Research Extracting biological information from DNA arrays: an unexpected link between arginine and methionine metabolism in Bacillus subtilis

Transcription

1 Research Extracting biological information from DNA arrays: an unexpected link between arginine and methionine metabolism in Bacillus subtilis )C EAI =5A MI = 5J FD= A4 >E V A= =?GKAI,=K@E V ) =E 0 =KJ W J E A,=?DE )@@HAIIAI 0 C C7 ELAHIEJO2=IJAKH4AIA=H?D+A JAH 2 BK = 0 C C V 1 IJEJKJ =JE = )CH EGKA2=HEI /HEC %#! 2=HEI+A@AN# H=?A W /A B H =JE?I 7 AI 5=E J 3KA JE %&!#8AHI=E AI+A@AN H=?A /A AJE?I B *=?JAHE= /A AI 1 IJEJKJ2=IJAKH %#% "2=HEI+A@AN# H=?A + ) J E A,=?DE - =E =@=?DE (D K?? D K D =@=?DE (F=IJAKH BH Published: 1 June 2001 Genome Biology 2001, 2(6):research The electronic version of this article is the complete one and can be found online at Sekowska et al., licensee BioMed Central Ltd (Print ISSN ; Online ISSN ) Abstract Background: In global gene expression profiling experiments, variation in the expression of genes of interest can often be hidden by general noise. To determine how biologically significant variation can be distinguished under such conditions we have analyzed the differences in gene expression when Bacillus subtilis is grown either on methionine or on methylthioribose as sulfur source. Results: An unexpected link between arginine metabolism and sulfur metabolism was discovered, enabling us to identify a high-affinity arginine transport system encoded by the yqixyz genes. In addition, we tentatively identified a methionine/methionine sulfoxide transport system which is encoded by the operon ytmijklmhisp and is presumably used in the degradation of methionine sulfoxide to methane sulfonate for sulfur recycling. Experimental parameters resulting in systematic biases in gene expression were also uncovered. In particular, we found that the late competence operons come, comf and comg were associated with subtle variations in growth conditions. Conclusions: Using variance analysis it is possible to distinguish between systematic biases and relevant gene-expression variation in transcriptome experiments. Co-variation of metabolic gene expression pathways was thus uncovered linking nitrogen and sulfur metabolism in B. subtilis. Background /A AFH CH= ID=LAK? LAHA@=L=IJ K >AH BCA AI B K M BK?JE FH FJE C AMJOFAI BBK?JE = E LAI JEC=JE 1? K?JE MEJD ANFHAIIE FH BE E C ANFAHE A JI M A@CA B CA A IAGKA?AI FH LE@AI = ANJHA A O F MAHBK =FFH =?D J E JAHFHAJE C C >= CA A ANFHAIIE F=JJAH I 2=H=@ NE?= OD MALAH E JAHFHAJE C= JDAE B H =JE F JA JE= O=L=E => ABH JDAIAANFAHE A JI Received: 26 March 2001 Revised: 4 May 2001 Accepted: 4 May 2001 EI >O JDA LAHO =??KH=?O B JDA FDOIE?= A=IKHA A JI E L LA@ I JD=J JDA E F HJ=?A B JDA B K?JK=JE I E DAHA JJ >E CE?= ANFAHE A JIEI 6DEI =?? K JIB HJDAA FD=IEIF =?A@>OI A IE F A@AI?HEF JELAIJK@EAI BCA AANFHAIIE MDAHAF=JJAH IIFA?EBE?J = F=HJE?K =HIAJ BCA AJE? HA LEH A I=HA@EI F =OA@ KIK= O B IJE? FKHF IAI HJK =JA O JH= I?HEFJE FH BE E C?= JA KI K?D HA DA F J comment reviews reports deposited research refereed research interactions information

2 2 Genome Biology Vol 2 No 6 5A MI =AJ= K? LAH AMCA ABK?JE I *AB HA K FE CJ?? KIE I D MALAH EJEI A?AII=HO MDA >IAHLE CL=HE=JE E = =HCA K >AH BCA AI J >A=> CKEIDIFA?EBE?>E CE?= ABBA?JIBH >=? CH =JE?L=HE=JE I@KA J JDAFH?A@KHAEJIA B 1 JDAFHAIA JM H MDE?DKIAI*=?E KIIK>JE EI MAD=LA= = O A@EJIJH= I?HEFJ AANFHAIIE I?D IA J ANJHA A O IE E =H JH= I?HEFJE F=JJAH I CH MJDE JDAFHAIA?A BAEJDAH AJDE E A H AJDO JDE HE > IA 64 =I JDA IK BKH I KH?A LAHA@ JD=J ALA MEJD = JDA >=? CH IJH=E JI =?JE C JDA ANFAHE A E =HEJOE JDACH I MA? ANJH=?J >E CE?= O IEC EBE?= J >IAHL=JE I = A O JD=J JDAHA ANEIJA@ = IJH C E >AJMAA IK BKH AJ=> EI E A AJ=> EI 9A= I ID MJD=J E =@@EJE J C >E CE?= O HA AL= J F=JJAH I JDA E L K J=HO JHEC CAHE C B JH= IEJE I E A LEH A I MEJD JDA HACK =OI= E F HJ= JH AE?HA=JE C IOIJA =JE? >E=IAI E JDA ANFHAIIE B I A FAH I 6DA? F AN>E?DA EIJHO B?, )IO JDAIEIBH 4 )EI= I =IEC EBE?= JI KH?A BIOIJA =JE?AHH HI MDE?D = AI EJ J J? F=HA ANFAHE A JI FAHB H A@ I ALAHJDA AII MAMAHA=> AJ IAJKF=IE F >KIJIJ=JEIJE?= FH J? B HJH= I?HEF J = = OIEI JD=J A => AI A CKEID B K?JK= JE IE ANFHAIIE BJDACA AI BE JAHAIJBH B K?JK=JE F=H=IEJE?ABBA?JI 6DEIFH AI JHAGKEHA>=? CH EIA IK>JH=?JE H H = E =JE B = J HE KI O@EBBE?K JH LAHIE= J=I! 9AD FAJD=J E =@@EJE J FH LE@E CK ANFA?JA@E B H = JE => KJ IK BKH AJ=> EI E *=?E KI IK>JE EI = FHALE KI O K HA? H@A@ HA =JE IDEF >AJMAA JDA AJ=> EI B =HCE E A JD=J B AJDE E A =I MA =I AJ=> EI B AJDE E A IK B NE@A JDEI M H JDA =JJA JE B JDA A?K =H >E CO? K EJO J JDA HEI I B E? HHA?J E JAHFHAJ=JE E DAHA J E JDA =HCA >E CE?= L=HE=>E EJO B JH= I?HEFJ AANFAHE A JI Results Effect of the sulfur source on gene expression 8=HE=?A= = OIEIID MA@JD=JJDA IJE F HJ= JI KH?A B >E=IE? F=HE C@EBBAHA JANFAHE A JIEI=@EBBAHA?AE JDA = K J B4 )KIA@J IO JDAIE A?, ) E =?AEI JDA@EBBE?K JO BAN=?J CANFAHE A I BH A ANFAHE A J J = JDAH IAA =JAHE= I )I ANFA?JA@ L=HE=?A = = OIEI ID MA@ JD=J?D= CE C JDA IK BKH I KH?A BH AJDE E A J 64 D=I EJJ AABBA?J JDAJH= I?HEFJE ALA B IJCA AI 1 JAHAIJ E C O D MALAH JDA EIJ B JDA IJ L=HE=> A CA AI D=I BA=JKHAI 1 JDEI CH KF CA AI B M BK?JE =HA IEC EBE?= J O HA K AH KI JD= ANFA?JA@ BH JDAEHFH F HJE E JDACA A MEJDF $ KHJDAH HAJDAIACA AI? HHA =JA@BK?JE I /H MJD E AJDE E A EI?D=H=?JAHE A@ > JD >O JDA DECDAH ANFHAIIE BJDA=HCE E A AJ=> B FAH OGE:; MDE?DEIJD H= = E =?E@ )*+ FAH A=IA BK?JE MDAHA=I CH MJD 64 =?JEL=JAI IALAH= CA AI E L LA@ E = ALE=JE C NOCA J NE?EJO?