A mini-promoter lacZ gene fusion for the analysis of fungal transcription control sequences

Gene, 158 (1995) 119 123 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50

119

GENE 08884

A mini-promoter lacZ gene fusion for the analysis of fungal transcription control sequences (Recombinant DNA; lacZ reporter gene; 13-galactosidase; targeted single-copy integration; transcription start point(s); gpd box; carbon regulation; nitrogen regulation)

Peter J. Punt, Anneke Kuyvenhoven and Cees A.M.J.J. van den Hondel Department of Molecular Genetic:; and Gene Technology, TNO Nutrition and Food Research Institute, 2280 HVRijswijk, The Netherlands

Received by J.R. Kinghorn: 26 May 1994; Revised/Accepted: 20 January/23 January 1995; Received at publishers: 28 February 1995

SUMMARY

A system for the in vivo analysis of fungal transcription control sequences, based on a mini-promoter, was designed. The mini-promoter, providing all sequences necessary and sufficient for transcription initiation, was derived from the Aspergillus nidulans gpdA promoter region. Transcription initiation was not affected by the introduction of transcription control sequences directly upstream from the mini-promoter. Furthermore, the expression of the mini-promoter was not affected by wide-dom~fin carbon or nitrogen control circuits. Using the mini-promoter vector, a previously identified upstream activating sequence from the A. nidulans gpdA gene was further characterized.

INTRODUCTION

The study of fungal gene expression is primarily aimed at an understanding of the molecular mechanisms of transcription regulation in these organisms. Research is largely focused on two aspects of gene regulation, namely (i) on the analysis of DNA sequences involved in transcription initiation and/or regulation, and (ii) on the characterization of regulatory proteins interacting with these sequences (for reviews, see Davis and Hynes, 1991; Punt and Van den Hondel, 19!)2). In both cases, approaches based on an in vivo analysis are of crucial importance for the understanding of the process. Correspondence to: Dr. P.J. Punt, TNO Nutrition and Food Research Institute, P.O. Box 5815, 2280 HV Rijswijk, The Netherlands. Tel. (31-15) 843-122; Fax (3t-15) 84!t-989; e-mail: [email protected]

Abbreviations: A., AspergiUus; [IGal, [3-galactosidase; bp, base pair(s); E., Eseherichia; gpdA, A. niduians gene encoding glyceraldehyde-3phosphate dehydrogenase; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; PCR, polymerase chain reaction; tsp, transcription start point(s); u, unit(s); UAS, upstream activating sequence(s); wt, wild type; XGal, 5-bromo-4-chloro-3-indolyl[3-D-galactopyranoside. SSDI 0378-1119(95)00166-2

In our laboratory an in vivo approach based on the use of reporter genes has been developed (van Gorcom et al., 1986). Mutation analysis based on this approach has led to a functional dissection of a number of different fungal promoter regions (for review, see Punt and Van den Hondel, 1992). A further step in this line of research will be the analysis of the in vivo activity of the identified promoter elements in more detail. In this paper we describe one approach to do this, namely by using a minipromoter. A mini-promoter is defined as that region of a promoter which consists of sequences necessary and sufficient for proper transcription initiation. In studies on gene expression in Saccharomyces cerevisiae this approach has already been very successful for the analysis of isolated promoter elements (e.g., Guarente et al., 1982; Stanway et al., 1989). The analysis of a previously identified UAS from the promoter region of the A. nidulans gpdA gene and its flanking sequences is described using a minipromoter::lacZ fusion gene. This 50-nt UAS, the so-called gpd box, was identified by sequence comparison of the upstream region of the A. nidulans and A. niger gpdA genes (Punt et al., 1990) and subsequent functional analysis (Punt et al., 1990; 1992).

120 EXPERIMENTAL AND DISCUSSION

(a) Construction of A. nidulans transformants carrying mini-promoter vectors which contain gpd box fragments To demonstrate the use of a mini-promoter approach for the analysis of transcription control sequences, DNA fragments containing the gpd box were introduced upstream from a mini-promoter derived from the gpdA promoter region. This mini-promoter consists of a C + T region (ct box) and other sequences required for transcription initiation (pAN5-dl0; Punt et al., 1990; Fig. 1). The level of expression obtained with this promoter is only 5% of that of the intact gpdA promoter (Punt et al., 1990; Fig. 1). A fragment comprising the gpd box with flanking Sau3A cloning-sites was previously synthesized (Punt et al., 1992). For subsequent cloning this fragment was treated with mung bean nuclease. Two additional DNA fragments containing the gpd box and flanking sequences were isolated as SstI-BamHI fragments from mAN5-11 and mAN5-21 (Punt et al., 1992) and blunted with T4 DNA polymerase for subsequent cloning. The fragments containing the 50-bp gpd box and flanking sequences have been included in this study, since they

