WELCOME TO BIO 130S!!!

MOLECULAR AND CELL BIOLOGY

THIS IS YOUR INTRODUCTORY COURSE TO THE MOLECULAR LIFE SCIENCES AT THE UNIVERSITY

OF TORONTO

1

YOUR BIO 130 TEACHING TEAM…

• Prof. Melody Neumann – January 6th to February 13th

Reading Week…..• Prof. Jennifer Mitchell – February 24th to

April 3rd

• Prof. Ken Yip – Evening section• Prof. Melody Neumann – Course and

Lab Coordinator2

Molecular Mycology and Plant-Pathogen Interactions

M. Neumann Research

Palm Valley, Australia

www.deichmann-photo.com

3

Molecular Mycology and Plant-Pathogen Interactions

M. Neumann Researchandiap1-1plants at 24 h post mock-inoculation with 10 mMMgCl2or inoculation withPst(106cfu/mL). This time point waschosen, as previous work has demonstrated that intercellular SAlevels increase in IWFs by 24 h post-inoculation (hpi) (Cameronand Zaton, 2004). Gas chromatography-mass spectrometry (GC-MS) analysis was employed to measure free SA levels in IWFscollected from ARR-competent Col-0 (in planta Pst~105cfu/leafdisc at 3 dpi) and ARR-incompetentiap1-1(in planta Pst~107cfu/leaf disc at 3 dpi) plants. Free SA was measured, asprevious experiments support the hypothesis that unconjugatedSA acts in the intercellular space as an antimicrobial agentduring ARR (Cameron and Zaton, 2004; Kuset al., 2002); more-over, experiments in tobacco indicate that conjugated SA is notbiologically active (reviewed in Ryalset al.,1994).IWFs collectedfrom Col-0 leaves inoculated withPstaccumulated~190 ng/mLSA, compared with~60 ng/mL in IWFs collected from mock-inoculated leaves (t-test<0.05;Fig. 4b).No significant differencein intercellular SA accumulation was observed in IWFs collectedfromiap1-1leaves that were mock-inoculated or inoculatedwithPst(Fig. 4b). These data demonstrate thatiap1-1does notaccumulate intercellular SA in response toPst, and suggest thatIAP1 is upstream of intercellular SA accumulation in the ARRpathway.Intracellular SA accumulation is reduced iniap1-1inresponse toPstTheiap1-1mutation results in a loss of accumulationof SA in the intercellular space in response toPst, suggestingthatIAP1is involved in SA biosynthesis or perhaps in thetransport of SA to the intercellular space. Ifiap1-1is involvedin SA biosynthesis, intracellular SA accumulation in response toPstshould also be reduced iniap1-1. Mature Col-0 andiap1-1intracellular SA levels were determined in leaves from whichthe IWFs were removed at 24 h post mock-inoculation orinoculation withPst(106cfu/mL). This time point was chosen,as previous work has demonstrated that intercellular SA levelsincrease by 24 hpi during ARR (Cameron and Zaton, 2004).GC-MS analysis was employed to measure total SA levels (SAplus SA glucosides) in ARR-competent Col-0 (Pst~105cfu/leafdisc at 3 dpi) and ARR-incompetentiap1-1(Pst~107cfu/leafdisc at 3 dpi) plants. Col-0 leaves inoculated withPstaccumu-lated~10 000 ng/g fresh weight SA compared with~3000ng/g fresh weight from mock-inoculated leaves (Fig. 4c). Totalintracellullar SA levels were reduced to 1000 ng/g fresh weightiniap1-1leaves inoculated withPstor mock-inoculated(Fig. 4c). These data demonstrate thatiap1-1does not accu-mulate intracellular SA in response toPst, and suggests thatIAP1 is upstream of intracellular SA accumulation in the ARRpathway.eds1-1is ARR defectiveEDS1is an essential regulator necessary for the accumulation ofSA, which, in turn, acts as a signalling molecule to up-regulatedownstream defences (Falket al., 1999; Feyset al., 2001, 2005).As EDS1 is involved in SA accumulation,it was hypothesized thatit may play a role in