=J= =IA DO@H FAH NE@AHA@K?J=IA NOCA =IA =IMA =I= C FAH C = FAH A=IA M J >A? A?JA@ MEJD IK BKH AJ=> EI OJ 1 DEI2OJ OJ 1 HE>4DEF OJ " # 6DKI IALAH= CA AICH KFJ CAJDAHE = FDOIE CE?= O? IEIJA J FE?JKHA NE@ HA@K?JE =HCE E A>E IO JDAIEI )*+FAH A=IAI 4A =H => O CA AIFKJ=JELA OANFHAIIA@=I= FAH $ =FFA=HJ CAJDAH E JDA EIJ OGE:; =HC/0OJ, =HC, OJ 1 2OJ E = O IALAH= CA AI>A CJ JDAFHA?A@E C I A >AHI B = FKJ=JELA FAH? /+? /, OG - 6=> A IK =HE AIJDAE B H =JE =L=E => A JDAIACA AI Arginine biosynthesis 1? JH=IJJ JDAIEJK=JE E = O JDAH>=?JAHE= JDA=HCE E A>E IO JDAIEICA AIE * IK>JE EI=HA IF EJE J JM F=HJI =HC- JD KCDJ B H =?AJO H EJDE =IA -+! # $ EIF=HJ B=JDEH@ FAH AII? A=H O E A@J =HCE E A AJ=> EI 6DEI IKCCAIJI I A? F=HJ A J= E =JE B JDA? C AJ=> EJAI =I E AK =HO JAI MDAHA =HCE E A >E IO JDAIEI EI IF EJ >AJMAA JDA JDA?OJ F =I 8=HE=?A = = OIEI ID MI JD=J > JD IAJI BCA AI=HAIJE K =JA@MDA?A ICH M AJDE E A =IJDAIK BKHI KH?A6=> A 7IE CJDA=>I KJAL= KAIB HDO>HE@E =JE E I MA B JD=J JDA F=JDM=O E? K@E C?EJHK E A H EJDE A=?AJO JH= IBAH=IA-+!!# =?AJO H EJDE A = E JH= IBAH=IA -+ $ H EJDE A?=H >= O JH= IBAH=IA-+!!M=IIJE K =JA@ $ JDA F=JDM=O A=@E C BH?EJHK E A E? K@E C =HCE E A =HCE E IK??E =JA IO JD=IA -+ $! " # =HCE E IK??E =JA O=IA -+ "! ;J, MDE?D D=I = K M HACK =J HO BK?JE M=I IJE K =JA@ % 6=> A ;J,EI=I = DO@H FDE E?FH JAE HA =H => AB H EJI DECD? JA J B AJDE E A EI JDAHAB HA K KIK= O HE?DE IK BKH 6DA FAH =HC/0OJ, EIB AE JDA EIJ E 6=> A MDAHA=I B H JDA FAH =HC+ *,?=H)*=HC A O CA AI =HC =HC, =HC E 6=> A 6DEIFH FJA@KIJ =JJDA=>I KJAL= KAIB B H= CA AI BJDA FAH? IEIJA JMEJD LAHANFHAIIE B JDA FAH MA B JD=J = JDAH CA AI E JDEI FAH AN?AFJ?=H) MAHA = I LAHANFHAIIA@ E AJDE E A 6=> A J ID M KF = C JDA BEHIJ LAHAN FHAIIA@CA AIIE F O ME CJ JDAB=?JJD=JJDAHAM=I HA L=HE=>E EJO BH ANFAHE A J J ANFAHE A J B H JDAIA CA AI HAIK JE C E = MAH F IEJE E JDA L=HE=?A = = OIEI EIJ 6= A J CAJDAH D MALAH JDAIA HAIK JI ID M JD=J > JD FAH LAHANFHAIIA@ =I=MD A E AJDE E A CH I )I=? JH MA ANFHAIIE BJDA=HC5 CA A MDE?DEIFHAIA J E = A JEHA O@EBBAHA J FAH BK M BK?JE

3 Table 1 Function of genes showing the largest differences as a function of the sulfur source Gene Mean Standard Function deviation argd Arginine biosynthesis argf Arginine biosynthesis argg Arginine biosynthesis argh Arginine biosynthesis argj Arginine biosynthesis comgc Late competence operon required for comgd Late competence operon required for levd PTS system - fructose-specific IIA component ycbd Probable aldehyde dehydrogenase yqix Probable amino-acid ABC permease (binding protein) yqiy Probable amino-acid ABC permease (permease) yqiz Probable amino-acid ABC permease (ATP-binding protein) yqze Hypothetical protein ytzd Hypothetical protein, downstream gene of operon arggh ahpf Alkyl hydroperoxide reductase (large subunit) - reduces organic hydroperoxides in their reduced dithiol form kata Vegetative catalase ytmi Possible methionine sulfoxide acetyl transferase (operon ytmijklmhispytmoytnijribrhipoytnm) ytmj Methionine - methionine sulfoxide ABC permease (binding protein) (this paper) ytml Methionine - methionine sulfoxide ABC permease (permease) (this paper) ytmo Methionine sulfoxide monooxygenase (this paper) The yqixyz permease 1J M=I ANFA?JA@ JD=J FAH A=>E EJO J AJDE E A M >A =BBA?JA@E KJ= JI BI A BJDACA AIE L LA@E CH MJD AJDE E A )I JDA OGE:; CA AI D=LA IEC EBE?= J IE E =HEJOJ = E =?E@FAH A=IACA AI MAE LAIJEC=JA@FAH A =>E EJO B KJ= J >=?JAHE= J AJDE E A =HCE E A J = E AJDE E A =L=E =>E EJO E KJ= JI I E JDA FKJ=JELA FAH A=IA CA AI OJ DEI2 JID M 1? JH=IJ MA B JD=J CH MJD B KJ= JI E = O B JDA OGE:; CA AI M=IIJH C OE F=EHA@E M??A JH=JE I B=HCE E A=I= I A EJH CA I KH?A 6DA >IAHL=JE JD=JCH MJD AJDE E A M=I HA =JA@ J =HCE E A?= A =I = IKHFHEIA 9A J =@L= J=CA B JDA B=?J JD=J =HCE E A EI = EJH CA I KH?A B H * IK>JE EI J ANF HAJDA=BBE EJO BJDEIFKJ=JELAFAH A=IA B H =HCE E I /H MJD =HCE E A=IJDA EJH CA I KH?A M=I E F=EHA@ E = B JDA JDHAA KJ= JI OGE: OGE; HOGE ECKHA JID M 6 AL= K =JAJDAABBE?EA?O BJDAFAH A=IA MA? F=HA@CH MJD E?HA=IE C=HCE E A??A JH=JE I ECKHA 6DAFAH A=IA = MI?A IJ CH M=JD= BJDA =NE K CH MJDH=JAMDA =HCE E AEI=J=??A JH=JE B! E JDAIAE I 6DEI FAH JDAHAB H=DECD =BBE EJO=HCE E A FAH A=IA IE E =H J JDA =HJ213 FAH B -I?DAHE?DE=? E % 1? JH=IJ JDA=BBE EJO BJDAHAIE@K= FAH A=IAIEI K?D MAH BJDA H@AH B# ECKHA Methionine sulfoxide metabolism: the ytmijklmhispytmoytnijribr operon 6DA AJ=> EI B AJDE E 64M=IBKHJDAHE LAIJE C=JA@ *A?=KIAJDAFAH A=IAB H 64EIK M = >LE AIOJ DEI2 /H MJD B KJ= JI E JDAIA CA AI 64 M=I K =BBA?JA@ D MALAH@=J= JID M )IE JDA?=IA BOGE:;? F=HE I MEJD M CA H=FAH A=IA E A O J JH= IF HJ = E =?E@I H JDAEH = = CI 6DA BEHIJCA AE JDEI FAH OJ 1 EI E A H= =?AJO =IA A?= ANFA?J JDAHAB HA JD=JJDAJH= IF HJA@IK>IJH=JA EI=?AJO =JA@>AB HA>AE C AJ=> E A@ 6DEIIKCCAIJIJD=JJDA IK>IJH=JA D=I J BH = IJH=ECDJB HM=H@ E L LA A JE =FH?AIIMDAHAEJM AJAHE KI 5AGKA?A= EC A JI BCA AIJD=J=HA=@ =?A J J JDA FAH A=IA CA A E JDA FAH KIE C FH JAE J ID M IKCCAIJA@ JD=J ;J ;J 1 4E>4 J NOCA =IA =?JE C = IK BKH? J=E E C IK>IJH=JA " # 6DEI A O A? L LA@E I AIJAF B 64@ACH=@=JE /H MJD 64 M=I D MALAH K =BBA?JA@ E JDA? C KJ= J ID M 9A JDAHAB HA =JJA FJA@ J IAA MDAJDAH JDEI FAH M=I E L LA@ E B AJDE E A AJDE E A IK B NE@A FH?AIIAI JD=J I?=L A CA IK BKH FHALA J AJDE E A IK B NE@A CAJJE C E J FH JAE IO JDAIEI /H MJD JOFA>=?JAHE= AJDE E A IK B NE@A M=I >AJJAH JD= AJDE E A = =HCAH CH MJD HACE BH E FHAC =JA@ MEJD µ B = I KJE IKCCAIJE C JD=J JDA B H AH EI =? IK>IJH=JA E JDA A LEH A J /H MJD M=I O I ECDJ O =BBA?JA@ E JDA KJ= JI OJ OJ MDA JDAO MAHA CH M E JDA FHAIA?A B = DECD??A JH=JE B AJDE E AIK B NE@A E = O CH MJDM=I >IAHLA@MEJD AJDE E AIK B A AEJDAHE HJDA KJ= JI AN? K@E CJDEIIK>IJ=?A=I= E JID M 9AJDAHAB HAE LAIJEC=JA@JDAIEJK=JE =J M??A JH=JE I BAEJDAH AJDE E A H AJDE E AIK B NE@A J O MEJD JDA FAH A=IA CA A KJ= JI >KJ = I comment reviews reports deposited research refereed research interactions information

