Developmentally regulated late mRNAs in the encystment of Physarum polycephalum plasmodia

Biochimica et Biophysica Acta, 1007 (1989) 264-269 Elsevier

264

BBA91937

Developmentally regulated late mRNAs in the encystment of Physarumpolycephalum plasmodia Louise Savard 1, Andr~ Laroche 1, G&ald Lemieux 2 and Dominick Pallotta 1 D~partements de t biologie et 2 biochimie, Facult~ des Sciences et de G~tnie, Universin; Laval, Quebec (Canada)

(Received 31 October 1988)

Key words: Spherulation; Spherulin 4: Plasma membrane protein: mRNA; Development: (P. polycephalum)

l~ysamm po~cepkalum plasmodia survive adverse conditions by transforming into encysted cells called sphemles, in thh work we a n a l y ~ the developmentally regulated mRNAs from the late stages of spherulation. A eDNA library was constructed and four abundant mRNAs were identified. One of the mRNAs was iwesent in trace amounts in early spherule~ while the other three were found only in late si~erules~ A eDNA clone for one of the late spherulation specific mRNAs was sequenced. It codes for a 332-amino-acid protein that did not show s|gnifleant similarities with any known intein. Since the mRNA for this protein accumulates during sphemlatton, the protein was called sphemlin 4. This Im~teln has many features of a plasma membrane protein; it contains a signal ~ptide and a long hydrophoble region, which could serve as a transmembrane anchor. Another interesting feature is the Imesence of seven consecutive glycinc residues in the N-terminal region. This is even more remarkable since the protein is not rich in glyeine.

Introduction Cellular encystment is a strategy used by a variety of prokaryotic and unicellular eukaryotic organisms to survive unfavorable conditions, For example, numerous parasites encyst to evade the natural defenses of their hosts and to propagate to other hosts, Cellular encystment involves the reprogramming of the general metabolic activities of the cells in order to synthesize a resistant ~tracellular structure. Although such a major reorganization of the intracellular activities requires a ¢ompl~ developmental genetic program, only a few genes associated with the encystment of eukaryotic cells have been identified, The slime mold Physarumpolycephalum is a useful organism in which to study the molecular genetics of encystment, The life cycle of this organism involves a transition between two vegetative stages, the amoeba and the plasmodium, The amoebae are uninucleated haploid cells which will encyst under adverse conditions, The plasmodium, a multinucleated diploid ~;ell, will also encyst when growth conditions are unfavora-

The sequence data in this paper have been submitted to the EMBL/ Gcabank Data Libraries under the accession number X14056 spheru. Correspondence: D. Palloua, ~ e m e n t de Biologic, Facuit~ des Sciences et de G~e., Universiltr: Laval, Quebec, Canada, G1K 7P4.

ble. The encystment of the two vegetative stages of Physarum is reversible and can be induced under controlled conditions in the laboratory. Starvation, or addition of high concentrations of some carbohydrates have been used succesfully by many workers who previously studied ultrastructural and biochemical variations associated with Physarumencystment [1]. Recently, it has been shown that the encystment of amoebae is accompanied by the synthesis of new poly (A) + RNAs and proteins, Two different mRNAs, specific to encysted amoebae, were isolated from a eDNA library. These mRNAs were not found in encysted plasmodia. Likewise, four mRNAs found in encysted plasmodia were absent from encysted amoebae. These results show that different developmental programs are used for amoebal and plasmodial encystment

[21. In other studies we demonstrated that major changes in mRNA populations occur during plasmodial encystment (spherulation). Studies on in vivo and in vitro synthesized proteins showed the presence of early and late spherulation specific mRNAs [3]. The abundant, early mRNAs were identified from a cDNA library I41. The nucleotide sequences of four of the mRNAs were determined. These mRNAs and the encoded proteins were named spherulins [5]. In this work, we have analysed the developmentally regulated mRNAs that accumulate in the late stages of spherulation; four abundant mRNAs were found. A

01674781/89~3.~ © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

265 cDNA clone for one of these mRNAs was sequenced. It codes for a protein containing a signal peptide and an unusual stretch of seven glycine residues. The codon usage suggests that this mRNA is translated by spherulation tRNA isoacceptors. Materials and Methods

Cell culture and poly(A) + RNA isolation Physarum polycephalum strain MaC was used. The plasmodia were grown in liquid semi-defined medium and induced to encyst by transfer to a balanced salt medium [6]. The formation of mature spherules was verified by microscopic examination and a viability assay, as described previously [3]. Under these conditions, the first resistant cells are observed after 24 h and the viability of the cell population reaches a plateau 24 h later. Total RNA was prepared from cells collected 72 h after the induction of encystment. The cells were rapidly frozen in liquid nitrogen and lyophylized. The RNA was extracted from the dried cells with phenol/ chloroform, in presence of SDS and EDTA [7]. The poly(A) + fraction was isolated by chromatography on oligo(dT)-cellulose. Construction and screening of a cDNA library A cDNA library was constructed with poly(A) + RNA extracted from encysted microplasmodia collected 72 h after the induction of encystment. The first strand was synthesized with AMV reverse transcriptase and the second strand was obtained by the use of DNA polymerase I and RNase H. The cDNA molecules were then oligo(dC) tailed and cloned into the oligo(dG) tailed Pstl site of pBR322. Details of these methods are found in Paliotta et al. [7]. The recombinant plasmids were used to transform CaCl2-treated E. coil cells, strain MC1061. The library (LAV6) was screened by differential hybridization [4] with 32P-labeled poly(A) + RNA ho,n cells collected after 24 and 72 h of encystment. The hybridization was done under the conditions described for the Northern blot analysis. Preparation of plasmid DNA Minipreparations of plasmid DNA were done by the boiling method [8], except that the DNA was recovered by spermine precipitation [9]. For large-scale preparation, the DNA was prepared by the alkaline lysis method and further purified by CsCI density gradient centrifugation. The cDNA inserts were excised by digestion with Pstl, separated by agarose gel electrophoresis and extracted by the phenol-freezing method [10]. Northern blot analysis Total RNA (10 lag) was denatured with 2.2 M formaldehyde and fractionated on 1.5~ agarose gels con-

raining 10 mM phosphate buffer, (pH 7.4). The RNA was transferred to Hybond-N membranes and hybridized with nick-translated DNA probes [11]. All hybridizations were done in 50% formamide, 5 × SSPE (1 ×SSPE: 180 mM NaCl/10 mM NaH2PO4 (pH 7.4)/1 mM EDTA), 5 × Denhardt's solution, 0.1% SDS, 100 lag/ml denatured herring sperm DNA and 20 pg/ml poly(A), at 42°C for 24-72 h. The filters were washed twice for 10 min in 2 × SSPE/0.2% SDS, a~ room temperature, and twice for 30 min in 0.1 × SSPE/0.2% SDS, at 55 o C.

Nucleotide sequo~cing Appropriate restriction fragments were subclon~d into M13mp18 and M13mp19 phages [12] and sequenced by the chain-termination method, using [a3sS]dATP and the Klenow fragment of Poll [13] or reverse transcriptase [14]. Results

In an earlier work from this laboratory we identified all of the abundant early spherulation mRNAs. The eigth mRNAs that were studied in detail were similar in their time of appearance. They were absent at 18 h, abundant at 24-36 h and present in trace amounts at 72 h of spherulation [4]. In the present work we are interested in the mRNAs that are synthesized and accumulate late in spherulation. To analyse these mRNAs a eDNA library was prepared from 72 h spherulation mRNA. A sampling of 960 tetracycline resistant clones from the library was analysed. First, 30 randomly selected clones were tested for their sensitivity to ampicillin. All of the clones failed to grow in presence of this antibiotic, suggesting that the clones in the library contained a pBR322 plasmid with an insert in the Pstl site. This lio, ary, designated LAV6, was screened by differential hybridization with labeled poly(A) + RNA collected from 24 and 72 h spherules. A fraction of the library (55 clones) hybridized specifically to the 72 h mRNA probe. The eDNA inserts from some of these stage-specific clones were isolated and individually hybridized to the LAV6 library. The results from this experiment enabled us to assign all of the 55 clones to one of four different groups. Since all of the clones in the library giving a specific hybridization signal were studied, it is likely that the four mRNAs identified represent the only abundant mRNAs found uniquely in 72 h spherules. The clones from each group containing the longest cDNA inserts were used for further studies. These clones were named LAV6..1 to LAV6-4. Their relative abundance in the library varied from 0.2~ to 3.0~ (Table 1). These stage specific cDNAs accounted for 5.6~ of the library, which suggests that a significant fraction of the

266 TABLE ! Characteristics of the late spherule.specific eDNA clones

So

E~. .

