of introns varies
• All globin genes organised the same way
• Implies ancient evolutionary relationship

16
Mammalian DHFR genes have common organisation

• Dihydrofolate reductase required for purine
  nucleotide biosynthesis
• Exon organisation is common to all mammals (To
  all vertebrates?)
• Introns vary greatly in length and sequence
  between species
• Typical of other genes

17
Intron and exon length distribution
18
Distribution of exon numbers

• Saccharomyces small genome, little repetitive
  sequence, very few introns, highly compact
• Flies and mammals, larger genomes, more
  repetitive sequence, more introns. Genes dispersed

19
Overall gene sizes when there are more introns

• Gene sizes a Normal Distribution in yeast
• But skewed distribution in flies, mammals (plants
  etc)
• Increase in gene size related to increase in
  presence of introns and number of introns

20
mRNA and gene sizes

• Yeasts few introns, mRNA mostly same size as
  genes
• Flies, mammals, plants etc mRNA much smaller
  than genes.
• hnRNA has same size as genes (from which is is
  transcribed)
• hnRNA spliced into mRNA

21
Alternative functions in genes

• Promoters
• Termination
• Splicing

22
Human Chromosome 16 - DWNN gene shows complex
organisation
DWNN gene is 35 000 bases long
23
Exons are conserved, introns vary

• Dotplots show similarity graphically
• Can show for DNA or protein sequences
• For two mouse alpha globin genes, can show exons
  are conserved, but introns do not show strong
  sequence relationship.
• Alpha globin genes recently duplicated

24
Exons show similarity between species

• Zoo Blots used to detect related DNA sequences in
  different species
• Detect exons by hybridisation
• Based on conservation of coding sequence
• More highly conserved genes imply more
  limitations on protein sequence variation
• Basis for computational identification of genes
  between species

25
Generation of new genes

• Diversification of species occurs by addition of
  new genes
• New genes occur from mutation and selection of
  existing genes
• Or gene duplication followed by mutation and
  selection
• Or recombination between two genes (gene
  shuffling)
• Or horizontal transfer of genes from other
  species
• New genes are very very rarely ( never) de novo
  events

26
Gene duplications

• Occur as the result of duplicating region of
  genome by recombination mechanisms
• From small duplication, to very large
• One gene duplicated or many
• Gene duplication generally not bad for organism
• Duplication of genes allow for divergence

27
cDNA to gene duplication

• Occurs by reverse transcription of mRNA into DNA
  by retroviral enzymes, in germ line
• Integration of cDNA into germline gives spliced
  copy in DNA
• Generally not functional copy of gene (no
  promoter)
• Provides sequences that could be fused to other
  genes

28
Pseudogenes

• Non functioning copies of genes
• Promoter, splicing, termination, translational
  mutations
• Various types, can be one or many mutations.
• Some transcribed, some not.
• Generated by recombination or cDNA insertion
• Can be close or distant in genome

29
Gene families

• Group of genes with related sequences and
  fucntions
• May be small or large family
• May be closely related (sequence /function)
• Or very distant
• Generated by duplication by recombination
• Level of relationship of products depends on
  extent of divergence

30
Protein Families

• The result of gene family divergence
• Must have related sequences, and thus related
  functions.
• May be related functions
• Thus may all be kinases, but all have different
  substrates
• Examples kinases, proteases, globins, DNA
  binding proteins, ATPases, NADP reductases

31
Protein Superfamilies

• Large family relationship
• May only be domains of the protein that are
  related
• Functions may be very different
• Evolutionarily very divergent
• Often the result of exon-shuffling
• Examples, Immunoglobulin superfamily, protein
  kinase superfamilies, blood clotting enzymes

32
Homologs, orthologs, paralogs
33
Exons and domains

• Can often relate exons to domains in proteins
• Domains are independently folding structures in
  proteins
• When exon boundaries coincident with the boundary
  of the domains then can plug together exons to
  create modular proteins
• Need the same reading frame at each exon boundary
• Many good examples of this, but not for every
  gene or domain.