4 4 Genome Biology Vol 2 No 6 5A MI =AJ= Table 2 Expression of arginine operons when cells were grown with methionine or with MTR as the sulfur source 10 WT yqix Gene and spot Met MTR Ratio argb a argb b argc a argc b argd a argd b arge a arge b argf a argf b argg a argg b argh a argh b argj a argj b args a args b cara a cara b carb a carb b yqze a yqze b yqix a yqix b yqiy a yqiy b yqiz a yqiz b cdna hybridization after growth in methionine or MTR. The conditions are chosen to display roughly identical values for most genes in both conditions. The values obtained for the arginine operons described in the text are shown here. They indicate a higher expression in methionine as compared to MTR, all things being otherwise equal. Each spot in the experiment is referred to as a and b. MEJD= KJ= J BCA A OJ )IID M E ECKHA KJ=JE I E JDAIA CA AI IEC EBE?= J O E F=EHA@ CH MJD > JD JDAIA IK>IJH=JAI ID ME CJD=JJDAFAH A=IAEI=JH= IF HJIOIJA B HJDAIA AJ=> EJAI CJD=J AJDE E FH?AA@I =J A=IJE F=HJ JDH KCD NOCA =JE OD 600nm Arginine concentration (mm) Figure 1 Comparison of the growth of B. subtilis wild type (WT) and yqix mutant (BFS4740) in different concentrations of arginine as the nitrogen source in glucose minimal medium (from 10 µm to 20 mm; note that arginine is toxic at 20 mm). Both strains were grown at 37 C with agitation (180 rpm). The OD was taken for both strains after 8 h growth, when they entered stationary phase (due to nitrogen limitation, except for the highest arginine concentrations). 6DEI >IAHL=JE M=I BKHJDAH IK>IJ= JE=JA@ >O JDA >IAHL= JE JD=JJDA KJ= JIMAHA HAHAIEIJ= JJ IA A AJDE E AJD= JID M Discussion 1 IJ>=?JAHE= IK BKH AJ=> EI D=I J>AA IJK@EA@E =I K?D@AFJD=I?=H> EJH CA HFD IFD HKI AJ=> EI KHHA?A J= = OIEI BJDA@EIJHE>KJE BIK BKH AJ=> EI CA AIE -? E ID MA@JD=JJDAO=HA? KIJAHA@J CAJDAH E J EI & 6DEI JD=J IA A?JE FHAIIKHA D=I B H?A@ JDA? C CA AI J CH KF J CAJDAH H JD=J JDAO D=LA HA =E A@ CH KFA@ J CAJDAH BH LAHO A=H O JE AI E JDA DEIJ HO B EBA 5K BKH HA?O? E C E F=HJE?K =H M=IB F =O= E F HJ= JH AE?A K =HIK BKH AJ=> EI MEJD AJDO JDE =@A IE A 6) >AE C E F HJ= J JDEIEI@AHELA@BH = O?A HA=?JE F=HJE?K =HJDA IO JDAIEI B F O= E AI " 6DA E B 6) 64 EI JHA?O? A@E -? E >KJEIABBE?EA J OHA?O? A@E * IK>JE EI FHAIK => O=I AJDE E A KH FHA E E =HO ANFAHE A JI ID MA@ JD=J?A I CH ME C 64 =I JDA IK BKH I KH?A >AD=LA@ I CH ME C AJDE E A 6DEI IKCCAIJA@ JD=J O = I = IAJ B CA AI M=IE L LA@E JDAHA?O? E CFH?AII 9 IIE> AJ E@A JEBOI A BJDA 9A D=LA KIA@ ANFHAIIE FH BE E C J E@A JEBO JD IA CA AI JD=J=HA@EBBAHA JE= OANFHAIIA@MDA AEJDAH 64 H AJDE E A EI KIA@ =I JDA IK BKH I KH?A J J FH LE@A = C >=

5 Percentage of wild type growth WT Met ytnj Met ytmj Met ytmk Met Met or MetO (100µM) WT MetO ytnj MetO ytmj MetO ytmk MetO Figure 2 Comparison of the growth of B. subtilis wild type (WT) with ytnj, ytmj and ytmk mutants (BFS70, BFS85 and BFS86, respectively) on methionine (Met) and methionine sulfoxide (MetO) at 100 µm concentration. Growth was measured after 7 h and is represented as a percentage of wild-type growth. FH BE AMDE?DM KIJ>AKIA@B H@E=C IJE?FKHF IAI 1 FHE?EF A ANFHAIIE FH BE E C= MI AJ E LAIJEC=JAJDA? A?JELA>AD=LE H B=CH KF BCA AI BE JAHAIJ>KHEA@E JDABK CA A 9DA JDAHAEI K?DK? JH A@L=HE=JE E JDA KJ? A B DO>HE@E =JE D MALAH EJ >A? B K?JK=JE BH =IFA?EBE?ABBA?J 6DA M H FHAIA JA@ DAHA M=I E J E@A JEBO =J A=IJ I A B JDA?=KIAI B L=HE=JE E ANFHAIIE FH BE E C E >=?JAHE= IE F A B K?JK=JE I IOIJA =JE? >E=IAI JHOE C J ANJH=?J BH CA AH= >=? CH EIA JD IA BAM CA AI JD=J@EIF =O@EBBAHA JE= ANFHAIIE 6DAB=?JJD=JMA?= AKF MEJD=I CE?= ANF = =JE BJDAL=HE=JE I@A IJH=JAIJD=J KHANFAHE A J= M H F = EIABBE?EA J F=LE C JDA M=O B H BKHJDAH HA? F AN HAIA=H?D KIE CJH= I?HEFJ A= = OIEI Statistics 9AD=LA?D IA J KIAL=HE=?A= = OIEI =IJ=JEIJE?= =FFH =?D JD=J D=I C >AA KIA@ >O A? EIJI I?EA JEIJI MD MEJD= =HCA K >AH I MDAHA JDA? JH B JDA A LEH A J EI F H 6DAIAJA?D EGKAID=LA>AA L= E@=JA@JDH KCD HAJD= $ OA=HI B D=H@ M H MA JDE JDAO ID >A HA BHA GKA J O? IE@AHA@ B H =HCA I?= A CA E?I ANFAHE A JI JDAHJA?D EGKAI IK?D=IH= = = OIEI =HA= I HA AL= J >KJ L=HE=?A = = OIEI EI KIABK B H = = O E C JDA =JKHA B IOI JA =JE? AHH HI=BBA?JE C@=J= MDA? IE@AHE C= =HCA K >AH B@=J=F E JI 6DA>=IEI BJDAIAJA?D EGKAIEIJD=J@=J=JHA=J A JFHE HJ = = OIEI KIJ>AHA@K?A@J = E E K B HAN= F A A ID JD=LAJ IK>JH=?J>=? CH KAI HKIAH=JE A=IKHA A JI >A?=KIAJDEI?= O>A@ AMEJD KJE C IFA?EBE? >E=I DECD O? IJH=E A@ I MDE?D =HA?AHJ=E O J BK BE A@ E JH= I?HEF J A ANFAHE A JI 5E E =H O A ID J E H = E I >=IA@ OE C >E CE?= DOF JDAIAIB HAN= F A KIE CJDA=LAH=CA BI AD KIA AAFE CCA AI MDE?D=HAIKFF IA@J >AANFHAIIA@=J=? IJ= J ALA >A?=KIAJDEIE =JE?>E=I MEJD JDA=IIK FJE JD=J B HJM CELA CA AJE? HA LEH A I JDAANFHAIIE ALA BIK?D? JH CA AIM JL=HO = OANFAHE A JI ' ID MJD=JJDEIEI?AHJ=E O JJHKAE CA AH= 6DA KJ? A B JDA L=HE=?A = = OIEI EI J E@A JEBO JD IA CA AI JD=J L=HO E =? IEIJA J = AH B H = CELA ANFAHE A J= IAJ KF?A JDAIA CA AI D=LA >AA E@A JEBEA@ B? KHIA EJ>A? AI A?AII=HOJ = A=GK= JEJ=JELAAL= K= JE B JH= I?HEFJE JH= I =JE E I B E JAHAIJJ AL= J Preparation of the cdna *=?JAHE= ANFHAIIE FH BE E CEIF=HJE?K =H O@EBBE?K JJ IAJKF >A?