8/9/2019 301Lect1 W2015

1/22

Controls F2 plants

CCLL LC 1 2 3

Welcome to BIOL301

Molecular and Cell Biology Laboratory

Hugo Zheng

8/9/2019 301Lect1 W2015

2/22

2

Lecture 1-5 and 12 (TBD)

Dr. Hugo Zheng

(focus on genes)

Lecture 6-10

Dr. Rodrigo Reyes-Ramothe(focus on proteins)

Lecture 11

Dr. Paul Harrison(bioinformatics)

Welcome to BIOL301

Molecular and Cell Biology Laboratory

8/9/2019 301Lect1 W2015

3/22

What you need: General Info Sheet + Syllabus (handout/myCourse)

Lab manual (McGill Bookstore)

Lab coat & safety goggles

Get a locker, no bags allowed in the lab

Reference texts Lodish (BIOL 200, 201 text book)

Griffiths (BIOL 202 text book)

Current Protocols Online eBooks (myCourse)

Context paper (myCourse)

3

Welcome to BIOL301

Molecular and Cell Biology Laboratory

8/9/2019 301Lect1 W2015

4/22

11 1-hour lectures

11 6-hour labs

ExperimentsEDMs experimental design/analysis modules

Lab coordinators: Anne-Marie Sdicu

Evaluation60% labs (Worksheet + Quiz)

10% midterm + 30% final problem solving based!

4

Welcome to BIOL301

Molecular and Cell Biology Laboratory

8/9/2019 301Lect1 W2015

5/22

Purpose of BIOL 301

You will be expected to combine what you have

learned in your genetic and developmental or pathological classes and current molecular and cell

biology techniques learned in this class, to design well-

controlled experiments to study a biological problem,

and to learn how to make a valid conclusion based on

the results

8/9/2019 301Lect1 W2015

6/22

Functions of genes, proteins, enzymes,

lipids, carbohydrates, hormones

What When Where

How?

(mechanism)

knowledge

8/9/2019 301Lect1 W2015

7/22

Todays discovery requires an integrated approach

Hypothesis generation- bioinformatics

- 2-hybrid

- genetic screens

Hypothesis testing- RNAi,

- site mutations

- homologous recombination

- gene expression

- localization & dynamics

- protein interaction

& modification

8/9/2019 301Lect1 W2015

8/22

ageneencodes aprotein, which is a kinase that modifies protein Ythat acts in intracellular signalingrequired for developmentroot

Basic Methods of Inquire in Biology

8/9/2019 301Lect1 W2015

9/22

bioinformatics

ageneencodes aprotein, which is a kinase that modifies protein Ythat acts in intracellular signalingrequired for development

mutant screen and

mappingbased cloning

1

2

3

4

5

root

expressed in root

meristem

Localized to PM

mutation phenotype

short roots

Basic Methods of Inquire in Biology

micro-array and/or

2D-PAGE analysis

- BiFC or FRET

- Co-purification

- Protein modification:- extraction of proteins from wt

and mutant kinase background

- 2D-PAGE

- Western blot using anti-protein Y

to see possible changes

in between wt and mutant kinase

background

regulate gene

expression,

protein

accumulation and

modification

6

7

8

8/9/2019 301Lect1 W2015

10/22

Focus of BIOL301 lectures

1. Gene identi ficat ion: forward/reverse genetics in model species

2. Molecular cloning of a gene: Map-based cloning andconfirmation of candidate gene

3. Study of gene expression: microarray, RT-PCR, Northern, in situ,

promoter-reporter gene fusion

4. Study of gene function: gene knockouts to analyze gene function,

RNA interference, over- and inappropriate gene expression

5. Protein localization and dynamics: GFP

6. Protein interaction: Methods for detection

7. Protein purification: tag-based protein purification to quantify

protein interaction in a complex8. Protein characterization: SDS-PAGE and 2D-PAGE for post-

translational modification

9. Protein characterization: Western blot

8/9/2019 301Lect1 W2015

11/22

Gene identification

genes are normally identified in a genetic model organism

Representative model to determine how things may work inlarger group of organisms focus on genetic models

