Characterisation of the gene encoding acetyl-CoA synthetase in Penicillium chrysogenum: conservation of intron position in plectomycetes

Gene, 130 (1993) 265-270 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119~93~SO6.00

265

GENE 07212

Characterisation

of the gene encoding acetyl-CoA synthetase in Penicillium chrysogenum: conservation of intron position in plectomycetes (Recombinant DNA; nucleotide sequence; cDNA cloning; introns; lariat; acetate utilisation; splicing; penicillin; Aspergillus; filamentous fungi)

Honorina Martinez-Blanco”,b, Margarita Orejasa, Angel Reglerob, JosC M. Luengob and Miguel A. Pefialva” Tentra de lnvestigaciones Biolbgicas de1 C.S.I.C., Vel6zquez 144,28006, Facultad de Veterinaria, Universidad de Lecin, 24007 Leh, Spain.

Madrid, Spain; and bDepartamento de Bioquimica y Biologia Molecular,

Received by M. Salas: 19 January 1993; Revised/Accepted: 5 March/S March 1993; Received at publishers: 15 April 1993

SUMMARY

Acetyl-coenzyme A synthetase (ACS; EC 6.2.1.1) from some plectomycete fungi is possibly involved in an accesory step of penicillin biosynthesis, in addition to its role in primary metabolism. We present the characterisation of the gene encoding this enzyme in Penicillium chrysogenum, which we designated acuA. Sequencing of genomic and cDNA clones showed that the coding region was interrupted by five introns, located at the same positions as those present in the Aspergil~us nodular homologue. This supports the possibility that the gene acquired its definitive mosaic organisation before the Pen~ci~~iumjAsperg~~~usdivergence. The mature transcript encodes a polypeptide with an kf, of 74 287 which is 89.4% identical to its A. nidu~a~s counterpart.

INTRODUCTION

Many microorganisms, including filamentous ascomycetes, can use acetate as sole C-source through the glyoxylate-cycle enzymes (Kornberg, 1966). To be further catabolised, acetate is first activated as a thioester of CoA by acetyl-CoA synthetase (ASC; EC 6.2.1.1). The genes encoding this enzyme in two filamentous fungi, N. crussa

Correspondence

to: Dr. M.A. Pefialva, Centro de Investigaciones Biol6gicas de1 C.S.I.C., Velazquez 144, 28006 Madrid, Spain. Tel. (341) 5611800, ext. 4358; Fax (34-l) 5627518.

Abbreviations: A., Aspergillus; aa, amino acid(s); ACS, acetyl-CoA synthetase; acuA, gene encoding ACS; bp, base pair(s); cDNA, DNA complementary to RNA, CoA, coenzyme A, kb, kilobase or 1000 bp; N., ~e~rospor~ nt, nucleotid~s) ORF, open reading frame; PAGE, polyacrylamide-gel ele~trophor~is; PCR, polymerase chain reaction; P., Pen~c~~l~urn; RT-PCR, reverse transcription followed by ~pli~~ation using polymerase chain reaction; S., Saccharomyces; SDS, sodium dodecyl sulfate.

and A. nidulans, have been characterised (Connerton et al., 1990). In addition to this role in primary metabolism, ACS has a possible role in secondary metabolism, particularly in those organisms which synthesise penicillins. The last step in the metabolic pathway leading to these compounds is a transacylation reaction in which the L-aminoadipic moiety of isopenicillin N is exchanged for other organic acids (A3-hexenoic, hexanoic, octanoic or phenylacetic) activated as CoA derivatives (for review see Luengo and Peiialva, 1993). We have previously shown that homogeneous ACS from two closely related Plectomycetes, P. chrysogenum and A. nidulans, is able to activate in vitro all these organic acids, suggesting that at least ACS (and perhaps other acyl-CoA synthetases) is able to carry out this accessory reaction of the penicillin pathway (Martinez-Blanc0 et al., 1992). As a prerequisite for reverse genetics to test this proposal, we describe here the molecular cloning and characterization of the gene encoding ACS in the penicillin-producing organism, P.

