259 Gene, 138(1994)259%260

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:<SDI 0378-1119 (93) E063h-R

GENE 07620

Nucleotide sequence corrections of the ~~~~ open reading frame encoding P-glucuronidase

(Plant reporter gene; GUS intron; transgenic gene expression)

Helmi R.M. Schlaman, Eddy Risseeuw, Marry E.I. Franke-van Dijk and Paul J.J. Hooykaas

Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, 2333 ALLeiden, The Netherlands. Tel. (31-71) 274700

Received by A.J. Podhajska: 23 June 1993; Revised/Accepted: 1 September/3 September 1993; Received at publishers: 1 October 1993

SUMMARY

Part of the open reading frame of uidA, encoding ~-glucuronidase, was sequenced and two differences were found with the previously reported nucleotide sequence [Jefferson et al., Proc. Natl. Acad. Sci. USA 83 (1986) 8447-84511. One is a silent mutation, the other results in the Gl~~‘~+Gln substitution.

The ~scherjchia coli uidA gene, coding for B-glucuronidase (GUS), is a widely used reporter gene in expression studies in higher plants (Jefferson et al., 1987). GUS is very stable and it has the advantage that most plants have little or no endogenous activity, and relatively easy assays make it possible to detect the enzyme in intact tissues, as well as to measure activities quantitatively (Jefferson, 1987). However, both methods require a dis- ruption of the material. Therefore, a chimaeric gene of uidA and nptll, coding for Km resistance, was con- structed recently (Datla et al., 1991). Transgenic plants carrying this fusion gene can initially be selected on media containing Km and expression of the gene can be visua- lised subsequently by performing GUS assays.

In the course of manipulating the uidA/nptZZ fusion gene (Datla et al., 19911, a 498-bp PCR fragment was amplified using standard technics ranging from nt 577 to nt 1075 in the uidA ORF (Fig. 1). Subsequent sequencing of three independent clones with the PCR product cloned

Correspondence to: Dr. H.R.M. Schlaman, Institute of Molecular Plant

Sciences, Leiden University. Clusius Laboratory, Wassenaarseweg 64,

2333 AL Leiden, The Netherlands. Tel. (31-71) 274-833; Fax (31-71)

274-999.

Abbreviations: aa, amino acid(s); bp, base pair(s): GUS, B-glucuro-

nidase; kb, kilobase or 1000 bp; Km, kanamycin; nptll, gene encoding

neomycin phosphotransferase II; nt, nucleotidefs); ORF, open reading

frame; PCR, polymerase chain reaction; aidA, gene encoding GUS.

in pIC2OR (Marsh et al., 1984) revealed two differences between the actual sequence and that previously reported (Jefferson et al., 1986): i.e., C?35+G and A903-+G. The first change results in a change of codon replacing GIu~‘~ by Gln in the resulting protein. The other difference is a silent mutation (Leu). None of the newly found differ- ences result in novel restriction sites.

To exclude that the differences found were the result of PCR artifacts, three other plasmids carrying the uidA gene were sequenced. These were pB1426 (a kind gift of W.L. Crosby), carrying the uidA/n~~ZZ fusion gene in pUC8, which was the template for the PCR reaction, pMOG416 (a kind gift of MOGEN Int.), carrying a gene which encodes the so-called GUS-intron (Vancanneyt et al., 1990) as a Hind111 fragment in PBS (Stratagene, La Jolla, CA, USA), and pB1221.1, harbouring the uidA gene under control of the CaMV 35s promoter in pUC19 (Jefferson, 1987). All these plasmids were obtained from independent sources and were proven to encode an enzy- matically active GUS protein. The same two differences in nt sequence were also found in these three plasmids. Additionally, in the gene encoding GUS-intron the BcEI site at the beginning of the ORF (Fig. 1) was absent due to a A92+G substitution. This results in a replacement of Asp 31 by Gly in the encoded protein. Apparently, this has no influence on the enzyme activity (Vancanneyt et al., 1990).

