Fall Semester 2002
Amino acid conservation
and replacement in Cytochrome c
R. E. Dickerson Sci. Am. 226(4):59 (1972)
This syllabus contains lots of information. Please read it carefully and refer to it frequently during the semester.
Table of Contents
About the Instructor
Meeting Time and Place
|Important General Information
Brief Course Description
Groups and Group Function
Case Study Problems
Case Study Option
Some Possible Topics
Prof. Harold B. White
Office: 123 Brown Lab
Office Hours: Normally, the hour after class on Wednesdays and Fridays will be available for office hours; however, you should feel free to contact me by phone or e-mail or to stop by my office at other times. If I do not have pressing business, I will be happy to meet on the spur of the moment.
Meeting Time and Place: 9:05 - 9:55 A.M., MWF in 109 Memorial Hall.
Prerequisite: CHEM-527 Introduction to Biochemistry or CHEM-642 Biochemistry
Text: Fundamentals of Molecular Evolution, Second Edition by Graur and Li (1999), Sinauer. This text provides an excellent background in population genetic principles as they apply to molecular evolution. It also provides an excellent introduction to the analyses of DNA and protein sequences as they apply to evaluating the evolutionary relationships among organisms. However, the authors are not biochemists and thus there is relatively little on the evolution of protein structure and function, the evolution of metabolism, or the origin of life. Consequently, it will be useful to have a good general biochemistry text such as that by Garrett and Grisham; Voet and Voet; Lehninger; Zubay; or Stryer around when you work on the Case Study Problems. A selection of biochemistry texts will be available for loaning.
Grading: Because CHEM-647 is a graduate-level course with a small enrollment, personal initiative in the form of outside reading and class participation is expected. A fundamental general background in biochemistry at the level of CHEM-527 or CHEM-642 is assumed. Classes will be structured around class and group discussion of problems, interactive lectures. There will be no formal examinations per se. Evaluation of each student's performance will be based on seven homework assignments (50%), two group assignments (15%), a case study problem (15%), and a follow up final interview based on your case study (10%). "Appa" (attendance, preparation, participation, and attitude) constitute the remaining 10%.
While your grade will be based on your performance, there is no grading curve in this course. If everyone does "A" work, everyone will get an "A." It is in your best interest to help your classmates; however, do it as a teacher. If you know something, don't just give the information. Explain it. Practice effective communication. If you don't know something, seek understanding rather than "the answer." Develop the skill to recognize and define what you don't know and learn not to be satisfied with superficial answers.
Important General Information
Course Description: Biochemical Evolution is a vast and rapidly growing subject. The recent literature displays a heavily emphasis on comparative analysis of both the primary and tertiary structures of proteins and the nucleotide sequences of genes and genomes. With the completion of the Human Genome Project and the rapid accumulation of other genomes, it is impossible to analyze all of the information available and it is easy to expend lots of time on trivial questions. Thus, it is absolutely essential that students learn to recognize and formulate interesting questions and become aware of the power and limitations of the methods. For that reason, the first two and a half months of this course (see schedule) will provide case study problems as examples of recently resolved issues and current controversy. Classes from late-November into December will be devoted to special topics lectures and a final group project that explore interesting problems in biochemical evolution.
Course Objectives: Education is subversive. It changes people. Thus students should be different as the result of taking this course. As a result of completing CHEM-647, Biochemical Evolution, I expect students will:
1. Acquire a better sense of the unity of life on earth and the relationships among major groups of organisms including humans.Teaching Philosophy: People learn best and almost effortlessly when they want to know something. Why else is it that many students (and some faculty) can recite for hours the details of television shows, the personal lives of celebrities, or baseball statistics without expecting to be examined on the information? Biochemical Evolution will never have a comparable general appeal; however, learning about it will come easier when there is a need to know that provides a focus for your learning. This is the essence of the problem-based approach to learning. Rather than lecture, I will conduct interactive discussions, ask questions, and have you work in groups during class time on case study problems. Hopefully you will find these problems interesting and they will stimulate you to ask questions - learning issues - in your pursuit of knowledge about biochemical evolution.
