Arabidopsis alternative oxidase sustains Escherichia coli respiration

Proc. Nati. Acad. Sci. USA Vol. 89, pp. 10842-10846, November 1992 Biochemistry

Arabidopsis alternative oxidase sustains Escherichia coli respiration (cyanide-resistant respiration/complementation/hemA mutation)

A. MADAN KUMAR AND DIETER SOLL* Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511

Communicated by Eric E. Conn, August 17, 1992 (received for review June 16, 1992)

Glutamyl-tRNA reductase, encoded by the ABSTRACT hemA gene, is the first enzyme in porphyrin biosynthesis in many organisms. Hemes, important porphyrin derivatives, are essential components of redox enzymes, such as cytochromes. Thus a hemA Escherichla col strain (SASX41B) is deficient in cytochrome-mediated aerobic respiration. Upon complementation of this strain with an Arabidopsis thaliana cDNA library, we isolated a clone which permitted the SASX41B strain to grow aerobically. The clone encodes the gene for Arabidopsis alternative oxidase, whose deduced amino acid sequence was found to have 71% identity with that of the enzyme from the voodoo lily, Sauromatum guam. The Arabidopsis protein is expressed as a 31-kDa protein in E. coil and confers on this organism cyanide-resistant growth, which in turn is sensitive to salicylhydroxamate. This implies that a single polypeptide is sufficient for alternative oxidase activity. Based on these observations we propose that a cyanide-insensitive respiratory pathway operates in the transformed E. colt hemA strain. Introduction of this pathway now opens the way to genetic/molecular biological investigations of alternative oxidase and its cofactor. The synthesis of ATP, an important carrier of free energy in the cell, is coupled in respiring organisms to the flow of electrons through the electron transport chain to a terminal electron acceptor such as oxygen. These organisms employ the well-characterized cyanide-sensitive, cytochromemediated respiratory pathway in which electrons flow from the substrates (e.g., metabolites of the citric acid cycle) to terminal oxidase via ubiquinone and cytochromes (Fig. 1A). In addition to this pathway, all higher plants contain a cyanide-resistant pathway involving an enzyme, alternative oxidase, that is sensitive to inhibition by substituted hydroxamates such as salicylhydroxamate (SHAM) (for review see ref. 1). In this route (Fig. 1B), electrons branch from the cytochrome pathway at ubiquinone (before cytochrome) and terminate with an alternative oxidase (2). Unlike the free energy from the cytochrome pathway, the free energy generated by flow of electrons from ubiquinone to alternative oxidase is generally believed not to result in the generation of ATP but instead is lost as heat (2). The alternative pathway is assumed to be operative when the conventional cytochrome pathway is either constricted by respiratory inhibitors or saturated with an excess of electrons generated during certain situations (2-4). Conditions such as germination, wound healing, flowering, and exposure to cold were shown to be associated with an increased alternative oxidase activity (3). A physiological role for the alternative pathway is well known in certain plants of the family Araceae. Due to an increase in the alternative oxidase activity in the flowers, heat is generated (up to 200C above ambient temperature) to volatilize foul-smelling attractants for insects engaged in the pollination process (5).

To study the biochemical aspects of alternative oxidase in more detail, attempts have been made to purify this enzyme from spadix mitochondria isolated from Arum maculatum (6); the voodoo lily, Sauromatum guttatum (7); and skunk cabbage, Symplocarpus foetidus (D. A. Berthold and J. N. Siedow, as reported in ref. 5). Analysis of the purified enzyme fractions showed three (D. A. Berthold and J. N. Siedow, as reported in ref. 5) or more (2) protein bands. These protein fractions were also used in the development of monoclonal antibodies (8), which identified a family of immunologically related proteins of 35-37 kDa. The antibodies also aided the isolation of a cDNA clone encoding alternative oxidase from S. guttatum with a predicted mature size of 32 kDa (9). Here we report the isolation, characterization, and functional expression of the gene for the alternative oxidase from Arabidopsis thaliana.t This was facilitated by the suppression by an Arabidopsis cDNA of a hemA mutation (10) that blocks the biosynthesis of 5-aminolevulinic acid in Escherichia coli. The rationale for the strategy of such suppression was originally designed to isolate genes involved in chlorophyll biosynthesis.

