Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

A 50-Year Personal Journey: Location, Expression, and Circadian Rhythms

Michael Rosbash

Howard Hughes Medical Institute, National Center for Behavioral Genomics and Department of Biology, Brandeis University, Waltham, Massachusetts 02454 Correspondence: [email protected]

I worked almost exclusively on nucleic acids and gene expression from the age of 19 as an undergraduate until the age of 38 as an associate professor. Mentors featured prominently in my choice of paths. My friendship with influential Brandeis colleagues then persuaded me that was an important tool for studying gene expression, and I switched my experimental organism to yeast for this reason. Several years later, friendship also played a prominent role in my beginning work on circadian rhythms. As luck would have it, gene expression as well as genetics turned out to be important for circadian timekeeping. As a consequence, background and training put my laboratory in an excellent position to contribute to this aspect of the circadian problem. The moral of the story is, as in real estate, “location, location, location.”

o how and why did I start working on circa- Prize with Arthur Kornberg (Grunberg-Ma- Sdian rhythms as a 38-year-old tenured pro- nago et al. 1955). My mentor in that laboratory fessor at Brandeis in 1982? At that time, many was her postdoc Cal McLaughlin, who later senior figures in circadian biology were either played a prominent role in yeast protein synthe- trained in that discipline or in neuroscience. sis in collaboration with Jon Warner and Lee My path was very different. I graduated from Hartwell. I then returned to the United States Caltech in 1965 with a BS in Chemistry. There to attend graduate school at Massachusetts I worked on nucleic acids in the laboratories of Institute of Technology (MIT). Although my Norman Davidson and then Robert Sinsheimer. PhD from there was officially in biophysics, Reg Kelly, who became a prominent cell biolo- I worked in the laboratory of Sheldon Penman; gist and neuroscientist at UCSF,was my mentor he was an ex-physicist turned cell physiologist in the Sinsheimer laboratory. I then went for a with an intense interest in the messenger RNA year to Paris, where I recuperated from 4 years at (mRNA) of higher cells. The Watson and Crick Caltech and studied protein synthesis in the epiphany in the mid-1950s had energized a laboratory of Marianne Grunberg-Monago. community of people like Sheldon and his Marianne had been a postdoc of Severo Ochoa former mentor Jim Darnell to expand from at NYU, where she did the lion’s share of the gene expression studies of viruses and bacteria work for which Ochoa shared the 1959 Nobel to RNA and protein synthesis of eukaryotes.

Editors: Paolo Sassone-Corsi, Michael W. Young, and Akhilesh B. Reddy Additional Perspectives on Circadian Rhythms available at www.cshperspectives.org Copyright # 2017 Cold Spring Harbor Laboratory Press; all rights reserved Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032516

1 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

M. Rosbash

I focused at MIT on membrane-bound protein I arrived at Brandeis in the fall of 1974 as a synthesis in tissue culture cells and viruses, how newly minted assistant professor. I was 30 years ribosomes engage in the synthesis of secreted old, and 9 years had passed since I graduated proteins and attach to the endoplasmic reticu- from Caltech. This was a standard trajectory in lum (Rosbash and Penman 1971a,b; Rosbash those days, when graduate work and postdocs 1972). MIT was chock-full of interesting stu- were much shorter than they are today and dents and faculty, including Sheldon of course. almost no one strayed from the straight and He was also a first-rate advisor with an amazing narrow: a direct path from an undergraduate cohort of students and postdocs, including Bob degree to graduate school, postdoctoral train- Weinberg and Rob Singer. ing, and then on to a faculty position. (My I then did a 3-year postdoc at the University 1-year detour in Paris was highly unusual.) of Edinburgh in the laboratory of John Bishop, The Western world was in full postwar expan- who was a young faculty member in the Depart- sion, and no one worried about employment. ment of . This was at the time a Weall assumed everything would work out, and modern spinoff from the Edinburgh Genetics it seemed like it did—for almost everyone. I Department, which was known for traditional received a birthday card from a close friend on quantitative genetics and was headed by Profes- my 30th birthday in Edinburgh. It said, “The sor Douglas Falconer. Epigenetics was a term world is your oyster.” Indeed it was. coined by the famous developmental biologist In this context (“the good old days”), it is C.H. Waddington. Once it became clear that notable that many prominent new professor gene amplification and deletion was not the instructors (PIs) had no publications during major mechanism by which different cells ex- their postdocs, or their papers were published press different , in time or in space, “epi- considerably after they took their first faculty genetics” or differential gene expression must be jobs and often without the names of their post- the responsible mechanism. The term has obvi- doc mentors. This was true for my Brandeis ously acquired a different meaning today. Wad, friend and collaborator Jeff Hall, and it was as he was known, had moved north from Cam- also the case for his good pal Doug Kankel, bridge, England to head this new department. who had a long career at Yale (Hall and Kankel My mentor Bishop was not only interested in 1976; Kankel and Hall 1976). Both Hall and eukaryotic gene expression like Penman but also Kankel were postdocs with Seymour Benzer in the development and application of nucleic at Caltech, and the work in these 1976 papers acid–centric methods. I learned a great deal was substantially performed in the Benzer from John, who worked at that time with his laboratory. It was also the case for other prom- own hands at the bench and was more of a bio- inent trainees from this era. For example, Mike chemist than Penman (Bishop and Rosbash 1973, Young left the Hogness laboratory at Stanford 1974; Bishop et al. 1974; Rosbash et al. 1974). In and joined the faculty at Rockefeller without addition to John and his other trainees (e.g., Nick a first author publication that also carried Hog- Hastie and Saveria Campo), Edinburgh was filled ness’ name. Although not true of everyone, the with interesting people and science, many of 1970s were clearly a different era from this pub- whom had a positive impact on my subsequent lication-CV point of view. career. For example, Ed Southern of Southern My plan at Brandeis was to continue the blot fame was an MRC (Medical Research work I had been doing during my postdoc, to Council) young laboratory head in an adjacent continue the study of gene expression changes building in Edinburgh. Ed was an inspiration during oogenesis and early development in both personally and professionally. Two other Xenopus laevis (frog) oocytes and early embry- prominent people from that era are os. I had begun this work in Edinburgh in and , who were both graduate collaboration with Peter Ford (Rosbash and students next door in ’s laboratory. Ford 1974; Ford et al. 1977). Peter was a postdoc Michael has become a lifelong close friend. in the neighboring laboratory of Max Birnstiel.

