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Finishing the cell cycle: control of mitosis and cytokinesis in fission yeast
OMMENT
Finishing the cell cycle: control of mitosis and cytokinesis in fission yeast FRED CHANG AND PAULNURSE IMPERIALCANCERRESEARCHFUND,LINCOLN'SINNFIEmS,LONDON,UK WC2 3PX. One of the key questions about the cell cycle is how the events leading up to cell division are timed and coordinated in an orderly progression. Work in fission yeast has identified a gene c d c 1 6 that may act as a molecular switch, coordinating the events at the end of the cell cycle: mitosis and cytokinesis. This switch may be regulated by 'checkpoint' controls that monitor the state of the mitotic spindle and septation, thereby linking regulators with structural events in cell division, c d c 1 6 has a functional homologue in budding yeast, the mitotic checkpoint gene BUB2, suggesting that these controls may be conserved in many organisms.
Cytoldnesis in fission yeast Fission yeast is a rod-shaped organism that divides by medial fission. The septation process is composed of three discrete events. In early mitosis, an actin ring forms at the middle of the cell 1. At the end of mitosis, the septum, composed of cellwall material, is formed at the site of the actin ring. Lastly, cell separation occurs by the digestion of septai material between the two daughter cells. Eight genes necessary for septation have been defined in fission yeas#. Mutants defective in these genes fail to divide, and accumulate as large, long cells with muhiple nuclei. These genes encode proteins such as tropomyosin3 that are involved in building the actin ring or septum. This article describes an additional gene, cdc16, which was identified as a negative regulator of cytokinesis.
accumulate multiple, often abnormal, septa (Fig. 1). The original c d c l 6 t-~ mutant is recessive, and a deletion of the gene causes a similar phenotype5. Analysis suggested that once a c d c l 6 t~ mutant enters cytokinesis, it cannot stop making septa, and the septation process goes on and on. The nuclear cycle, however, is not arrested and continues for one generation, stopping in G2 with two nuclei in each cell 4. Thus, cell-cycle regulation of septation is perturbed, so that septa form even when the nuclear cycle is in G2. However, a c d c 1 6 t.~ cell must enter mitosis before it can begin to make septa. A c d c 2 t s c d c 1 6 ts double mutant, which cannot enter mitosis, fails to form any septa 4. The phenotype of the c d c 1 6 ts mutant suggests that the product of c d c 1 6 is a negative regulator of septation that turns off the septation process after one septum has been formed.
cdcl6, spindles and mitosis In a recent report, Fankhauser etai.5 demonstrate that c d c 1 6 appears to
(~
(a) wildtype
C°
(o
cdc16
o) Co
l I
o) Col
cdcl 6 and cytokinesis c d c l 6 - 1 1 6 was isolated as a temperature-sensitive (ts) lethal mutant that showed strikingly uncontrolled septation at the restrictive temperature 4. cdc16 ts mutant cells make septa that do not separate, and they continue making these about every 45 minutes (approximately one-third of a normal generation time). The cells thus
have an additional function, 'checking' on mitotic spindles and regulating the end of mitosis. Cloning and sequencing of c d c 1 6 revealed significant similarity (39% identity) to the budding yeast gene BUB2, a homology further strengthened by the finding that the BUB2 gene complements the cdc16 ts fission yeast mutant. BUB2 is one of the BUB/MAD family of genes implicated in a mitotic spindle checkpoint 6,7. A cell that has been depleted of its microtubules, as the result of a nmtation or of drug treatment, arrests in mitosis. In a b u b - m u t a n t , there is no cell-cycle arrest; the cell proceeds into the next cell cycle, undergoes budding and DNA replication, and quickly dies. The original interpretation of these mutants was that the products of the BUB/MAD genes check that the mitotic spindle is in place. If the spindle is not assembled, then the BUB/MAD proteins are involved in arresting the cell in mitosis until spindle is completed. Mitotic arrest may be caused by the p34 cdc2/CDC28 mitotic protein
oD
i o)
(4 Ill No) FIGn cdcl6- mutant fission yeast cells make multiple septa. (a) Diagram of septation and cell separation in wild-type and cdcl6 ts mutant cells. Bold lines in the middle of the cells represent septa; ovals represent nuclei. (b) cdc16 ts mutant cells grown at restrictive temperature. Septa have been stained with calcofluor.