ARR.eds1-1and wild-type Ws plants wereassayed for ARR by inoculating young and mature plants withPst.A significant decrease in bacterial growth (t-test<0.05) wasnot observed ineds1-1, as the bacterial density remained high(~107) in both young and mature plants (Fig. 5a). Conversely,inplantabacterial levels were reduced 17-fold in mature relative toyoung wild-type plants.These data suggest thatEDS1is requiredfor the ARR response.AsEDS1is involved in SA accumulation and intercellular SAinfiltration partially rescues theiap1-1ARR defect, a similarexperiment was performed witheds1-1.The addition of SA to thea)YoungMature10810710610bacterial density (cfu/ld)105in planta108H20SAb)7SA101065bacterial density (cfu/ld)10Col-0104in plantaFig. 5(a)In plantabacterial levels ineds1-1,jar1-1,jin1-1, Col-0 andWs. Plants were inoculated with 106colony-forming units (cfu)/mLPseudomonas syringepv.tomato(Pst) at 3 and 6 weeks post-germination(wpg).In plantabacterial levels [cfu/leaf disc (cfu/ld)] were monitored at 3days post-inoculation (dpi) and are presented as the meanstandarddeviation (SD) of three samples. This experiment was repeated once forjin1and twice foreds1-1andjar1-1with similar results. (b)eds1-1and Col-0plants (5-week-old) were infiltrated with water (H,Pst-5) or salicylic acid(SA) (0.1 mM) (SA,Pst-5) , followed by inoculation withPst5 h later, as inFig. 4. This experiment was repeated twice with similar results.Genes involved in age-related resistance inArabidopsis625© 2009 THE AUTHORSJOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTDMOLECULAR PLANT PATHOLOGY(2009)10(5), 621–634�intercellular space ofeds1-1prior to inoculation withPstresulted in a two-fold reduction (t-test not statistically signifi-cant) in bacterial density relative to water-infiltrated controlplants; however, these plants still supported high levels ofPst(~2x107cfu/leaf disc) (Fig. 5b). It is interesting to note that, inone of two other experiments,there was a statistically significantreduction (t-test<0.05) inPstgrowth in SA-infiltratededs1-1plants; however,Pstlevels remained high (107cfu/leaf disc, datanot shown).Thus, the addition of SA to the intercellular space ofeds1-1plants did not result in the rescue of theeds1-1ARRdefect.The ARR response occurs injar1-1andjin1-1Jasmonic acid (JA) and related metabolites are lipid-derivedcompounds which act as signals in defence to some pathogens(often necrotrophic) and in the wound response to insects, aswell as in plant growth and development (reviewed in Waster-nack, 2007). Work to date suggests that SA acts as an anti-Pstagent in the intercellular space during ARR, rather than as asignal for the up-regulation of defence genes such asPR1(Cameron and Zaton, 2004; Kuset al., 2002). Therefore, othersignalling molecules, such as JA, may be required for ARR sig-nalling. To test this hypothesis, two JA mutants,jar1-1(jasmonate-resistant 1) andjin1-1(jasmonate-insensitive 1),were assayed for ARR competence.JAR1encodes a JA-amino synthetase that activates JA foroptimal signalling inArabidopsisby conjugating it to aminofore,jar1-1mutants do not accumulate activated JA-Ile and aredefective for downstream JA-Ile-induced defence signalling(Staswick and Tiryaki, 2004). Col-0 andjar1-1mutants wereassayed for ARR competence by inoculating both young andmature plants withPst(Fig. 5a). A 10-fold decrease in bacterialdensity was observed in mature relative to young Col-0 andjar1-1plants.This significant decrease in bacterial growth (t-test<0.05) in mature plants suggests thatjar1-1mutants maintain afunctional ARR pathway