=KIA E? JH=IJ J AK =HO JAI J = A IJ=> AF O=@A O =JA@ 4 ) 1 B=?J JDA=LAH=CAJKH LAH B 4 ) KIK= CH I B HC= EI I IK?D=I-? E IK>JE EI EI BJDA H@AH B E KJAI )I ID M E KH= = OIEI ANFAHE A J= >E=IAI EA=JJDA ALA B 4 ) HA? LAHO IO JDAIEI B = =>A A@?, )? FO B H DO>HE@E =JE J JDA, )=HH=OI 6DA= K J B?, )IO JDAIE A@EI JFH F HJE = J JDA= K J B4 HIA B?, )IO JDAIEI=FFA=HIJ L=HOBH CA AJ CA A =LAH=CA= K JI B 4 )IFA?EAICELE CJDA IJL=HE=> A?, ) KJ? A 1 CA AH= JDAHA=I CELA B HKIE C =HCAH = K JI B 4 ) EI J =L E@ JDA IEJK=JE MDAHA JDA MAIJ ANFHAIIE ALA I=HADE@@A E JDA>=? CH EIA 1? JH=IJ EJ EI JDA BA=H JD=J JDA A >H= A M >A I=JKH=JA@ A F=H@E AJDA=??KH=?O BJDAL= KAI? C J JDA DECDAIJ ANFHAIIE ALA I JD=J CKE@AI JDA?D E?A B = I = = K J B4 )E I AANFAHE A JI 9AAL= K=JA@JDAE F HJ=?A BJDA>E=IAI@KAJ JDA>=? CH EIA JDAI=JKH=JE FH > A JDA JDAH>OIJK@OE CJDA? HHA =JE IE A=IKHA A JI FAHB H A@ KIE C AEJDAH µc H µc 4 ) BH JDA I= A ANJH=?J ) II BIA IEJELEJO@KAJ JDA>=? CH EIAME = EBAIJEJIA B=I= M? HHA =JE >AJMAA JDAH= H@AH B JDACA AIE A=?DANFAHE A JCA AID=LE C>AA H= A@=I= BK?JE B JDAEH IEC = E JA IEJO B H JDA CA AI CELE C M = K JI B?, ) LAHIA O I=JKH=JE ME A=@J = comment reviews reports deposited research refereed research interactions information

6 6 Genome Biology Vol 2 No 6 5A MI =AJ= F H? HHA =JE B H CA AI CELE C =HCAH = K JI B?, ) A BJDAIA>E=IAID=@=IEC EBE?= JE B KA?AE KHANFAH E A JI 1 JDA=>IA?A BIK?D>E=I JDAH= E C BJDACA AI EIJA@=I=BK?JE BJDAIEC = E JA IEJO KIJ>AJDAI= A E A=?D ANFAHE A J 6DEI EI J MD=J D=I >AA >IAHLA@ BJDAI?= A ) ANF = =JE B H JDEI =? B? HHA =JE D=I >AA FH F IA@ E I A CA AI?, ) IO JDAIEI =O >A =BBA?JA@ >O JDAB H =JE JDA 4 ) H>OJDA FHAIA?A B4 )IAC A JI BDECD O>E=IA@ K? A JE@A? F IEJE 6DAIA I MDAHA JDA ABBE?EA?O B?, )IO JDAIEI ECDJ>A=BBA?JA@B H =HCA4 )??A JH= JE I )JD H KCDIJK@O BJDAIAGKA?A BCA AICELE CJDA IJAHH=JE?DO>HE@E =JE L= KAI =O= M AJ AIJE =JA JDAL= KA BJDEIDOF JDAIEI IJH=ECDJB HM=H@HA=I? >A B E JDA FHAIA J ANFAHE A JI IJH=JA@ M H AHI JDAKJE E =JE DAN= K? A JE@A FHE AHI ID = ALE=JA I A B JO = A O 4 C A=HJDA! BJDA 4 ) 6DAEH M J D MALAH ANF IJDAABBA?J B4 ) GK= JEJEAI JDA KJ? A BJDADO>HE@E =JE F=JJAH EJ =O MA >A JD=J >E=IAI IE E =H J JD DAHA M >A K? LAHA@ E = O JDA H A B? FAJEJE B = =HCA K >AH BFHE AHI B@EBBAHA JIAGKA?AI =IMA =I B JDA =HCA GK= JEJO B H4 ) FHAIA J E JDA?, ) HA=?JE ENJKHA D=I JOAJ>AA = = O KHJDAHM H EI AA@A@ >AB HA MA M JDA HA =JELA? JHE>KJE I B JDAIA F=H= AJAHI EJ?= >A ANFA?JA@ JD=J E JDA A=H BKJKHA =FFH FHE=JAFH J? IME = ALE=JAI A BJDAIADOF JDAJE?= >KJF =KIE> A?=KIAI B>E=I Growth culture conditions 6DAANFAHE A JI@AI?HE>A@DAHA= MA@KIJ FHE HEJE AJDA I KH?AI JDA= = OIEI BJDA>=?JAHE= JH= I?HEFJ A 1 =@@EJE J? JH E C?, )IO JDAIEI MA B JD=J? JH E C?K I M=I HA E F H J= J JD= ANFA?JA@ 7 E?K IIAA J HAIK JE? H@E =JA@L=HE=JE I BCA A ANFHAIIE MA >IAHLA@@EBBAHA?AIE JDAANFHAIIE B? FA JA?A FAH 6DEI JD=J IK>J E CH I HAIK J E GKEJA LEIE> A IA B? IEIJA E CA A ANFHAIIE + FAJA?A =IMA =IIF HK =JE EI EJ HA@>OJDA?A KIE CIK>J A?KAI IJ B MDE?D =HA IJE K M 6DEI ANFAHE A J ID MIJD=JJDAIAFH?AIIAI=HAANJHA A OIA IEJELAJ CH IANFAHE A JID=LA>AA O=I F IIE> A E? K@E C? JH B =CEJ=JE EJ HE C CH MJD CJ AAFALAHOJDE C=I? IJ= J=IF IIE> A 6DEI M=H I =C=E IJ FHA =JKHA =IIEC A J B ABBA?JI? FA JA?A HIF HK =JE FH?AIIAIBH ANFAHE A JIMDE?DD=LA J>AA IJ=JEIJE?= O? JH A@ 1JEI? A=HJD=JIE F O?=HHO E C JE= ANFHAIIE ANFAHE A JI M D=LA A@ KI J E JAHFHAJ = ABBA?J JD=J EI IE F J JDA ANJHA A IA IEJELEJO BJDEIFH?AIIJ IK>J A?D= CAIE A LEH A I =I = ABBA?J B 64? FAJA?A IAA JDA? CA AI E 6=> AI! 2HALE KI HAIK JI JDAIA E JAHAIJE CFDA A =ID IE@AHA@E JDEI ECDJ -LAHO>E CEIJ MID M@EBBE?K A? IK E CEJEI J AN=?J O E = DECD O GK= JEJ=JELA = AH J O JDA ANFAHE A JI FAHB H A@ E = JDAH => H=J HO >KJ = I JD IAFAHB H A@E IE@A A\I M => H=J HO EJ EI H=HA JD=J = ANFAHE A I?= >A? JH A@ BE A O A KCD J = M B H = HEC H KI O AN=?J E FHAIIE I J JDA? JH=HO H AN= F A E BK? JE = >=?JAHE= CA E?IANFAHE A JI A@ AI JANF E? EJ O? JH MD=JD=FFA IJ JDA?A I>AJMAA FHA?K CH MJDE JDABE = A@EK ) JD KCDJDAIAJM IJAFI??KH KIK= O=J!% + IJ BJA >=?JAHE==HAF =?A@B H=L=HE=> A JE A E = H MDAHA JA FAH=JKHA EI IEC EBE?= J O MAH KIK= O J? JH A@ = O AFECA AJE? FDA A = =HAJHECCAHA@@KHE CJDEIJH= IEJE =O A=@ E CA A ANFHAIIE ME >A >KJME? K? JH A@@EBBAHA?AI >AJMAA ANFAHE A JIJD KCDJJ >AJ J= OE@A JE?= 6 CELA KIJ AAN= F A = OANFAHE A JI=HAFAHB H A@=J[H JA FAH=JKHA\ MDE?DEI=LAHOF H O@ABE A@FDOIE?= F=H= AJAH 6DAHA =HA?AHJ=E O JDAH B=?J HI IAA B H AN= F A JDAH A BC EJO B JDAM=JAHMDE?D=HA@EBBE?K JJ? JH = AJDA= = O IEI BJDAANFAHE A JI CA J Methionine and methylthioribose (MTR) 6DA ANEIJA?A B =HCA E JAH=?JE I B JDA JOFA CA JE E EJIJDAE B H =JE JD=J?= >AANJH=?JA@BH =JH= I?HEFJ A = = OIEI 6DA IJ=JEIJE?= = = OIEI D MALAH A => A@KIJ E@A JEBOCA AIJD=JMAHA= IJ?AHJ=E O=II?E=JA@MEJDCELA FDOIE I 64 EI ABBE?EA J O HA?O? A@ E * IK>JE EI KIE C = OAJ K M F=JDM=O 1J IAA I HA=I => A J E BAH JD=J JDEI HA?O? E C EI JDH KCD AJDE E A =I E A>IEA = F AK E=A? K JAHF=HJI BJDA? C F AK E=A CA AI IAA J >A FHAIA J E * IK>JE EI D MALAH 6DA FHAIA J ANFAHE A J M=I JDAHAB HA E J E@A JEBO JDA BEHIJ A A A J B JDA F=JDM=O AJDO JDE HE> IA FAH A=IA HJDACA AIHAIF IE> AB HEJIHA?O? E C >O? F=HE C CH MJD 64 MEJD JD=J AJDE E A EJI E 6DA? CCA @ KJ EB JDAEH ANFHAIIE EI A >O 64 =JKH= O >A?=KIA 64 EI = H = B JDA?A JDA? C CA AI ECDJ>AANFHAIIA@? IJEJKJELA O E MDE?D?=IAJDAO ME J>AE@A JEBEA@>OJDEIFH?A@KHA IEC EBE?= M=I >IAHLA@ >KJI AHAFHAIIE BJDA=HCE E A>E IO JDAJE?F=JDM=OM=I? IEIJA J OB E CH MJD 64 6DEI E JAHAIJE C E >AJMAA EJH CA IK BKH AJ=> EI =O >A =?? K JA@ B H = AI 64 H A B EJI E = E JAHAIJE C

