ice-like proteases in apoptosis

REVIEWS APOM~:~, or programmed cell death, is a naturally occurring process of cell suicide that plays a crucial role in the development and maintenance of meta- zoans by eliminating superfluous or unwanted cells (reviewed in Refs 1, 2). Despite its importance in normal physi- ology, the molecular and biochemical mechanisms of apoptosis are poorly understood. Recently, much of the knowledge on the genetic regulation of cell death has emerged from using the nematode Caenorhabditis elegans as a model organism. In this worm, 1090 cells are generated during development, of which 131 cells die by an intrinsic death program (reviewed in Ref. 3). Genetic analyses have identified 14 genes that function in different steps of programmed cell death in C. elegans. Three of these, ced-3, cod-4 and ced-9, play crucial roles in executing and regulating cell death, while others are needed for engulfment and disposal of dead cells (reviewed in ReL 4). Both ced-3 and cod.4 are essential for cell death to occur and, consequently, mutants ~ lcking either of these genes have extra cells s. The ced-9 gene antagonizes the function of cod-3 and ced.4 by pro- tecting cells from death 6. Conversely, in cod-9 loss-of-function mutants, cells that normally live undergo programmed cell death early In development, result- ing in embryonic lethality 6.

The first important clue indicating that the pathways regulating apoptosis are similar In C elegans and mammals came from the discovery that the function of the cod.9 mutation can be par- tlally restored by the expression of the human bcl.2 gone 7. Indeed, subsequent cloning of cod-9 showed that its product is similar to mammalian Bcl-2 protein s, bcl.2, originally cloned from the breakpoint of a t(14;18) chromosomal translocation in human B<ell lym- phomas, belongs to a family of related proteins known to modulate apoptosis in a wide variety of cells, by an as yet unknown mechanism (reviewed in Ref. 9). The cod-4 gene encodes a novel 631rDa protein with a potential Ca2"-bindtng domain m, but to date the mechanism of its function in apoptosis is completely obscure and no mammalian homologues of cod-4 have been found.

On the other hand, the cloning of cod-3 has revealed that its product is

$, Kumar is at the Hanson Centre for Cancer Research, Box 14, Rundle Mall Post Office, Adelaide, $A 5000, Australia.

TIBS 20 - MAY 1995

ICE.like proteges in apoptosis

Sharad Kumar

The discovery of structural and functional similarities between the product of the nematode cell-death gone ced-3 and mammaUan interleukin-/~- converting enzyme (ICE) is providing important insights into the molecular mechanism of apoptosis. This article summarizes the current knowledge of ICE and its homologues, and how these may be invoSved in regulating apoptosis.

similar to the mammalian cysteine protease interleuldn-ll3-converting enzyme (ICE) n, providing further evidence of evolutionary conservation of the apoptotic pathways. The predicted Cod-3 protein is 503 amino acids in length and contains a serine-fich region of approximately 100 amino acids in the middle of the protein ||. The expression of ced-3 is most abund- ant during embryonic development of C. elegans, when most programmed cell death occurs n. Animals carrying mutations in ced.4 show no effects on the levels of ced-3 mRNA, suggesting that ced-4 function is not required for ced.3 expressionlL Overexpression of ced.3 in Rat-1 cells results in apoptosis, which |urther suppor~s the role of ced.3 in programmed cell death ~2. The finding that one of the C elegans ceil-death proteins, Ced-3, is similar to ICE, a mammalian protein of known biochemical function, is an important step towards understanding the pathway(s) mediating cell death in mammals.