despite the disruption in JA signalling.Studies have indicated that the phytotoxin coronatine, pro-duced by theP. syringaegroup of pathovars (includingPst), actsas a molecular mimic of JA-Ile (Brookset al., 2005; Krummet al.,1995; Staswick and Tiryaki, 2004) to elicit the JA signallingpathway downstream of JAR1 and JIN1, leading to the suppres-sion of SA-mediated defences, and thereby favouringPstgrowth(Laurie-Berryet al., 2006). Therefore, although studies withjar1-1andPstwill reveal whether a JA-dependent responserequires JA-Ile, it cannot establish whether an intact JA signal-ling pathway is required (Laurie-Berryet al., 2006). Instead,studies with jasmonate-insensitive mutants, such asjin1-1, arenecessary.JIN1is a MYC2 transcription factor (Lorenzoet al.,2004) that functions downstream of JAR1-synthesized JA-IleandPst-produced coronatine, and therefore is required for JAdefence signalling, even in the presence of coronatine-producingpathogens (Laurie-Berryet al., 2006). Young and mature Col-0andjin1-1plants were tested for their ability to display ARR byinoculation withPst(Fig. 5a). A significant 10-fold decrease(t-test<0.05) in bacterial levels in mature relative to young Col-0andjin1-1plants confirms thatjin1-1mutants have an intactARR pathway, suggesting that JIN1-dependent JA signalling isnot required for ARR.Identification of ARR-associated genes usingmicroarray analysisA microarray experiment was conducted to identify genes thatare differentially regulated during ARR. Gene expression wascompared between mature Col-0 plants that were mock-inoculated or inoculated withPst. It was possible to analyse onlyone time point after inoculation, and therefore samples werecollected at 12 hpi on the basis of previous intercellular SAreduction experiments using salicylate hydroxylase, which sug-gested that intercellular SA negatively affectsPstgrowth duringARR between 5 and 24 h after inoculation (Cameron and Zaton,2004). RNA extraction and cDNA synthesis were performed onthree replicates of each treatment, and each replicate was analy-sed using an Affymetrix GeneChip microarray (see SupportingInformation). The signal strength of each gene was averagedover the three replicates of each treatment, and the averagesignal strength of each gene was compared between samplesthat were mock-inoculated or inoculated withPst. Significanceanalysis of microarrays (SAM; Tusheret al., 2001) determinedwhich genes were significantly up- or down-regulated. SAMidentifies fewer false-positive up- or down-regulated genes thanan averaget-test when dealing with microarray data (Tusheret al., 2001).A delta value of 1.483 resulted in the identificationof 231 differentially expressed genes with a corresponding falsediscovery rate (qvalue) of<15%. SAM identified 125 signifi-cantly up-regulated and 105 significantly down-regulated genes.The complete list of genes identified in this microarray isavailable at http://bar.utoronto.ca/affydb/cgi-bin/affy_db_proj_browser.cgi. Genes up-regulated greater than two-fold are listedin Table 1 and significantly down-regulated genes are listed inTable S2 (see Supporting Information).Three members of the CYTOCHROMEP450(CYP)gene familywere differentially regulated in the microarray. The mosthighly up-regulated microarray gene,CYP71A13(~3.5-foldup-regulation, Table 1), plays a role in camalexin production inArabidopsis(Nafisiet al.,2007).CYP85A2was also up-regulated(~1.4-fold, http://bar.utoronto.ca/affydb/cgi-bin/affy_db_proj_et al., 2005; Nakashita626J. L. CARVIELet al.© 2009 THE AUTHORS(2009)10(5), 621–634

