1) Learning Molecular Tools

Week #1 (Jan 11 - 15)

Week #2 (Jan 18 - 22)

Week #3 (Jan 25 - 29)

Week #4 (Feb 1- Feb 5)

2) Virtual Reprints

A. Structure - Function: How do Calcium Pumps of the SR/ER work?

B. What is a Green Fluorescent Protein (GFP) and why is it so popular?

C. How do proteins "know" when to stay in the ER?

D. Methods that have produced a big impact.

E. How does a mammalian fetus know if it is supposed to be male or female?

F. How do cells in an embryo know which type of cell they should be?

Starting at Square One - learning the tools

Molecular Biology Lecture Schedule for First 3 Weeks and First Exam

Readings come from Life8^th Ed., by Purves et al.

Week #1 (Jan 11 - 15) (return to the begining)

Wed. Feb 3

• Test due Wed. Feb. 3 class time followed by optional WWW Workshop
  How to make your own web page and post on server; Dana 256.

HTML Helpful Hints

Virtual Reprints for Molecular Biology - Spring 2005
(Preliminary Readings come from Life, by Purves et al., 8^th edition)

(return to the beginning)

Fri. Feb 5 and Mon. Feb. 8

Section I (How do Calcium Pumps of the SR/ER work?)

Preliminary Reading

a) Homology

• pg 525 - 526 Evolution of genomes... stop@ Genes and proteins
• pg 537 Molecualr sequence... stop@ FIgure 24.12 What's the difference between homologues, orthologs and paralogs?
• pg 551 Molecular Clocks... stop@ Section 25.3
• Figure 24.3 - Does shared sequence mean shared function?

b) Inhibitors

• pg. 131 - 135 Section 6.5. - What does an inhibitor do and how does it do it?

c) Calcium pump role in cell physiology

• What does the calcium pump do? (gif animation)
• What role does calcium play in the muscle? (QT cartoon)
• How does the pump affect relaxation? (QT cartoon)
• See a quick movie of muscle contracting. (QT movie)

One panel from a poster describing how immunofluorescence labeling is done.
See immunofluorescence labeling of calcium pump in skeletal muscle.

Paper

• Campbell, Kessler, Sagara, Inesi, and Fambrough. (1991) Nucleotide Sequences of Avian Cardiac and Brain SR/ER Ca²⁺-ATPases and Functional Comparisons with Fast Twitch Ca²⁺-ATPase. Journal of Biological Chemistry. 266 (24): 16050-16055.
  
  • Optional - Learn about cyclopiazonic acid

Preliminary Reading

a) See immunofluorescence labeling of calcium pump in non-muscle cell grown in culture.
Where is SERCA2 located in non-muscle cells?

b) Use Streptolysin-O (SLO) to selectively permiabilize the plasma membrane.
What is SLO? What does it do? How does it work?

c) Jmol images of SERCA1a prior to and after the phosphate binding.

d) Hydropathy plots online

Paper

• Campbell, Kessler, and Fambrough. (1992) The Alternative Carboxyl Termini of Avian Cardiac and Brain Sarcoplasmic Reticulum/Endoplasmic Reticulum Ca²⁺-ATPases Are on Opposite Sides of the Membrane. Journal of Biological Chemistry. 267 (13): 9321-9325.
  
  • Good follow up paper but not assigned reading:
    • Linu M. John, James D. Lechleiter, and Patricia Camacho (1998) Differential Modulation of SERCA2 Isoforms by Calreticulin. Journal of Cell Biology 142: 963 - 973.

Preliminary Reading
a) See a cartoon showing sodium pump subunit assemply.
How do the two sodium subunits fit together?

b) Immunopecipitation Method - Animation

c) liposome method

Paper

• Lemas, Hamrick, Takeyasu, and Fambrough. (1994) 26 Amino Acids of an Extracellular Domain of the Na,K-ATPase a-Subunit are Sufficient for Assembly with the Na,K-ATPase ß-Subunit.Journal of Biological Chemistry. 269 (11): 8255-8259.

