Identification of an Artemia franciscana retropseudogene containing part of the last exons of the sarco/endoplasmic reticulum Ca-ATPase-encoding SERCA gene

Gene, 149(1994)377-378 0 1994 Elsevier Science B.V. All rights reserved. 0378-l 119/94/$07.00

377

GENE 08329

Identification of an Artemia franciscana retropseudogene containing part of the last exons of the sarco/endoplasmic reticulum Ca-ATPase-encoding SERCA gene* (Crustacean; molecular cloning; nucleotide sequence; retrosequence; pseudogene; SERCA)

Ricardo Escalante and Leandro Sastre Instituto de Investigaciones Biomedicas de1 CSIC, C/ Arturo Duperier 4, 28029-Madrid, Spain

Received by G. Bernardi: 30 May 1994; Revised/Accepted: 7 July/l1 July 1994; Received at publishers: 4 August 1994

SUMMARY

A genomic clone has been isolated which contains sequences highly homologous to part of exon 14 and exons 15, 16 and 17 of the Artemiufranciscuna sarco-endoplasmic reticulum Ca-ATPase(SERCA)-encoding gene, but none of the introns. The homologous region extends to the 3’ end of the mRNA, although the poly(A) tail is not present. The structure of this clone suggests that it represents a S-end-truncated retropseudogene (rY).

The crustacean Artemia franciscana contains a single SERCA gene in those cDNA (Palmer0 and Sastre, 1989) and genomic (Escalante and Sastre, 1994) clones that have been isolated and characterized. Screening of the genomic libraries allowed the isolation of a positive clone, gArATCa16, whose restriction map (Fig. 1) did not overlap with that of the other genomic clones isolated for this gene. A MI-Hind111 fragment from this clone was the only one hybridizing with SERCA cDNA clones and was cloned in pUC18 for further characterization and sequencing (Fig. 1A). The nt sequence of this fragment (Fig. 1B) showed the existence of a region of DNA highly homologous to SERCA cDNA clone, pArATCa604, from nt 1503 to the 3’ end of this clone (nt 2675), except for the poly(A) tail (Escalante and Sastre, 1993). This region extends from the end of exon 14 to exons 15, 16 and 17 of the de Investigaciones Correspondence to: Dr. L. Sastre, Instituto Biomtdicas, C/ Arturo Duperier 4, 28029-Madrid, Spain. Tel. (34-l) 585-4626; Fax (34-l) 585-4587. *On request, the authors will supply experimental evidence for the conclusions reached in this Brief Note. Abbreviations: A., Artemia; bp, base pair(s); kb, kilobase or 1000 bp; nt, nucleotide(s); rY, retropseudogene; SERCA, sarco/endoplasmic reticulum Ca’+-ATPase; SER CA, gene (DNA) encoding SERCA. SSDI 0378-1119(94)00540-O

previously characterized SERCA gene (Escalante and Sastre, 1994). The structure of this genomic clone suggests that it codes for a 5’-truncated SERCA rY. We have not been able to identify any repeats at the ends of the rY but the absence of repeated sequences has been also described for other retrogenes (Bernstein et al. 1983). The comparison of the nt sequence of the rY with that of SERCA cDNA clones (Fig. 1B) shows the existence of a number of scattered point mutations at 4.4% of the nt residues. The mutation frequency is similar in protein coding and 3’ untranslated regions, as would be expected for a non-functional rY. Some of these mutations introduce in frame stop codons in the coding region of the cDNA. A high proportion of these (27%) are C-T transitions in the rY. Other frequent changes are T-C (15%), T-A and G-A (12% each). Besides these point mutations, there are a few deletions and insertions. The biggest deletion in the rY comprises 27 nt (between nt 875 and 876) and corresponds to a direct repeat present in SERCA cDNA and genomic clones that can be seen just upstream from the deleted fragment. The presence of retrogenes has been described for many mammalian genes but the number of retrogenes seems to be much smaller in other organisms such as

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INTRON

POSITION

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EXON

birds or insects (Weiner et al. 1986). The rY described here is the first one found in crustaceans, showing their existence in this group of animals. Since the number of genes characterized in Artemia is small (Marco et al. 1991), the finding of a rY might indicate that they are not rare in these animals. However, the study of many other genes is required to determine if retrogenes are frequent in crustaceans, as found in mammals or are as scarce as in insects and other animals.

