mutant, arrhythmic Drosophila melanogaster.
4.

5.

6.

7.

of phosphorylation and degradation of hPER

Bargiello, T. A., Jackson, F. R. & Young, M. W.

protein oscillation in normal human fibroblasts.

Restoration of circadian behavioral rhythms byIn Molecular Clocks and Light Signalling (edsgene transfer in Drosophila. Nature 312, 752–

Chadwick, D. J. & Goode, J. A.) 253, 238-249

754 (1984).

(2003) Novartis Found. Symp.

Bargiello, T. A. & Young, M. W. Molecular

12. Nagase, T. et al. Prediction of the coding

genetics of a biological clock in Drosophila.

sequences of unidentified human genes. VII.

Proc. Natl. Acad. Sci. USA. 81, 2142–2146

The complete sequences of 100 new cDNA

(1984).

clones from brain which can code for largeJackson, F. R., Bargiello, T. A., Yun, S. H. &proteins in vitro. DNA. Res. 4, 141-150 (1997).

Young, M. W. Product of per locus of Drosophila

13. Tei, H. et al. Circadian oscillation of ashares homology with proteoglycans. Nature

mammalian homologue of the Drosophila

320, 185-188 (1986).

period gene. Nature 389, 512-516 (1997).

Bargiello, T. A. et al. The Drosophila clock geneperaffects

intercellularjunctionalcommunication. Nature 328, 686-691 (1987).
8.

9.

11. Miyazaki, K., Mezaki, M. & Ishida, N. The role

Cell 39, 369–376 (1984).

14. Sun, Z. S. et al. RIGUI, a putative mammalianortholog of the Drosophila period gene. Cell 90,1003-1011 (1997).

Hardin, P. E., Hall, J. C. & Rosbash, M.

15. Sakamoto, K. et al. Multitissue circadian

Feedback of the Drosophila period gene

expression of rat period homolog (rPer2) mRNA

product on circadian cycling of its messenger

is governed by the mammalian circadian clock,RNA levels. Nature 343, 536-540 (1990).

the suprachiasmatic nucleus in the brain. J.

Sehgal, A., Price, J. L., Man, B. & Young, M. W.

Biol. Chem. 273, 27039-27042 (1998).

Loss of circadian behavioral rhythms and per

16. Kondo, T. A cyanobacterial circadian clock

RNA oscillations in the Drosophila mutant

based on the Kai oscillator, In Clocks andtimeless. Science 263, 1603-1606 (1994).

Rhythms (eds Stillman, B., & Stewart, D.) 47-

10. Price, J. L. et al. double-time is a novel

55 (Cold Spring Harbor Laboratory Press,

Drosophila clock gene that regulates PERIOD

2008).

protein accumulation. Cell 94, 83-95 (1998).

A speedy version of the double-time story
Justin Blau1✉, Brian Kloss2 and Adrian Rothenfluh3

1: Department of Biology, New York University, 2: New York Structural Biology Center
3: Departments of Psychiatry, Neurobiology & Anatomy, Human Genetics, University of Utah
With the recent Nobel prize awarded to Jeff Hall,

1950s and ‘60s as a fascinating phenomenon.

Michael Rosbash and Mike Young, we were invited

However, it was not until Seymour Benzer and his

to retell the story of double-time (dbt), a key

lab bravely attempted to identify clock mutants

component of the circadian clock that was identified

that the molecular analysis of circadian rhythms

in the Young lab in the 1990s 1,2. Hopefully, it willopened up, culminating in the 2017 Nobel prize.

become clear that Nobel-prize winning science is a

Ron Konopka, a PhD student in Benzer’s lab,

mixture of bold vision, persistence and lucky breaks.

identified the first mutants that altered circadianbehavior in a forward genetic screen 3. All three

Circadian (~24hr) rhythms had been studied in themutations mapped to the same chromosomal✉justin.blau@nyu.edu
時間生物学Vol. 24, No. 1 (2018)45

location and Konopka and Benzer called theto its final location further along the second

affected gene period (abbreviated to per) since thechromosome

mutations had such dramatic effects on the period

tagging approach did not work as intended, thelength of fly rhythms. The pershort

induced mutation was sufficiently large to identify(perl)(pers) and perlong

mutants changed the fly’s normal 24hr

7.