The developmentally regulated mouse gone Nedd2 was also shown to encode a protein similar to Ced-3 and ICE, and to induce apoptosis in mouse fibroblasts and neural cells 13. Sub- sequently, the human homo!ogue of this gone was cloned and named ICH.! (Ref. 14). The gene encoding another ICE-like protein, CPP32, has been cloned recently ~s, and a cysteine protease resembling ICE (priCE, for protease resembling ICE) has been detected in extracts from cells committed to apoptosis ~6. Emerging evidence suggests an important role for the ICE family of cysteine proteases in the active phase of mammalian cell death. This article reviews the current state of structural and functional understanding of these proteins.

iCE Interest in ICE goes back several

years because of its function in processing interleukin-l[3 0L-l[3), a cytokine that plays a key role in inflammation and in a variety of other physiological and pathological processes. First isolated from monocytic cells, ICE was shown to cleave the 31 kDa IL-I[~ precursor (pro-IL-lJ3) at Aspll6-Alall7 to generate the 17.5 kDa mature form of IL- li3 ~7,~8. Purification of ICE and cloning of its gone revealed that it was not related to any known proteaseslg,20. The mature active form of human ICE is derived from a 404 amino acid precursor (p45) by proteolytic cleavage at Aspl03, Aspllg, Asp297 and Asp316 (Ref.19; Fig. l). The protease involved in the cleavage of p45 may be ICE itself l:~. The active enzyme consists of two subunits, p20 (residues 120=297) and pl0 (residues 31'1-404), both of which are essential for enzymatic activity ~9. Inhibitor studies indicate that ICE is a cysteine protease, with Cys285 serving as the catalytic residue ~9. Mutations of this residue result in complete loss of enzyme activity ~9. Two groups have recently reported the crystal structure of ICE, which indicates that the catalytically active form of the enzyme is a tetramer consisting of a (p20-p10)2 homodimer 2|,22. The model proposes that each p20=pl0 hereto- dimer in the tetramer ,s probably derived from two different p45 precursor molecules 2L22. The active site of ICE spans both p20 and pl0 subunits, which explains the requirement for both subunits for catalysis. The structure also indicates that the interac- tions between the p20 carboxyl terminus and the pl0 amino terminus are major contributory factors to the stab- ility of the dimer. In the crystal structure, covalent binding of the ICE

© 1995, Elsevier Science Ltd 0968-0004/95/$09.50

T ~ S 20 - MAY 1995 REVIEWS inhibitor AcoTyr-VaJ-A~a-Asp~cNoromethyl- ketone (Ac-YVAD=CMK) at Cys285 con- [h'med that iCE is a cysteine protease. In addition to Cys285, His237, with its imidazole ring close to the sidechain of Cys285, is also likely to have a ~'ole in catalysis 21,22. Target cleavage by ICE requires Asp in the P1 position and a small hydrophobic amino acid residue at P(. The minimum peptide substrate for ICE contains four amino adds before the cleavage site, the optimal peptide sequence being Ac-YVAD m. The requirement of ICE for Asp at the P~ position is shared with serine proteases of the granzyme B family, which are needed for the cyto= toxic activity of lymphocytes and natu- ral kil~er cells~,~! Although there is no apparent structural similarity between ICE and granzymes, these serine proteases are known to induce apoptosis, suggesting that the activation of these Asp-spedfic proteases plays an important role in the mediation of apoptosis.

Several lines of evidence suggest that ICE or its homologues have a direct role in the regulation of apoptosis. The protein sequence of ICE is 2870 identical to that of Ced-3 (Ref. 11), and overexpres- s~on of the gene encoding ICE ~n Rat-I cells was shown to induce apoptosis ~. Mutations in the catalytic Cys residue of ICE completely abolish its apopto.'is- indudng activity. Microin]ection of ICE- eDNA expression vectors into chicken dorsal-mot-ganglion neurons also results in cell death ~, while micro- injection of CrmA, a viral inhibitor of ICE ~, prevents neuronal cell death induced by deprivation of nerve growth factor ~5. Expression of the gene encoding CrmA in RaM cells prevents apoptosis induced by serum depletion ~4, pre- sumably by inactivating ICE or a related protease. Increase in ICE activity has been reported in phagocytic cells stimu- lated with agents that promote apoptosis ~7. The majority of ICE in monocytic cells is in the form of the inactive 45k De precursor ~s, which is probably cleaved to generate active enzyme under conditions that promote apoptosis. ICE has also been implicated in the extracellular-matrix-reguiated apoptosis of mammary epithelial cells ~9. The only known substrate of ICE is pro-IL-l[~, yet ICE expression has been detected in many cell types that do not ap- pear to produce IL-1B, suggesting that there may be additional substrates and that IL-113 itself is not required for apoptosis.

