1. Genetics II (eukaryotes) IT Carlow Bioinformatics September 2006

2. Homo sapiens That’s us. 3.1 Gbases, 25,000 genes Genetic code same as E.coli –Hence “universal” DNA replication (DNApol) Transcription RNApol Ribosomes, translation So “essentially” the same?

3. Other Eukaryotes Mouse, Rat, Cow, Chimp etc. –Chimp human 5mya L.C.Ancestor –Mouse rat 30mya LC Ancestor –Mouse human 100mya LC Ancestor –Chicken human 300mya LC Ancestor C.elegans 19,000 genes, 300 cells, 97Mbase Drosophila 14,000genes, 180Mbase S. cerevisiae 6,000genes, 12Mbase

4. Eukaryotes have nucleus DNA bundled in discrete units – chromosomes –Ends need capping, telomerase issues Bundling = additional access complications –histones, supercoiling Nucleus forces decoupling transscr translat Two way traffic in/out nucleus - –NF  B - Transcriptional regulators

5. Operons? In general not. But yeast often has common promoters on divergent (opp strand) genes Singer Lloyd Humniecki Wolfe 2005 –Find tissue specific clusters – spleen expressed –Chance or “design” –Compare human and mouse cluster breaks

6. Operons in Mammals?

7. Telomeres Eukaryotic chromosomes are linear chromosomes seem to have fixed location. Telomeres have characteristic # of repeats –Human TTAGGG, Oxytricha TTTTGGGG Chrs get shorter each generation –Priming for Okazaki fragments –Telomerase adds repeats –Telomerase fails: cancer, senescence

8. How similar is the machinery? DNA polymerase size % ID RNA polymerase Ribosomes –rRNA bigger 5S, 5.8S, 18S and 28S –Bases: 120bp,160bp,1900bp,4700bp –Protein count 50 rplX & 33 rpsX

9. tRNA Essential mediators of translation 74-90 base in size clover-leaf stucture Anti-codon loop –Curved so “wobble” is possible at third posn – –One anti-codon can serve 2 or 3 codons XXG can pair with C … Or U XXI (inosine) can pair with A, C or U

10. Introns About 5% of yeast genes Most mammalian genes Alternative splicing –Explain why we are more complex than worms –Challenges dogma 1 gene = 1 protein –Accounts for 80,000 diff proteins

11. Intron splice site

12. Alternative splicing 1 Splice / don’t splice If stop codon in frame in intron then truncated protein. Can be used as a genetic switch to control production of two alternative proteins

13. Alternative splicing 2 Competing 5' or 3' Splice Site Here two different 3’ splice sites Proximal, distal

14. Alternative splicing 3 Exon skipping Could be more than one exon skipped Lots of potential for variant transcripts Slightly different enzymes Missing protein domains

15. Alternative splicing 4 Mutually exclusive exons Here exons 1, 2, & 4 or 1, 3, & 4 Two different forms of protein

16. Alternative splicing 5 That’s just 1 classification –Can you think of another? Binf consequences –Gene prediction difficult in eukaryotes –No one answer in any one case –EST as binf tool for prediction

17. Junk? Human genome 3Gb but only 25K genes Even when introns accounted for 3% genome coding for “genes” 1% is actual codons The rest?

18. Pseudogenes Defined as gene inactivated because of mutation –Most obviously by nonsense/stop codon mutation –Genetic code arranged so many mutations tolerable –Once inactivated more mutations accumulate Processed pseudogene –Reverse transcriptase copy of mRNA –Lacks introns, 5’ upstream control regions 1/3 rd of human genome gene and gene related –pseudogenes, –gene fragments, truncated genes –introns/UTRs

19. Repetitive elements 2/3 rd of genome “intergenic” –1400Mb interspersed repeats (transposable elements) 44% of genome 640Mb LINES, LINE-1 420Mb SINES, Alu million copies 250Mb LTR, ERV 200,000 copies 90Mb DNA transposons, PiggyBac 2000 copies –600Mb Microsatellites etc. 90Mb CACACA and other repeats (forensics)

20. A bit of history Darwin Origin of Species 1860s Mendel sends ms to Darwin (ignores) 1909 Gene “invented” 1910 Genes sit on chromosomes, in order 1941 One gene = one enzyme 1944 Genes definitely DNA 1953 Double helix 1977 Splicing 1993 MicroRNA identified

21. What is a gene? Nature 25 May 2006 News Feature p399-401 Plants (Hothead), now mice may hold RNA copy of gene to “correct” DNA! ENCODE project Encyclopedia of DNA elements –Close look at 1% of human genome Alternative splicing (1977) can be fitted in. 5% of genome transcribed as read-through! Exons can combine with exons many genes away! 63% of mouse genome transcribed! 8/500 non-coding RNAs essential for signalling and growth

22. Bioinformatic consequences Pseudogenes a bioinf problem –Transcribed? See ESTs Alternative splicing a gene prediction prob –Exon prediction “easy” –Gene prediction harder Careers in RNA bioinformatics.