Gene, 127 (1993) 145-148

0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-I 119/93/$06.00 145

GENE07016

Analysis of 5’ flanking sequences from the Schizosaccharomyces pombe cdc2 gene

(Cell cycle; fission yeast; mRNA promoter; TATA box; cut assay)

Anna K. Meddins”, Paul Nurseb and Kathleen L. Goulda3b

“Department of Cell Biology, School of Medicine, Vanderbilt University, Nashville, TN 37232, USA; and bCell Cycle Group, Microbiology Unit, Department of Biochemistry, University of Oxford, Oxford OXI 3QU, England. Tel. (44-0865)275296

Received by D.T. Denhardt: 5 November 1992; Accepted: 9 November 1992; Received at publishers: 29 December 1992

SUMMARY

Previously, the sequence of the Schizosaccaromyces pombe cdc2 gene was reported to begin at a Hind111 site, 141 nucleotides (nt) upstream from the ATG start codon [Hindley and Phear, Gene 3 1 (1984) 129-1341. We have extended the sequence of the 5’ untranslated region of the gene to a PstI site at -822 nt. We demonstrate by primer extension analysis that transcription of the gene initiates at one major point 180 nt upstream from the ATG start codon. Since the 822-nt fragment extending from the PstI site to the start codon has been used in many studies as the promoter for cdc2 [Booher and Beach, Mol. Cell. Biol. 6 (1986) 3523-3530; Carr et al., Mol. Gen. Genet 218 (1989) 41-49; Gould and Nurse, Nature 342 (1989) 39-451, we investigated the strength of this promoter element relative to the SV40 early promoter, a promoter known to work very well in S. pombe [Jones et al., Cell 53 (1988) 659-6671. We confirm that the cdc2 gene fragment has significant promoting activity, albeit 20-to 60-fold less than the SV40 early promoter, when assayed in S. pombe.

INTRODUCTION

The cdc2 gene encodes a 34-kDa protein kinase which controls both the initiation of DNA replication and the entry into mitosis in S. pombe (reviewed in Norbury and Nurse, 1992). In this organism, the cdc2 protein kinase and the mRNA encoding it are present at constant levels throughout the cell cycle and during exit from the cell cycle (Durkacz et al., 1986; Simanis and Nurse, 1986). The activity of cdc2 appears to be regulated solely at the

Correspondence to: Dr. K. L. Gould, Department of Cell Biology,

School of Medicine, Vanderbilt University, Nashville, TN 37232, USA.

Te1.(615)343-9502; Fax(615)343-4539.

Abbreviations: bp, base pairs(s); CAT, chloramphenicol acetyltranfer-

ase; cat, gene encoding CAT, cdc, cell division cycle gene; cdc2, S. pombe protein kinase encoded by cdc2; CDC2, human protein kinase ana-

logous to cdc2; kb, kilobase( nt, nucleotide(s); oligo, oligodeoxyribo-

nucleotide; ORF, open reading frame; S., Schizosaccharomyces; SV40, simian virus 40; tsp, transcription start point(s).

post-translational level by phosphorylation and subunit interactions in S. pombe (Norbury and Nurse, 1992). This constrasts with the regulation of cdc2 homologues in higher eukaryotic cells. In mammalian cells, the abun- dance of the CDC2 mRNA does vary during the cell cycle and as cells exit and re-enter the cell cycle (Lee et a1.,1988; McGowan et al., 1990; Dalton, 1992; Welch and Wang, 1992). This variation in abundance is mediated in part by changes in the rate of CDC2 mRNA transcription (Dalton, 1992).

A 3.4-kb PstI fragment from the S. pombe cdc2 locus is able to complement temperature-sensitive cdc2 mutants when present on a multi-copy plasmid (Booher and Beach, 1986; Carr et al., 1989; Gould and Nurse, 1989). This genomic fragment contains the coding region, a 3’ untranslated region (3’ UTR), and presumably a pro- moter. Although there is no detectable transcriptional regulation of the cdc2 gene (Durkacz et al., 1986), we have analyzed the region 5’ of the ATG start codon in this

146

gene fragment for the presence of transcriptional control elements since it appears to constitutively promote cdc2 mRNA production.

The cdc2 gene was sequenced beginning at a Hind111 site 141 bp upstream from the ATG start codon (Hindley and Phear, 1984). The aims of the present study were to (I) extend the sequence of the cdc2 locus upstream to a PstI site at -822 nt, to (2) map the tsp of the cdc2 mRNA by primer extension, and (3) to compare the relative pro- moter strength of this region of the cdc2 gene with the SV40 early promoter in S. pombe.

