The Treasure of the Humble: Lessons from Baker s Yeast*

Transcription

1 Q 2011 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 39, No. 4, pp , 2011 Article The Treasure of the Humble: Lessons from Baker s Yeast* Received for publication, October 29, 2010, and in revised form, December 19, 2010 Ramakrishnan Sitaraman From the Department of Natural Resources, TERI University, New Delhi , India The study of model organisms is a powerful and proven experimental strategy for understanding biological processes. This paper describes an attempt to utilize advances in yeast molecular biology to enhance student understanding by presenting a more comprehensive view of several interconnected molecular processes in the overall functioning of an organism, and introducing the concept of the inverse problem as it relates to biology. Keywords: Biotechnology education, PBL, biological system, inverse problem, model organism. Using tractable model organisms to better understand biological processes is a well-established strategy in research, harking back to Mendel s famous genetic experiments using peas. I have taught molecular and cell biology to students enrolled in the 2-year long (4 semesters) Master s program in Plant Biotechnology at our university that commenced in August, I started with an introductory course titled Molecular and Cell Biology Part 1 (MCB-1) in the first semester, followed by Molecular and Cell Biology Part 2 (MCB-2) in the second (Jan May, 2009). MCB-2 aims to familiarize the student with a wide variety of advanced concepts and techniques in modern molecular biology, and also emphasizes the integration of various biological processes at the level of an organism. Therefore, I chose some topics from yeast biology that could be included under the categories of DNA rearrangements, cell differentiation, gene regulation, cell signaling, and secretion. These general categories were illustrated by a fairly detailed account of various aspects of the mating types (and their switching) in yeast and the basis of the physical and physiological asymmetry underlying the process of budding (cell division). These topics were only a subset of those constituting the entire course. If all topics were to be covered using a single model organism, this course would cease to address its original objectives in terms of breadth of coverage (see discussion). To summarize, the objectives of this exercise were: 1. To give students a coherent picture of the actual and possible interactions between different processes within the cell. 2. To enable students to apply concepts and approaches learnt to model organisms of their choice. 3. To promote an integrated (systems/organism level) approach to biological problems in a very basic and intuitive manner. 4. To promote an understanding of the inverse problem in biology. Although there are several excellent textbooks available on the topics mentioned earlier, I consciously decided to use either original research articles or reviews. Information may be obtained from either source, and perhaps more easily from a textbook, but journal articles offer additional advantages, especially for postbaccalaureate programs. First, the review articles and research papers focus solely on particular facets of yeast biology, or use yeast biology to gain insight into particular biological processes. Each article is effectively a snapshot of the state of a field of study at a given time. Therefore, the students have to read the material carefully and somewhat imaginatively, to integrate the information presented in disparate sources and derive therefrom a more holistic view of the organism. Second, and more importantly, the use of journal articles highlights the problems encountered during actual research and the relative value of competing explanations for a given data set. After all, training students in the systematic application of the scientific method is the more intangible, yet vital, aspect of any postbaccalaureate science and technology program that decisively determines the quality of our scientific workforce. *This work is supported by Department of Biotechnology, Government of India. To whom correspondence should be addressed. Department of Natural Resources, 10 Institutional Area, Vasant Kunj, New Delhi , India. Tel.: Fax: This paper is available on line at The Different Facets of Yeast When confronted with unfavorable conditions for growth, diploid Saccharomyces cerevisiae is capable of forming durable spores to tide over the lean period. During sporulation, meiosis results in a halving of the chromosome number, generating four haploid spores. Upon DOI /bmb.20503

2 262 BAMBED, Vol. 39, No. 4, pp , 2011 germination, haploid yeast can be one of two mating types a or a, depending on the parental type. Homothallic strains however, can switch mating types owing to the action of HO (homothallic), a site-specific endonuclease that creates a double-stranded (ds) break at the MAT (mating-type) locus. This is followed by degradation of a- or a-encoding DNA and strand invasion by the HMLa or HMRa (Hidden MAT Left or Right) silent cassettes, respectively at the 3 0 end of the cleaved MAT locus. At the end of this process, termed synthesisdependent strand annealing, the mating type of the cell is found to have switched to the opposite type relative to its initial state. Although yeast can also survive in the haploid state, greatly facilitating genetic experiments, the diploid state is restored by the mating of a-type haploids with a-type ones, forming