MATERIALS AND METHODS General. A series of overlapping DNA fragments was generated by the exonuclease III/nuclease S1 deletion method (11). Nucleotide sequences were determined by the dideoxy method (12) and analyzed by the Wisconsin Genetics Computer Group programs (13). Plaque hybridizations and genomic blot analyses were carried out according to published protocols (14). An A. thaliana genomic DNA library (in AGEM-11) was a gift from R. W. Davies (Stanford University). Strains and Growth Conditions. The E. coli strains used were DH5a and the 5-aminolevulinic acid auxotroph SASX41B (HfrPO2A hemA41 metBI relAI) (15). The strains were grown on M9 minimal medium (14) with glycerol (0.2%) as a carbon source and supplemented with 5-aminolevulinate (50 Ag/ml) and methionine (40 j.g/ml) when required. Ampicillin (100 tug/ml) was added to the growth medium when

indicated. Complementation. Plasmid DNA (100 ng) from an Arabidopsis cDNA library in plasmid pcDNAII (gift from R. Meagher, University of Georgia) was used to transform competent SASX41B cells by electroporation. Aliquots were plated on M9/ampicillin medium and incubated at 370C to select colonies that contained putative hemA-complementing plasmids. Immunoblotting. E. coli lysates were fractionated by electrophoresis in SDS/15% polyacrylamide gels. Blotting was Abbreviation: SHAM, salicylhydroxamate. *To whom reprint requests should be addressed at: Department of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 6666, New Haven, CT 06511. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M96417).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 10842

10843

Proc. Natl. Acad. Sci. USA 89 (1992)

Biochemistry: Kumar and S611

A

SUCCINATE

Q5INON

-H 2

O

CYANIDE SENSITIVE RESPIRA TION

NADH & NADH LINKED SUBSTRATES SUCCINATE

B

(7777(24% C°2

H20 NADH & NADH LINKED SUBSTRATES

°2 A LT ERNATIEOD

-H20

aA---I

CYANIDE SENSITIVE RESPIRA TION

CYANIDE RESISTANT RESPIRA TION

C

SUCCINATE

02 H2 0

NO RESPIRA TION

NADH & NADH LINKED SUBSTRATES

SUCCINATE

D ..

FP -:

H2 0 NADH & NADH IN¶'ED SUBSTt ATES

fALTERNATIVE

2 OXIDAS -

_---..

H 20

NO RESPIRA TION

CYANIDE RESISTANT RESPIRA TION

FIG. 1. Features of the cytochrome-mediated respiratory pathway. Electrons generated from substrates of the citric acid cycle flow through flavoproteins (FP), ubiquinones, and cytochromes and finally are oxidized to form water (A), and in cyanide-resistant respiration, electrons deviate from the cytochrome pathway at ubiquinone and are oxidized by alternative oxidase (B). The cytochrome-mediated respiratory pathway is nonfunctional in SASX41B cells because the hemA mutation leads to inability to synthesize heme, an essential cofactor for cytochromes (C). Introduction of an active alternative oxidase gene permits hemA cells to overcome the need for the functional cytochrome for aerobic respiration. In this pathway electrons diverge at ubiquinone and are oxidized by alternative oxidase to yield water (D).

10844

Biochemistry: Kumar and Soil

Proc. Nad. Acad. Sci. USA 89 (1992)

done exactly as described (16) except that the second antibody used was conjugated with horseradish peroxidase. AOA monoclonal antibodies against S. guttatum alternative oxidase made by T. Elthon (8) were a gift of J. N. Siedow (Duke University). Growth of E. coli in the Presence of Inhibitors. To monitor the effect of respiratory inhibitors on growth, E. coli cells were prepared as follows. E. coli DH5a cells transformed with either empty pcDNAII vector or with pAOX were grown into logarithmic phase in minimal medium, pelleted at 4000 x g for 10 min, washed three times with 10 mM MgSO4, and resuspended in minimal medium at a cell density of 10 OD6w units/ml. For growth-inhibition studies, 0.2 ml of this suspension was added to 20 ml of M63 medium (17) containing 0.5% succinate, 0.1% Casamino acids, 0.2% glycerol, thiamin (1 pug/ml), ampicillin (100 ,g/ml), and various concentrations of KCN and SHAM and incubated at 370C. Fresh KCN and SHAM solutions were prepared as described (18).

