Bio Sci 145 B Lecture 1 462004 Bruce

Bio. Sci 145 B Lecture #1 4/6/2004 • Bruce Blumberg – 2113 E Mc. Gaugh Hall - office hours Wed 11 -12 AM (or by appointment) – phone 824 -8573 – blumberg@uci. edu • TA – Curtis Daly – 2113 Mc. Gaugh 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/04 s/05705/ - link only here – http: //blumberg-serv. bio. uci. edu/bio 145 b-sp 2004 – http: //blumberg. bio. uci. edu/bio 145 b-sp 2004 Bio. Sci 145 B lecture 1 page 1 ©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 Bio. Sci 145 B lecture 1 page 2 ©copyright Bruce Blumberg 2004. All rights reserved

Class requirements • Grading Midterm 40% Final exam 40% Term paper 10% Participation 10% 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. Bio. Sci 145 B lecture 1 page 3 ©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 Bio. Sci 145 B lecture 1 page 4 ©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 Bio. Sci 145 B lecture 1 page 5 ©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) Bio. Sci 145 B lecture 1 page 6 ©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 Bio. Sci 145 B lecture 1 page 7 ©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 Week 2000 6 –(10) Hsu et al. , 2002 (11) Tyson et al. , 2004 (12) Venter et al. , 2004 7 – (13) Cawley et al. , 2004 (14) Kapranov et al. , 2002 (15) Schena et al. , 1996 8 – (16) Boutros et al. , 2004 (17) Carlson et al. , 2003 (18) Golling et al. , 2002 9 – (19) Fields and Song, 1989 (20) Ito et al. , 2001 (21) Uetz et al. , 2000 10 - (22) Gavin et al. , 2002 (23) Hamadeh et al. , 2002 (24) Hoffmeyer et al. , Bio. Sci 145 B lecture 1 page 8 ©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) Bio. Sci 145 B lecture 1 page 9 ©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? ) Bio. Sci 145 B lecture 1 page 10 ©copyright Bruce Blumberg 2004. All rights reserved Phylum Chordata Phylum Arthropoda

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 • Cot½ - 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 Bio. Sci 145 B lecture 1 page 11 ©copyright Bruce Blumberg 2004. All rights reserved

Organization and Structure of Genomes (contd) • Assumptions – Cot½ 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 ? Bio. Sci 145 B lecture 1 page 12 ©copyright Bruce Blumberg 2004. All rights reserved

Organization and Structure of Genomes (contd) • Measure eukaryotic DNA – Multiple components – Can calculate more than 1 Cot½ 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 Bio. Sci 145 B lecture 1 page 13 ©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 -50 K Bio. Sci 145 B lecture 1 page 14 ©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 – Rot½ analysis shows that RNA does not hybridize with highly repetitive DNA – What does this mean? • Most of m. RNA is transcribed from non-repetitive DNA • Moderately repetitive DNA is transcribed • Highly repetitive DNA is probably not transcribed into m. RNA – Key argument why genome sequencers do not bother with “difficult” regions of repetitive DNA Bio. Sci 145 B lecture 1 page 15 ©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? • • • Repetitive DNA Telomeres Centromeres Transposons Junk of all sorts – Where did all this junk come from and why is it still around? • DNA replication is very accurate • Selective advantage? • OR Bio. Sci 145 B lecture 1 page 16 ©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 Bio. Sci 145 B lecture 1 page 17 ©copyright Bruce Blumberg 2004. All rights reserved

Transcription of Prokaryotic vs Eukaryotic genomes • Prokaryotic genes are expressed in linear order on chromosome – m. RNA corresponds directly to g. DNA • 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? Bio. Sci 145 B lecture 1 page 18 ©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 P 450 s Bio. Sci 145 B lecture 1 page 19 ©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? Bio. Sci 145 B lecture 1 page 20 ©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 Bio. Sci 145 B lecture 1 page 21 ©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 Bio. Sci 145 B lecture 1 page 22 ©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) Bio. Sci 145 B lecture 1 page 23 ©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, Hox. A) • Dispersed – on various chromosomes (actin, tubulin) • Both – related clusters on different chromosomes (α, β-globins, Hox. A, B, C, D) – Members of clusters may show stage or tissue-specific expression • Implies means for coregulation as well as individual regulation Bio. Sci 145 B lecture 1 page 24 ©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 Bio. Sci 145 B lecture 1 page 25 ©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 Bio. Sci 145 B lecture 1 page 26 ©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 Bio. Sci 145 B lecture 1 page 27 ©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 Bio. Sci 145 B lecture 1 page 28 ©copyright Bruce Blumberg 2004. All rights reserved

Types and origin of repetitive elements – dispersed repeated sequences Bio. Sci 145 B lecture 1 page 29 ©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, P 1, etc) – LINE (long interspersed nuclear elements) can mediate movement of exons between genes • Pick up exons due to weak polyadenylation 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 Bio. Sci 145 B lecture 1 page 30 ©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! Bio. Sci 145 B lecture 1 page 31 ©copyright Bruce Blumberg 2004. All rights reserved