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GENES, CELLS AND EVOLUTION (BIOL1020) MODULE 5 – GENOMICS

What is Genomics? -

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We can look historically that most genetic studies have involved studying the functions and structures of individual genes – or small families of genes. i.e. Taking a gene of interest, mutating it, seen what happens to the animal – or look at a mutated animal and find out more about the related gene. Genomics is largely dependent on more recent technologies – and is the study of the structure and function of ALL of the genes – the Genome – or the genes collectively as a group. There are different fields of Genomics which arise from addressing everything in the genome:o Structural Genomics – looking at the structure of the chromosomes, the order and spacing of genes. How the genes all fit together, where they all are in relation to one another, and essentially the structure of the genome. o Functional Genomics – What genes are expressed and when? What genes are transcribed and translated – and under what circumstances? What are the functions of the proteins which are encoded by those genes? o Comparative Genomics – Comparing the genomes of different species through evolution. i.e. Having very similar genomes between 2 species would suggest a close relationship.

The Structure of Genomes - Prokaryotes -

Recall that Prokaryotes are SINGLE CELLULAR organisms – and relatively simple cells – so not as complex structurally as eukaryotes are. Accordingly – their genomes are more simple. o The prokaryotic genome is compact – the genes are tightly spaced together – i.e. 1 gene per 1-2Kb in a prokaryotic genome. o The genomes are circular as plasmids - they are looped rather than being multiple chromosomes which are linear. o These plasmids are associated with protein – but are NOT as heavily associated with protein as the eukaryotic chromosomes are.

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For example, E.coli – the entire genome is 4.6million bp – 4400 genes – and if we took the plasmid and stretched it out – it would be 1mm long. Packaging this length is NOT as large as a challenge as the eukaryotes face when they have to package their chromosomes…

The Structure of Genomes - Eukaryotes -

Moving onto Eukaryotes – i.e. Humans – the picture becomes much more complex. In humans, we have ~3000MB of chromosomal DNA – approximately 1000x more than a bacterium. We have 3 questions to address:-

1. What is all that extra DNA? -

Why is it that a human being needs a 1000x the number of bases to encode its proteins and express them properly?

2. What advantages does added complexity provide? -

What are advantages provided by that complexity?

3. How can cells contain, package, and control so much DNA? -

How do we manage all this DNA with such a structural and logistical challenge? A single copy of the human genome – in almost all cells of the body – would stretch to 2m long. All the genes along that 2m would have to be accessible – despite being coiled and packaged…

Question 1 – What is all that extra DNA? -

The 4 factors which make the eukaryotic genome so vast include:o Eukaryotes have more genes than bacteria. (Human ~23,000, E.coli ~4,400) o Eukaryotic Genes consist of Introns – bacteria do NOT have introns, they have a single open reading frame encoding for a protein and that’s it.

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Eukaryotes have long, complex enhancers – the regions which the eukaryotic genome needs to direct expression of genes is much broader for each gene than for bacteria. The repetitive sequences for each gene – the purpose which is not clear – but take up quite significant stretches of our genome.

Question 1 – The Structure of Genomes - Eukaryotes -

The Eukaryotic Genome is contained in Multiple Chromosomes – with more genes than prokaryotes – humans having ~23,000 whilst bacteria having ~4,000. There are more non-protein coding DNA. There is a lower gene density – i.e. typically we have one gene per 1-2Kb in bacteria – genes are much less tightly packed in eukaryotic chromosomes. They are very highly associated with proteins – i.e. getting packaged tightly with Chromatin. 1 Copy of the Human Genome is equivalent to 2 linear metres of DNA – hence seemingly difficult to handle in a single cell – i.e. packaging with chromatin – where chromatin organizes and packages the human genome.