T1GOCTOBER1993 VOL.9 NO. 10 © 1993 Elsevier Science Publishers Ltd (I IK) 0168 - 9525/93/$06.(~)
E ~
i
~OMMENT
of one another. Septation would kinase. Activation of mitotic # spindle-~._ require only tile presence of a kinase is necessary for entry spindle, and would not depend into mitosis, and destruction of on the destruction of cdc2 kinase. mitotic kinase activity is required I m,,o.,s , I c,'o '°es"l Unfortunately, little is known for exit from mitosis. In a budabout the role of cdc2 kinase ding yeast cell arrested with no destruction at the end of mitosis spindle, mitotic kinase (CDC28) in fission yeast. The existence of activity remains high. In a bub2mutants that arrest in mitosis with mutant, mitotic kinase activity is "earlymitosis" ] condensed chromosomes and a not maintained, but slowly no spindle septum s,9 suggests that fission decays 6. Thus, the BUB2 protein cdc16 yeast can foma a septum without may cause mitotic arrest by precytokinesisOFF cdc2ON ON exiting from mitosis; however, venting inactivation of the mitotic cdc2 kinase activity has not been kinase. measured in these mutants. Fanldaauser et al. s tested [latemitosis I Clearly, the relationship between whether the cdc16 t~ fission yeast [spindlepresent septation and cdc2 kinase activity mutant acts in the same way as the 6 cytokinesisON remains to be elucidated. hub2- mutant. Fission yeast cells cdoaOFF OFF A second question is how cells that lack microtubules arrest in check for a mitotic spindle. What mitosis, with high cdc2 mitotic feature of the mitotic spindle is kinase activity and no septum. In septation ] completed actually being monitored? A fission c d c l 6 Lsmutants that lack microyeast cell that lacks microtubules tubules, there is no mitotic arrest; cdc16 ON cytokinesisOFF arrests in mitosis without forering cdc2 kinase activity falls, and the a septum. However, mutants with cells septate. Therefore, cdc16 in an abnomlal spindleg.10, such as fission yeast may play a role analthe kinesin mutant cutT, do form ogous to that of BUB2 in budding /~TGgl septa. Thus the switch into cytoyeast: in the absence of a mitotic spindle, cdc16may maintain high Model fbr a cdcl6switch that coordinates mitosis and kinesis does not require a fully cdc2 kinase activity and delay the cytokinesis. See text ff)r details. intact spindle, but may sinlply reexit from mitosis, cdcl6 is probquire some aspect of spindle forably not a simple activator of cdc2 mation, a prtxzess which is missing kinase, since cdc16 t.~mutant cells can (Fig. 2). In early mitosis (or in the in cells that are completely devoid of still enter mitosis; rather, it may be absence of a mitt)tic spindle), cdc16 functional micmtubules. necessary tt) prevent the premature is on, maintaining cdc2 kinase activity When pondering these complex destruction of cdc2 kinase activity anti inhibiting cytokinesis. At the end genetic circuits, it is important not to during mitosis. of mitosis, forrn:ltion of the mitotic forget the cell biology. For example spindle inhibits cdc16; its activity is in fission yeast, at least part of the Tying it all together: a model turned off, cdc2 kinase activity cdc2 kinase localizes on spindle pole In trying to work out the role of decays, cytokinesis is turned on, and bodie# l st) perhaps it is not surpriscdc16, we are faced with the enigma the cell septates. After a septum has ing that the state of microtubules might that a single gene product appears to formed, c d c l 6 is turned on, and affect activity or regulation of the be doing several different things: cytokinesis is turned off. kinase. Further studies of cdc16 and monitoring microtubules, regulating Of course, at the moment this checkpoints may help us appreciate the cdc2 kinase and regulating cyto- model is a genetic fantasy that awaits the importance of the cellular enkinesis. Here, we suggest a relatively biochemical tests. One genetic pre- vironment in which these cell-cycle simple model, which builds on others diction is that a cell that expresses a regulators act. proposed previously ~5. constitutively active foma of cdcl6, cdc16may be a central switch that owing to overexpression or mutation controls both cytokinesis and the end of cdcl6, would arrest in mitosis with References 1 Marks,J. and Hyams, J.S. (1985) Eur. of mitosis, acting as a positive regulator high cdc2 kinase activity, a spindle J. CellBiol. 