2 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032516 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

A 50-Year Personal Journey in Circadian Rhythms

(My postdoctoral advisor John Bishop was lowered the energy barrier to my adopting this remarkably generous in letting me pursue technology. whatever scientific problems interested me, in The second factor was my seduction by the whatever collaborative arrangement I chose to “the awesome power of genetics.” Contempo- make.) I started working on this problem as rary practitioners now quite rightly view any soon as I arrived at Brandeis in October of study of gene expression as indistinguishable 1974 and had even written and submitted an from genetics. (Genetics ¼ the study of genes ¼ NIH R01 grant on this topic before leaving the the study of genomes ¼ the study of gene United Kingdom. Its funding began coincident expression.) However, this was not the case with my arrival at Brandeis, which is another 40 years ago before the advent of recombinant indication of how much more straightforward DNA technology. Genetics was then more the practical aspects of scientific life were 40 narrowly defined, for example, the study of years ago. My first students and postdocs chromosomes or meiosis, and it also referred worked on this topic, and the enterprise was to a strategy (i.e., using and making mutants quite successful. Nonetheless, my own restless- to study a particular problem). I had been ness and two external factors encouraged me to studying gene expression at MIT and then in expand my horizons. (The restlessness could be Edinburgh, but I had no expertise or any serious more positively described as a function of my interest in using genetics as a strategy (e.g., broad interests.) broadly mutagenizing a strain with little or The first factor was the advent of recombi- no specificity, selecting for a phenotype, and nant DNA technology. It had appeared in the characterizing the mutagenized strain and genes literature during my postdoc. With great fanfare that underlie the phenotype). and not inconsiderable controversy, it pro- Many people today refer to this aspect of gressed in just a few years from a concept, and genetics as “forward genetics.” This term was then a technology for a select few, to an essential coined to distinguish it from “reverse genetics,” tool for much of the research community and in which DNA sequences of specific genes can pharmaceutical industry. Of contemporary in- be deliberately mutated. As reverse genetics terest perhaps, the current hoopla, promise, generally requires cloned and sequenced DNA and publicity surrounding genome editing are and/or specific oligonucleotides to carry out not dissimilar from how recombinant DNA the mutagenesis, it did not exist in the pre- or technology was received in the 1970s. That early recombinant DNA era. Then there was advance meant that one could study in detail only forward genetics or its original term, ge- specific individual genes and abandon the less- netics. Importantly, the organism and system I precise study of collections of genes or RNA had been using in Scotland and then in my early populations. years at Brandeis—frog oocytes and embryos— There was a local aspect to my embrace of was opaque to genetic strategies. recombinant DNA technology. Peter Wensink My religious conversion to genetics began was hired at Brandeis as an assistant professor shortly after my arrival at Brandeis in the fall and had been trained in recombinant DNA of 1974. It was because of my friendship with technology during his postdoc in the Hogness two evangelists, Jeff Hall and Lynna Hereford. laboratory at Stanford. Stanford was the mecca Jeff had begun as a newly minted Brandeis as- of this technology, where it had been invented sistant professor a few months before I arrived, and then put into practice. Peter’s arrival at and Lynna moved to my laboratory in the spring Brandeis coincided with mine, and his labora- of 1975. Jeff and Lynna had both been trained as tory was also adjacent to mine in the open PhD students in the Department of Genetics at floor plan that was and still is the Rosenstiel the University of Washington. This department Center for Basic Medical Sciences at Brandeis. was the first “genetics department” in the Peter brought the required reagents and exper- United States, with that word in its title. I imag- tise with him from Stanford, which significantly ined for some years after meeting Jeff and Lynna

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032516 3 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

M. Rosbash

that the University of Washington Genetics some form of posttranscriptional rather than Department conferred divinity as well as PhD transcriptional regulation. Second, the mutants degrees and then released these graduates to probably affected pre-mRNA splicing and po- spread the genetics gospel around the country tentially identified a set of genes and proteins to life science Philistines like me. important for this recently discovered and then To enable my learning about yeast and at largely enigmatic process. Third, ribosomal pro- the same time immerse Lynna in nucleic acid tein genes must be enriched for intron-contain- studies, we first collaborated to address general ing genes, which would explain the selectivity of issues of yeast gene expression (e.g., how many the mutants for ribosomal protein synthesis. At protein-coding genes are encoded in the yeast that time, the actin gene was the only previously genome). To these ends, Lynna and I used identified yeast gene to have an intron (Ng and nucleic acid reassociation kinetics (Hereford Abelson 1980). and Rosbash 1977a,b), methods I had helped All of these three predictions turned out develop and used to study higher eukaryotic to be correct. For reasons that are still uncertain, gene expression during my postdoc in Scotland about 2/3 of ribosomal protein genes contain (Bishop and Rosbash 1974; Bishop et al. 1974; introns, whereas only about 5% of total Rosbash et al. 1974). We also used an aspect of yeast genes contain introns (i.e., about 3% of reassociation kinetics to develop a method for nonribosomal protein genes have introns) cloning specific genes (Rosbash et al. 1979). It (Neuveglise et al. 2011); this accounts for the was used to clone genes (Hereford et al. apparent specificity of the mutants for ribosom- 1979) as well as ribosomal protein genes (Wool- al protein synthesis. Most important, the RNA ford et al. 1979). Lynna went on to study the mutants, which were subsequently renamed former, whereas I focused on the latter. PRP mutants for pre-mRNA processing mu- I had already been interested in ribosomal tants, turned out to be important entry points protein gene expression for some time when I for identifying the splicing machinery and the began working on this problem in the late detailed contributions of different splicing 1970s. There are about 80 of these proteins in proteins and complexes to this complicated eukaryotes (Ben-Shem et al. 2011). How do pathway. The RNA/PRP mutants were also my they get to be stoichiometric in the ribosome? entre´e into the splicing problem, which I con- (i.e., how is this regulation achieved?) There tinued to study for more than 20 years after that was an intriguing hint from the literature. initial paper (Rosbash et al. 1981). John Warner and his colleagues had shown The ability to subject mutant strains to a that a set of very interesting yeast temperature- temperature shift and then examine events in sensitive mutants—dubbed at the time RNA vivo as a function of time after the shift proved mutants—had a selective effect on the synthesis remarkably rewarding. Equally important, the of ribosomal proteins (Gorenstein and Warner mutant strains proved invaluable when com- 1976). bined with in vitro splicing assays. Extracts With our newly cloned ribosomal protein could be used for mechanistic studies, which genes and the then just emerging northern were difficult if not impossible in vivo. For ex- blot technology, we could address what hap- ample, extracts from strains that were deficient pened to the RNA profiles when the mutant in specific proteins and steps could be assayed in yeast strains were shifted to the nonpermissive vitro and used for reconstitution with recombi- temperature. Although the mature ribosomal nant proteins or protein complexes. Recogniz- protein mRNA rapidly disappeared as ing this possibility, John Abelson came to visit predicted, new higher molecular weight species me at Brandeis in 1980 or 1981, shortly after he appeared. They turned out to be intron- heard about our in vivo experiments. He and his containing species, leading to several tentative close colleague Christine Guthrie became lead- conclusions (Rosbash et al. 1981). First, the ers in this field and trained many of the current regulation identified by the mutants indicated senior people working in the yeast and splicing