contain (additional) inverted repeats sequences (Fig. 1), which may be of relevance for promoter activity. The three gpd box fragments were introduced in two orientations directly upstream from the ct box in pAN5-dl0 (at the PvuII site 29 nt upstream from the major tsp; Punt et al., 1990), resulting in plasmids pAN5-dl0.1A/B, -dl0.2A/B, -dl0.3A/B. Plasmids carrying the gpd box in the same orientation as in the intact upstream region of the gpdA gene are labelled 'A'. The opposite orientation is labelled 'B' (Fig. 1). Due to progressive action of mung bean nuclease some of the clones obtained with the 50-bp gpd box, contained deletion derivatives of the gpd box. Two of these derivatives were included for further analysis (pANS-dl0.1A1 and pAN5-dl0.1A2; Fig. 1). Furthermore, a clone was obtained carrying two copies of the 50-bp gpd box (in tandem) in front of the minipromoter (pAN5-dl0.1AA; Fig. 1). Also, this clone was used for further analysis. The plasmids obtained were introduced into A. nidulans (argB2, biA1, methG2). A. nidulans transformants were analyzed using a previously described lacZ plateassay (Punt et al., 1992) to identify transformants that have incorporated the lacZ-fusion gene. Southern analy-

BGal Activity ct pANS-d10

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2370

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pAN5-dl 0.2AIB

3320

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4620

( 70)

pANS-dl0.3A/B

4090

(420)

3800

(430)

pANS-dl0.1A1

2030

(210)

ND

pAN5-dlO.1A2

890

(150)

ND

pAN5-dlO.1AA

4200

(440)

ND

Fig. 1. [3Gal activity levels obtained with transformants carrying a single copy of one of the various gpd box plasmids cultivated for 16-20 h at 35°C in supplemented minimal growth medium with 1% glucose and 10 mM NaNO3 as carbon and nitrogen source, respectively. The different gpd box containing fragments and their position in the intact gpdA upstream region (relative to the major tsp) are indicated schematically. Nucleotides deleted in the gpd box deletion derivatives pAN5-dl0.1A1 and -d10.1A2 are indicated by -pAN5-insert under the sequence of the gpd box. P,~i,~ and Pgpda indicate the mini-promoter and the intact gpdA promoter. The position of the ct box is indicated by ct. The location and extend of inverted repeat sequences is given by converging arrows. The levels of [3Gal activity (given in u/mg protein) and the standard errors, indicated in parentheses, are obtained from 5-7 experiments. A and B columns give the [3Gal activities for single copy transformants carrying gpd box fragments in the A or B orientation (see section a). ND, not determined. Methods: Unless indicated otherwise, all methods were essentially as described previously (Punt et al., 1992).

121 box results in a further increase of the level of gene expression, indicating that each copy is capable of increasing gene expression in a more or less additive fashion. Introduction of the gpd box with flanking gpdA sequences results in a higher level of [3Gal activity than without flanking sequences (compare pAN5-dl0.2A/B and -dl0.3A/B transformants with pAN5-dl0.1A/B transformants; Fig. 1) suggesting that, besides the gpd box, additional functional elements are present in these larger promoter fragments. However, as no major differences in the level of gene expression are observed between pAN5-dl0.2A/B and pAN5-dl0.3A/B transformants, the additional inverted repeat sequences present in pAN5-dl0.3A/B but absent in pAN5-dl0.2A/B (Fig. 1) appear not to be involved in gene expression under the conditions tested. Deletion of 18 bp at the 3' end of the gpd box clearly reduced the activity of the gpd box (pAN5-dl0.1A2 transformants; Fig. 1), whereas deletion

sis was used to identify transformants containing a single copy of the vector at the argB locus and PCR analysis with two oligos flanking the gpd box sequences (using a Perkin Elmer Cetus DNA thermal cycler) was used to verify the presence of the expected gpd box variants in the different transformants (results not shown). Two independently isolated single copy transformants for each plasmid were used for further analysis.

