Gene, 104 (1991) 47-54 0

1991 Elsevier

GENE

Science

Publishers

B.V. All rights reserved

47

0378-l 119/91/$03.50

05087

Expression of lad (Recombinant

DNA;

gene fusions affects downstream transcription in yeast Saccharomyces

cerevisiue; URA3; promoter

Christine A. Barnes a, Gerald C. Johnston” Departments

of” Microbiology,

b Biochemistry,

occlusion;

heat shock)

and Richard A. Singer bTc and

’ Medicine, Dalhousie University, Hal$ax, Nova Scotia, B3H 4H7 (Canada)

Received by J. Gorman: 9 October 1990 Revised/Accepted: 25 March /18 May 1991 Received at publishers: 7 June 1991

SUMMARY

Chimeric genes containing Escherichia co/i IacZ sequences are often used to characterize gene expression in yeast cells. By Northern analysis, we found that such genes produce multiple transcripts due to inefficient 3’-end formation. The same transcript pattern was found for two related chimeric genes when these genes were cloned separately into the commonly used vector, YIPS, and integrated into the yeast genome at two different locations. Each chimeric gene was composed of promoter and N-terminal coding regions from the yeast SSAI or SSA2 genes fused in-frame to the lac operon. Transcripts were shown to initiate within the yeast promoter fragment, but transcript size indicated that 3’ ends were localized to three different regions: within the luc operon near the 3’ end of the IacZ gene; near a terminator region previously identilied upstream of the URA3 gene in YIPS; and at the URA3 terminator region. Readthrough transcription of the URA3 promoter from upstream lac sequences decreased the basal activity of the URA3 promoter, although induced URA3 transcription levels were unaffected. This readthrough transcription also resulted in a novel, longer URA3 transcript.

INTRODUCTION

Fusions of yeast genes to lac operon sequences derived from the bacterium Escherichia coli have considerable utility in the study of gene expression. These gene fusions usually involve the IacZ gene, leading to the synthesis in yeast cells of hybrid mRNA molecules containing sequences from both yeast and ZacZ DNA. Many gene-fusion transcripts encode chimeric proteins that retain the /?Gal activity of the native IacZ gene product, a situation that simplifies

Correspondence co: Dr. R.A. Singer, Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, B3H 4H7 (Canada) Tel. (902)494-8847;

Fax (902)494-1355;

E-Mail ‘[email protected]’.

Abbreviations: aa, amino acid(s); /?Gal, B-galactosidase; bp, base pair(s); Hsp, heat-shock protein; kb, kilobase or 1000 bp; nt, nucleotide(s); ORF, open reading frame; r, ribosomal; S., sedimentation transcription start point(s); ::, novel joint (insertion).

constant;

rsp,

measurement of the activity of the yeast promoter driving the chimeric gene (Rose and Botstein, 1983a). Transcript abundance for genes such as these is often quantified by nuclease protection or primer extension, procedures that do not display full-length transcripts. Here we present a preliminary characterization by Northern analysis of the transcript pattern for a typical type of 1ucZ chimeric gene in the yeast Saccharomyces cerevisiae. Two chimeric genes were studied, with the same IacZ sequences fused to two related promoters and N-terminal coding sequences (Ellwood and Craig, 1984). Complex transcript patterns were found: several regions specifying mRNA 3’-end formation in yeast were identified within and beyond Iac operon sequences, leading to multiple transcripts. Furthermore, the ratio of long to short transcripts within this transcript family was affected by mild heat shock. These fusion transcripts had unexpected effects on neighboring gene expression: transcriptional readthrough from the chimeric gene inhibited basal promoter activity of the

48 flanking URA3 gene, and led to a novel URA3 transcript, perhaps by activating cryptic promoter sequences found in the plasmids.

RESULTS

transcript initiation sites identified by Ellwood and Craig (1984). These findings suggest that, for each transcript that contains lac sequences, transcription is initiated within the SSA2 promoter fragment that is fused to the lac operon (Fig. 5).