266 c~~ysoge~~m. RT-PCR has been used to characterise introns splitting the coding region, whose positions show a remarkable conservation with those of their A. nidulans counterparts.

EXPERIMENTAL

AND DISCUSSION

(a) Obtaining genomic clones by beterologous bybridisation A 528-bp probe (nt 1252 to 1880 in the A. ~iduluns j&A sequence published by Connerton et al., 1990) was PCR-amplified from A. nidulans DNA. This fragment corresponds to exonic sequences coding for a segment highly conserved between the N. crassa and A. nidulans polypeptides. The fragment was used to screen a P. chrysogenum (strain Wis 54-1255) genomic library in h EMBL4. Positive clones were purified, and restriction mapping combined with Southern hybridisation with the probe mentioned above was used to delimit a fragment containing the putative ACS-encoding ORF. By using a combination of fragment subcloning and specific synthetic primers, a total of 2868 bp (including the crosshybridising region) were sequenced on both strands (see Fig. 1). Sequence comparison of polypeptides encoded by this fragment to the A. nidulans FacA polypeptide strongly indicated that it contains the gene encoding ACS in P. chrysogenum, which we will refer to as acuA. The nt sequence of the gene is shown in Fig. 1. The coding region contains five introns whose position can be almost unambiguously inferred from homology breaks delimited by splicing consensus sequences. (b) Characterisation of intronic sequences To rigorously confirm the position of the intronic sequences, mRNA isolated from mycelia grown in acetate was RT-PCR-amplified using primers corresponding to exonic regions flanking the putative introns. In all cases cDNA sequencing confirmed the position of introns in the acuA gene. The structure of the acuA coding region is shown in Fig. 2. Remarkably, the position of all introns in the coding regions of the genes encoding ACS in A. njdul~ns and P. chrysogenum (both P~ectffmycetes) coincides exactly. It should be noted that the possibility of a sixth intron present in the 5’-non coding sequence as occurs in the A. nidulans gene has not been explored in the P. chrysogenum counterpart characterised here and cannot be discounted. The observed difference between the organisation of genes encoding ACS in N. crassa and A. niduians (Connerton et al., 1990) and the similarities of the latter with the P. chrysugenum acuA (this work) does not favor intron addition (in the genes from

P~ectomycetes~ nor

intron loss (in Pyrenomycetes). However, conservation of introns in Plectomycetes suggests strongly that none of these events occurred after the PenicilliumlAspergiilus divergence. Not only do introns have identical locations but sequence comparisons (Fig. 2C) reveal a high degree of conservation in intronic sequences (in which most nt changes are neutral), indicating that they are closely related to an ancestor sequence. This further supports our interpretation that the mosaic organisation of the gene encoding ACS in P~ectomycetes had acquired its definitive confo~ation before the Penie~ll~um/As~ergillus divergence took place. P. chrysogenum introns described here (see Fig. 2B) are typical of filamentous fungi (reviewed in Gurr et al., 1987). Their borders conform to the GT...AG rule. There is preference for A in the third position of the 5’-splice site, but one of the introns differs from the consensus, in contrast to the situation in the A. nidulans gene. The introns contain a recognisable lariat box (Parker et al., 1987), much more relaxed that the one found in S. cerevisiue, a common situation in introns from filamentous ascomycetes (Gurr et al., 1987). Conservation is greater in the second half of the box, where the consensus CTAAC can clearly be seen, consistent with other P. chrysogenum introns from primary metabolism genes (Cantoral et al., 1988; Van Solingen et al., 1988). In contrast, lariat boxes in introns from a penicillin biosynthetic gene (Barredo et al., 1989) are more divergent. It is not known whether this fact reflects functional differences. (c) Analysis of the acukencoded ~ly~ptide The deduced polypeptide encoded by P. c~rys~gen~m acuA is 669 aa long, just one residue shorter then its A. nidulans close relative and 23 aa shorter than the N. crassa acu-5 gene product (see Fig. 3). The M, is 74 287, which is consistent with 70 kDa estimated from SDS-PAGE of the purified enzyme (Martinez-Blanc0 et al., 1992). In addition, the deduced protein shows an 89.4% identity with the A. nidulans FacA polypeptide. These results strongly indicate that the cloned gene encodes P. chrys~genum ACS, previously characterised biochemically by us. Sequence comparisons (Fig. 3) between available microbial polypeptides (three from filamentous fungi and one from archaebacteria) reveal boxes of complete identity which might be functionally relevant for ACS activity. These boxes may be used as ‘tags’ to isolate genes encoding these enzymes by using PCR approaches with degenerate oligodeoxynucleotides. While reviewers’ comments were being introduced into the manuscript, others (Gouka et al., 1993) have independently reported the sequence of a genomic DNA fragment

