1. BioSci 145B lecture 1 page 1 © copyright Bruce Blumberg 2004. All rights reserved BioSci 145B Lecture #1 4/6/2004 Bruce Blumberg –2113E McGaugh Hall - office hours Wed 11-12 AM (or by appointment) –phone 824-8573 –blumberg@uci.edu TA – Curtis Daly –2113 McGaugh Hall, 924-6873, 3116 check e-mail and noteboard daily for announcements, etc.. –If you do not have ready access to e-mail or the web speak with me ASAP –Please use the course noteboard for discussions of the material I will post all questions received via e-mail on the course noteboard If you object to your question being posted please indicate this clearly in the message.. lectures will be posted on web pages after lecture –http://eee.uci.edu/04s/05705/ - link only herehttp://eee.uci.edu/04s/05705/ –http://blumberg-serv.bio.uci.edu/bio145b-sp2004http://blumberg-serv.bio.uci.edu/bio145b-sp2004 –http://blumberg.bio.uci.edu/bio145b-sp2004http://blumberg.bio.uci.edu/bio145b-sp2004

2. BioSci 145B lecture 1 page 2 © copyright Bruce Blumberg 2004. All rights reserved Introductions and Goals Let’s introduce each other –Name –Major –Favorite thing about UCI –Least favorite thing about UCI On a 3 x 5 card write –a sentence or two describing what you want to get out of this class. –times when you would participate in an online chat Q&A session

3. BioSci 145B lecture 1 page 3 © copyright Bruce Blumberg 2004. All rights reserved Class requirements Grading Midterm40% Final exam40% Term paper10% Participation10% attendance, class discussion, paper presentation 20-30 minute presentation and discussion of a journal article is required These will be randomly assigned There are more papers than students – possibility for extra credit Meet with me during week 1 or 2 to discuss and get paper topic approved Attendance and participation is important Please come to class having read assigned material Final examination will not be cumulative, however, understanding of concepts and techniques from first part of course is required.

4. BioSci 145B lecture 1 page 4 © copyright Bruce Blumberg 2004. All rights reserved General comments Overall philosophy –This class is about understanding genomic and proteomic approaches to problems of biological interest. –Intended to be informative and cutting edge but also interesting and relevant, even fun. –Office hours are Wednesdays 11-12 but I am always around I will try to hold virtual office hours at least one evening/week via chat –Questions are welcome. Please stop me if something is unclear. Letters of recommendation –If you want more than a form letter I need to know you as more than a student number and grade come to office hours participate in class discussions make your interest in the subject apparent

5. BioSci 145B lecture 1 page 5 © copyright Bruce Blumberg 2004. All rights reserved Requirements for the term paper Goals –Analytical thinking –Improved writing Select a topic related to genomic or proteomic analysis of an interesting problem –Talk with me about your topic Write a short paper (~5 pages) in the style of a research grant describing how you will attack this problem (I will post an example). –Specific aims – questions, hypotheses –Background and significance What is known, what remains to be learned why should someone give you money to study this problem? –Research plan – specific experiments to answer the questions posed in specific aims Expected vs unexpected results

6. BioSci 145B lecture 1 page 6 © copyright Bruce Blumberg 2004. All rights reserved Requirements for the oral presentation Goal – again to get you to think more analytically –Exposure to literature (classic and current) –Learn critical reading –Discuss practical applications of what we are learning Powerpoint (“journal club”) presentation – as a presenter –20 minutes with time allowed for discussion (max of 15 – 20 slides) –Frame the problem – what is the big picture question? What was known before they started? What was unknown? Present background (few slides), handouts helpful but not required –What are specific questions or hypotheses to be tested Discuss figures –What is the question being asked in each figure or panel? –What experiments did the authors do to answer questions? –Do the data support the conclusions drawn? »Were controls done? What did they conclude overall? What could have been improved? –Point out a few papers for further reading (reviews, followups, etc)