(b ) lacZ expression analysis Quantitative 13Gal assays were carried out in mycelial extracts from single copy transformants grown in minimal medium (Fig. 1). From the data presented it is clear that introduction of gpd box containing fragments (in either orientation) upstream from the mini-promoter results in a significant increase in the level of gene expression compared to the low level obtained with the minipromoter alone. Introduc,tion of two copies of the gpd A GATC

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Fig. 2. Primer extension analysi:; of transformants carrying a single copy of one of the various gpd box plasmids cultivated in minimal growth medium with 1% glucose and 10mM NaNO 3, (A) Primer extension analysis was performed as described previously (Punt et al., 1990) using an oligo complementary to the 5' part of the coding region of the lacZ gene. Lanes G, A, T, C show the products of dideoxy sequencing reactions with pAN5-d8827 DNA (Punt et al., 1990; the position of the extension products corresponds directly to that of the sequencing products obtained with this vector). The location of the ATG and the ct box (CT) are indicated. Lanes 1 to 13 give the extension products obtained with RNA of transformants obtained with (1) pAN5-dl; (2) pAN5-dl0; (3) pAN5-dl0.1A2; (4) pAN5-dl0.1A1; (5) pAN5-dl0.1A; (6) pAN5-dl0.1B; (7) pAN5-dl0.1AA; (8) pAN5-dl0.2A; (9) pAN5-dl0.2B; (10) pAN5-dl0.3A; (11) pAN5-dl0.3B; (12) ArgB-; (13) without Aspergillus RNA. The position of the major tsp as identified for the intact promoter region (lane 1; Punt et al., 1990) is indicated by an arrow. Minor tsp are indicated by arrowheads. The two minor tsp which are used with an increased relative frequency are indicated with solid arrowheads. The positions of three not lacZ-specific extension products, which are observed in all lanes with Aspergillus RNA, are indicated with asterisks (differences in intensities of these signals are probably due to the use of different amounts of RNA). (B) Nucleotide sequence around the major (+ 1) and minor tsp. The various tsp are indicated as in panel A.

122 of 7 bp at the 5' end of the gpd box (pAN5-dl0.1A1 transformants; Fig. 1) did not significantly affect the level of gene expression. Corroborating previous results (Punt et al., 1990), this result indicates that the 3' end of the gpd box, but not the 5' end, is involved in promoter activity. Taken together, these results clearly demonstrate the usefulness of the mini-promoter lacZ-fusion approach for the analysis of a fungal transcription control sequence.

(c) Transcription initiation analysis To verify that the differences in lacZ expression levels obtained with the various plasmids reflect differences in transcription activation it is essential to know that in all transformants identical tsp are used. The use of different tsp leads to different mRNA molecules which could have different properties concerning stability and/or translation efficiency. To analyze the tsp used in the various transformants primer extension experiments were carried out with total RNA using a lacZ-specific primer. As was already found previously, transcription initiation (major and minor tsp) in pAN5-dl0 transformants occurred at the same positions as observed for the wt gpdA gene and the lacZ fusion gene in transformants carrying the intact promoter (Punt et al., 1990; pAN5-dl transformants; Fig. 2). Also for all gpd box containing transformants similar transcription initiation sites were observed. It should be noted that for pAN5-dl0.2A/B and -dl0.3A/B transformants some of the minor tsp are used with increased relative frequencies (Fig. 2). However, it is not expected that these relatively minor changes significantly bias the results obtained with lacZ expression analysis as these minor tsp are still far upstream from the ATG codon.

TABLE I 13Gal activity levels in a pAN5-dl0 transforrnant grown under different culture conditions Growth medium a

13Gal activity (u/mg protein) b

GLUNO3 FRUNO3 GLUNOaNH 4 FRUNO3NH 4

390 320 260 430

(120) (< 10) (20) (130)

a Spores were inoculated in minimal growth medium with 1% glucose (GLU) or 0.1% fructose (FRU) as carbon source and 10 m M NaNO3 (NO3) or 10 m M N a N O 3 + 10 m M ammonium tartrate (NO3NH4) as nitrogen source. For further details, see legend to Fig. 1. b The levels of ~Gal activity and the standard errors (in parentheses) were obtained from 2-7 separate cultures.

promoter may also be used to analyze sequence elements involved in these control circuits.