7 Table 3 Function of genes showing the largest differences between days A and B Gene Mean Standard Function deviation comea Late competence protein required for comfa Late competence protein required for DNA uptake comfc Late competence protein required for DNA uptake comga Late competence operon required for comgb Late competence operon required for comgc Late competence operon required for comgd Late competence operon required for comge Late competence operon required for nuca Competence-specific nuclease raph Response regulator: aspartate phosphatase (sporulation) ypze Hypothetical protein yqze Hypothetical protein ysha Conserved hypothetical protein yvrp Conserved extracytoplasmic component of a permease (hypothetical) cotg Spore coat protein glga Bacterial glycogen synthase rplv Ribosomal protein L20 ybfo Similar to erythromycin esterase (detoxification) ylla Hypothetical protein yojl Similar to cell wall-binding B H =? HAFHAII H EB A JAI JD=J =HCE E A EI JDAFHA?KHI H BF O= E AIE * IK>JE 6) JDA E A@E=JAFHA?KHI H B 64 =IJ E?DE AJHE? B=IDE MDA JDAF O= E AIFAH E@E AEIIO JDAIE A@ " 1J =O >A HA AL= J J HA =H JD=J JDA EI =JA@ =HC- CA A EI FHAIA J E = FAH B K M BK?JE MEJD IALAH= CA AI D=LE C IEC EBE?= J D CO MEJD CA AI E L LA@ E IK BKH AJ=> EI HA LAH JDA OJ, CA A E JDA =HC/0OJ, FAH = IAGKA?A HE?D E IK BKH? J=E E C= E =?E@I MDE?DEIK KIK= E * IK>JE EI FH LE@ E C = BKHJDAH B = IEC EBE?= J? KF E C >AJMAA =HCE E BKH AJ=> EI E JDEI HC= EI 1 =@@EJE ANFHAIIE BJM FAH CFAH A=IAI OGE:; DEI2L=HEAIE FF IEJAM=OIMDA?A I=HACH M AJDE E A H 64 6DEIFH FJA@KI J A=IKHAJDACH MJD B KJ= JI BA=?D BJDAIACA AI L=HE KI IK BKH I KH?AI IEC EBE?= J CH MJD E F=EH A J M=I >IAHLA@ MDA??A JH=JE B JDA AJ=> EJAI M=I DECD 6DA IJ F =KIE> A ANF = =JE EI JD=J 64 AJDE E A?= >AJH= IF HJA@E J JDA?A >OIALAH= FAH A=IAI FAH A=IAI JH= IF HJE C AJDE E A =HA = HA=@O M I JD=JE =?JEL=JE B A AI J= JAH CH MJD KHJDAHM H ID MA@JD=JJDAOGE:; CA M=IJH= IF HJE C=HCE E A 6 >A? IEIJA JMEJDJDA-? E A? =JKHA IKFF HJA@ >O 5MEII2H J MA FH F IA J HA = A JDAIA CA AI =HJ23 B H =HCE E A JH= IF HJ 6DA A?K =H =JKHA B JDA E >AJMAA IK BKH =HCE E A HA =E IJ >AE LAIJEC=JA@ D MALAH )IE E =HE L LA A J E JH= IF HJ M=I MEJ AIIA@ MEJD JDA OJ DEI2 CA AI 6DAIACA H=FAH A=IAB H AJDE E A AJDE E A IK B NE@A JDA FAH EI A= J AJDE J NOCA =IA FHAIK => O =BJAH =?AJO =JE 5 MA D=LA JA J=JELA O HA = A@ JDA CA AI OJ 1 OJ DEI2 OJ OJ 1 AJDE E A IK B FAH A=IA JH= IF HJ N)*+ N AJDE E A IK B NE@A NE@=IA H>B H>B HE> B =LE E =IA JDKI=L E@E C[HE>\ MDE?DKIK= B HHE> IA 6DAANEIJA?A BJDEI FAH EIA=IOJ =?? K J B H ME C JD=J JDA KIK= >E J FA B * IK>JE EI EI JDA FDO F = A EB AJDE E A EI ANK@A@ BH F = J A=LAI JDA JDEI??KHI E JDA FHAIA?A B = DECD ALA NOCA MDE?DME ABBE?EA J O AJ=> E AJDA AJDE E A J AJDE E AIK B F IIE> O=?AJO =JE BJDAIK>IJH=JAME FHALA JEJBH CAJJE CE J JDAFH JAE IO JDAIEIF=JDM=O E = O B CA AI E L LA@ E I?=LA CE C NOCA H=@E?= IBKHJDAHIKCCAIJIJD=J=FH?AIIE L LE C NOCA EI?=HHEA@ KJE JDAHA?O? E C B 64 6DEIEIE E AMEJDJDA E L LA A J B=@E NOCA =IAE 64HA?O? E CE F AK E=A! Materials and methods Bacterial strains and growth conditions 6DAB ME C* IK>JE EIIJH=E IMAHAKIA@E JDEIM H JDA JOFA IJH=E $& " * 5% OJ =? * 5&# OJ =? * 5&$ OJ =? * 5"%" OGE: =? * 5"%!' OGE; =? * 5"%!& OGE =? 6DAIA IJH=E I D=LA >AA? IJHK?JA@ MEJDE JDA -+ FH A?J B H JDA BK?JE =?D=H=?JAHE =JE B JDA CA A B * IK>JE EI E -KH FA* 5% * 5&# * 5&$ # =F= * 5"%" * 5"%!' * 5"%!& $ *=?JAHE= MAHA CH M E -, E E = A@EK 02 " & 0 2 " " " C K? IA % =!?EJH=JA! C KJ= E A # JHOFJ FD= "" BAHHE??EJH=JA!! # µ C+ $ +=+ "' # µ A+! "' ' µ + # # µ comment reviews reports deposited research refereed research interactions information

8 8 Genome Biology Vol 2 No 6 5A MI =AJ= + " µ +K+ # µ + + # µ = " "& µ MEJD AJDE E A H AJDO JDE HE> IA 64 HAIFA?JELA OE?K JKHAIB H4 )FHAF= H=JE HMEJD AJDE E A H AJDE E AIK B NE@A??A JH=JE I E JDA JANJ =I I A IK BKH I KH?A % 9DA =HCE E AM=IJAIJA@=I EJH CA I KH?A C KJ= E AM=I EJJA@BH JDA A@EK =?A@MEJD=HCE E A??A JH=JE JDAJANJ C5 " =J M=IKIA@ =IIK BKHI KH?A +A IMAHACH M E # B-, A@EK E # -H A AOAH B =I I =J!% + MEJD? IJ= J =AH=JE H CH MJD F =JAI E E = A@EK M=I IKFF A A JA@ MEJD%C B=C=H > A,EB? M =J!% + ) ANFAHE A JI MAHA FAHB H A@ E =?? H@=?A MEJD JDA -KH FA= HACK =JE HAGKEHA A JI??AH E C JDA KIA B CA AJE?= HC= EI I ALA? J=E A J =CHAA A J K >AH %!# Isolation of total RNA 6 J= 4 ) M=I EI =JA@ BH?A I =J =, $ B & 5= F AI BCH ME C?A I# MAHA?A JHEBKCA@B H E =J & HF + 6DAFA AJIMAHAE A@E=JA OBH A E = E?A >=JD *AB HA 4 ) FHAF=H=JE FA AJI MAHA >HEAB O L HJANA@ µ B OI O A I KJE L C K? IA L 6HEI # -,6) OI O A " C M=I=@@A@ 6DAIKIFA IE IMAHAJH= IBAHHA@J IJAHE A HB JK>AI AFJ B H E =J H JA FAH=JKHA 6DAI= F AIMAHAIK>IAGKA J O ENA@KFMEJD!% # µ B # -,6) JDA!%# µ B OIEI >KBBAH 5,5 =?AJ=JAF0" ENA@ 6DAI= F AIMAHAE?K>=JA@B H! E =J$# +E =@HO >=JD )BJAH E?K>=JE % µ B D J =?E@ FDA M=I =@@A@ I= F AIMAHAL HJANA@B H! E E =$# +M=JAH >=JD 6DA I= F AIMAHA? A@@ M E?A B H! E JHEBKCA@ B H # E =J! HF )BJAH?A JHEBKC=JE JDA=GKA KIFD=IAM=IJH= IBAHHA@J =BHAID HB JK>A JDA D J =?E@ FDA FH?A@KHA M=I HAFA=JA@JME?A 6DAJDEH@=GKA KIFD=IAM=IANJH=?JA@MEJD!# µ B =?E@ FDA!!