7. BioSci 145B lecture 1 page 7 © copyright Bruce Blumberg 2004. All rights reserved Requirements for the oral presentation (contd) Powerpoint presentation – as a listener –READ THE PAPERS –Study the figures What points don’t you understand? –Make notations, ask the speaker to clarify these –Listen to the speaker If presentation is unclear, ask the speaker to elaborate Always feel free to ask questions – we want an open discussion Papers are posted on the web sites listed –I will burn CDs for everyone when the course roster is finalized Logistics –Prepare presentation and either e-mail to me or bring it on a CD-ROM Floppy disk USB flash memory drive –Or bring your own laptop

8. BioSci 145B lecture 1 page 8 © copyright Bruce Blumberg 2004. All rights reserved Presentation schedule Week 1 – Berget et al., 1977; Chow et al., 1977 - Curtis Week 2 – (1) Geisler et al., 1999 (2) Maniatis et al., 1978 (3) Osoegawa et al., 2000 Week 3 – (4) Adams et al., 1991 (5) Sargent and Dawid, 1983 (6) Wang and Brown, 1991 Week 4 - (7) Innis et al., 1988 (8) Myers et al., 2000 (9) Sanger et al., 1977 Week 5 – midterm, no presentations Week 6 –(10) Hsu et al., 2002 (11) Tyson et al., 2004 (12) Venter et al., 2004 Week 7 – (13) Cawley et al., 2004 (14) Kapranov et al., 2002 (15) Schena et al., 1996 Week 8 – (16) Boutros et al., 2004 (17) Carlson et al., 2003 (18) Golling et al., 2002 Week 9 – (19) Fields and Song, 1989 (20) Ito et al., 2001 (21) Uetz et al., 2000 Week 10 - (22) Gavin et al., 2002 (23) Hamadeh et al., 2002 (24) Hoffmeyer et al., 2000

9. BioSci 145B lecture 1 page 9 © copyright Bruce Blumberg 2004. All rights reserved Lecture Outline 4/6/2004 – Organization and Structure of Genomes Today’s topics –Genome complexity –Implications of split genes for protein diversity –Repetitive elements and gene evolution The big picture – how are genomes similar and different? This week’s papers – Two groups discover introns and win the Nobel prize in Physiology or Medicine (1993) –Authors of your book do not cite either paper - Hmm what’s up with that? Beget et al., August (PNAS) (adenovirus) Chow et al., September (Cell) (adenovirus) Breathnach et al., November (Nature) (chicken) Jeffreys and Flavell, December (Cell) (rabbit)

10. BioSci 145B lecture 1 page 10 © copyright Bruce Blumberg 2004. All rights reserved Organization and Structure of Genomes (contd) Genome size –i.e. total number of DNA bp –Varies widely - WHY? C- paradox –i.e., what is the source of the differences? Do the number of genes required vary so much? –(how many “phyla” are represented at the right?) Phylum Chordata Phylum Arthropoda

11. BioSci 145B lecture 1 page 11 © copyright Bruce Blumberg 2004. All rights reserved Organization and Structure of Genomes (contd) How to measure genome complexity? –Hybridization kinetics –Shear and melt DNA –Allow to hybridize and measure ds vs ss by spectrophotometry C o t ½ - measures genome size and complexity –Larger value – longer to hybridize Smaller k –Longer to hybridize – more unique sequences, larger genome –Much of what we knew about genome size and complexity comes from these studies

12. BioSci 145B lecture 1 page 12 © copyright Bruce Blumberg 2004. All rights reserved Organization and Structure of Genomes (contd) Assumptions –C o t ½ measures rate of association of sequences –Simple curves at right suggest simple composition No repetitive sequences (graphed inverse to book) What would a more complex genome look like? –Would it be just shifted further to the right? –Or ?