4 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032516 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

A 50-Year Personal Journey in Circadian Rhythms

fields. My own trainees also became important contributions on pseudoobscura contributors to these fields. (Pittendrigh 1954; Pittendrigh 1966). The Ko- Shortly after establishing yeast as an impor- nopka and Benzer paper also initiated my inter- tant model for studying ribosomal protein gene est in the circadian field, as I often heard about it expression, I wanted to examine ribosomal pro- as well as stories about Ron from Jeff. However, tein gene expression regulation in higher eu- there was no professional common ground that karyotes and decided to do this in Drosophila. might have encouraged a collaboration between To this end, we cloned the first ribosomal pro- my laboratory and Jeff ’s. Several circumstances tein gene in metazoans, the Drosophila rp49 then came together at more or less the same gene (Vaslet et al. 1980; O’Connell and Rosbash time and changed the landscape. 1984); its mRNA is still a standard used in many First, Bambos Kyriacou, a postdoc in Drosophila gene expression studies all over the Jeff ’s laboratory, made an experimental bridge world. This gene was also used to show that a between Drosophila courtship and circadian minute mutation encodes a ribosomal protein rhythms. Bambos remains a terrific guy to this (Kongsuwan et al. 1985). This study was the first day, with a great sense of humor. He had dis- link between this important set of Drosophila covered that the courtship song, what the male mutants and a gene product. “sings” to the female by vibrating a wing, was Although this work on rp49 bridged to the affected by the Konopka period mutants (Ky- organism studied by my friend and colleague riacou and Hall 1980). This paper showed that Jeff Hall, he had never been seriously interested the song not only had a species-specific sound in my work on nucleic acids or gene expression, (frequency) but also a rhythm; the frequency whether in frogs, yeast, or flies. (“Seriously in- was about 40 msec, and the rhythmic oscilla- terested” means seriously thinking of working tions in frequency occurred with a period of on the problem.) Nor had I been seriously in- about 1 min. Remarkably, that period was un- terested in Drosophila courtship behavior and der control of the period mutants; for example, neuroscience, which his laboratory had been the short-period variant had a period of about studying since his arrival at Brandeis. So our 40 sec and the long-period variant a period friendship remained just that: we had mutual of about 80 sec. The key point here is not the interests in sports (including playing basketball courtship song but that circadian rhythms were for many years in a traditional noon pickup being actively investigated in Jeff ’s laboratory. game at Brandeis), music, left-wing politics, This heightened his interest in circadian biology and general scientific issues and gossip. More- beyond courtship as well as my own interest in over, our pre-Brandeis backgrounds had multi- the problem. ple overlaps. I also came to know many of Jeff ’s Second, recombinant DNA technology was pals from his time in Seymour’s laboratory. making rapid progress. It became feasible by They included Doug Kankel who went to Yale the early 1980s to imagine cloning Drosophila after Caltech as I mentioned above, Chip Quinn period by “chromosome walking,” a strategy pi- who went to Princeton, and Bill Harris. Doug oneered by the Hogness laboratory at Stanford and Chip were fellow postdocs, whereas Bill had (Bender et al. 1983a,b; Spierer et al. 1983). Gene been a Seymour graduate student and went to identification might then be performed by Harvard to do a postdoc. Jeff also got to know genetic rescue, which had just been accom- Ron Konopka at Caltech, who was finishing his plished by Rubin and Spradling (Rubin and PhD with Seymour when Jeff arrived. Jeff and Spradling 1982; Spradling and Rubin 1982). Al- Ron maintained a close relationship until Ron though my laboratory was not engaged in these died in 2015. It was of course the landmark procedures, we were deep into other recombi- Konopka and Benzer paper published in 1971 nant DNA studies as mentioned above. The gap that arguably initiated the Drosophila circadian was therefore not huge. I thus suggested to Jeff field (Konopka and Benzer 1971). I use the in the spring of 1982—after one of our basket- word “arguably” to note the earlier Pittendrigh ball games—that we consider collaborating.

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032516 5 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

M. Rosbash

The dream: cloning and sequencing might pro- for the first few years, and the cloning and vide some insight into what period actually does rescue of period was performed independently and therefore how the circadian clock actually in both places (Bargiello and Young 1984; Bar- ticks. There was, however, still an energy barrier giello et al. 1984; Reddy et al. 1984; Zehring et al. to overcome as well as new personnel required 1984); Mike and his colleagues deserve high to begin this project. marks for their accomplishments. Although Third, I was denied tenure in the Rosenstiel unpleasant, the competition contributed to a Center, where my laboratory was located in the fast-paced focus, which probably contributed 1970s and early 1980s. Although this is a much to some of our successes. longer story, the relevant part here is that my There were also some errors. In my opinion, laboratory was forced to move to the only avail- our biggest one during these early years was a able Biology Department space, which was biochemistry paper suggesting that period en- adjacent to Jeff ’s laboratory. It goes almost coded a proteoglycan (Reddy et al. 1986). The without saying that this proximity, including gene encoded an orphan protein in those early a shared conference room where we had joint days of DNA sequencing; it contained no fea- laboratory meetings for many years, catalyzed tures of interest that could connect to proteins our collaborative efforts. of known functions. Being desperate, we were Fourth, I had a serious health crisis in the seduced/hoodwinked by the only possible ex- summer of 1982. Suffice it to say that this crisis ception: glycine-threonine (GT) repeats within lowered the energy barrier to making serious the period protein (Reddy et al. 1986). They changes to my life. They included deciding to connected to proteoglycans, which often have work on the cloning of period as soon as some- similar repeats within their sequences and are one appeared who was interested. sites of O-linked glycosylation. If the fly period Fifth, a much more serious health crisis protein were a proteoglycan, it might have provided the required personnel and therefore biochemical features consistent with this post- the chance to begin the period cloning project. translational modification (e.g., a high apparent Vivian Ernst was a wonderful young Assistant molecular weight). Although I was more molec- Professor of Biochemistry at Brandeis, who ularly sophisticated than Jeff or anyone else in tragically succumbed to breast in early our groups, I was woefully deficient in biochem- September of 1982. Her memorial service was ical expertise, meaning protein biochemistry. my very first day back at work after those 6 Taken together with the competition issue re- weeks at home. I walked gingerly across campus ferred to above (the Young laboratory was also to the chapel and then slowly and very sadly working on this proteoglycan hypothesis) and returned back to my laboratory. I was greeted a lack of prudence, I was much too hasty in there by a second-year graduate student of accepting and pushing forward our modest Vivian’s, Pranitha Reddy. With tears in her biochemical experiments. I still do not know eyes, she asked if she could do her thesis in my whether those experiments were in error or laboratory. I gave her the period cloning project, just their interpretation; I suspect the results and this is how my collaborative work with Jeff were correct but a result of protein aggregation. Hall on circadian rhythms began in the early fall In any case, the proteoglycan hypothesis seemed of 1982. to have legs at the time. Importantly, there was The first decade of published research from neither strong data to the contrary nor any com- our laboratories, 1984–1994, is well document- peting hypotheses at that time. ed in the literature and even described in many That situation changed the following year secondary sources. In my view, there is therefore with the publication of the protein sequence little material to add. There are, however, a few encoded by singleminded, a gene encoding a nu- personal anecdotes that might be of interest. clear transcription factor. It had homology with We were locked in an intense battle for pri- the period protein (Crews et al. 1988). Although macy with the Young laboratory at Rockefeller the homology was quite modest and could have