39, 27-32 of mitosis and a negative regulator of and no septum. 2 Nurse, P., Thuriaux, P. and Nasmyth, cytokinesis. According to this model K. (1976) Mol. Gen. Genet. 146, (Fig. 2), when cdc16is switched on, Further issues 167-178 the cell is in mitosis and cdc2 kinase is Important issues remain to be ad3 Balasubramanian, M.K., Helfman, on. When cdc16is switdled off, the cell dressed. The relationships between D.M. and Hemmingsen, S.M. (1992) Nature 360, 84--87 exits mitosis and enters cytokinesis. the major players, cdc16, cdc2 4 Minet, M., Nurse, P., Thuriaux, P. Two checkpoint controls may govern activity and cytokinesis, must be and Mitchison, J.M. (1979) tl~e activity of cdcl6: (1) c d c l 6 is in- clarified. In the model presented J. Bacteriol. 137, 440-446 hibited by the presence of a mitotic by Fankhauser et al. 5, destruction of 5 Fankhauser, C., Marks, J., Reymond, spindle and (2)cdc16 is activated by cdc2 kinase activity may bring about A. and Simanis, V. (1993) EMBOJ. the presence of a completed septum. cytokinesis. Alternatively, cdc2 kinase 12, 2697-2704 cdc16 activity is predicted to activity and cytokinesis may be 6 Hoyt, M.A., Totis, L. and Roberts, switch on and off during the cell cycle regulated by cdc16 independently B.T. (1991) Cell66, 507-517
-se.,umJ
icdc1
Tit; OCTOnER1993 VOL.9 NO. 10
~3~
~OMMENT 7 Li, R. and Murray, A.W. (1991) Cell 66, 519-531 8 Hirano T., Hiraoka, Y. and Yanagida, M. (1988) J. Cell Biol.
Nature 347, 563-566 11 Alfa, C.E., Duconunun, B., Beach, D. and Hyams. J.S. (1990) Nature 347, 680-682
106, 1171-1183 .9 Uzawa, S. etal. (1990) Ce1162. 913-925 10 Hagan, I. and Yanagida, M. (1990)
aECHNICAL HIPS A q u i c k m e t h o d f o r i m m t m o s c r e e n i n g r e c o m b i n a n t bacterial c o l o n i e s When working with E. coli expression vectors, it is often helpful to check quickly for proper expression of cloned DNA fragments that contain an open reading frame (ORF). The usual procedure I is time consuming and involves unusual buffers and exposure to chloroform vapours. We describe here a quick protocol, based on commonly used techniques1, that is particularly useful for directly screening for peptide production in plated transformation reactions or in plasmid cDNA libraries. This method also allows the integrity of a cloned ORF to be rapidly checked after transfer from one vector to another, or when it has been cloned after PCR amplification. Nitrocellulose filters are soaked for 5 min in 5 mM IPTG, 5 mg m1-1 ampicillin, air dried for 5 min in a sterile hood, and placed onto plated bacterial cultures; small colonies (<0.3 mm diameter) yield the sharpest signals. Plates are incubated at 37°C for 3--5 h, then cooled at 4°C for 10 min. Filters are removed and placed, with the bacterial colonies facing up, onto 3MM Whatman paper soaked with 0.5 M NaOH, 1.5 i NaCl for 5 min. This lysis step is repeated. Plates are incubated at 37°C for 3--5 hours to allow recovery of clones. Filters are neutralized by placing onto 3MM Whatman paper soaked with 1 i TrisHCI pH 7.5 for 5 min. The neutralization step is repeated, and the filters are washed briefly in PBS, 0.05% Triton-Xl00. Subsequent steps are identical to those described previously1, and use PBS, 0.05% Triton-Xl00, 5% non-fat dried milk as a blocking buffer, and PBS, 0.05% Triton-Xl00, 0.5% non-fat dried milk during incubation with the antibody and during washing. Antigen-antibody complexes are revealed using the appropriate chromogenic stain. We routinely use this protocol for screening ORF constructs cloned in amp R vectors that carry [PTG-inducible promoters (e.g. Bluescript or pGEX) (Fig. la), but it can easily be adjusted for use with other vectors, or for other purposes, such as screening cDNA libraries. When monoclonal antibodies are used this method gives specific signals with a remarkably low level of background: we routinely check 6-10 antibodypositive clones from each transformation by western blotting and have never found a false positive. Nonspecific background is not increased when working in conditions that mimic cDNA library immunoscreening, using Petri dishes that contain >103 colonies (Fig. 2a,b). Moreover, background signal is very low even when the protocol is used on large solid cultures obtained from dotted inocula (Fig. 2b). ACKNOWLEDGEMENT
The pGEX-SCR plasmid was a gift of P. de Zulueta.