(e) Construction of a versatile mini-promoter vector The results described in this paper clearly indicate the usefulness of a mini-promoter vector to analyze fungal promoter sequences. Therefore, a more versatile derivative of vector pAN5-dl0 was constructed which carries convenient KpnI and Nod cloning sites upstream from

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(d) Analysis of the effects of culture conditions on the activity of the mini-promoter To determine whether the activity of the mini-promoter is affected by carbon and nitrogen control circuits in A. nidulans (e.g., Davis and Hynes, 1991), a single-copy transformant carrying pAN5-dl0 was tested under various growth conditions. The effect of carbon catabolite repression was analyzed by growing the strains in minimal growth medium ( 10 mM NaNO3) with 0.1% fructose (derepressed) instead of 1% glucose (repressed). To analyze the effects of nitrogen metabolite repression, the strains were grown in minimal nitrate medium to which 10mM ammonium tartrate was added. As shown in Table I, the expression level obtained for the minipromoter transformant is not significantly affected by either carbon catabolite repression or nitrogen metabolite repression (or both). Based on this result the mini-

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Fig. 3. Mini-promoter vector pAN5-mini. For the construction of this vector KpnI and NotI cloning sites were introduced between the SstI and PvulI site in the gpdA upstream region in pAN5-23 (Punt et al., 1990) using synthetic oligos. From the resulting vector the BamHI-BgllI fragment was cloned into the unique BamHI site of pAN923-42BBsu~ (Punt et al., 1990). The nt sequence around the KpnI and Nod cloning sites in pAN5-mini is given (it should be noted that the SstI site indicated is not unique in pAN5-mini). B, BamHI; E, EcoRI; K, KpnI; Nc, NcoI; N, NotI; Xb, XbaI; X, XhoI; argB*, mutant argB allele; P,,i,~, mini promoter; ttrpc, terminator region of the trpC gene.

123 the mini-promoter sequences (pAN5-MINI; Fig. 3), allowing introduction of different promoter sequences.

(f) Conclusions In this paper a mini-promoter/reporter system for the analysis of fungal expression signals is described. This system has a number of important characteristics: (1) The mini-promoter contains all sequences necessary and sufficient for transcription initiation. Introduction of an activating sequence upstream from the mini-promoter does not alter the transcription initiation sites used. (2) Transcription activity originating from the minipromoter without any additional promoter element is low, but significant. The basal expression level allows the analysis of both positive and negative promoter elements. (3) The activity of the mini-promoter is not affected by nitrogen and carbon control circuits, allowing the study of sequences involved in these control circuits. (4) Clearly, more extensive analysis of the minipromoter/reporter system using sequence elements from a variety of fungal prome, ter regions will be required to evaluate the full potential of the system described.

ACKNOWLEDGEMENT

The authors wish to acknowledge Prof. Dr. P.H. Pouwels for critical evaluation of the manuscript.

REFERENCES Davis, M.A. and Hynes, M.J.: Regulatory circuits in Aspergillus nidulans. In: Bennett, J.W. and Lasure, L.L. (Eds.), More Gene Manipulations in Fungi. Academic Press, New York, NY, 1991, pp. 151-189. Guarente, L., Yocum, R.R. and Gifford, P.: A GALIO-CYC1 hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site. Proc. Natl. Acad. Sci. USA 79 (1982) 7410-7414. Punt, P.J., Dingemanse, M.A., Kuyvenhoven, J., Soede, R.D.M., Pouwels, P.H. and Van den Hondel, C.A.M.J.J.: Functional elements in the promoter region of the Aspergillus nidulans gpdA gene, encoding glyceraldehyde-3-phosphate dehydrogenase. Gene 93 (1990) 101-109. Punt, P.J., Kramer, C., Kuyvenhoven, A., Pouwels, P.H. and Van den Hondel, C.A.M.J.J.: An upstream activating sequence from the Aspergillus nidulans gpdA gene. Gene 120 (1992) 67-93. Punt, P.J. and Van den Hondel, C.A.M.J.J.: Analysis of transcription control sequences of fungal genes. In: Verma, D.P.S. (Ed.), Molecular Signals in Plant-Microbe Communications. CRC Press, Boca Raton, FL, 1992, pp. 29-48. Stanway, C.A., Chambers, A., Kingsman, A.J. and Kingsman, S.M.: Characterization of the transcriptional potency of sub-elements of the UAS of the yeast PGK gene in a PGK mini-promoter. Nucleic Acids Res. 17 (1989) 9205-9218. Van Gorcom, R.F.M., Punt, P.J., Pouwels, P.H. and Van den Hondel, C.A.M.J.J.: A system for the analysis of expression signals in Aspergillus. Gene 48 (1986) 211-217.