6 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032516 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

A 50-Year Personal Journey in Circadian Rhythms

reflected a motif shared by a large number of compatible with proteoglycans. I pushed hard proteins with different functions, it offered an to distinguish between the two possibilities. important competing hypothesis to proteogly- We had already shown that the GT region, the cans: the period protein could be a transcription hallmark of the proteoglycan hypothesis, is factor. This possibility coincided more or less not conserved nor is that region necessary for with the first data from my postdoc Paul Har- PER circadian function (Yu et al. 1987). Paul din, indicating that the period mRNA under- Hardin showed that feedback regulation de- went circadian oscillations in fly heads (Hardin pended on per 50 flanking sequences (i.e., it et al. 1990). Paul gets full credit for making this predominantly reflected transcriptional regula- important finding, including the important tion) (Hardin et al. 1992). Perhaps because it idea to use fly heads rather than whole bodies had been assumed that this was the case (there for the source of RNA. (The lack of cycling in was not much appreciation at that time for certain body tissues reduces the cycling ampli- posttranscriptional regulation), we had serious tude in whole flies to below credibility.) Impor- trouble publishing this important paper. These tantly, the phase of RNA cycling was affected by difficulties make for an interesting backstory, the per missense mutations identically to their but I will save that for another time and place. effects on behavioral phase, suggesting that the A graduate student, Xin Liu, went to Seymour protein directly affected its own gene expression Benzer’s laboratory to do immunoelectron mi- (Hardin et al. 1990). As the clock works up- croscopy and showed that PER was a nuclear stream of behavior, this result might also reflect protein (Liu et al. 1992). Last, another excellent the influence of a clock operating upstream of graduate student Honkui Zeng then showed gene expression. Circadian behavior might even that PER overexpression led to low per RNA be upstream of the gene expression regulation, levels, consistent with negative feedback (Zeng and this possibility was included in the initial et al. 1994). model figure (Fig. 5 in Hardin et al. 1990). The transcriptional feedback loop hypothe- Taken together with the homology to the sis was therefore quite robust by 1994. Although single-minded transcription factor, however, I will not belabor the subsequent more than 20 the Hardin work suggested a more parsimoni- years of work from all over the world and in ous hypothesis: the per protein was engaged in a many systems, their findings generally support negative feedback loop that affects its own this transcriptional feedback loop view of circa- expression and that this loop was “the clock” dian rhythms in eukaryotes. There are, however, or at least central to timekeeping. The negative a few landmark accomplishments that deserve feedback loop would therefore also be upstream further mention. of the behavioral rhythms that are usually mea- There was the identification of the timeless sured by circadian biologists. gene by the Young laboratory in a forward ge- Notably, Paul’s success was helped by his netic screen (Sehgal et al. 1994, 1995; Gekakis local environment, the fact that a good fraction et al. 1995; Myers et al. 1995; Price et al. 1995). of his colleagues in my laboratory studied yeast timeless was the second important clock gene, RNA and were facile with state-of-the-art RNA the first identified since the Konopka and methods. As polymerase chain reaction (PCR) Benzer paper. There was also the identification did not exist at this time, Paul had to use RNase and cloning of the mammalian Clock by Taka- protection to assay-specific per mRNA levels. hashi and colleagues, also in a forward genetic This method is not terribly difficult but also screen (Vitaterna et al. 1994; Antoch et al. 1997; not trivial, so the Brandeis circadian work ben- King et al. 1997), as well as their identification efited from being adjacent to—in effect embed- of its partner BMAL1 in collaboration with the ded within—a nucleic acid–centric research Weitz laboratory (Gekakis et al. 1998). Ravi program. Allada and Joan Rutila spearheaded an effort in This new working model of gene expression my laboratory to identify a positive transcrip- feedback was very attractive. It also seemed in- tion factor in the circadian system (Allada et al.

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032516 7 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