(a)
(b)
1
,
kD
.116.2 .97.4
.66.2
.45
FIGO (a) Immunodetection of peptide-producing colonies from a plated transformation mixture. The ORF of the modulo (rood) gene2 was PCR-amplified from cDNA, cloned in pGEX-2T and transformed into E. coli XL1. Nitrocellulose fdters carrying bacterial transformants were screened using the mod-specific monoclonal antibody (Mab La9), an alkaline-phosphatasecoupled secondary antibody and NBT/BCIP chromogens. (b) One of the positive clones, indicated in (a) by an arrow, was isolated, and correct expression of the ORF confirmed by SDS-PAGE. Lane 1, Coomassie Blue stained, no induction; lane 2, Coomassie Blue stained, 2 h induction with 0.1 m i IPTG; lane 3, mod revealed by Mab La9 a~er western blotting and 2 h induction with 0.1 mMIPTG.
(a)
1
=
.
....
~
:•
:" .."
.
3
(la)
2
1
3
,
'
.
~
.
•
~,~Ex.sc,
..
FIGM
(a) Colonies of E. coli XL1 (approximately 3.5 X 103 colonies) transformed with a mixture of pGEX-2T(97.8%), pGEX-MOD(2O~)and pGEX-SCR(0.2%) were plated to demonstrate immunodetection of clones producing mod and scr (sex-combs reduced protein). A nitrocellulose replica of the culture was cut into three pieces, and these incubated as follows: (1) Mab La9; (2) SCR-specific Mab 6H4 (Ref. 3); (3) Mab La9 and Mab 6H4. The 42 positive clones can be seen in (1), 8 in (2) and 65 in (3). (b) Cells transformed with either pGEX-MOD, pGEX-SCRor pGEX-2T were dotted on a plate, cultured overnight at 37°C and replicated onto nitrocellulose. Fusion proteins were identifiedusing: (1) Mab La9;(2) Mab 6H4; (3) Mab La9 and Mab 6H4.
TIG OCTOBER1993 voL. 9 No. 10
I
Finishing the cell cycle: control of mitosis and cytokinesis in fission yeast FRED CHANG AND PAULNURSE IMPERIALCANCERRESEARCHFUND,LINCOLN'SINNFIEmS,LONDON,UK WC2 3PX. One of the key questions about the cell cycle is how the events leading up to cell division are timed and coordinated in an orderly progression. Work in fission yeast has identified a gene c d c 1 6 that may act as a molecular switch, coordinating the events at the end of the cell cycle: mitosis and cytokinesis. This switch may be regulated by 'checkpoint' controls that monitor the state of the mitotic spindle and septation, thereby linking regulators with structural events in cell division, c d c 1 6 has a functional homologue in budding yeast, the mitotic checkpoint gene BUB2, suggesting that these controls may be conserved in many organisms.
Cytoldnesis in fission yeast Fission yeast is a rod-shaped organism that divides by medial fission. The septation process is composed of three discrete events. In early mitosis, an actin ring forms at the middle of the cell 1. At the end of mitosis, the septum, composed of cellwall material, is formed at the site of the actin ring. Lastly, cell separation occurs by the digestion of septai material between the two daughter cells. Eight genes necessary for septation have been defined in fission yeas#. Mutants defective in these genes fail to divide, and accumulate as large, long cells with muhiple nuclei. These genes encode proteins such as tropomyosin3 that are involved in building the actin ring or septum. This article describes an additional gene, cdc16, which was identified as a negative regulator of cytokinesis.
accumulate multiple, often abnormal, septa (Fig. 1). The original c d c l 6 t-~ mutant is recessive, and a deletion of the gene causes a similar phenotype5. Analysis suggested that once a c d c l 6 t~ mutant enters cytokinesis, it cannot stop making septa, and the septation process goes on and on. The nuclear cycle, however, is not arrested and continues for one generation, stopping in G2 with two nuclei in each cell 4. Thus, cell-cycle regulation of septation is perturbed, so that septa form even when the nuclear cycle is in G2. However, a c d c 1 6 t.~ cell must enter mitosis before it can begin to make septa. A c d c 2 t s c d c 1 6 ts double mutant, which cannot enter mitosis, fails to form any septa 4. The phenotype of the c d c 1 6 ts mutant suggests that the product of c d c 1 6 is a negative regulator of septation that turns off the septation process after one septum has been formed.