RESULTS Complementation of the E. coil hemA Mutation by Arabidopsis cDNA. Electroporation of Arabidopsis cDNA into SASX41B cells, auxotrophic for 5-aminolevulinic acid, resulted in the appearance of two colonies (out of about 108 transformants) on M9/ampicillin plates after 72 hr at 370C. Plasmids purified from these transformants conferred 5-aminolevulinic acid prototrophy on SASX41B cells upon reelectroporation. Restriction analysis of the inserted DNA suggested these plasmids to be identical (data not shown), and one of them (pAOX) was selected for further analysis. The 1.1-kilobase (kb) cDNA insert in pAOX was completely sequenced. It contained an incomplete open reading frame (see legend to Fig. 2) and a 0.2-kb 3' untranslated region. This cDNA fragment was used as a probe to isolate a larger genomic clone as a 6.3-kb BamHI fragment. This clone contained the complete 5' end of the open reading frame (see legend to Fig. 2). Characterization of the Arabidopsis Alternative Oxidase Gene. Initial analysis of the DNA sequence indicated an open Ataaox

..........

..........

..........

..........

reading frame of 918 nucleotides. The deduced amino acid sequence (Fig. 2) of the open reading frame did not show any homology with the known genes involved in the porphyrin biosynthesis (reviewed in ref. 10). Instead, a search of the GenBank data base (release 70.0) revealed that the open reading frame displayed significant nucleotide homology with alternative oxidase of S. guttatum (9). However, there was no homology with the 5' and 3' flanking regions, nor were consensus promotor elements or polyadenylylation consensus sequences found. The open reading frame encodes a 306-amino acid protein of about 33 kDa. The deduced Arabidopsis alternative oxidase amino acid sequence displayed 71% identity with that of the S. guttatum enzyme, with the highest homology present in the C-terminal half (Fig. 2). Although significant sequence homologies between terminal oxidases are known (19, 20) the Arabidopsis alternative oxidase does not share any of these features. To explore the arrangement of the alternative oxidase gene(s) in the genome of Arabidopsis, genomic blots of total Arabidopsis DNA digested with BamHI and EcoRI were hybridized with the complete cDNA fragment of pAOX under stringent conditions. In addition to a faint hybridizing signal, the digests showed a single strong band (BamHI, 6.3 kb; EcoRI, 12 kb) (data not shown). Hybridization of the cDNA fragment of pAOX with random amplified polymorphic DNAs from recombinant inbred Arabidopsis lines (21) indicated that the alternative oxidase gene is located on chromosome III, -8 centimorgans below the AAT 255 region on the A. thaliana genome. Arabidopsis Aternative Oxidase Is Expressed in E. coi. The isolation of the pAOX clone by suppression suggested that there was functional expression of the A. thaliana gene in E. coli. To demonstrate the presence of the protein in E. coli extracts, we performed immunoblot analysis with antibodies against S. guttatum alternative oxidase. It had been observed earlier that the AOA monoclonal antibody reacted with the corresponding protein from different tissues of a variety of plants (8). As can be seen in Fig. 3, a cross-reacting protein (apparent molecular mass of 27 kDa) was present in all

1 ........

Suraox

MMSSRLVGTA LCRQLSHVPV PQYLPALRPT ADTASSLLHG CSAAAPAQRA

Ataaox Suraox

*J v 51 DTRAPT.IGG MRFASTITLG EKTPMKEEDA NQKKTENEST GGDAAGGNNK GLWP-SWFSP P-H---LSAP AQDGG--KA- G.... AGKVP P-EDG-AEK.