M. Rosbash

1998; Rutila et al. 1998). The screen was success- teins (Kai A, B, and C) and ATP (Nakajima ful and identified loss-of-function mutants in et al. 2005). It therefore can function without Drosophila Clock (protein ¼ CLK ¼ ortholog transcription, and this simple in vitro clock is of mammalian Clock) and Cycle (protein ¼ even temperature-compensated (i.e., the circadi- CYC ¼ ortholog of BMAL1). The approach an period is essentially temperature-invariant). incorporated an interesting subscreen; the mu- This work was elegant and compelling beyond tants were assayed for their effects on period words (in a word, “breathtaking”). transcription. We recovered many arrhythmic Notably, there is still no bona fide parallel to mutants, but only three showed little or no the Kai system in the eukaryotic world, even period transcription; two turned out to be alleles after more than 15 years of having these protein of CYC and the other an allele of CLK. sequences and now substantial mechanistic Patrick Emery and Ralf Stanewsky collabo- information on the cyanobacterial clock. It rated to clone fly cryptochrome (CRY) to show therefore remains likely that the cyanobacterial its importance for light regulation of the circa- and eukaryotic systems arose independently dian clock and to identify and characterize a (i.e., circadian clocks appeared multiple times mutant loss-of-function strain of flies (Emery in evolution (Rosbash 2009). Moreover, bona et al. 1998, 2001a,b; Stanewsky et al. 1998). fide and well-characterized circadian clocks Ironically, it is still not known how CRYactually have not been reported outside of cyanobacteria works and how it collaborates with the eyes to (Ma et al. 2016). This is despite the fact that the perceive “circadian” light. three different Kai genes are found distributed Circadian regulation is not only transcrip- among archaeal and protobacterial species as tional (i.e., there are many important studies well as all cyanobacterial species (Dvornyk showing the importance of posttranscriptional et al. 2003) and the fact that the cyanobacterial regulation for circadian timekeeping). In my clock can be transplanted to Escherichia coli view, the original and key set of publications (Chen et al. 2015). Some cyanobacterial species came from the Young laboratory, which identi- even solve the sensitivity of nitrogen fixation fied the Drosophila doubletime gene. It encodes to oxygen problem with spatial rather than the kinase CKI, which is a crucial regulator of temporal segregation (Ernst et al. 1992). An fly circadian period (Kloss et al. 1998; Price et al. implication of this prokaryotic digression is 1998), and its ortholog plays an equally impor- that eukaryotic transcription–translation clocks tant role in mammals (Lowrey et al. 2000). may have had broader phylogenetic impact than Notably, PER appears to be a very important the cyanobacterial clock. circadian substrate (Ko et al. 2002), which can A more recent challenge to the PER-CLK be interpreted to reinforce the centrality of the transcription-centric model is the pro- transcriptional feedback loop. posed role of metabolism and specifically per- Although this story focuses on animal oxiredoxin hyperoxidation in circadian rhythms. clocks and especially on flies, a robust and bio- A set of crucial experiments shows that these logically relevant transcription–translation cir- rhythms are independent of transcriptional cadian clock functions in plants (Nohales and rhythms in human red blood cells as well as in Kay 2016). In addition, there is a quite different fly and mouse systems (O’Neill and Reddy 2011; circadian clock in a prokaryotic photosynthetic O’Neill et al. 2011; Edgar et al. 2012). Impor- organism, namely cyanobacteria. This clock in tantly, key aspects of the red blood cell experi- its simplest form requires three genes: kaiA, ments were independently replicated (Cho et al. kaiB, and kaiC (Ishiura et al. 1998). It is also of 2014). As mammalian red blood cells lack adaptive significance (i.e., the clock contributes nuclei and therefore transcription, aspects of to fitness) (Ouyang et al. 1998). A landmark this very important hypothesis are likely to be biochemical study from Kondo and colleagues correct. However, very specialized conditions showed that the circadian clock could be recon- were necessary to repeat the in vitro peroxire- structed in vitro with three recombinant pro- doxin cycling, explaining perhaps why there was

8 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032516 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

A 50-Year Personal Journey in Circadian Rhythms

no peroxiredoxin cycling observed in red blood tissue that is not well explored from this point of cells taken directly from in this study view. In the fly, only a small number of neurons, (Cho et al. 2014). Importantly, no laboratory about 75 pairs of cells bilaterally located in the has independently replicated the experiments central brain, contain a functional clock by im- showing the cycling of hyperoxidized peroxire- munohistochemical criteria (i.e., staining for doxin in flies and mice bearing arrhythmic clock proteins) (Tataroglu and Emery 2014). mutations in key clock proteins (Edgar et al. Most fly neurons may therefore be “clock- 2012). These stunning results suggested that less,” of which a specific example is the dopami- these rhythms truly run independently from nergic system. Most of these neurons seem to the core clock transcriptional machinery. Al- be missing clock gene expression (Abruzzi et al. though animal metabolic rhythms are expected, 2017); mammalian dopaminergic neurons also their complete independence from the core may not express clock gene transcripts (S Nelson, clock machinery and the downstream behavio- pers. comm.). Single-cell sequencing should ral rhythms is surprising and therefore very im- eventually address the general question about portant if true, hence, the desire for replication. brain neurons and circadian clocks in mammals For example, sleep–wake as well as food-con- as well as flies. sumption rhythms are under clock control in Looking toward the future, eukaryotic sys- ad lib–fed animals, and one imagines that met- tems in my opinion are still in search of abolic rhythms might predominantly follow satisfactory and general explanations for tem- food ingestion. A key experiment to distinguish perature compensation, notwithstanding the between the possibilities has not been per- publication of some elegant studies that have formed in my view; metabolic and peroxire- addressed this problem (Mehra et al. 2009). As doxin rhythms should be assessed in fast and mentioned above, this mechanistic term refers slow running circadian mutants in constant to the fact that circadian period is essentially darkness. Are the peroxiredoxin rhythms truly temperature-invariant. I might add in this con- independent or are they influenced by the core text that we also do not know the rate-limiting clock transcriptional machinery? step or steps that govern circadian timekeeping. Another fly in the ointment is/was the male In other words, what determines the pace of courtship song rhythm (Kyriacou and Hall the clock? Is there only one rate-limiting step 1980). How can transcriptional feedback ac- or are there more than one, each governing a commodate a rhythm with a 1 min period? discrete portion of the cycle. Mutants are an Perhaps the courtship song rhythm really does unreliable guide to defining the pacesetters reflect another function of PER, which does or rate-limiting steps in normal (wild-type) not involve transcription. Although this view animals (Rosbash 2009). I suspect that these of the courtship song rhythm is not without two problems, rate-limiting steps and tempera- controversy (Stern 2014), the story is ongoing ture compensation, are related; every rate-lim- (Kyriacou et al. 2017). iting step should be temperature-compensated. There are other big-picture questions that This prediction assumes that there is no com- still remain unanswered. What fraction of the pensation at some gross level, for example, that genome is really controlled by the clock? As the transcription and accumulation of PER and the current estimate is at least 50% (Zhang TIM accelerate, whereas their subsequent pro- et al. 2014), might it be 100% (i.e., might every tein turnover slows down at elevated tempera- gene be controlled by the circadian clock some- tures. Surprisingly, this has never been tested to where)? my knowledge. Work in the mammalian system indicates The fly has also become an attractive model that many or most, perhaps even all, cells and for sleep studies. This is a result of pioneering tissue contain a canonical circadian clock (Bal- studies by two groups more than 15 years ago salobre et al. 1998; Zhang et al. 2014). Yet there (Hendricks et al. 2000; Shaw et al. 2000). The may be important exceptions, and the brain is a purpose of sleep as well as its regulation is one of

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032516 9 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