cdcl6, spindles and mitosis In a recent report, Fankhauser etai.5 demonstrate that c d c 1 6 appears to
(~
(a) wildtype
C°
(o
cdc16
o) Co
l I
o) Col
cdcl 6 and cytokinesis c d c l 6 - 1 1 6 was isolated as a temperature-sensitive (ts) lethal mutant that showed strikingly uncontrolled septation at the restrictive temperature 4. cdc16 ts mutant cells make septa that do not separate, and they continue making these about every 45 minutes (approximately one-third of a normal generation time). The cells thus
have an additional function, 'checking' on mitotic spindles and regulating the end of mitosis. Cloning and sequencing of c d c 1 6 revealed significant similarity (39% identity) to the budding yeast gene BUB2, a homology further strengthened by the finding that the BUB2 gene complements the cdc16 ts fission yeast mutant. BUB2 is one of the BUB/MAD family of genes implicated in a mitotic spindle checkpoint 6,7. A cell that has been depleted of its microtubules, as the result of a nmtation or of drug treatment, arrests in mitosis. In a b u b - m u t a n t , there is no cell-cycle arrest; the cell proceeds into the next cell cycle, undergoes budding and DNA replication, and quickly dies. The original interpretation of these mutants was that the products of the BUB/MAD genes check that the mitotic spindle is in place. If the spindle is not assembled, then the BUB/MAD proteins are involved in arresting the cell in mitosis until spindle is completed. Mitotic arrest may be caused by the p34 cdc2/CDC28 mitotic protein
oD
i o)
(4 Ill No) FIGn cdcl6- mutant fission yeast cells make multiple septa. (a) Diagram of septation and cell separation in wild-type and cdcl6 ts mutant cells. Bold lines in the middle of the cells represent septa; ovals represent nuclei. (b) cdc16 ts mutant cells grown at restrictive temperature. Septa have been stained with calcofluor.
T1GOCTOBER1993 VOL.9 NO. 10 © 1993 Elsevier Science Publishers Ltd (I IK) 0168 - 9525/93/$06.(~)
E ~
i
~OMMENT
of one another. Septation would kinase. Activation of mitotic # spindle-~._ require only tile presence of a kinase is necessary for entry spindle, and would not depend into mitosis, and destruction of on the destruction of cdc2 kinase. mitotic kinase activity is required I m,,o.,s , I c,'o '°es"l Unfortunately, little is known for exit from mitosis. In a budabout the role of cdc2 kinase ding yeast cell arrested with no destruction at the end of mitosis spindle, mitotic kinase (CDC28) in fission yeast. The existence of activity remains high. In a bub2mutants that arrest in mitosis with mutant, mitotic kinase activity is "earlymitosis" ] condensed chromosomes and a not maintained, but slowly no spindle septum s,9 suggests that fission decays 6. Thus, the BUB2 protein cdc16 yeast can foma a septum without may cause mitotic arrest by precytokinesisOFF cdc2ON ON exiting from mitosis; however, venting inactivation of the mitotic cdc2 kinase activity has not been kinase. measured in these mutants. Fanldaauser et al. s tested [latemitosis I Clearly, the relationship between whether the cdc16 t~ fission yeast [spindlepresent septation and cdc2 kinase activity mutant acts in the same way as the 6 cytokinesisON remains to be elucidated. hub2- mutant. Fission yeast cells cdoaOFF OFF A second question is how cells that lack microtubules arrest in check for a mitotic spindle. What mitosis, with high cdc2 mitotic feature of the mitotic spindle is kinase activity and no septum. In septation ] completed actually being monitored? A fission c d c l 6 Lsmutants that lack microyeast cell that lacks microtubules tubules, there is no mitotic arrest; cdc16 ON cytokinesisOFF arrests in mitosis without forering cdc2 kinase activity falls, and the a septum. However, mutants with cells septate. Therefore, cdc16 in an abnomlal spindleg.10, such as fission yeast may play a role analthe kinesin mutant cutT, do form ogous to that of BUB2 in budding /~TGgl septa. Thus the switch into cytoyeast: in the absence of a mitotic spindle, cdc16may maintain high Model fbr a cdcl6switch that coordinates mitosis and kinesis does not require a fully cdc2 kinase activity and delay the cytokinesis. See text ff)r details. intact spindle, but may sinlply reexit from mitosis, cdcl6 is probquire some aspect of spindle forably not a simple activator of cdc2 mation, a prtxzess which is missing kinase, since cdc16 t.