2. Be able to read and understand the significance of most articles published in the Journal of Molecular Evolution and Molecular Biology and Evolution.
3. Understand and be able to communicate Darwin's theory of evolution by natural selection.
4. Appreciate the relevance of biochemical evolution to understanding and treating infectious diseases.
5. Be able to construct phylogenetic hypotheses and test them by analyzing sequence information using standard tree-building software.
6. Identify and articulate personal knowledge gaps and efficiently locate the information needed to fill them.
7. Scientifically critique "intelligent design" and "irreducible complexity."
8. Be able to describe in their own words and discuss using examples concepts and ideas such as:
9. Recognize and be familiar with the contributions to biochemical evolution by some of the following scientists:
- the molecular clock hypothesis
- homology, orthology, and paralogy
- molecular divergence and convergence
- neutral and selected mutations
- alleles in populations
- endosymbiotic theory
- the "RNA World"
- radioisotopic dating
- "Selfish Gene" hypothesis
- gene duplication and the origin of new functions
- allopatric and sympatric speciation
- classification of major groups of organism
- maximum parsimony and neighbor joining methods
Benner, Steven A.
Doolittle, Russell F.
Szostak, Jack W.
Biochemical Evolution is not about memorization. This course is about understanding, thinking, pursuing knowledge, identifying resources, and communicating. It is about making evolution understandable at the molecular level, hopefully interesting, and possibly exciting enough that you will want to continue learning about it for the rest of your life. (Two former students have gone on to do graduate work with evolutionists on the list above.)
I enjoy teaching this course because I decide the content and can select topics that I find (and hopefully you will find) interesting. Biochemical Evolution is not a prerequisite for any other course. It is not structured around a particular text and it lacks a defined body of material "to cover." This allows me to include an eclectic mix of topics that range from natural history to geochronology along with evolution and biochemistry. This is a course that in broad view can provide a unifying perspective on life and the world around us.
Groups and Group Function:
Each student will be assigned to a group of about 4 students. These groups will function independently during class and outside of class for the whole semester. The collective resources and efforts of the group will be used to deal with the case-study assignments. For example, several learning issues may be identified in group discussion during class and group members will be assigned or volunteer to investigate particular issues and report back to the group. The goal is to have everyone learn more than they would have working alone. Nevertheless, individual work (often 8 - 12 hours/week) provide the foundation for productive and synergistic group work.
In order to promote effective group function, each group will discuss and agree upon a list of ground rules at the beginning of the semester. All students will evaluate themselves and their fellow group members with respect to contributions to group function at least twice during the semester. This evaluation will contribute to the "appa" (attendance, participation, preparation, and attitude) portion of the course grade and will be used primarily in deciding borderline grades.
Case Study Problems (50%):
There will be six case study problems during the course. Each deals with an important issue or controversy in biochemical evolution. The purpose of these exercises is to promote understanding of biochemical evolution by thinking about and analyzing real problems. In general, case studies reports will be due every week or two until Thanksgiving break.
Because my objective is your understanding, I encourage you to use the library and discuss these case study problems with other students in and outside of class. Consider diverse resources including faculty here and elsewhere (via e-mail) after you have spent some time analyzing the problems on your own. While free exchange of information is expected both within and outside of groups, case study reports must be written individually as an opportunity for you to demonstrate to me and to yourself that you understand the concepts and issues involved.
Collaboration with other students does not include copying or paraphrasing of their answers. Plagiarism will not be tolerated. If you are uncertain about what this means, review the University's policy on academic dishonesty. Write up your own answer in your own words. I look for well-thought-out answers that are clearly and neatly presented. I expect acknowledgment of the resources you use (books, articles, web sites, and people). Also, assignments for this course cannot be submitted for credit in another course.
Case Study (15%):
Your case-study topic must be submitted to me on the "Request for Case Study Topic Form" by Friday, October 11. While you should consider topics among those listed below, other topics are acceptable with the my approval.
Perhaps the best way to construct a case study problem is to learn as much as you can about your topic, take notes, then organize your knowledge in the form of a concept map (Due Friday, November 1). Decide what is most important and interesting to you and for others to know. Your concept map should help define major concepts and subdivisions of your topic. Then think about ways you could get students to discover, experience, learn and remember that information without being told. Creative approaches may include historical anecdotes, illustrative objects, or in class activities as part of a classroom presentation.
Your case study should have an informative title and present an original synthesis of information in three or more stages. Creating an original synthesis presents formidable challenges for most students when writing term papers but is easier with a case study. What is an original synthesis? Firstly, it must be a scholarly document that reveals some depth of inquiry. For example, it should have firm grounding in the primary literature rather than relying heavily on secondary or questionable sources. Consider using key data or information from classic articles. Original syntheses often play with ideas, provide an overview of the subject, critique and evaluate research results, and generally display personal input. Such case studies show a clear understanding of the topic and define the important concepts in the field. In other words, the voice of the author is evident throughout.
Your case study problem should be well-organized and clearly written. Remember, CHEM-647 is a biochemistry course. Relevant compounds, pathways, and biochemical processes should be central. Biology can provide interest and relevance but molecules cannot be avoided. References should be cited in the format of Biochemistry or the Journal of Biological Chemistry. Late papers will preclude an "A" in the course!
yourself with the meaning of plagiarism and the University's policies on academic
dishonesty. A case study should be your synthesis rather
than a paraphrased version of others' work. Don't rely heavily on one or
two secondary sources. Read the original sources. You may use the Writing
Center or have a friend critique your case study; however, writing and revision
are your responsibilities.
Your case study should be at most six pages long, including figures, with an appendix in the form of a thorough set of "teaching notes." This is where you provide an overview of the relevant literature and show or explain what you expect students to do and get out of your case study problem. This section should be on the order of six single-spaced pages and include citations to 20 or more good sources. As with a term paper, this should be a scholarly document. Case studies can take many forms, but good ones have intrinsic general interest, tell a story, and often involve a current controversy or dilemma that requires a decision based on incomplete information. Pedagogically, they should involve higher order thinking skills (analysis, evaluation, and judgement), stimulate group discussion, and require collaborative effort.
Each student must schedule a one-hour meeting with me between December 4 and 13. At that meeting, your graded case study problem will be returned and we will discuss issues related to your topic. You will be expected to be able to discuss your topic in an evolutionary context and be able to relate it to topics disdcussed in the course.
Each person has distinctive knowledge, experiences, learning styles, and communication skills. The person who knows the most may not be the person who explains things best. Success in life often depends on the ability to work together and tap the different strengths of coworkers. In order to contribute to the learning of your classmates and to learn from them, you should attend class regularly and be on time, arrive prepared, participate in discussions, and generally have a constructive attitude. To encourage these traits, 10% of your grade will depend on them.
Some Possible Case Study Topics:
Your case study and class the interview based on it constitute 25% of your grade and can be considered as a type of final examination. Because you will become an expert on your topic by the end of the semester and because a considerable part of your grade will be related to how well you develop your topic, pick something that interests you. Some subjects you might consider are listed below; however, please feel free to request other topics. A good place to start is to search Medline via PubMed, a search engine provided by NIH. The links to article abstracts in the list below were obtain easily in one afternoon using Medline.
a) Where do enzymes with new activities come from? There is abundant evidence that many enzymes that catalyze a particular type of reaction are homologous, i.e., one was derived from the other or from a common ancestor enzyme. Taxonomic characters such as substrate specificity, inhibitors, mechanism, stereospecificity, molecular weight, subunit structure, crystal structure, amino acid sequence, phylogenetic distribution, etc., can be brought to bear on this problem. Much of this literature is not written in an evolutionary context, so pertinent data must be ferreted out and organized into a coherent picture. Papers in the European Journal of Biochemistry 214, 549-561 (1993) and 215, 687-696 (1993) are good examples of original syntheses for PLP-dependent enzymes and biotin-dependent enzymes, respectively. The evolution of P-450 enzymes is discussed in FEBS Letters 332, 1-8 (1993).
b) Are nuclear genes encoding mitochondrial and chloroplast enzymes really derived from the genome an ancient endosymbiont? Mitochondria and chloroplasts are considered to be derived from endosymbiotic bacteria. Such a relationship could explain the many cases of enzyme duplication. For instance, the citric acid (TCA) cycle operates inside the mitochondria; however, most of the TCA enzymes occur as mitochondrial and cytoplasmic isoenzyme pairs. Are there any features of the mitochondrial or chloroplast enzymes which make them more similar to their bacterial counterparts than to their cytoplasmic neighbors? Some possible points of departure can be found in Cell 47, 73-80 (1986), Biochem. J. 270, 651-657 (1990), and Eur J Biochem 228(3):551-61 (1995).
c) Evolution of the genetic code. Using information on the x-ray structures of amino acid-activating enzymes and the base sequences of various tRNAs, can one deduce a phylogeny of tRNAs and tRNA synthetases? Would this indicate the order in which different amino acids were incorporated into the code? Or is the question of order meaningless for what may be a "frozen accident" in which a sloppy, complex coding system got better and then became fixed? See Trends in Biochemical Sciences 16, 1-3 (1991); 17, 159-164 (1992); Proc. Natl. Acad. Sci. 88, 8121-8125 (1991); Cell 81, 983-986 (1995); and J. Mol. Evol. 40, 545-50 (1995).
d) Why are there so few monomeric enzymes? A large number of enzymes in intermediary metabolism are composed of identical subunits. Such a general phenomenon would seem to imply an evolutionary significance to this sort of molecular organization. How general is this phenomenon, and is there any evolutionary explanation? Use Comp. Biochem. Physiol. 54B, 1-6 (1976) as a point of departure and relate it to the structures of the many enzymes now known.
e) Selective advantage of isozymes and allozymes. Why are isozymes and allozymes so common in multicellular organisms? Their existence can be rationalized in part by tissue-specific regulation of differentiation and by heterosis, respectively. Do these arguments apply to microorganisms (prokaryotes and unicellular eukaryotes)?
f) Evolution of aerobic metabolism - How to deal with a poisonous gas. Aerobic metabolism as exemplified by the TCA cycle coupled to the electron transport system in mitochondria is a relative latecomer as metabolic processes go. Propose a rational sequence of evolutionary events that could have transformed an anaerobic process or system into an aerobic system. The proposal should be documented by an analysis of existing systems in living anaerobic and aerobic organisms.
g) Polymorphism, neutrality, and the rates of molecular evolution. One might expect that a rapidly evolving protein (1 amino acid substitution/100 residues/106 yr.) would show a relatively high amount of polymorphism in natural populations if nonselective evolution is important. Can such a generalization be made based on information available? See Proc. Natl. Acad. Sci. USA 90, 7475-7479 (1993) as a place to start.
h) Evolutionary assembly of metabolic pathways. It has been argued that metabolic pathways evolved in reverse order, i.e., the last enzyme in a biosynthetic sequence was the first to evolve [Proc. Natl. Acad. Sci. 31, 153 (1945)]. The coevolution of insect-plant relationships might predict that the biosynthesis of secondary plant substances (e.g., alkaloids), insect pheromones, etc., have evolved in the order they occur in a pathway, i.e., the last enzyme was the last to evolve. Analyze and provide evidence for or against these proposals.
I) Molecular opportunism and the rate of protein evolution. Is there any evidence that a protein assuming a new function evolves very rapidly? See J. Biol. Chem. 264, 11387-11393 (1989) and J. Mol. Evol. 28, 528-535 (1989) for lysozyme, and Mol. Biol. Evol. 10, 873-891 (1993) for cytochrome b.
j) Convergent and parallel evolution of enzymes. It is often assumed that enzymes catalyzing the same reaction are homologous having diverged from an ancestral enzyme with the same activity. Yet, there are some indications that certain activities arose independently several times, e.g., alcohol dehydrogenase. Is this true for alcohol dehydrogenase? Are there other activities that display such a convergent pattern? Does comparison of the tertiary structure of proteins unambiguously distinguish convergent from divergent evolution? See J. Mol. Biol. 151, 179-197 (1981) and Proc. Natl. Acad. Sci. USA 92, 6793 (1995)
k) Origin and evolution of introns. The phenomenon of split genes is fairly general among eukaryotes. It is not uncommon to have the coding region represent <10% of the total length of a gene and have 50 or more intervening noncoding regions. It has been proposed that such an organization is of evolutionary significance. How does the evolutionary argument compare with other suggestions for the function of intervening sequences? Did the last common ancestor of life on earth have introns? See Science 257, 1489-1490 (1992); J. Theor. Biol. 151, 405-416 (1992) and Science 250, 1377-1382 (1990).
l) Origin and evolution of new and complex biochemical functions. Michael Behe, a biochemist from Lehigh University, recently published Darwin's Black Box — The Biochemical Challenge to Evolution (for and against). In it he claims that biochemical systems such as blood clotting or flagella are too complex, "irreducibly complex," to have evolved and therefore must be the products of "intelligent design." Pick some taxon-specific biochemical molecule, structure, or process that interests you and examine its origins and evolution, e.g., histones, antibodies, photosynthesis, embryonic development, insulin, electron transport chain, nuclear pores, chaperonins, methane production, keratin, visual pigments, a secondary metabolite, synthesis of certain amino acids or cofactors, etc. Try to postulate and justify reasonable intermediate stages.
m) What selective forces drive codon usage patterns? Examine codon utilization patterns for genes expressed in different tissue of the same organism or in response to different stimuli. Evaluate the selective factors that might lead to differences where they are found. See Proc. Natl. Acad. Sci. 83, 8132-8136 (1986).
n) Identifying primitive molecular traits. Cairns-Smith in his book, Seven Clues to the Origin of Life, concludes that the oldest components of biological systems are the least variable. Is this a fundamental principle of biochemical evolution that can guide one in seeking the origins of any biochemical system? Can one determine the biochemical characteristics of the most recent common ancestor of all life? Proc Natl Acad Sci 86:7054-8 (1989).
o) Evolution of lipid membranes. Archaebacterial membranes contain glygerolipids possessing ether-linked isoprenoid hydrocarbons. Eukaryotes and other prokaryotes contain glycerolipids with ester-linked fatty acids. Both isoprenoids and fatty acids are biosynthesized from acetyl-CoA. Are the isoprenoid lipids a primitive or derived characteristic? Are the biosynthetic pathways related by more than their precursor? What circumstances/conditions would favor one pathway or end product over the other?
p) Applying selection for in vitro evolution. Until recently, biochemists had to be content with laborious mutational approaches to modifying proteins and nucleic acids for medicinal or other commercial use if the natural products had some undesirable properties. The recent introduction of strategies to evolve (rather than design) useful molecules permits the production of useful molecules that never before occurred in nature. The limits are one's imagination. What does the future hold? See Science 249, 505-510 (1990), Science 265, 1032-1033 (1994) and Scientific American Dec. 1992 page 90 for a start.
q) Looking backward in the future. The first complete nucleotide sequence of the genome of a free-living organism, Hæmophilus influenzae, was published in 1995, all 1.8+Mbp [Science 269, 496-512 (1995)]. Many more genome sequences are know known and many others will appear in the next few years. It has been said that an organism's evolutionary history is written in its genes. How should this information be analyzed? What are the important unresolved evolutionary questions that will soon be addressed?
r) Phylogenetic nonsense generated by protein sequences. Relaxin, a peptide hormone homologous with insulin, appears to defy expected phylogenetic patterns [FASEB J 8, 1152-1160 (1994)]. How seriously does this challenge evolutionary theory? Is this an isolated example?
s) Molecular Mimicry. Mimicry is a well known phenomenon at the organismal level. Some spectacular examples among many include chemical mimicry by flowers that produce pheromones that attract male pollinators of a single insect species or visual mimicry by insects that look like leaves. Recently, two quite different crystal structures of translation factors (proteins) reveal an overall size and shape of tRNAs. Is this really an example of molecular mimicry as the authors think? If so, can the evolutionary forces at the molecular level be analogized to those that select for mimicry on the organismal level? [Science 270, 1464 - 1471 (1995); 286, 2349 - 2352 (1999)]