NUMBER

B cl%&Z%ZTr TATUVLWLCT m_cAA A_ lcexc%?Al.-Z -cc c-CT.& -31 -l%T_lTM~cc~occ *mVx c-m TET&lcAlcT cAcccAcloo 37 1503 ....... .......... ..c ....... .......... 107 AeccocTrTf MTmTAcC =-CA -cTrcM ?cITAcAccTc __A _T .......... .......... .......... ................ ..T. ..T....... .......... *llGs-mTITy: Ac&cccm cm T&--xc ADTZ,_Tc ACT&_ 177 .......... .......... .......... ............ ..* ..... T......... .......... mm= cAm_ TIOTC- WT cm?!A&: cm _TA%W 247 .......... .......... .......... .................... ......... . .......... Ge_VZrrTGeC~~VxC TIT-eA- VZiVZ&Zm _P.!rrcVZT ATOTCOIVVLT &Zm-&?r 316 ..C...... ............. . ..c ...... ........... .......... ..C..T. .......... GrrrlVC~ TlToTrcccT G&ZA!r- wu_% mGACT&Z_ 386 .C...A.... .................. . ..o ....... .......... ..c ....... .......... TpcTAo(ITvL -mCTlYTCA -TAT= CamAn? CCGeTTAGW CATAn.l-lVx 456 .......... .......... .......... .......... .......... .... ..+.... .......... ZsmeP.Ta:a;rcrroTeeTeculOar ClTlrmm BTAT rr=mccc -AT&& 526 ..A...CT. .................... G......... ........... .......... .......... loMcoAmTliOOMCrn An%+AWCcT ml.cxc cm ZvZ.?&.-VZ rr_A 596 .......... .......... ................ ..c. ... ..c ........ ..c ... .......... s m-m GmAsm ATTpATTTluL AILrr?c=CCGWUCAM 665 ........... ..T...... .......... .......... ............. ..- .... ........i. TOAIC- m=CATZT ArrliclwLhT -.4.%m AAMTMm CT-m& AC-II% 735 .......... ...... ..T. .......... .T........ CLCIU .......... ..A.. .A...T.... sNe,eAToT T-m CATR1CGTin OToaTGM TAAT.sscTIcO CrrCAACcAT msmGTAT805 .......... .......... .......... .c .......... .......... .......... ..AT.... Icc?VRc=luumTcA=-= ACT__ AMWATAIT c&&G-WA RA_TAsA 875 .......... .......... .......... .......... .......... .... ..c ... .......... -----_-___ __________ _______mXmweM TpAluAAoAT OTILULMTAT m 918 c .......... TTATATrGAs GTAAWMT wm ......... T..... . ................ &TTIcCTTIIT m= ~CAlUl%Gc TIIThZD?.i?% TcAeN?V1T CrnAMOCT fixm=m 988 ..T....... ..T............. c ......... T..T...... G........... . .......... xGesse_ -TilQI wMTITITrlG _% McTrm TTACWTGT 1058 .......... .O..... ..T G-........ .......... .......... .......... .......... m= &mAT- CCT_hn;TATrl.Zc_ TCT-TFTTAT AT mc CcAmA 1127 . .......... ........... ..c ...... ................... .......... .......... IUVLTAAMIY: TTAollTlTA TA_ AcclyTcAAc a=ATAm AcAMTCN.?LCT TATl.l%%xcT 1197 .. ..- ............ ..2675 ATMTAAIUIT TAeccVZAAG cG&?%Mcx: B B TceAMm Oc~TrMllV 1267 AT%ZE:TM Acel?&%he TMTMrrlT n\rr%.ZGVZ kcT=TKx TATluhMM AC0 1330 Fig. 1. Restriction map and partial nt sequence of the phage h clone gArATCa16, containing a Artemia franciscana SERCA rY. (A) The hgArATCa16 was isolated from an A. franciscana genomic library, using SERCA cDNA clones as probe, as described previously (Escalante and Sastre, 1994). The restriction map of the clone was determined by single and double digestions with EcoRI (E), Hind111 (H) or Sal1 (S) and the SalI-Hind111 fragment hybridizing with the cDNA was cloned into the plasmid vector pUC18. The diagram shown under the restriction map of the phage represent the position of the rY as a box. The numbers above the box indicate the first and last nt of the cDNA clone pArATCa604 that are homologous to the rY. The hatched box represents protein coding regions of the cDNA and the black box the 3’ untranslated region. The positions of the introns in the corresponding genomic clones is indicated by arrows under the diagram box. The number of the corresponding exons are also indicated between the intron arrows. (B) The nt sequence of rY was determined by the dideoxy chain-termination method (Chen and Seeburg, 1985) using a Applied Biosystem 373A Sequencer and the Taq Dye Dideoxy terminator Cycle Sequencing Kit. The nt sequence of the rY is compared with that of SERCA cDNA clone pArATCa604 (Escalante and Sastre, 1993) that is placed underneath. The rY sequence has been numbered from the first nt homologous to the cDNA. Identical nt are indicated by dots and deletions by dashes. The first and last nt of the cDNA that are homologous to the rY (nt 1503 and 2675) are indicated. The two nt surrounding each intron position are underlined (nt 62-63; 196-197 and 313-314 of the rY). The three nt homologous to the cDNA stop codon are also underlined (nt 521 to 523 of rY). This nt sequence will appear in the EMBL database under accession No. X79331.

ACKNOWLEDGEMENTS

This work was supported by a Grant from the Direccibn General de Investigation Cientifica y Tecnica (PB92-0076). R.E. is the recipient of a pre doctoral fellowship from the Comunidad Autonoma de Madrid.

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