Question 1 – Eukaryotes have more genes than Bacteria + Introns -

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Part of the explanation why our genomes are larger than bacteria can be attributed to the fact that we have MORE GENES. (Human ~23,000, E.coli ~4,400) – BUT recall that a human needs 1000x the number of bases to encode its proteins…so where does the ~200x fold come from? The presence of Introns – the regions which do NOT encode proteins. Eukaryotic genes in general compose of both introns and exons. Recall… o A Promoter – the elements which allow the gene to be recognized by the transcriptional machinery and transcribed. o Exons – the protein-encoding regions of the gene. o Introns – which are of a much larger ratio – hence the majority of the gene is not encoding for a protein. This gene is transcribed into a primary transcript or pre-mRNA – where all the introns and exons are both present – and this is spliced into a mature transcript – the mRNA – where only the exons are expressed. o The protein we get is relatively small compared to what we would expect from all this DNA – this is because of the removal of Introns.

Question 1 – Eukaryotes have genes with Long Complex Enhancers

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The Lac Operon is a common example for the expression of a set of genes in bacterium – and it works very simple, efficient and compact. It has a short regulatory region – with a few protein binding domains - which controls the expression of multiple genes – the 3 genes – which are all “protein-coding”. In Eukaryotes – looking at 2 Zebra Fish chromosomes – we look at a particular gene called PAX6 – the “blues” are the actual genes itself, the introns and exons – the “greens” however, those spanning from the left and right in 100’s of mb – are regulatory elements – which are complex, numerous and often very far from the actual gene – i.e. the transcription of which they are controlling. Recall that most of the gene does not encode for the protein – now we can see that the stretch of DNA that contains a gene – most of that stretch of DNA actually isn’t even the gene…

Question 1 – Repetitive Sequences -

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Repetitive Sequences are spaced throughout the eukaryotic genome – ranging in size from 5bp to 300kb and appear over and over in these stretches – stacked on top of each other. Through natural history, they have resulted from duplications in the genome – i.e. stretches resulting from an error in replication or in chromosome sorting to be copied… and some of them are derived from DNA and transposable elements – removing themselves and inserting themselves into a genome – they leave something behind when they remove themselves. They don’t have an obvious function – but are prominent around the centromeres – i.e. where the X meets in the chromosome – important when the cells are dividing, because the chromosomes need to be sorted properly, and the cytoskeleton grabs onto the centromere in order to sort the chromosomes. They are also prominent around the telomeres – which are regions which do not encode many genes – but are involved in protecting the rest of the chromosome from degradation. (i.e. Structural Functions?)

Repetitive Sequences – Alu Repeats -

Repetitive Sequences may have a Structural Function – but alternatively may be simply unwanted DNA which is normally very difficult to remove through evolution… they can actually be harmful – for example Alu Repeats – which are implicated in BRCA 1- Breast Cancer.

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Alu Repeats are one of the most prominent repeats in the Human Genome – there are about 500,000 copies of the Alu Repeat – hence about 5% of your genome as this Alu Repeat. It can multiply over generations by the mechanisms mentioned earlier – and when it moves around the genome – it can interfere with genes and their expression – implicated in cancer, muscular dystrophy and other diseases. But How? o Even if the gene itself is intact – the regulatory elements can be disrupted - the promoter is controlling when and where this gene is expressed – but having a huge set of base pairs which have no relation to the promoter – we would interfere with proper expression – it may not be expressed, or be expressed at the wrong place or time. o Next, we know the genes encode proteins – and such coding is highly delicate – the sets of 3 base pairs associated with an amino acid… if we disrupt this by adding a bunch of repeats which are not a part of the coding sequence – we will interfere with the protein formed as a result. o Next, if the repeat is inserted at one of the seams between the introns and exons – these have to be carefully stitched when mRNA is processed – but if one of those seams which have a characteristic specific sequence recognized by the machinery – if that is disrupted with the insertion of a repeat – we will get improper splicing of the introns – hence the wrong protein. o Some of these repeats – keeping in mind that we all have them and they’re quite large – some of those don’t really cause any disruptions – when they are not near any genes – i.e. no problems with the promoters, enhancers or the open reading frame itself…