a zygote that multiplies by mitosis and is genotypically a/a. For teaching purposes, it is helpful to frame topics in the form of biological problems that an organism has to surmount, to be normal that is, behave the way we know and expect it to. This problem-based approach also enables the teacher to introduce the important concept of the inverse problem in biology, that is, inferring the parameters of a system from a set of observed experimental values and/or behaviors. As analysis of an inverse problem is often accompanied by the loss of data, multiple possibilities (i.e., nonunique solutions) have to be considered and reviewed in the light of available information. This typically requires design of experiments to distinguish between the alternates. Therefore, aside from promoting coherence within the course, the use of a model organism, especially one in which welldeveloped genetic and biochemical tools exist, such as yeast, can actively promote systems level thinking. At a more practical level, such exercises can also develop and hone the skills required to creatively and objectively approach a research problem. Problem 1: Preventing Daughters from Imitating Mothers [1 3] When the four haploid spores within the yeast ascus germinate, they are either mating type a or a. Each spore gives rise to a mother cell that, in turn, buds off a smaller daughter cell during the first mitotic division. Interestingly, the daughter cell is prevented from switching mating type before a second mitotic division, whereas the mother cell can do so. Thus, there is an element of differentiation in yeast that is more commonly associated with higher (multicellular) organisms a visible morphological difference based on physiology. This differentiation in terms of HO expression is brought about by the action of a specific repressor Ash1 (asymmetric synthesis of HO). However, it must be noted that this does not solve the problem of asymmetric HO expression, but merely transforms it into one of asymmetric ASH1 expression or localization. It turns out that a unique mechanism is at work here, working in exquisite synchrony with the process of budding (cell division). ASH1 mrna is uniformly distributed in both M and D when budding begins and the cells still share a nucleus and cytoplasm. However, it is bound by specific proteins (mainly the She 1 proteins) forming the ribonucleoprotein complex (RNP). Finally, the RNP associates with Myo4 (a type V myosin motor), which is capable of traveling along actin cables from the mother to the daughter cell, resulting in the localization of the RNP, and hence ASH1 mrna, exclusively in the daughter cell [1]. The localization of ASH1 mrna to the bud suggests that the daughter-specific repression of HO is just one manifestation of the general process of differentiating the mother cell from the daughter cell. A crucial role in this process is played by the kinase Cbk1 (cell-wall biosynthesis kinase), which preferentially localizes to the bud early in cell division. Cbk1 is a part of the RAM 1 signaling network that is involved in the observed developmental asymmetry during budding. Another player in this process is the transcription factor Ace2 (activator of CUP1 1 expression) that promotes expression of daughter-specific genes involved in the separation of mother and daughter cells at the end of mitosis. Interestingly, Ace2 is found in both cell types, but is localized predominantly in the nucleus of daughter cells and predominantly in the cytoplasm of mother cells as mitosis nears completion. Ace2 is expressed during the G2 phase, but is phosphorylated at the nuclear localization signal (NLS) preventing it from entering the nucleus. Toward the end of the M stage, it gets dephosphorylated and is able to enter the nucleus. However, Ace2 possesses a nuclear export signal (NES) as well that allows it to exit the nucleus. Therefore, in the mother cell, the majority of Ace2 molecules are found in the cytoplasm at equilibrium. But, in the daughter cell nucleus (in which Cbk is preferentially localized), Cbk1 phosphorylates the NES of Ace2, effectively preventing its egress and effectively leading to an accumulation of Ace2 within the nucleus. This ensures that Ace2-regulated genes are expressed in daughter nuclei alone [2]. It must also be remembered that the expression and localization of Cbk1 must necessarily be synchronized with the cell cycle, both spatially and temporally. This is achieved by its interaction with the RAM network proteins and by its phosphorylation. Cbk1 has two phosphorylation sites with regulatory functions one near the C-terminus (the CT-motif ) and another in the kinase domain activation loop ( T-loop ). Some RAM proteins initially prime Cbk1 by phosphorylating it at other sites which, in turn, allows for phosphorylation at the CT-motif and dephosphorylation of the priming sites in an Ace2- dependent manner. Cbk1 phosphorylated at the CT-motif associates with Ace2 forming a complex that enters the daughter cell nucleus. Phosphorylated Cbk1 that is not associated with Ace2 is dephosphorylated, and is a substrate for priming again. Notably, in contrast to this fluctuation in the phosphorylation of the CT-domain, the T-loop is autophosphorylated at practically constant levels in the 1 The abbreviations used are: SNAP, soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein; She, Swi5p-dependent HO expression; Swi, switching deficient; CUP1, copper (Cu) promoter; RAM, regulation of Ace2 activity and cellular morphogenesis.