267

AAGtTTAtttCGGAGtAACQGAAAGAACetCCGCATGGC~GAACCCAAA~TCGTATGGG~CAAGGCAAT~TACTGAAAT~TACTGAAAT~TACTGAATT~GACCGTATT~GGAATGTAT~

120

TTATTCCTG;TTeGGAGATEAGAGTGGATCGlCCGAATG~CCAATGCAC~ATGTACTTT~TCTAGGCCG~CTGCGGCTA~CGAGACAGC~GGAGTTGGG~AGTTTGAAG~GGTATTGTAA

240

CTTATTGTA~TTTATTGTA;GtGGGCACGG~CCACTGATG~AAAGGGAAG~GGCACATCC~CCCGGGACA~CTGGACTAC~AATATTGTC~CGAGTCCCC~CTCCTTGAG~TTCTTTTTC~

360

tTTTCTCTT~TtTAATATCiTCT~TAATTQCTATACATA~CCTGTTTGA~CATTACTCT~AGTATATTA~ATAGTTCAT~CCCCACATT~ATTATTCCC~TTGGACTAC~GCAATCA~G~

48p .

. . . . CGGACGGCCCAATTCAGCCTCCCAAGCCCGCAGTGgt~~ga~tcaccg~cctcc8gaccgag~tgacc8gacccgtgtcgc~ctggtg~Ccg8agtatc~tgggctaactggtg~t~t~g SDGPIPPPKPAV

. '

600 13

GTGCATGAGteACACGAGGiCtACACTTTttACGTCCCC~AGGCGTTCC~CGATAAGCA~CCCTCCGGC~CTCACATCA~GGACATTGA~GAGTACAAG~AGCTTTACG~AGAATCAAT~ VHEAHEVDTFHVPKAFHDKHPSGTHIKDIEEYKKLYEESI

7 0 33

AAGAGtCCCtAiCAtCTTCTGGtCACtCATttCCCGCGAG~TCCTCACAT~TGACAAGGA~TTTGAAACC~CACATCACG~CTCGTTTGA~AACGGCGAC~ATGCCTGGT~CGTCGAGGG~ KSPDTFYARHARELLTFDKDFETTHHGSFENGDNAUFVEG

"$8

eGGTTGAAtGCATCGTTCAACTGTGTttA;CCATGCC~TCAAGAACC~AGATAAGGT~GCCATTATT~ATGAGGCCG~CGAGCCCAA~GAGGGCCGT~AGATCACCT~CGGAGAGCT~ RLNASFNCVDRHALKNPDKVAIIYEADEPNEGRKITYGEL

'#

ATGtGtGAGCTGTtCCGGGiTGCCTGGAC;tTGAAGGACtTCGCTTTCC~GGCTTGCTC~ HREVSRVAUTLKERGVKKGDTVGIYLPMI

'!!$I

PEAVIAFLACS

tGTATTGGT;tCGTGCACTtCGTTGTCTTEtCTGGTTTC~CTTCCGACT~CCTCCGGGA~CGTGTCCTG~ACGCCTCCT~CAAGGTCAT~ATTACCTCC~ACGAGGGCA~GCGCGGTGG~ RIGAVHSVVFAGFSSDSLRDRVLDASSKVIITSDEGKRGG

l$$

AAGATCATTtGtACTAAGAAGATTGTGGAtGAGGCCATG~AGCAGTGCC~CGATGTGCA~ACCGTGCTG~TGTACAAGC~CACCGGTGC~GAGGTGCCC~GGACCGCTG~CCGTGACAT~ KIIGTKKIVDEAMKPCPDVHTVLVYKRTGAEVPVTAGRDI