The late spherule.specific eDNA clones were isolated from a eDNA library by differential hybridization. The relative abundance of the clones was determined by individually hybridizing the eDNA inserts to the library. The sizes of the inserts were established by gel electrophoresis of Psti-digested p~asmids, and the lengths of the corresponding mRNAs were calculated from Northern blot hybridizations by using Haeill fragments of @X174 RF as standards. Clone

Abundance in library (~)

Length of mRNA (nucleotides)

Length of eDNA (basepairs)

LAVB-I LAVBo2 LAVB.3 LAVB,4

0.8 0,2 3,0 1,6

1250 > 2100 2100 t0$0

1250 800 900 700

mRNA population of mature spherules is composed of late RNAs (synthesized after 24 h). To confirm the stage specificity of these clones, their eDNA inserts were hybridized to Northern blots containing RNA from growing plasmodia, early spherules (24 h) and late spberules (72 h) (Fig. 1). A eDNA, LAV2N-I, corresponding to a mRNA present in these three developmental stages was used as a posittve control. The results showed that the mRNAs corresponding to LAVB-1, LAVB-2 and LAVB-3 are not found in growing plasmodia or early spherules, but are abundant in late spherules. The mRNA corresponding to LAVB-4 is absent in growing plasmodia, present in trace amounts in 24 h spherules, and is abundant in 72 h spherule~ This mRNA, therefore, begins to accumulate slightly before the three other late spherule mRNAs, The clone LAVB,I, which contained a near full-length copy of a late spherule specific mRNA, was retained for further analysis, A detailed restriction map of the LAVB, I eDNA insert was established and the appropriate fragments were subeloned into M13 and sequenced (Fig, 2 and 3), The nucleotide sequence of A BC

ASC

A BC

Q~ e

g

ABC

AB C

kb

t

u~e~

, 1

2

~iii ¸ 3

4

5

F~s, 1, Nocthemblots of poly(A)÷ RNA from plasmodiaand spherules, $ ~g of RNA from 72 h spherules(A), 24 h spherules (B) and phsmodia (C) weredenatured with 2,2 M formaldehyde,fractionated on I,$~ agarosegels~and transferred onto nitrocellulosefilters, The filterswerehybridizedwith nic1:,translatedeDNA inserts from clones LAVB-I(I)~ LAVB-2(2), LAVB-3(3), LAVB.4(4) and LAV2-N1(5). Size markers (in kilobases, kb) are Haelll fragments of OX174 RF DNA.

Sc

.

HI

.

Sc

HI

.

_j

IP ,Q

-'~ .~

qo,

4~

Fig. 2. Restriction map and sequencing strategy of the cDblA insert LAV6-1. The solid box indicates the translated sequence and the open boxes the 5' and 3' untranslated sequences. The restriction fragments indicated by the arrows were subcloned into M13mp18 and M13mp19 phages and were sequenced by the dideoxynucleotide chain-termination method. The restriction enzyme sites are: Hi, H/nell; Sa, Sau3A; S¢, Sacl,

LAV6-1 showed that this cDNA corresponds to a messenger of 1172 nucleotides with a single, long open reading frame of 996 nucleotides. The 5' untranslated region is A / C rich and contains the sequence CGAACA immediately upstream of the presumed AUG initiator codon. This sequence more resembles the yeast consensus sequence for transcription initiation, .,':'"uAA AA"C A , . , C'~' than its mammalian counterpart, 5'-GCC~CC [15]. The 3'.noncoding region is 110 nucleotides long and contains a putative polyadenylation signal, AAUAGA located nine nucleotides upstream of the poly(A) tail. This cDNA encodes a 332-amino-acid protein. The first 20 amino acids at the N-terminal extremity conform perfectly to a typical signal peptide [16-18]. This sequence is composed of a short basic region, a hydro. phobic central region of 11 amino acids, and a polar region which defines the cleavage site (Fig. 3). According to the model of yon Heijne, the signal peptide is most probably cleavgd after the alanine residue at position 20 [19]. The amino-acid sequence of this protein showed three other interesting features, A sequence of seven consecutive glycine residues, bordered on each side by a basic residue is present in the N-terminal region of the protein. A repeat of the hexapeptide (Asn-Thr-Pro-Ala-Pro.Thr) in the N-terminal region is also observed. Finally, a sequence of 11 consecutive hydrophobic amino acids is found in the central region of the protein. The hydropathy profile showed that the LAV6-1 protein has four distinct domains (Fig. 4). The first 20 amino acids define the hydrophobic signal peptide re. glen, which forms the first domain. Amino acids 21-69 comprise the only long hydrophilic region in the protein. This second domain contains the oligo-glycine stretch and the two hexapeptide repeats. The third domain, which is defined by amino acids 70-128, conrains the only long hydrophobic region of the protein. The sequence of 11 consecutive hydrophobic amino acids is found in this region. The fourth domain, which includes amino acids 129-332, has short, interspersed hydrophobic and hydrophilic regions. The three cysteine residues in the protein are found in this region.

267

I

GGG GGG G~T TGT AAA ATC CGT TCT ACC AAT YTT TCA TCT TTC TTC

91

GT$ ATC TTT GCT ATC TTG CTT GGC AGT GCT CTG GCC TGG CAT GG$ Val l i e Pt,e Ala I l e Leu Leu Gly S e t Ala L eu AlaITr p His Gly

136

TCT ~

CAC CAT ~AT CCC ACA ~,G GCG CCA ACT G~. GCC CCT CAC

Ser Ly$ 8is H i s A~n P r o Thr Lys Ala P r o Thr Glu Ala P r o His 181 ~.6

A~A GGA GGA GGA GGC GGC GGA GGG CAC ~ C

ACG CCA GCA CCT ACT

CAG CCT CCA CGC CAA AAC ACT CCC GCT CCC Ace TTC CAA GCC CTC

Gin Pro P r o Arg GIn.AI.~..,TJIr...IP.E~.~0..p.~.~T/4/~.Phe Gln Ala Leu 271

AAC ~TC TGG TTC TTC TTC TGG CTT CTT CTT CTG GCT CGA GCT CCT Ash Val T~p Phe Pbe Phe Trp Leu Leu Leu Leu Ala Arg Ala Pro

316

CTG ACT TAT GTG CAC ATC ACT GCC ACA AGC TCC GAG CCC ATC AAG

Leu Thr Tyr Val 8~s l i e Thr Ala Thr S e t S e t Glu P r o I l o Lys 361

CTG TTG GTT CCC TTG TAC GTC T~T CCC GOT GCT GCT TOG G~T TCC

Leu Leu Val P r o Leu Tyr Val Tyr P r o Gly ~ l a Ala T r p Asp S e t 406

GTA GCT AAC GCT GCC AAG ACT GGC GTC AAA ATC ATC GCC ATC ATC V~I A ~ ~ n AI~ AI~ Lys Thr Gly Vel Lys ~le ~le AI~ 11o I1¢

451

AAC CCC AAC AGC GGA CO& GCT TCC TCT GGA CCA GAC TCT TCC TAC ASh P r o ASh S e t Gly Pro Ale SeT SeT Gly P r o Asp S e t S e t Tyr

496

ACT ACC TAC ATG RAC AAG TTG ACC GCT GCT GGT GTC GAC ATG GTG Thr Thr Tyr Me~ Ash Lys Leu Thr Ala Ala Gly V~I Asp He~ V~I

541

GGA TAC GTA CAC ACC TCC TAC GGA GCT COT OCT GTT GGT G&T GTG Gly Tyr Val H i s Th~ SeT T y r Gly Ala Arg k l a Val G1¥ Asp Val

586

AAT GCT G~C ATT GAC ACG TAC GCC TCT AAA TAC CCA GGC CTC AAG Ash Ala Asp lle Asp Thr Tyr Ala Set Lys ~¥r Pro Gly Leu Lys

631

GGT ATC TTT TTG GAT GAG GCC TCT GCT TCA GCC AGC GAG ATT TCT Gly l l e Phe Leu Asp Glu AI~ Se~ Ala SeT AI~ Se~ GIo l i e SeT

6~6

TAT TAC ACC AAC GTA TAC AAC CAC ATC AAG TCC AAG TCC GGA TAC ~yr Tyr Thr ASh Val Tyr Ash H~s l i e Lys Set Lys SeE G l y Tyr

721

GTC AAC TCC ATT TTG SAC CCC GGA ACT CAA CCC G&C CAA GG& TAC Val ASh $e~ l l e Leu ASh Pro Gly Thr Gln Pro Asp Gln Gly Tyr

