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Kingsborough Community College of The City University of New York
Department of Biological Sciences

Biology 65: Molecular and Cellular Biology

Bulletin Description of the Course

Principles and problems of the structure and functions of cell components are covered. Emphasis will be placed on the molecular composition of cells and the molecular mechanisms a cell uses to grow and divide. Experiments and computer exercises are designed around fundamental questions in eukaryotic cell biology with a strong emphasis on contemporary, sophisticated biochemical and molecular biological techniques.

Syllabus (week-by-week)

Week 1 Lecture: Introduction to the Cell
a. Cells and genomes
-universal features of cells
-the diversity of genomes
-genetic information in eukaryotes (orthologs, paralogs and homologs)
b. Biochemistry of the cell
-covalent and noncovalent interactions bring molecules together
-macromolecules of the cell and their contribution to the cell
c. Proteins
-Importance in anabolic and catabolic reactions of the cell
-Other functions of proteins

Week 1 Lab: The Cell
1. Microscopy
-phase contrast
2. Methods in Sterile Technique
-cell spreading
-sterile inoculation onto solid medium and into liquid medium
3. Measurements and Quantitation
-Appropriate use of micropipettes and pipettes
-Basic laboratory math: How to make a buffer from stock solutions
4. The laboratory notebook

Week 2 Lecture: DNA
a. DNA and Chromosomes
-topography of a chromosome in prokaryotes and eukaryotes
-gene arrangement in prokaryotes and eukaryotes
-heterochromatin organization versus euchromatin organization
-covalent modification of histone tails
b. DNA Replication
-a comparison of prokaryotic and eukaryotic DNA polymerases
-a comparison of prokaryotic and eukaryotic Origins of Replication
-DNA replication and nucleosomes in eukaryotes
-the necessity of telomerases
c. DNA Repair and Recombination
-DNA repair models
-general and site specific recombination
-the contribution of DNA recombination to molecular biology

Week 2 Lab: DNA I
a. Plasmid Transformation of E. coli in preparation for isolation next week.
b. Computer Exercise: Information Literacy- an introduction to E-journals, PubMed, Genbank
and other related links

Week 3 Lecture: From DNA to Protein I
a. Transcription
-principal types of RNAs produced in cells
-mRNA, rRNA, tRNA, snRNA, snoRNA, other noncoding RNAs
-a comparison of eukaryotic and prokaryotic RNA promoters and polymerases
-initiation, elongation and termination of transcription
b. Regulation of Gene Expression
- DNA binding motifs in gene regulatory proteins
-how genetic switches work
-regulation of the prokaryotic operon
-Regulatory DNA sequences in eukaryotes
-activators, repressors, enhancesomes and combinatorial control
c. Comparison of mRNAs between prokaryotes and eukaryotes
-processing and export of eukaryotic mRNA

Week 3 Lab: DNA II
a. Isolation of plasmid DNA from transformed E. coli
b. Quantitation of recovered DNA: A260/A280
c. Computer Exercise: Translation of DNA Sequence I

Week 4 Lecture: From DNA to Protein II
a. The Players
-rRNA, tRNA, proteins
-tRNA synthetases
-initiation and elongation factors
b. The Mechanics of Translation in Prokaryotes and Eukaryotes
c. Molecular chaperones
-hsp 60 and hsp 70
d. The ubiquitin conjugating system and the proteasome
e. Regulated destruction of proteins via ubiquitin ligase or activation of a degradation signal

Week 4 Lab: DNA III
c. Restriction Enzyme digestion of plasmid DNA
a. Gel Electrophoresis
b. Determining size of DNA fragments
d. Computer Exercise: Translation of DNA Sequences II: Where’s the Intron?

