BioSci 145B lecture 1 page 1 © copyright Bruce Blumberg 2004. All rights reserved BioSci 145B...

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– [email protected]

• 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 here– http://blumberg-serv.bio.uci.edu/bio145b-sp2004– http://blumberg.bio.uci.edu/bio145b-sp2004

http://eee.uci.edu/04s/05705/

http://blumberg-serv.bio.uci.edu/bio145b-sp2004

http://blumberg.bio.uci.edu/bio145b-sp2004


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


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.


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


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


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)


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


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


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)


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


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


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 ?


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


Organization and Structure of Genomes (contd)• What does it mean?

Genetic complexity is not directly proportional to genome size!

• Increase in C is not alwaysaccompanied by proportionalincrease in number of genes– Note incorrect numbers

in chart• Drosophila

– ~14,000• human

– Controversial– ~30-50K


Organization and Structure of Genomes (contd)• What can we learn by hybridizing RNA back to the genomic DNA?

– Label RNA and hybridize withexcess DNA – measure formationof hybrids over time

– Rot½ analysis shows that RNA doesnot 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


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


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


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 priorto transport out of nucleus

– Posttranscriptional processingin an important feature ofeukaryotic gene regulation

• Why do eukaryotes have introns?– What are they good for?


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


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?


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


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


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)


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


Evolution of gene clusters (contd)• multigene families (contd)

– Gene number tends to increase withevolutionary complexity

• Globin genes increase in number from primitive fish to humans

– Clusters evolve by duplication and divergence


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


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 contentdiffers 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


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


Types and origin of repetitive elements – dispersed repeated sequences


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 betweengenes

• Pick up exons due to weak poly-adenylation signals

• The new exon becomes part of LINEby reverse transcription and isinserted into a new gene alongwith 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 mechanismfor generating new genes


Next Class• Thursday

– Papers about introns– They are posted on web sites (but large downloads)– Please read them and be prepared to discuss!

BioSci 145B lecture 1 page 1 © copyright Bruce Blumberg 2004. All rights reserved BioSci 145B...

Documents

Transcript of BioSci 145B lecture 1 page 1 © copyright Bruce Blumberg 2004. All rights reserved BioSci 145B...