AND DISCUSSION

The transcript

patterns

in yeast cells were determined

for

two related 1acZ fusion genes. Each fusion gene had been assembled in the same way: the E. coli lac operon containing a truncated ZacZ gene, lacking a promoter and the first seven codons, was fused in-frame to a yeast promoter and N-terminal coding sequences (Ellwood and Craig, 1984). The promoters were from the SSAI and SSA2 genes, two members of the yeast Hsp70 heat-shock gene family (Ingolia et al., 1982; Werner-Washburne et al., 1987). We integrated these fusion genes along with flanking lacYA and plasmid-borne URA3 sequences into the yeast genome by homologous recombination at different sites (Fig. l), a situation that proved useful in this analysis. (a) Multiple ZucZ transcripts from SSAZ-ZucZ Total RNA from two SSAZ-1acZ integrants was analyzed by Northern hybridization, using the 1acZYA probe shown in Fig. 1A. This probe hybridized to transcripts containing lacZ, lacy or 1acA RNA, and was specific for transcripts from integrant strains (data not shown). Surprisingly, the transcripts of the fusion genes identified by the ZacZYA probe were of three distinctly different sizes (Fig. 2). The patterns were virtually identical for a single plasmid integrated at the ura3-52 locus (Fig. 2B) and for tandem SSA2-1acZ plasmids integrated elsewhere (Fig. 2A). Each transcript was polyadenylated, as evidenced by Northern analysis of poly(A) + RNA (data not shown). Two additional probes were used to characterize the transcripts from the SSAZ-1acZ fusion genes (Fig. 1A). One probe was derived from vector sequences upstream from the fusion gene, and indicated the 5’ limits of transcripts. The other probe was derived from URA3 sequences, and was used to identify transcripts from the plasmid-borne URA3 gene and the chromosomal ura3-52 locus. The transcript from the ura3-52 locus was relatively unaffected by most manipulations used here (Fig. 4A), and thus provided an internal control for RNA levels. None of the three ZacZYA-hybridizable transcripts (see Fig. 2) hybridized with the upstream vector probe (Fig. 3). Those transcripts that were identified with this upstream probe were also found in untransformed cells (Fig. 3, lane 3), or contained pBR322 sequences expressed in yeast (Marczynski and Jaehning, 1985). Therefore, all three lac transcripts most likely originated within the SSA2 promoter fragments fused to the lac operon, presumably at the

(b) Multiple sites for 3’ -end formation Figs. 2A and 2B show the transcript

pattern

from the

integrated SSAZ-1acZ fusion gene for cells grown at 23’ C. The smallest transcript hybridizing to the 1acZYA probe, as seen earlier for a similarly constructed 1acZ fusion gene (Clements et al., 1988), was approx. 3.4 kb. A transcript of this size could encode the entire E. coli flGa1 protein of 102 1 aa and the additional 10 aa encoded by the SSA2 gene that are included in this fusion gene (Ellwood and Craig, 1984). The size of this transcript indicates that sequences that can function as 3’ processing (transcript cleavage and polyadenylation) signals in yeast cells lie within the E. coli lac sequences, near the C terminus of the 1acZ coding region. Various sequences have been proposed to direct 3’-end formation in yeast cells. A search of the lac operon sequence (GenBank) indicated that one of these proposed terminator elements, 5’-TTTTTATA (Henikoff et al., 1983; Henikoff and Cohen, 1984), is not found in these lac fusion sequences. A second proposed terminator element is encoded by the multipart sequence element TAG . . . . . TAGT . . . . . (A + T-rich). . . .TTT (Zaret and Sherman, 1982). A variation of this sequence (TAAG . . . TATGT . . . [ 16 of the next 20 nt either A or T] . . . TTT) is found 40-80 nt beyond the C terminus of the 1acZ gene; the remainder of the IaL DNA contains no other sequences that resemble as closely this proposed 3 ’ -end formation signal. Another suggestion is that 3’-end formation, at least for the CYCl gene, relies on the A + T-richness (80%) surrounding this multipart element (Osborne and Guarente, 1989). The suggested region of 3’-end formation in lac DNA is also A + T-rich (78% A + T over 77 nt). In any case, termination within 100 bp downstream from this putative element in lac DNA, as found for the CYCI gene (Zaret and Sherman, 1982; Russo and Sherman, 1989), would result in a transcript the size of the smallest fusion-gene transcript (Fig. 2). The longer lac fusion transcripts (Fig. 2; Clements et al., 1988) indicate that this region is only partially effective at specifying 3’-end formation. The size of the second transcript (7 kb) suggests that it terminates downstream from the 1acA gene. This transcript did not hybridize to the URA3 probe, which contains URA3 N-terminal coding sequences but does not contain sequences from the URA3 promoter (Fig. 4). Thus the 7-kb transcript may terminate at a 3’-end site that has been shown to lie within the cloned URA3 promoter (Yarger et al., 1986), at an appropriate distance downstream from