3 263 presence of some RAM components [3]. This control of Cbk1 by phosphorylation via the RAM network ultimately couples the asymmetric localization process to the cell cycle. Thus, mothers and daughters develop and exhibit distinct phenotypes over the duration of the cell cycle. Problem 2: Is it a, a, or Both, and Therefore Neither? [4, 5] Although it is clear enough that genotypically a or a haploid cells should be phenotypically a or a as well, it is less clear what the phenotype of a diploid a/a zygote and its diploid descendants should be. If it were to express either phenotype, then there are chances of its actually mating with a diploid of the opposite type, leading to a tetraploid zygote. Therefore, it stands to reason that the mating type-related genes and haploid-specific genes need to be turned off in a diploid by some mechanism. In actuality, this is accomplished by the repression or nonactivation of a- and a-specific genes (asgs and asgs) as well as haploid-specific genes (hsgs) by the products of the genes at the two MAT loci. To start with, a regulatory protein, Mcm1 (minichromosome maintenance), present in both haploids and diploids, influences the expression of both a- and a-specific genes. In a-type haploids, Mcm1 binds to its cognate regulatory DNA sequences, leading to the expression of asgs. In the a- type haploid, the Mata1 protein, expressed from the MATa locus, in combination with Mcm1, acts as an activator of asgs. However, in the diploid, the a2 protein, expressed from the MATa locus, binds to DNA sequences flanking the Mcm1-binding sequences of asgs, effectively repressing them. Because of simultaneous expression of Mata1 (from the MATa locus), an a1-a2 complex is also formed that represses hsgs as well as MATa1. The absence of the Mata1 activator ensures that the asgs are not expressed. By using regulatory proteins in a combinatorial fashion to control gene expression (combinatorial gene control), yeast diploids ensure repression of haploid-specific and mating type-specific genes [4, 5]. Problem 3: Calling and Listening [6, 7] The existence of two distinct mating types of haploid yeast raises the question as to how they indicate their mating type as well as sense the presence of the opposite type. Haploid yeast secrete the a or a pheromone. The a- factor is synthesized as a large precursor, is isoprenylated, methylated, proteolytically cleaved, and finally, exported out of the cell by the ABC transporter protein Ste6 (sterility). By contrast, secretion of the a-factor involves trafficking of a precursor protein-bearing vesicles from the endoplasmic reticulum through the Golgi complex where glycosylation and proteolytic processing take place. Transit through the golgi network requires the fusion of vesicles in a target-specific, rather than random manner. How this specificity is attained may be illustrated using the yeast vacuolar fusion system as a model. Vacuoles (and other vesicles as well) display a characteristic set of surface proteins termed v-snares and t-snares (vesicular- and target-snap 1 Receptors). Vesicles dock only when they display SNAREs belonging to a cognate pair of v-snares and t-snares. The process of fusion is mediated by calmodulin, a protein that is capable of binding Ca 2þ ions and interacting thereafter with several target proteins. Ultimately, the vesicles may ultimately fuse with the plasma membrane, whereby their contents are either secreted or displayed on the cell surface [6]. The pheromones are secreted into the medium and bind to a receptor expressed on the surface of haploid cells of the opposite mating type, initiating a cascade of signaling events. These involve several MAPKs (Mitogenactivated Protein Kinases) some of which are localized on a specific scaffolding protein Ste5. The net result of this signaling, which essentially involves phosphorylation of downstream members of the cascade by upstream ones, is the transcription of several genes, the 5 0 untranslated sequences of which possess a regulatory DNA sequence motif termed the pheromone response element (PRE) [7]. When yeast cells sense pheromone, their growth is arrested, and they undergo morphological changes resulting in the formation of so-called shmoos. The shmoos (cf. gametes) mate with shmoos of the opposite mating type, resulting in diploid yeast cells. Sample Evaluation Questions The information required to attempt the following questions is available in the references cited within each of the foregoing sections, or in standard textbooks. However, as noted earlier, the goal of this exercise is to recognize connections between the various processes studied in these reference materials. In addition, as these questions aim not only to test the students understanding of the yeast module, but also their ability to apply concepts covered in other parts of the course. 1. Draw an input output (I/O) diagram depicting the control of the HO endonuclease, the asgs and asgs [4, 5, 8]. Rationale: The students were already familiar with I/O conventions used in systems biology from an earlier part of the course. We followed conventions given in Chapter 20 of Molecular Biology of Gene by Watson et al. [8]. Briefly, in an I/O diagram, genes are placed at the nodes of the diagram. Inputs and outputs are placed in rectangles. The symbol? denotes activation, and the symbol, repression. When two or more activators/ repressors are involved in a particular outcome, their effects are combined with an AND gate. 2. Given that signaling kinases can phosphorylate multiple targets across several signaling pathways, how is specificity achieved [7]? Rationale: This requires the student to understand the isolation of different signaling pathways with shared kinases by having different kinase subsets bind to pathway-specific scaffolding proteins. Therefore, the localization of kinases to a pathwayspecific scaffolding protein produces a pathway-