Thus, although the P elementtim.

rhythms to 19hr and 28hr respectively, while theper0 null mutant abolished behavioral rhythms

per and tim null mutations do not affect thealtogether. These mutants were also important for

viability of flies. This facilitated the experiments

the field of behavioral genetics as a whole since theythat led to the discovery of the negative feedback

demonstrated that animal behavior could beloop that is found at the heart of all circadian clocks

profoundly affected by a single gene.

– even though clock genes are quite different
between animals, fungi and plants. In flies, per andThe period gene was cloned thirteen years later in

tim are transcribed, translated and then Per andthe Hall and Rosbash labs at Brandeis University

Tim proteins enter the nucleus, where they repress

and independently in the Young lab at Rockefeller

further transcription of per and tim 8,9. Thus per

University 4,5. Both groups used a new technology:

and tim are components of a molecular clock that

per was cloned by transforming per0 null mutantgives 24hr rhythms in the levels of per and tim RNA

flies with P element transposons containing DNAand proteins.

that restored behavioral rhythms, making per the
first animal gene identified in this way.

This was the state of our understanding of the clockin 1996, but two major questions remained

However, the sequence of per gave no clues for howunanswered:it functioned in the fly’s internal clock. It alsotranscription? And how does one cycle of such aseemed highly unlikely that per was working alone.

simple feedback loop take 24hr to complete?

So in the late 1980s, Mike Young’s lab re-embraced

Encouraged by their success in identifying tim, thethe genetic approach to rhythms and started usingYoung

P elements to screen for new clock mutants. Whenchromosome for new clock mutants. However, they

P elements insert into the genome, they often

reverted to using EMS as a mutagen since the P

disrupt gene function and the P element thenelement screen had not made it particularly easy tomarks the affected gene by its position in theidentify tim and also since EMS can generate point

genome. In theory, this makes it much easier tomutations that alter period length – like theidentify the affected gene than trying to find aoriginal pers and perl alleles. A new postdoc, Jeff

single base pair change caused by chemicalPrice, a PhD student, Adrian Rothenfluh, and two

mutagens such as EMS that Konopka and Benzer

technicians, Amy Kiger and Marla Abodeely,

had used to isolate the original per mutants.

mutagenized cn bw flies, made them homozygous

lab

What

activates

continued

toperscreen

andthetimsecondand screened them for altered circadian behavior.
The P element screen was a success: 23 years after

Jeff soon found a new mutation with a short period

the per mutants were published, Amita Sehgal andof 21hr. He named the mutant Speedy after theJeff Price, two postdocs in Mike Young’s lab,

cartoon character Speedy Gonzales, although Mike

described a null mutation in a new clock gene –later renamed it double-timeShort (dbtS). Mike wastimeless (tim) – that made flies arrhythmic just like

immediately excited by the dbtS phenotype, since a

the original per0 null mutation 6. However, in

fast-running clock is unlikely to be caused by a non-cloning the gene, Mike Myers, another Young lab

specific defect and Jeff proceeded to map thepostdoc, found that the P element that caused themutation.

arrhythmic phenotype was no longer inserted in thetim locus but had deleted 70bp of tim as it jumped

時間生物学

Vol. 24, No. 1 (2018)

To everyone’s surprise, dbtS mapped to the third46

chromosome even though the screen was designed

Notch 11. Justin focused on late stage embryos sincetothat is when per and tim are expressed inisolatehomozygous

secondchromosome

mutations. Jeff was able to isolate homozygous dbtSembryogenesis

flies, which had an even shorter period of 18hr. Jeff

sloppy collections of late embryos included a few

and a new postdoc in the lab, Brian Kloss, thenanimals that had already hatched into larvae.

homed in on the genetic locus first using deletions

Strikingly, Justin could see tim RNA and protein inof the third chromosome and then using P element-a few cells in the center of the larval brain. Mike

containing lines in the affected region. Ultimately,

immediately realized that if the molecular clock

they found one P element(dbtP),

12.