Week 5 Lecture: The Compartmentalization of Cells
a. Intracellular Compartments
b. Transport of Proteins between the Nucleus and Cytoplasm
-The nuclear pore
-Nuclear Localization Receptors (NLSs) and nuclear import receptors
-Directional transport of proteins through Ran-GTPases
c. Transport of Proteins into Mitochondria
-protein translocators: the TOM and TIM complexes
-signal sequences and signal peptidases
-hsp 70
-the OXA Complex
d. Transport of proteins into the Endoplasmic Reticulum
-SRPs and SRP receptors; start transfer and stop transfer signal sequences
-Single-pass versus multi-pass proteins
-ER retention signals
-glycosylphosphatidylinositol (GPI) anchor
-phospholipid assembly
-scramblases and flippases

Week 5 Lab: DNA IV
a. Chromosomal DNA isolation from yeast
b. Restriction Enzyme Digestion of chromosomal DNA
c. Electrophoresis of chromosomal DNA
c. Computer Exercise: RFLPs: a case study of Huntington’s chorea

Week 6 Lecture: The Cytoskeleton
a. The Dynamic Structure of Cytoskeletal Filaments
-nucleation, elongation and equilibrium phases
-directionality of filaments
-plus and minus ends
-treadmilling and dynamic instability
b. Molecular Motors
-the myosin family
-kinesins and dyneins
c. Regulation of Cytoskeletal Components
-g microtubule-organizing center
-the ARP complex and actin filament growth
-profilin and thymosin

Week 6 Lab: Protein I
a. Introduction to Green Fluorescent Proteins (GFPs)
b. Protein extract of yeast transformed with a GFP
c. Determination of protein concentration
d. Computer Exercise: Designing primers for PCR

Week 7: The Cell Cycle I
a. Overview of the Cell Cycle
b. Molecular Components of the Cell Cycle
-cyclin-dependent kinases
-cyclin/cdk complexes
c. Regulation of the Cell Cycle
-Protein kinases and phosphatases that modify Cdks
-Wee 1
-Cdc25 phosphatase
-Cdk-activating kinases
-Cdk inhibitory proteins
-p21, p27, p16 in mammals
-Ubiquitin ligases and their activators
-APC and SCF
-Gene regulatory proteins

Week 7 Lab: Proteins II
a. PCR
b. SDS PAGE of yeast protein extract
c. Staining of protein gel
d. Drying gel
e. Localizing GFP

Week 8: The Cell Cycle II
a. Overview of Mitosis and Cytokinesis
b. Regulation of Mitosis and Cytokinesis
-MAP kinases
-the contractile ring
c. Cell Growth and Apoptosis
-replicative cell senescence
-PI 3 and S6 kinases
-the apoptotic pathway
d. Cancer
-causes of cancer
-cancer-critical genes
-molecular basis of cancer behavior
-cancer treatments

Week 8 Lab:
a. Electrophoresis of PCR product
b. Another Kind of Transformation: Infection of Arabadopsis with Agrobacterium
c. Computer Exercise: Sequence comparisons: What's the difference between identical and homologous sequences? Let’s Blast!

Week 9: Membranes
a. Membrane structure
-functional importance of lipid bilayer assymmetry
b. Membrane proteins
-various ways proteins associate with the membrane
-GPI anchor

Week 9 Lab: Enzymes I
a. Protein extract of yeast
b. Determining trehalase activity in yeast
c. Computer Exercise: Blast analysis of trehalose-6-phosphate-synthases

Week 10: Intracellular Vessicular Traffic
a. Clatharin-coated vesicles
b. Monomeric GTPases
c. SNARE protein
d. Regulation of intracellular transport
-ER to Golgi
-trans Golgi to lysosomes
e. Endocytosis

Week 10 Lab: Enzymes II
a. Partially purifying trehalase through ion exchange chromatography
b. Determining the Specific Activity for trehalase
c. Computer Exercise: Three dimensional analysis of the Ras protein

Week 11: Cell Communication
a. General mechanisms of extracellular signaling through cell receptors
-phosphorylation cascades
-intracellular signaling proteins
-relay proteins
-messenger proteins
-adapter proteins
-amplifier proteins
-transducer proteins
-bifurcation proteins
-integrator proteins
-latent gene regulatory proteins
b. G-protein-linked cell-surface receptors
-Protein Kinase A (PKA)
-Ras pathway
c. Enzyme-linked receptors
-receptor tyrosine kinases
-tyrosine-kinase-associated receptors
-receptor serine/threonine kinases
-histidine-kinase-associated receptors

Week 11 Lab: Immunoflourescence
a. Determining tumor formation in Arabadopsis
b. Immunoflourescence: Looking at localization of GFP-tagged actin in yeast and DAPI
stained nuclei

Week 12: Development of Multicellular Organisms: Drosophila
a. General mechanisms of all animal development
b. HOX Genes
c. Positional Signals

Lab 12: Written Exam
Student Presentations

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