M. Rosbash

the great mysteries of neuroscience, indeed of Ben-Shem A, Garreau de Loubresse N, Melnikov S, Jenner L, biology writ large. Classical studies in mammals Yusupova G, Yusupov M. 2011. The structure of the eukaryotic ribosome at 3.0 A˚ resolution. Science 334: indicate that sleep is regulated by the circadian 1524–1529. system as well as by a much more mysterious Bender W,Akam M, Karch F,Beachy PA, Peifer M, Spierer P, homeostatic regulatory system (Borbely 1982; Lewis EB, Hogness DS. 1983a. Molecular genetics of Daan et al. 1984; Borbely et al. 2016). The for- the bithorax complex in . Science 221: 23–29. mer reflects the internal timekeeper that gener- Bender W, Spierer P, Hogness DS. 1983b. Chromosomal ates our regular daily changes in sleepiness and walking and jumping to isolate DNA from the Ace alertness, whereas the latter keeps track of sleep and rosy loci and the bithorax complex in Drosophila need and responds to external events like sleep melanogaster. J Mol Biol 168: 17–33. Bishop JO, Rosbash M. 1973. Reiteration frequency of duck deprivation. We have recently shown that spe- haemoglobin genes. Nat New Biol 241: 204–207. cific circadian neurons in the fly brain promote Bishop JO, Rosbash M. 1974. Polynucleotide sequences in sleep by inhibiting other wake-promoting cir- eukaryotic DNA and RNA that form ribonuclease- cadian neurons, and that this interplay between resistant complexes with polyuridylic acid. J Mol Biol circadian neurons is a major feature of the 85: 75–86. Bishop JO, Morton JG, Rosbash M, Richardson M. 1974. circadian regulation of sleep (Guo et al. 2016). Three abundance classes in HeLa cell messenger RNA. Although these studies do not impact on ho- Nature 250: 199–204. meostatic regulation, the fly is also beginning to Borbely AA. 1982. A two process model of sleep regulation. illuminate the principles that underlie this mys- Hum Neurobiol 1: 195–204. terious process (e.g., Donlea et al. 2014). As we Borbely AA, Daan S, Wirz-Justice A, Deboer T. 2016. The two-process model of sleep regulation: A reappraisal. J originally learned from developmental biologi- Sleep Res 25: 131–143. cal studies and then from circadian biology, the Chen AH, Lubkowicz D, YeongV,Chang RL, Silver PA. 2015. fly may once again provide insights that will Transplantability of a circadian clock to a noncircadian illuminate the mammalian landscape, in this organism. Sci Adv 1: e1500358. case for the function and regulation of sleep. Cho CS, Yoon HJ, Kim JY, Woo HA, Rhee SG. 2014. of hyperoxidized peroxiredoxin II is And to finish more or less where I began, it determined by hemoglobin autoxidation and the 20S will not surprise me if gene expression regula- proteasome in red blood cells. Proc Natl Acad Sci 111: tion is a prominent feature of sleep regulation. 12043–12048. Crews ST, Thomas JB, Goodman CS. 1988. The Drosophila single-minded gene encodes a nuclear protein with se- quence similarity to the per gene product. Cell 52: 143– REFERENCES 152. Daan S, Beersma DG, Borbely AA. 1984. Timing of human Abruzzi KC, Zadina A, Luo W,Wiyanto E, Rahman R, Guo F, sleep: Recovery process gated by a circadian pacemaker. Shafer O, Rosbash M. 2017. RNA-seq analysis of Dro- Am J Physiol 246: R161–R183. sophila clock and non-clock neurons reveals neuron- specific cycling and novel candidate neuropeptides. Donlea JM, Pimentel D, Miesenbock G. 2014. Neuronal PLoS Genet 13: e1006613. machinery of sleep homeostasis in Drosophila. Neuron 81: 860–872. Allada R, White NE, So WV, Hall JC, Rosbash M. 1998. A mutant Drosophila homolog of mammalian Clock dis- Dvornyk V, Vinogradova O, Nevo E. 2003. Origin and evo- rupts circadian rhythms and transcription of period and lution of circadian clock genes in prokaryotes. Proc Natl timeless. Cell 93: 791–804. Acad Sci 100: 2495–2500. Antoch MP, Song EJ, Chang AM, Vitaterna MH, Zhao Y, Edgar RS, Green EW,Zhao Y,van Ooijen G, Olmedo M, Qin Wilsbacher LD, Sangoram AM, King DP, Pinto LH, X, Xu Y, Pan M, Valekunja UK, Feeney KA, et al. 2012. Takahashi JS. 1997. Functional identification of the Peroxiredoxins are conserved markers of circadian mouse circadian clock gene by transgenic BAC rescue. rhythms. Nature 485: 459–464. Cell 89: 655–667. Emery P,So WV,Kaneko M, Hall JC, Rosbash M. 1998. CRY, Balsalobre A, Damiola F,Schibler U. 1998. Immortalized rat a Drosophila clock and light-regulated cryptochrome, is a fibroblasts contain a circadian clock. Cell 93: 929–937. major contributor to circadian rhythm resetting and Bargiello TA, Young MW. 1984. Molecular genetics of a photosensitivity. Cell 95: 669–679. biological clock in Drosophila. Proc Natl Acad Sci 81: Emery P,Stanewsky R, Hall JC, Rosbash M. 2000a. A unique 2142–2146. circadian-rhythm photoreceptor. Nature 404: 456–457. Bargiello TA, Jackson FR, Young MW. 1984. Restoration of Emery P, Stanewsky R, Helfrich-Fo¨rster C, Emery-Le M, circadian behavioural rhythms by gene transfer in Dro- Hall JC, Rosbash M. 2000b. Drosophila CRY is a deep sophila. Nature 312: 752–754. brain circadian photoreceptor. Neuron 26: 493–504.

10 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032516 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