34
Exon shuffling

• If exons have the same reading frame
• because splicing is independent of coding
  sequence
• Can splice together whatever set of exons is
  transcribed.
• If splicing assembles fused open reading frame
• Then can translate protein
• If DNA recombination assembles two exons together
  and they can translate into a protein that can
  fold correctly into it domains, then this protein
  is highly likely to be functional

35
Uniqueness of Exon shuffling

• Exons only in eukaryotes
• Exon shuffling only in eukaryotes
• One mechanism for acceleration of gene
  diversification in eukaryotes
• Provides engine to power evolutionary change
• Is a selective advantage for presence of
  introns/exons

36
Globin gene family

• In humans have a and b globin gene clusters
• In adults form haemoglobin, a2b2 hetero-tetramers
  which bind oxygen in the blood
• Foetal and Embryonic forms bind oxygen more
  tightly to transfer oxygen from maternal blood
• Gene expression developmentally regulated

37
Developmental expression of globins

• Embryonic z2e2 z2?2 and ?2e2
• Foetal z2?2
• Adult ?2?2 and ?2?2
• Sequential replacement of gene expression
• Genes activated along a and b clusters (see
  figure 23.2)

38
Beta-globin genes in other vertebrates

• Find varying numbers of genes
• However, have embryonic, foetal and adult forms
• Find pseudogenes in many clusters
• Can trace evolutionary history of genes by
  analysing sequence relationships

39
Evolution of globin gene families

• Evolution of both a and b globin gene families is
  by duplication
• Result of unequal crossing over events
• Fixation of additional genes in populations
  allows divergence of function
• Loss of function results in psuedogenes
• Divergence of function resulted in development of
  three different types (embryonic, foetal and
  adult)
• See figure 23.7 for examples of diverence

40
Why increased globin gene diversity?

• To generate globin proteins with different oxygen
  binding affinities
• Selective advantage to get more oxygen to
  developing embryo/foetus
• As development becomes more complex and embryo
  develops in maternal body, then need more complex
  oxygen transport, and thermodynamics

41
Globin genetic diseases mutations

• Find many different mutations in a and b globins
• Class of diseases termed thalassaemias
• Point mutations that affect oxygen binding, and
  regulation of oxygen binding (Hill effect etc)
• Also promoter, splicing and poly A mutations
• Must be able to generate enough hemoglobin to
  transport oxygen at all times though

42
Globin genetic diseases deletions

• Also have major deletion events in a and b globin
  clusters
• Result from deletions occurring in unequal
  crossing over events, between regions of homology
  in globin clusters
• Deletion of different combinations of genes gives
  different phenotypes
• See figures 23.5 and 23.6

43
Why so many globin mutations

• In certain human populations globin mutations are
  extremely common
• Reduced hemoglobin function causes red blood
  cells to be fragile
• Fragile RBCs break open more easily when infected
  with malaria parasite (Plasmodium)
• So people with thalassaemia have increased
  resistance to malaria, when heterozygous for
  globin mutations

44
Heterozygous Advantage

• Termed Heterozygous Advantage, so selects for
  people with globin mutations in presence of
  malaria
• Results very high of thalassaemia in West
  Africa, Mediterranean area, South East Asia.
• Is a directly observable effect of selection
  pressure on human populations resulting in
  evolutionary divergence on historical timescales

45
Recombination events

• Crossing over - homologous recombination
  occurring normally during meiosis
• Unequal crossing over resulting from mismatching
  regions of homology, that are incorrectly aligned
  during meiosis
• Results in generation of additional sequence in
  one chromosome, and loss of sequence in the other.

46
Homologous Recombination

• Two genes in parental chromosomes
• Unequal crossing over
• Results in generation of 1 and 3 genes

47
Gene duplications and divergence

• Generation of 2 genes from 1 gene occurs when
  sequences outside gene allow unequal crossing
  over
• Once two copies of a gene exist on a chromosome
  selection can occur
• Multiple copies must be compatible with life,
  true for many genes
• Then copies can diverge by accumulating mutations
  and developing new functions

48
Divergence and selective advantage

• With divergence of function, have the possibility
  that new function will provide advantage to
  organism
• Most mutations will destroy function of gene
• Small proportion will improve it
• These can be selected for in populations
• Many only give real advantage in unusual
  environments
• May gain frequency from population bottleneck

49
Evolutionary or molecular clock

• Rate at which molecular changes are fixed in a
  population is measurable
• Can measure length of time since divergence of
  species or genes using molecular clock
• Can compare data with fossil record and isotopic
  dating systems to calibrate clock

50
ReplacementRates

• Replacement rates for non-coding positions faster
  than for coding positions
• Effect of selection on protein function
• Use non-coding for recent divergence, coding
  sequence for older divergence

51
Evolutionary divergence of globins

• Can measure evolution of globin gene clusters, in
  terms of molecular clocks, and which species have
  which globin genes
• Increased complexity of globin gene clusters in
  higher vertebrates
• Present in lower vertbrates, invertebrates, and
  distantly related protein found in plants
• See Figures 23.7 and 23.9