However, Justin’s somewhat

which failed to

was functional in larvae – as his earlier work withcomplement dbtS and behaved like a null allele.

Amita Sehgal and Jeff Price had suggested 13 – thenThis time, the P element proved invaluable inJustin should be able to study how the clock isidentifyinginto.

affected in dbtP homozygous larvae before they diedInterestingly, homozygous dbtP animals did notas pupae. Justin found that dbtP mutants massively

survive to adulthood, indicating that dbtP was aover-accumulate Per protein, indicating that Dbt’s

lethal mutation and that dbt had additional

normal function is to destabilize Per. Furthermore,functions outside the clock 2.

rhythms of Per and Tim proteins stopped in thethegeneithadinserted

pacemaker neurons in the central brain in dbtP
Meanwhile Adrian continued to screen for newlarvae, allowing us to conclude that dbt is essential

mutations.

for the molecular clock to run 2.

Encouragedbytheimpressive

phenotype of dbtS heterozygotes, Mike and Adrian
decided that screening single flies in an F1

The data from dbtP larvae complemented thedominant screen would be a more efficient way tocareful and painstaking time courses of per and tim

identify mutants. This made sense since all of theRNA and proteins that Adrian performed in dbtS

period-altering mutations identified so far wereand dbtL adult flies. The great advantage ofsemi-dominant, meaning their phenotype could be

studying adult flies is the abundance of per and tim

detected even as heterozygotes. And the precision

RNA and proteins in the fly eye, which permits

and robustness of the automated locomotor activity

biochemical assays. Adrian found that Per protein

assay – and of the flies’ internal clock – meant that

accumulation and degradation cycles were alteredit was easy to reproducibly detect a fly with lessin dbtS and dbtL flies – and so too were Per’s

than a 5% change in its period length. Sure enough,rhythmic phosphorylation cycles 2. Alterations toAdrian found many new mutants including a new

Per phosphorylation made even more sense when(dbtL)

Brian used the P element in dbtP to identify thelong-period dbt allele

with a heterozygous

period of 25hr and a homozygous period of 27hr 2.

affected gene and found that dbt encoded the fly
version of Casein Kinase ,H– finally, a fly clock gene

Meanwhile a new postdoc, Justin Blau, had joinedwith a known function and an obvious mammalian

the Young lab. Mike suggested that Justin study acounterpart 1. Importantly, Brian identified amino

set of genes that potentially interacted with Peracid substitutions in dbtS and dbtL mutants as well

protein, which had been identified in a yeast two-as major reductions in dbt expression levels in dbtP

hybrid screen performed in Chuck Weitz’s lab atmutants. And Lino Saez, a long term researcher inHarvard 10. However, it was unclear whether these

the Young lab, demonstrated that Dbt stably

genes were expressed in the same cells as Per inassociates with Per. Together, these data showedvivo or whether their in vitro interaction was anthat Dbt regulates several steps in the clock andartifact of forcing them together in yeast. Justin

helps extend the simple negative feedback loop to

started by asking where these genes were expressed

last 24hr 1.

in Drosophila embryos, which the Young lab had
extensive expertise with from their studies of

時間生物学

Vol. 24, No. 1 (2018)

Despite early difficulties in finding mammalian47

orthologs of the fly per gene, CK1H and its close

RNA processing and Jeff Hall’s lab studied

relative CK1G were obvious orthologs of dbt. Not

Drosophila courtship. Perhaps this diversity ofsurprisingly, Mike was very excited to learn thatinterests allowed them all to sustain their long-the tau mutation in Syrian hamsters that shortens

term excitement for circadian rhythms.