$ µ B?D H B H " µ B EI = O =? D >O#IA?L HJANE CB MA@>O# E?A JHEBKC=JE =J! HF 6DA =GKA KI FD=IA M=I JH= I BAHHA@ J = BHAID HB JK>A $% µ B?D H B H & µ B EI = O =? D M=I =@@A@ 6DA I= F AI MAHA L HJANA@B JHEBKCA@B H# E =J! HF )BJAH JH= IBAHHE C JDA =GKA KI FD=IA J = BHAID HB JK>A L =?AJ=JAF0# M=I=@@A@ B MA@ >O =@@EJE B # L B =>I KJA AJD= 6K>AI MAHA ENA@>OE LAHIE =?A@=J` +B HD 6DA I= F AIMAHA?A JHEBKCA@B H! E =J! HF + JDAIKFAH =J= JM=IHA LA@ JDAFA AJIMAHAM=IDA@MEJD % AJD= JEBKC=JA@ =I => LA )BJAH HA LE CJDAIKFAH =J= J JDAFA AJIMAHA@HEA@E IFAA@ L=? B H! E!µ B6HEI -,6) 6-M=I=@@A@ =?A@E =!% +M=JAH >=JDB H# E 6 HA LAJDACA E?, ) µ B6- $µ B # +=+ µ B, =IA1, =IA1 4 =IABHAA 4?DAMAHA H! E E =!% +M=JAH >=JD 6DEIJHA=J A JM=IB MA@>OANJH=?JE MEJD%µ =?E@ FDA # IA? L HJANE C # E?A JHEBKC=JE =J! HF )BJAH?A JHEBKC=JE JDA =GKA KI FD=IA M=I JH= IBAHHA@J =?E@FDA H B H M=I=@@A@ 6K>AIMAHAL HJANA@B H JHEBKCA@B H# E =J! HF )BJAH?A JHEBK C=JE JDA=GKA KIFD=IAM=IJH= IBAHHA@J HB JK>A % µ?d H B H M=I =@@A@ 6K>AI MAHA L HJANA@B JHEBKCA@B H# E =J! HF 6DA =GKA KI FD=IA M=I JH= IBAHHA@ J = BHAID HB =?AJ=JAF0# ENA@ B MA@>O=@@EJE B B=>I KJAAJD= 6K>AIMAHA?A JHEBKCA@ B H! E =J! HF " + JDA IKFAH =J= J M=I HA LA@ JDA FA AJI MAHA M=IDA@ MEJD JEBKC=JA@=I=> LA )BJAHHA LE C JDA IKFAH =J= J JDA FA AJI E IFAA@ L=? B H! E µ =?A@B H# E E =!% +M=JAH>=JDB H? F AJAI K>E E =JE 6DA4 )??A JH=JE E A@ >O ECDJ =>I HFJE =J $ & 4 ) I= F AI µc MAHA =@A@ J =C=H IACA J?DA? JDA4 JACHEJO cdna synthesis and labeling H?, ) IO JDAIEI +,5 IFA?EBE? FHE AHI?, ) =>A E C FHE AHI FJE E A@ B H * IK>JE EI 5EC = /A 5OI *E JA?D GK= JEJEAI BJ J= 4 KIA@ 0O>HE@E =JE FH >AIBH µcj J= 4 )MAHACA AH =JA@E =BE = L K A B!µ >O ENE CJ CAJDAH4 ) "µ B* IK>JE EI =>A E CFHE AHI!! 6K>AIMAHADA=JA@=J' +E =@HO>=JDB H E I M? A@ J " + )BJAH =@@EJE B µ # 7 B ) 8 HALAHIA JH= I?HEFJ=IA 4 =IA0 4?DA µ µ+e!! 2 +E AM - C K? A=H JDAHA=?JE ENJKHAM=IE?K>=JA@=J" +B H! D E =M=JAH>=JD 0O>HE@E =JE FH >AIBH µc4 )MAHA CA AH=JA@E JDAI= AM=OAN?AFJJD=JJDAHA=?JE M=IFAH B H A@ E = BE = L K A B µ >O ENE C 4 ) " µ B * IK>JE EI =>A E C FHE AHI # K =>A A@ K? A JE@AI 6K>AI MAHA DA=JA@ KF =J ' + B H E I M O? A@@ M J " + )BJAHHA=?DE C" + $ µ # 7 B) 8HALAHIAJH= $µ+e B!! 2 M=I =@@A@ JDA HA=?JE M=I = MA@ J FH?AA@ => LA 7 E? HF H=JA@ K? A JE@AI MAHA HA LA@ BH =>A A@?, )>OCA BE JH=JE JDH KCD=/ #5AFD=@AN? K 4?DA )FFH NE =JA O $ %# E? HF H=JE B =>A A@ K? A JE@AIM=I=?DEALA@E I DNA array hybridization -NFHAIIE FH BE E C A=IKHAI C >= JH= I?HEFJE O 4 ) DO>HE@E =JE H O DO>HE@E =JE B?, )IO JDAIE A@=BJAHJ J= 4 )D=I>AA ANJH=?JA@BH JDA?A I J >A KIA@ E CA AH= J=> O >A?=KIA 4 ) EI LAHO K IJ=> A EI F=HJE= CDO>HE@E =JE HA LAH JDA4 ) KIJ>A =>A A@ >AB HA DO>HE@E =JE 6DEI E = BKHJDAH

9 I KH?A BL=HE=>E EJO=I =>A E C=BJAH4 )ANJH=?JE EI J LAHOIA AJ IOIJA =JE?>E=I KHJDAH =>A E C@KHE CCH MJD BJDA?K JKHA AA@I = EFK = JE B LAHO =HCA GK= JEJEAI B H=@E =?JELEJO CA AH=JE C = E F HJ= J>=? CH EIAJ I=O JDE C BI=BAJOFH > A I 6DEI A@KI E A IJ B KH? A=CKAI J FHABAHJDA IO JDAIEI B = IE C A?, )? FO B H A=?D 4 ) A?K A KIE C =FFH FHE=JA FHE AHI H=@E =?JELA H B K HAI?A J K? A JE@AI 6DA FHE AHI MAHA FHAIA J E AN?AII MA?=HABK O =L E@A@ HAE EJE=JE E HALAHIA JH= I?HEFJE =I 2+4 = F EBE?=JE =J JDEI IJ=CA M CHA=J O A D=?A JDA HEI BE C =HCA>E=IAI )? AH?E= =HH=O O A >H= AIM=I?D IA B HJDA DO>HE@E =JE IKFF HJ >A?=KIAJDEIEIJDAE ANFA IELAI K JE?D IA >O => H=J HEAIMDE?D@ JD=LA=??AIIJ =HCA B=?E EJEAIB H = E CC =II=HH=OI HA LAH JDEI= MI A J? F=HAHAIK JIMEJDJD IA >J=E A@E JDAH => H=J HEAI 1 =@@EJE KIE C O =HH=OI= MI AJ HAKIAJDAI= A =HH=O IALAH= JE AI MDE?D FH LE@AI E JAH =? IEIJA?O J JDAANFAHE A JI 1J KIJ= I >AIJHAIIA@JD=JDO>HE@E =JE O A >H= A IE = FH?AII B JDA DO>HE@E E C A?K A EI HA =JELA O BHAA E? JH=IJJ JDA?H M@A@IEJK=JE C =III E@AI E = O JDA K >AH B CA AI E = >=?JAHE= CA AI J HAGKEHA ANJHA A E E=JKHE =JE 2= H= = O BE JAHI * IK>JE EI CA A =HH=OI 5EC = /A 5OI *E JA?D CEAI MAHA I = A@ E N 552- N 552- & =+ FD IFD=JA F0 % % -,6) B H # E FHADO>HE@E A@ E # DO>HE@E =JE I KJE 552- #N 5,5,A D=H@J\I HA=CA I E?=JA@I= IFAH, )B H D=J$# +KIE CH AH> JJ AI 6DAA JEHA?, )FH >AM=I DA=JA@B H E =J'# + H=FE@ O? A@@ M E?A =@@A@ J B D J DO>HE@E =JE I KJE 6DA FHADO >HE@E =JE I KJE M=I HA LA@ HAF =?A@ MEJD DO>HE@E =JE I KJE 0O>HE@E =JE M=I?=HHEA@ KJ B H $ % D =J $# + ME C DO>HE@E =JE A=?D BE JAH M=I HE IA@ MEJD # B M=IDE C I KJE 552- #N 5,5 B H! E =J H JA FAH=JKHA 6DEI FAH=JE M=I HAFA=JA@ JME?A B MA@ >O JDHAA M=IDAI E B M=IDE CI KJE =J$# +B H E A=?D E JAHIMAHAJDA >HEAB O@HEA@E =9D=J = F=FAH I = A@MEJDM=IDE CI K A@E =@= F5=H= BE #µ * M E JAHI MAHA ANF IA@ J = 2D IFD H1 =CAH I?HAA A?K =H,O = E?I B H D )HH=OI MAHA IJHEFFA@ MEJD # B > E E =JE I KJE 6HEI F0 % $ -,6) 5,5B KHJE AIB H# E M=IDA@ MEJD M=IDE CI KJE HA@=JH JA FAH= JKHAE =IA= A@F =IJE?>=CMEJD BJDAI= AI KJE ME CJDEIFH?A@KHA JDAI= A=HH=O? JE AI -=?DCA ADO>HE@E =JE M=IFAHB H E?=JA F=EHI BIF JI@AF IEJA@IE@A>OIE@A JDA O A >H= A 6DEI FH LE@A@ = =II=O B H HAFA=J=>E EJO E =@@EJE J =? JH B H EJO JAIJA@ > JD E?=JE C ANFAHE A JI F=HE CFHAF=H=JE J4 )??A JH=JE I 6DA E A=HEJO BJDAHA =JE >AJMAA JDA?, )??A JH=JE C4 )M=I= = O A@>O? F=HE CIO JDAIAIFAHB H A@KIE CAEJDAH HµC4 ) ANJH=?JA@BH JDAI= A4 )FHAF=H=JE 6DEIIJAFE JDA FH J? = I = MA@KIJ LAHEBOJD=JJDAI=JKH=JE BJDA A >H= AIEI JHA=?DA@ 6DAL=HE=>E EJOE HJDAK? JH A@B=?J HIE JDA?K I JDA4 )FHAF=H=JE IM=IAIJE C JDA ANFAHE A J LAH= =II=O B JDA EJO M=I BKHJDAH A IKHA@ >O JDA B=?J JD=J > JD A LEH A I MAHA M J LAHO IE E =H>AD=LE HI BJDA?A I Image acquisition and data analysis -NF IA@2D IFD H1 =CAHI?HAA IMAHAI?