Nedd2 was originally identified by a subtraction-cloning approach as a developmentally regulated gene in the mouse central nervous sys- ocE tern 3°, and was subsequently shown to encode a protein similar to ICE and Ced-3 (Re ~ 13). Using a mouse Yedd2 eDNA probe, Wang et el. ~4 isolated the human homologue of Yedd2 and named i t ]CH-I (in this article, Nedd2 and iCHJ refer to mouse and human homologues, respectively). Most tissues express two forms of Yedd2/lCH4 mRNA derived from the alternative splicing of a 61 base-pair exon. This splicing results in frameshift and thus truncation of the 51kl)a Nedd2/ICH-IL protein to generate a 39kDa Nedd2s/EH-Is protein (Ref. 14; S. Kumar, unpublished; Fig. 2). Nedd2/ICH-I is a somewhat closer relative of Ced-3 (31% identity) than ICE (29% identity), suggesting that Nedd2/ICH-I and ICE genes probably evolved in- dependently of each other from ced-3. Sequence alignment shows that all of the residues implicated in substrate binding and catalysis of ICE are conserved in Nedd2/ICH-1 (Fig. 2), suggesting that Nedd2/ICH-I is also a cysteine protease. Overexpression of Nedd2/ICH-l/. in a number of cell types results in apoptosis, although not all cells are equally sensitivem! Mutations of the Cys319 residue of Nedd2 or Cys320 of ICH-1L, which correspond to the catalytic Cys285 in ICE, completely abolish the apoptosisqnducing activity, which is consistent with the hypothesis that the protease activity of these proteins is responsible for mediating cell death m J4. In addition, inhibition of Nedd2/ICH-1L- induced apoptosis by Bcl-2 (Refs 13, 14), and to some extent by CrmA ~4, implies that its mechanism of action is similar to Ced-3 and ICE.

The smaller Nedd2s]ICH-Is protein lacks the pl0 subunit and is structurally similar to the putative p20 subunit con- talning the conserved catalytic residues (Fig. 2). The only difference between the p20 subunit and Nedd2s]iCH-1 lies in the carboxy-terminal sequence, where Nedd2s]ICH-ls contains 21 amino

D D D9

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DD DD Nedd2s/| I! HI gCH- l s r - .......................

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Ced-3 and its mammalian homoiogues. Active human interleukin-l[3-converting enzyme (iCE) is generated by the proteoDytic cleavage of a 45 kDa precursor and consists of two subunits, p20 and plO. The cleavage of iCE occurs at specific Asp (D) residues as indi- cared. Cod-3, Nedd2/iCH-1 and CPP32 contain several Asp residues that can act as possible cleavage sites. The smaller Nedd2s/ICH-ls protein is derived from an alternatively spliced mRNA and lacks the region corresponding to the plO subunit of ICE. A pentapep- tide sequence, GIn-Aia-Cys-Arg-Gly (blue) is conserved in all these proteins. ~n ICE, the Cys contained with;n this pentapoptide sequence acts as the catalytic residue. The residue numbers shown are for human iCE, iCH-1 and CPP32 proteins.

acids derived from the alternative]y spliced exon, in place of the last ten residues of the putative p20 subunit of Nedd2/lCH-lc (Fig. 2). 'Expression of ICH-ls was shown to prevent serum- deprivation-induced apoptosis of RaM cells, suggesting that it may act as an antagonist of ICH-b functionl4; however, the mechanism of this is not clear. Mammalian cells constitutively express- ing Nedd2s show no protection against apoptosis induced by overexpression of Nedd2 (S. Kumar, unpublished). Whether ICH-Is prevents cell death in RaM cells by interacting directly with ICH-lc in a dose-dependent manner, or acts on some other positive regulator of apoptosis, remains to be established.