Ataaox Suraox

101 GDKGIASYWG VEPNKITKED GSEWKWNCFR PWETYKADIT IDLKKHHVPT ..EAVV---A -P-S-VS--- ----R-T--- -----Q--LS ---H------

Ataaox Suraox

Suraox

201 KSLRRFEQSG GWIKALLEEA ENERMHLMTF MEVAKPKWYE RALVITVQGV -------H-- ---R------ ---------- ----Q-R--- ----LA----

Ataaox Suraox

FFNAYFLGYL ISPKFAHRMV GYLEEEAIHS YTEFLKELDK ---------- L-------V- ---------- ------DI-S

Ataaox Suraox

IAIDYWRLPA DATLRDVVMV VRADEAHHRD VNHFASDIHY --L------Q GS------T- ---------- -------V--

Ataaox Suraox

PIGYH

Ataaox

FIG. 2. Alignment of alternative oxidase

amino acid sequences deduced from cDNA se151 quences of A. thaliana (Ataaox) and voodoo lily TFLDRIAYWT VKSLRWPTDL FFQRRYGCRA MMLETVAAVP GMVGGMLLHC S. guttatum (Suraox). The sequence of the Ara-I--KL-LR- --A------I ------A--- ---------- -----V---L bidopsis enzyme is presented in one-letter amino acid symbols; a dash indicates the same

-L---

amino acid in the S. guttatum enzyme. The S. guttatum enzyme has a 49-amino acid extension at the N terminus compared with the Arabidopsis enzyme. Gaps (dots) have been introduced 251 by BESTFIT for maximal alignment (13). Stars GNIENVPAPA indicate arginine residues in the putative signal -A-QDC----peptide, and the open arrow denotes the putative N-terminal amino acid of the Arabidopsis 301 and S. guttatum alternative oxidase. v, Probable internal start site for the protein expressed in QGRELKEAPA -DL---TT-E. coli. Amino acids 1-12 in the Ataaox sequence (boldface characters) are derived from the genomic clone sequence; they are absent from the cDNA clone, which is incomplete at the 5' terminus.

Biochemistry: Kumar and S611 3

2

1

Proc. Nati. Acad. Sci. USA 89 (1992)

10845

4

,;h

8 .

2'6'e 'MR

0 ~3.J ~ ~ ~ ~ ~ ~ ~

0102.0 0

nid.

by Western blot analysis using the AQA monoclonal antibody to S.

guttatum alternative oxidase. For each lane, an equal concentration of cells was pelleted, solubilized, and separated by SDS/15% PAGE. The proteins were electrophoretically transferred to nitrocellulose

was detected by anti-mouse antibody conjugated with horseradish

DH5ar cells containing empty DH~a cells containing pAOX; lane 4, DH5a

peroxidase. Lane 1, DH5a cells; lane 2, pcDNAII vector; lane 3,

cells containing pAOX grown in the presence of 0.5 mM KCN. Large arrows indicate alternative oxidase; small arrow denotes the position of carbonic anhydrase (CA, 29 kDa).

extracts

and

thus

represented

a nonspecific

reaction.

(indicated by large arrows) of 31 kDa and 26 kDa in the lysates

DH~a

0

5

15

10

_- _,

_ _

20

To

25

Time, hr FIG. 4. Cyanide-resistant growth of E. coli strains. Cell growth was monitored by checking the optical density at 600 nm. E. coli DH5a/pcDNAII (empty vector) (U, *, *) and DH5a/pAOX (o, A, o) cells were grown at 370C in medium without KCN (n, o) or with KCN at 0.2 mM (A, A) or 0.5 mM (e, o).

In

addition, the antibody recognized specifically two proteins

of E. ccli

_

0.0

and probed with the antibody. The presence of the bound antibody

cells transformed with pAOX and grown in

the absence (lane 3) or presence (lane 4) of 0.5 mM KCN. The intensity of the bands in lane 4 shows that the expression of alternative oxidase was enhanced when E. ccli was grown in the presence of KCN. The smaller protein (26 kDa) probably

concentrations of this compound should abolish cyanideresistant growth of DH5a/pAOX cells. This prediction is borne out by the results shown in Fig. 5. Clearly, the growth of DHSa/pAOX cells in the presence of 0.5 mM KCN was abolished in a concentration-dependent manner by SHAM. This again demonstrates the functional expression of the plant enzyme in E. coli.

represents a partial degradation product of the 31-kDa protein, as no protease inhibitor was used in the extract prepa-

DISCUSSION

ration. We attempted to compare the molecular mass and

immunological reactivity of the native plant protein by similar analysis

of an

extract of Arabidopsis

leaf mitochondria.