A 50-Year Personal Journey in Circadian Rhythms

Ernst A, Black T, Cai Y, Panoff JM, Tiwari DN, Wolk CP. Ko HW, Jiang J, Edery I. 2002. Role for Slimb in the degra- 1992. Synthesis of nitrogenase in mutants of the cyano- dation of Drosophila Period protein phosphorylated by bacterium Anabaena sp. strain PCC 7120 affected in het- Doubletime. Nature 420: 673–678. erocyst development or metabolism. J Bacteriol 174: Kongsuwan K, Yu Q, Vincent A, Frisardi MC, Rosbash M, 6025–6032. Lengyel JA, Merriam J. 1985. A Drosophila Minute gene Ford PJ, Mattieson T, Rosbash M. 1977. Very long-lived encodes a ribosomal protein. Nature 317: 555–558. messenger RNA in ovaries of Xenopus laevis. Dev Biol Konopka RJ, Benzer S. 1971. Clock mutants of Drosophila 57: 417–426. melanogaster. Proc Natl Acad Sci 68: 2112–2116. Gekakis N, Saez L, Delahaye-Brown A-M, Myers MP,Sehgal Kyriacou CP,Hall JC. 1980. Circadian rhythm mutations in A, Young MW, Weitz CJ. 1995. Isolation of timeless by Drosophila melanogaster affect short-term fluctuations in PER protein interactions: Defective interaction between the male’s courtship song. Proc Natl Acad Sci 77: 6729– L timeless protein and long-period mutant PER . Science 6733. 270: 811–815. Kyriacou CP,Green EW,Piffer A, Dowse HB. 2017. Failure to Gekakis N, Staknis D, Nguyen HB, Davis CF,Wilsbacher LD, reproduce period-dependent song cycles in Drosophila is King DP,Takahashi JS, Weitz CJ. 1998. Role of the CLOCK due to poor automated pulse-detection and low-intensi- protein in the mammalian circadian mechanism. Science ty courtship. Proc Natl Acad Sci 114: 1970–1975. 280: 1564–1569. Liu X, Zwiebel LJ, Hinton D, Benzer S, Hall JC, Rosbash M. Gorenstein C, WarnerJR. 1976. Coordinate regulation of the 1992. The period gene encodes a predominantly nuclear synthesis of eukaryotic ribosomal proteins. Proc Natl protein in adult Drosophila. J Neurosci 12: 2735–2744. Acad Sci 73: 1547. Lowrey PL, Shimomura K, Antoch MP, Yamazaki S, Zeme- Grunberg-Manago M, Oritz PJ, Ochoa S. 1955. Enzymatic nides PD, Ralph MR, Menaker M, Takahashi JS. 2000. synthesis of nucleic acidlike polynucleotides. Science 122: Positional syntenic cloning and functional characteriza- 907–910. tion of the mammalian circadian mutation tau. Science Guo F, Yu J, Jung HJ, Abruzzi KC, Luo W, Griffith LC, Ros- 288: 483–492. bash M. 2016. Circadian neuron feedback controls the Ma P,Mori T,Zhao C, Thiel T,Johnson CH. 2016. Evolution Drosophila sleep–activity profile. Nature 536: 292–297. of KaiC-dependent timekeepers: A proto-circadian tim- Hall JC, Kankel DR. 1976. Genetics of acetylcholinesterase ing mechanism confers adaptive fitness in the purple in Drosophila melanogaster. Genetics 83: 517–535. bacterium Rhodopseudomonas palustris. PLoS Genet 12: Hardin PE, Hall JC, Rosbash M. 1990. Feedback of the Dro- e1005922. sophila period gene product on circadian cycling of its Mehra A, Shi M, Baker CL, Colot HV, Loros JJ, Dunlap JC. messenger RNA levels. Nature 343: 536–540. 2009. A role for casein kinase 2 in the mechanism under- Hardin PE, Hall JC, Rosbash M. 1992. Circadian oscillations lying circadian temperature compensation. Cell 137: in period gene mRNA levels are transcriptionally regulat- 749–760. ed. Proc Natl Acad Sci 89: 11711–11715. Myers MP,Wager-Smith K, Wesley CS, YoungMW,Sehgal A. Hendricks JC, Finn SM, Panckeri KA, Chavkin J, Williams 1995. Positional cloning and sequence analysis of the JA, Sehgal A, Pack AI. 2000. Rest in Drosophila is a sleep- Drosophila clock gene timeless. Science 270: 805–808. like state. Neuron 25: 129–138. Nakajima M, Imai K, Ito H, Nishiwaki T, Murayama Y, Hereford LM, Rosbash M. 1977a. Number and distribution Iwasaki H, Oyama T, Kondo T. 2005. Reconstitution of of polyadenylated RNA sequences in yeast. Cell 10: 453– circadian oscillation of cyanobacterial KaiC phosphory- 462. lation in vitro. Science 308: 414–415. Hereford LM, Rosbash M. 1977b. Regulation of a set of Neuveglise C, Marck C, Gaillardin C. 2011. The intronome abundant mRNA sequences. Cell 10: 463–467. of budding yeasts. C R Biol 334: 662–670. Hereford L, Fahrner K, Woolford JLJ, Rosbash M, Kaback Ng R, Abelson J. 1980. Isolation and sequence of the gene for DB. 1979. Isolation of yeast histone genes H2A and H2B. actin in Saccharomyces cerevisiae. Proc Natl Acad Sci 77: Cell 18: 1261–1271. 3912–3916. Ishiura M, Kutsuna S, Aoki S, Iwasaki H, Andersson CR, Nohales MA, Kay SA. 2016. Molecular mechanisms at the Tanabe A, Golden SS, Johnson CH, Kondo T. 1998. Ex- core of the plant circadian oscillator. Nat Struct Mol Biol pression of a gene cluster kaiABC as a circadian feedback 23: 1061–1069. process in cyanobacteria. Science 281: 1519–1523. O’Connell P, Rosbash M. 1984. Sequence, structure, and Kankel DR, Hall JC. 1976. Fate mapping of nervous system codon preference of the Drosophila ribosomal protein and other internal tissues in genetic mosaics of Dro- 49 gene. Nucl Acids Res 12: 5495–5513. sophila melanogaster. Dev Biol 48: 1–24. O’Neill JS, Reddy AB. 2011. Circadian clocks in human red King DP,Zhao Y,Sangoram AM, Wilsbacher LD, Tanaka M, blood cells. Nature 469: 498–503. Antoch MP,Steeves TDL, Vitaterna MH, Kornhauser JM, O’Neill JS, van Ooijen G, Dixon LE, Troein C, Corellou F, Lowrey PL, et al. 1997. Positional cloning of the mouse Bouget FY,Reddy AB, Millar AJ. 2011. Circadian rhythms circadian clock gene. Cell 89: 641–653. persist without transcription in a eukaryote. Nature 469: Kloss B, Price JL, Saez L, Blau J, Rothenfluh A, Wesley CS, 554–558. Young MW.1998. The Drosophila clock gene double-time Ouyang Y, Andersson CR, Kondo T, Golden SS, Johnson encodes a protein closely related to human casein kinase CH. 1998. Resonating circadian clocks enhance fitness I1. Cell 94: 97–107. in cyanobacteria. Proc Natl Acad Sci 95: 8660–8664.

Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032516 11 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

M. Rosbash

Pittendrigh CS. 1954. On temperature independence in the circadian rhythmicity and transcription of Drosophila clock-system controlling emergence time in Drosophila. period and timeless. Cell 93: 805–814. Proc Natl Acad Sci 40: 1018–1029. Sehgal A, Price J, Man B, Young M. 1994. Loss of circadian Pittendrigh CS. 1966. The circadian oscillation in Drosoph- behavioral rhythms and per RNA oscillations in the ila subobscura pupae: A model for the photoperiodic Drosophila mutant timeless. Science 263: 1603–1606. clock. Z Pflanzenphysiol 54: 275–307. Sehgal A, Rothenfluh-Hilfiker A, Hunter-Ensor M, Chen Y, Price JL, Dembinska ME, Young MW, Rosbash M. 1995. Myers M, Young MW. 1995. Rhythmic expression of Suppression of PERIOD protein abundance and circadi- timeless: A basis for promoting circadian cycles in period an cycling by the Drosophila clock mutation timeless. gene autoregulation. Science 270: 808–810. EMBO J 14: 4044–4049. Shaw PJ, Cirelli C, Greenspan RJ, TononiG. 2000. Correlates Price JL, Blau J, Rothenfluh A, Abodeely M, Kloss B, Young of sleep and waking in Drosophila melanogaster. Science MW. 1998. double-time is a novel Drosophila clock gene 287: 1834–1837. that regulates PERIOD protein accumulation. Cell 94: Spierer P,Spierer A, Bender W,Hogness DS. 1983. Molecular 83–95. mapping of genetic and chromomeric units in Drosophila Reddy P, Zehring WA, Wheeler DA, Pirrotta V, Hadfield C, melanogaster. J Mol Biol 168: 35–50. Hall JC, Rosbash M. 1984. Molecular analysis of the pe- Spradling AC, Rubin GM. 1982. Transposition of cloned P riod locus in Drosophila melanogaster and identification elements into Drosophila germ line chromosomes. Sci- of a transcript involved in biological rhythms. Cell 38: ence 218: 341–347. 701–710. Stanewsky R, Kaneko M, Emery P, Beretta M, Wager-Smith Reddy P, Jacquier AC, Abovich N, Petersen G, Rosbash M. K, Kay SA, Rosbash M, Hall JC. 1998. The cryb mutation 1986. The period clock locus of D. melanogaster codes for identifies cryptochrome as a circadian photoreceptor in a proteoglycan. Cell 46: 53–61. Drosophila. Cell 95: 681–692. Rosbash M. 1972. Formation of membrane-bound poly- Stern DL. 2014. Reported Drosophila courtship song ribosomes. J Mol Biol 65: 413–422. rhythms are artifacts of data analysis. BMC Biol 12: 38. Rosbash M. 2009. The implications of multiple circadian Tataroglu O, Emery P. 2014. Studying circadian rhythms in clock origins. PLoS Biol 7: e62. Drosophila melanogaster. Methods 68: 140–150. Rosbash M, Ford PJ. 1974. Polyadenylic acid-containing Vaslet CA, O’Connell P, Izquierdo M, Rosbash M. 1980. RNA in Xenopus laevis oocytes. J Mol Biol 85: 87–101. Isolation and mapping of a cloned ribosomal protein Rosbash M, Penman S. 1971a. Membrane-associated pro- gene of Drosophila melanogaster. Nature 285: 674–676. tein synthesis of mammalian cells. I: The two classes of Vitaterna MH, King DP, Chang AM, Kornhauser JM, Low- membrane-associated ribosomes. J Mol Biol 59: 227– rey PL, McDonald JD, Dove WF, Pinto LH, Turek FW, 241. Takahashi JS. 1994. Mutagenesis and mapping of a Rosbash M, Penman S. 1971b. Membrane-associated pro- mouse gene, Clock, essential for circadian behavior. Sci- tein synthesis of mammalian cells. II: Isopycnic separa- ence 264: 719–725. tion of membrane-bound polyribosomes. J Mol Biol 59: Woolford JL Jr, Hereford LM, Rosbash M. 1979. Isolation of 243–253. cloned DNA sequences containing ribosomal protein Rosbash M, Ford PJ, Bishop JO. 1974. Analysis of the C-val- genes from Saccharomyces cerevisiae. Cell 18: 1247–1259. ue paradox by molecular hybridization. Proc Natl Acad Yu Q, Colot HV, Kyriacou CP, Hall JC, Rosbash M. 1987. Sci 71: 3746–3750. Behaviour modification by in vitro mutagenesis of a var- Rosbash M, Blank D, Fahrner K, Hereford L, Riccardi R, iable region within the period gene of Drosophila. Nature Roberts B, Ruby S, Woolford JL Jr. 1979. R-looping and 326: 765–769. structural gene identification of recombinant DNA. Zehring WA, Wheeler DA, Reddy P, Konopka RJ, Kyriacou Methods Enzymol 68: 454–469. CP,Rosbash M, Hall JC. 1984. P-element transformation Rosbash M, Harris PKW,Woolford JL Jr, TeemJL. 1981. The with period locus DNA restores rhythmicity to mutant, effect of temperature-sensitive RNA mutants on the tran- arrhythmic Drosophila melanogaster. Cell 39: 369–376. scription products from cloned ribosomal protein genes Zeng H, Hardin PE, Rosbash M. 1994. Constitutive over- of yeast. Cell 24: 679–686. expression of the Drosophila period protein inhibits peri- Rubin GM, Spradling AC. 1982. Genetic transformation of od mRNA cycling. EMBO J 13: 3590–3598. Drosophila with transposable element vectors. Science Zhang R, Lahens NF, Ballance HI, Hughes ME, Hogenesch 218: 348–353. JB. 2014. A circadian gene expression atlas in mammals: Rutila JE, Suri V, Le M, So WV, Rosbash M, Hall JC. 1998. Implications for biology and medicine. Proc Natl Acad Sci CYCLE is a second bHLH-PAS clock protein essential for 111: 16219–16224.

12 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032516 Downloaded from http://cshperspectives.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press

A 50-Year Personal Journey: Location, Gene Expression, and Circadian Rhythms

Michael Rosbash

Cold Spring Harb Perspect Biol published online June 9, 2017

Subject Collection Circadian Rhythms

Circadian Posttranscriptional Regulatory Coordination between Differentially Regulated Mechanisms in Mammals Circadian Clocks Generates Rhythmic Behavior Carla B. Green Deniz Top and Michael W. Young Design Principles of Phosphorylation-Dependent Introduction to Chronobiology Timekeeping in Eukaryotic Circadian Clocks Sandra J. Kuhlman, L. Michon Craig and Jeanne F. Koji L. Ode and Hiroki R. Ueda Duffy Interplay between Microbes and the Circadian Cellular Timekeeping: It's Redox o'Clock Clock Nikolay B. Milev, Sue-Goo Rhee and Akhilesh B. Paola Tognini, Mari Murakami and Paolo Reddy Sassone-Corsi A 50-Year Personal Journey: Location, Gene Molecular Mechanisms of Sleep Homeostasis in Expression, and Circadian Rhythms and Mammals Michael Rosbash Ravi Allada, Chiara Cirelli and Amita Sehgal Regulating the Suprachiasmatic Nucleus (SCN) Membrane Currents, Gene Expression, and Circadian Clockwork: Interplay between Circadian Clocks Cell-Autonomous and Circuit-Level Mechanisms Charles N. Allen, Michael N. Nitabach and Erik D. Herzog, Tracey Hermanstyne, Nicola J. Christopher S. Colwell Smyllie, et al. Systems Chronobiology: Global Analysis of Gene The Plant Circadian Clock: From a Simple Regulation in a 24-Hour Periodic World Timekeeper to a Complex Developmental Manager Jérôme Mermet, Jake Yeung and Felix Naef Sabrina E. Sanchez and Steve A. Kay

For additional articles in this collection, see http://cshperspectives.cshlp.org/cgi/collection/

Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved