letters to nature 20 . Katz , L. C. & Shatz , C. J. Synaptic activity and the construction of cortical circuits . Science 274 , 1133 1138 -LRB- 1996 -RRB- . 21 . Sawatari , A. & Callaway , E. M. Diversity and cell type specificity of local excitatory connections to neurons in layer 3B of monkey primary visual cortex . Neuron 25 , 459 471 -LRB- 2000 -RRB- . 22 . Schubert , D. , Kotter , R. , Zilles , K. , Luhmann , H. J. & Staiger , J. F. Cell type-specific circuits of cortical layer IV spiny neurons . J. Neurosci . 23 , 2961 2970 -LRB- 2003 -RRB- . 23 . Agmon , A. & Connors , B. W. Correlation between intrinsic firing patterns and thalamocortical synaptic responses of neurons in mouse barrel cortex . J. Neurosci . 12 , 319 329 -LRB- 1992 -RRB- . 24 . Gibson , J. R. , Beierlein , M. & Connors , B. W. Two networks of electrically coupled inhibitory neurons in neocortex . Nature 402 , 75 79 -LRB- 1999 -RRB- . 25 . Gonchar , Y. & Burkhalter , A. Connectivity of GABAergic calretinin-immunoreactive neurons in rat primary visual cortex . Cereb . Cortex 9 , 683 696 -LRB- 1999 -RRB- . 26 . Gonchar , Y. & Burkhalter , A. Distinct GABAergic targets of feedforward and feedback connections between lower and higher areas of rat visual cortex . J. Neurosci . 23 , 10904 10912 -LRB- 2003 -RRB- . 27 . Meskenaite , V. Calretinin-immunoreactive local circuit neurons in area 17 of the cynomolgus monkey , Macaca fascicularis . J. Comp . Neurol . 379 , 113 132 -LRB- 1997 -RRB- . 28 . Staiger , J. F. et al. . Innervation of interneurons immunoreactive for VIP by intrinsically bursting pyramidal cells and fast-spiking interneurons in infragranular layers of juvenile rat neocortex . Eur . J. Neurosci . 16 , 11 20 -LRB- 2002 -RRB- . 29 . Girman , S. V. , Sauve , Y. & Lund , R. D. Receptive field properties of single neurons in rat primary visual cortex . J. Neurophysiol . 82 , 301 311 -LRB- 1999 -RRB- . 30 . DeAngelis , G. C. , Ghose , G. M. , Ohzawa , I. & Freeman , R. D. Functional micro-organization of primary visual cortex : receptive field analysis of nearby neurons . J. Neurosci . 19 , 4046 4064 -LRB- 1999 -RRB- . Supplementary Information accompanies the paper on www.nature.com/nature . Acknowledgements We are grateful for support from the National Institutes of Health . We thank Y. Komatsu and F. Briggs and members of the Callaway laboratory for discussions . Competing interests statement The authors declare that they have no competing financial interests . Correspondence and requests for materials should be addressed to E.M.C. -LRB- callaway@salk.edu -RRB- . ... . Different time courses of learning-related activity in the prefrontal cortex and striatum Anitha Pasupathy & Earl K. Miller The Picower Center for Learning and Memory , RIKEN-MIT Neuroscience Research Center and Department of Brain and Cognitive Sciences , Massachusetts Institute of Technology , Cambridge , Massachusetts 02139 USA ... . control of volitional movements1 3,16 18 . Both of these areas receive inputs from many brain systems -LRB- for example , sensory , motor and reward -RRB- , which makes them well suited for roles in learning . Their anatomy also suggests a close relationship -- the PFC and basal ganglia are interconnected in cortico-basal ganglionic ` loops ' 19,20 -- but the nature of this interaction is still unclear . Some results have led to the suggestion of a sequential relationship , in which the PFC is involved in new learning and the basal ganglia are subsequently involved in consolidating familiar routines into automatic habits21 ,22 . Another hypothesis , not necessarily incompatible with the one above , suggests a dominant role for the basal ganglia in new learning15 ,23 due to its anatomical architecture and the membrane properties of striatal spiny neurons . These hypotheses lead to specific predictions about the time course of learning in these areas : based on the first hypothesis , the PFC is predicted to lead the basal ganglia ; based on the second hypothesis , the basal ganglia lead the PFC. Here , we report evidence in favour of the latter event ; that is , learning-related changes appear sooner and progress more rapidly in the striatum than the PFC. To test these hypotheses , we simultaneously recorded neural activity from the dorsolateral PFC -LRB- areas 9 and 46 -RRB- and the head and body of the caudate nucleus , a part of the striatum that receives direct projections from , and indirectly projects to , the PFC19 ,20 -LRB- see Methods -RRB- . Monkeys learned associations between each of two visual cues and two saccadic eye movements -LRB- right and left , Fig. 1a -RRB- . Monkeys were familiar with the task , but each day two novel cues were used and their associations learned by trial and error using juice reward as feedback . Once the cue saccade associations had been learned , they were reversed without warning and the opposite pairing was then learned -LRB- see Supplementary Note 1 -RRB- . Figure 1b -LRB- left -RRB- shows the average behavioural performance before and after the reversals . Saccade choices dropped to about 0 % correct for the first few trials after the reversal because the previous associations were still being followed . Then , performance jumped to chance -LRB- 50 % -RRB- followed by a slow increase with trial number . Likewise , reaction time increased by an average of about To navigate our complex world , our brains have evolved a sophisticated ability to quickly learn arbitrary rules such as ` stop at red ' . Studies in monkeys using a laboratory test of this capacity -- conditional association learning -- have revealed that frontal lobe structures -LRB- including the prefrontal cortex -RRB- as well as subcortical nuclei of the basal ganglia are involved in such learning1 5 . Neural correlates of associative learning have been observed in both brain regions6 14 , but whether or not these regions have unique functions is unclear , as they have typically been studied separately using different tasks . Here we show that during associative learning in monkeys , neural activity in these areas changes at different rates : the striatum -LRB- an input structure of the basal ganglia -RRB- showed rapid , almost bistable , changes compared with a slower trend in the prefrontal cortex that was more in accordance with slow improvements in behavioural performance . Also , pre-saccadic activity began progressively earlier in the striatum but not in the prefrontal cortex as learning took place . These results support the hypothesis that rewarded associations are first identified by the basal ganglia , the output of which ` trains ' slower learning mechanisms in the frontal cortex15 . The prefrontal cortex -LRB- PFC -RRB- is a cortical area important for the organization of goal-directed , rule-based behaviours ; the basal ganglia are a group of subcortical nuclei long associated with the NATURE | VOL 433 | 24 FEBRUARY 2005 | www.nature.com/nature Figure 1 Task and behaviour . a , One of two initially novel cues was briefly presented at centre of gaze followed by a memory delay and then presentation of two target spots on the right and left . Saccade to the target associated with the cue at that time was rewarded . After this was learned , the cue saccade associations were reversed and re-learned . b , Average per cent correct performance -LRB- left -RRB- and reaction time -LRB- right -RRB- across sessions and blocks as a function of trial number -LRB- left : all trials ; right : correct trials only -RRB- during learning for two monkeys . Zero -LRB- indicated by red arrow -RRB- represents first trial after reversal . Error bars show standard error of the mean . 873 2005 Nature Publishing Group letters to nature Figure 2 Change in peri-cue saccade direction selectivity in prefrontal cortex and caudate nucleus with learning . a , b , Population strength of direction selectivity -LRB- PEVdir : proportion of explainable variance by direction factor -RRB- -LRB- colour scale -RRB- shown as a function of correct trials and time from cue onset for PFC -LRB- a -RRB- and caudate nucleus -LRB- Cd -RRB- -LRB- b -RRB- during cue -LRB- white lines -RRB- and delay periods . Black dots indicate ` rise time ' -LRB- time to half-maximum selectivity -RRB- . Selectivity strength increases and appears earlier in both areas as learning takes place . Changes appear earlier and reach an asymptote sooner in the caudate nucleus than the PFC. c , Rise times for PFC -LRB- blue -RRB- and Cd -LRB- red -RRB- . Dotted lines show sigmoids of best fit . Data shown in a c are based on correct trials collapsed across all blocks -LRB- reversals -RRB- . 50 ms in the first few correct trials after reversal and then gradually decreased as the reversed associations were learned -LRB- Fig. 1b -LRB- right -RRB- , see Supplementary Note 2 -RRB- . Because the monkeys did not instantly reverse the associations , we could examine learning across multiple trial blocks in each recording session . Across 51 sessions , we examined the activity of 432 PFC and 279 caudate nucleus neurons -LRB- see Methods -RRB- . Many PFC and caudate nucleus neurons showed activity that reflected the saccade direction -LRB- PFC : 39 % or 168/432 ; caudate nucleus : 36 % or 101/279 of all recorded neurons -RRB- , especially around the time of its execution . Neuronal activity also reflected the cues or their associations with the saccades -LRB- see Supplementary Note 3 -RRB- . As the monkeys learned which associations would yield reward , there was an increase in early-trial activity -LRB- that is , activity around the time of cue presentation -RRB- that predicted the direction of the saccade to be made after the delay . Examples of single PFC and caudate nucleus neurons with this ` prospective ' activity and the development of this activity with learning are shown in Supplementary Fig. 1 . We assessed learning-related changes in saccade direction selectivity for the 168 PFC and 101 caudate nucleus neurons showing such selectivity during any trial period -LRB- analysis of variance -LRB- ANOVA -RRB- , P , 0.01 -RRB- . Selectivity was quantified with a regression analysis that measured the proportion of explainable variance in activity accounted for by saccade direction -LRB- PEVdir , see Methods -RRB- . PEVdir is shown for the PFC and caudate nucleus populations as a function of time during cue and delay periods and number of correct trials -LRB- Fig. 2a , b -RRB- . During the first few correct trials early in the learning process , both populations showed relatively weak earlytrial direction selectivity . This strength of selectivity increased with the number of correct trials -LRB- especially near the end of cue presentation -RRB- , albeit at different rates in the PFC and caudate nucleus neurons : selectivity increased sooner and more abruptly in the caudate nucleus compared with the PFC -LRB- see Supplementary Note 4 -RRB- . This faster increase in early-trial direction selectivity in the caudate nucleus can be seen in Fig. 2c , which shows the time when half-maximum selectivity was reached -LRB- the ` rise time ' -RRB- for each neuron population on each trial . In the first few correct trials , rise time is late in the delay , near the time of saccade execution . After just a few correct trials , rise time in the caudate nucleus is much earlier as the strength of ` prospective ' direction selectivity rapidly increases in the cue period -LRB- see Supplementary Note 5 -RRB- . In contrast , the more gradual increase of early-trial direction selectivity in the PFC results in a slower leftward shift in rise time . Best-fitting sigmoidal curves -LRB- dashed lines -RRB- confirmed that the shift in rise Figure 3 Change in saccade direction selectivity at the time of saccade execution during the learning process . The same neuron populations and conventions were used as in Fig. 2 . a , b , White dashed lines denote the onset of target spots . Grey lines represent average reaction time . Peak direction selectivity -LRB- colour scale -RRB- in PFC -LRB- a -RRB- and Cd -LRB- b -RRB- remains relatively constant , but appears progressively earlier with learning in the caudate 874 nucleus , as illustrated by rise time -LRB- black dots -RRB- . Note that the colour scales here are higher than in Fig. 2 -LRB- peri-cue period -RRB- because direction selectivity is much stronger during saccade execution . c , Rise times for the PFC -LRB- blue -RRB- and Cd -LRB- red -RRB- populations relative to saccade onset . Black dashed line represents time of saccade onset . NATURE | VOL 433 | 24 FEBRUARY 2005 | www.nature.com/nature 2005 Nature Publishing Group letters to nature time was faster and reached an asymptote sooner in the caudate nucleus -LRB- maximum slope -LRB- at trial 4 -RRB- 480 ms per trial , asymptote at trial 6 -RRB- than the PFC -LRB- maximum slope -LRB- at trial 7 -RRB- 108 ms per trial , asymptote at trial 12 -RRB- -LRB- see Supplementary Note 6 -RRB- . This sudden increase in early-trial direction selectivity after only a few correct trials could even be seen in some single neurons from the caudate nucleus -LRB- see Supplementary Fig. 2 -RRB- . This is in sharp contrast to the monkeys ' more gradual improvement in performance : 95 % of the change in correct choices and reaction time was only achieved after 20 correct trials per cue . Indeed , PFC rise times showed a significantly stronger correlation with per cent correct performance -LRB- linear correlation coefficient r 20.96 -RRB- than caudate nucleus rise times -LRB- r 20.79 -RRB- -LRB- see Supplementary Notes 7 and 8 -RRB- . Figure 3a , b shows the average saccade direction selectivity for the same neuron populations before and after saccade execution . Here , both areas showed strong selectivity , but with clear differences . For each trial , rise time and peak were earlier in the caudate nucleus than in the PFC. Additionally , with increasing numbers of correct trials , selectivity became increasingly pre-saccadic in the caudate nucleus but not in the PFC ; it began and peaked progressively earlier relative to target onset -LRB- ` go ' signal -RRB- -LRB- Fig. 3a , b -RRB- and saccade initiation -LRB- Fig. 3c -RRB- . In the first few correct trials , rise time in the caudate nucleus was about 60 ms before the saccade , but occurred 250 ms before the saccade after 20 correct trials -LRB- Fig. 3c -RRB- . In contrast , PFC rise times were relatively stable during learning and were centred closely around saccade initiation . These results illustrate differences between PFC and caudate nucleus activity during conditional association learning . Earlytrial caudate nucleus activity quickly reflected the forthcoming saccade , whereas such activity appeared more slowly in the PFC. This fits with observations of a striatal infrastructure ideal for rapid , supervised -LRB- reward-based -RRB- learning24 ,25 . One possibility is that the PFC and caudate nucleus are components of different learning systems that are set in opposition in our task . The cue response associations may have engaged the caudate nucleus ; the striatum is thought to be central to the ` habit memory ' that establishes such links . However , flexibility -LRB- for example , reversal -RRB- has been associated with the PFC , so perhaps activity in the caudate nucleus was ` ignored ' in favour of PFC mechanisms with slower plasticity , but consequently greater flexibility . Also , our results may support hypotheses that learning in the frontal cortex could be ` trained ' by the basal ganglia15 ,26 . Dopaminergic reward-prediction error signals from the midbrain27 ,28 may allow rapid formation of rewardrelevant associations in the striatum23 , which over a course of trials might train slower , and more graded , hebbian mechanisms in the PFC via the output nuclei of the basal ganglia and the thalamus15 -LRB- see Supplementary Note 9 -RRB- . Behaviour may follow changes in the PFC or a combination of the PFC and striatum -LRB- and other areas29 -RRB- ; this would explain the overall slower time course of behavioural improvement relative to changes in the caudate nucleus . Although pre-saccadic activity might also be linked to the improvement in choices with learning , it seems likely to reflect the decrease in reaction time ; a correlation between caudate nucleus -LRB- and frontal cortex -RRB- pre-saccadic activity and reaction time has been previously demonstrated10 ,30 . These results indicate that during conditional visuomotor learning , changes in caudate nucleus activity can lead those in the PFC. A reversals -LRB- four to nine trial blocks -RRB- per recording session -LRB- average six trial blocks -RRB- . For each session , two new cues -LRB- complex , multi-coloured images -RRB- were selected at random . In addition , there were two highly familiar , unchanging cue response associations that were randomly intermingled and presented half as often . Results using these familiar cues will be reported in future publications . Data collection Neural activity was recorded from the dorsolateral prefrontal cortex -LRB- areas 9 and 46 -RRB- and the head and body of the caudate nucleus . Recording wells were positioned stereotaxically based on images obtained using magnetic resonance imaging . All animal procedures conformed to NIH guidelines and the MIT Committee on Animal Care . Arrays of 12 24 -LRB- 8 16 in the PFC and 4 8 in the caudate nucleus -RRB- dura-puncturing tungsten microelectrodes -LRB- FHC Instruments -RRB- , were mounted on custom-made , independently adjustable microdrives . All isolated neurons were accepted for study without being prescreened . Waveforms were digitized , stored and sorted offline based on waveform shape characteristics . Data analysis Two-way balanced analyses of variance -LRB- ANOVAs -RRB- were conducted on the average neuronal activity during each of four periods : ` cue ' -LRB- 500 ms , starting 100 ms after cue onset -RRB- ` delay ' -LRB- end of cue period to 150 ms before saccade onset -RRB- , ` saccade ' -LRB- 300 ms centred on saccade onset -RRB- and ` reward ' -LRB- 250 ms , starting 50 ms after reward onset -RRB- . As in previous work7 , selectivity after learning was based on the last 10 correct trials per association before reversal in each block , but results were similar when selectivity was based on all correct trials . All tests were evaluated at P , 0.01 . Saccade direction selectivity was quantified as the fraction of each neuron 's variance explained by saccade direction . The total variance -LRB- j2 -RRB- was partitioned into object -LRB- j2 -RRB- , obj direction -LRB- j2 -RRB- , interaction -LRB- j2 -RRB- and error -LRB- j2 -RRB- terms . Selectivity strength -LRB- R 2 for the dir int err 2 2 direction factor -RRB- was quantified as -LRB- jdir/j -RRB- . The proportion of total explainable variance was -LRB- 1 2 j2 / j2 -RRB- . R 2 was computed for each neuron over a 100 ms centred window , slid in err 10 ms steps throughout the trial -LRB- see Supplementary Note 11 -RRB- . To quantify changes across trials , R 2 was calculated for each neuron across an eight-trial window , slid in one-trial steps over the first 30 correct trials per cue per trial block , collapsed across blocks . Analysis was restricted to the first 30 correct trials per association , the minimum block length , because block length varied with learning rate . We compared direction selectivity across the PFC and caudate nucleus populations -LRB- Figs 2 and 3 -RRB- by computing the proportion of explainable variance accounted for by the direction factor -LRB- PEVdir -RRB- as the ratio of average R 2 and the average total explainable variance across cells . Thus PEVdir represents the population strength of direction selectivity . Using R 2 instead of PEVdir yielded similar results , but PEVdir is advantageous because it expresses saccade direction selectivity as a proportion of explainable variance without including unexplained variance -LRB- that is , that due to uncontrolled variables and noise -RRB- . To assess the trend in direction selectivity with learning , we determined the halfmaximum -LRB- across all trials -RRB- PEVdir for each population separately and then calculated rise time as the time at which PEVdir in each trial reached that value -LRB- see Supplementary Note 12 -RRB- . Received 31 August ; accepted 17 December 2004 ; doi : 10.1038 / nature03287 . 1 . Petrides , M. in Handbook of Neuropsychology -LRB- eds Boller , F. & Grafman , J. -RRB- 59 82 -LRB- Elsevier , Amsterdam , 1994 -RRB- . 2 . Passingham , R. E . The Frontal Lobes and Voluntary Action -LRB- Oxford Univ. . Press , Oxford , 1995 -RRB- . 3 . Fuster , J. M . The Prefrontal Cortex : Anatomy , Physiology , and Neuropsychology of the Frontal Lobe -LRB- Lippincott-Raven , Philadelphia , 1997 -RRB- . 4 . Wise , S. P. , Murray , E. A. & Gerfen , C. R . The frontal cortex-basal ganglia system in primates . Crit . Rev. Neurobiol . 10 , 317 356 -LRB- 1996 -RRB- . 5 . Murray , E. A. , Bussey , T. J. & Wise , S. P. Role of prefrontal cortex in a network for arbitrary visuomotor mapping . Exp . Brain Res . 133 , 114 129 -LRB- 2000 -RRB- . 6 . Tremblay , L. , Hollerman , J. R. & Schultz , W. Modifications of reward expectation-related neuronal activity during learning in primate striatum . J. Neurophysiol . 80 , 964 977 -LRB- 1998 -RRB- . 7 . Asaad , W. F. , Rainer , G. & Miller , E. K. Neural activity in the primate prefrontal cortex during associative learning . Neuron 21 , 1399 1407 -LRB- 1998 -RRB- . 8 . White , I. M. & Wise , S. P. Rule-dependent neuronal activity in the prefrontal cortex . Exp . Brain Res . 126 , 315 335 -LRB- 1999 -RRB- . 9 . Toni , I. & Passingham , R. E. Prefrontal-basal ganglia pathways are involved in the learning of arbitrary visuomotor associations : a PET study . Exp . Brain Res . 127 , 19 32 -LRB- 1999 -RRB- . 10 . Lauwereyns , J. , Watanabe , K. , Coe , B. & Hikosaka , O . A neural correlate of response bias in monkey caudate nucleus . Nature 418 , 413 417 -LRB- 2002 -RRB- . 11 . Hadj-Bouziane , F. & Boussaoud , D. Neuronal activity in the monkey striatum during conditional visuomotor learning . Exp . Brain Res . 153 , 190 196 -LRB- 2003 -RRB- . 12 . Schumacher , E. H. , Elston , P. A. & D'Esposito , M. Neural evidence for representation-specific response selection . J. Cogn . Neurosci . 15 , 1111 1121 -LRB- 2003 -RRB- . 13 . Brasted , P. J. & Wise , S. P. Comparison of learning-related neuronal activity in the dorsal premotor cortex and striatum . Eur . J. Neurosci . 19 , 721 740 -LRB- 2004 -RRB- . 14 . Hoshi , E. & Tanji , J. Area-selective neuronal activity in the dorsolateral prefrontal cortex for information retrieval and action planning . J. Neurophysiol . 91 , 2707 2722 -LRB- 2004 -RRB- . 15 . Houk , J. C. & Wise , S. P. Distributed modular architectures linking basal ganglia , cerebellum , and cerebral cortex : their role in planning and controlling action . Cereb . Cortex 5 , 95 110 -LRB- 1995 -RRB- . 16 . Miller , E. K. & Cohen , J. D . An integrative theory of prefrontal cortex function . Annu . Rev. Neurosci . 24 , 167 202 -LRB- 2001 -RRB- . 17 . Graybiel , A. M . The basal ganglia and the initiation of movement . Rev. Neurol . -LRB- Paris -RRB- 146 , 570 574 -LRB- 1990 -RRB- . 18 . Hikosaka , O. , Takikawa , Y. & Kawagoe , R. Role of the basal ganglia in the control of purposive saccadic eye movements . Physiol . Rev. 80 , 953 978 -LRB- 2000 -RRB- . Methods Behavioural task Each trial began with the presentation of a fixation spot , followed by 500 ms of cue presentation , and 1000 ms of memory delay . Monkeys were required to maintain gaze within 1.58 of the fixation spot during these periods . After the delay , the fixation spot was extinguished and two targets appeared on the right and left . A direct saccade to the target associated with the cue yielded reward -LRB- see Supplementary Note 10 -RRB- . After performance reached criterion -LRB- $ 90 % correct over 10 trials per cue -RRB- and there were at least 30 correct trials for each cue , the associations were reversed . Monkeys completed three to eight NATURE | VOL 433 | 24 FEBRUARY 2005 | www.nature.com/nature 2005 Nature Publishing Group 875 letters to nature 19 . DeLong , M. R. & Georgopoulos , A. P. in Handbook of Physiology -- Nervous System -LRB- eds Brookhart , J. M. & Mountcastle , V. B. -RRB- 1017 1061 -LRB- American Physiological Society , Bethesda , 1981 -RRB- . 20 . Middleton , F. A. & Strick , P. L. Basal-ganglia ` projections ' to the prefrontal cortex of the primate . Cereb . Cortex 12 , 926 935 -LRB- 2002 -RRB- . 21 . Packard , M. G. & Knowlton , B. J. Learning and memory functions of the basal ganglia . Annu . Rev. Neurosci . 25 , 563 593 -LRB- 2002 -RRB- . 22 . Graybiel , A. M . The basal ganglia and chunking of action repertoires . Neurobiol . Learn . Mem . 70 , 119 136 -LRB- 1998 -RRB- . 23 . Bar-Gad , I. , Morris , G. & Bergman , H. Information processing , dimensionality reduction and reinforcement learning in the basal ganglia . Prog . Neurobiol . 71 , 439 473 -LRB- 2003 -RRB- . 24 . Reynolds , J. N. , Hyland , B. I. & Wickens , J. R . A cellular mechanism of reward-related learning . Nature 413 , 67 70 -LRB- 2001 -RRB- . 25 . Wilson , C. J. & Kawaguchi , Y . The origins of two-state spontaneous membrane potential fluctuations of neostriatal spiny neurons . J. Neurosci . 16 , 2397 2410 -LRB- 1996 -RRB- . 26 . O'Reilly , R. C. & Munakata , Y. Computational Explorations in Cognitive Neuroscience : Understanding the Mind by Stimulating the Brain -LRB- MIT Press , Cambridge , Massachusetts , 2000 -RRB- . 27 . Schultz , W. & Dickinson , A. Neuronal coding of prediction errors . Annu . Rev. Neurosci . 23 , 473 500 -LRB- 2000 -RRB- . 28 . McClure , S. M. , Berns , G. S. & Montague , P. R. Temporal prediction errors in a passive learning task activate human striatum . Neuron 38 , 339 346 -LRB- 2003 -RRB- . 29 . Wirth , S. et al. . Single neurons in the monkey hippocampus and learning of new associations . Science 300 , 1578 1581 -LRB- 2003 -RRB- . 30 . Hanes , D. P. & Schall , J. D. Neural control of voluntary movement initiation . Science 274 , 427 430 -LRB- 1996 -RRB- . Supplementary Information accompanies the paper on www.nature.com/nature . Acknowledgements We thank M. H. Histed for valuable discussions ; K. J. MacCully for technical assistance ; W. F. Asaad , A. J. Bastian , T. Buschman , A. C. Diogo , J. Feingold , D. J. Freedman , M. Machon , J. McDermott , J. E. Roy and M. Wicherski for helpful comments . This work was supported by a grant from the N.I.N.D.S. A.P. was supported by the Tourette 's Syndrome Association . Competing interests statement The authors declare that they have no competing financial interests . Correspondence and requests for materials should be addressed to A.P. -LRB- anitha@mit.edu -RRB- . CFTR 's cytoplasmic nucleotide-binding domains to opening of the ion channel in the transmembrane domains . This establishes a molecular mechanism , involving dynamic restructuring of the NBD dimer interface , that is probably common to all members of the ABC protein superfamily . Crystal structures of most ABC-protein NBDs determined so far share the same fold7 ,8 with a core subdomain -LRB- ` head ' -RRB- that binds the ATP , and an a-helical subdomain -LRB- ` tail ' -RRB- that includes the ABCspecific signature sequence -LRB- LSGGQ -RRB- . Dimeric structures revealed nucleotide-bound NBD homodimers in rotationally symmetric ` head-to-tail ' arrangement , enclosing two ATP molecules within interfacial composite sites , each comprising conserved ATP-binding motifs from the head of one monomer and signature sequence residues from the tail of the other3 ,5,9,10 . On the basis of this structural evidence and biochemical studies of reversible dimerization of isolated NBDs4 ,5,11 13 , opening and closing of CFTR channels can be interpreted14 in terms of cycles of NBD1 NBD2 dimerization and dissociation , induced by ATP binding and hydrolysis , respectively -LRB- Fig. 1a -RRB- . Opening of a phosphorylated CFTR Cl2 channel seems to require ATP binding to both composite sites because , at low -LSB- ATP -RSB- , mutations expected to weaken ATP binding can make nucleotide occupancy at either site rate-limiting for channel opening14 . In addition , interfering with hydrolysis prevents the normal rapid closing of CFTR channels1 ,2,14 . Because photolabelling studies show that ATP can remain at the NBD1-head site for several minutes without being hydrolysed15 ,16 , whereas a CFTR-channel gating cycle lasts only seconds , channel opening and closing seem to be timed by nucleotide binding and hydrolysis at the composite site ... . CFTR channel opening by ATP-driven tight dimerization of its nucleotidebinding domains Paola Vergani1 , Steve W. Lockless2 , Angus C. Nairn3 ,4 & David C. Gadsby1 1 Laboratory of Cardiac/Membrane Physiology , 2Laboratory of Molecular Neurobiology and Biophysics , and 3Laboratory of Molecular and Cellular Neuroscience , The Rockefeller University , New York , New York 10021 , USA 4 Department of Psychiatry , Yale University , New Haven , Connecticut 06519 , USA ... . ABC -LRB- ATP-binding cassette -RRB- proteins constitute a large family of membrane proteins that actively transport a broad range of substrates . Cystic fibrosis transmembrane conductance regulator -LRB- CFTR -RRB- , the protein dysfunctional in cystic fibrosis , is unique among ABC proteins in that its transmembrane domains comprise an ion channel . Opening and closing of the pore have been linked to ATP binding and hydrolysis at CFTR 's two nucleotidebinding domains , NBD1 and NBD2 -LRB- see , for example , refs 1 , 2 -RRB- . Isolated NBDs of prokaryotic ABC proteins dimerize upon binding ATP , and hydrolysis of the ATP causes dimer dissociation3 5 . Here , using single-channel recording methods on intact CFTR molecules , we directly follow opening and closing of the channel gates , and relate these occurrences to ATP-mediated events in the NBDs . We find that energetic coupling6 between two CFTR residues , expected to lie on opposite sides of its predicted NBD1 NBD2 dimer interface , changes in concert with channel gating status . The two monitored side chains are independent of each other in closed channels but become coupled as the channels open . The results directly link ATP-driven tight dimerization of 876 Figure 1 Open CFTR channels correspond to dimerized NBDs . a , Diagram illustrating the proposed mechanism coupling the opening of the Cl2 channel pore -LRB- Cn , closed states ; O , open -RRB- in the transmembrane domains -LRB- converging , or semi-parallel , straight lines -RRB- to the hydrolysis cycle through the dimerization of NBDs -LRB- green , NBD1 ; blue , NBD2 -RRB- . The dynamic formation and disruption of a tight NBD dimer interface are represented by major changes in shape and position simply for clarity -LRB- see text -RRB- . b , Mutating the ` Walker B ' glutamate , Glu 1371 , in NBD2 markedly increases the stability of the Cl2 channel 's open burst state . Records from patches containing hundreds of channels , activated by exposure to 5 mM ATP and 300 nM cAMP-dependent protein kinase -LRB- PKA , red -RRB- . Time constants for current decay fit lines -LRB- blue -RRB- : WT , t 0.45 s ; E1371Q , t 476 s. Note the fivefold expanded timescale for the WT record . NATURE | VOL 433 | 24 FEBRUARY 2005 | www.nature.com/nature 2005 Nature Publishing Group

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

striatum
monkey
reversal
prefrontal cortex
learning


Widespread

Lateral

Gene

Transfer

from

Intracellular

Bacteria

to

Multicellular

Eukaryotes

Julie

C.

Dunning

Hotopp

et

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thank

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Rust

for

initial

discussions

of

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W.

Wang

for

help

with

analysis

on

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antibodies

and

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Liu

for

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some

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constructs

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Julie

C.

Dunning

Hotopp

Michael

E.

Clark

Deodoro

C.

S.

G.

Oliveira

Jeremy

M.

Foster

Peter

Fischer

M

nica

C.

Mu

oz

Torres

Jonathan

D.

Giebel

Nikhil

Kumar

Nadeeza

Ishmael

Shiliang

Wang

Jessica

Ingram

Rahul

V.

Nene

Jessica

Shepard

Jeffrey

Tomkins

Stephen

Richards

David

J.

Spiro

Elodie

Ghedin

Barton

E.

Slatko

Herv

Tettelin

John

H.

Although

common

among

bacteria

lateral

gene

transfer

the

movement

of

genes

between

distantly

related

organisms

is

thought

to

occur

only

rarely

between

bacteria

and

multicellular

eukaryotes

However

the

presence

of

endosymbionts

such

as

Wolbachia

pipientis

within

some

eukaryotic

germlines

may

facilitate

bacterial

gene

transfers

to

eukaryotic

host

genomes

We

therefore

examined

host

genomes

for

evidence

of

gene

transfer

events

from

Wolbachia

bacteria

to

their

hosts

We

found

and

confirmed

transfers

into

the

genomes

of

four

insect

and

four

nematode

species

that

range

from

nearly

the

entire

Wolbachia

genome

-LRB-

megabase

-RRB-

to

short

-LRB-

base

pairs

-RRB-

insertions

Potential

Wolbachia-to-host

transfers

were

also

detected

computationally

in

three

additional

sequenced

insect

genomes

We

also

show

that

some

of

these

inserted

Wolbachia

genes

are

transcribed

within

eukaryotic

cells

lacking

endosymbionts

Therefore

heritable

lateral

gene

transfer

occurs

into

eukaryotic

hosts

from

their

prokaryote

symbionts

potentially

providing

a

mechanism

for

acquisition

of

new

genes

and

functions

T

he

transfer

of

DNA

between

diverse

organisms

lateral

gene

transfer

-LRB-

LGT

-RRB-

facilitates

the

acquisition

of

novel

gene

functions

Among

Eubacteria

LGT

is

involved

The

Institute

for

Genomic

Research

J.

Craig

Venter

Institute

Medical

Center

Drive

Rockville

MD

USA

of

Biology

University

of

Rochester

Rochester

NY

USA

Parasitology

Division

New

England

Biolabs

Incorporated

County

Road

Ipswich

MA

USA

of

Internal

Medicine

Infectious

Diseases

Division

Washington

University

School

of

Medicine

St.

Louis

MO

USA

Clemson

University

Genomics

Institute

BRC

New

Cherry

Street

Clemson

SC

USA

Genome

Sequencing

Center

Baylor

College

of

Medicine

Houston

TX

USA

of

Infectious

Diseases

University

of

Pittsburgh

School

of

Medicine

Pittsburgh

PA

USA

These

authors

contributed

equally

to

this

work

To

whom

correspondence

should

be

addressed

E-mail

jhotopp@som.umaryland.edu

Present

address

Institute

for

Genome

Sciences

University

of

Maryland

School

of

Medicine

Health

Sciences

Facility

II

Room

Penn

Street

Baltimore

MD

USA

Present

address

Brown

University

Providence

RI

USA

Present

address

Pace

University

New

York

NY

USA

These

authors

contributed

equally

to

this

work

in

the

evolution

of

antibiotic

resistance

pathogenicity

and

metabolic

pathways

-LRB-

-RRB-

Rare

LGT

events

have

also

been

identified

between

higher

eukaryotes

with

segregated

germ

cells

-LRB-

-RRB-

demonstrating

that

even

these

organisms

can

acquire

novel

DNA

Although

most

described

LGT

events

occur

within

a

single

domain

of

life

LGT

has

been

described

both

between

Eubacteria

and

Archaea

-LRB-

-RRB-

and

between

prokaryotes

and

phagotrophic

unicellular

eukaryotes

-LRB-

-RRB-

However

few

interdomain

transfers

involving

higher

multicellular

eukaryotes

have

been

found

Wolbachia

pipientis

is

a

maternally

inherited

endosymbiont

that

infects

a

wide

range

of

Fig.

Fluorescence

microscopy

evidence

supporting

Wolbachia/host

LGT

DNA

in

the

polytene

chromosomes

of

D.

ananassae

were

stained

with

propidium

iodide

-LRB-

red

-RRB-

whereas

a

probe

for

the

Wolbachia

gene

bound

to

a

unique

location

-LRB-

green

arrow

-RRB-

on

chromosome

SCIENCE

VOL

SEPTEMBER

Downloaded

from

on

May

Widespread

Lateral

Gene

Transfer

from

Intracellular

Bacteria

to

Multicellular

Eukaryotes

arthropods

including

at

least

of

insect

species

as

well

as

filarial

nematodes

-LRB-

-RRB-

It

is

present

in

developing

gametes

-LRB-

-RRB-

and

so

provides

circumstances

conducive

for

heritable

transfer

of

bacterial

genes

to

the

eukaryotic

hosts

Wolbachia-host

transfer

has

been

described

in

the

bean

beetle

Callosobruchus

chinensis

-LRB-

-RRB-

and

in

the

filarial

nematode

Onchocerca

spp.

-LRB-

-RRB-

We

have

found

Wolbachia

inserts

in

the

genomes

of

additional

diverse

invertebrate

taxa

including

fruit

flies

wasps

and

nematodes

A

comparison

of

the

published

genome

of

the

Wolbachia

endosymbiont

of

Drosophila

melanogaster

-LRB-

-RRB-

and

assemblies

of

Wolbachia

clone

mates

-LRB-

-RRB-

from

fruit

fly

whole-genome

shotgun

sequencing

data

revealed

a

large

Wolbachia

insert

in

the

genome

of

the

widespread

tropical

fruit

fly

D.

ananassae

Numerous

contiguous

overlapping

clone

sequences

-LRB-

contigs

-RRB-

were

found

that

harbored

junctions

between

Drosophila

retrotransposons

and

Wolbachia

genes

The

large

number

of

these

junctions

and

the

deep

sequencing

coverage

across

the

junctions

indicated

that

these

inserts

were

probably

not

due

to

chimeric

libraries

or

assemblies

To

validate

these

observations

we

amplified

five

DrosophilaWolbachia

junctions

with

polymerase

chain

reaction

-LRB-

PCR

-RRB-

and

verified

the

end

sequences

for

three

of

them

Fluorescence

in

situ

hybridization

-LRB-

FISH

-RRB-

of

banded

polytene

chromosomes

with

fluorescein-labeled

probes

of

two

Wolbachia

genes

-LRB-

-RRB-

revealed

the

presence

of

Wolbachia

genes

on

the

chromosome

of

D.

ananassae

-LRB-

Fig.

-RRB-

We

found

that

nearly

the

entire

Wolbachia

genome

was

transferred

to

the

fly

nuclear

genome

as

evidenced

by

the

presence

of

PCRamplified

products

from

of

physically

distant

Wolbachia

genes

in

cured

strains

of

D.

ananassae

Hawaii

verified

by

microscopy

to

be

lacking

the

endosymbiont

after

treatment

with

REPORTS

antibiotics

-LRB-

fig

-RRB-

-LRB-

-RRB-

In

contrast

only

spurious

incorrectly

sized

and

weak

amplification

was

detected

from

a

cured

control

line

lacking

these

inserts

-LRB-

Townsville

-RRB-

The

genes

assayed

-LRB-

table

-RRB-

are

spaced

throughout

the

Wolbachia

genome

Thus

the

high

proportion

of

amplified

genes

suggests

gene

transfer

of

nearly

the

entire

Wolbachia

genome

to

the

insect

genome

A

region

containing

four

Wolbachia

genes

with

two

retrotransposon

insertions

was

sequenced

-LRB-

-RRB-

from

a

single

bacteria

artificial

chromosome

-LRB-

BAC

-RRB-

constituting

an

independent

source

of

DNA

as

compared

with

the

largely

plasmid-derived

whole-genome

sequence

of

D.

ananassae

The

two

retroelements

each

contained

base

pair

-LRB-

bp

-RRB-

target

site

duplications

-LRB-

bp

identical

-RRB-

long

terminal

repeats

and

gag-pol

genes

-LRB-

Fig.

-RRB-

indicating

that

the

Wolbachia

insert

is

accumulating

retroelements

Insertion

of

this

region

appears

to

be

recent

as

shown

by

the

nearly

identical

target

site

duplications

and

the

nucleotide

identity

between

corresponding

endosymbiont

genes

and

sequenced

homologs

in

the

D.

ananassae

chromosome

Crosses

between

Wolbachia-free

Hawaii

males

-LRB-

with

the

insert

-RRB-

and

Wolbachia-free

Mexico

females

-LRB-

without

the

insert

-RRB-

revealed

that

the

insert

is

paternally

inherited

by

offspring

of

both

sexes

confirming

that

Wolbachia

genes

are

inserted

into

an

autosome

Because

Wolbachia

infections

are

maternally

inherited

this

also

confirms

that

PCR

amplification

in

the

antibiotictreated

line

is

not

due

to

an

undetectable

infection

Furthermore

the

Hawaii

and

Mexico

crosses

revealed

Mendelian

autosomal

inheritance

of

Wolbachia

inserts

-LSB-

paternal

N

proportion

of

offspring

with

Wolbachia

genes

-LRB-

k

-RRB-

maternal

N

k

-RSB-

Six

physically

distant

inserted

Wolbachia

genes

perfectly

cosegregated

in

maternal

inheritance

crosses

-LRB-

-RRB-

suggesting

they

also

are

closely

linked

PCR

amplification

and

sequencing

-LRB-

-RRB-

of

Wolbachia

loci

in

D.

ananassae

lines

from

widely

dispersed

geographic

locations

revealed

large

Wolbachia

inserts

in

lines

from

Hawaii

Malaysia

Indonesia

and

India

-LRB-

table

-RRB-

Sequence

comparisons

of

the

amplicons

from

these

four

lines

revealed

that

all

open

reading

frames

-LRB-

ORFs

-RRB-

remained

intact

with

identity

between

inserts

This

is

compared

to

an

average

of

identity

for

the

inserts

compared

with

wMel

the

Wolbachia

endosymbiont

of

D.

melanogaster

These

results

indicate

the

widespread

prevalence

of

D.

ananassae

strains

with

similar

inserts

of

the

Wolbachia

genome

probably

because

of

a

single

insertion

from

a

common

ancestor

In

addition

reverse

transcription

PCR

-LRB-

RTPCR

-RRB-

followed

by

sequencing

-LRB-

-RRB-

demonstrated

that

of

Wolbachia

genes

-LRB-

of

genes

assayed

table

-RRB-

are

transcribed

in

cured

adult

males

and

females

of

D.

ananassae

Hawaii

The

complete

sequence

of

one

of

the

transcripts

was

obtained

with

amplification

of

cDNA

ends

-LRB-

RACE

-RRB-

on

uninfected

flies

-LRB-

-RRB-

suggesting

that

this

transcript

has

a

mRNA

cap

a

form

of

eukaryotic

posttranscriptional

modification

Analysis

of

the

transcript

quantities

of

inserted

Wolbachia

genes

with

quantitative

RT-PCR

-LRB-

qRT-PCR

-RRB-

-LRB-

-RRB-

revealed

that

they

are

times

to

times

less

abundant

than

the

fly

's

highly

transcribed

actin

gene

-LRB-

table

-RRB-

There

is

no

cutoff

that

defines

a

biologically

relevant

amount

of

transcription

and

assessment

of

transcription

in

whole

insects

can

obscure

important

tissue-specific

transcription

Therefore

it

is

unclear

whether

these

transcripts

are

biologically

meaningful

and

further

work

is

needed

to

determine

their

importance

Screening

of

public

shotgun

sequencing

data

sets

has

identified

several

additional

cases

of

LGT

in

different

invertebrate

species

In

Wolbachiacured

strains

of

the

wasp

Nasonia

six

small

Wolbachia

inserts

-LRB-

bp

-RRB-

were

verified

by

PCR

and

sequencing

-LRB-

-RRB-

that

have

nucleotide

identity

to

native

Wolbachia

sequences

in

some

cases

with

short

insertion

site

duplications

These

include

four

in

Nasonia

vitripennis

one

in

N.

giraulti

and

one

in

N.

longicornis

-LRB-

table

and

Fig.

-RRB-

Amplification

and

sequencing

of

to

geographically

diverse

strains

of

each

species

indicated

that

the

inserts

are

speciesspecific

For

example

three

Wolbachia

inserts

in

N.

vitripennis

are

not

found

in

the

closely

related

A

wMel

Mel

Ana

wAna

BAC

Dana

wMel

Mel

B

wMel

C

wBm

Bm

NG

NV

D.

immitis

NV

NG

mRNA

NL

NL

Fig.

Schematics

of

Wolbachia

inserts

in

host

chromosomes

-LRB-

A

-RRB-

Contigs

containing

Wolbachia

sequences

generated

from

the

D.

ananassae

Hawaii

shotgun

sequencing

project

are

segregated

into

sequences

coming

from

the

endosymbiont

-LRB-

wAna

-RRB-

or

from

the

D.

ananassae

chromosome

-LRB-

Dana

-RRB-

on

the

basis

of

presence

or

absence

of

eukaryotic

genes

in

the

contigs

These

are

compared

to

those

from

the

reference

D.

melanogaster

Wolbachia

genome

-LRB-

wMel

-RRB-

and

a

D.

ananassae

BAC

NAD

nicotinamide

adenine

dinucleotide

-LRB-

B

-RRB-

Fragments

of

the

Wolbachia

gene

gene

have

inserted

into

different

positions

in

the

N.

giraulti

-LRB-

NG

-RRB-

and

N.

vitripennis

-LRB-

NV

-RRB-

genomes

with

unique

insertions

in

each

lineage

including

N.

longicornis

-LRB-

NL

-RRB-

-LRB-

C

-RRB-

A

region

in

the

D.

immitis

genome

-LRB-

-RRB-

that

is

transcribed

has

introns

similar

to

sequences

from

the

Wolbachia

infecting

B.

malayi

-LRB-

wBm

-RRB-

All

matches

in

-LRB-

A

-RRB-

and

-LRB-

B

-RRB-

have

nucleotide

identity

those

in

-LRB-

C

-RRB-

have

nucleotide

identity

TPR

tetratrico

peptide

repeat

CDS

coding

sequence

VOL

SCIENCE

SEPTEMBER

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from

on

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REPORTS

Table

Summary

of

Wolbachia

sequences

and

evidence

for

LGT

in

public

databases

Junctions

were

validated

by

PCR

amplification

and

sequencing

-LRB-

-RRB-

with

the

number

of

successful

reactions

compared

to

the

number

attempted

Species

marked

with

a

plus

sign

are

described

in

the

literature

as

being

infected

with

Wolbachia

All

whole-genome

shotgun

sequencing

reads

were

downloaded

for

arthropod

and

nematode

genomes

-LRB-

-RRB-

Organisms

identified

as

lacking

Wolbachia

sequences

either

had

no

match

or

matches

only

to

the

prokaryotic

ribosomal

RNA

Because

the

Nasonia

genomes

are

from

antibiotic-cured

insects

they

were

identified

as

having

a

putative

LGT

event

merely

on

identification

of

Wolbachia

sequences

in

a

read

All

other

organisms

were

considered

to

have

putative

LGT

events

if

the

trace

repository

contained

read

with

-LRB-

i

-RRB-

nucleotide

identity

over

of

the

read

to

a

characterized

eukaryotic

gene

-LRB-

ii

-RRB-

identity

over

of

the

read

to

a

Wolbachia

gene

and

-LRB-

iii

-RRB-

manual

review

of

the

BLAST

results

for

to

reads

to

ensure

significance

-LRB-

-RRB-

not

applicable

Organism

Total

traces

screened

Wolbachia

traces

LGT

Junctions

validated

Wolbachia

infection

References

and

Notes

Y.

Boucher

et

al.

Annu

Rev.

Genet

-LRB-

-RRB-

S.

B.

Daniels

K.

R.

Peterson

L.

D.

Strausbaugh

M.

G.

Kidwell

A.

Chovnick

Genetics

-LRB-

-RRB-

K.

E.

Nelson

et

al.

Nature

-LRB-

-RRB-

J.

O.

Andersson

Cell

Mol

Life

Sci

-LRB-

-RRB-

W.

F.

Doolittle

Trends

Genet

-LRB-

-RRB-

R.

Stouthamer

J.

A.

Breeuwer

G.

D.

Hurst

Annu

Rev.

Microbiol

-LRB-

-RRB-

N.

Kondo

N.

Nikoh

N.

Ijichi

M.

Shimada

T.

Fukatsu

Proc

Natl.

Acad

Sci

U.S.A.

-LRB-

-RRB-

K.

Fenn

et

al.

PLoS

Pathog

-LRB-

-RRB-

M.

Wu

et

al.

PLoS

Biol

-LRB-

-RRB-

S.

L.

Salzberg

et

al.

Genome

Biol

-LRB-

-RRB-

Materials

and

methods

are

available

as

supporting

material

on

Science

Online

B.

C.

Campbell

J.

D.

Steffen-Campbell

J.

H.

Werren

Insect

Mol

Biol

-LRB-

-RRB-

J.

Foster

et

al.

PLoS

Biol

-LRB-

-RRB-

S.

Sun

K.

Sugane

J.

Helminthol

-LRB-

-RRB-

S.

H.

Sun

T.

Matsuura

K.

Sugane

J.

Helminthol

-LRB-

-RRB-

K.

Sugane

K.

Nakayama

H.

Kato

J.

Helminthol

-LRB-

-RRB-

J.

H.

Werren

Annu

Rev.

Entomol

-LRB-

-RRB-

J.

H.

Werren

D.

M.

Windsor

Proc

Biol

Sci

R.

Soc

London

Ser

B

-LRB-

-RRB-

The

D.

ananassae

BAC

sequence

is

deposited

in

GenBank

-LRB-

-RRB-

the

D.

ananassae

sequence

comparisons

from

the

four

lines

are

deposited

in

GenBank

-LRB-

to

-RRB-

the

D.

ananassae

RACE

sequence

is

deposited

in

dbEST

-LRB-

-RRB-

and

GenBank

-LRB-

-RRB-

the

Nasonia

sequences

are

deposited

in

This

isolate

was

previously

shown

to

have

Wolbachia

reads

in

its

trace

repositories

that

are

contaminating

reads

from

the

D.

ananassae

genome

sequencing

project

-LRB-

-RRB-

species

N.

giraulti

or

N.

longicornis

which

diversified

million

years

ago

-LRB-

-RRB-

These

data

suggest

that

the

Wolbachia

gene

inserts

are

of

relatively

recent

origin

similar

to

the

inserts

in

D.

ananassae

Nematode

genomes

also

contain

inserted

Wolbachia

sequences

Because

Wolbachia

infection

is

required

for

fertility

and

development

of

the

worm

Brugia

malayi

the

genomes

of

both

organisms

were

sequenced

simultaneously

complicating

assemblies

and

leading

to

the

removal

of

Wolbachia

reads

during

genome

assembly

-LSB-

identity

over

of

the

read

length

on

the

basis

of

the

independent

BAC-based

genome

sequence

of

wBm

the

Wolbachia

endosymbiont

of

B.

malayi

-LRB-

-RRB-

-RSB-

Despite

this

the

genome

of

B.

malayi

contains

contigs

with

Wolbachia

sequences

-LRB-

e

value

-RRB-

nine

of

which

were

confirmed

by

long-range

PCR

and

end-sequencing

-LRB-

-RRB-

These

include

eight

large

scaffolds

containing

Wolbachia

fragments

within

kb

of

a

B.

malayi

gene

-LRB-

table

-RRB-

Comparisons

of

wBm

homologs

to

these

regions

suggested

that

all

of

these

Wolbachia

genes

within

the

B.

malayi

genome

are

degenerate

In

addition

a

single

region

kb

was

examined

that

contains

a

degenerate

fragment

of

the

Wolbachia

aspartate

aminotransferase

gene

-LRB-

-RRB-

Its

location

was

confirmed

by

PCR

and

sequencing

in

B.

malayi

as

well

as

in

B.

timori

and

B.

pahangi

-LRB-

-RRB-

Of

the

remaining

arthropod

and

nematode

genomes

in

the

trace

repositories

-LRB-

-RRB-

we

found

six

containing

Wolbachia

sequences

Potential

Wolbachia-host

LGT

was

detected

in

three

D.

sechellia

D.

simulans

and

Culex

pipiens

-LRB-

Table

-RRB-

as

revealed

by

the

presence

of

reads

containing

homology

to

both

endosymbiont

and

host

genomes

-LRB-

-RRB-

SCIENCE

VOL

SEPTEMBER

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from

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Trace

repository

sequences

Acyrthosiphon

pisum

-LRB-

aphid

-RRB-

Aedes

aegypti

-LRB-

mosquito

-RRB-

Anopheles

gambiae

-LRB-

mosquito

-RRB-

Apis

mellifera

-LRB-

honeybee

-RRB-

Brugia

malayi

-LRB-

filarial

nematode

-RRB-

Culex

pipiens

quinquefasciatus

-LRB-

mosquito

-RRB-

Daphnia

pulex

-LRB-

crustacean

-RRB-

D.

ananassae

-LRB-

fruit

fly

-RRB-

D.

erecta

-LRB-

fruit

fly

-RRB-

D.

grimshawi

-LRB-

fruit

fly

-RRB-

D.

melanogaster

-LRB-

fruit

fly

-RRB-

D.

mojavensis

-LRB-

fruit

fly

-RRB-

D.

persimilis

-LRB-

fruit

fly

-RRB-

D.

pseudoobscura

-LRB-

fruit

fly

-RRB-

D.

sechellia

-LRB-

fruit

fly

-RRB-

D.

simulans

-LRB-

fruit

fly

-RRB-

D.

virilis

-LRB-

fruit

fly

-RRB-

D.

willistoni

-LRB-

fruit

fly

-RRB-

D.

yakuba

-LRB-

fruit

fly

-RRB-

Ixodes

scapularis

-LRB-

tick

-RRB-

N.

giraulti

-LRB-

wasp

-RRB-

N.

longicornis

-LRB-

wasp

-RRB-

N.

vitripennis

-LRB-

wasp

-RRB-

Pediculus

humanus

-LRB-

head

louse

-RRB-

Pristonchus

pacificus

-LRB-

nematode

-RRB-

Tribolium

castaneum

-LRB-

beetle

-RRB-

GenBank

sequence

Dirofilaria

immitis

-LRB-

filarial

nematode

-RRB-

The

sequencing

of

wBm

also

facilitated

the

discovery

of

a

Wolbachia

insertion

in

Dirofilaria

immitis

-LRB-

dog

heartworm

-RRB-

The

D.

immitis

chromosomal

region

encoding

the

immunodominant

antigen

-LRB-

-RRB-

contains

Wolbachia

DNA

within

its

introns

and

in

the

untranslated

region

-LRB-

-RRB-

-LRB-

Fig.

-RRB-

These

Wolbachia

genomic

fragments

have

maintained

synteny

with

the

wBm

genome

-LRB-

-RRB-

suggesting

they

may

have

inserted

as

a

single

unit

and

regions

were

replaced

by

exons

of

A

second

gene

-LRB-

DgK

-RRB-

has

been

identified

in

other

D.

immitis

lines

that

has

nucleotide

identity

in

the

exon

sequences

but

contains

differing

number

position

size

and

sequence

of

introns

-LRB-

-RRB-

and

has

no

homology

to

known

Wolbachia

sequences

Whole

eukaryote

genome

sequencing

projects

routinely

exclude

bacterial

sequences

on

the

assumption

that

these

represent

contamination

For

example

the

publicly

available

assembly

of

D.

ananassae

does

not

include

any

of

the

Wolbachia

sequences

described

here

Therefore

the

argument

that

the

lack

of

bacterial

genes

in

these

assembled

genomes

indicates

that

bacterial

LGT

does

not

occur

is

circular

and

invalid

Recent

bacterial

LGT

to

eukaryotic

genomes

will

continue

to

be

difficult

to

detect

if

bacterial

sequences

are

routinely

excluded

from

assemblies

without

experimental

verification

And

these

LGT

events

will

remain

understudied

despite

their

potential

to

provide

novel

gene

functions

and

affect

arthropod

and

nematode

genome

evolution

Because

W.

pipientis

is

among

the

most

abundant

intracellular

bacteria

-LRB-

-RRB-

and

its

hosts

are

among

the

most

abundant

animal

phyla

the

view

that

prokaryote-to-eukaryote

transfers

are

uncommon

and

unimportant

needs

to

be

reevaluated

REPORTS

GenBank

-LRB-

to

-RRB-

the

Brugia

malayi

contigs

are

available

in

GenBank

-LRB-

and

-RRB-

the

Brugia

malayi

scaffolds

are

available

in

GenBank

-LRB-

and

-RRB-

and

the

microarray

primers

sequences

that

were

used

for

RT-PCR

are

deposited

in

ArrayExpress

-LRB-

-RRB-

This

work

was

supported

by

an

NSF

grant

to

J.H.W.

and

H.T.

a

National

Institute

of

Allergy

and

Infectious

Diseases

grant

to

E.G.

and

New

England

Biolabs

Incorporated

support

to

B.E.S.

Fig

Tables

to

References

March

accepted

July

Published

online

August

science

Include

this

information

when

citing

this

paper

Supporting

Online

Material

Materials

and

Methods

Draft

Genome

of

the

Filarial

Nematode

Parasite

Brugia

malayi

Elodie

Ghedin

Shiliang

Wang

David

Spiro

Elisabet

Caler

Qi

Zhao

Jonathan

Crabtree

Jonathan

E.

Allen

Arthur

L.

Delcher

David

B.

Guiliano

Diego

Miranda-Saavedra

Samuel

V.

Angiuoli

Todd

Creasy

Paolo

Amedeo

Brian

Haas

Najib

M.

El-Sayed

Jennifer

R.

Wortman

Tamara

Feldblyum

Luke

Tallon

Michael

Schatz

Martin

Shumway

Hean

Koo

Steven

L.

Salzberg

Seth

Schobel

Mihaela

Pertea

Mihai

Pop

Owen

White

Geoffrey

J.

Barton

Clotilde

K.

S.

Carlow

Michael

J.

Crawford

Jennifer

Daub

Matthew

W.

Dimmic

Chris

F.

Estes

Jeremy

M.

Foster

Mehul

Ganatra

William

F.

Gregory

Nicholas

M.

Johnson

Jinming

Jin

Richard

Komuniecki

Ian

Korf

Sanjay

Kumar

Sandra

Laney

Ben-Wen

Li

Wen

Li

Tim

H.

Lindblom

Sara

Lustigman

Dong

Ma

Claude

V.

Maina

David

M.

A.

Martin

James

P.

McCarter

Larry

McReynolds

Makedonka

Mitreva

Thomas

B.

Nutman

John

Parkinson

Jos

M.

Peregr

n-Alvarez

Catherine

Poole

Qinghu

Ren

Lori

Saunders

Ann

E.

Sluder

Katherine

Smith

Mario

Stanke

Thomas

R.

Unnasch

Jenna

Ware

Aguan

D.

Wei

Gary

Weil

Deryck

J.

Williams

Yinhua

Zhang

Steven

A.

Williams

Claire

Fraser-Liggett

Barton

Slatko

Mark

L.

Blaxter

Alan

L.

Parasitic

nematodes

that

cause

elephantiasis

and

river

blindness

threaten

hundreds

of

millions

of

people

in

the

developing

world

We

have

sequenced

the

megabase

-LRB-

Mb

-RRB-

genome

of

the

human

filarial

parasite

Brugia

malayi

and

predict

protein

coding

genes

in

Mb

of

robustly

assembled

sequence

Comparative

analysis

with

the

free-living

model

nematode

Caenorhabditis

elegans

revealed

that

despite

these

genes

having

maintained

little

conservation

of

local

synteny

during

million

years

of

evolution

they

largely

remain

in

linkage

on

chromosomal

units

More

than

conserved

operons

were

identified

Analysis

of

the

predicted

proteome

provides

evidence

for

adaptations

of

B.

malayi

to

niches

in

its

human

and

vector

hosts

and

insights

into

the

molecular

basis

of

a

mutualistic

relationship

with

its

Wolbachia

endosymbiont

These

findings

offer

a

foundation

for

rational

drug

design

he

phylum

Nematoda

is

speciose

and

abundant

and

although

most

species

are

free-living

many

are

parasitic

Over

onethird

of

all

humans

mainly

in

the

developing

world

carry

a

nematode

infection

Parasitic

worms

typically

cause

chronic

debilitating

infections

that

are

often

difficult

to

treat

and

that

despite

the

high

cost

to

human

health

have

been

neglected

in

biomedical

research

Current

knowledge

of

nematode

molecular

genetics

and

developmental

biology

is

largely

based

on

extensive

studies

of

the

free-living

bacteriovorous

species

Caenorhabditis

elegans

Here

we

present

the

initial

analysis

of

the

genome

of

the

human

filarial

parasite

Brugia

malayi

Brugia

malayi

is

endemic

in

Southeast

Asia

and

Indonesia

Like

other

filarial

nematodes

B.

malayi

develops

through

four

larval

stages

into

an

adult

male

or

female

-LRB-

fig

-RRB-

entirely

within

one

of

two

host

species

a

mosquito

vector

-LRB-

Culex

Aedes

and

Anopheles

-RRB-

and

humans

where

adult

worms

can

live

for

more

than

a

decade

B.

malayi

was

chosen

for

wholegenome

sequencing

-LRB-

-RRB-

because

it

is

the

only

T

major

human

filarial

pathogen

that

can

be

maintained

in

small

laboratory

animals

Most

filarial

nematodes

including

B.

malayi

carry

three

genomes

nuclear

mitochondrial

-LRB-

available

at

GenBank

accession

no

-RRB-

and

that

of

an

alphaproteobacterial

endosymbiont

Wolbachia

We

present

here

the

draft

assembly

and

annotated

genome

of

the

TRS

strain

of

B.

malayi

We

provide

comparative

analyses

with

Caenorhabditis

and

another

well-annotated

member

of

the

superphylum

Ecdysozoa

Drosophila

melanogaster

to

further

illuminate

the

origins

of

novelty

and

loss

of

ancestral

characters

in

the

model

species

and

the

parasite

Comparative

genome

analysis

reveals

key

features

of

Nematoda

that

define

the

scope

of

molecular

diversity

that

has

contributed

to

the

success

of

the

phylum

The

analysis

also

uncovers

adaptations

that

appear

to

have

evolved

in

the

B.

malayi

genome

in

response

to

the

pressures

of

parasitism

and

to

the

presence

of

the

parasite

's

Wolbachia

endosymbiont

wBm

The

B.

malayi

nuclear

genome

is

organized

as

five

chromosomes

-LRB-

-RRB-

including

an

XY

sexVOL

SCIENCE

determination

pair

and

has

been

estimated

to

be

to

megabases

-LRB-

Mb

-RRB-

-LRB-

-RRB-

The

sequence

of

the

B.

malayi

nuclear

genome

was

obtained

to

coverage

with

the

use

of

whole-genome

shotgun

-LRB-

WGS

-RRB-

sequencing

-LRB-

-RRB-

The

sequences

were

assembled

into

scaffolds

totaling

Mb

of

data

with

a

further

Mb

of

contigs

not

integrated

into

any

scaffold

-LRB-

orphan

contigs

-RRB-

The

repeat

content

of

the

B.

malayi

genome

estimated

at

-LRB-

-RRB-

may

have

contributed

significantly

to

assembly

difficulties

-LRB-

-RRB-

From

these

sequence

data

we

estimate

that

the

B.

malayi

genome

is

to

Mb

-LRB-

Table

-RRB-

-LRB-

-RRB-

In

comparison

the

C.

elegans

genome

is

Mb

and

the

Caenorhabditis

briggsae

genome

Mb

The

overall

G

C

content

-LRB-

-RRB-

is

lower

than

that

of

C.

elegans

-LRB-

-RRB-

or

C.

briggsae

-LRB-

-RRB-

-LRB-

-RRB-

The

complement

of

protein-coding

genes

was

derived

by

automated

gene

prediction

from

the

assembly

and

by

manual

annotation

of

selected

gene

families

-LRB-

table

-RRB-

The

robustly

predicted

gene-coding

regions

occupy

of

the

sequence

at

an

average

density

of

genes/Mb

-LRB-

Table

-RRB-

After

inclusion

of

genes

estimated

to

be

found

in

the

unannotated

portion

of

the

genomic

sequence

-LRB-

-RRB-

we

infer

that

B.

malayi

has

between

and

protein-coding

genes

agreeing

with

previous

estimates

-LRB-

-RRB-

Even

the

higher

estimate

is

lower

than

the

-LRB-

WormBase

data

release

-RRB-

and

-LRB-

-RRB-

genes

reported

for

C.

elegans

and

C.

briggsae

respectively

which

suggests

that

parasitic

nematode

genomes

have

fewer

genes

than

their

free-living

counterparts

echoing

a

pattern

observed

in

bacterial

pathogens

For

the

six

scaffolds

longer

than

Mb

totaling

Mb

of

the

genome

the

arrangement

of

B.

malayi

genes

was

compared

with

that

of

their

C.

elegans

orthologs

-LRB-

Fig.

-RRB-

Linkage

is

in

general

conserved

For

large

regions

of

the

B.

malayi

genome

orthologs

map

predominantly

to

one

-LRB-

or

in

the

case

of

scaffold

two

-RRB-

C.

elegans

chromosome

-LRB-

s

-RRB-

-LRB-

Fig.

A

to

C

-RRB-

which

indicates

maintenance

of

linkage

of

these

genes

despite

million

years

of

separation

-LRB-

-RRB-

However

local

gene

order

is

not

conserved

-LRB-

Fig.

-RRB-

The

largest

scaffold

contains

interdigitating

blocks

of

genes

that

map

to

chromosomes

and

X

of

C.

elegans

which

suggests

there

were

ancient

breakage

and

fusion

events

between

linkage

groups

These

data

support

a

model

where

within-linkage

group

rearrangements

have

been

many

times

more

common

than

between-linkage

group

transloca

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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