Easy to work with

Grow fast

Lots of progeny

Small genome

Most have sequenced genomes

Easy to be manipulated to make transgenic individuals

Have international community of scientists working on same

system meetings, databases, stock centers, genetic & genomicresources available

8/9/2019 301Lect1 W2015

12/22

1. Protein secretion and membrane biogenesis

2. Function of the cytoskeleton

3. Gene regulation and chromosome structure

4. Control of cell cycle and cell division

1. single-celled eukaryotic organism

2. Undergo mitosis (90min) and meiosis

3. Grow rapidly in simple nutrient medium

4. Genome has 12.5mbp

5. ~6000 protein-coding genes

6. Easy genetic manipulation

Saccharomyces cerevisiae has been used as a

minimal model eukaryote

8/9/2019 301Lect1 W2015

13/22

Arabidopsis thaliana is chosen as a model

plant species

~20cm

1. Small in size, mature plants ~20cm

2. Can grow indoors in large number

3. Short life cycle, 8-10 weeks

4. Genome has 140mbp

5. ~20,000 protein-coding genes

6. Easy genetic manipulation

1cm

1. Cell biology

2. Physiology

3. Gene regulation

4. Tissue patterning

5. Plant immunity

8/9/2019 301Lect1 W2015

14/22

Forward versus reverse genetics in gene

identification

Forward genetics = classical genetics

going from phenotype to gene

widely used to identify genes acting in process of

interest

Reverse genetics

going from gene to function

requires a priory knowledge that makes you suspect a

gene is working in your process of interest

8/9/2019 301Lect1 W2015

15/22

Gene identification with forward genetics

Hunt mutant -- look for individuals with defects &/or

changes in your process of interest Conduct a genetic analysis for mutated gene(s):

recessive or dominant, single or multiple genes

Identify and clone gene(s) altered and/or involved inthat process

wild type mutant

8/9/2019 301Lect1 W2015

16/22

Mutagens used to increase frequency of

finding mutations & thus the number of genes

identified

chemical mutagens e.g. ethylmethane sulfonate (EMS)

point mutations -- affect proteins through truncation or loss of

key amino acids

radiation e.g. X-rays, fast neutrons leads to small or large deletions of DNA or even chromosome

breakage & rearrangements

insertional mutagens

mutagenize genes by sticking a large chunk of DNA in themiddle of it or its regulatory sequences

e.g. transposable elements, T-DNA in plant

Gene

T-DNA

8/9/2019 301Lect1 W2015

17/22

You are interested in how plants control their

growth (comprised of many processes)

17

How do you find out which gene controls what process?

Arabidopsis thaliana

8/9/2019 301Lect1 W2015

18/22

18

How do you find out which switch controls what?

???

8/9/2019 301Lect1 W2015

19/22

Random mutating and isolating mutants with

phenotype of interest in the next generation

Random mutations

Individual with

a mutation in

a gene that is

needed for

proper growth

8/9/2019 301Lect1 W2015

20/22

Lab #1

Part One: Introduction to lab

satety intro: how to handle yourself, equipment & chemicals in thelab

Techniques/skills required for mutant isolation in Arabidopsis

Part Two: Using learned techniques/skills mutant identification shorty mutants ofArabidopsis

quick plant genomic DNA extraction (will be used for PCR in Lab #2)

20

wild type shorty wild type shorty

8/9/2019 301Lect1 W2015

21/22

Goals of Lab #1-#5

21

In the Lab #1, you will identify an Arabidopsis mutant with a

growth defect, which you namedshorty: In the Lab #2-#5, you

will map (Lab #2), clone (Lab #3), confirm (Lab #4), and study

the expression pattern (Lab #5) of the gene.

wild type shorty

8/9/2019 301Lect1 W2015

22/22

Context Paper used throughout Lecture 2-3

Will be using this as an example

throughout Lect 2-3

Available on myCourse

22