13. BioSci 145B lecture 1 page 13 © copyright Bruce Blumberg 2004. All rights reserved Organization and Structure of Genomes (contd) Measure eukaryotic DNA –Multiple components –Can calculate more than 1 C o t ½ value –Either means starting material is not pure (i.e., multiple types of DNA) –Or means different frequency classes of DNA Highly repetitive Moderately repetitive Unique –Very big surprise

14. BioSci 145B lecture 1 page 14 © copyright Bruce Blumberg 2004. All rights reserved Organization and Structure of Genomes (contd) What does it mean? Genetic complexity is not directly proportional to genome size! Increase in C is not always accompanied by proportional increase in number of genes –Note incorrect numbers in chart Drosophila –~14,000 human –Controversial –~30-50K

15. BioSci 145B lecture 1 page 15 © copyright Bruce Blumberg 2004. All rights reserved Organization and Structure of Genomes (contd) What can we learn by hybridizing RNA back to the genomic DNA? –Label RNA and hybridize with excess DNA – measure formation of hybrids over time –R o t ½ analysis shows that RNA does not hybridize with highly repetitive DNA –What does this mean? Most of mRNA is transcribed from non-repetitive DNA Moderately repetitive DNA is transcribed Highly repetitive DNA is probably not transcribed into mRNA –Key argument why genome sequencers do not bother with “difficult” regions of repetitive DNA

16. BioSci 145B lecture 1 page 16 © copyright Bruce Blumberg 2004. All rights reserved Organization and Structure of Genomes (contd) Gene content is proportional to single copy DNA –Amount of non-repetitive DNA has a maximum,total genome size does not –What is all the extra DNA, i.e., what is it good for? –Where did all this junk come from and why is it still around? Repetitive DNA Telomeres Centromeres Transposons Junk of all sorts DNA replication is very accurate Selective advantage? OR

17. BioSci 145B lecture 1 page 17 © copyright Bruce Blumberg 2004. All rights reserved Organization and Structure of Genomes (contd) What is this highly repetitive DNA? Selfish DNA? –Parasitic sequences that exist solely to replicate themselves? Or evolutionary relics? –Produced by recombination, duplication, unequal crossing over Probably both –Transposons exemplify “selfish DNA” Akin to viruses? –Crossing over and other forms of recombination lead to large scale duplications

18. BioSci 145B lecture 1 page 18 © copyright Bruce Blumberg 2004. All rights reserved Transcription of Prokaryotic vs Eukaryotic genomes Prokaryotic genes are expressed in linear order on chromosome –mRNA corresponds directly to gDNA Most eukaryotic genes are interrupted by non-coding sequences –Introns (Gilbert 1978) –These are spliced out after transcription and prior to transport out of nucleus –Posttranscriptional processing in an important feature of eukaryotic gene regulation Why do eukaryotes have introns? –What are they good for?

19. BioSci 145B lecture 1 page 19 © copyright Bruce Blumberg 2004. All rights reserved Introns and splicing Alternative splicing can generate protein diversity –Many forms of alternative splicing seen –Some genes have numerous alternatively spliced forms Dozens are not uncommon, e.g., cytochrome P450s

20. BioSci 145B lecture 1 page 20 © copyright Bruce Blumberg 2004. All rights reserved Introns and splicing Alternative splicing can generate protein diversity (contd) –Others show sexual dimorphisms Sex-determining genes Classic chicken/egg paradox –how do you determine sex if sex determines which splicing occurs and spliced form determines sex?