= A@MEJD=FENA IE A B %% µ = ""#51 2D IFD H1 =CAH A?K =H,O = E?I )? AH?E= I BJM=HA :@ JI4A=@AH + 5- M=I KIA@ J CHE@ JDA FD IFD HE =CAH E =CA 61 E =CA BE A=IKHAJDAE JA IEJO BA=?D@ J Statistical techniques 6DAIJK@OF = M=I A= JJ E@A JEBO> JDJDAABBA?J BJDA IK BKH I KH?A CA A ANFHAIIE JDA = H I KH?AI B IOIJA =JE?AHH HIE >=?JAHE= ANFHAIIE FH BE E C Distribution of random fluctuations in gene expression = OIEI BL=HE=?AMEJDBELAB=?J HI JDA C=HEJD BJDACA AANFHAIIE 6DA C=HEJD JH= I B H =JE EIKIA@B H H = E E E E CJDAL=HE =?A B JDA HAIE@K= I IAA & ' JDEI?= >A KIJEBEA@ >O JDA K JEF E?=JELA =JKHA B JDA JH= I?HEFJE IEC = I 6DEI IJ=JEIJE?= D=I >AA KIA@ E = E?H JANJ & F IIAIIAI JDA B ME C FH FAHJEAI 1J EI MA IKEJA@J JDEIJOFA B@=J=MEJD=B=?J HE= IJHK?JKHA 1J E? K@AI= JDA@=J=E AI=@L= J=CA BJDA IE A B JDA I= F A J E FH LA JDA F MAH B JDA IJ=JEIJE?= JAIJIE? JH=IJJ JDAKIA BIAF=H=JA5JK@A JJAIJI 6DAHA EI= CANFAHEA?A @ M A@CA B EJI E EJI FEJB= I 1J EI A=IO J E L= E@=JA JDA >=IE? DOF JDAIEI IAAMDAJDAHEJEI = =FFH FHE=JA = IMAHHA AL= JGKAIJE I=> KJ =IFA?EBE?@=J=IAJ 8=HE=?A = = OIEI ID MA@ = IJH C E JAH=?JE >AJMAA JDA = K J B4 )KIA@ JDA@=JA BJDAANFAHE A J JDAIK BKH I AI 1 JDAHM H@I I ACA AI=HA HA IA IEJELAJD= JDAHIJ?D= CE I 6DEIEIMD=JEI A@ B H MDA A?D= CAI JDA IK BKH I KH?A =I E@A= O O = BAM CA AI ID D=LA JDAEH E JDEI?=IA 6DEI HAIK J EI D MALAH K ANFA?JA@ MDA JDA = K J B 4 ) EI?D= CA@ =I JDEI A= I JD=J JDA H=JE >AJMAA JDAGK= JEJO JEJO B 4 )L=HEAIBH ACA AJ = JDAH comment reviews reports deposited research refereed research interactions information

10 10 Genome Biology Vol 2 No 6 5A MI =AJ= 6DA@EBBE?K JEAI>ACE MDA JAH=?JE I>AJMAA JM B=?J HI CA AI CA A N IK BKH I KH?A CA A N = K J B4 )NIK BKHI KH?A AN@=JAN= K J B 4 B=?J HI=?JIE K J= A KI O JDA L= KAI A=IKHA@ B H I A CA AI 1 JDAH M H@I A?=? FKJA >AJMAA JM A= IKHA A JI JDAI= ACA A >KJEJEI JF IIE> AJ =IIEC JDA L= KA B J JDA I A ABBA?J B?D= CAI I >AJMAA JDA JM A=IKHA A JI 6=> AI EIJE C HAIK JI B JDEI JOFA KIJ JDAHAB HA >A E JAHFHAJA@ MEJD ANJHA A?=KJE EJ M >A ALA M HIA B? KHIA EB A KIA@H=JE I =IEIK B HJK =JA O A )FHK@A JI K JE EIJ??A JH=JA JDACA AIJD=J@ JID ME F H J= JL=HE=JE I AN?AFJB H A BJDAB=?J HI 1 FH=?JE?A A? FKJAIB HA=?DCA AJDA@EBBAHA?A B A=IKHA A JIB H= CELA B=?J HB HAN= F AJDAIK BKHI KH?AE JDAB I H* JDA A?=?K =JAIJDA B= B KH@EBBAHA?AI 6DA IJ E JAHAIJE C CA AI =HA JD IA JD=J? >E A JDA =HCAIJ A= JDA 6DA CA AH= I KJE J JDAFH > A ALA MDA JDAB=?J H BE JAHAIJD=I HAJD= JM L=HE=> AIB HAN= F AJDAIE K J= A KIIJK@O BJDHAA H HA IK BKH I KH?AI EI J FAHB H B H A=?D CA A = L=HE =?A= = OIEIMEJDJM B=?J HIJDAB=?J H BE JAHAIJ JDA A = JDA JDAH AI CH KFA@ J CAJDAH B H AN= F 4 )= K J JDA JDAH JDA J HAJ=E JD IA CA AI =O > JD = MA= E JAH=?JE >AJMAA B=?J =HCAIK BJDAIGK=HAI BJDA@ALE=JE IBH JDA A= B HJDAB=?J H BE JAHAIJ Controlled factors A?= HA=I => O=IIK AJD=JJDA >IAHLA@DO>HE@E =JE IEC = EI FH F HJE = J JDA?, ) = K J IF JJA@ >O JDA H > J =I JDA A=IKHA A J B JH= I?HEFJE ANFHAIIE EI >=IA@ JDA DO>HE@E =JE B? F AN FH >AI =J M + I MDAHA JDA J=HCAJ IAGKA?AI =HA E =HCA AN?AII JD=J EI B=H BH JDA I=JKH=JE 9A D=LA J= A BELA B=?J HI E J =?? K J EHIJ B JDACA JA@IK BKHI KH?A MEJDJM ALA I JDA?K B 4 ) ANFAHE A JI MEJD I= A =JAHE= FH J? MAHA =@A =J 6DEH@ JDA= K J B4 ) µc HµC KHJD JDAH > J =OI JM FI JDA A >H= A ANJ J A=?D JDAH JDEIB=?J HEI@A JA@@H FMEJDJM ALA I -NFAHE A JI MEJD ALA =HA?= A@ HAF E?=JAI EBJD JDACA A JDAHA=HA" %CA AI 9A JDKI D=LA " N " % $# % JHA=J A J K EJI -NFAHE A JID=LA>AA =@AMEJDJM A >H= AI 6DA A >H= A B=?J H EI IKFAHE F IA@ JDA E N = K J B 4 )ANJH=?JE I MA=HA J=> CKEIDJDAIAJM I KH?AI BL=HE=JE 6=> A "ID MIJDA?D=H=?JAHEIJE?L= KAIB HJDADO>HE@E =JE E JA E J ANFAHE A JI 6DA BECKHAIB HA=?DCA A? JDA=LAH=CA BJDAE JA IEJO BA=?D A BJDA@KF E?=JA@H FIE =H>EJH=HOK EJI JDA L= KACELA EIJD=J BJDA@H FMEJDJDADECDAIJL= KAE A=?D GK=HJE A 6DA@EIJHE>KJE EILAHO=IO AJHE?= 6DAIFHA=@ B =I A=IKHA@ >O >AJMAA JDA BEHIJ JDA JDEH@ GK=HJE A L=HEAI =I = BK?JE B JDA = K J B 4 ) KIA@ J IO JDAIE A?, ) µc "%' ± & µc %&#± # 6DAI= HJDA=LAH=CAL= KA MEJD = B=?J H $ >AJMAA JDA JM FH J? I MDE A JDA 4 ) = K JKIA@L=HEA@>O=B=?J HµC #!± && µc %&% ±!$ 6DAHAANEIJI=? A=H@EBBAHA?A>AJMAA JDA A=IKHA A JIFAHB H A@KIE 6DEI@EB BAHA?AEI J >IAHLA@E JDAFHAF=H=JE IFAHB H A@KIE C JDALAHOI= AANJH=?JI MEJDµC4 ) 8=HE=?A = = OIEI ID MI JD=J JDA E JAH=?JE I E L LE C JDA = K J B 4 ) =HA HA E F HJ= J JD= JD J = JDAHB=?J HI 6D=JEI L=HE=JE IBH CA AJ CA A MEJDE = I= AANFAHE A J BJDAH=JE >AJMAA JDAGK= JEJO B?, ) B 4 ) =HA HAE F HJ= JJD= JDAL=HE=JE I HAIK JE CBH JDA?K JKHA@=O HJDAIK BKHI KH?A 6DEIEI D MALAH =B=?J H BJA AC A?JA@E ANFAHE A J= FH J? I HA@AIAHLAIIFA?E= IJK@O 6 JDEI JDA CA AI MAHA H@AHA@ =I = BK?JE B JDA =LAH=CA E JA IEJO B JDA IEC = E J BELA? =IIAI MEJD JDA I= A K >AH B A A A JI 6DA BEHIJ? =II?A JE AIBH J? J=E A@JDA&!CA AID=LE CJDA MAIJIEC = E JA IEJO A=IKHA@ JDAJ J= EJO BJDAAECDJ ANFAHE A JI + =II "? J=E A@ JDA IK>IAGKA J &! CA AI MDE?DD=LA=IEC = MEJD= E JA IEJODECDAHJD= JD=J Table 4 Characteristic values for the distribution of intensities (arbitrary units) in different experimental conditions Met A 1 MTR A 1 Met B 1 MTR B 1 Met A 10 MTR A 10 Met B 10 MTR B 10 First quartile Second quartile Third quartile ,230 1,371 1,335 1,294 The genes were ranked according to the intensity of the corresponding hybridization; the values were then arranged into four sets of equal numbers, with increasing hybridization values. The value for the highest hybridization in each quartile is given here. Met, methionine; MTR, methylthioribose; A and B refer to different days, and 1 and 10 refer to the amount of RNA (see text).