49

Yip102

ura3-52

C URAZ: lNTEGRATED[URAJ]

5

2

7

1

1

Fig 1. Integration of pZA102

of SSA24acZ

(kindly supplied

et al., 1979). The SSAI-IacZ

plasmid

gene SSAZ are fused in-frame contained

plasmids.

to the IacZ gene (Ellwood

gene are fused in-frame

and Craig,

fusion-gene

by E.A. Craig, University

and Craig,

YIplOO was derived

to IacZ (Ellwood

1984). In each case, the upstream onlyLEU2

(Panel A) The SSA2-IacZ of Wisconsin),

DNA (Casadaban

21R (MATa adel leu2-3,112 (Orr-Weaver

SSA2 or upstream

genes boxed, plasmid

sequences

flanking

ditype (PD) tetrads,

chromosomal

locus and the vector-encoded

C) Integration

ofSmaI-cleaved

genomic

DNA resolved

fragment.

Untransformed

two bands

of approx.

confirmed

that plasmid

YIplO

uru3; Johnston sequences

two nonparental

through

sequences

analysis

as sawtooth

(NPD), showed

initiation

immediately

tandem

band, indicating

were tightly linked to ura3-52 (data not shown).

upstream

indicated

in panel A. The vector probe was a 2.8-kb PstI fragment

The three probes

Hsp70 (Ellwood

from the chimeric

with plasmid

DNA linearized

YIplO

(TT), indicating

to direct

cleavage

are diagrammed ofApaI-cleaved

VRA3 locus and the integrated no linkage

(Feinberg

and Vogelstein,

between

used to detect

plasmid

YIplO

RNA from vector, sequences

at

UK43

the URA3 (Panel

blot ofBamHI-digested

1983) 0.9-kb ApaI-PstI ZJRA3 at ura3-52.

(lane 1) showed Genetic

at uru3-52 of a single copy of YIplOO, linearized

that contains

luc DNA downstream

integration

with

with the

vector-borne

1984), while an SSAZ-IacZ transformant

single-copy

gene

analysis.

copies of YIplO2 at a single locus (data not shown).

Integration

verified (data not shown).

containing

Hsp70

YIp5 (Struhl

of transcription

both the ura3-52 and lJRA3 alleles, was shown by the Southern

13.8-kb plasmid

fragment

were verified by restriction

lines. (Panel B) Integration

and seven tetratypes

1% agarose and probed with a radioactive

IacZYA fragment

lJRA3 plasmid

either at the SmaI site in URA3, or by partial

of the chromosomal

ApaI site within URA3, was similarly

same ApaI-PstI URA3 fragment

direct the correct

1982) were transformed

an 11.6-kb URA3 band (Rose and Winston,

16 kb and 9 kb and no unit-length

The IacZYA probe was a 6.2-kb EamHI-Sa/I

of the integrative

al., 1986). Constructions

was linearized

pattern

ditypes

UR.43 gene. Physical

21R (lane 2) contained

under the plasmid sequences

fragment

of yeast LEU2 origin. The two forms of integrated

the segregation

by ligating the EcoRI-Sal1

from the ATG) from the SSA2 cognate

(E.A. Craig), in which nt -499 to + 285 from the heat-inducible

and Hopper,

et al., 1981). YIplO

at ura3-52, which recreates

by electrophoresis

sequences

similarly from pZAlO0

as thin lines, and chromosomal

a site other than was-52 was shown by genetic analysis: gene to yield five parental