(return to the beginning)

Mon. Feb. 15

Papers

• Prasher, Eckenrode, Ward, Prenderast, and Cormier. (1992) Primary Structure of the Aequorea victoria green-fluorescent protein. Gene. 111: 229-233.
  
• Amino acid structures - Jmol table

Wed. Feb. 17

First Web Page Due at Class Time
optional help session on Orthologs

Fri. Feb. 19

• Jmol Image of GFP (shown as a monomer)
  
• Chalfie, Tu, Euskirchen, Ward, and Prasher. (1994) Green Fluorescent Protein as a Marker for Gene Expression. Science. 263: 802-805.
  (see news story and cover shot from same issue)
• See movie of microinjection of C. elegans.

Preliminary Reading
What is FRAP?

Nobel Prize for GFP

Student Web Page on GFP

Optional Reading

• Cole, Smith, Sciaky, Terasaki, Edidin, and Lippincott-Schwartz. (1996) Diffusional mobility of Golgi proteins of membranes of living cells. Science 273: 797-801.

Required Reading

• For summary of this paper and related movies, click here

Optional Reading

• Presley, Cole, Schroer, Hirschberg, Zaal, and Lippincott-Schwartz. (1997) ER to Golgi traffic visualized in living cells. Nature 389: 81-85. (see commentary)

Required Reading

• For summary of this paper and related movies, click here.

Additional Sources of Information

• A new way to get DNA into cells (while they are still a part of the organism!)
• Another way to get DNA into some organisms (QT movie with C. elegans)
  (Movie generated by Paul Muhlrad in the lab of Samuel Ward, Dept. Molecular and Cellular Biology, Univ. Arizona)

(return to the beginning)

Section III (How do cells know to keep some proteins in the ER?)

Wed. Feb. 24 - Fri. Feb. 27

Preliminary Reading

• pg 272 - 274 Figures 12.15 - 12.17

Papers

• Munro and Pelham. (1987) A C-Terminal Signal Prevents Secretion of Luminal ER Proteins. Cell. 48: 899-907.

Saturday February 27 - (spring break) - Sunday March 7

Second Web Page Due March 10
Mon. March 8 - Fri. March 12

Preliminary Reading

• FACS method
  
• Calculating Molecular Weights

Papers

• Vaux, Tooze, and Fuller. (1990) Identification by Anti-Idiotype Antibodies of an Intracellular Membrane Protein that Recognizes a Mammalian Endoplasmic Reticulum Retention Signal. Nature 345: 495-502. (see cover photo)

Mon. March 15 - Wed. March 17

• Semenza, Hardwick, Dean, and Pelham. (1990) ERD2, a Yeast Gene Required for the Receptor-Mediated Retrieval of Luminal ER Proteins from the Secretory Pathway. Cell. 61: 1349-1357.

• Lewis, Sweet, and Pelham. (1990) The ERD2 Gene Determines the Specificity of the Luminal ER Protein Retention System. Cell. 61: 1359-1363.
  
• E.P. (1997) Prepaper Publicity Ignites Race to Publish. Science. 275: 1877.
  
  • Interesting follow up story: A Di-Acidic Signal Required for Selective Export from the Endoplasmic Reticulum (Nishimura and Balch, 1997. Science 277: 556 - 558.)

Amino Acid Structures for in-classs discussion

---

Second Exam due Monday March 22 at class time

• Optional Jmol Workshop

(return to the beginning)

Section IV (Methods that Push the Envelope)

Wed. March 24

1) New Cloning Strategies

Preliminary Reading

• pg 295 Transposable elements...
• pg 311 - 312 Transponsons

A. Enhancer Traps

• O'Kane and Gehring. (1987) Detection in situ of genomic regulatory elements in Drosophila.. PNAS 84: 9123-9127.
• Plasmid Rescue Method

Fri. March 26

B. Yeast Two-hybrids

• Chien, Bartel, Sternglanz, and Fields. (1991) The Two-Hybrid System: A Method to Identify and Clone Genes for Proteins that Interact with a Protein of Interest. PNAS. 88: 9578-9582.
  
