Characterization of the Dictyostelium discoideum cellulose-binding protein CelB and regulation of gene expression.
Similar to other stages of Dictyostelium development, spore germination is a particularly suitable model for studying regulation of gene expression. The transition from spore to amoeba is accompanied by developmentally regulated changes in both protein and mRNA synthesis. A number of spore germination-specific cDNAs have been isolated previously. Among these are two members of the 270 gene family, a group of four genes defined by the presence of a common tetrapeptide repeat of Thr-Glu-Thr-Pro. celA (formerly called 270-6) and celB (formerly 270-11) are expressed solely and coordinately during spore germination, the levels of the respective mRNAs being low in dormant spores, rising during germination to a maximum level at about 2 h, and then rapidly declining as amoebae are released from spores. The mRNAs are not found in growing cells or during multicellular development. The rapidity with which these transcripts accumulate and then disappear during germination implies that the respective products may be important for the process. We reported previously that the CelA protein is a cellulase (endo-1, 4-beta-glucanase (EC 3.2.1.4)). In the present investigation, properties of the CelB protein, a glycosylated protein of 532 amino acids, 36% of which are serine or threonine, were examined, and the upstream sequences involved in the developmental regulation of the expression of the gene have been determined. The CelB protein does not demonstrate cellulase activity, but it has a cellulose-binding domain. Its role, if any, in degradation of the cellulose-containing spore wall is unknown. To identify cis-acting elements in the celB promoter, unidirectional 5' deletions of the celB upstream noncoding region were constructed and used to transform amoebae. Analysis of promoter activity during different stages of development shows that a short, very A/T-rich sequence of approximately 81 base pairs is sufficient for spore-specific celB transcription. Contained in this sequence is the Myb oncogene protein binding site, TAACTG, which was shown previously to be a negative regulator of celA transcription. Dictyostelium and mouse Myb proteins bind to this region of the promoter, suggesting that Myb might regulate celB gene expression negatively as it does in celA.
Ramalingam
R
R
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110, USA.
Ennis
H L
HL
D000818:Animals; D001483:Base Sequence; D004271:DNA, Fungal; D004023:Dictyostelium / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D006031:Glycosylation; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D014170:Transformation, Genetic
D004271:DNA, Fungal; D012333:RNA, Messenger; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C083711:cellobiose phosphotransferase B