was constructed

EcoRI site used was within the LELI2 gene, and sequences

recombination

relevant

1984), with the large EcoRI-Sal1

et al., 1983; Ellwood and Craig, 1984; see Uemuraet

integration

ApaI within plasmid-borne

YIplO

in which nt -755 to + 30 (numbered

and Craig, 1984). These SSA2 and SSAZ sequences

Cells of S. cerevisiae strain by homologous

plasmid

2

analysis at the

IucZYA, and (IRA3 sequences upstream

are

from the SSA2 promoter.

from the site of gene fusion. The URA3 probe was the

as used above.

the fusion-gene promoter. The sequence of this particular region does not resemble any of the proposed 3’-end formation signals, and is only 63 y0 (70/l 11) A + T-rich (Yarger et al., 1986). The sequences in this region are also only partially effective at specifying 3’-end formation, as indicated by the presence of an even longer SSAZ-IacZ transcript. Certain reported results (Fig. 5 of Yarger et al.,

1986) also suggest incomplete transcription termination in this region. A third transcript (8 kb) hybridized to both the lucZYA and URA3 probes, and therefore most likely terminates at the 3’ end of the URA3 gene. This region is relatively A + T-rich and contains the putative terminator element 5’-TTTTTATA (Rose et al., 1984) but not the multipart

50 I

A

SSA2 - /acZ

B

I

not at ura3 Temp. Time

Fig. 2. Transcripts at 23°C to a density tryptophan, amounts

indicated

( min )

Gaithersburg,

32

37

23

32

32

37

23

32

32

37

-

30

60

15

-

30

60

15

-

30

60

15

(all at 40 ng/ml) plus adenine

fusion genes. Total RNA was isolated

to GeneScreen

membrane

and uracil(20

(New England

Nuclear,

integrant

(Johnston

(Li et al., 1985) from cells that had been grown overnight

et al., 1977) supplemented

pg/ml), or after incubation

at an elevated

at 32 or 37°C for the indicated

resolved by electrophoresis

on the left margin. (Panel A) Cells with the SSAZ-IucZ

cells; (panel C) ura3::SSAl-1acZ

medium

at 23°C or incubated

MD), were separately

at ura3

32

and SSAI-IacZ

of total RNA from cells growing

SSA7 -/acZ

at ura3

23

of 2-4 x lo6 cells/ml in YNB glucose-based

and tyrosine

Laboratories, transferred

of the SSA2-lad

( ‘C )

C

SSA2 - /acZ

Boston,

through

1.2% agarose

MA), and probed

plasmid tandemly

integrated

times,

histidine,

2 M formaldehyde

fragment

leucine, lysine,

for the indicated

and RNA size markers

containing

with the IacZYA

with arginine, temperature

time. Equal

(Bethesda (Maniatis

(Fig. 1A). Transcript

Research et al., 1982),

sizes in kb are

at a site other than uru3; (panel B) ura3::SSA2-1acZ

integrant

cells.

terminator element. Readthrough of this region of 3’-end formation to the limited extent seen at high transcript levels (Buckholz and Cooper, 1983) would not be evident in this analysis. The transcript pattern for the SSAZ-1acZ fusion gene is summarized in Fig. 5. (c) Thermally induced changes in transcript abundance Elevated temperatures caused significant changes in the relative abundances of the three transcripts from the chimeric SSAZ-1ucZ fusion genes. This effect is best seen after transfer of cells to 37°C. This 37°C heat shock had little effect on the total abundance of the three transcripts, in keeping with the limited induction of SSA2 transcript seen by nuclease protection and primer extension (Ellwood and Craig, 1984). However, the amount of 3.4-kb transcript increased, and that of the 8-kb transcript decreased, relative to the abundance of the 7-kb transcript (Fig. 2A, B). The lower temperature of 32°C elicited similar but less pronounced changes in relative transcript abundance (as well as a surprising decrease in SSA2 promoter activity; Fig. 2). The longer fusion transcripts may be selectively destabilized by heat shock. A selective destabilization is seen upon heat shock for transcripts encoding r-proteins

(Herruer et al., 1988), but there is no evidence for a similar selective destabilization of other transcripts (for example Herrick et al., 1990). Alternatively, there could be increased efficiency of 3’ -end formation at elevated temperatures; this interpretation is prompted by the effects of readthrough transcription on URA3 expression, as described below. (d) Similar transcripts from SSAl-1acZ The transcript pattern was also determined for the SSAl1acZ gene. This chimeric gene is analogous to the SSAZ1ucZ gene described above, except that the promoter and N-terminal coding sequences were derived from the SSAl gene. This Hsp70 gene is expressed at low basal levels at 23’ C and is induced upon heat shock (Ellwood and Craig, 1984; Slater and Craig, 1987) so that transcript patterns were more readily assessed for this gene after transfer of cells to elevated temperatures (Fig. 2C). For SSAI -1acZ the pattern of transcript abundance at 37’ C was similar to that for the SSAZ-1ucZ gene (Fig. 2). Therefore, the pattern of transcripts is not affected by the promoter used to direct transcription. The three transcripts of SSAl-1ucZ were coregulated: the thermal induction of these transcripts mirrored the induction of mRNA from this gene fusion as

51 12

B

SSA2 - IacZ

SSA2 - IacZ

not at ura3

kb remp. rime

6.7-

/_

)

23

32

32

37

( min

)

-

30

60

15

Integration

Site

ura3

Uracil

-

Elsewhere +

-

+

255

2.71.7-

( Y

1

185

l.O0.8-

1.2-

is,*

1.0

-

0.6

-

._A I

1

2

0.6

3

1

initiate within cloned promoter

integrants

(lane 2), and untransformed fragment

Total RNA was extracted

vector

Fig. 4. Transcripts

of URA3. (Panel A) The blot shown in Fig. 2A was stripped integrated

(Fig. IA). Transcript

fragments.

cells (lane 3) growing

with the upstream

Transcript

at uru3 or elsewhere

sizes in kb were determined

sizes and positions

(not at ura3) growing

kb

Fig. 5. Transcripts

3

4

ia-

“!?A3

of the SSA-IacZ

as in Fig. 2 from ura3::SSAl-1uc.Z

at 23°C. Total RNA resolved of the rRNA

bands

and reprobed with or without

by electrophoresis

integrants

(lane l),

as in Fig. 2 was probed

are indicated.

with the VRA3 probe (Fig. 1A). (Panel B) Total RNA from added

uracil was similarly

probed

for URA3 sequences.

as in Fig. 2.

kb SSA2

2

Fig. 4.

Fig. 3. SSA-1acZ transcripts

cells with SSAZ-IacZ

m

4

Fig. 3.

uru3::SSA2-lacZ

-

and URA3 genes.

shown on the left for transcripts

initiated

and on the right for transcripts

of URA3 (Fig. 4).