• (see QuickTime Movie on Two-Hybrid Method)
• Plasmid Shuffle Method

Monday March 29

2) Building a Better Mouse

• homologous recombination
• FACS method revisited
• Cre - loxP cell-specific knockouts

cell-specific knockouts

• Wade Roush. (1997) New knockout mice point to molecular basis of memory. Science 275: 32-33.
• Gu, Marth, Orban, Mossmann, and Rajewsky. (1994) Deletion of a DNA polymerase ß gene segment in T cells using cell type-specific gene targeting. Science 265: 103-106.

Optional Reading: single-copy insertions

• unknown author (1997) Hitting the spot with genes. Science 273: 32.
• Bronson, Plaehn, Kluckman, Hagaman, Maeda, and Smithies. (1996) Single-copy transgenic mice with chosen-site integration. PNAS 93: 9067-9072.

Wed. March 31

3) Faster Knockouts or Knockdowns

Preliminary Reading

• pg 365 Antisense RNA
• in situ method
• See movie of microinjection of C. elegans.

Papers

• Wagner and Sun. (1998) Double-stranded RNA poses puzzle. Nature. 391: 744-745.
• Fire, Xu, Montgomery, Kostas, Driver and Mello. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391: 806-811.

Friday April 2

Papers

• Hannon. 2002. RNA Interference. Nature. 418: 244 - 251.
• Shi. 2003. Mammalian RNAi for the masses. Trends in Genetics. 19 (1): 9-12.

(return to the beginning)

Section V (Sex determination in Mammals)

Wed. April 7

Preliminary Reading
in situ hybridization
a) cartoon of in situ hybridization method
b) FISH Read 3 links under FISH in left frame (near bottom)
c) RT-PCR - requires Flash Plug-in

Papers

• Koopman, Munsterberg, Capel, Vivian, and Lovell-Badge. (1990) Expression of a candidate sex-determining gene during mouse testis differentiation. Nature 348: 450-452.
  
  

Fri. April 9

Third Web Page Due
(no class)

Mon. April 12

• Comparison of Mouse and Human Sry / SRY protein sequences using MacDNAsis
  
• Koopman, Gubbay, Vivian, Goodfellow, and Lovell-Badge. (1991) Male development of chromosomally female mice transgenic for Sry. Nature 351: 117-121.

Optional Reading

• Berta, Hawkins, Sinclair, Taylor, Griffiths, Goodfellow, and Fellous. (1990) Genetic evidence equating SRY and the testis-determining factor. Nature 348: 448-450.

Wed. April 14

Preliminary Reading
SSCP (Read points 1, 2, and 3 under the heading of "Differences in electrophoretic mobilities of wildtype and mutant nucleic acids") - written by John A. Wagner at School of Medical Sciences, Cornell University.

JMol tutorial of SRY binding to DNA (Note the shape of the DNA.)

Karyotyping and G-banding (click on the word "G-banding" and read the text above)

Papers

• Lida, Nakahori, Komaki, Mori, Haysahi, Tsutsumi, Taketani, and Nakagome. (1994) A novel nonsense mutation in the HMG box of the SRY gene in a patient with XY sex reversal. Human Molecular Genetics. 3 (8): 1437-1438.

• Bernstein, Koo, and Wachtel. (1980) Abnormality of the X chromosome in human 46,XY female siblings with dysgenetic ovaries. Science 207: 768-769.