~mutant cells can (Fig. 2). In early mitosis (or in the in cells that are completely devoid of still enter mitosis; rather, it may be absence of a mitt)tic spindle), cdc16 functional micmtubules. necessary tt) prevent the premature is on, maintaining cdc2 kinase activity When pondering these complex destruction of cdc2 kinase activity anti inhibiting cytokinesis. At the end genetic circuits, it is important not to during mitosis. of mitosis, forrn:ltion of the mitotic forget the cell biology. For example spindle inhibits cdc16; its activity is in fission yeast, at least part of the Tying it all together: a model turned off, cdc2 kinase activity cdc2 kinase localizes on spindle pole In trying to work out the role of decays, cytokinesis is turned on, and bodie# l st) perhaps it is not surpriscdc16, we are faced with the enigma the cell septates. After a septum has ing that the state of microtubules might that a single gene product appears to formed, c d c l 6 is turned on, and affect activity or regulation of the be doing several different things: cytokinesis is turned off. kinase. Further studies of cdc16 and monitoring microtubules, regulating Of course, at the moment this checkpoints may help us appreciate the cdc2 kinase and regulating cyto- model is a genetic fantasy that awaits the importance of the cellular enkinesis. Here, we suggest a relatively biochemical tests. One genetic pre- vironment in which these cell-cycle simple model, which builds on others diction is that a cell that expresses a regulators act. proposed previously ~5. constitutively active foma of cdcl6, cdc16may be a central switch that owing to overexpression or mutation controls both cytokinesis and the end of cdcl6, would arrest in mitosis with References 1 Marks,J. and Hyams, J.S. (1985) Eur. of mitosis, acting as a positive regulator high cdc2 kinase activity, a spindle J. CellBiol. 39, 27-32 of mitosis and a negative regulator of and no septum. 2 Nurse, P., Thuriaux, P. and Nasmyth, cytokinesis. According to this model K. (1976) Mol. Gen. Genet. 146, (Fig. 2), when cdc16is switched on, Further issues 167-178 the cell is in mitosis and cdc2 kinase is Important issues remain to be ad3 Balasubramanian, M.K., Helfman, on. When cdc16is switdled off, the cell dressed. The relationships between D.M. and Hemmingsen, S.M. (1992) Nature 360, 84--87 exits mitosis and enters cytokinesis. the major players, cdc16, cdc2 4 Minet, M., Nurse, P., Thuriaux, P. Two checkpoint controls may govern activity and cytokinesis, must be and Mitchison, J.M. (1979) tl~e activity of cdcl6: (1) c d c l 6 is in- clarified. In the model presented J. Bacteriol. 137, 440-446 hibited by the presence of a mitotic by Fankhauser et al. 5, destruction of 5 Fankhauser, C., Marks, J., Reymond, spindle and (2)cdc16 is activated by cdc2 kinase activity may bring about A. and Simanis, V. (1993) EMBOJ. the presence of a completed septum. cytokinesis. Alternatively, cdc2 kinase 12, 2697-2704 cdc16 activity is predicted to activity and cytokinesis may be 6 Hoyt, M.A., Totis, L. and Roberts, switch on and off during the cell cycle regulated by cdc16 independently B.T. (1991) Cell66, 507-517
-se.,umJ
icdc1
Tit; OCTOnER1993 VOL.9 NO. 10
~3~
~OMMENT 7 Li, R. and Murray, A.W. (1991) Cell 66, 519-531 8 Hirano T., Hiraoka, Y. and Yanagida, M. (1988) J. Cell Biol.
