Biochimica et Biophysica Acta, 1173 (1993) 357-359 357 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00

BBAEXP 90513 Short Sequence-Paper

Identification of a ras gene in the slime mold Physarum polycephalum

Piotr Kozlowski, Jan Fronk and Kazimierz Toczko Institute of Biochemistry, Warsaw University, Warsaw (Poland)

(Received 1 March 1993)

Key words: GTP-binding protein; Palmitoylation; Prenylation; Sequence homology; (P. polycephalum)

A ras homologue was identified in the cDNA library from the slime mold Physarum polycephalum. The cDNA codes for a protein of 189 amino acids, showing high homology to ras genes from other organisms, especially to these from Dictyostelium discoideum. Amino acid sequence at the C-terminus of the putative protein suggests that unlike most other ras proteins, it is not palmitoylated and bears a geranylgeranyl rather than farnesyl chain.

Ras proteins are evolutionarily conserved signal- transducing GTP-binding proteins believed to play some fundamental role in the cell [1,2]. In search for a convenient model to study their role in cell functioning and differentiation we turned to the simple eukaryote Physarum polycephalum. Natural cell-cycle synchrony and capability of undergoing several easily manipulated differentiation programmes make this organism ideally suited for this purpose [3], provided its ras gene is identified. Especially interesting are the transitions among different cell types: uninucleate amoebae and flagellates and the multinucleate plasmodium. In this paper we showthe nucleotide sequence of a P. poly- cephalum ras gene and discuss its hypothetical post- translational modifications and its relation to homo- logues from other organisms.

The following strategy was used to identify a ras homologue in the P. polycephalum cDNA bank. ML6 bank containing cDNA inserts > 1 kbp cloned in Agtl0 from mixture of amoebae and flagellates [4] was pro- vided by Dr. Timothy Burland. The bank was divided into 32 subbanks, each containing ca. 30 000 sequences, from each of the subbanks a DNA miniprep was di- gested with EcoRI and analyzed by Southern blotting at low stringency, using as a probe a mixture of ras genes from Drosophila melanogaster (Drasl [5]) and from Neurospora crassa [6]. One subbank exhibited

Correspondence to: J. Fronk, Institute of Biochemistry, Warsaw University, Zwirki i Wigury 93, 02 089 Warsaw, Poland. The nucleotide sequence data reported in this paper have been deposited in the EMBL/GenBank Data Libraries under the'acces- sion number L10344.

significant hybridization with the ras probes. This sub- bank was further analyzed by several rounds of plaque hybridization, until one clone was isolated containing a 2.1 kbp insert. HindlII cuts the 2.1 kbp insert in two fragments of 1.4 and 0.65 kbp. These fragments were subcloned in pUC18 and sequenced (the smaller of the fragments was sequenced in its entirety, the larger one only partially). An open reading frame of 190 codons (570 nucleotides), beginning with an ATG and ending with a TAA, was found extending across the HindlII site: the ATG and three adjacent codons in the 1.4 kbp fragment and the remaining 186 codons in the 0165 kbp fragment. In the 3' trailer a putative polyadenylation signal was identified, although there was no poly(A) tail in the isolated clone (Fig. 1). A search for homolo- gous nucleotide sequences among GenBank entries identified numerous ras genes, with D. discoideum DdrasG [7] exhibiting the highest homology. Nu- cleotide sequence analysis was performed by NCBI with the BLAST algorithm [8].

Translation of this sequence gives a putative protein of 189 amino acids. This P. polycephalum protein ex- hibits extensive homology to bona-fide ras sequences from other organisms (Fig. 2). As could perhaps be expected, it is the most homologous to ras genes from another slime mold, D. discoideum [7,9], with the first 107 amino acids being indentical between Pprasl and DdrasG. All three slime mold genes show the extent of homology to human ras [2,10] surprisingly high for organisms so distant evolutionarily. Features character- istic for ras proteins are present in the P. poly- cephalum sequence: a conserved N-terminal stretch of 167 amino acids which includes several patches of

358

I TAAcAcCTTGTTAATGTGTGTcCAccTcCCcGCccTCcTCTcAATTTcTcccCcTCTTcTTTTcTGGTAcAAcACATTCTGTATTAAATT

91 ATGAcTGAATACAAGCTTGTAATTGTcGGTGGTGGTGGTGTTGGcAAGAGTGcCTTGAcTATTCAACTcATccAGAAccATTTcATTGAc 1 MetThrG~uTyrLysLeu~alIle~a~GtyGtyG~yG~y~atG~yLys~erAlaLeuThrI~eGtnLeuI~eG~nAsnHisPheIleAsp

181GAGTATGACCCCACCATTGAGGACTCTTACCGCAAG•AAGTGAC•ATTGATGAGGAGACATGCTTGCTTGACATTCTTGACACTGCCGGT 31 G•uTyrAspPr•ThrI•eGtuAspSerTyrA•gLysGtnVa•ThrIteAspGtuGtuThr•ysLeuLeuAspIteLeuAspThrA•aG••

271 •AAGAAGAGTACAGCGCCATGAGAGATCAATACATGCGTACTGGCCAGGGTTTCTTGTGTGTCTACTC•ATCACATCCCGTAGCAGTTTT 61 GtnG~uG~uT~r~erA~aMetArgAspG~nTyrMetArgThrG~yG~nGtyPheLeucys~a~Tyr~erIteThr~erArg~erSerPhe

361GATGAAATTGCTTCCTTCAGAGAGCAAATCCTCCGTGTGAAGGACAAGGACAAGGTTCCCATGATTGTTGTTGGAAACAAATGTGACTTG 91 As~G~uIteA~a~erPheArgGtuGtn~teLeuArgVa~LysAspLysAspLysVa~P~HetIteVatVa~G~yAsnLysCysAspLeu

451GAGGGCGAGAGACAAGTTACTACTGGTGAGGGTCAAGACCTCGCAAGGTCCTTCGGATGCCCCTTCATGGAGACATCCGCCAAGAGCAGA 121G•uGtyG•uA•gG•n•a•ThrTh•G•yGtuGlyG•nAspLeuA•aA•g•e•PheG•yCysP••PheMetGtuTh•Se•A•aLysSe•A•g

541GTGAACG~TGAGGAGTCGTTCTACCAGTTAGTG~GCGAGATCCGCAAGGACT~CCG~ACCGATAC~AAGGG~C~CGGCGGAAAGGGTGGC 151ValAsnVatG~uGtu~erPheTyrGtnLeuVa~ArgG~uIteArgLysAspSerArgThrAspThrLysGly~r~G~yG~yLysGlyG~y

631AAGAAGA•A•TCAAGTG••T•CT••TGTAAATGGTAATATGCTTGTA•CTG••TTGT•TGTGTTGTATTCGTTAGCTATTGAAAAAAAAT 181LysLysThrLeuLysCysLeuLeuLeuTER

721 TGAAAAATAAATGTGTATTTTTGTA

Fig. 1. Nucleotide sequence of the Pprasl cDNA. The 5' end of the sequence is arbitrarily set 90 nucleotides upstream of the ATG start codon and the 3' end is at the end of the original clone. HindIII site and the putative polyadenylation signal are underlined.

absolute homology, corresponding to functionally im- portant regions of the protein, a highly variable C- terminal region and a conserved CAAX tetrapeptide (where C is cysteine, A is an aliphatic amino acid and X is any amino acid) at the very C-terminus [1,2]. Based on all these features we conclude that the se- quence identified is of a true ras gene and propose to

name it Pprasl (the ' l ' in the anticipation of a likely identification of other ras genes in this organism).

All ras proteins have a CAAX motif at their C- termini, in which the cysteine is the site of prenylation [1,2]. While in other GTP-binding proteins the prenyl moiety may be a farnesyl or a geranylgeranyl chain, ras proteins are believed to be farnesylated [1]. The choice

Pprasl (100) DdrasG(94.6) Ddras (84.9) H-rasl (74.1) Ncras (72.3) Drasl (68.7)

10 20 30 40 50 60 70 M . . . . . TEYKLVIVGGGGVGKSALTIQLIQNHFIDEYDPTIEDSYRKQVTIDEETCLLDILDTAGQEEYSAMRDQ

• . . . . . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

*************************************************************************** . . . . . . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

Pprasl DdrasG Ddras H-rasl Ncras Drasl

80 90 100 110 120 130 140 150 YMRTGQGFL••YSITSRSSFDEIASFREQILR•KDKDK•PMI••GNK•DLEGERQvTTGEGQD•ARSFGcPFMETSAKSR **** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * ********************************************************************_********* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * - * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