their behavioral rhythms to 20hr 14 is a missense
mutation in CK1H15. Similarly, mutations in humanFor us lab members, the Young lab was a stable andCK1G alter period length and cause familial

stimulating environment with an exciting biologicaladvance sleep phase syndrome 16. Indeed, CK1Gandquestion at its heart and with just enoughCK1H phosphorylate mammalian Per proteins andcompetition to drive us forward. Despite thisthus perform a similar function to Dbt in flies. Thus

competition, Mike always gave us the freedom tothe bold behavioral genetic approach in Drosophila

explore our own ideas and he was never forceful ininitiated by Konopka and Benzer 3, and continued

insisting on a particular research avenue – insteadby the Hall, Rosbash and Young labs, has not only

he presented his ideas in such an appealing way

shed light on an interesting biological phenomenon

that it was hard to disagree! Certainly, Mike’s

invision, instincts and persistence paved the way forflies,

but

has

also

led

understandingof

mammaliantoanin-depth

behavior

andphysiology and even human dysfunction.

his lab’s seminal contributions to the field. We neverimagined that we might be doing Nobel prizewinning work and were just excited to figure out

The late 1990s were a very exciting time to be in the

how the clock ticks in the tiny fruitfly brain. The

Young lab as pieces of the clock started appearing

same is true of Mike, who was always excited by theever more rapidly and were fitted together into alatest discoveries in the lab and is a genuinely

coherent whole. In the Young lab, we were not only

humble Nobel prizewinner. This was echoed by Rickdriven by our own excitement but also by rumors

Lifton, the President of Rockefeller University, whothat the Rosbash lab had also identified mutations

said on the day of the Nobel prize announcement,in new clock genes on the third chromosome. In the“The universal comment in my Inbox this morning

end, the Rosbash and Hall labs focused on the Clockhas been: I don’t know if the Nobel prize has everand cycle mutations that encoded the missing

been given to a nicer person.” To win the Nobel prize

transcriptional activators of per and tim

and maintain such modesty is a lesson for us all.

17,18. And

when we met members of the Rosbash lab atconferences, we realized that the “evil empire” wasReferences:in reality a friendly group of postdocs and PhD

1.

Kloss, B. et al. The Drosophila clock gene

students like ourselves who were easy to get along

double-time encodes a protein closely related towith.

human casein kinase I epsilon. Cell 94, 97-107(1998).

Although Jeff Hall, Michael Rosbash and Mike

2.

Drosophila clock gene that regulates PERIOD

Young all have very different personalities, one

protein accumulation. Cell 94, 83-95 (1998).

commonality is that they all remained at the same
university for their entire careers as Professors.

Price, J. L. et al. double-time is a novel

3.

Konopka, R. J. & Benzer, S. Clock mutants ofSuch loyalty and such unwavering institutional

Drosophila melanogaster. PNAS 68, 2112-2116

support is unusual in modern academia and meant(1971).

that the three prizewinners never had to rebuild a4.

Bargiello, T. A., Jackson, F. R. & Young, M. W.

lab from scratch and could stay focused on theirRestoration of circadian behavioural rhythms

research. Additionally, all three winners also had

by gene transfer in Drosophila. Nature 312,

other research interests: Mike Young’s lab also752-754 (1984).

studied Notch, conveniently located near per on theX chromosome; Michael Rosbash’s lab also studied

時間生物学Vol. 24, No. 1 (2018)

5.

Zehring, W. A. et al. P-element transformation
with period locus DNA restores rhythmicity to48

6.

mutant, arrhythmic Drosophila melanogaster.

