* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download The human genome - The Galton Institute
List of types of proteins wikipedia , lookup
Genomic imprinting wikipedia , lookup
Gene expression profiling wikipedia , lookup
Molecular cloning wikipedia , lookup
Gene expression wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Community fingerprinting wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Transposable element wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Whole genome sequencing wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Genomic library wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Non-coding DNA wikipedia , lookup
Genome organisation, DNA fingerprinting, VNTRs… Andrew P Read Emeritus Professor of Human Genetics, University of Manchester Centre for Genomic Medicine, St Mary’s Hospital, Manchester Galton Institute teachers’ day, Nowgen, 30th June 2015 The human genome: things to ask • How big is it ? • Why is it organised into chromosomes? • What is in it ? • How does it work? • How does my genome compare to others? How big is it? Weight of DNA in a (diploid) cell = 6.9 pg Mol wt of a base pair (incl phosphates) ≈ 650 6 x 1023 bp would weigh 650 g Wikipedia (6 x 1023/650) x 6.9 x 10-12 bp would weigh 6.9 x 10-12 g No. of base pairs in a diploid cell = 6.3 x 109 Human genome is ca.3,200,000,000 base pairs of DNA The human mitochondrial genome 16,569 bp DNA 13 protein-coding genes 2 ribosomal RNA genes 22 transfer RNA genes The great majority of mitochondrial proteins are encoded in the nucleus. www.mitomap.org The human genome: things to ask • How big is it ? • Why is it organised into chromosomes? • What is in it ? • How does it work? • How does my genome compare to others? Why is it organised into chromosomes? 3,200,000,000 base pairs of DNA Wikipedia Base pairs are 0.34 nm apart Length of DNA in a (diploid) cell: Length = (3.2 x 109) x 2 x (0.34 x 10-9) m = 2 metres !! Why is it organised into chromosomes? A normal diploid human cell contains 2m of DNA Magnify 1,000,000 x Human cell Lecture room nucleus 10 x 10 x 10 μm 10 x 10 x 10 m 2m of DNA 2,000 km of string! The human genome: things to ask • How big is it ? • Why is it organised into chromosomes? • What is in it ? • How does it work? • How does my genome compare to others? Protein-coding genes… 3’UT 5’UT DNA → RNA → protein Where is….. the promoter ? the transcription start site ? the transcription termination site ? the 5’ end of exon 3 ? the 3’ end of intron 3 ? The number of exons in a gene varies widely with no evident logic Table 3.1 Our genome looks chaotic… • genes seem to be randomly arranged across the chromosomes • there are very few functional clusters (like E.coli operons) • e.g. haemoglobin: α-chain gene is on c’me 16, β on c’me 11 • e.g. Fanconi anaemia • People with a balanced chromosomal translocation are usually normal and healthy (though they are at risk of having miscarriages or abnormal babies) NCG3 Fig 2.14 Fanconi anaemia An autosomal recessive condition with developmental abnormalities and high risk of cancer Due to malfunction of a multiprotein complex that repairs damaged DNA Locations of genes for 13 of the proteins in the complex Genome browsers Ensembl UCSC browser http://www.ensembl.org http://genome.ucsc.edu/ Genome statistics www.miRBase.org The transcriptome and the proteome are much more complex than the genome • At least 20,389 protein-coding genes …… …. but 194,353 gene transcripts. • One gene can encode multiple proteins by: • Alternative splicing (average 6.3 splice isoforms per gene) • Using alternative promoters (70,292 promoter-like sequences in the genome) • Differential post-translational modification • Special mechanisms e.g. immunoglobulin genes One gene – more than one protein Alternative splicing Including / skipping an exon Alternative splice donor sites Alternative splice acceptor sites Alternative exons The neurexin B gene can encode ca. 1,000 different proteins Promoters Exon that can be included or skipped Exon with 2 alternative 5’ splice sites Exon with 3 alternative 5’ splice sites Exon with 2 alternative 3’ splice sites Exon with 2 alternative 5’ splice sites, includes an alternative stop codon, producing a protein lacking the transmembrane and cytoplasmic domains encoded by exon 24. What is in it? • ca. 20-25,000 protein-coding genes • Suppose a typical protein is made of 500 aminoacid residues • It would need 1,500 nucleotides of messenger RNA to encode it • So our genome might