CPP32 The gone encoding CPP32 (32kDa

putative cysteine protease) was recently cloned from a human T-cell line 15. Although CPP32 is considerably shorter than the other ICE homologues, it con- talns regions corresponding to both the p20 and the pl0 subunits and shows conservation of the amino acid residues

~ 9

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REVIEWS I ' iBS 20 - MAY ~995

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Figure 2 An alignment of the protein sequences of Ced-3, interleukln-213-converting enzyme (ICE), Nedd2/ICH-1 and CPP32. Nedd2 and Nedd2s rep- resent two forms of mouse proteins derived from alternatively spliced mRNA species (Ref. 13; S. Kumar et al. unpublished). Human homologues are shown as ICH-1L and ICH-ls (Refs 14, 35). The amino-terminal sequence in ICH-1L and ICHols is derived from Ref. 35. hlCE and mICE indicate human 19,2o and mouse 36 ICE, respectively. Conserved residues are ~oxed. Blue boxes indicate the residues conserved between two of the four proteins (ICE, Cede, Nedd2/ICH-1 and CPP32). The residues conserved between all proteins are shown in yellow. Residues corresponding to the catalytic Cys285 of ICE are shown in red. His237 of ICE, shown in purple, has also been implicated in cata- lysls =l,12. Arg179, Gin283, Arg34:l. and Ser347, marked by red dots, form the Asp pocket in ICE 2i,22 and are conserved in all these proteins, The locations of Asp cleavage sites in human ICE to generate p20 and plO subunits are indicated. Note that the regions corresponding to the p20 and plO subunits of ICE are the most conserved regions in these proteins.

required for substrate binding and catalysis of ICE (Fig. 2). Expression of full-length CPP32 using a baculovirus expression vector results in death of Sf9 insect cells. Co-expression of the regions corresponding to the two subunits (Fig. I) also induces apoptosis, suggesting that CPP32 is both structurally and functionally similar to ICE. The CPP32 transcript is detected in various cell lines of different origins, with cells of haemopoietlc lineages showing higher expression ~s.

pdCE A cysteine protease activity with a

role in apoptosis was identified in cyto- plasmic extracts prepared from chicken DU249 cells committed to apoptosis.

2OO

This enzyme was named priCE for protease resembling ICE 16. priCE cleaves the nuclear enzyme poly(ADP-ribose) polymerase at a tetrapeptide sequence Ac-YVAD, which is identical to one of the two ICE cleavage sites in pro-IL-ll3. priCE is distinct from ICE as poly(ADP-ribose) polymerase is not cleaved by ICE and priCE does not cleave pro4L-l[~. Cleavage of ll6kDa poly(ADP-ribose) polymerase to 85 kDa and 25kDa fragments is an early process in drug- and ~radiation-induced apoptosis 3l, although it is not clear whether this is necessary for apoptosis to proceed. Significantly, the inhibition of priCE by the ICE inhibitor Ac-YVAD-CMK blocks the biochemical and morphological changes seen in a

cell-free apoptosis system developed by Lazebnik et al. 32, suggesting that priCE or a homologue lies near the apex of an apoptotic pathway. Although priCE has not been purified and no information on its sequence is currently available, its functional similarity indicates that it is likely to be an ICE homologue. Further work is required to establish whether priCE represents a novel cysteine protease or whether it is the chicken homologue of Nedd2/ICH-1 or CPP32.