?66

TTG OCt ATC TCT TCC AAC ATC GTG &TT TTC GAG G~T GCA GGC TCT Leu Al~ I l e Set SeT ASh I l e V~l l l e Phe GIo Asp &l~ G l y Set

811

RAT CTC RAG RAT AAC T&C GCC TCT TGG GTT AAG TGC GCT CCC TCG

&sn Leu Lys ASh &sn Tyr &la S e t T r p Val Lys Cys k l ~ P r o S e t 856

GCC TCC CAG AAG AGT GGC TAC AAG TAC RAA TTC TCC GGA ATT GCT k l ~ Set Gln ~ s Set Gly Tyr Lys Tyr Lys Phe Set Gly l i e &la

901

CAC &GC ACT TCC TCG GGT > ATG TCA GG& ~TC &TT RAC &CA ATG HAs S e z Thr S e t S e t Gly S e t He~ S e t GIy ~ l e I l e &sn Thr He~

946

GTT TCC GTC CTG GCA &TG GG& CTG GTT T~C GTT &CG G&T GGT GCT Val SeT V~l Leo k l ~ He~ Gly Leu Val T y r Val Thr Asp Oly k l a

991

GCT GGT TGC TGC &C& TAC RAC &C& TTG ACT TCT TAT TTG TCC C~.k Ala Gly Cys Cys Thr ~yr Asn Thr Leu Thr Set Tyr Leu Set Gln

I~36

GAA GCC TCT GCG GTC CAC GCT CTG AAC TAA AAA RAG GTT TTT AGT GIo A ~ Set AI~ ~al HAs kl~ Leo ASh

1081

CC& GTA CAT TTT TTT TTT TTT GTA ACT TCG TTC GTO GTT CCA GTC

~(

TTG TAT TAT GTA PLAT ~CO ~AT CAA ACC TTA GTA ATA G~A TGT AAT

Fig. 3. The nucleotide and deduced amino-acid sequences of LAV6-1. The putative signal peptide sequence at the N.terminal region is enclosed in a box. The seven consecutive glycine residues are underlined with a bold line. The two hexapeptide repeats (Asn, Thr, Pro, Ala, Pro, Thr) are indicated with a broken line. The hypothetical polyadenylation signal (AATAGA) is underlined with a thin line.

268 ,1 v J"

2

v

.

3 .

,,

0

.,y •

.....

4

,.,

.

100

,

.

r

,

200

• .

,

~2

300

Fig. 4. The hydopathy profile of the amino-acid sequence derived from the LAV6-1 mRNA (spherulin 4) was traced by the method of Kyte and D Jofittle [26]. Hydrophobicity is plotted as a function of the residue number; hydrophobic regions are above zero. while hydrophilic regions are below zero. The four putative domains are numbered and separated by arrowheads.

The derived amino-acid and nucleotide sequences of "LAV6-1 were compared to the sequences in GenBank and NBRF libraries.. No significant similarities were seen with any known sequence, including the 24 h spherulin proteins. Since the LAV6-1 mRNA accumulates during spherulation, the protein encoded by this protein was called spherulin 4. In a previous work from this laboratory it was shown that codon usage differs in mRNAs expressed in plasmodia and in 24 h spherules [5]. In gen~.:,al, codon usage is more random in early spherule mRNAs. Bernier et al. [5] identified ten codons which are rarely used in plasmodia but are frequently used in 24 h spherule mRNAs. These codons are shown in Table 11. To determine whether the codon usage of 24 h spherules is mainrained in 72 h spherules the codon frequency of the spherulin 4 mRNA was determined. The majority (70%) of the key codons are used in 24 and 72 h spherule mRNAs but are rarely used in plasmodial mRNAs, indicating that codon usage is similar in early and late spherule mRNAs (Table 11). These results suggest that

all the spherulation mRNAs are translated with the same population of tRNA isoacceptors.

Discussion Previous work from this laboratory showed that plasmodial encystment (spherulation) is under the control of a complex genetic program, involving the coordinate and temporal expression of a large number of genes. During the early stages of encystment the composition of the mRNA population is drastically modified. A large number of mRNAs present in growing plasmodia are degraded and replaced by the early spherulation mRNAs. An estimated 25-70 different mRNAs accumulate, and they account for approx. 30% of all the mRNAs in s~herulating cells [3,4]. As the encystment process continues, the early spherulation mRNAs are in turn degraded and replaced by a smaller group of late mRNAs that are the subject of the present work. Eight early and four late spherulation mRNAs have been cloned in our laboratory. These cDNAs

TABLE !!

Codon usageof spkendln 4 mRNA (S~) and compa~on wtO,codonfrequency oj earl), spherulin mRNAs (S¢) and plmmodi¢l mRNAs (P) ~a

Codon ~

Sob

$4 ~

~

aa

Codon

So

S,

P

aa

Codon

SO

$4

P

aa

Met lie

AUG AUU AUC AUA UUAe UUG CUU CUC CUA CUG GUU GUC GUAe GUG UUUe UUC

100 38 $8 4 9 23 25 23 $ 14 35 21 16 28 46 $4

|00 38 62 0 0 38 17 !7 0 29 ,'g) 30 17 22 25 75

100 29 69 2 0 19 ? 6"/ 0 7 25 35 2 38 It 89

Set

UCU UCC UCA UOG AGU® AGCe CCU

21,$ 24 4 16 13 21,$ 37 22 37 4 38 38 19 $ 36 64

31 39 6 6 8 il 20 50 30 0 43 22 22 13 29 80

19 61 8 l0 ! i 18 75 7 0 21 64 II 4 II 89

Ala

GCU GCC GCAO GOG CAU CAC CAP, CA(] AAUO AAC AAAO AAG GAU GAC GAP, GAG

38 26 24 12 68 32 62 38 45 55 56 44 64 36 42 58

~i4 33 8 5 20 80 71 29 19 81 31 69 50 50 33 67

37 48 5 10 23 77 53 4"/ 12 88 3 97 38 62 21 "/9

C.vs UGUo UGC Trp UGG Arg GCU CGC CGA CGG AGA AGG Gly GGU GGC GGA GGG end UAA UAG UGA

Leu

Val

Phe

Pro

CCC Thr

Tyr

CCAe CCG ACU ACC ACA ACG UAU UAC

His Gin Asn Lys Asp Glu

Codon

Se

S,~

P

45 55 100 18 21 15 0 18 28 20 23 45 12 75 0 25

0 100 100 25 25 25 0 25 0 23 23 47 7 100 0 0

6 94 100 42 42 2 0 3 11 32 13 53 2 100 0 0

a Codon usage is e . x p r e ~ in percent. Codons commonly used in the spherulin mRNAs but very rarely found in actin and tubulin mRNAs are indicaled by a dot, b Early spherulin codon frequencies are calculated from Bender et al. [5]. c ~ 4 codlon frequency calculated from results in the present work. Plasnmdial codon frequencies are calculated from actin ardB, actin ardC and a-tubulin mgNAs.

269 represent useful molecular markers for the study of plasmodial encystment. The nucleotide sequences of four eDNA copies of the early mRNAs were determined. The encoded proteins were named spherulin l a, l b, 2 and 3. Spherulins l a and lb are 81% identical and possess putative signal peptides and N-glycosylation sites. Spherulins 2 and 3 do not contain signal peptides and are probabl: intracellular porteins [4,5]. The nucleotide sequence of one late spherulation mRNA, which we called spherulin 4, was deter~ined. This mRNA encodes a protein that has many features of a plasma membrane protein. It has a typical signal peptide, which is probably cleaved during transport through the endoplasmic reticulum. This would create a mature protein composed of a short N-terminal hydrophilic region, a central hydrophobic segment, and a long C-terminal region. The central region with the series of 11, 5 and 5 consecutive hydrophobic amino acids is a possible transmembrane anchor. The N-terminal and C-terminal regions could extend into the extraceUular or cytoplasmic spaces. Another interesting feature is the presence of seven consecutive glycine residues. This is even more remarkable since this protein is not rich in glycine (9%). Several examples of glycine stretches in proteins have been reported. Three developmentally regulated Drosophila genes (Ubx, Dfd and fshl ) plus P9 gene, which codes for a proiein similar to the rat helix-destabilizing protein found in hnRNP particles, code for glycine-rich domains [20-22]. In addition, a dispersed Drosophila gene family containing multiple GGX triplets has been described [23]. Other glycine repeats were found in the proteins encoded by the grp-I gene of petunia [24] and the MAH9 gene of maize [25]. Although the role of the oligo-glycine residues in these proteins is not known, it was suggested that in Ubx the glycine stretch favors the formation of a flexible globular region in the protein that may serve to separate distinct functional domains [21]. It is interesting to note that the oligoglycine stretch in spherulin 4 is located between two apparently distinct domains: a signal peptide and a long hydrophobic region. Studies of in vivo protein synthesis indicate that although the general rate of protein synthesis is low in late spherules, a few new proteins are synthesized [3]. The spherulin 4 mRNA appears late in spherulation and is most likely translated at this time. These late proteins appear after dehydration-resistant cysts are formed and are therefore probably not involved in this process. It was noted, however, that late cysts (72 h) had a more rapid germination time than early cysts [3]. It is likely that the late proteins are involved in the final

stages of cyst maturation, which would include the ability to germinate. Acknowledgements We thank Francois Bernier for furnishing LAV2N-1. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Fonds pour la Formation de Chercheurs et rAide/~ la Recherche du Quebec (FCAR).

References 1 Rusch, H.P. (1980) in Growtll and Differentiation in Physarum polycephalum (Dove, W.F. and Rusch, H.P., eds.), pp. 1-8, Princeton University Press, Princeton. 2 Binette, F., Pallotta, D. and Lemieux, G. (1988) Curr. Genet. 13, 151-157. 3 Bernie., F., Seligy, V.L., Pallotta, D. and Lemieux. G. (1986) Biochem. Cell Biol. 64, 337-343. 4 Bernier, F., Pailotta, D. and Lcmieux, G. (1986)Biochim. Biophys. Acta 867, 234-243. 5 Bernier, F., Lemieux, G. and Pailotta, D. (1987) Gene 59, 265-277. 6 Daniels, J.W. and Baldwin, H.M. (1964) in Methods in Cell Physiology (Prescott, D.M., ed.}, pp. 9-41, Academic Press, New York. 7 Pallotta, D., Bernier, F., Hamelin, M., Martel, R., and Lemieux, G. (1986) in The Molecular Biology of Physarum polycephalum. pp. 315-327, Plenum. New York. 8 Holmes, D.S. and Quigley, M. (1981) Anal. Biochem. 114, 193-197. 9 Hoopes, B.C. and McCiure, W.R. (198i) Nucleic Acids Res. 9. 5493-5504. 10 Silhavy, T.J., Berman, M.L. and Enquist, L.W. (1984). Experiments with Gene Fusion, pp. 164-165, Cold Spring Harbor Laboratory, NY. I1 Rigby, P.W.J., DiecLmann, M., Rhodes, C. and Berg, P. (1977) J. Moi. Biol. 113, 237-251. 12 Yanisch-Perron, C., Vieira, J and Messing, J. (1985). Gent 33, 103-119. 13 Sanger, F. and Coulson, A.R. (1975) J. Mol. Biol. 113, 237-251. 14 Graham, A., Steven, J. McKechnie, D. and Harris, W.J. (1980) Focus 82, 4-5. 15 Hamilton, R., Watanabe, C.K. and DeBoer, H.A. (1987) Nucleic Acids Res. 15, 3381-3393. 16 Von Heijne, G. (1981) Eur. J. Biochem. 120, 275-278. 17 Von Heijne, G. (1985) J. Mol. Biol. 184, 99-105. 18 Von Heijne, G. (1986) J. Mol. Biol. 189, 239-242. 19 Von Heijne, G. 0983) Eur. J. Biochem. 133, 17-21. 20 Beachy, P.A., Helfland, S.L. and Hogness, D.S. (1985) Nature 313, 545-551. 21 Laughan, A., Carroll, S.B., Storfer, F.A., Riley, P.D. and Scott, M.P. (1985) Cold Spring Harbor Syrup. Quant. Biol. 50, 253-262. 22 Haynes, S.R., Rebbert, M.L., Mozer, B.A., Forquignon, F. and David, I.B. (1987) Proc. Natl. Acad. Sci. USA 84, 1819-1823. 23 Flaveli, A.J., Dyson, J. and ish-Horowicz, D. (1987) Nucleic Acids Res. 15, 4035-4048. 24 Condit, C.M. and Meagher, R.B. (1986) Nature 323, 178-181. 25 Gomez, J., Sanchez-Martinez, D., Stiefe!, V., Rigau, J., Puigdomenech, P. and Pages M. (1988) Nature 334, 262-264. 26 Kyte, J. and Doolittle, R.F. (1982) J. Moi. Biol. 157, 105-132.