Nature 347, 563-566 11 Alfa, C.E., Duconunun, B., Beach, D. and Hyams. J.S. (1990) Nature 347, 680-682
106, 1171-1183 .9 Uzawa, S. etal. (1990) Ce1162. 913-925 10 Hagan, I. and Yanagida, M. (1990)
aECHNICAL HIPS A q u i c k m e t h o d f o r i m m t m o s c r e e n i n g r e c o m b i n a n t bacterial c o l o n i e s When working with E. coli expression vectors, it is often helpful to check quickly for proper expression of cloned DNA fragments that contain an open reading frame (ORF). The usual procedure I is time consuming and involves unusual buffers and exposure to chloroform vapours. We describe here a quick protocol, based on commonly used techniques1, that is particularly useful for directly screening for peptide production in plated transformation reactions or in plasmid cDNA libraries. This method also allows the integrity of a cloned ORF to be rapidly checked after transfer from one vector to another, or when it has been cloned after PCR amplification. Nitrocellulose filters are soaked for 5 min in 5 mM IPTG, 5 mg m1-1 ampicillin, air dried for 5 min in a sterile hood, and placed onto plated bacterial cultures; small colonies (<0.3 mm diameter) yield the sharpest signals. Plates are incubated at 37°C for 3--5 h, then cooled at 4°C for 10 min. Filters are removed and placed, with the bacterial colonies facing up, onto 3MM Whatman paper soaked with 0.5 M NaOH, 1.5 i NaCl for 5 min. This lysis step is repeated. Plates are incubated at 37°C for 3--5 hours to allow recovery of clones. Filters are neutralized by placing onto 3MM Whatman paper soaked with 1 i TrisHCI pH 7.5 for 5 min. The neutralization step is repeated, and the filters are washed briefly in PBS, 0.05% Triton-Xl00. Subsequent steps are identical to those described previously1, and use PBS, 0.05% Triton-Xl00, 5% non-fat dried milk as a blocking buffer, and PBS, 0.05% Triton-Xl00, 0.5% non-fat dried milk during incubation with the antibody and during washing. Antigen-antibody complexes are revealed using the appropriate chromogenic stain. We routinely use this protocol for screening ORF constructs cloned in amp R vectors that carry [PTG-inducible promoters (e.g. Bluescript or pGEX) (Fig. la), but it can easily be adjusted for use with other vectors, or for other purposes, such as screening cDNA libraries. When monoclonal antibodies are used this method gives specific signals with a remarkably low level of background: we routinely check 6-10 antibodypositive clones from each transformation by western blotting and have never found a false positive. Nonspecific background is not increased when working in conditions that mimic cDNA library immunoscreening, using Petri dishes that contain >103 colonies (Fig. 2a,b). Moreover, background signal is very low even when the protocol is used on large solid cultures obtained from dotted inocula (Fig. 2b). ACKNOWLEDGEMENT
The pGEX-SCR plasmid was a gift of P. de Zulueta.
(a)
(b)
1
,
kD
.116.2 .97.4
.66.2
.45
FIGO (a) Immunodetection of peptide-producing colonies from a plated transformation mixture. The ORF of the modulo (rood) gene2 was PCR-amplified from cDNA, cloned in pGEX-2T and transformed into E. coli XL1. Nitrocellulose fdters carrying bacterial transformants were screened using the mod-specific monoclonal antibody (Mab La9), an alkaline-phosphatasecoupled secondary antibody and NBT/BCIP chromogens. (b) One of the positive clones, indicated in (a) by an arrow, was isolated, and correct expression of the ORF confirmed by SDS-PAGE. Lane 1, Coomassie Blue stained, no induction; lane 2, Coomassie Blue stained, 2 h induction with 0.1 m i IPTG; lane 3, mod revealed by Mab La9 a~er western blotting and 2 h induction with 0.1 mMIPTG.
(a)
1
=
.
....
~
:•
:" .."
.
3
(la)
2
1
3
,
'
.
~
.
•
~,~Ex.sc,
..
FIGM
(a) Colonies of E. coli XL1 (approximately 3.5 X 103 colonies) transformed with a mixture of pGEX-2T(97.8%), pGEX-MOD(2O~)and pGEX-SCR(0.2%) were plated to demonstrate immunodetection of clones producing mod and scr (sex-combs reduced protein). A nitrocellulose replica of the culture was cut into three pieces, and these incubated as follows: (1) Mab La9; (2) SCR-specific Mab 6H4 (Ref. 3); (3) Mab La9 and Mab 6H4. The 42 positive clones can be seen in (1), 8 in (2) and 65 in (3). (b) Cells transformed with either pGEX-MOD, pGEX-SCRor pGEX-2T were dotted on a plate, cultured overnight at 37°C and replicated onto nitrocellulose. Fusion proteins were identifiedusing: (1) Mab La9;(2) Mab 6H4; (3) Mab La9 and Mab 6H4.
TIG OCTOBER1993 voL. 9 No. 10
I