260

ATGTTACGT&TGTAGAAA&CCA?+CCCG+GAUTC M L R PVBTPTREI

~~TCGACGGCCTGT~GCATTC~ATCGCG~CTGT~~~CGTT~~~T~~GCGCGTTAC~ KKLDGLWAFSLDRENCGIDQRWWB S A L Q

GAAROCCGGG~TTGCTGTGCCAGGCAGTTT~~CGAT~GTTCGCCGATG~GATA~CGT~TTATGCGffi~CGT~~TATCAGCGCG~GTCTTTATACCG~~TTG~~ BSRAIAVPGSFNDQFADADI RNYAGNVWYQR 6 V F I P K G W A GGCCAGCGTATCGTGCTGCGTTCGATGC~TCACT~TTAC~~GTGT~T~T~T~GG~GT~AT~AGCATCA~C~CTATACGC~TTTG~GCCGATGTCACGCCG G Q R I VLRFDAVTHYGKVWVNNQBVM E H Q G G Y T PFBADVTP TATGTTATTGCCGGGAAAAGTGTACGTATCACCGTTTGTGTG~~CG~C~~CTGG~GACTATCCCGCC~~T~TGATTACCGACG~C~C~G~G~GTCTTAC YVIAGKSVRITVCVNNBLNWQTI PPGMVITD ENGKKKOSY TTCCATGA~TCTTTAACTATGCCGGAATCCATCCATCGCAGCGT~TGCTCTACACCACGCCG~~CCT~TGGACGATATCACCGT~TGACG~TQTCaCQC~A~TMCCACQCQ FHDFFNYAGI HRSVMLYTTPNTWVDDITVVTHVAQDCNHA

TCTGTTGACTGGCAGGTGGTGGCCAATGGTGATGTCAGCGTTGA?, CTGCGTGATGCGGATCAACA~T~TTGGACARGGC SVDWQVVANGDVSVELRDADQQVVATGQGTSGTLQVVNPH

~ff~C~CC~TG~~~ATCT~ATG~~GTGCGTCACAGC~ *

GCCAGACAGAGTGTGATATCTACCCGCTTfGCGCGTC~CATCC~TCAGT~~GTG~~CG~CAG L W Q PGBGYLYELCVTAKSQ TECDIYPLRVGIRSVAVKGEQ

* TTCCTGA~~CCACAAACCGTTffACTTTACTGGCTTT(;GTCGTCATG~GATGC~ACTT~CGT~~~ATTCGAT~CGTG~GAT~TGCACGACCACGCATT~T~A~~ FLINiiKPFYFTGFGRHEDADLRGKGFDNVLMVHDHALMDW

ATTGGGGCC~CTCCTACCGTACCTCGCATTACCCTTACCCTTACGCTG~GAGATGCTCGA~~CAGATG~CAT~~TCGT~TQA~A~~C~CTQC~TCQQC~MCCTCTCT I GANSYRTSHY P Y A B B M L D WAD 6: H G I VV I DE TAAV G F N T. S

AACCACCCAAGCGTGGTGATGT~AGTATTGC~CG~CC~ATACCCGTCCGC~~TGCAC~~TATTTCG~CCACT~~~G~CGCGT~CTCGACCCGACGCGTCCG NHPSVVMWSIANBPDTRPQGARE YFAPLAEATRKLDPTRP ATCACCTGCGTCAATGTAATGTTCTGCGACGCT~CACC~TACCATCAGCGATCTCT~GATGTG~GTGCCTG~CCGTTATTAC~T~TATGTCC~GCGGCGATT~CG I TCVNVM FCDAHTDTI SDLFDVLCLNRYYGWYVQSGDLBT

GCAGAGAAGGTACTGGAAAAAGAACT~C~~~~A~~G~TCAGCCGATTAT~T~CCG~TA~CGT~ATACGTTAGCC~~GCA~~TGTACACCGACATG AEKVLBKELLAWQEKLHQPII ITKYGVDTLAGLHSMYTD”

TGGAGTG~GAGTATCAGTGTGCATGGffM;ATATGTAT~CCG~T~GATCGCGT~GCGCCGT~T~T~~~TAT~~T~CGCCGA~TGCGACff~C~~ATA WSEBYQ CAWLDMYHRVPDRVSAVVGEQVWNFADFATSQGI

TTGCGCG~~C~T~~G~~TCTTCACTCGCGACCGCAAACCGAAGTCGGCG LRVGGNKKGIFTRDRKPKSAAFLLQKRWTGMNFGEKPQQG

GGCAAACAATGA 1811 G K 0 f 603

120

40

240 80

360 120

480 160

600 200

720 245

840 280

960 320

1080 360

1200 400

1320 440

1440 480

1560 520

1680 560

1800 600

Fig. 1. The nt sequence of the uidA ORF coding for GUS as reported by Jefferson et al. ( 1986) including the corrections on the sequence as reported

by Farrell and Beachy (1990) which are indicated by the underlined sequences. The two nt corrections are indicated by asterisks above the sequence,

The sequences in bold faces correspond with the two primers which were used for the amplification of the PCR product and which were subsequently

used for the sequencing of different plasmids carrying uidA sequences. The &II site is indicated by the boxed sequence. The star at the end of the aa

sequence marks the stop codon.

Both the results presented here and those of Farrell

and Beachy (1990), who also published a correction on

the sequence of the GUS ORF (Fig. 1 ), indicate the neces-

sity to m-determine published sequences now and then,

ACKNOWLEDGEMENTS

We thank Jiirgen Groet for technical assistance. This

work was supported by The Netherlands Foundation of

Technical Sciences (STW) with financial aid from the

Netherlands Organisation of Scientific Research (NWO).

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