R~=~l~ion of pmteM~ in almptosis The presence of several Ced-3-1ike

proteins in mammals provides evidence that the cellular regulation of ICE-like proteases in apoptosis is likely to be

TOBS 20 - MAY 1 9 9 5 REVIEWS complex, but at present there is little information available on this. Many cells express more than one of these cysteine proteases, but whether all are required in a single pathway is not known. Rt is pRausible that each of these proteases works on a different set of targets. This is supported by the fact that, although ICE and priCE recognize the same tetrapeptide cleavage site, they act on different target proteins. Significant sequence divergence be= tween ICE, Nedd2/lCH-1 and CPP32 also suggests different target specificities for these proteins, ff this is true, all these proteases should be required for apoptosis in a single cell type, and dis- ruption of any one should block cell death. These proteases might regulate the activity of each other in a hierarchi- cal pathway or, conversely, apoptotic signals may activate all these proteases simultaneously. Since activation of these proteases requires cleavage into p20- and pl0-1ike subunits, they might also be regulated by forming cross- species heterodimers.

Individually, the regulation of these proteases might be achieved at the level of transcription, post-translational modifications and/or interaction with other modulators. Nedd2 expression is high in several embryonic mouse tissues undergoing programmed cell death ~3, and overproductiot~ of both Nedd2/ICH-1L and ICE induces apoptosis m4. A recent report indicates that the mRNA encoding ICE is induced in mammary epithelial cells under conditions that promote apoptosis zg. Base- ment-membrane extraeellular matrix, which suppresses the apoptosis of mammary epithelial cells, negatively regulates ICE expression ~9. Activation of ICE by proteolytic cleavage in apoptotic cells has been documented ~-7, and priCE activity is detected only in cells committed to apoptosis ~6, suggesting that post-translational processing is also an important step in the regulation of these proteases. Proteins such as members of the Bcl-2 family, Nedd2s/ICH-ls, and possible cellular homologues of baculovirus protein p35 (Ref. 33) and cowpox virus CrmA, both of which can suppress apoptosis in mammalian cells, are all likely candidates in modulating the activity of these proteases. In C. elegans, both cod-3 and ced4 genes act at the same general step in the apoptotic pathway. Thus, the so-far undiscovered mammalian homologue of Ced-4 may modulate the activities of ICE-like mammalian proteases positively. Cod.4 might

be the target for Ced-3, or it might stimulate the proteolytic activation of Ced-3 (Reds 4, 12). The modem of apoptosis shown in Fig. 3 tries to in- corporate various regulatory aspects of ICE-like proteins and is based on the assump- tion that all cell-death pathways converge onto a single apical reaction - the activation of cysteine protease(s)- that initiates apoptosis by cleaving some crucial cellular protein target(s) z.

Concluding remaiks There is growing support

for a central role for ICE-like cysteine proteases in apop tosis. However, many important questions about their biology and regulation re- Bcl-2 main. The immediate priority is to determine the downstream targets of various ICE-like proteases. The specificity of the proteolytic cleavage of ICE suggests that the number of targets for the ICE family of proteins may be limited to certain crucial proteins, but we need to know whether these targets are intermediate signalling molecules or the end points in the apoptotic pathway. At present, it is also not clear whether these proteases me- diate apoptosis induced by all types of signals. For in- stance, cytotoxic-T-cell me- diated apoptosis is resistant to Bcl-2, so it should be o| interest to check whether the ICE family of proteases is involved. Mouse-gone knock- out mutants should help to establish the role of these cysteine proteases in apoptosis, although further biochemical and molecular analyses will be critical in delineating the functional aspects of these proteins. There is emerging evidence suggesting that aberrant regulation of apoptosis plays an important part in a number of human disorders, such as Alzheimer's disease, acquired immune deficiency syndrome and cancer (reviewed in Ref. 34). If ICE-like cysteine proteases do play a fundamental role in apoptosis,

r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,

Signal.

eca-e ~ t ~ F==- ps5 t?t

ICE-tike proteins

A L ~ Autoactivation

- - I ~ ~ p35(?)