Rosbash, M. Embryonic expression of theCell 39, 369-376 (1984).

period clock gene in the central nervous system

Sehgal, A., Price, J. L., Man, B. & Young, M. W.

of Drosophila melanogaster. EMBO J. 5, 2313-

Loss of circadian behavioral rhythms and per

2320 (1986).

RNA oscillations in the Drosophila mutant
7.

8.

9.

13. Sehgal, A., Price, J. & Young, M. W. Ontogeny

timeless. Science 263, 1603-1606 (1994).

of a biological clock in Drosophila melanogaster.

Myers, M. P., Wager-Smith, K., Wesley, C. S.,

PNAS 89, 1423-1427 (1992).

Young, M. W. & Sehgal, A. Positional cloning

14. Ralph, M. R. & Menaker, M. A mutation of theand sequence analysis of the Drosophila clock

circadian system in golden hamsters. Sciencegene, timeless. Science 270, 805-808 (1995).

241, 1225-1227 (1988).

Hardin, P. E., Hall, J. C. & Rosbash, M.

15. Lowrey, P. L. et al. Positional syntenic cloning

Feedback of the Drosophila period geneandproduct on circadian cycling of its messenger

mammalian circadian mutation tau. ScienceRNA levels. Nature 343, 536-540 (1990).

288, 483-492 (2000).

functionalcharacterizationoftheVosshall, L. B., Price, J. L., Sehgal, A., Saez, L.

16. Xu, Y. et al. Functional consequences of a CKIG& Young, M. W. Block in nuclear localization ofmutation causing familial advanced sleep

period protein by a second clock mutation,timeless. Science 263, 1606-1609 (1994).

phase syndrome. Nature 434, 640-644 (2005).
17. Allada, R., White, N. E., So, W. V., Hall, J. C. &10. Gekakis, N. et al. Isolation of timeless by PER

Rosbash, M. A mutant Drosophila homolog ofDefective interaction

mammalian Clock disrupts circadian rhythms

between timeless protein and long-period

and transcription of period and timeless. Cellmutant PERL. Science 270, 811-815 (1995).

93, 791-804 (1998).

protein interaction:

11. Kidd, S., Baylies, M. K., Gasic, G. P. & Young,

18. Rutila, J. E. et al. CYCLE is a second bHLH-

M. W. Structure and distribution of the Notch

PAS clock protein essential for circadianprotein in developing Drosophila. Genes Dev. 3,rhythmicity and transcription of Drosophila

1113-1129 (1989).

period and timeless. Cell 93, 805-814 (1998).

12. James, A. A., Ewer, J., Reddy, P., Hall, J. C. &Mike Young の「狡兎三窟」
松本顕✉
順天堂大学医学部
Mike Young の研究室（ロックフェラー大学）に読の批判内容でだいたい彼と判る）とは対照的とい留学していた縁でこの記事の執筆依頼を頂いた。まえる。脱線ついでに。私の中では、ノーベル賞の 3ずは御依頼に従って Mike の人柄を紹介したい。

氏のイメージは日本の 3 大戦国武将と重なる。革新

Mike は常に紳士的で普段からポーカーフェイス。

的で好戦的な Rosbash は信長。喜怒哀楽が激しく人自分からはあまりジョークを言ったりせず、どちら情家で変わり者の Jeff は秀吉。ポーカーフェイスでかといえば物静かな人物である。講演も淡々として戦略的な Mike は家康… みなさんはどんな印象でおり、私信のメールでさえビジネスライク。必要なしょうか？（受賞講演は YouTube で。3 氏ともいつ時に必要な事項のみ。この点で、難解な単語が連発もより緊張している感じですが）する Jeff Hall の長文（Jeff が論文の査読をするとすそんな Mike に今回の受賞のお祝いメールを送っぐバレる、と評判）や、好き嫌いの感情むき出しのMichael Rosbash の手紙（ハエのリズム屋なら、査✉amatsumo@juntendo.ac.jp
時間生物学Vol. 24, No. 1 (2018)49