132 259

TGGTGGCAttAGGAGGTCG;GAAGTACCCCAACTACCTC~CCCCTGAGT~GGTCAGCTC~GAGGATCCT~TCTTCCTGT~GTACACCTC~GGTTCCACC~GTAAGCCCA~GGGTGTTAT~ UYHEEVEKYPNYLAPESVSSEDPLFLLYTSGSTGKPKGVH

1:;:

CAtACCACTGtCGGTTACC;GCTCGGTGCtGCCATGACT~GAAAGTACG~GTTTGATAT~CACGACGAT~ATCGCTACT~CTGCGGTGG~GATGTCGGT~GGATTACAG~TCACA~CTA~ HTTAGYLLGAAHTGKYVFDIHDDDRYFCGGDVGUITGHTY

I@

GTCGTGTACbtCCCTtTAT;GCTTGGCTG~GCCACCGTC~TGTTCGAGA~TACCCCCGC~TACCCTAAC~TCTCGCGCT~CTGGGATGT~ATTGACAAG~ACGACGTCA~ACAATT~TA~ VVYAPLLLGCATVVFESTPAYPNFSRYUDVIDKHDVTQFY

I#8

GTTGCACCC~CCGCTCTGC~TCTGCTGAAGCttCCTGGA~ATGAGCACA~TCACCACAA~ATGCACAGT~TGCGTATTC~TGGCTCCGT~GGAGAGCCC~TTGCCGCGG~AGTCTGGAA~ VAPTALRLLKRAGDEHIHHKMHSLRILGSVGEPIAAEVUK

ltfg

TGGTACTTCEAGTGTGTTGGCAAGGAGGA~GCTCACATC~GCGACgtt~~tt~~~~~tt~~~~ttgg~~~ttttgg~~t~~~tt~t~~t~tttgg~t~t~t~gACATA~~GG~AAA~~G~ UYFECVGKEEAHICD GACCGGCTCACATGTCATCACCCCTCTCG~CGGTATCAC~CCCACCAAG~CCGGCAGTG~CTCCCTACC~TTCTTCGGT~TCGAGCCTG~~ATTATCGA~CCCGTCT~~~GAGAGGAGA~ T G S H V I T P L G GIT PTK P G S A S L P F F G I E P A I I

D

P

T

Y

UP

V

S

G

E

1920 434

T

E

E

2400 1474

TGTCGGCAA~GATGTCGAG~GTGTTTTGG~CTTCAAGCA~AGCGTTACA~GGA~ACTTA~TTGAACGTG~A~AAGGGTT~ VGNDVEGVLAFKPPUPSMARTVUGAHKRYMDTYLNVYKGY

2 60 1 14

CTCCgtolg;cgsttcgc.~~~tg~~ttg~~gggttg~t~~t~~~t~~t~t~t~gTTCA~CGGAGATGG~GCTGGCCGT~ACCACGACG~CTATTACTG~ATCCGCGGT~GTGTTGA~G~ F T G D G A G R D H 0 G Y Y

U

I

R

G

R

..crgCTTCaGTTGCCGAG;CTGCTGTCt;TGGTATTtCtGACGAGCTG~CCGGTCAGG~TGTCAATGC~TTTGTCTCT~TCAAGGAGG~CAAGCCCAC~GAACAGATC~GCAAGGACC~ P S V A E A A V V G I A D E L T G g A V N A F V S L K E G K P

T

E

0

I

SK

I?

I

L

R

TGtAATGtAAGTTCGCAAG;CCATTGGTC~CTTCGCCGC~CCCAAGGCT~TCTTCGTCG~GGATGACCT~CCCAAGACC~GCAGTGGCA~GATCATGCG~CGAATCCTC~GGAAGATT~~ A HP V R K S I G P FAA P K AV F V V D D L P K T R S G K I MR

V

K

D

D

2 GO $ 37

D

L

2320 597

I

L

2640 637

Fig. 1. Nucleotide sequence of the P. chrysogenum acuA gene and deduced aa sequence [one letter code for aa shown below the middle nt of each triplet, except in intronic regions (lower case letters for nt)]. Three asterisks mark the stop codon. Positions in the aa and nt sequences are indicated on the right margin of the corresponding lines, The nt sequence starts at a Hind111 located 477 nt upstream from the coding region. Dots over nt sequence lines mark each tenth nt. Sequence submitted to GenBank and assigned accession No. L09598.

containing the acuA gene in a different P. chrysogenum strain. This nt sequence shows no differences with the one reported here. (d) Conclusions

(1) The gene encoding acetyl-CoA synthetase from P. It has been desig-

chrysogenum has been characterised. nated acuA.

(2) Five short introns interrupt

the coding region of

this gene, Their positions exactly match those observed for introns within the coding region of the A. nidulans homologous gene. (3) The M, of the deduced polypeptide (669 aa) is 74 287. (4) Sequence comparison between fungal and bacterial ACS polypeptides identifies boxes of identity which should be useful to clone genes from other organisms encoding these enzymes,

268

A VAL

t

TYR

VAL 428 42s

515 516 556 55s

WI

649 6W 669 6

8CCi

db T-AA-***

-*q-Al-G 56

51

60

76

B

IVS4 IVS5

l****GGCTAACT*5*=*TATAG GTAAGA*****62 *b***~T~CT*8=~*TGTAG GTTCGT--1;; l****TACTAACT*$***TATAG ,....3g... l*AACTAACT*3***AACAG izs% GTAAGC**-0.46 l-•**AGCTAACG*lO**CACAG