4 264 BAMBED, Vol. 39, No. 4, pp , 2011 specific complex, circumventing the problem of cross-talk across pathways if the kinases were freely diffusible within the cytoplasm. 3. How could one construct a yeast strain that produces light in response to pheromone? Rationale: The students had already studied bacterial quorum sensing and the luciferase reaction. This question tests not only their knowledge of genetic engineering, placing the lux genes downstream of the PRE in yeast. An additional consideration is the anticipation of practical roadblocks in actually carrying out this work. For one, luciferase, derived from the firefly (an insect) could prove toxic to the heterologous host (yeast, a fungus). A subtler problem arises during protein expression. Differences in the GC contents of genomes in different organisms lead to the preferential usage of particular synonymous codons, which is known as codon bias. This bias is correspondingly reflected in the varying relative abundances of isoaccepting trnas in different organisms. Therefore, translation of the mrna from the luciferase gene may not be very efficient in yeast unless codon usage is optimized to reflect the situation in yeast. 4. Explain how you could couple the process of vesicle fusion in yeast to a specified external chemical signal [6]. Rationale: This requires students to understand that the interaction of a ligand with a receptor results in the release of second messengers, specifically Ca 2þ ions, from cellular stores. This question is more in the nature of a thought experiment wherein they have to review the signal transduction systems they have studied to suggest appropriate ones that could be adapted for this purpose. As stated before, vesicle fusion is mediated by calmodulin, which is the calcium sensor. Therefore, the signal transduction system chosen will have to be modified, and perhaps hybrid proteins with appropriate interacting domains generated, so that the signal in question can result in the release of Ca 2þ ions. 5. It is found that in certain haploid, temperature-sensitive mutant strains of yeast, schmoo formation is inhibited when the temperature is elevated to 358C even in the presence of pheromone. Explain how this might occur, and how you would test your hypothesis. Rationale: This is an example of an inverse problem, where a phenotype has to be traced back to the dysfunction of a physiological process. A precondition for schmoo formation is cell cycle arrest. Therefore, given the signaling and response pathways, students are expected to infer that a repressor, probably involved in cell cycle arrest, is inactivated by a temperature-sensitive mutation. One standard strategy to identify the gene(s) involved would be to complement the defect with a functional copy. Therefore, a genomic library from wild-type yeast DNA may be constructed in the mutant strain using appropriate vectors. When the recombinants are transferred to the restrictive temperature, only those complemented with a wildtype copy of the defective gene will be able to form schmoos, enabling the identification of the gene involved. Analysis of Student Feedback At the end of the semester, students were distributed a questionnaire (Table I) to evaluate the utility of this module and the quality of its contents. The answers indicate that most of the material was new for the students, their understanding of molecular and cell biology was significantly enhanced by this module, and a more integrated view of at least one organism was presented. The last outcome is usually missed when using the more conventional approach of using different model organisms to illustrate various topics in molecular and cell biology (see Table I, Q.5 and 6). Although most of the students aver that this module did enhance the value of the Master s program in Plant Biotechnology, they remain unsure as to whether such an approach would be as effectively in other life sciences/biotechnology programs (Table I, Q. 9, 10). The numbers of responses in different categories for Q.11 and 12 indicate that the students have also appreciated the view of science that it has given them, regardless of their future plans and their proclivity for higher studies. Regarding Q.13, students generally felt that this module had enabled them to think in an integrated view of an organism, and also felt confident in applying the general principles learned here to other situations. Replies to Q.14 indicate that this module had sparked students enthusiasm for science and research. Most students have suggested the inclusion of more research articles (especially classic papers) and more topics in yeast