Read Abstract Only

• Bardoni, Zanaria, Guioli, Floridia, Worley, Tonini, Ferrante, Chiumello. McCabe, Fraccaro, Zuffardi, and Camerino. (1994) A dosage sensitive locus at chromosome Xp21 is involved in male to female sex reversal. Nature Genetics 7: 497-501.
• OMIM entry for DSS
• DAX1 or NROB1 on genome browser
  
• Valerie J. Grant. (1996) Sex determination and the maternal dominance hypothesis. Human Reproduction. 11 (11): 2371-2375.
• Can you bias the gender of your baby?
• Money back if not right.

(return to the beginning)

Fri. April 16

Conversation with Jef Boeke, JHU

Image courtesy of Stephen W. Paddock, Eric J. Hazen, and Peter J. DeVries (Univ. Wisconsin - Madison)

Section VI (Homeobox Genes and Their Functions)

Mon. April 19

Preliminary Reading

a) development of axis

• pg 441 - 445 Morphogen gradients... to end of chapter
• Drosophila gastrulation QT movie (13 MB)
  

Fly Parts

• All the Parts of a Fly
• FlyMove Images
• The Interactive Fly

b) heat shock

• pg 48 - 49 Chaperonins...
• pg 847 Plants have....

c) CAT assay

Paper

• Driever and Nüsslein-Volhard. (1989) The bicoid protein is a positive regulator of hunchback transcription in the early Drosophila embryo. Nature 337: 138-143.
  • Fly embryogenesis
  • Virtual Cell Zoom in on nucleus and look at nucleosomes
  • nucleosome structure

• Read For Highlights Only
  Wang and Lehmann. (1991) Nanos is the localized posterior determinant in Drosophila. Cell. 66: 637-647.

Optional Reading

• Struhl, Johnson, and Lawrence. (1992) Control of Drosophila body pattern by the hunchback morphogen gradient. Cell. 69: 237-249.

• Chromosomal Walking Method
• Drosophila gastrulation QT movie (13 MB)

Fri. April 23

Preliminary Reading
Antennapedia and body parts

• The Head of a Fly
• A Leg of a Fly (from the back)
• Sex comb of male flies
• Antenna
• Wild-type head - nice EM image vs.
• Antp phenotype - very nice EM image
• Molecular Information on Antp

Read For Highlights Only

• Furukubo-Tokunaga, Flister, and Gehring. (1993) Functional specificity of the Antennapedia homeodomain. PNAS 90: 6360-6364.
  
• 3D structure of Antp
• Site-Directed Mutagenesis (Kunkel Method)
• Ultrabithorax phenotype
• Sonic Hedgehog presents clinical problems

Mon. April 26

Preliminary Reading

• digoxigenin

Read For Highlights Only

• Shimell, Ferguson, Childs, and O'Connor.(1991) The Drosophila dorsal- ventral patterning gene tolloid is related to the human bone morphogenetic protein 1. Cell 67: 469-481.

Wed. April 28

Preliminary Reading
Bone formation

• pg 1017 - 1018 Bone also contains.... stop @ the structure of bone may be compact....
• Figures 47.16, 18.2

Papers

• Wade Roush. (1996) Protein builds second skeleton. Science 273: 1343.
  
• Shafritz, Adam B., Eilleen M. Shore, Francis H. Gannon, Jichael A. Zasloff, Rebecca Taub, Maximillian Muenke, and Frederick S. Kaplan. (1996) Overexpression of an Osteogenic Morphogen in Fibrodysplasia Ossificans Progressiva. New England Journal of Medicince335 (8): 555 - 561.
• Eileen M Shore, Meiqi Xu, George J Feldman, David A Fenstermacher, The FOP International Research Consortium, Matthew A Brown, & Frederick S Kaplan. (2006) A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva. Nature Genetics. 38: 525 - 527.

Final Web Page Due Friday April 30

no class meeting

Wed. May 5

Final Exam Distributed and Fellowship Slide Show

Course Evaluations

(return to the beginning)

Final Exam Due May 10, 9am

Graduation, May 16