EXPERIMENTAL AND DISCUSSION

(a) Sequence analysis of cdc2 promoter element The region of the cdc2 locus that contains the coding

region has been reported previously (Hindley and Phear, 1984). We extended this sequence upstream from the ATG start codon to a PstI site at nt -822 to include possible transcriptional control elements (Fig. 1). As expected, this region did not contain any significant ORFs. The sequence was analyzed for the presence of reported eukaryotic transcriptional control sequences (Locker and Buzard, 1990). Seven potential TATA boxes were located at positions -103, -232, -289, -455, -581, -687 and -813. No exact match to any other promoter or

enhancer element described (Locker and Buzard, 1990) was detected. The possibility remains that novel elements are located in this region and/or that recognized or unrec- ognized transcription control elements might be present more than 822 nt upstream from the ATG start codon.

(b) Location of the tsp

The tsp was determined by primer extension. Previous studies had indicated that transcription of the cdc2 gene initiated upstream from the Hind111 site in a region not previously sequenced (P. N., unpublished data). There- fore, an oligo (number 1274) that anneals 3’ of the Hind111 site was chosen for primer extension (see Fig. 1). With this oligo as the primer, there appeared to be a single tsp corresponding to nt - 180 (Fig. 2). A mRNA leader sequence of 180 nt is considered typical for S. pombe; the distance between the tsp and the ATG start codon is generally less than 200 nt (Russell, 1989). A putative TATA box lies 53 nt upstream from the tsp (Fig. 1). The distance from the TATA box to the tsp ranges from 34-46 nt in several other S. pombe genes (Russell, 1989).

(c) Promoter activity The upstream PstI-Hind111 fragment of cdc2 has the

ability to promote transcription of the gene to levels ade- quate for complementation of temperature sensitive cdc2

mutants (Booher and Beach, 1986; Carr et al.,

PstI Sau3A Him11 XmI ssp1

I I I I I CTGCAGAAATATAAGATG4~A~A~C~TGTGT -822

HinfI/PleI HinfI TthlllII

I I I GTAATAGTTATTATITATATTAACTTITTRTACCTTT'X TCCTICGCAATGCTFITTATTITITRT ATGACE'ETAACI'IYXGACATACACACGGA'TTCTGCITG~

DraI BspMI I I

?TATAAGTG~ATA~A~A~A~~T~~~ GAGCAGGIEGAGAAGTACAGTmTACAA'I?TCACAA~ACATlTAW'ITAAAGG

ssp1 We1 I I

G'lTAG'KTATZUATATl'AGACAACTCAGGATAA'lWAACCTACCGTAAA'lTTATlTATIT?&TAATAGCACTITFIX ACl'ZATATl-CACATTTACAATI'lTIT AA'I'EACGGTTATACA

Mb011 Mb011 BclI/Sau3A/HphI I I II I

ATGTCTITAGATGATGI?TTG ATI'ATGTAG- TATCTGATCACCTAGTATA4ATKXAAGTAATGAG'ITA-iGTCCTACTTI-EI AAT-lTCA~GATAACA#=~ATA

Ha&I NheI We1 MnlI I I v I

spe1 Hind111 I I

AG~AG~G4TGAAGCGCTA~C~~G~~~AT~TAG~~~G~CA~AG~TA~~AG~AGC~G~ACCCACAG~C~CAC~CAT~GC~~T

Sau3A SfaNI I I

ATAAACGTA~AGATCA'ITCTCGC4TCTCTATTAA

1989;

-703

-583

-463

-343

-223

-103

+3

Fig. 1. Nucleotide sequence of the 5’ untranslated region of the cdc2 from a PstI site to the ATG start codon. The nt sequence of the PstI-Hind111

fragment was determined on both strands by the dideoxynucleotide method using Sequenase (US Biochemical, Cleveland, OH, USA) and a series of

oligo primers. The positions of primers corresponding to the DNA strand shown are indicated with a line above the sequence and the positions of

primers corresponding to the complement of the sequence shown are indicated by underlining. The tsp is indicated with a downward arrowhead. A

putative TATA box 53 bp upstream from the tsp is boxed. Both the sequence determined in this study and the sequence determined previously (from

the Hind111 site) (Hindley and Phear, 1984) are shown. The sequence will appear in the GenBank/EMBL Nucleotide Sequence Databases with the

accession No. LO7304.