biology, and having tutorial sessions for analytical problem solving. One student ventured to suggest that similar treatment of a plant model system would be beneficial, given that MCB-2 is part of a program in plant biotechnology. Finally, most students did not have any general suggestions or comments (Q. 15) except for one who suggested that some laboratory work using yeast could also be included in the lab course that is offered concurrently. In summary, the student comments indicate their overall satisfaction with this module, and their enthusiasm for studying model organisms of their choice in a research-oriented environment. DISCUSSION The inclusion of a module based on a single model organism within a course on molecular and/or cell biology gives the students an idea of the process of research specialization. In addition, it also affords an opportunity for the instructor to invite researchers with expertise in the field and integrate their lectures on advanced topics into the academic program in a formal sense. In departments with a large number of faculty members involved in research, this could be one method of increasing the involvement of postdoctoral fellows/faculty members in teaching without making potentially burdensome demands on their time. The use

5 TABLE I Questionnaire and student responses. The class size is 10 and the numbers indicate the number of students giving a particular response. The answers to questions 13, 14 and 15 are reviewed in the section on analysis of student feedback Responses Question Yes No Can t Say Background 1. Did you already have some knowledge of the material covered? Most of it (0) A little (5) None at all (5) Teaching objectives 2. Has your knowledge of the possibilities of molecular biology/cell biology 10 increased significantly as a result of this module? 3. Do you feel that this module added value to the course? Compared to the situation before participating in this module, do you feel 10 better equipped with conceptual and experimental approaches to conduct research on a model organism of your choice? 5. Were you better able to identify likely interactions between various cellular 9 1 processes as a result of this module as compared to the conventional approach? 6. Does this module give you a more integrated view of at least one model 10 organism when compared with the conventional approach? 7. Is this approach more effective in clarifying concepts/paradigms/phenomena 9 1 as compared to the conventional one? 8. In the conventional approach, there is more variety in terms of subject matter, while the yeast module offers greater coherence and continuity. Would you opt for the yeast module, if given a choice? Other considerations 9. Do you feel that this module added value to the program? If a basic course on molecular and cell biology were part of the curriculum in different programs e.g., microbiology, biotechnology, animal biotechnology etc., would students benefit from such a focused study of a model organism? 11. Is this module relevant to your career plans? If given the opportunity, would you opt for higher studies? 6 4 Scientific content 13. How did the overall content of the module help in improving the course, program and in furthering your career plans? 14. How can the content be improved so that its contribution to the course, program and your career plans is enhanced? General 15. Any other comments/suggestions 265 of review and research articles as core course material improves the students analytical ability and enhances class participation. The organization of the yeast module may be depicted in the form of a hierarchical diagram (Fig. 1). This emphasizes three levels of analysis observation, the implications in terms of traditional, descriptive biology, and the processes and the molecules involved in producing the observed effect. Incidentally, this mirrors the process of scientific discovery, proceeding from phenomenon to mechanism. Instructors may start by placing any topic at the topmost tier, and progressively include details as warranted at each tier. In practice, this would mean that the teacher pay more attention to the first two levels in the diagram that of observation and inference for classes K10 K12. One could also review the results of classic experiments conducted using a chosen organism and elaborate on its impact on the field. The composition of the lower tiers would be dictated by the contents of the specific course in question and the academic level at which it is being offered. At the undergraduate level, more detail can be supplied and more attention could be paid to the two lower tiers. For instance, students could be given an appropriate research article to read and summarize in the form of a term paper, or make a class presentation based on carefully chosen parts of a review article. In general, students in high school should be taught with a view to making them appreciate the FIG. 1.Different levels of analysis of the yeast system represented as a hierarchical structure, starting with visually observable biological phenomena at the top, and proceeding thence to inferences and actual molecular details. The reader is also reminded that there are inverse problems to be dealt with en route to the lowest tier of molecular processes.