Sizes in kb are

at the SSA2 promoter

(Fig. 2)

measured by primer extension and nuclease protection (Ellwood and Craig, 1984). This finding provides evidence that all three transcripts of SSAI-ZucZ, and by extension SSAZ-IacZ, originated from the SSA promoter sequences of the fusion genes. Furthermore, the sizes of the transcripts suggest that transcripts from both fusion genes were cleaved for 3’-end formation at the same sites. In particular, the SSAI sequences fused to IacZ are 0.25 kb longer than the fused SSA2 sequences (Ellwood and Craig, 1984) and, as

expected, the 3.4-kb transcript from the SSAI-IacZ fusion gene migrated somewhat more slowly than the analogous transcript from the SSAZ-IucZ fusion gene. This decreased migration also resolved the SSAl-1ucZ fusion-gene transcript from the position of 28s rRNA, dispelling any notion that the 3.4-kb band resulted from trapping by the abundant rRNA. (e) Promoter occlusion at URA3 At 23 “C the major species of SSAZ-IacZ mRNA was the 8-kb transcript, which includes the URA3 gene (Fig. 4). Under these conditions, the usual l.O-kb URA3 transcript (Buckholz and Cooper, 1983; McKnight and McConaughy, 1983) was virtually undetectable (Fig. 4A, lane 1). However, the l.O-kb URA3 transcript was of much greater abundance after shift to higher temperatures (Fig. 4A, lanes 2-4). This increased abundance of the l.O-kb URA3 transcript was correlated with decreased

52 abundance of the 8-kb SSAZ-1acZ transcript that extends into URA3 (Fig. 4A), suggesting that readthrough transcription from the upstream fusion gene may inhibit URA3 promoter activity. Under other conditions transcription from the URA3 promoter took place despite continued readthrough transcription. Induction of the l.O-kb URA3 transcript, along with a parallel induction of the 0.6-kb transcript from the ura3-52 gene (Nakayama et al., 1985) was seen during growth in uracil-free medium, even though the levels of 8-kb transcript, and consequent readthrough transcription, remained unchanged (Fig. 4B). The transcription of URA3 is activated from basal levels by the PPRl gene product (Loison et al., 1980; Losson and Lacroute, 1983; Losson promoter activity et al., 1985). The PPR I -mediated indicated in uracil-free medium is therefore resistant to this form of promoter occlusion, suggesting that it is the PPRlindependent (Losson et al., 1985) basal activity of the URA3 promoter that is inhibited. Analogous effects have been described for other promoters. For example, the promoter activity of a solo 6 element inserted upstream from HIS4 inhibits the nearby HIS4 promoter (Silverman and Fink, 1984). However, this effect is better termed promoter competition, because cisacting mutations in the downstream HIS4 promoter that increase promoter activity also decrease the activity of the 6 promoter inserted upstream (Hirschman et al., 1988). In E. coli cells, transcription from the pL promoter of a 2 prophage continues into bacterial DNA and interferes with the gal promoter far downstream (Adhya and Gottesman, 1982). For avian retroviruses, transcription that continues into a long terminal repeat of the virus decreases transcription initiation within that long terminal repeat (Cullen et al., 1984); similarly, for plasmid-borne genes arranged in tandem the downstream promoter is inhibited by transcription extending from the promoter of the upstream gene (Proudfoot, 1986; Kadesch and Berg, 1986). The mechanism of promoter occlusion may be through disruption of DNA-protein complexes, exemplified by the displacement of bound transcription factors from the occluded downstream promoters of tandemly arranged rRNA genes (Bateman and Paule, 1988; Henderson et al., 1989). (f) A novel URA3 transcript The decreased abundance of basal-level l.O-kb URA3 transcript due to promoter occlusion was accompanied by an increased abundance of a 1.2-kb URA3 transcript (Fig. 4A). This longer URA3 RNA has only been detected in these SSA-facZ transformants (data not shown), and therefore may be a feature of these plasmids. The 1.2-kb transcript was prominent only under conditions of abundant 8-kb SSA24acZ mRNA, and was unaffected by PPRI activation of the URA3 promoter (Fig. 4B).

