The levels of mRNA expressed by gene palF of A. nidulans do not appear to be pH regulated.

FGN 47:72-73

Walter Maccheroni Jr.1, Gregory S. May2, Nilce M. Martinez-Rossi3 and Antonio Rossi4 - 1Dept Genetics, ESALQ-USP, Piracicaba, Brazil, 2Division of Pathology and Laboratory Medicine, University of Texas, M.D. Anderson Cancer Center, Houston, USA, 3Dept Genetics, FMRP-USP, Ribeirão Preto, Brazil and 4Dept Química, FFCLRP-USP, Ribeirão Preto, Brazil.

Although pal genes are putative members of a signaling cascade involved in ambient pH sensing and in the consecutive activation of PacC protein, recent findings show that most of them (palA, B, C, H and I) do not respond to ambient pH at the transcriptional level. Here, we show that mRNA levels of the remainding palF gene are also constant at various growth pH values.

In Aspergillus nidulans ambient pH-regulated gene expression is under the control of a complex genetic circuit comprising at least seven genes that have been cloned and sequenced (Negrete-Urtasun et al. 1999. Mol. Microbiol. 33: 994-1003). The gene pacC codes for a transcription factor that undergoes proteolysis at alkaline growth pH, yielding a functional protein responsible for the induction of genes expressing enzymes with optimal activity at alkaline pH (e.g. alkaline phosphatase) and repression of those with optimal activity at acidic pH (e.g. acid phosphatase). Transcription of pacC is itself pH regulated, being induced under alkaline growth conditions. The genes palA, B, C, F, H, and I are putative members of a signalling cascade involved in ambient pH sensing and the consecutive activation of PacC protein. Although the role of pacC in gene regulation has been well defined in A. nidulans, no function has yet been assigned to any of

the pal genes. The palB gene codes for a calpain protease that is not involved directly in PacC processing, whereas the other genes have revealed, solely by sequence prediction analysis, a few features concerning cell localization signals and putative protein structural motifs. Thus, much effort is needed to elucidate the role of pal genes in the transduction of the ambient pH signal to PacC. An interesting finding however is that the transcription of the genes palA, B, C, H, and I is not pH regulated like pacC. In the present study, we completed the transcriptional analysis by studying the influence of ambient pH on the transcription of the uncharacterized palF gene.

Strain pabaA1 (109 spores) was grown with shaking in 100 ml of liquid minimal medium (Cove, 1966. Biochim. Biophys. Acta 113: 51-56) buffered at pH 5.0 and pH 6.3 with 50 mM sodium citrate, and at pH 8.0 with 50 mM Tris-HCl for 12 h at 37C. Mycelia were harvested and ground in liquid N2 and total RNA was extracted with the Trizol reagent (Gibco BRL) according to manufacturer's instructions. Northern blot was carried out as follows: total RNA (10 µg) was subjected to electrophoresis in a 1% agarose/formaldehyde gel, stained with ethidium bromide, photographed, and blotted onto a nylon membrane as previously described (Maccheroni et al. 1997. Gene 194: 163-167). A 3.3-kb DNA fragment corresponding to a palF cDNA (100 ng) was labeled by random priming with [-32P]dCTP (>3000 Ci/mmol; 10 mCi/ml) to a specific activity of 109 CPM/µg. The membrane was hybridized overnight at 65oC in a solution containing 250 mM NaH2PO4, 7% SDS and washed once at 37oC (40 mM NaH2PO4, 5% SDS) for 1 h and once at 55oC (40 mM NaH2PO4, 1% SDS) for 30 min. Hybridization signals were detected after 5-days exposure to X-ray film.

As shown in Figure 1, a single transcript (3.5-kb) is observed at similar abundance under all growth conditions, indicating that palF transcription is also unchanged in response to ambient pH. This raises the possibility of post-transcriptional processing exerted by pal genes as the mechanism of ambient pH signal transduction or even that other pal genes are still waiting to be identified.

Acknowledgement

This work was supported by FAPESP, CNPq and CAPES (Brazil) and by NIH and NSF (USA).


Figure 1. Influence of ambient pH on palF gene transcription. Ribosomal RNAs were used as control to indicate that equal loads of total mRNA, obtained at different growth pHs, were used in each lane.

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