147

L

Fig. 2. Primer extension. An antisense oligo, No. 1274, (S-AGA- GATGCGAGAATGATCT) corresponding to nt -70 to -89 (Fig. 1) was hybridized to a single-stranded cdc2 DNA template and extended in the presence of [a35S]dATP using DNA polymerase. The hybrid DNA was cleaved with Hind111 and the labeled 70-nt fragment was purified away from the template DNA by electrophoresis through a denaturing 6% polyacrylamide gel. Following elution from the gel and ethanol precipitation, this ?+labeled 70-mer was annealed to 6 pg of S. pombe poly(A)+RNA for 1 h at 50°C. Deoxynucleotides and reverse transcriptase were then added to initiate the extension reaction as pre- viously described (Jones et al., 1988). The extension mix was extracted with phenol:chloroform, ethanol precipitated and analyzed by denatur- ing 8% polyacrylamide gel electrophoresis adjacent to a dideoxy sequencing reaction primed with oligo number 1274. The position of the extension product corresponding to tsp at -180 nt is indicated with an arrow (and an arrowhead in Fig. 1). The position of the primer is indicated with a heavy dot on the left margin.

Gould and Nurse, 1989). To determine at what general level this promoter fragment is active, its activity towards the reporter gene, cat, was compared with the SV40 early promoter.

The pTRSV40 vector (Toyama and Okayama, 1990) was used for examining relative CAT activities. pTRSV40 contains polyadenylation signals both upstream from the cat gene driven by the SV40 early promoter and down- stream from the cat gene (Toyama and Okayama, 1990).

ac-Cm

ac-Cm

Cm

origin

Fig. 3. Activity of the PstI-Hind111 cdc2 gene fragment. CAT activities provided by the pTRSV40 vector (SV40), pTRASV40cdc2 vector (cdc2), and the pTRASV40 vector (none) are assayed as conversion of [14C]chloramphenicol (Cm) to its acetylated forms (ac-Cm). Methods: The SV40 promoter in pTRSV40 (Toyama and Okayama, 1990) was replaced by the PstI-Hind111 fragment of cdc2 as follows. The SV40 early promoter was removed from pTRSV40 by cleaving with BamHI, filling in the BamHI site and cutting with HindIII. After cleavage with PstI, blunt-ending, and cleavage with HindIII, the cdc2 gene fragment was ligated to the pTRASV40 vector fragment. The pTRASV40 vector was also blunt-ended after cleavage with Hind111 and ligated to itself to create a promoter-less version of the vector. The three vectors, pTRSV40, pTRASV40cdc2 and pTRASV40 were transformed into an S. pombe strain with the ura4-D18 mutation and Ura+transformants were selected. Protein extracts were prepared as described (Toyama and Okayama, 1990) and protein concentrations were determined using the BCA protein assay kit (Pierce, Rockford, IL). CAT assays were initially performed using 10 pg of total protein and analyzed by thin-layer chro- matography, as described (Gorman et al., 1982). More or less protein was used in subsequent experiments to accurately quantitate the results.

The additional polyadenylation site upstream from the promoter prevents transcription read-through from start points elsewhere in the vector. pRTSV40 also contains a S. pombe ars and the Saccharomyces cerevisiae URA3 gene (Toyama and Okayama, 1990). To test relative pro- moter activities, the SV40 promoter was removed from pTRSV40 to produce a promoterless vector and was also replaced with the PstI-Hind111 cdc2 fragment.

CAT activity following transformation of the three

148

vectors into S. pombe cells was measured from lysates of pooled transformants in two separate experiments. A typ- ical CAT assay using 10 c(g of S. pombe protein extract from each transformation is shown in Fig. 3. To obtain accurate measurements of promoter efficiencies, addi- tional experiments were performed with 30 ug of protein to bring the lower activities into the linear range (> 10% conversion to acetylated forms). We found that the c&2 promoter fragment had ten- and 30-fold greater activity than the promoterless construct but 16- and 60-fold less activity than the SV40 early promoter. Although there are stronger promoters (Toyama and Okayama, 1990), the SV40 early promoter is an efficient promoter in S. pombe (Jones et al., 1988). Thus, the activity of the cdc2 gene fragment is significant but should be considered rela- tively weak.

ACKNOWLEDGEMENTS

The authors thank Dr. Stuart MacNeill for providing oligo 1274, Dr. Sergio Moreno for S. pombe poly(A)+RNA, Dr. Tony Weil for supplying the pTRSV40 vector, and Drs. MaryAnn Thompson and Steve Hann for advice on CAT assays. This work was supported by awards from Boehringer-Ingelheim Pharmaceuticals, Inc., the Lucille Markey Charitable Trust, and the Searle Scholars Program of the Chicago Community Trust to K.L.G. and the ICRF and MRC to P.N.

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