11 B? =II MAHJD= JD=J B? =II" $ 6DA? HHA =JE >AJMAA JDAL= KAI >J=E A@KIE JD IAFAHB H A@KIE CµC B4 )M=I?=?K =JA@B HA=?D? =II6=> A # A HHA =JE >AJMAA JDA A=IKHA A JIFAHB H B4 ) MDA JDAIEC = M=IAEJDAHLAHO M HLAHODECD 6DEIID MA@BEHIJ JD=J JDA AN?AII B IAGKA?A J=HCAJ, ) EI IKBBE?EA J B H DO>HE@E =JE J >AB=HBH FAHD=FI K ANFA?JA@ O JD=J JDA >=? CH EIA JDA HAIE@K= H=@E =?JELEJO BJDA A >H= AI J= JAHJDA A=IKHA A J BDO>HE@E =JE B M?, )= K JI 1? JH=IJ JDA? HHA =JE M=I LAHO F H B H JDA A@EK DO>HE@E =JE L= KAI 6 LAHEBOJD=JJDEIM=I J= JDA MAHL=HE=?AE JDA?A JH=? =IIAI MA?=?K =JA@ JDA? HHA =JE >AJMAA JDAL= KAI >J=E A@KIE CJDAI= A = K J B4 )ANJH=?JA@BH =?K JKHACH M MEJD AJDE E A = JDAH MEJD AJDO JDE HE> IA 6=> A $ 6DA Table 5 Variation of the correlation in the measurements performed with 1 µg and 10 µg RNA Centile Met A 1 x Met B 1 x MTR A 1 x MTR B 1 x Met A 10 Met B 10 MTR A 10 MTR B The results for experiments with 1 or 10 µg RNA were compared after ordering the genes with the corresponding hybridization values in increasing order. A high correlation indicates that the results are very similar for both conditions. The correlation is very good for highly expressed or poorly expressed genes whereas it is low for those with medium expression. Met, methionine; MTR, methylthioribose; A and B refer to different days, and 1 and 10 refer to the amount of RNA (see text). Table 6 Variation of the correlation in the measurements performed with two sources of sulfur Centile Met A 1 x Met A 10 x Met B 1 x Met B 10 x MTR A 1 MTR A 10 MTR B 1 MTR B The data are arranged as in Table 5. Correlation is good, indicating that there is not a large difference between growth on methionine and growth on MTR (as expected).? HHA =JE IMAHA K?DDECDAHJD= JD IAB 6=> A =IIAI? J=E A@AN=?J OJDAI= A CA AI A=IKHA A JIL=HEA@LAHO EJJ A B HJDAL=IJ = HEJO BJDACA AI BH AIK BKHI KH?AJ JDA JDAH 6DA =>IA?A B = HHA =JE >AJMAA JDA A=IKHA A JI FAHB H A@ KIE C µc H µc 4 ) B H A@EK DO>HE@E =JE L= KAIEIJDKIHA= 1 JDAHJAH I JDAAIJE = JE BJDAANFHAIIE B=CA IE@AH=> O JDA = K J B 4 ) KIA@ J IO JDAIE A EJI?, )? F A A = EGK JI B=K EGKA4 ) FHAF=H=JE 6DEIFDA A EIF=HJE?K =H OE F HJ= JB H JDA CA AI JD=J CELA = A@EK DO>HE@E =JE JDAHAB HA JH= I?HEFJE IEC = HJDEIHA=I MA?= =IIAHJJD=JJDA >IAHL=JE I =@A E JDA FHAIA J JDA E E =?? KIE IJD=J?= >AANJH=?JA@BH JDA@=J= E A OJD=JJDAHA=HABKHJDAHABBA?JI>KHEA@E JDA@=J= E F=H JE?K =HE L LE CCA AIMEJD A@EK ANFHAIIE )H=FE@>H MIAJDH KCDJDA EIJ BJDA IJL=HE=> ACA J HALA= = O IFA?E= BA=JKHA = A O JDAHA EI = FAH BA?J O H = HAFHAIA J=JE BK M [O\CA AI" =I? F=HA@J $' "E JDACA IAMAHA JJDA I= ACA AI=IE JDAJM FHA?A@E C EIJI 6DA HAFA=J=>E EJO B JDA H > J IF JJE C EI JDEI I KH?A B =HJEB=?J?= JDAHAB HA '# B JDA CA =O >AJMAA > JD IF JI MAH JD= 1JEI JF IIE> AJ M MDA =IF JEIIOIJA =JE?= O HAE JA IAJD= EJI? K JAHF=HJ I AIFA?EBE?BA=JKHA BJDACA A H@KAJ JDA A >H= A B H AN= F A EHHACK =HEJO B JDA H > J DAJAH CA AEJO BJDA>=? CH A AAFIJDAI= AF IE JE E JDA A >H= AE = ANFAHE A JI Uncontrolled factors JO B BE A O? JH E C JDA ANFAHE A JE IBH A@=OJ JDA KH?A BIOI JA =JE? AHH HI 6DA EIJ B JDA IJ L=HE=> A CA AI =?? H@E CJ JDEIF=H= AJAHD=IIALAH= E JAHAIJE CBA=JKHAI /A AI B M BK?JE MAHA=> H = J! BH JDA FH F HJE E JDA CA A EI DECD O IEC EBE?= J F C= CA A@EIJHE> KJE KHJDAH A@>OJDAANFHAIIE B= A IA > A BCA AIE L LA@E? >OJDAANFHAIIE BCA AIE L LA@E IF HK =JE Unidentified factors 6DAIA? JH JH A@B=?J HIIK?D=I? JHE>K JE B JDA GK= JEJO B HAIE@K= H4 ) E JDA E EJE= 4 ) FHAF=H=JE B K?JK=JE E JDAIJ=JA BJDA?K JKHA = EBAIJ JDA IA LAIE 1 JDA HO JDAHAHA =E I=HAIE@K= JAH E )IJDAHAIE@KAEILAHOI = E JDAFHAIA JIJK@O E@A JEBEA@B=?J HI@ JF =O= E F HJ= JH AE JDAANFAHE A JFHAIA JA@ comment reviews reports deposited research refereed research interactions information

12 12 Genome Biology Vol 2 No 6 5A MI =AJ= Acknowledgements This work benefited from the support of the BACELL European Union program. Some of the mutants used for confirmatory studies came from the collection of the Japanese-European consortium for functional analysis of the Bacillus subtilis genome: we are grateful to N. Ogasawara and S.D. Ehrlich for providing access to the strains. We thank I. Martin-Verstraete for her interest and her help in supplying the strains. References 1. Sekowska A, Danchin A: Identification of yrru as the methylthioadenosine nucleosidase gene in Bacillus subtilis. DNA Res 1999, 6: Danchin A, Sekowska A: Expression profiling in reference bacteria: dreams and reality. GenomeBiology 2000, 1: Glantz SA: Primer of Biostatistics. 4th edition. New York: McGraw Hill, Sekowska A, Kung HF, Danchin A: Sulfur metabolism in Escherichia coli and related bacteria: facts and fiction. J Mol Microbiol Biotechnol 2000, 2: Coppée J-Y, Auger S, Turlin E, Sekowska A, Caer J-PL, Labas V, Vagner V, Danchin A, Martin-Verstraete I: Sulfur-limitation-regulated proteins in Bacillus subtilis: a two-dimensional gel electrophoresis study. Microbiology 2001, 147: Bacillus subtilis Genome Sequencing Project [ 7. Wissenbach U, Six S, Bongaerts J, Ternes D, Steinwachs S, Unden G: A third periplasmic transport system for L-arginine in Escherichia coli: molecular characterization of the artpiqmj genes, arginine binding and transport. Mol Microbiol 1995, 17: Rocha EP, Sekowska A, Danchin A: Sulphur islands in the Escherichia coli genome: markers of the cell s architecture? FEBS Lett 2000, 476: Gourse RL, Gaal T, Bartlett MS, Appleman JA, Ross W: rrna transcription and growth rate-dependent regulation of ribosome synthesis in Escherichia coli. Annu Rev Microbiol 1996, 50: Arfin SM, Long AD, Ito ET, Tolleri L, Riehle MM, Paegle ES, Hatfield GW: Global gene expression profiling in Escherichia coli K12. The effects of intergration host factor. J Biol Chem 2000, 275: Bigay J, Deterre P, Pfister C, Chabre M: Fluoroaluminates activate transducin-gdp by mimicking the gamma-phosphate of GTP in its binding site. FEBS Lett 1985, 191: Myers RW, Abeles RH: Conversion of 5-S-methyl-5-thio-Dribose to methionine in Klebsiella pneumoniae. Stable isotope incorporation studies of the terminal enzymatic reactions in the pathway. J Biol Chem 1990, 265: Wray JW, Abeles RH: The methionine salvage pathway in Klebsiella pneumoniae and rat liver. Identification and characterization of two novel dioxygenases. J Biol Chem 1995, 270: Anagnostopoulos C, Spizizen J: Requirements for transformation in Bacillus subtilis. J Bacteriol 1961, 81: Micado Release 6.1 [ 16. JAFAN: Japan Functional Analysis Network for Bacillus subtilis [ 17. Spizizen J: Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc Natl Acad Sci USA 1958, 44: Kerr MK, Martin M, Churchill GA: Analysis of variance for gene expression microarray data. 2001, preprint available online at [ 19. Efron B, Tibshirani R, Goss V, Chu G: Microarrays and their use in a comparative experiment. 2001, preprint available online at [ 20. Bertucci F, Bernard K, Loriod B, Chang YC, Granjeaud S, Birnbaum D, Nguyen C, Peck K, Jordan BR: Sensitivity issues in DNA array-based expression measurements and performance of nylon microarrays for small samples. Hum Mol Genet 1999, 8: