Two genes encode related cytoplasmic elongation factors 1α (EF-1α) in Drosophila...

Volume 16 Number 8 1988 Nucleic Acids Research

Two genes encode related cytoplasmk elongation factors la (EF-la) in Drosophila melanogasterwith continuous and stage specific expression

Bernd Hoveraann, Sabine Richter, Uwe Walldorf' and Celina Cziepluch

Zentrum fur Molekulare Biologie Heidelberg (ZMBH), Universital Heidelberg, Im Neuenheimer Feld282, D 6900 Heidelberg, FRG and 'Biozentrum, Klingelbergstrasse 70, CH 4056 Basel, Switzerland

Received February 4, 1988; Accepted March 18, 1988 Accession nos: Fl=X06869, F2=X06870

ABSTRACTWe have characterized two previously cloned genes, Fl and F2(l)that code for elongation factor EF - la of Drosophilamelanoaaster. Genomic Southern blot hybridization revealed thatthey are the only gene copies present. We isolated cDNA clonesof both transcripts from embryonal and pupal stage ofdevelopment that cover the entire transcription unit. The 5'ends of both genes have been determined by primer extension andfor Fl also by RNA sequencing. These start sites have beenshown to be used consistently during development. Comparisonof cDNA and genomic sequences revealed that EF - la,Fl consistsof two and EF - la,F2 of five exons. The two describedelongation factor genes exhibit several regions of strongsequence conservation when compared to five recently clonedeucaryotic elongation factors.

INTRODUCTION

The process of translation of genetic information from mRNA to

protein follows a distinct pathway. One step, the elongation of

the amino acid chain, involves a series of protein components

that have been classified according to their function as

elongation factor 1 (EF - 1) and elongation factor 2 (EF - 2).

The cytoplasmic EF - 1 complex is thought to consist of three

proteins: EF - la, 6 and 7(2). EF - la facilitates the GTP

dependent binding of charged tRNAs to the acceptor site of the

ribosomes. This process includes the binding and hydrolysis of

GTP, the binding of aminoacyl tRNA and the recognition and

interaction with the 80S ribosome. The multifunctional demand

on EF - la obviously leaves little room for an evolution of

divergent protein structures. It is therefore not surprising

that the eucaryotic cytoplasmic la elongation factors isolated

to date (3-10) show a high degree of homology that is indicated

by physical properties like pi and molecular weight. Their

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close relationship also becomes evident by comparing the gene

structure of various recently cloned and seguenced EF - la

genes. Saccharomvces cerevisiae for example contains two genes

for the cytoplasmic factor (11-13), either one is sufficient

for cell viability. The fungus Mucor racemosus has three

closely related EF - la genes (14), and the brine shrimp

Artemia salina may even contain four copies of this gene (15).

The number of genes detected in mouse (our unpublished

observation) might exceed ten, including possible pseudogenes.

The one human (16) and part of a mouse EF - la gene sequence

known to date (17-19) all share a very high degree of homology

within the protein coding portion with all cytoplasmic EF - la

genes analysed so far.

Here, we present evidence that Drosophila melanoaaster contains

only two different gene copies of the cytoplasmic factor. We

report the isolation and sequence of cDNA clones covering the

entire mRNA for both EF - la,Fl and F2. Transcription start

and processing sites are determined by comparison of cDNA and

genomic sequences and primer extended RNA sequencing.

MATERIALS AND METHODS

DNA sequencing was performed according to the chemical cleavage

method of Maxam and Gilbert (20) and the chain termination

method of Sanger (21). For RNA sequencing we followed the

protocol of Geliebter (22) using a 20mer oligonucleotide primer

and AMV reverse transcriptase. 10 pq poly (A)* RNA and 0.5

pmol 32P labelled oligonucleotide primer were coprecipitated

and taken up in annealing buffer (250 mM KC1, 10 mM Tris/Cl pH

8.3). After heating to 80"C for 3 min the mixture was kept at

45*C for 30 min. The individual reactions were carried out at

43'C for 15 min.

For primer extended cDNA synthesis the reaction was set up as

described previously (23). For Southern analysis of chromosomal

DNA and for the screening of cDNA libraries we followed the

standard protocols as summarized by Maniatis (24). Poly (A)*

RNA was prepared as described by Hovemann et al. (23).

Oligonucleotide primers were obtained from the service unit of

the ZHBH. cDNA libraries were used that were originally

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constructed by the laboratory of T. Kornberg. We screened batch

E6 and Q4. Drosophila melanoaaster strains Canton S (CS) and

Oregon R (OR) were grown as mass population on standard

cornmeal agar medium in fly cages. (25).

RESULTS AND DISCUSSION

In an approach to clone sex dependently regulated genes of

Drosophila melanoctaster we had previously described the

isolation of two closely related genes that were arbitrarily

named Fl and F2. According to Northern blot analysis, Fl RNA

was found in all stages of development whereas F2 RNA was not

present in early embryos and was peaking in pupae. We now

identified the function of their putative protein products by

comparison of their gene sequences with those of a series of

recently published elongation factor (EF - la) genes. The

genomic sequences of both genes (the Fl sequence has partly

been published by us (1)) have now been determined in full

length and are shown in Figure 1 and 2. In order to determine

the length of the 51 untranslated leader sequences preceeding

the translational reading frame of Fl and F2 we used two

independent approaches. We determined the 51 end of the RNA

indirectly by oligonucleotide primed cDNA synthesis and, in

addition, we isolated and sequenced cDNA clones in an attempt

to obtain full length RNA copies (Fig. 3, 4).

Gene structure of Dm EF - la.Fl

For indirect RNA sequencing and primer extension we used a

synthetic oligonucleotide complementary to positions -14 to +6

relative to the translational start site (Fig. 1 and 3). The

sequence determined by reverse transcription of the Fl RNA

deviated from the corresponding strand of the genomic DNA after

the C at position -20 (Fig. 1). Indirect RNA sequencing as well

as the primer extension experiments suggested that the 5' mRNA

leader sequence was encoded by at least one additional miniexon

of together 60 nucleotides (Fig.3). This was corroborated by

the fact that the sequence difference started with an AG

dinucleotide flanked by the splicing acceptor site consensus

sequence observed in Drosophila (26). At the same time we

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1 CAATTCCACA AGAAAAAGTA TTTAAACCTT AT1TAAATTT AGTTATAAAT TTTTACAATA TTGTATAAAT TGATACAATA CCAAATTATT

• 1 TCTACTTTTA CTTOTAGCAT ATTTTAATTO TAGCAACGCA CGCAAAAAAA TCTATAAATA AATOAAACGT ATTACATAAA TTCAAACOAT

l t l CTCAAOCTTC CATTCTTATT TAAAOTTCTA TTACGTTAGO OTTCACATAC AAATTAAAGT GGCAGOTTCT ATCTCAAAAC ATTCCTTCAA

2 7 1 AATGCGGACT ACTAATGCAA TTOTTATTOT TTTTACATAT TAAAAOATAT OTCTTCCAAT ATTACCTATA OAAATTATAC ACATCOTTTT

3 6 1 GTAGAAAATA CTTTTGOAAT CACTGATTAT TTACTTTTTC ATATAAAAAC AATCTCOAOC AAACAAGCTT TTTTAAATTC CTCAATCTTT

4 51 AOOTTATTGT ATTTTOCCAC TTTCAATCAC TT1AATTTCA ATAAAATOAA OTOCTTCATT CCCCCOTAOT GGAAACACCG CAGTGOOAAC

3 4 1 ACOGTTTCTG CTCTTTTGAC AOTTOCOTAO CTTCOCTCAC ACCATGTGTC AAACGACGCT TCCTCTGCTC AOCTCTOCCO AACOCTCCTT

6 3 1 CACTTTGTTC GAATCCGTCG CCGCTTAGAC TTCGTGATTT CTCATTCAGC TTATTAOAOA OTAAGTTTTA CCTOCOAOOC TATAATTAAO

TOATTTCTOC AAAAAAACTO CAOQGGOCAA AC1ATTIATA AACAAATATG CACCTOAOAC OCCOAATTTG TCCATATTTC CAGTGTTTTT

CCTUTVTUH. TGTAATAAAC CCOOAOATAA CCTCTAACTO CGCTTTTCCA AAGTGAAAGG TGOCCATAOA AOCAAACACG TOOCAAOTCT

GCAAAOOCAA AAATTTTAAC TOOCOTTCCC AOTTAAAOTT CCCAOCATTC TCAAAATAAT TTTCCGOCTT TTCCGCCCGC A1II1COCCC

TOCAATATOO TGCACTTAOC OTOTAATTAC TTTOCCACGC CCACGCCGGA CACAGAOGTC ATCCACCAOA TGTOCTCATT AACCOAOAAA

AAAAAACGTG CTTTCTCTCT TOCCTTTOTC ATCGCCTATA OATATTCCTT A l H . l l l l . i l TTTGCGGCAT GCAATTCTAA AATOGCGACC

CASTOGCOTO AOTCAAOTOC OCGAAAAAAT TCOCCTOOCA ACAAGCGAAA AAATGTOCTT TTTTOOOTTT CCAOCCCATT AOCATATCTO

OTOTAATCGC ACTCOCATCA OCTATTTCGC C1TTTCCAAC COACTCAATA ATTGGTTTTO OTAAAATOOC TOCCGCTOCA CTACOTTCTT

OATTAATTCO TTOTOTOCCC L l U L I l l l l CATTTCTTTC CAATTACCAA TTGTGCCACC SCOGCOGACA C5CTT0CATT TGTACAAGTC

ACACACGCAC ACTAATCCAC ATCCOCCATT TTGGTCTCTC TCTCTTCCTC TCTTACTTTT TCCOOCCOOC AACAGCGTCA CACAAATACA

CAGGCATAGA TATACACACO CATAGOCAGA TAAGCACATO TGTATTTOCO AATTAAATTT GCTGCAATTT TCCTTTOOAC TCTTCOATTT

AACATOATOA TGATTTTTCA OTTCTGCTAC TOAAGAOAOT TGACAOAAAO CAAAAATACC AAAATCACTG AAACAAAATC OAGTTTCCAT

ATCOAATTTT ATTTOCACGC TCTTTTCTOT AGTTOCOCCC CACTCGTTTT ACCCACACCC CTACATOCOO OCACTOaTCC TAACCTCAAA

AAACACGTTT TGTACOGCTG CAAGAGTTTO AOGTTAGGTr OTOCTCOCOC ATGCAAACAA AAGTCOAACG TACOCTAOGG AAATGAGAAA

OTCTTATACC CACTAATAAT rGTAOTTOTA ATCCCACCGA ATTCTTTTAC CCTTTGTTTA TTCCAACCTC TCTTOCTCOC CAACCCGCCG

AACCCTOCAA CCTTCCAATG TTCCAACGTT CCOTTAATCC AACACTCOAA TACACACAAC AO :CATAGTO TAATCATCCA AC

3 0 6 3 ATO HOC AAG GAA AAO ATT CAC ATT AAC ATT OTC OTO ATC GGA CAC OTC CAT TCC OOT AAG TCO ACC ACC ACC GOAH « t GLr L r « G l u Lys 1 1 « H i s z l « A«n I I * V a l V a l I I * G I T H i * V a l Aap 3 * r G l r Lys 8 * r Ttar T h r Tbr G l r

2 1 3 1 CAC TTO ATC TAC AAO TGC GGT OOT ATC OAC AAO CCT ACC ATC GAG AAG TTC OAG AAO OAO OCC CAG CAO ATG OOAB i * L*u I I * T r r Lr* C y s G l y G l r I I * Aap Lya A x o Tbr I l a G l u Lya Ph* G l u Lya O l u A l a G l a O l u H*t G l r

2 3 1 3 AAG GGA TCC TTC AAG TAC OCC TOO OTT TTG OAT AAO TTG AAO OCT OAG CCC OAO COT OOT ATC ACC ATC OAT ATCLya O l y S a r Pha Lya T y r A l a T r p v a l L « u i A a p L r a L«a Lya Ala Olu Arg Gla Arg Oly I l a Thr I l a Aap I l a

2311 GCC CTO TOG AAO TTC GAA ACT GCC AAO TAC TAC OTG ACC ATC ATT OAT GCC CCC GOA CAC AGO OAT TTC ATC AACAla L«u Trp Lya Pba Olu Tbr Ala Lra Tyr Tyr Val Thr I l a I l a Aap Ala Pro Gly Hi* Arg Aap Ph* I l a Lya

2163 AAC ATO ATC ACT OOT ACC TCO CAO OCC OAT TGC GCC CTO CAG ATT OAC OCC OCC OGA ACC OOA OAA TTC OAO GCCAan H*t I I * Thr Glr Thr f a r OlD Ala Aap Cya Ala Val Gin I I * Aap Ala Ala Gly Thr Oly Olu Pba Gla Ala

2431 OOT ATC TCO AAO AAC CAC CAO ACC COC OAO CAC CCC CTO CTC CCC TTC ACC CTO OCT OTO AAO CAO CTO ATC OTTOly I I * » . r Lya Aaji Aap Gin Thr Arg Olu I l a Ala L*u L*u Ala Pha Thr Lam Oly Tal Lya Cln Lau I I * Val

2913 CGT OTG AAC AAO ATO OAC TCC TCC GAS CCA CCA TAC AOC GAG GCC COT TAT OAG OAA ATC AAO AAO OAA GTG TCCOly Val Aan Lya H«t Aap f*r l « r Ola Pro Pro Tyr l * r Gla Ala Arg Trr Ola Glu I I * Lya Lya Olu Val f a r

2 J I I TCT TAC ATC AAO AW GTC COC TAC AAC CCA OCC CCC GTT OCC TTC OTO CCC ATT TCC OOA TOO CAC OGC CAC AACf a r Tyr I I * Lra Lya Val Gly Tyr Ajn Pro Ala Ala Val Ala Pha Tal Pro I I * f a r Oly Trp HI* Oly Aap Aan

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1 6 ( 3 ATC TTO O U CCC TCT ICC U C 1TO CCC TOO TTC U O OOA TOO O U OTO OGA COC U S GAG SGT U C OCT OAC COC• a t Lau O l s f r o I . r Thr U l Hat Pro Trp Ph . Lra Olr Trp Olu V«l O l r Are ! • ! • Olu S i r U n Ala U p O i l

2731 U O ACC CTO OTC S i T SCC CTC OAT OCC ATC CTT CCC CCA OCC COT CCC ACC OAC AAO SCC CTO COT CTO CCC CTOLra Thr Lau Val Asp Ala Lau Aap Ala I l a Lau r r o r r o Ala Arg Pro Ttar A l p Lia Ala Lau Arg Lau f r o ban

2113 CAC BAT OTO TAC AAA ATT OOC OOT ATT OOA ACA OTA CCC OTO SOT COT OTO OAO ACT OOT CT6 CTO AAO CCC COTOln Aap Val TTT Lra I l a Olr Olr I l a S i r Thr Val Pro Val S i r Are Val S l u Thr O l r Val Lau Lra r r o C l r

2 M I ACC CTT OTO OTC TTC CCC CCT OCT AAC ATC ACC ACT CAS OTC AAO TCC OTO OAO ATO CAC CAC OAO OCC CTO CAOThr Val Val Val Pba Ala Pro Ala Aan I l a Thr Thr Olu Val Lra f a r Val Olu Hat I l a I l a S l u Ala M i Oln

1 H ] SAO SCC OTT CCC OOA OAC AAC OTT SCC TTC AAC GTC AAO U C OTO TCC OTO AAO OAO CTO COT COT CCC TAC GTTOla Ala Val Pro S i r Aap Aan Val S i r Pha Aan Val Lra Aan Val Sar Val Lra S l u Lau Arg Arg S i r Trr Val

3 t 3 l SCC SOT SAC TCC AAO SCT AAC CCC CCC AAC CSA SCC OCC SAC TTC ACC CCC CAS GTC ATC STG CTS AAC CAC CCCAla Olr Aap Sar Lra Ala Aan Pro Pro Lra Olr Ala Ala Aap Pha Thr Ala Cln Val I l a Val Lau Aan I l a Pro

3113 SOT CAO ATT CCC AAC SOC TAC ACC CCA OTC TTO SAT TSC CAC ACC SCT CAC ATT SCT TCC AAO TTC SCT CAS ATCS i r Gin I l a Ala Aan Olr Trr Thr Pro Val Lau Aap Cra l i a Thr Ala I l a Zla Ala Cra Lra Pha Ala Olu I l a

31SS TTO OAO AAO OTC OAC COT COT TCC OOC AAO ACC ACC CAC SAC AAC CCC AAG TTC ATC AAG TCT OCC OAT OCT SCCLau Ola Lra Val Aap Arg Arg Sar Olr Lra Thr Thr Olu S l u Aan Pro Lra Pha I l a Lra Sar S i r Aap Ala Ala

32C3 ATC OTC AAC CTO OTO CCC TCT AAO CCC CTO TOC STG GAG CCC TTC CAC GAG TTC CCC CCT CTC SST CSC TTC SCTI l a Val Aan Lau Val Pro Sar Lra Pro Lau Cra Val Olu Ala Pha Oln Ola Pha Pro Pro Lau Olr U g Pha Ala

333S GTO COT OAC ATC ACS CAS ACC STO SCT STC SGT STC ATT AAS SCT OTC AAC TTC AAO SAT SCC TCC OST COC AASVal Arg Aap Hat Arg S i n Thr Val Ala Val Olr Val I l a Lra Ala Val Aan Pha Lra Aap Ala t a r Olr Olr Lra

3413 GTC ACC AAO OCC GCC SAS AAS SCC ACC AAG CCC AAO AAO TAO CT00TTT0CT TCCACTCAAC ' " • i i c u n ACACSCASTAVal Thr Lra Ala A la S l u Lra Ala Thr Lra S i r Lra Lra • • •

3495 STAOCAGCAA CAACAAOCAT ATAACCAACA TCATAATCCA CCCAACAACA CCACTCAATA ATACCASCAA CAOCAOCAOC OUCACAATA

3515 OTAOTATAAC ACCAACACCT OTCCTSCOCA AOATOACCOA TAASATSATS TTTCACCACA ACCATAAGTT TUTTTCTTC CATCOAAAOG

3675 AGTTTCGACO OATACCUTG CTAAATCCAC ACCASCCCGC CTTCACTOOS AAATCOSTCS ATCCCAAOOA TAAOAOTSCA CACTSSCAAA

3765 ACACTTOCAT TTATOCATCC ACTCCTCATC CACTTCCCCO TC01H.1I I t OTTTACTAAA TATGCTATOA TSCACSCAOT TOACTTCOTT

3155 TTATCATATC ATATATAOOA ATCCTCTOTA OCATTTATOA TATCOTTTU ATTAACCTTT ATACTTTOAT ATOTATCATT TATCTTACCC

3945 TACTTTTOCA CACACTACTT TSTACACAAC AAAAOUCCA 0UT1OAAOC OATAAACTAT ATTTACAAAA AAUTAAAAA CCCTATTTTT

4*35 OTATTTCTTT TSTTTTTACC ACCCAOCCCO TAAAAGACCA CTCTCTTTTT OOTTOTTOCC TCCCOATTT

FIGURE 1Sequence of the EF - la,Fl gene region. Throughout the codingportion aminoacids are written below the sequence. The intronarea is boxed. Transcription start and polyadenylation site areindicated by an arrow and a dot, respectively. A sequenceheterogeneity observed in cDNA cDml9 is indicated at position686. The oligonucleotide primer i s shown by an arrow (Pos.2049-2068). Conserved sequence blocks are underlined.

isolated several Fl cDNA clones from a AgtlO library which had

been prepared from embryonal poly (A)* RNA. By comparison of

their restrict ion s i t e patterns with the genomic map some

isolates seemed to contain a mRNA copy including the very

5'end. One such cDNA (cDm 19, Fig.3) was subsequently seguenced

and showed the same 5' end portion when compared to the

indirect RNA sequencing results. The heterogeneity observed at

nucleotide 56 of the f i r s t exon may be attributed to a

difference in the inbred strains Canton S and Oregon R that

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were used for genomic and cDNA library construction,

respectively (Fig. 1).

In order to locate the 5' end of EF - la,Fl mRNA within the

genomic sequence, primer extended 32P - labeled cDNA was

hybridized to a blot of DNA containing the subcloned BamHI and

two Hindlll fragments proceeding about 10 kb upstream of the

EF - la,Fl coding portion. Alternatively, cDNA clone cDml9 was

hybridized to a blot of the Fl gene and its upstream sequences

included in phage ACS1 (Fig. 3). Consistently, we observed

hybridization to the 1.3 kb and the neighbouring 1.2 kb EcoRI

fragments. In order to be able to precisely localize the 51

exon(s) in the genomic DNA we subsequently sequenced both EcoRI

fragments (Fig. 1). The resulting data showed that the first

exon is contained within the 1.2 kb EcoRI fragment. EF - la,Fl

thus extents over 3.4 kb and consists of two exons that are

separated by a 1.3 kb intron (Fig 3).

Structure and sequence of the EF - la.F2

An EF - la,F2 containing 4.0 kb EcoRI fragment of genomic DNA

that had been identified by crosshybridization to the EF -

lcr,Fl cDm49 probe (1) has been sequenced using both the methods

of Maxam and Gilbert and of Sanger (Fig. 2). Comparison of this

genomic sequence with those of a series of cDNA clones isolated

from cDNA libraries of pupal RNA by hybridization with the same

4.0 kb EcoRI fragment revealed a single long open reading

frame. It encoded a second, related elongation factor protein

that is one amino acid shorter and shows 90% homology when

compared to EF - la,Fl (Fig. 8). Two introns that interrupt the

coding portion of the F2 gene had already been mapped (1). To

determine the transcriptional start of the EF - la,F2 gene we

proceeded the same way as described for Fl and used a synthetic

oligonucleotide primer complementary to nucleotides -14 to +6

relative to the translation start. The major cDNA product

primed with this oligonucleotide on pupal RNA was 142

nucleotides long (Fig. 4). In addition, cDNA clones that

possibly reached the 5' end of the F2 mRNA were isolated from a

library prepared from pupal poly (A)* RNA. cDNA pc3 was

selected as the clone that extended farthest to the 5' end.

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1 TAAGCCAATA GTGTGCACAA I H l l u l l C AATTAOTOGT CAATOTGCAT ACTTTAOTGA CAGTCCCTGA AACTACTATA TTATTTTATC

»1 TOCAAAAGAC TCAOTTTAAO U U T t T U I ATATTCCATO AATOOTAOTA AAATTGTATT ACTAI1UIA TTTTGOTACO TTTTATACTT

1 1 1 AAOOCATOOA AACTTTATTT AAGTCAAGAA ATCCGCATAA TOCAATAGOA AACCCAAOGC CIUU11ATA CATGCAATCC TGTGCCATCT

271 CTAGOTCGGA ATCAOTTCAG CTCCGTTCAC CTCACCATCO U U l i U U L OQTCTTTCCG TTTTCTOATT TCOAobTAAG TGCACGCAGA

361

7 2 1

• 11

OAODT

OCTCCCOTTA AAATTOTGAA AATATTAATA OGCATTOATT ASMJTOOAA ATGTAAAAAO OGAAAGTCCC AGAATTCCCT ACCCTGCATT

ATTAOOCGAA TTTCOOTTCG ATTTCCAACC TAAAGAAACT TCTAAAGTAA ACAAAOTTCC 0AAAASTOA0 AOACTCTAAC TOATTTOCOC

TCCCOOCCCO TCTTCTCATT CCTTTTCCAT AATAOCTGTO TAAATCOATT CCAATTOOAA ATTOCTTTTC CAGCOACCTT AAATTGCAAG

TAAATTAATA AACTTCCATA OACTTTCOAA TTCCAACATG OCOACCGGCT GCATGTOTCT GCOCOTTCOA TTPTOCCTOO ATTOTACCCG

TTTCTCCTTC CCOTTCTCAA GCCGTTTATT CCCOAGTAGT TTCTATTOOA ATTCGCAGOC u m i n n TATCCGCGGC ATGATGOCAC

ATOOTTAOCA OATTATTTTC TTCCCCTCCA TCTCTOACOA AOTATTTTOC ATATTCTTTC CCCCTTCATT CCCATTCCTT CTTCCAATTT

OCACTTCOAT OCAAATACAA AGATTTAAAA ATOGCATCCA GOAAAATCOG CAAOTOAAAC TOTCACTOOG OTAOAAAATA AATCACAACG

CCCTOCAOTT CTCGCCGTCT CTTTCCCTTC CTTCTCTOCA TGACCAGCAA GTGCACTOCO CCCGTTCOCC GTCCCTTTCT CTCCCOCTCT

CTCCATCTCC CTCTACAOTT TTTCACCCTT TGOAATCGCG OOATTTTCOC COCACOACCC CCACCOAATC CCGATOCTTT TOOCCATTTC

CCTTTOOATT TTCTTCCACC GTGCTGCGAA AOTTGCCAAA TTTCGGCATT TCQACA111U OCTTAATTOA AATCCGTTTO GGTGTOCOAT

TTTCATTOGT TTTCCCACTA AAAACGCCGG CCOOCACATT TTCOCCATOC ACTOCCOCAC TTCCCOOCTT TCCOACOAOO U111CTCTTC

OGCTTAATCC TCTCCAOCCG AGGAGAGTGC ATTTTCCCAO TACOCACACT TCOOCTCCAT TCOTTTCTGT CT1WGOCTCO TTATTGATTT

TTCGCCCOGT GCACTTCGGC AGAGGATATA CACOOCAOTC TTTAACCAAC AGACACTTOG CCCOGTCOTO OTCCOOCTOC AGAGTACGGA

AOATCCGCAT AGAGTTTAAA AACTOCCATT TTTATGACAA CGATTTCCTT CTAATTCTAO OATATAOCOT COCOTOOOTT TOTOATCAOT

1 6 2 1 TTCTAAGTCC GCCAOTTGCC OAOTAATAAG AAACTCTAGA AAOTCTCOTO AAAACAG )TO A 0 I 1 U I I . H . CTTGTAAATT CTTGCTGCAT

AOATTTOTOO OCAAAAATAT TATGGOAATA TOOOTOTATT TCTCAATCGT ACA

OTATTTATAT TTTTCCTATT ATTCAGTATA AOOCTTAATT TOAACTAATT OOTAAACTTT TCGCGTOATT TTCGTCTTTA CTCTTOAATT

OTTTAAAATT CGTATTTTCO AAATATAAAA OTTCAACOOT TTTCCCTGTC TACGTTTOTO CCOTCCOTAT OAAOTOTOCT 1"1TUU1\.T<.G

CCACCACGAT GACACGACCC ACAGCATACA OACOTCACTC GTCTOCACCA CCCA1TAAGT TCAGACCCAC ATTOOCATGC TACCTCCCCO

ACTACGGAAA CCACCCACTT TGCTCATCCO AATACCTOCA TCCCTTCTGT CTCCCAC:AG CTCTAAAAAA TAGCTTAATC TGCAAGG

2 1 5 1 ATG OOC AAO GAG AAG ATC CAT ATT AAC ATT GTG GTC ATT OGC CAT OTO OAC TCC GGC AAG TCO ACO ACC ACC GGCK.t Glr Lr> Olu Lra I l« l i a I l « kmo I I . V.I V.I I I . GlT His Val Asp l . r Glr Lrs » .r Tbr Tbr Tbr Oly

2333 CAC TTO ATC TAC AAA TOC GGC GGC ATC GAC AAO CGT ACO ATT GAG AAG TTC OAG AAO OAG GCC CAG GAA ATO GGAH i s L«u U s Trr Lrs Crs Olj Clj U s Asp Lrs Arg Thr I I . Olu Lrs Ph. Olu Lrs Olu Ala Oln Glu Hat Olr

3 3 « « AAA OOC TCC TTT AAO TAC OCT TGO CTA CTO GAC AAG CTG AAO OCA GAG COO CAG CGO COC ATC ACC ATC GAC ATTL T S G l r Ssr Pha Lra Trr Ala Trp Val Lau Asp Lrs L«u Lrs Als Olu Arg Olu Arg Glr I l a Thr I l a Asp I l a

33*3 OCC CTA TOO AAO TTC GAG ACG TCC AAO TAC TAT OTO ACC ATC ATC OAT OCC CCT OOT CAC AOO OAT TTC ATC AAOAls Lau Trp Lra Ph. Glu Thr l ar Lrs Trr Trr Vsl Thr U s U s Asp Als Pro Olr l l s Arg Asp Fha U s Lrs

2431 AAC ATO ATT ACC GGT ACC TCT CAO OCC OAT TOT GCG CTG CTG ATC GAC GCC OCC GOA ACT OOA GAO TTC OAO OCCAsn H a t I la Thr Glr Thr far OlD Ala Asp Crs Ala Val Lau Ila Asp Ala Ala Olr Thr Olr Glu Pha Olu Ala

2 3 3 3 GOO ATC TCO AAO AAC OOC CAO ACC COC CAO CAC GCC CTT CTO OCA TTC ACG CTO OOC OTO AAO CAO CTT ATT OTOGlr I la far Lrs Asa Olr Oln Thr Arg Glu His Ala L*u L«u Ala phs Thr Lau Olr Val Lrs Gin Lmu Zla Val

26«I OOC OTC AAC AAG ATO GAC TCC ACT OAO CCO CCO TAC AGC OAO GCC CGC TAC OAO GAG ATC AAO AAO OAO GTG TCCOlr Vsl Aso Lrs Hat Asp Isr Thr Oln Pro Pro Trr Ssr Olu Ala Arg Trr Olu Olu I la Lrs Lrs Olu Val Ssr

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2613 TCC TAC ATC AAG AAG ATC OGC TAC AAT CCC GCC»ar Tyr Il« Lya Lya Ila Gly Tyr Ajn Pro Ala