52
Gene correction

• Poorly understood mechanism by which duplicated
  genes are corrected against each other
• Can result in the maintenance of many identical
  copies of a gene over many generations
• Correction event may be rare. May replace many
  divergent copies with one specific type.
• Loss of gene correction will allow evolution of
  duplicated genes

53
Divergence or gene death

• When genes diverge can get loss of function
  creation of a pseudogene.
• Or generation of mutated functional version that
  can be selected for or against
• Selection over long period can result in the
  emergence of a novel protein with changed
  function
• Duplication is essential for this divergence of
  gene function

54
Gene duplication central to evolution

• Require increased genetic complement to allow for
  selection of novel biological functions
• Gene duplication is the simplest and most common
  way to create this
• When selective pressure on one copy of the gene
  is lost, then can accumulate mutations, and
  select for new functions
• Overall result is diversification of function and
  creation of gene families

55
Repetitive DNA Sequences

• Classes of repetitive sequences found in all
  higher eukaryotes
• Simple sequences found in satellite regions and
  centromers
• Middle repetitive sequences in several classes
• Many repetitive sequences are mobile genetic
  elements (eg transposons)

56
Repetitive sequences expand in higher eukaryote
genomes

• As genomes get larger, proportion of repetitive
  sequence increases
• Repetitive sequences fall into various classes by
  sequence relationship and mode of amplification
• Amount of unique sequence is still high even in
  large genomes

57
Simple sequence Satellites

• Defined by unique density caused by unusual
  sequence composition
• Made up of very short sequence repeats

58
Satellites localise to centromers

• Sequences found in satellite regions
• And centromers
• Localised by in situ hybridisation
• Define centromeric structure and function

59
Mammalian satellites

• Mouse satellites made up of 238 bp repeat
• This is made up of internally repeated sequences
• Result from repeated duplication of 9 bp unit

60
Mini and micro satellites

• Generated from repeated sequences
• Repeat units from 2 to gt50 bp
• May result in many alleles at a locus
• May be coding or non-coding
• Valuable for genetic markers and genotyping
  experiments
• Used in DNA fingerprinting (eg in forensics)

61
Microsatellite allele analysis
62
Microsatellites and disease

• Microsatellites with 3 bp repeats common in human
  genetic disease, where amplification generates
  expanded protein sequences
• Causes expansion of regions of poly-glutamine,
  which cause protein precipitation and cell death
• Expansion observed in families
• Disease gets worse with additional generations as
  the microsatellite expands
• Examples Huntingdons Disease, myotonic dystrophy

63
Middle Repeat Sequences

• Can be caused by expansion of classes of larger
  sequence elements
• 300 - 20 000 bp
• Scattered through the genome
• Not caused by unequal crossing over
• Generally transcribed into RNA and converted to
  DNA and integrated into the genome
• Some elements can insert and excise, and are
  therefore considered to be mobile elements

64
Alu sequences

• Largest class of middle repetitive sequences in
  humans
• About 300 bp long
• Have polyA tract at one end
• Are related to 7SL RNA sequence
• Contain internal RNA Pol III promoter
• Thus when reverse transcribed and reintegrate
  create a new Pol III promoter
• Can cause mutations by integration into genes

65
LINEs

• Genetic structure similar to retrovirus
• Long terminal repeats (LTR) at each end
• Internal sequences code for transposase proteins
• Transcribed by RNA Pol II
• Reverse transcribed and integrated into genome
• Cannot be excised
• Do not appear to be packaged as a virus

66
Transposons

• Similar to LINE and retroviruses, except that can
  insert and excise from genome
• Some excisions leave small number of bases
  inserted into genome
• Others excise cleanly
• These are true mobile elements
• Can be the cause of frequently mutating and
  reverting genotypes
• Reponsible for classic maize colour mutations

67
Spontaneous mutations in pears
68
Distribution of repetitive sequences

• Distribution of middle repetitive sequences in
  animal and plant genomes is completely random
• Occur in introns, inter-genic regions, highly
  transcribed and transcriptionally inactive
  regions
• May be transcriptionally inactive or active
• May be complete copies or partial copies

69
Function of repetitive sequences

• junk DNA - implies has no function?
• Mobile elements generates
• Genome rearrangements
• Duplications
• Mutations and gene inactivation
• Gene activation
• Generation of additional DNA in genome

70
BLAST and repetitive human sequences

• Repetitive sequences in your sequence will result
  in large numbers of matches to genome data
• Solution check the filter human repeats check
  box in the search set up.
• This will remove repeats based on a database of
  repeat sequences.
• With the switch on, you can detect the repeats
  and identify them