Gene Density in Prokaryotes and Eukaryotes

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Looking at the bacterium – we see many green – the actual genes – which constitutes the majority of the ~70000bp portion taken from their genome (i.e. 57 genes in 70000bp) – and some blue – the intergenetic sequences – little spaces in between the genes, and RNA polymerase gene in red – which we shall follow and compare between these species. Introns appear in eukaryotes – i.e. the yeast cell – a single cellular eukaryote fungus. It is still predominantly green, hence still many genes (i.e. 31 genes in 70000bp) – and the RNA polymerase gene is somewhat similar. The genes aren’t really much different in size – it’s just a little more space between them – i.e. intergenetic sequences - and the appearance of a few introns which lessens gene density here. Dramatic change is visible when we get to fruit flies – (i.e. 9 genes in 70000bp) – this drop in gene density is apparent because of the presence of MORE introns – the RNA polymerase gene has expanded, the intergenetic spaces are larger, and we see some repeated sequences. Finally, at humans – we see only 2 genes per 70000bp – we have enormous sets of introns, larger intergenetic spaces and many repeats in both the intergenetic spaces and introns.

Genome Size ≠Organism Complexity -

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Humans are not the leaders of inefficient gene regulation or packaging… E.coli has 85% of its genome encoding for proteins – however, eukaryotes can still be higher than this… Some animals – Newt and Lungfish have genomes many times longer than the human’s – these would be extending extremely long and yet be packaged in the nucleus of a cell. Why these genomes are so large is not clear…

Question 2 – What Advantages does Added Complexity Provide? -

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Why do we bother to have all this extra DNA? Clearly, it makes it more difficult for the cell to replicate as it must deal with more DNA, it would be more difficult to package and to control during transcription, so why do we bother? Eukaryotes have more genes – having approximately 5 times as many genes – which provides a greater variety of proteins for the production of our more complex bodies as compared to a bacterium – and also the diversity of our cell types – and the functions which they perform demand this – it requires more flexibility in the proteins we can produce. Eukaryotes possess Introns – even if we have the same number of genes - we can produce many more proteins because we can alternatively splice genes which possess introns – hence there are more variations on the same gene. Eukaryotes have Long Complex Enhancers – they have greater control of where and when the genes are expressed – and this is needed because eukaryotes are so much more complex. A bacteria never needs to decide what cell it has to be – it doesn’t have to be a red blood cell, neurone, skin cell, etc. o Each of these cells are structured differently with different biochemical mechanisms going on within it and all of this complexity requires the expression of different proteins and different forms of proteins from the same gene… we need all these different programs in the genome to run these different cells… o So this means, we need more genes – for a variety of proteins along with the variety we get from having introns to produce all these different specialized cells – and then the decision for what genes to express and where becomes much more complicated when we have all these different types of cells. The Repetitive Sequences are a lot less clear… they may have a structural role, or just excess baggage that evolution hasn’t swept away yet…

Question 3 – How can cells contain, package and control so much DNA? -

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Although genes are much more spread out in Eukaryotes – there are mechanisms for “nesting” the genes a little more closely to make the genome somewhat smaller. The Genes can Overlap – it happens because not the entire gene encodes for the protein - so we can have one gene orientated in one direction where the intron isn’t part of the final product – but if we read the sequence in the opposite orientation – it is part of the final product for the next gene over. This means that the first exon of one gene is located in the last intron of the neighbouring gene. Another bit of regulation – the ability to regulate a gene’s expression without having to expand the genome – comes from the fact that we have 2 strands of DNA – and we can potentially transcribe them both as the gene is expressed. o In any double-stranded DNA molecule – only one of the strands typically encodes for protein – the Plus-Strand – hence when transcription takes place, the two strands are unzipped – and mRNA is made off of the coding strand via a Template Minus Strand. Hence we can make mRNA here, which basically matches the Plus Strand with U instead of T.