Target{s)

Col| death

Rgure 3 A model showing the involvement of ICE-like cysteine proteases as ~ffectors of apoptosis. This model is based on an earlier proposal by Vaux et al. (Ref. 2). Central to this model is the activation of cysteine protease(s) by cleavage into p20 and plO subunits to generate active enzyme(s). The initial activation of protease(s) would lead to further autoactivation to drive the pathway into cell death. This model argues that once the proteases have been activated, no new protein synthesis is required for the execution of cell death. Based on the structure of ICE, the active pretense consists of a dimer of two p20-plO heterodimers, each heterodimer derived from a p20 and a plO subunit from two different precursor molecules 21,22. The negative regulation of these proteases may be achieved by interaction with proteins such as Bcl-2, Nedd2s/ICH-ls, and possible cellular homologues of the viral proteins such as p35 and CrmA. Although both Bcl-2 and p35 are known to inhibit mammalian cell death, as yet it is not clear at what specific step in the apoptotic pathway they might act. These proteins may inhibit the activation of the cysteine proteases by blocking proteolytic cleavage, or by sequestering the downstream targets. The so-far undiscovered mammalian homologue of C. elegans Cod-4 (Ced-4H) may either interact directly with, or act as a target for, the cysteine protease(s). On the basis of the work with C. elegans 4 it has been placed parallpl to the Cod-3 homologues. However, its actual role and location in the apoptotic pathway remain to be established.

it may be possible to regulate ceil death by using specific protease inhibitors. This may have important therapeutic implications in the treatment of dis- eases that arise from excess or prema- ture cell death.

COMPUTER CORNER ~ m

Li et a/. 37 recently reported that mice deficient in ICE are apparently normal: although they show a defect in the pro- duction of mature IL-I[5 after stimu- lation with lipopolysaccharide, thymo- cytes and macrophages from these animals undergo normal apoptosis, indicating that ICE does not have an autonomous function in programmed cell death. These results suggest that multiple ICE-like cysteine proteases in mammalian cells are redundant in their apoptotic function. Alternatively, ICE itself might not play a role in apoptosis. In this case, one or more of the other ICE family of proteins may be involved in mediating apoptosis.

Aclmowledgemen~ 1 thank L. Ashman and N. Harvey

for comments on the manuscript, D. Vaux for stimulating discussions and A. Cambareri for help with the figures.

Re~nc~ 1 Raft, M. C. (1992) Nature 356, 397-400 2 Vaux, D. L., Haecker, G. and Strasser, A. (1994)

Cell 76, 777-779 3 Sulston, J. (1988) in The Nematode

Caenorhabditis e/egans, pp. 123-155, Cold Spring Harbor Laboratory Press

4 Hengartner, M. O. and Horvitz, H. R. (1994) Cur[. Opin. Genet. Day. 4, 581-586

5 Ellis, H. M. and Horvitz, H. R. (1986) Cell 44, 817-829

6 Hengartner, M. 0., Ellis, R. E. and Horvitz, H. R. (1992) Nature 356, 494-499

7 ;~ux, D. L., Weissman, I. L. and Kim, S. K. (1992) Science 258,1955-1957

8 Hengartner, M. O. and Horvitz, H. R. (1994) Cell 76, 665-676

9 Nufiez, G. and Clarke, M. F. (1994) Trenas Cell BioL 4, 399-403

10 Yuan, J. and Horvitz, H. R. (1992) Development 116, 309-320

11 Yuan, J. et aL (1993) Cell 75, 641-652 12 Miura, M. et aL (1993) Cell 75, 653-660 13 Kumar, S. et al. (1994) Genes Day. 8,1613-1626 14 Wang, L. et aL (1994) Cell 78, 739-750 15 Femandes-Alnemri, T., Litwack, G. and Alnemri,

E. S. (1994) J. BioL Chem. 269, 30761-30764 16 Lazebnik, Y. A. et aL (1994) Nature 371, 346-347 17 Black, R. A., Kronheim, S. R. and Sleath, P. R.

(1989) FEBS Lett. 247, 386-390 18 Kostura, M. J. et aL (1989) Proc. Natl Acad. Sci.

