INTERPRETACIÓN DE RESULTADOS:

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4.1.0. INTRODUCTION

176

4.2.0. REGULATION OF cmA, niiA AND niaD

GENE EXPRESSION

176

4.3.0. UPSTREAM SEQUENCE ANALYSIS

183

4.4.0. STRUCTURAL ANALYSIS OF THE cmA GENE

185

4.5.0. STRUCTURAL ANALYSIS OF THE CRNA

POLYPEPTIDE

186

4. 6. 0. THE EFFECT EXERTED BY THE nit-4 GENE ON

A. NIDULANS NR ACTIVITY

188

4.7.0. PROSPECTS

190

1

4.7.1. Analysis of the 1.1 kb transcript

190

4.7.2. Regulation of cmA, niiA and niaD

gene expression

191

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4.1.0. INTRODUCTION

The discussion expands on some of the points raised in the RESULTS and will attempt to further explain them. In addition, an outline of ideas for future work is provided.

Initially, the role of the smaller 1.1 kb transcript is discussed followed by a detailed examination of the mechanism of NR autoregulation (4.2.0). The following section (4.3.0.) relates the regulation of the nitrate gene cluster by the areA and nirA gene products with the conserved motifs within the 5' non-coding regions of the A.nidulans crnA, niiA and niaD genes in addition to the A.oryzae and A.niger niiA

and niaD genes.

An extension of the results regarding the structure of the crnA

gene and polypeptide is detailed in sections 4.4,0 and 4.5.0, respectively. Section 4.6.0 discusses possible explanations for the observed changes of NR regulation by the N. crassa nit-4 gene compared with wild type

A.nidulans.

Finally, proposals for further work to extend our knowledge of the 1.1 kb crnA transcript, the regulation of the nitrate gene cluster and the structure of the crnA encoded polypeptide are reviewed (4.7.0).

4.2.0. REGULATION OF cmA, mTA AND maD GENE

EXPRESSION

The regulation of crnA , niiA and niaD gene expression was investigated by studying the mRNA levels of each gene on Northern blots (3.4.0 and 3.5.0) by hybridisation to individual gene-specific probes.

The crnA cDNA hybridises to two messages on Northern blots (3.4.0). The larger, inducible message of 1.8 kb, is in agreement with the expected size of the crnA mRNA from the sequence analysis. The smaller

1.1 kb message is constitutively expressed and its precise origin not yet- determined.

The existence of more than one mode of nitrate uptake has been suggested (Brownlee and Arst, 1983). It was proposed that one system is active in conidia and young mycelia with a second becoming active in older mycelia. Initially, the crnA gene was shown to be expressed in young mycelia, i.e. 12 h, (Fig 3.6) leading to the consideration that the smaller message may be transcribed from the second nitrate transport gene of A.nidulans, active in older mycelia. However, the abundance of the two transcripts does not alter with respect to each other as the age of the mycelia increases (3.4.5) suggesting that the two transcripts are not from developmentally regulated genes. An alternative explanation is that the mechanism of nitrate uptake in A.nidulans is similar to that of barley, in which there are considered to be two systems. One system is constitutive with high affinity and responsible for initial nitrate uptake. Once inside the cell, nitrate is then proposed to induce the synthesis of a second transport protein (reviewed in Wray, 1989). The abundance of the two crnA transcripts was not analysed immediately after the addition of nitrate preventing confirmation of this theory. However, the manner in which the two transcripts are expressed, i.e. one constitutive and the other inducible, suggests similarities with the proposed mechanism of

nitrate uptake in barley. j

That the crnA cDNA hybridises equally to the two messages under

j

j

from within the region of the crnA gene (3.4.0). The crnA anti-sense sequence includes many stop codons decreasing the probability of the presence of a gene. Consequently, it is presumed that the smaller mRNA

"il

is transcribed off the crnA gene (3.4.0). This does not lead to the rejection

of the possibility of tw o transport systems as the two transcripts m ay § encode tw o functional proteins w ith related but not identical function.

The protein encoded by the 1.1 kb message, if functional, w ould consist of similar m em brane-spanning dom ains as described for the crnA encoded polypeptide (Fig 3.5), only fewer. It is possible that this protein may also be capable of nitrate transport but with high affinity thereby allowing initial, low level nitrate uptake w hen nitrate first becomes available in the im m ediate environm ent. Perhaps this leads to the induction of crnA, niiA and niaD gene expression as a result of NR and NIRA dissociation (3.5.4). Similarly, it is possible that the two mRNAs transcribed off the phosphate transport gene, pho-4, of N.crassa (Mann et al, 1989) encode two separate proteins.

Several mechanisms are know n to produce transcripts of different function from the sam e gene b u t rarely are both synthesized sim ultaneously (1.3.2). A lternative splicing results in tw o transcripts from the niiA gene (Johnstone et al, 1990) but this w ould not account for a 700 bp difference betw een crnA transcript sizes. A transcriptional term ination site w ithin the third intron w ould result in a transcript of approxim ately 1.54 kb. D espite this, differential splicing and 5'

heterogeneity rem ain feasible explanations for the existence of the 1 . 1 kb |

transcript. In contrast, there is the possibility that the smaller message m ay be the result of a processing phenom enon of the larger crn A

transcript or simply an artefact derived from the crnA cDNA probe. (Such as a short stretch of DNA that hybridises to a c r w A - u n r e l a t e d , constitutively expressed transcript). The latter seems unlikely due to the loss of the 1.1 kb transcript in the deletion m utants, A506 and A507 (3.4.0) and also in wild type cells grown in glucose limiting conditions (3.4.2).

The expression of the crnA gene is similar to the niiA and niaD