However, we failed to detect the protein, presumably because of the

very low levels

of enzyme

reported in the

mitochondria from non-spadix sources (2). The Arabidopsis alternative oxidase gene was cloned in

pcDNAII, a lacZ fusion vector. Sequence analysis of the pAOX clone showed an in-frame stop codon between the

In an attempt to isolate genes operating in the C5 pathway of chlorophyll biosynthesis, we have fortuitously isolated a gene for alternative oxidase from Arabidopsis by suppression of a hemA mutation of E. coli. Although the alternative oxidase has been known to be involved in cyanide-resistant respiration, very little information is available at the molecular level about this enzyme in plants and other organisms (2). Like other enzymes involved in mitochondrial electron trans-

lacZ and the alternative oxidase coding sequence. Because the enzyme was clearly expressed in E. ccli, translation is

probably initiated from an internal ATG codon. Based on the 31-kDa size of the protein expressed in E. ccli (see Fig. 3), we

2.0

speculate that translational initiation occurs at the first methionine in the deduced amino acid sequence (v in Fig. 2) and leads

to a protein

consisting of 280 amino acids with a

1.5-

molecular mass of 30,800 Da.

Alternative Oxidase Confers Cyanide-Resistant E. coli. As manifested by the suppression of mutation, the Arabidopsis alternative oxidase

Arabidopsis

Respiration the hemA

on

o 1.0

expressed in E. ccli can substitute for the bacterium's normal respiratory pathway. However, the latter is more efficient, as

wild-type cells grow approximately 4 times glycerol medium

than

SASX41B

cells

faster in minimal

transformed

with

0.5

pAOX. Functional expression of the Arabidcpsis alternative oxidase in E. ccli would be expected to confer cyanide-resistant

and SHAM-sensitive respiration.

anide concentration of 0.5 DHSa transformed with

The optimal inhibitory cy-

mM

suppressed

the

empty

growth

vector

of

strain

(pcDNAII),

whereas cells transformed with pAOX grew well (Fig. 4). Thus, the alternative oxidase does confer cyanide-resistance

E. coli. Since SHAM is a well-known inhibitor of alternative oxidase, and thus of the alternative pathway (22), increasing on

0.0. 0 5 10

15

20

25

Time, hr FIG. 5. Effect of SHAM on KCN-resistant growth of DH5cx/ pAOX cells. E. coli DH5a/pAOX cells were grown at 370C in medium containing 0.5 mM KCN without SHAM (o) or with SHAM at 60 ,uM (i), 120 AtM (o), or 300 AuM (e). In the absence of KCN, SHAM (1.2 mM) had no effect on E. coli growth.