21. BioSci 145B lecture 1 page 21 © copyright Bruce Blumberg 2004. All rights reserved Origins of intron/exon organization Introns and exons tend to be short but can vary considerably –“Higher” organisms tend to have longer lengths in both –First introns tend to be much larger than others – WHY? Often contain regulatory elements –Enhancers –Alternative promoters –etc

22. BioSci 145B lecture 1 page 22 © copyright Bruce Blumberg 2004. All rights reserved Origins of intron/exon organization Exon number tends to increase with increasing organismal complexity –Possible reasons? Longer time to accumulate introns? Genomes are more recombinogenic due to repeated sequences? Selection for increased protein complexity –Gene number does not correlate with complexity –Ergo, it must come from somewhere

23. BioSci 145B lecture 1 page 23 © copyright Bruce Blumberg 2004. All rights reserved Origins of intron/exon organization When did introns arise –Introns early – Walter Gilbert There from the beginning, lost in bacteria and many simpler organisms –Introns late – Cavalier-Smith, Ford Doolittle, Russell Doolittle Introns acquired over time as a result of transposable elements, aberrant splicing, etc If introns benefit protein evolution – why would they be lost? –Which is it? Introns late (at the moment)

24. BioSci 145B lecture 1 page 24 © copyright Bruce Blumberg 2004. All rights reserved Evolution of gene clusters Many genes occur as multigene families (e.g., actin, tubulin, globins, Hox) –Inference is that they evolved from a common ancestor –Families can be clustered - nearby on chromosomes (α-globins, HoxA) Dispersed – on various chromosomes (actin, tubulin) Both – related clusters on different chromosomes (α,β-globins, HoxA,B,C,D) –Members of clusters may show stage or tissue-specific expression Implies means for coregulation as well as individual regulation

25. BioSci 145B lecture 1 page 25 © copyright Bruce Blumberg 2004. All rights reserved Evolution of gene clusters (contd) multigene families (contd) –Gene number tends to increase with evolutionary complexity Globin genes increase in number from primitive fish to humans –Clusters evolve by duplication and divergence

26. BioSci 145B lecture 1 page 26 © copyright Bruce Blumberg 2004. All rights reserved Evolution of gene clusters (contd) History of gene families can be traced by comparing sequences –Molecular clock model holds that rate of change within a group is relatively constant Not totally accurate – check rat genome sequence paper –Distance between related sequences combined with clock leads to inference about when duplication took place

27. BioSci 145B lecture 1 page 27 © copyright Bruce Blumberg 2004. All rights reserved Types and origin of repetitive elements DNA sequences are not random –genes, restriction sites, methylation sites Repeated sequences are not random either –Some occur as tandemly repeated sequences –Usually generated by unequal crossing over during meiosis –These resolve in ultracentrifuge into satellite bands because GC content differs from majority of DNA –This “satellite” DNA is highly variable Between species And among individuals within a population Can be useful for mapping genotyping, etc –Much highly repetitive DNA is in heterochromatin (highly condensed regions) Centromeres are one such place

28. BioSci 145B lecture 1 page 28 © copyright Bruce Blumberg 2004. All rights reserved Types and origin of repetitive elements (contd) Dispersed tandem repeats are “minisatellites” 14-500 bp in length –First forensic DNA typing used satellite DNA – Sir Alec Jeffreys –Minisatellite DNA is highly variable and perfect for fingerprinting

29. BioSci 145B lecture 1 page 29 © copyright Bruce Blumberg 2004. All rights reserved Types and origin of repetitive elements – dispersed repeated sequences

30. BioSci 145B lecture 1 page 30 © copyright Bruce Blumberg 2004. All rights reserved Types and origin of repetitive elements – dispersed repeated sequences Book discusses types of elements, similarities, differences at great length –Main point is to understand how such elements can affect evolution of genes and genomes –Gene transduction has long been known in bacteria (transposons, P1, etc) –LINE (long interspersed nuclear elements) can mediate movement of exons between genes Pick up exons due to weak poly- adenylation signals The new exon becomes part of LINE by reverse transcription and is inserted into a new gene along with LINE –Voila – gene has a new exon –Experiments in cell culture proved this model and suggested it is unexpectedly efficient –Likely to be a very important mechanism for generating new genes

31. BioSci 145B lecture 1 page 31 © copyright Bruce Blumberg 2004. All rights reserved Next Class Thursday –Papers about introns –They are posted on web sites (but large downloads) –Please read them and be prepared to discuss!