6 266 BAMBED, Vol. 39, No. 4, pp , 2011 process of discovery, the development of ideas, and making inferences, but without invoking too many scientific concepts and details that could be beyond the scope of the curriculum. The undergraduates, on the other hand, could be asked to delve into greater scientific detail and evaluate alternate explanations for the same data, and sensitized to the importance of good experimental design incorporating appropriate controls. Also this will enhance their laboratory experience by giving it a sound conceptual basis, especially for those who do wish to get involved in research at the undergraduate level and beyond. Although the feedback is mostly positive insofar as it addresses the primary teaching objectives specified in the course outline, it must be emphasized that the model organism approach is presented here solely as a complement to the conventional approach, especially in postbaccalaureate programs, wherein a research-based project is usually a pre-requisite for graduation. Therefore, there is no case for supplanting the traditional approach. If anything, the distribution of answers to Q.8 (Table I) indicate that a significant proportion of students (4/10) did not indicate a strong preference for this approach versus the conventional one. One preferred the conventional approach, whereas the remaining five preferred the model organism-based curriculum. This broad distribution of responses supports my initial contention that, while the study of a model organism in some depth to illustrate some biological phenomena is of much utility, especially as a prelude to systems biology, its exclusive use throughout a course of this sort has the potential to detract from the breadth of knowledge that we would like our students to acquire, and could well end up prematurely encouraging too narrow a focus. Finally, while curricula, teaching methods and program objectives vary widely across academic levels and institutions, not to mention national boundaries, it good to remember that the ultimate objective of all science teaching is the awakening of the student to the wonders of the natural world, kindling their curiosity and creativity, and promoting insightful questioning. The detailed study of a model organism may be used with good effect to this end but, like all medicines, the dosage has to be carefully set and scrupulously adhered to if undesirable side effects are to be avoided. Acknowledgment I would like to thank the scientists whose work has been used in formulating this module (see list of references) and also to two anonymous reviewers for their constructive critiques. I would also like to thank the students whose active participation and honest feedback constitutes a vital component of this work: Isha Atray, Anjali Bala, Anuradha Bansal, Aastha Jain, Aditi Jain, Bhavya Khullar, Farzana Kolyariwala, Abhishek Saxena, Kshipra Sharma and Mohita Sharma. Dr. David Bedwell (University of Alabama at Birmingham) and Dr. Vytas Bankaitis (University of North Carolina, Chapel Hill) are thanked for introducing me to yeast genetics. The first half of the title is inspired by the identical title of a collection of essays by the Belgian philosopher-naturalist Maurice Maeterlinck. This paper is dedicated to my parents Mr. G. Sitaraman and Ms. Indu Bala. REFERENCES [1] M. P. Cosma (2004) Daughter-specific repression of Saccharomyces cerevisiae HO: Ash1 is the commander. EMBO Rep. 5, [2] E. Mazanka, J. Alexander, B. J. Yeh, P. Charoenpong, D. M. Lowery, M. Yaffe, E. L. Weiss (2008) PLoS Biol 6, [3] J. M. Jansen, M. F. Barry, C. K. Yoo, E. L. Weiss, E. L. (2006) Phosphoregulation of Cbk1 is critical for RAM network control of transcription and morphogenesis. J. Cell. Biol. 175, [4] R. Jensen, G. F. Sprague, I. Herskowitz, (1983) Regulation of yeast mating-type interconversion: Feedback control of HO gene expression by the mating-type locus. Proc Natl. Acad. Sci. USA, 80, [5] A. Rokas (2006) Different paths to the same end. Nature 443, [6] H. R. B. Pelham (1999) The Croonian lecture. Intracellular membrane traffic: Getting proteins sorted. Phil. Trans. R. Soc. Lond. B 354, [7] M. C. Gustin, J. Albertyn, M. Alexander, K. Davenport (1998) MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 62, [8] J. D. Watson, T. A. Baker, S. P. Bell, A. Gann, M. K. Levine, R. Losick (2008) Molecular biology of the gene, 6th ed., Pearson Education Inc., San Francisco, pp