Reverse Genetics to discover genes upregulated in an Age-Related Response to a plant pathogen

4

Curriculum Design and Improving Student Learning

M. Neumann Research

• Cell and Molecular Biology Laboratories• Hybrid and Online Learning

Inverted Classrooms and Fully Online • Writing in Biology• Critical Analysis of Primary Literature• Animations for Cell and Molecular Biology

5

The Rest of the BIO130 Team…

Ms. Nyla Maharaj Cabrera– bio130@utoronto.ca– ESC 3053

Graduate Student TAs— from CSB— all pursuing MSc and PhD degrees in related fields

Course Lab Technicians– Tatjana Vasic, Karen Xu, Cathy Guo, and Jane Guo

6

Your BIO130 Learning Tools…

7

Lecture Notes

Guided notes Fill‐ins of key points (provided in lectures)

You will need to add additional notes on your own

BIO130H

Provide knowledge & preparation for 3rd and 4th

year courses in this discipline

8

Textbook

• Molecular Biology of the Cell, 5th ed. Alberts et al. 2008. Garland Science, New York. 

• Free 2‐year E‐text for new books and rental textbooks (UofT bookstore)

• 11 copies on Reserve at Gerstein, 3at Noranda Library (ESC)

9

iClickers

• Not for marks• Encourage active learning in class• Exploration of a wide range of topics• Available at UofT Bookstore—can buy used and get money back when returned

10

Textbook Reading Quizzes

• 5 questions on Bb for each week’s readings

• Objectives of quizzes are to help you to:– learn course materials– gauge depth required– keep up with lecture materials

• First quiz (available now) is for practice

11

Course Website

Blackboard (Bb) site on Portal

– Course News– Lecture Notes– Textbook Reading Quizzes– Audio Lecture Recordings– Extra Lab Notes– Electronic Discussion Board for Questions

12

Labs

• 4 wet labs, 1 tutorial‐style computer lab

• Lab topics:

•DNA isolation•Restriction Enzymes and Electrophoresis•Bioinformatics•Microscopy•Microtubule Dynamics

13

What you need for labs…

2014 Course ManualU of T Bookstore $ 11.65

Safety goggles/glasses$12

Both: $25

Lab coat$16

14

Evaluation

30% Laboratories and Textbook Readings 14%  lab work9%  quizzes— best 3 out of 4 quizzes (3% each)5% library assignment2%  textbook reading quizzes 

70% Exams 30%  Mid‐term (March) covering Section 1 of the lectures40%  Final exam (April) covering Section 2 lectures and all 

labs15

When do labs start?

• Lab section Pxx01x – cycle 1 – week of Jan 13th

• Lab section Pxx02x – cycle 2 – week of Jan 20th

• All labs start promptly at 10 minutes after the hour

• No Quiz in Lab 1!

16

Lab conflict?  No lab?

• All lab enrollments and changes have to be requested through the BIO130 office (ES3053) this week

• Mon 2‐4, Tues – Thurs 10‐12 am and 2‐4 pm

• You should have 2 potential lab times

17

Where can I get help?

• Discussion Board‐‐monitored by course staff• Thursday lecture tutorials with course Professors• Tuesday lecture drop‐in with course Professors—starts next week

• Ms. Maharaj Cabrera‐‐administrative issues• Prof. Melody Neumann‐‐lab content and other academic issues related to BIO130

18

19

BIO 130SLecture 1 - Introduction

Overall objective of this course:• Provide knowledge & preparation for 2nd, 3rd

& 4th year courses in this discipline• Examine some highlights of the “biological

revolution” underway• Discover some connections between basic

molecular and cell biology research and its application in our lives

Overall topic:• Life inside a cell

20

So…………what are we going to talk about in my section?Cellular and Genomic Diversity• Prokaryotes and Eukaryotes

– differences and similarities– general organization– origins

• Model Organisms- examples and how they are used

21

Typical Model Organisms

Caenorhabditis elegans

Drosophila

E.coli

Yeast

Arabidopsis

Mouse22

The Molecules of LifeGeneral concepts about:• DNA• RNA• Proteins

All about their nomenclature (names), important structural components, and the bonds that hold them together

23

All about DNA………

• Nuclear and Organellar Genomes

• Chromosomes and Chromatin

• DNA replication and Repair

• Genes and Genomics

24

…..a fair bit about RNA

• General characterization of transcription

• RNA processing• Transcriptomics

25

….and even something about proteins

• Translation• Post-translation

processing• Proteomics

26

Final Reminders• Course manual and i‐clickers at U of T bookstore 

• Textbook with free E‐book at UofT bookstore• Check Blackboard site regularly• Have lab coat and goggles for wet labs• Check lab group under My Grades on Blackboard to get room # for lab

Again, welcome to BIO130 and see you Thursday!!!