USA 86, 5227-5231

TIBS 2 0 - MAY 1995

t9 Thornberry, H. A. et al. (1992) Nature 356, 768-774

20 Cerretti, D. P. et al. (1992) Science 256, 97-100

21 Wilson, K. P. et aL (1994) Nature 370, 270-275 22 Walker, N. P. C. et al. (1994) Cell 78, 343-352 23 Odake, S. et al. (1991) Biochemistry 30,

2217-2227 24 Shi, L. et al. (1992) J. Exp. Mad. 176,1521-1529 25 Gagliardini, V. et al. (1994) Science 263, 826-828 26 Ray, C. A. et al. (1992) Cell 69, 597-604 27 Nett-Rordalisi, M. A., Berson, D. R. and Chaplin,

D. D. (1993) J. Cell Biochem. 17B, 117 28 Ayala, J. M. et aL (1994) J. Immunol. 153,

2592-2599 29 Boudreau, N. et aL (1995) Science 267,

891-893 30 Kumar, S. Tomooka, Y. and Noda, M. (1992)

8iochem. Biophys. Res. Commun. 185, 1155-1161

31 Kaufmann, S. H. et al. (1993) Cancer Res. 53, 3976-3985

32 Lazebnik, Y. A. et al. (1993) J. Cell BioL 123, 7-22

33 Clam, R. J., Fechheimer, M. and Miller, L. K. (1991) Science 254, 1388-1390

34 Barr, P. J. and Tornei, L. D. (1994) Biotechnolo~ 12, 487-493

35 Kumar, S. et al. Human Genet. (in press) 36 Nett, M. A. et al. (1992) J. Immunol. 149,

3254-3259 37 Li, R et al. (1995) Cell 80, 401-411

Methods and reagents Agarose gel electrophoresis in your kitchen

Methods and reagents Is a unique monthly column that highlights currer:' ,~i~,~ ~ :~::= ,, th¢ newsgroup blonet.molbio.methds-reagnts, available on the Internet. This month's column describes how to make an electrophoresis system from common houc3hold items. For details on how to partake in the newsgroup, see the accompanying box.

Hme. m electmphoresb Characterization of DNA molecules is usually done by electrophoretic separ- ation through a gel matrix. Since some researchers are constantly trying to get

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For more details, see also T/BS 18 (1993). ~ .

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away with the least expensive way to make their own equipment, and one thread of discussion this month sur- rounded the construction of an agarose gel electrophoresis box from scratch for under a dollar, this led me to recall some past discussions based on a similar premise - a low-cost method of doing molecular biology in your kitchen or basement. Here i've compiled some of the ideas posted to the net for doing simple and cheap lab experiments at home, or as a demonstration in a high- school classroom.

To construct a gel box for under a dollar, first you'll need a plastic box. A lid from an old molecular-biology-kit box, a toolbox, a fishing box, a pipet- tip rack, or a small (6 x 8 inches) Tupperware TM sandwich-saver will suf- fice. You'll also need a smaller box and a makeshift comb for casting the gel.

The 2-3 mm wide plastic combs that come with disposable pre-cast gels are usually meant to be thrown away; however, they can be trimmed down with a razor blade and smoothed over with a nail file to the desired size and will probably last the lifetime of your home- made gel system.

Electrodes can be made out of two paper clips, but the cathode will pit and eventually erode away to nothing after a few gel runs. Although it costs a little more, a better choice is platinum wire, which can be purchased in various gauges and lengths from many scientific supply companies. To cut the cost, a cheaper nickel-chromium wire may be used as the anode since the anode will not pit in the same way as the cathode.

Weird science Chulho Kang (kang~nsvax.mssm.edu)

wrote that he has designed a gel box with carbon rods as the electrodes. For his system, two pencil cores act as the electrodes. To remove the core from a pencil, he flamed it with a bunsen burner to burn off the wood until only the core remained. He then made two holes a little larger than the carbon core diameter at opposite ends of a plastic box and pushed the pencil core through, allowing about 0.5 cm to stick

ice-like proteases in apoptosis

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