2751 ATC CTC CAO CCO TCC OAC AAO ATC CCC TCC TTCHat L«u Olu Pro far Olu Lya Hat Pro Trp Pba

2133 AAC TCC TTC ATC CAC CCO CTC OAC OCO ATC CTTLys Cya L«u Ila Aap Ala Lau Aap Ala Ila Lau

2908 CAC CAC CTC TAC AAC ATC CCA COC ATC COA ACCCln Aap Val Tyr Lya Ila Cly Oly lit Oly Tbr

TCO OTC CCC TTC OTO CCC ATC TCC CCA TCC CAC CCC CAC AATfx Val Ala Pba Val Pro Ila far Gly Trp Hla Gly Aap Aan

AAO OCA TCG TCC CTC OAO COC AAG GAA COC AAC CCA OAC CCCLya Oly Trp lar Val Clu Arg Lya Olu Oly Lya Ala Glu Cly

CCA CCC CAO COT CCC ACC OAC AAC CCC CTC CCC CTG CCC CTCPro Pro Cln Arg Pro Tbr Asp Lya Pro Lau Arg Lau Pro Lau

CTA CCA CTA COT COT CTO OAO ACT CCT CTC CTC AAC CCA CVal Pro Val Oly Arg Val Glu Thr Cly Lau Lau Lya Pro

2*11

3071

3161

3251

3341

3431

GTAAGCCTCC

GGGCTGGCAT

AOCOAGACCC

ATAACCCTAG

CTCTCTCTCT

TTTCAC

GGCTTGATCA

CCAACACCCA

TCCACCTCAT

CTAACATATT

ATCCACCCAC

CC ATC OTC

GGTCGGGTCT

CCOCCTCCAA

OCACTTOCTC

AATATATOTA

CCCCATCCAA

OTC AAC m

OOOCCCTCTT

AOCOOAOCCO

AAATTCTCAC

OAOCTATTOA

ATOTCTACAC

TTCTCTTTGG

CAACGAAGTC

TCCGAAGAGC

TTCAAATAAA

ATAACCTCCC

P CCO CCO OTC AAC CTC

OCACTTCATA

TTOCOCATOT

TTCCATTTTC

AATAAATTCG

CATATGTAAC

CTC ACC CAA

CATCTATTCT

ATOCATTATT

AACATOAAAC

AGTTACTTCC

rrcorrrcoo

OCAAAATTTO

CAOCCAACCT

TGAAAGGCCA

AATAATATCG

TCOCTTCCTT

OOTCOACACT

CTTCOTCGAC

TTAAAATAAA

CTCCACOTTT

TCCCCTTTCC

OTA AAC TCT CTO CAC ATC CAC CAC

3502 CAC OCT CTC ACC OAA OCC ATO CCC GGC GAC AAC CTT CCC TTC AAC OTO AAO AAC CTC TCC CTC AAC CAC CTC CCTClu Ala L.u Thr olu Ala IMt Pro Cly Asp Asn Val Clr Pba Aan Val Lys A«n Val lar Val Lys Clu Lau Arg

3577 CCT CCC TAT OTC OCC OCC OAT TCC AAC AAC AAT CCT CCT AOC OCA OCA CCC CAC TTT ACC CCT CAGArg Gly Tyr Val Ala Cly Aap Sar Lys Asn Aan Pro Pro Arg Gly Ala Ala Aap Pha Thr Ala Cln

GTAGCGTAAC

3653 AAACATCACA AATCTTTOAT ACTTCAACTC ATCTTTCTTT GTO ATT GTC CTC AACVal Ila Val Lau Asn

373. CAT CCO OOC CAO ATC OCC AAT OOO TAC ACT CCC GTC TTO OAT TOC CAC ACO OCO CAC ATT OCC TOC AAG TTT TCCHis Pro Oly Oln Ila Ala Asn Oly Tyr Thr Pro Val l*u Aap Cys lis Thr Ala Hia Ila Ala Cys Lys Pha lar

3111 GAO ATC AAO OAO AAG TAC OAC COC COT ACO GGC GGA ACC ACC OAA OAC OGO CCO AAO OCT ATC AAO TCC OGG GATGin Ila Lya Olu Lys Tyr Asp Arg Arg Thr Oly Oly Thr Thr Olu Asp Oly Pro Lys Ala Ila Lys lar Oly Asp

3116 OCO OCC ATC ATT GTG CTG CTO CCC AGC AAO CCO TTO TOC OTA OAO AOC TTC CAO GAG TTC CCA CCO CTO OOA COOAla Ala Ila Ila Val Lau Val Pro Sar Lya Pro Lau Cya Val Olu Sar Pha Oln Glu Pha Pro Pro Lau Oly Arg

39*1 TTC OCT GTG CCC OAC ATO AOO CAO ACC OTO OCC GTG OOC GTC ATC AAG TCG GTO AAC TTT AAA OAG ACG ACC TCGPha Ala Val Arg A«p Mat Arg Oln Thr Val Ala Val Oly Val Il« Lys Sar Val Asa Pha Lys Olu Thr Thr lar

4t3i CGC AAO GTG ACA AAA OCC OCT OAO AAO OCA CAO AAO AAO AAA TAA CTAGCCTACC ACCAGAACAA CCTCATCACTOly Lya Val Thr Lys Ala Ala olu Lys Ala Oln Lys Lys Lys •••

4111 COAACCCAAC AACAACAAAA ACAGACGGCT AOAGCAACAO CAOCAACAAC ACACAACAAC AATACACATO TCAAAATTAT AATACCCACT

42tl CGACGATCAA ATTCACACCT TOACTCCATO OCAAOAOAOA CACCAATTAC TACTATTACT AOCTOCTCOO AOAAOCOOCA OATATTAACC

42«1 OAAATCOAOC AOATTATACC CTATATAATA ACCACACGTA COATTAOCOA OOAOAOOAOC ATCAOOTOCA OCOAOOATOC CAAGOAGOAO

4311 CCCTTCCAOC CTCOCCOOOT COOTTTTOOT COCCTTCCCC OTOGTOOTCT ACTCCAOCTA TCTOAACATO TATCOTCACC GCAAGTCCTT

4471 TCOTAGGAAA CCACCCOCTA OCCACTCCOC AGAGTOGATA GOOGCCTCCO GAGCACTOCT OTAOCCCOCC CCTTCOATAT ATACTCATCT

45*1 CTAAAACTAA CCTTACACTT OATTAGCAGC CACACATCCG OTCGCATCCA CCTOTTTCOA ATCCATTTTA AACACTTTTT ATACTTTTOA

4«51 TAAOTCAAOT COOAOOCATT COATTTAAAA TCTATTOAAA TATOTAATTT CCOAATTTAO TTTTAAACCA COTCCOCOCT CCCAAAAATC

4741 CCCCOAACCO AAAAGACTAC ATTCOCOATO AATTCAAAAT TTCTCTTOAA ACCAAAAAAA ACAAATOCTT AACAAOTATT ACAAAAAAOA

4131 AATCAACATT ACACACATAA TCATOCOOTT TTTGAAAACA TTATAAATOT TTAATCOAGC CTCATTTGCA TTTOCATATT ACATAATATA

4»21 COTTA«CCAC ATOTCATCTC ATTOCCCATA ATAACCTOCA TCCTOCATAT TATACACOTT AATCTCACAC TCTOAATTTA TACAAACCOA

5»11 AOACAATTOT AACCOACACC AOAACAATTC TTOOATACAO AACATCTTCC CTTGATAAAA OATCTTTTAA ATOATCAOAA AAATAAAGGA

51»1 AOCTTAACCO TAAAATACCA CACACGAACO CCTTTTAATT OAAAAATACT TOAATATCTA TOAAOAAAAT CAATTC

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FIGURE 2Sequence of the EF - la,F2 gene region. The localization oftranscription start, polyadenylation site, intron, exon, codingand noncoding portions is denoted as in Figure 1. According tocDNA pC3 and pC4 different splice acceptor sites at position2129 and 2132, respectively, are used. Oligonucleotide primersequences are indicated by an arrow above the sequence. Notethat due to the intron the arrow representing the primer closeto the 5'end is split. A conserved sequence motif isunderlined.

Hybridization of the oligonucleotide primed cDNA of 142

nucleotides in length to an EcoRI Xbal digest of the subcloned

fragment 1 (Fig.4) and of the 5'EcoRl - Xbal fragment of cDNA

pc3 to a blot of A17 DNA containing 10 kb sequence upstream of

the F2 reading frame gave rise to hybridization signals in two

different regions (Fig.4). Extending the sequence analysis of

the genomic DNA to about 2.5 kb upstream from the translation

start and determination of the cDNA pc3 sequence resulted in

the localization of two 51 miniexons. They were separated from

the coding portion of the gene by a 0.45 kb intron at position

-27 and a 1.25 kb intron at position -114 relative to the

translational start. When compared with the length of the

oligonucleotide extension product of 142 nucleotides, cDNA pc3

was missing 1 nucleotide at the 51 end. In order to map the

transcription start of F2 to the nucleotide precisely, we

repeated the primer extension experiment with an

oligonucleotide complementary to position

-124 to -104 relative to the translational start (position 336

in Fig: 2). An extension product of 12 nucleotides (data not

shown) confirmed the result already obtained with the first

primer. In summary: EF - la,F2 is organized in five exons

separated by intron sequences of 1.24, 0.45, 0.45, and 0.08 kb

in length and extends over 4.8kb.

Stable promoter elements

The use of alternative promoter elements has been shown to be a

means of differential gene regulation. In order to find out

whether alternative promoters were also used for the expression

of EF - la genes during p_. melanoaaster development, we

performed primer extension experiments using poly (A)* RNA from

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T EE E EI I 1.2 I 1.3 I 3 .9 1

Hu B B B BB BH H

1kb

•« P

cDm 49

cDm 19

EF-1 ,F1

II

— 66

FIGURE 3Structure of the EF - lcr,Fl gene locus .Part I shows a r e s t r i c t i o n map of phage x CS1 ( %J) containingthe Fl gene. Res tr ic t ion s i t e s are shown for EcoRI (E), Hindll l

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(H), and BamHI (B). The size of the EcoRI fragments harbouringthe Fl gene is indicated in kilobases. Subcloned Hindlll andBamHI fragments used to localize exon I are numbered 1-3. Belowthe restriction site map the localization of cDNAs 19 and 49 isdenoted by horizontal lines. The complete Fl gene structure isgiven as thin line and boxed area for intron and exonsequences, respectively. The open box confines the coding part.P together with a filled triangel denotes the position fromwhich the oligonucleotide primed cDNA synthesis is started.Part II: Panel A shows the cDNA synthesis product and theindirect RNA sequencing result. Panel B shows an Ethbr stainedagarose gel of the subcloned Hindlll and BamHI fragments 1-3digested with Hindlll and EcoRI, respectively, together with anSouthern blot of this gel hybridized with the "P - labelledoligonucleotide primed cDNA. Panel C: Agarose gel separation ofthe EcoRI fragments of the genomic DNA cloned in phage X CS1.Hybridization of 32P labelled cDNA cDM 19 gives rise to thepattern shown to the right.

0 - 16 hr embryos (E), climbing third instar larvae ( L ) , 2 - 4

day old pupae (P), and of adult flies (A). Since both primers

were complementary to sequences encompassing the beginning of

the respective EF - la protein coding regions (Fig.l and 2), a

switch to an alternative promoter would have been reflected in

the appearance of cDNAs exhibiting different lengths. As can be

seen in Figure 5, the primer extension products remained of the

same length throughout development therefore indicating, that a

promoter switch is obviously not apparent during either Fl or

F2 expression. Since the radioactive label incorporated into

the cDNAs represents the relative amount of RNA available for

primer extension at the respective stage of development, our

results indicate that EF - lo,Fl RNA is present in all stages

of development, although to a variable amount. EF - la,F2 RNA,

on the other hand, is highly expressed in the pupal stage but

is also present to a lesser extend in third instar larvae and

flies. In addition to the major cDNA product of 142 nucleotides

in length a weak additional band of about 160 nucleotides

appeared possibly indicating the existence of a minor

transcription start site for EF - la,F2.

Only two gene copies for cvtoplasmic EF - la exist in

Drosophila melanooaster

E. coli or Saccharomvces cerevisiae have been reported to

contain two nearly identical gene copies of EF - la. In

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St

IBXh S B EEE Xb S S E E E

i1 kb

Pc3

Pc4

Pc21

EF-1, F2

U2 - I

D

/.o -

2 1 - • «3

02

FIGURE 4S t r u c t u r e of t h e EF - l a , F 2 g e n e l o c u s . Part I shows ar e s t r i c t i o n map o f t h e DNA c l o n e d i n phage A17 ( U ) .Res tr i c t ion s i t e s are indicated for Styl (S t ) , Sa i l (S) , Xhol(Xh), Xbal (Xb) and as in Figure 3 . 1-3 denote s subcloned

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fragments used for localization of exon 1 and 2. Primersequence, cDNA localization and resulting gene structure areshown as in Figure 3.Part II: In panel A the cDNA synthesis products are shown.In panel B,C, and D Ethbr stained agarose gels are showntogether with their Southern blot hybridization results: Bsubcloned fragment 1 digested with EcoRI and Xbal andhybridized with primer extended cDNA, C X17 digested with EcoRIand hybridized with the 5'EcoRI-XbaI fragment of cDNA pC3. Dsubcloned fragment 3 digested with EcoRI and Styl hybridized asin in C. Only the Styl site that gives rise to the hybridizingfragment is indicated.

contrary, our results for Drosophila melanoaster suggest the

existence of two different elongation factor genes. In order to

decide whether the two identified gene copies are the only ones

that code for cytoplasmic elongation factor EF - la in

Drosophila. we performed genomic Southern blot hybridization

experiments using EF - la,Fl cDNA cDm49 (Fig. 3) as a probe.

DNA was isolated from Canton S embryos, digested with either

EcoRI or Hindlll, blotted and hybridized. EcoRI digested DNA

gave rise to a 4.0 kb hybridization signal of twice the

intensity when compared to a second signal of 1.3 kb. Two bands

appeared with Hindlll digested genomic DNA (Fig 6). In each

case, the hybridizing DNA fragments could be assigned to the

map of the already cloned Fl and F2 DNA (Fig. 3 and 4). The

part of the Fl gene, for example, that is contained in cDm49,

spans a portion of the 1.3 kb and the 4.0 kb EcoRI fragments

whereas the crosshybridizing part of the F2 gene is also

F 1 F 2

E L P A E L P A

8 6 - 4 ^ ^ ^ ^ _ M.2

FIGURE 5Primer extension products obtained using Fl and F2 specificoligonucleotides (see Fig.l and 2). Poly (A)* RNA from 0-16hour embryos (E), third instar larvae (L) , two to four day oldpupae (P) and flies (A) is used.

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U 5 -

63-1 4 0 -

13-

IHtndBI Eco RI

FIGURE 6Genomic Southern hybridization with cDm49 (see Fig.3) DNA ashybridization probe. The size of the identified DNA fragmentsis indicated in kilobases.

located on a 4.0 kb EcoRI fragment. Titration experiments with

gene specific hybridization probes revealed that both genes are

present as single copy per haploid genome (34) . Therefore, we

can conclude that additional related gene copies encoding

cytoplasmic EF - la do not exist in Dj. melanoaaster•

Codon usage in the EF - la.Fl and F2 gene.