27

28

Introduction to Cells and DiversityLecture 2

29

1) Prokaryotic and Eukaryotic cells

2) Origins3) Diversity and Model

Systems 4) Introduction to Nucleic

Acids

Readings:Alberts - MBoC,Chapter 1

Chapter 2Pgs 116-117

Lecture 2

Introduction to Cells, Diversity and Nucleic Acids

30

Guide to note taking

1)Write a definition, concept, key point, or procedure next to each number in parenthesesFill in blank spaces with a word or phrase to complete a definition, concept, key point, or procedureAnnotate your notes with the content from the lecture and relevant portions of textbook

4

31

Tree of Life

Alberts, Figure 1-21

32

Two main types of cells:

1) Prokaryotic cells

Eubacteria, and archaea2) Eukaryotic cells

Plants, fungi, animals, humans

33

Prokaryotic Cell

Alberts, Figure 1-18a

34

Eukaryotic CellAlberts, Figure 1-30

So did eukaryotes evolve from prokaryotes, and if so, HOW?

35

36

Origins of mitochondria

Alberts, Figure 1-34

Anaerobic ancestor

37

A carnivorous, single-celled eukaryote

Alberts, Figure 1-32

So How Do We Study All This Diversity???

• 19th and Early 20th century approach

• Late 20th and 21st century approach

38

Model Organisms

General Attributes of Model Organisms• Rapid development with short life cycles• Small adult (reproductive) size• Readily available (collections or wide-

spread)• Tractability – ease of manipulation or

modification• Understandable Genetics

39

Model System ExamplesProkaryote• E. coli, a heterotrophic eubacterium found in the

human gut• Synechocystis, a free living phototrophic

cyanobacterium (also a eubacterium)

40

Model System ExamplesEukaryote• Yeast – (Saccharomyces cerevisiae), a minimal

eukaryotic model• Arabidopsis – a model flowering plant species• Caenorhabditis elegans – a nematode worm

(with 959 body cells!!)• And also Drosophila, Mouse and now Humans

41

Let’s Just Back Up a Bit….

How does the diversity we see in the world around us happen and how is it passed on??

42

The Central Dogma

Information flow in the cell

DNA RNA ProteinTranscription Translation

43

44

Central Dogma - refinedDNA

mRNA tRNA rRNA

- transport AA’s- for protein synthesis

Messenger RNA (mRNA)Transfer RNA (tRNA)Ribosomal RNA (rRNA)

translation

Our Elaborated Central DogmaGenome Transcriptome Proteome

DNA RNA ProteinTranscription Translation

Interactome

Metabolome

Phenome

Organization

Replication

45

DNA is made with starstuff…..

46

Phosphorus in the Young Supernova Remnant Cassiopeia ABon-Chul Koo1,Yong-Hyun Lee1, Dae-Sik Moon2,3,4, Sung-Chul Yoon1, John C. Raymond5 Science 13 December 2013: Vol. 342 no. 6164 pp. 1346-1348

47

DNA, RNA and proteins are all:

Info in nucleic acid sequence is translated into an AA sequence via a genetic code which is essentially universalamong all species

Overview

Alberts, Figure 6-2

48

The genetic code

Functional consequences of a degenerate genetic code are:

A. All 20 amino acids are encoded by the DNA

B.There are opportunities for the synthesis of novel amino acids

C.Base-paring between codon and anti-codon structures occurs more easily

D.Random mutations resulting in amino acid changes are reduced

49

50

What are Nucleic Acids?

1) The genetic material in a cell

2) DNA =

3) RNA =

Organism’s blueprints

Ribonucleic acid

Deoxyribonucleic acid

51

Pentose sugar

Nitrogenous base

Phosphate group

scaffold for base

backbone

Three Parts of a Nucleic Acid:

1.

2.

3.

Alberts, Panel 2-6

52Alberts, Panel 2-6

Bases

purinepyrimidine

53

What are the differences between DNA and RNA?

DNA:

RNA:

Alberts, Figure 6-4b

54

1) Nucleoside monophosphate:

2) Nucleoside diphosphate:

3) Nucleoside triphosphate:

Nucleic Acid Nomenclature