10846

Biochemistry: Kumar and Soil

port, it is found in association with the inner mitochondrial

membrane in Arum maculatum (23). As the gene is encoded in the nucleus (24), the gene product is expected to have an N-terminal extension to translocate it into the mitochondrion. Examination of the amino acid sequence deduced from the open reading frame of the Arabidopsis alternative oxidase (Fig. 2) revealed two arginine residues positioned with a gap of 7 amino acids close to the N-terminal end of the protein. If it is assumed that the preprotein of alternative oxidase is cleaved at the position of alanine (indicated by an open arrow in Fig. 2), then the relative positions of the arginines (-2 and -10) fit well with one of the most characteristic features of the mitochondrial transit peptide (25). The same alanine residue has also been proposed as the N-terminal amino acid of the S. guttatum enzyme (9). Based on this assumption we speculate that the mature Arabidopsis alternative oxidase protein contains 293 amino acids, with a molecular mass of -32 kDa. The S. guttatum enzyme is 7 amino acids smaller because of two deletions of 4 and 3 amino acids, as deduced from the gene sequence (Fig. 2). The functional expression of the Arabidopsis alternative oxidase in E. coli demonstrates that a single polypeptide chain (of 31 kDa) is sufficient for enzyme activity. This fact should clarify the currently somewhat confused picture derived from results of enzyme purification (reviewed in ref. 2). The most interesting observation of the present study is the isolation of plant alternative oxidase by suppression of a hemA mutation in E. coli. The use of a hemA E. coli strain for the isolation of glutamyl-tRNA reductase, the first enzyme in chlorophyll and also heme synthesis (in many organisms), is well known (26-29). The hemA mutation makes E. coli auxotrophic for 5-aminolevulinic acid, a precursor for both chlorophyll and heme biosynthesis (10). The inability to synthesize heme in this strain renders the cytochromemediated respiration pathway inoperative. For this reason hemA strains fail to grow aerobically (15). Introduction of the plant alternative oxidase, a protein not found in E. coli, restores respiration by bypassing the inactive cytochrome apoenzymes in the transfer of electrons to oxygen. This unintended "engineering of a new pathway" in E. coli aids the study of many aspects of the alternative oxidase. For example, the ability of Arabidopsis alternative oxidase to retain its biological activity in E. coli could be exploited to design an experimental strategy to define the nature of the cofactor present in alternative oxidase. As in other waterforming oxidases, a metal cofactor has been implicated (2). Should iron be involved it must be of the non-heme type. Other oxidases are copper enzymes (e.g., ref. 30). If flavoproteins were candidates for alternative oxidase cofactors (31), studies with E. coli rib mutants (32) deficient in riboflavin might be useful. In addition, the presence of the cloned Arabidopsis alternative oxidase in E. coli strains will allow the creation and examination of mutants in cytochromes under aerobic growth conditions. The hemA strain of E. coli could also be recommended as yet another approach for the cloning of alternative oxidase genes from other organisms. Creation of new metabolic pathways is intellectually appealing and has interesting scientific and potential commercial uses. Previous studies have described generation of new metabolic pathways in bacteria in which a desired product [e.g., naphthalene-degrading enzymes (33), indigo (34), or 2-ketogluconate, an intermediate in L-ascorbic acid synthesis (35)] is obtained in vivo by introducing a gene for an enzyme from a different bacterium. The new enzyme utilizes the end product of the pathway in the host as a substrate and releases a product otherwise not produced by the host bacterium. The success in such endeavors rests on many factors (e.g., proper gene expression, protein stability, proper biosynthesis and subcellular location) and has often involved closely related

Proc. Natl. Acad. Sci. USA 89 (1992)

organisms. It is intriguing that a plant enzyme can restore E. coli growth by substituting for an inoperative pathway. We are indebted to Dr. Pablo Scolnik (DuPont) for mapping the pAOX clone to the Arabidopsis genome. We thank Drs. R. Meagher (University of Georgia) and R. W. Davies (Stanford) for gifts of Arabidopsis libraries and Dr. J. N. Siedow for the antibodies. We are grateful to Drs. M. Berlyn, J. N. Siedow, E. Verkamp, and I. Zelitch for many discussions. This work was supported by grants from the Department of Energy and the National Institutes of Health. 1. Henry, M. F. & Nyns, E. J. (1975) Sub-Cell. Biochem. 4, 1-65. 2. Moore, A. L. & Siedow, J. N. (1991) Biochim. Biophys. Acta 1059, 121-140. 3. Day, D. A., Arron, G. P. & Laties, G. G. (1980) The Biochemistry of Plants, ed. Davies, D. D. (Academic, New York), Vol. 2, 197-241. 4. Lance, C., Chauveau, M. & Dizengremel, P. (1985) in Encyclopedia of Plant Physiology, eds. Douce, R. & Day, D. A. (Springer, Berlin), Vol. 18, pp. 202-247. 5. Meeuse, B. J. D. (1975) Annu. Rev. Plant. Physiol. 26, 117-126. 6. Huq, S. & Palmer, J. M. (1978) FEBS Lett. 95, 217-220. 7. Elthon, T. E. & McIntosh, L. (1987) Proc. Natl. Acad. Sci. USA 84, 8399-8403. 8. Elthon, T. E., Nickels, R. L. & McIntosh, L. (1989) Plant. Physiol. 89, 1311-1317. 9. Rhoads, D. M. & McIntosh, L. (1991) Proc. Natd. Acad. Sci. USA 88, 2122-2126. 10. Jahn, D., Verkamp, E. & Soil, D. (1992) Trends Biochem. Sci. 17, 215-218. 11. Henikoff, S. (1987) Methods Enzymol. 155, 156-165. 12. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 13. Devereux, J., Haeberli, P. & Smithies, 0. (1984) Nucleic Acids Res. 12, 387-395. 14. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold

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