Transcription through the URA3 terminator is unlikely to account for this longer transcript. Although such extended transcription has been seen under conditions of markedly increased URA3 expression, it accounted for only 8 y0 of the URA3 transcript (Buckholz and Cooper, 1983) while virtually all URA3 transcript was altered by readthrough from the upstream SSAZ-IacZ promoter (Fig. 4A). The 1.2-kb URA3 RNA seen here is also smaller than the 1.5-kb transcript resulting from transcription through the usual region of 3’-end formation (Buckholz and Cooper, 1983). The longer URA3 transcript was also independent of sequences downstream from URA3 (Fig. 4B). Immediately downstream from the sites of URA3 3’ ends (Buckholz and Cooper, 1983) are vector sequences in one integrant, while in the other integrant URA3 is flanked by normal chromosoma1 sequences (Fig. 1A); nevertheless, the same longer URA3 transcript was seen in each case. These observations all suggest that the 1.2-kb transcript did not result from 3’-end formation beyond the usual URA3 terminator region. Therefore, the additional sequences in this longer RNA are probably derived from transcription upstream from the usual region of URA3 transcript initiation. The transcript size locates the 5’ end near the junction of URA3 5’-flanking sequences and pBR322-derived vector sequences (Struhl et al., 1979; Fig. 5). The 1.2-kb URA3 RNA could be a 3’ cleavage product left over from the production of the 7-kb transcript. However, transcript cleavage and transcript termination in yeast are usually closely associated (Osborne and Guarente, 1988; Snyder et al., 1988; Russo and Sherman, 1989) suggesting that after 3’-end formation at the 7-kb site any residual elongation of the remaining transcript downstream from the 7-kb site would not be extensive enough to produce a 1.2-kb RNA. In addition, 3’ cleavage products produced by mRNA 3’-end formation in yeast have not been detected directly and are thought to be unstable, perhaps because they would not have a 5’ triphosphate that would permit RNA capping (Banerjee, 1980). Circumstantial evidence therefore suggests that this longer URA3 transcript is probably not a 3’ cleavage product, although this interesting idea has not been ruled out. Alternatively, the longer URA3 transcript may result from the activation, by readthrough transcription from the SSAZ-ZacZ promoter, of a cryptic promoter upstream from the plasmid-borne URA3 gene. Inspection of the URA3 sequence (Rose et al., 1984) suggests that a URA3 transcript containing sequences upstream from the usual tsp, such as the 1.2-kb transcript, would not be translated to yield URA3 protein because of additional AUG start codons in the 5’-extended transcript. Two ATGs are found within 225 nt upstream from the URA3 ORF, and at least one of these upstream ATGs, encoded in a minor URA3 transcript, has been shown to initiate translation (Rose and Botstein, 1983b). Because

53 neither of these upstream ATGs is encoded in the major family of URA3 transcripts (Rose and Botstein, 1983b; Losson et al., 1985), they do not affect translation of the usual l.O-kb URA3 mRNAs. In a 5’-extended RNA, however, these 5’ AUGs would be preferential sites of translation initiation (Sherman and Stewart, 1982; Donahue and Cigan, 1988). Both of these start codons are out of frame with respect to the URA3 ORF, which is itself immediately preceded by an in-frame stop codon. Therefore, an upstream AUG, by specifying translation initiation at an inappropriate site, could inhibit URA3 gene expression from a 5’-extended mRNA. Because of these structural considerations, the functional significance of the 1.2-kb URA3 transcript was tested by altering the exogenous uracil supply. In uracil-containing medium, in which URA3 gene expression is dispensible, the integrants studied here contained the 1.2-kb URA3 transcript and low levels of l.O-kb URA3 transcript. Under uracil-free conditions, however, the URA3 promoter was activated to produce signi~~ant levels of 1.O-kb transcript, even though the 1.2-kb transcript was still present (Fig. 4B). Such activation of URA3 is typical of cells starved for uracil (Rose and Botstein, 1983b). This finding suggests that induction of the l.O-kb URA3 transcript was necessary to supply adequate URA3 enzyme activity, which in turn suggests that the 1.2-kb transcript of the same region does not provide URA3 function.

Council of Canada. C.A.B. was supported by an MRC Studentship.

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