*****E***L*FA*N*AK**ED*GTY****KH***AEE***vLA******-ASWN*NNEQAREv*KQY*I*YI*****T*

Pprasl DdrasG Ddras H-rasl Ncras Drasl

160 170 180 VNVEESFYQLVREIRKDSRTDTKGPGGKGGKKTLKCLLL *****A**S********LKGDSKPEKGKKKRPLKACTLL I****A**S*******ELKGDQSSGKAOKKKKQCLIL QG**DA**T******QHKLRKLNPPDESGPGCMSCKCVLS H***NA**D*******EDKKLGEKVGGTSFANNNGAVKQMDVGDEDVQAGCCAKCIMM MG*DDA**T********KDNKGRRGRKMNKPNCRFKCKML

Fig. 2. Pairways amino acid sequence comparisons between Pprasl and 5 other ras proteins. Numbering is according to the Pprasl sequence. Numbers in brackets after the name of a protein indicate percentage amino acid identity within the GTP-binding domain (amino acids 2 through 167) between Pprasl and the respective protein. Identical amino acids are indicated by *, and gaps introduced to maximize homology are indicated by - . No at tempt was made to align sequences beyond the 167th amino acid. The proteins are from the following organisms: DdrasG

[7] and Ddras [9] from D. discoideum, H-rasl [10] from man, Ncras [6] from N. crassa, Drasl [5] from D. melanogaster.

between the two isoprenols is apparently determined by the C-terminal amino acid, with geranylgeraniol being specified by C-terminal leucine [11]. It is of significant interest in this context that slime molds' ras genes belong to the minority of ras proteins which have a leucine at their C-termini. It remains to be seen whether these ras proteins are indeed geranylgerany- lated in vivo. Unlike the vast majority of ras proteins [1], Pprasl cannot be palmitoylated, as it lacks a cys- teine near its C-terminus other than that in the CAAX tetrapeptide; it does have, however, a lysine-rich stretch close to the CAAX motif, which has been shown to be sufficient for membrane-targeting of unpalmitoylated ras proteins [11]. The presence or absence of the palmitoyl chain and the kind of the prenyl chain on the ras protein are very likely to influence its membrane attachment [1,11] and thus also its functioning in the signal transduction. Yet another interesting feature of the Pprasl cDNA is its remarkably long (1400 nu- cleotides) 5' leader.

As an initial attempt to study the genomic organiza- tion of Pprasl we used the 0.65 kbp HindlII-EcoRI fragment to probe P. polycephalum genomic DNA blots. DNA was purified from two strains of P. poly- cephalum, digested with restriction nucleases having no sites within the 0.65 kbp fragment, and analyzed by Southern hybridization at high stringency (Fig. 3). In the MCC strain single hybridizing bands are seen in each of the digests, while M3CIV gives rise to two bands in both digests (indicated in Fig. 3 by their respective sizes). Several additional, weak bands are also seen (indicated by bars). The most plausible expla- nation of these, results is that Pprasl is probably a single copy gene, with strain M3CIV being heterozy- gous for a polymorphism in the gene's flanking se- quences. The weakly hybridizing bands may represent partial digestion products and/or Pprasl-related genes. The data available allow no conclusions regarding the presence of introns to be drawn.

This study was supported by grant No. PB 2213/4/91 from KBN. We are grateful to Drs. Eileen Paul, Bill Dove and Tim Burland for providing the ML6 library prior to its publication, to Drs. Ben-Zion Shilo and Hector Torres for the D. melanogaster and N. crassa ras clones, respectively, and to NCBI for sequence analysis. Ms. Zuzanna Tymowska working

359

3.9- 3.4-

11.7- 11.7-i

1.6-

Fig. 3. Genomic organization of the Pprasl gene. DNA purified from P. polycephalum strain M3CIV (lanes 1 and 3) and MCC (lanes 2 and 4) was digested with EcoRI (lanes 1 and 2) or Hindlll (lanes 3 and 4) and Southern hybridized with the 0.65 kbp Pprasl fragment at high stringency. Strongly hybridizing bands are indicated by their respective sizes in kbp, and weak bands are indicated by unmarked

bars. See text for details.

towards her MSc degree helped in the early phases of this work.

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