contain around 1,500 x 25,000 bp of coding sequence = 37 million bp. • This is only 1.16% of the total DNA of our genome! The best current real estimate is 1.22% So what about the 98.8% of DNA that is noncoding? • some is in introns of protein-coding genes • much of it is in repetitive sequences … … there are interspersed repeats… From the initial report of the Human Genome Project (Nature 409: 860–921; 2001) … and tandem repeats. Tandem repeats Jobling et al. Human Evolutionary Genetics 2nd edn Fig 3.16 Tandem repeats • usually non-coding • satellite DNA (173 bp repeat unit) in centromeric heterochromatin • minisatellites particularly near telomeres of chromosomes • microsatellites scattered throughout the genome • number of repeat units in a given mini / microsatellite often varies between individuals – variable number of tandem repeats (VNTR) 3 alleles of a (CA)n microsatellite DNA fingerprinting Southern blot, hybridising to a probe that recognises a whole series of different minisatellite VNTRs DNA profiling • DNA fingerprinting has been superseded by DNA profiling • DNA profiling uses a panel of microsatellites, amplified by PCR and genotyped on an automated genome sequencer • A profile using the UK SGM+ panel of 10 microsatellites plus a sex marker. The human genome: things to ask • How big is it ? • Why is it organised into chromosomes? • What is in it ? • How does it work? • How does my genome compare to others? Epigenetics: making it work The 2m of DNA in a cell nucleus is packaged, primarily by the 5 histone proteins, H1, H2A, H2B, H3, H4. Variable covalent modification of histones (methylation, acetylation, phosphorylation…), also methylation of DNA, controls the packaging and hence gene activity. The ENCODE project Aim: to delineate all functional elements encoded in the human genome 2003 Pilot phase: 44 genomic regions, totalling 30 Mb, examined using a wide range of experimental and computational methods, with the aim of identifying all functional elements. Nature 447: 799 - 816 ; 2007 2007 Production phase: extend to whole genome. 30 papers in September 2012 – see Nature 489: 57; 2012 The Nature ENCODE Explorer: www.nature.com/encode/ 5 papers in August 2014 – see Nature 512: 374; 2014 Major findings of the ENCODE project • The great majority of all the genome is transcribed, at least at some times and in some types of cell. • Chromatin can be classified into ‘flavours’ based on patterns of DNA methylation and covalent modification of histones, and these flavours correlate with biochemical activity. • Some sort of function (coding, transcribed, protein-binding…) can be assigned to 80% of nucleotides in the genome – but see Graur et al., Genome Biol Evolution 5: 578-590; 2013 for a dissenting interpretation. Regulatory elements: enhancers Enhancers are promoter-like sequences that may be brought into contact with the promoter by DNA looping. Promoters and enhancers bind transcription factors (DNA-binding proteins that help turn genes on). Tissue-specific gene expression is largely controlled by enhancers. ENCODE identified 399,124 sequences with enhancer-like features The human genome: things to ask • How big is it ? • Why is it organised into chromosomes? • What is in it ? • How does it work? • How does my genome compare to others? Differences between genomes of normal healthy people • There would typically be 3-4 million differences: single nucleotide variants, indels (small insertions/deletions), variable number tandem repeats, copy number variants (kilobases to megabases) … • A very few of the differences directly determine a characteristic: e.g. sex, eye colour, blood groups… • A larger number of the differences contribute to a characteristic, along with other genetic and environmental factors: e.g. height, blood pressure, liability to diabetes… • The overwhelming majority have no apparent effect. Summary and conclusions • We are vastly more complex than Drosophila flies or Caenorhabditis worms, but we don’t have vastly more protein coding genes. • Only 1.2% of our genome is protein-coding sequence. Much of the rest is involved in control (there may also be some ‘junk DNA’). • Coding sequence is controlled by an interacting network of DNA methylation (on CpG), histone modifications and non-coding RNAs. • Cells are awash with non-coding RNAs, few of which have well understood functions. • There’s still a very long way to go in understanding how our genomes work – but the bit we do understand is already fascinating and inspiring..