Reading Schedule - Davidon College Student Access ONLY

Non-journal readings come from Life 6 ^th Ed., by Purves et al.

1) Learning Molecular Tools

Week #1

Week #2

Week #3

Week #4

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. New Methods that are begining to have 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 Life 6 ^th Ed., by Purves et al.

Week #1 (return to the begining)

Virtual Reprints for Molecular Biology - Spring 2003
(Preliminary Readings come from Life, by Purves et al., 6^th edition)

(return to the begining)

Wed. Feb 5

• Test due Wed. Feb. 5 class time followed by WWW Workshop
  How to make your own web page and post on server; location Watson 247

WWW workshop

1. Web Standards for this course
2. How to create web pages
3. A template for your use
4. How to load your pages onto the BioServer
5. How to create relative links
6. How to make Greek letters in html
7. How to find a Chime File
8. How to embed a Chime File in your web page

Fri. Feb 7and Mon. Feb. 10

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

Preliminary Reading

a) Homology

• pg 435-437 Current... stop@ Summary What's the difference between homology and similarity?
• pg 441-445 Molecular Clocks... stop@ How do.. Can you measure the evolution of function?
• Figure 24.5 - Does shared sequence mean shared function?

b) Inhibitors

• pg. 105-108 Where are... stop@ Metabolism.... - What does an inhibitor do and how does it do it?

c) Localization

• figures 47.5, 47.7, 47.9, 47.10

d) 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) Chime images of SERCA1a prior to and after the phosphate binding. (work in progress)

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.

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Mon. Feb. 17

Papers

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

Wed. Feb. 19

Web Page Due at Class Time
optional review session in class

Fri. Feb. 21

• Chime 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.

Student Web Page on GFP
Commercial web site for GFP

Preliminary Reading
What is FRAP?

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.

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 begining)

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

Wed. Feb. 26 - Fri. Feb. 28 (no class 28th) + Mon. March 10

Preliminary Reading

• pg 64-67 The Endomembrane System- where do proteins go inside a cell?

Papers

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

Saturday March 1 - (spring break) - Sunday March 9

Mon. March 10 - Fri. March 14

Preliminary Reading

• FACS method

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.

Mon. March 17 - Wed. March 19

• 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

---

Exam due March 24 at class time

• Exam due and Chime Workshop

 

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Section IV (Amazing Methods that Push the Envelope)

Wed. March 26

1) New Cloning Strategies

Preliminary Reading

• pg 264-265, 249 - Transposons

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 28

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

Mon. March 31

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. April 2

3) Faster Knockouts

Preliminary Reading

• pg 322 Antisense
• 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.

Fri. April 4

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 begining)

Section V (Sex determination in Mammals)

Mon. April 7 - Wed. April 9

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.

Wed. April 9

• 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.

Fri. April 11

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.

RasMol Image 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.
  
• Valerie J. Grant. (1996) Sex determination and the maternal dominance hypothesis. Human Reproduction. 11 (11): 2371-2375.

(return to the begining)

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 14 - Wed. April 16

Preliminary Reading
a) development of axis

• 300-301 The role of Polarity...
• 305-309 The role of Differential.... to end of chapter
• Drosophila gastrulation QT movie (13 MB)

Fly Parts
All the Parts of a Fly

b) heat shock
pg 42 Chaperonins and 690 Plants have....

c) CAT assays

Papers

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

Fri. April 18 - Wed. April 23 (and next reading)

• 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)

Monday April 21 Easter Break!
Wed. April 23

Preliminary Reading
Antennapedia and body parts
The Head of a Fly - parts labeled
A Leg of a Fly - parts labeled
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.
  
• RasMol image of Antp
• Site-Directed Mutagenesis (Kunkel Method)
• Ultrabithorax phenotype

Fri. April 25

Read For Highlights Only

Preliminary Reading
digoxigenin

• 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.

Preliminary Reading
Bone formation

• pg 843 - 845 Vertebrate... stop @ Bones that have....
• Figures 19.2, 19.5
• pg 354 White blood cells...

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. Overexpression of an Osteogenic Morphogen in Fibrodysplasia Ossificans Progressiva. New England Journal of Medicince 335 (8): 555 - 561.

Course Evaluation

Wed. April 30

Preliminary Reading
Figures 45.14, 45.15, 45.16, 45.17, 45.19

Papers

• Marcia Barinaga (1995) Focusing on the eyeless gene. Science 267: 1766-1767.
  
• Halder, Callaerts, and Gehring. Induction of ectopic eyes by targeted gene expression of the eyeless gene in Drosophila . Science 267: 1788-1792.
  
• unknown author (1997) On the path of the primordial eye. Science 275: 1885.
  
• Steven Dickman. (1997) HOX gene links limb, genital defects. Science 275: 1568.
  • Not assigned but interesting follow up news item about the molecular reason snakes have no legs (Science 283:475. 1999)

Fri. May 2
Final Exam Distributed and Slide Show

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Lab Schedule In Context of Research Project (not for 2003)