CONSENSUS

GTaaG-

EE3 IVS3

tayAG

rRcTAACt

C PCIVSl GTAAGAAT09CCGACCTC~CCGAGATU\CCAGACCCGTOTC*~ +t++ t+ +t+ tt+ +tttt + + + +++++ ANIVSZ

GTAAG

CACCGGCTCCTACTTCGAACTGATT

PCIVSZ GTTCGTTCCCCCTTACCCTTGGAC tt AND'S3

+ ++ ++t+t

t+

++ t+

t++++

+

GTAAGTTTTATC

t+

+

t

+

tt AG64

AG58 +t

CiAATCTTTGTACAG62

TT GCAGGGTTGATACTAACTCATAT

ATAGs

tt ++

++++

+ tt

+++ +

TTGTGGTCGCTC

t t++ t +t

CATTCTATCAATTCGCTCACTCGAAGGTGACTGACATCGTATAG56

+t+t + ++

++++++

++t+ ++

TTTTTGACTGA

CTTTTGGAATARCTT CTAATTTTTGGATCTGT f +t++ + + + ++ +t+t t+t

PCIVS4 GTAAGTCCAACCACAGTATCTGCCFWLRA ANIVSS

t G

GTAAGT CATTTATGCCTTTGGAGTCCTGTCTTCTCATTTACTARCGGT

PCIVS3 GTAAGACGCTTCGCAGCCTGCC ANIVS4

GACCAATG

GTARGTTATACXAAA

t+

TTGCAACTGAGCCCAAACTAACTATGAKAG60 ++ + + ++ ++ + ++ ++

++ ++

TAAGAGCGCGAACTT

TGTGGCTAA

CTCATCAT

AG49

PCIVSS GTAAGCAT~TCTCTCAO~~T~TACCCGCAATCGTATCGTCCGIV\,6 ANIVSC

t++tt

+

++tt+ +

t+

GTAAGG

C

CTCTC G

ACTTGACTTCGT

+

+++t

+

t++

tt

TGAGTACG TGG

+++ +t + ++

+

AACTAKTTGTTAC

CTAAGs4

+t

Fig. 2. Introns in the coding region of the P. c~~ys~gen~rnacuA gene. (A) Structure of the coding region (genomic). Open boxes indicate exons, whereas inverted carets represent intronic sequences, with the numeral below indicating their length (in bp). The borders of the exons are indicated by downward open arrows and numbers which represent the aa position in the sequence. Filled arrows indicate the precise position of splicing junctions in each triplet, with the three letters corresponding to the three nt of the codon. (B) Schematic comparison of the five introns characterised in this work, showing only the nt sequences of splicing borders and lariat boxes. A consensus sequence derived for these elements is shown in bold. Numbers indicate distances (in bp). (C) The nt sequence comparison of P. chrysogenum (PCIVS) and A. niduhns (ANIVS) introns. Note significant sequence conservation. There is no correspondence in intron numbering (which indicates the relative order of a given intervening sequence), because we have not determined in this work whether an intron present in the S-non coding region of the A. nidulans facA gene also exists in the P. chrysogenum homologue. Symbols (+) mark the nt identities.

269 ANFACA PCACUA ACUS NTXACS

94 94 57 9%

ANFACA PCACUA ACUS WXACS

192 192 153 19%

AWFACA PCACUA ACUS XTX?kCS

292 292 253 29%

ANFACA PCACUA ACUS MTXACS

391 391 349 39%

ANFAUl PCACUA ACUS _S

491 491 449 495

ANFACA PCACUA ACUS NTXACS

585 585 442 545

ANFACA PCACUA AC115 NTXACS

of deduced aa sequences from microbial genes encoding acetyf-CoA synthetases. Data of nl. crussa (indicts ACUS) and A. polypeptide were from Connerton et al. (1990), whereas the sequence of the ~et~~ot~rj~ soehngenii polypeptide (MTXACS) is from Eggen et al. (1941). Numbers at the right indicate aa positions. Dashes and asterisks befow alignments indicate a gap or the presence of an identical residue in ah four sequences, respectively. Fig. 3. Comparison ~i~~~a~s(ANFACA)

ACKNOWLEDGEMENTS

H.M-B thanks A~~bi~ticos S.A. for support. This work was supported by grants BIO 671/91 (CICYT) and BRIIXE CT90-169 (C.E.E.) to M.A.P. and by grants PB89-0387 (DIGICYT) and from the Consejeria de Cultura {JCL) to J.M.L.

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S-monophosphate decarboxylase) gene. Nucleic Acids Res. 16 (1988) 8177. Connerton, I.F,, Fincham, J.R.S., Sandernan, R.A. and Hynes, M.J.: Compa~so~ and cross-species expression of the acetyl-CoA synthetase genes of the ascomycete fungi Asp&&s nidulens and Neurospara crassa. Moi. Microbial. 4 (1990) 451-460. Eggen, R.I.L., Geerling, ACM., Boshoven, A.B.P. and de Vos, W.M.: Cloning, sequence analysis and functional expression of the acetyf coenzyme A synthetase gene from ~etka~a~krjx soekngettii in Esckerickia caii. J. Bacterial. t73 (1991) 638336389. Got&a, R.J., Van H~~~veidt, W., Rove&erg R.A.L., Van Zeijl, C.M.J., Van den Handel, C.A.M.J.J. and Van Gorcom, R.F.M.: Development of a new transformant selection system for Penicitlium ckrysagenum: isolation and characterization of the P. ckrysogenum acetyl-coenzyme A synthetase gene (facA) and its use as a homologous transformation system. Appl. Microbial. Biotechnol. 38 (1993), 514-519. Gurr, S.J., Unkless, SE. and Kinghorn, JR.: The structure and organisation of n&ear genes of tih?mentousfungi. In: Kiighom, JR. (Ed.), Gene Structure in Eukaryotic Microbes. IRL Press, Oxford, 1987, pp. 93-139.

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Parker, R., Siciliano, P.G. and Guthrie, C.: Recognition of the TACTAAC box during mRNA splicing in yeast involves basepairing to the UZ-like snRNA. Cell 49 (1987) 229-239. Van Solingen, P., Muurlin, H., Koekman, B. and Van der Berg, J.A.: Sequence of the Penicillium chrysogenum phosphoglycerate kinase gene. Nucleic Acids Res. 17 (1988) 11823.