Since Fl (464 aminoacids) expression is generally markedly

stronger when compared to F2 (463 aminoacids), we examined the

codon usage of both coding regions in light of the assumption

that highly expressed genes are subject to a more extreme codon

bias (27-29). Consistent with the hypothesis, codon preference

in Fl is restricted to 44 triplets whereas in F2 55 different

codons are used (Fig. 7 ) . Moreover, if one neglects those

triplets that are used only once or twice in Fl, codon usage in

the residual 97% of the reading frame is biased to 34

triplets. This is different in the F2 gene where codon usage is

random with a slight bias against A-T richness.

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I T Fl IF2 I C Fl IF2 I A Fl IF2 I C Fl |F2 I

ITTTTITTCITTAITTC

ICTTCICTCICTAICTG

IATTAIATCIATAIATO

IGTTGIGTCIGTAIGTG

PHBPBELEULEU

LEULEULIULEU

1LEILBILEMET

VALVALVALVAL

017

e6

12•15

1120

e8

7141

22

511

e4

361

12

ie23•ie

I95

25

ITCTITCCITCAITCG

ICCTICCCICCAICCG

IACTIACCIACAIACG

IGCTIGCCICCAIGCG

SERSIRSERSER

PROPROPROPRO

THRTHRTHRTHR

ALAALAALAALA

411

e3

21750

42310

113400

1 211211

111

e6

374

111

1 411611

1

17

61191156

ITATITACITAAITAG

ICATICACICAAICAG

IAATIAACIAAAIAAG

IGATIGACIGAAIGAG

TYRTYROCHAMB

HISHISGLNGLN

ASNASNLYSLYS

ASPASPGLUGLU

11001

011011

0181

45

1212725

1 2110I 11 0

1 31 81 0111

1 41121 5142

1 61161 5129

ITGTITGCITGAITGG

ICGTICGCICGAICGG

IAGTIAGCIAGAIAGG

IGGTIGGCIGGAIGGG

CYSCYSOPATRP

ARGARGARGARG

SERSERARGARG

GLYGLYGLYGLY

0505

11400

0102

1812130

1 15 10 15 1

6 16 10 13 1

0 13 10 13 1

4 125 1104

FIGURE 7Codon usage as deduced from the nucleotide sequence of the Fland F2 reading frames.

Evolutionary stability of EF - la genes

When we compared the amino acid sequences of EF - la,Fl and F2

with all eucaryotic cytoplasmic elongation factors (Fig.8)

known to us, we observed a strong conservation of their primary

structure. Some of the highly conserved regions have already

been correlated with functional domains. The most strongly

conserved NH2 - terminal end sequence of EF - la had been

assigned to GTP binding activity. EF - la also shares homology

with several classes of nucleotide binding proteins and even

the procaryotic elongation factor genes (37,38). Another highly

conserved region further substantiated in our comparison is

comprised in the sequences around Ala 92, Lys 244 and Lys 273.

The corresponding amino acids in EF - Tu of £_,_ coli were

considered to be important for tRNA binding. This remarkable

degree of sequence conservation is most easily explained if one

considers the multifunctional nature of the EF - la protein. In

the future, cloning, in combination with site directed

mutagenesis, will open the way to design modified factors that

can be tested for single functional steps in the elongation

process.

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UXUIirYWIOHTDSOKVTTTGILIYKCWIIDU'iKEUHICTYVTIGETDSGirTTTCHLIYlCaCIDU'

IKCKIHIMIWICETDtCKnTTOHLIYZCOOIDEl'

IKEIZHIIflVVlOVTDSOESTTTOaLIYECCGIDat-iKEXnilBIVTIGHYDSGlCnTTGHLIYKCOOIDER1

I EXmXAAKLG KG!11EXTKUAAXLC EGIEXTSKXAQEKCKCIEKFKKEAQEHOKGIEXTSKKAgEHCKC

'IEXFEKEAAEKOKG

L WLOIOJUEU101'

, WIDIOJU EJLEXG I

.trVLDIOJLAXREXCI

ALtnUTTP KTOVTTI DA PCHU) r:ALVXTBTFETOVTVIDAPOBRDFAL WOXTPCnrVTV ID A PCH£D FALVXFETAXYTVTIIDAPGERDF

K HUCOK LACE

K D. HEL FIALTCFETSETTVTIIDAPGHRDFAnnrrrAxyrvTiiDAPc MOUSEILWFETfKTTVTIIDAFGlfltDFIi: HUMAfl

\xitiuiMrwiownMirrrtcuLiricGGiDiM.TittnrxxxinGKGtTirrk\riu}r&EJ&MXxoitt KYTVTIIDAPGBRDFIK COWJEHSUS

Xl^#TSOADCAILIIAOGTGEF*EAGISECCOTVXEJll<IJ^TLOVRQLITAVpijQSVK--VDESR:FÊIVKETSJ(FIUVGYHFrTVPFVPISCUVGra YEASTITVTtOADCAILIlAOGTGEFXAOISEOO^n»EBAUJirnjVIQLIYAVinaa)SVIC--WDESIFOEIVIXT««FIKEVOYHPETTFF^ YEAST

[ I T t ^ O A P C A I L I I A O C T O E r i A O I f C M O T i n t T J i i r n ^ F R Q L I T A I i n M ^ HUCO* RACEMHI TtJT»OADC ATLI'm ITCT»O AIX: 1VQ11

JCHHCDJI AJtTEHlAtGVHGDH 0 . HIL FISCWHGOD D. HEL FIItCVVGDN HOUSESOWnGDfl HUWAJI

MITCTtQADCATLI AJLCTGITEAGISnrCOTREHALLAFTLCVKOLITOVIfXKOSTEFPTII RYEItTEIVS YIKXIOYBP TTAFVPISGW CDK CtWIEHSUS

MEATTW^WYIOWEirriJLOVVKOETlAEAIDAIEQPIIFTOItFLRMFWDVTItlGClOTVPVORVrreVII^^ YEASTIUIJ^TWJmmDO¥EXITXAOVTKOKTU*EAIDAIEgF«PTDMLaiJLO^VTKIO^ YEAST

. _ TIRAGKWHFAPAAVTTEVKSVEIIHHETLT HUCOR RACE

TFAFAJIirraVUVIHHHESLE AJtTEHIA

•OTVWFAPAHITTEVKIVEJUniEALO D- MEL "HLEPSE»t»lffI0»STERXXOBJJ0ECLIDAI*OAIlJF^FTOltPLRL*LQDVTEI0OI0TVFV0RVirrGLlJCPCMVVHFA^ D. HEL F I IIl^PSAJIMPVrimnrrTljaWSlICTTUJLfclJ>CILPPT»FTOE*LJUjPLQirVTEIGOIGTVFV HOUSE

201 mjW!AJWFWIO¥ITT*HKIULS<nTUJyUJ>CIlJPTtPTDItPL*lJLO.DVT^ HUHAJI

T1JUC 0 A QETLLXALDAILPP IPTDltFLJILFLflDVTEICCICTVPVCRVITOVSITCWTVTFAPAirVTTIVlUTKHHHEAL COMSEUSUS

IUUJ*VCGDJJOrDPPIUK:MF1U^TVIYlJral^ YEAST(»VF«Dir/orilVDfVSTEEItR<nr/CODAIOrDPPi:0<ÛFl(ATTITljniPOOIIAOY«PVUKBTAflIAC^^ YEAiTEGLF^^WVGrirVDrriTEI)ItR0inrCtDfl3fDPAKE«ASrrAQTIILJrHPO0ISAGYA^VU}CHTAHlACB7SELIIKIDRRS0iaQtID8 HUCOC RACEQASFGIXVGnnrnr/tTK£IJULGTVAJDSIDnn>ARGlvrTAÎVljniPOQISirOYTPTUXntTA£UC17UIU AJtTEHIAIATP<IDirVGrirnVVfTUUUlGTVACDSIUJ^FKOAADrrAOÎTLjraP<ÎAjrOYTPTU>CaTAHIACRFUILE D. HEL FImMMrwvanfvyyvfTyTHrFWTTVft""!Fin^FB?ftAPrrAQ*r*LJ"tK^TiKnvTPTTjy'WT*HTftr|^>iTFT»>n»»^^TTTgnti>»tTt^cnAA 0 . HEL F I IEALPgCWVGrilTRjrVSfEPVtJtGyVACDSlOntT HOUIE

3*1 EALP<H»nwrinri3rVSTIPYER0MVJUlDliarDPFTiEAJ^rTA0VIIIjraFqqi RUHAM

EA PCnrVCnmanrSTKI RXOWVAODSEJfDPPKO A FTAOVTVLiniPOQmOY P T L O C H T A H I A C K F EL EK ORRSGKX ED PKF MQDAA COHSEMSUS

L VltFTP IKFMCTVAFIET F:L VIFT P f KFHC V U F • EYT:IVXKVP S UPMCVKATTDT PIHITLTPIEPLCVEAFIDTTiIVWL TP SK P LCVEAFQETH

L^ftFATRDtaOTVAVOTIUVD-KTXKAAJCVTIUUkQXJLAKK*'URFAVR^taOTVAVOVIUVD-ETEKAAJCVTIUUQCAJUCE-'LOEFAVRCTttOTVAVCVIEAVE-KTDEAGKVTtAAArAIIlI'

' AVRDMRQTV AVOTIMYWT KBTTSGEVTILAUKAQICU •

IV VPSEFKCTKAFSEYPPLORFATROtaOTVAVOTIUT KJC AOKVTTUU EA K

YIASTHUCCHt RACEARTEHIAD. HBL FI

D. HEL F I IHOUSEHUMAN

COHSSMSUI

FIGURE 8Comparison of the aminoacid sequences deduced from cloned EF -la gene sequences (11-19). For mouse only part of the sequenceis known (17,18). Asterisks denote identical aminoacids, pointsindicate a conservation of at least 50% between the differentproteins,

CONCLUDING REMARKS

cDNA clones were isolated that span the complete mRNA sequences

of the two n^ melanogaster elongation factors EF - la (FI and

F2). Using these clones in addition to oligonucleotide primer

extended cDNA probes, we were able to characterize miniexons

encoding the 5'end of the untranslated leader of both EF - la

mRNAs. By comparison of cDNA and genomic sequences the gene

structures could be determined. In contrast to the two nearly

identical genes present in fij. coli or Saccharomvces cerevlsiae.

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Drosophila contains two copies that are clearly different from

each other with respect to sequence and structure. Even though

it is not proven yet that both messages are actually translated

in vivo, we detect both sequences in the high molecular weight

fraction of polysomal gradients at their respective time of

expression indicating active translation (Richter and Hovemann,

unpublished result). The amount of cDNA synthesized using Fl-

and F2 specific oligonucleotide primers reflects the extent of

RNA expression in the various developmental stages. According

to this expression profile, Fl should represent the

housekeeping gene that gives rise to the elongation factor

needed in all growing cells. F2 transcription, peaking in pupal

stage, represents on the other hand an elongation factor gene

that is specifically expressed in certain developmental stages,

possibly,even in a tissue specific manner. It is also

conceivable that EF - lo,F2 exerts a specific function by

preferring its own pool of aminoacyl tRNAs. This would then

mean that it would by itself be involved in some kind of

translational control.

The complete gene structures allow us now to perform a

comparison at the nucleotide level. The coding region of Fl and

F2 differ in sequence between each other to a degree that is

similar when compared with the Artemia salina or the human

gene (15,16). We, therefore, conclude that the genetic

separation of Fl and F2 is not a recent event. Since both genes

established themselves with their unique expression profile,

they could express independent functions. A stage specific EF -

la like activity was also observed in Xenopua laevis

previtellogenic oocytes (35,36). However, a molecular analysis

of the oocyte specific activity is still missing.

Promoter regions of housekeeping function genes have been

characterized of being devoid of the TATA motif (31). We did

not recognize the TATA box sequence in either one of the two

EF - la genes. Still, there is little sequence homology at all

in front of the transcription start of Fl and F2. A comparable

promoter motif, however, that is common to housekeeping genes

is not yet established. Since EF - la belongs to the group of

proteins that are engaged in protein synthesis, we searched for

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common sequence motifs that might be indicative for the

concerted regulation of genes encoding translation factors and

ribosomal proteins. In the yeast Saccharomvces cerevisiae a

general promoter enhancer motif, the HOMOL box (32) , has been

identified in front of the EF - la and several ribosomal

protein genes. We find strong homology to this motif 373

nucleotides in front of the EF - la,Fl gene (position 258 in

Fig.l). To our knowledge this is the first case that the yeast

consensus sequence has been observed at the right distance in

front of this group of genes in Drosophila. A sequence homology

between Fl and F2 that might also be worth mentioning is

located at the end of the intron that is preceeding the

translational reading frame. The sequence of 12 nucleotides at

position 1566 in Figure 1 and position 1797 in Figure 2 is

completely conserved. In light of the highly divergent

surrounding this consensus sequence is very unlikely to have

evolved by accident.

EF - la,Fl transcription strength is comparable with that of

the induced vitellogenin I gene (33). The usage of a limited

number of codons in the Fl message, therefore, supports the

hypothesis of a codon bias for such highly expressed genes. In

Drosophila heat shock exerts a strong transcriptional and

translational control. Translation of nonheatshock message is

restrained by a slow down of initiation and elongation (34).

The availability of both elongation factor genes will allow us

to address questions regarding the possible involvement of one

of the EF - la factors in this regulation phenomenon.

Acknowledgment

We are grateful to T. Kornberg for making cDNA libraries

available to us. We would like to thank I. Sures for helpful

criticism in the preparation of this manuscript. This work was

supported by the Deutsche Forschungsgemeinschaft (SFB 229/C3).

S. Richter is recipient of a G.F.G stipend of the Land Baden-

Wurttemberg.

REFERENCES1 Walldorf,U., Hovemann,B., and Bautz, E.K.F. (1985) Proc.

Natl. Acad. Sci. USA SZ, 5795-57992 Kaziro, Y. (1978) Biochim. Biophys. Acta 505. 95-127

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3 Drews,J., Bednarik,K., and Grasmuk,H. (1974) Eur. J.Biochem. 41, 217-227

4 Kemper,W.H., Merrick.W.C., Redfield,B., Liu, C.-K.,andWeissbach,H. (1976) Arch. Biochem. Biophys. 174. 603-612

5 Nagata,S., Motoyoshi,K., and Iwasaki,K. (1978) J. Biochem.fil, 423-429

6 Slobin,L.I., and Holler,W. (1978) Eur. J. Biochem. Q£, 69-77

7 Murakami,K., and Miyamoto,K. (1982) J. Neurochem. 38.1315-1322

8 Lauer,S.J., Burks,E., Irvin,J.D., and Ravel,J.M. (1984) J.Biol. Chem. 252, 1644-1648

9 Thiele,D., Cottrelle,P., Iborra,F., Buhler,J.-M.,Sentenac.A., and Fromageot,P. (1985) J. Biol. Chem. 260.3084-3089

10 Crechet,J.-B., Canceill.D., Bocchini,V., and Parmeggiani,A.(1986) Eur. J. Biochem. 1£1, 635-645

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Two genes encode related cytoplasmic elongation factors 1α (EF-1α) in Drosophila...

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