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The Top Red Strand – as it is the same sequence as the mRNA – we refer to as the Coding Strand and the Non-Coding Strand is the opposite strand. It is also the NonTemplate Strand – but the Bottom Blue Strand is the Template Strand – complementary to the mRNA. This can be useful for the regulation of genes is that some genes encode both sense and antisense transcripts – kinda going against what we’ve been saying… If we transcribe off the other strand – we’ll get an mRNA which is complementary to our coding mRNA – and hence it can bind to the coding mRNA to prevent its translation into a protein. In some parts of the genome – BOTH strands of DNA are transcribed into RNA – the “Sense” Transcript encodes a functional coding mRNA, the “Anti-Sense” Transcript controls the transcription of the “Sense” Transcript by binding to it to regulate the gene by preventing the synthesis of the protein. This gene regulation mechanism requires NO extra base pairs for it to work.

Question 3 – Levels of Chromatin Packing

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Finally, we have the structural challenge of packaging all this DNA (~2m) into the nucleus which is a small part of the cell. We do this through Chromatin Packaging – packaging with proteins which specifically bind to DNA into one another, and instruct the coiling and packaging of the DNA. We start with the DNA double helix – and then we have Histone Proteins which bind to the DNA and form “bead” like structures on a string, allowing the DNA to coil around the protein – and these protein Histones nest with one another to form a “cable” The double-helix DNA is starting to get packaged very effectively through protein scaffolds – which are subsequently packaged again with one another and essentially repackaging a packed structure. Eventually, we form a chromosome short enough to fit into the nucleus of a cell.

Prokaryotic Genomics – A Quote…

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Microbes – the oldest form of life on Earth, inhabit nearly every environment and can thrive under extreme conditions of heat, cold, pressure and radiation. Although microbes represent the vast majority of life on the planet – more than 99% have not been cultured, and consequently – their genomic diversity has been largely unrecognized and unutilized. By studying their DNA – scientists hope to find ways to use microbes to develop new pharmaceutical and agricultural products, energy sources, industrial processes, and solutions to a variety of environmental problems. Because they have such diversity in the environments they can live – and the biochemical processes which they carry out – they potentially have utility for all types of applications. (i.e. PCR) As shown, we have almost 400 prokaryotic genomes sequenced entirely. It is so much easier to sequence them as they are simple – the genes are all packed on top of each other and it is simply an open reading frame.

The Extremes which Bacteria can exist in… -

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Colwellia is a genus of bacteria which live in marine ecosystems in the Antarctic under ice. The challenges they face include freezing cold temperatures – and hence they adjustment their biochemistry – i.e. the structure and the processes the cell carries out so that it can function at a lower temperature. They effectively have anti-freeze proteins, and all their metabolic processes – such as enzyme structure and function are all shifted to work at lower temperatures. Chemosynthetic Bacteria occupy vents on the ocean floor. Hence they must deal with heat – i.e. 350-400°C – and also significant pressure. They have made a series of adaptation mainly related to their biochemistry to sustain there. They are also in total darkness – hence they do not feed the way most organisms feed – they are chemosynthetic – feeding directly off the vents. Helicobacter Pylori occupies the inside of the human stomach – and is the cause of stomach ulcers. Therefore, they have to deal with low pH – acidity which normally destroys bacteria, however – these are adapted by tightly regulating the flow of protons across its membrane. Deinococcus Radiodurans can withstand the sterilization irradiation process of food processing, withstanding incredible amounts of heat and radiation. Radiation breaks up DNA, which is what makes it lethal, the systems break down as gene expression is disturbed, mRNA’s are incorrect and the organism may die. This organism has a very robust DNA-repair machinery – looking out for mutations and fixing them, and it can repair the DNA just as quickly as the DNA is damaged by radiation.

Bacteria as an Experimental System – the power of Manipulation -

Bacteria have features which make them ideal for study in a laboratory. o Bacteria have a fast generation time – we can get many of them in a short period of time, and put them through many consecutive generations in a short period of time to examine evolutionary processes. (i.e. typically dividing every 20-30 minutes) o A single bacterium can be plated out, and the subsequent colony represents ONE original descendant bacterium – which has divided and divided until there is a “large pile of them” forming a colony. Every bacterium within that dot are genetically IDENTICAL – hence if we are examining genetic manipulation – we don’t need a single cell, we can examine a whole colony. o We can have an enormous num...