Beatrice Felenbok

Correct intron splicing generates a new type of a putative zinc-binding domain in a transcriptional activator of Aspergillus nidulans

alcR is the pathway‐specific transcriptional activator of the ethanol regulon in the filamentous fungus, Aspergillus nidulans. The deduced amino acid sequence of a cDNA clone, including the 5′ part of the alcR‐mRNA, shows that a putative Zn‐binding of the all‐cysteine class, exemplified by GAL4 is present. This structure presents some striking features. At variance with other structures of this class, the binding domain is strongly asymmetrical. Model building indicates that the zinc‐binding motif of alcR could adopt an helix‐turn‐helix structure. We propose that the DNA binding motif of alcR could participate in two types of DNA‐binding structures: the zinc‐cluster and the helix‐turn‐helix.

Effect of the thymidine analogue 5-bromo-2′-deoxyuridine on dictyostelium discoideum development

Normal morphogenesis of Dictyostelium discoideum is prevented when 5-BUdR is added to growing vegetative amoebac at least one doubling before the onset of differentiation. The mode of action of the analogue on Dictyostelium closely parallels its mode of action on the metazoans.

Differential chemical labeling of the AlcR DNA-binding domain from Aspergillas nidulans versus its complex with a 16-mer DNA target: Identification of an essential tryptophan involved in the recognition and the interaction with the nucleic acid

DNA binding of the ethanol regulon transcription factor AlcR from Aspergillus nidulans was shown to involve a consensus basic region as in the other zinc cluster proteins. However, additional interactions between some residues and DNA were suspected, among which were a hypothetic hydrophobic interaction between Trp45 and the T residue of the consensus TGCGG sequence. In the present study, the differential chemical labeling of both the free protein and the protein/DNA complex showed significantly different behaviors of the three tryptophan residues comprised in the AlcR sequence toward the Koshland reagent. The spectacular decreased reaction rate for Trp45 within the complex confirmed the location of this residue at the protein/DNA interface. A similar result obtained with Trp53, an amino acid present at the C‐terminal side of AlcR, also indicated its involvement in the DNA recognition. In contrast, the formation of the complex accompanied by an allosteric rearrangement allowed the Trp36 to be much more exposed to the solvent than in the free protein. These data provide additional evidence that the unique specificity of AlcR among the zinc binuclear cluster family results in new types of interactions between AlcR and its cognate targets. From a methodological point of view, the approach of differential chemical labeling combined with mass spectrometric analyses proved to be an interesting tool for the recognition of hydrophobic interactions between the tryptophan residues of a protein and its macromolecular target.

Kinetic of 5-bromodeoxyuridine action on Dictyostelium discoideum growth and development.

We have shown that 5-BUdR affects growth and differentiation in Dictyostelium discoideum, by two different mechanisms. The growth, upon addition of the drug, is decreased to a lower rate which remains constant for at least two generations. Whatever the mechanism of 5-BUdR action on growth, it cannot be through incorporation of the drug into definite targets in the DNA. This is in contrast to the mode of action of the analog against differentiation of Dictyostelium spores. Kinetic data show also that inhibition of development occurs when either of the two DNA strands is substituted by 5-BUdR at a particular target and that 5-BUdR incorporation is semi-conservative. But it is not possible at present to distinguish whether the analog action is an all-or-none inhibition of two or more independent targets, or a dose-dependent inhibition of one (or more) target(s).

Structural analysis of a developmentally regulated sequence encoding for a cysteine proteinase in Dictyostelium discoideum

SummaryA developmentally regulated DNA sequence was isolated from a Dictyostelium discoideum genomic library by cross hybridization with a cDNA clone of cysteine proteinase I. The genomic fragment represents a different but related gene, designated cysteine proteinase II. This sequence is unique in the genome and it hybridizes to a polyA+ RNA of 1.5 kb length, which is produced after the aggregation period.From the determination of polarity of transcription and from the nucleotide sequence, it was shown that the isolated fragment representing about a third of the gene includes its 3′ end. The open reading frame ends with a termination codon TAA and is not interrupted by intervening sequences. The flanking untranslated sequence beyond the 3′ terminus is very A+T rich (91%) and includes a sequence, ATTAAA, known to be important in polyA addition.A striking homology in the corresponding amino acid sequence was found not only with cysteine proteinase I of Dictyostelium discoideum but also with other known cysteine proteases from plants and from mammals. The strongest homology is observed especially around the cysteine at the active site identified in some of these enzymes. Interestingly the organization of cysteine proteinase I and II of Dictyostelium discoideum differs considerably. The C terminal region of the latter corresponds to the N terminal one of the former.

Incorporation of 5-bromo-2'deoxyuridine (BUdR) in Dictyostelium discoideum DNA.

Abstract The modality of incorporation of 5-bromo-2′deoxyuridine (BUdR) in the DNA of Dictyostelium discoideum was studied after one generation of growth of the amoebae in the presence of different concentrations of the drug. The analog was incorporated following the semiconservative pattern of DNA replication. BUdR incorporation in monosubstituted DNA has been measured both by CsCl isopycnic centrifugation or by base analysis chromatography; substitution of thymidine by its analog reaches a maximal value of 30% (60% in the substituted strand). Up to 20% substitution it is proportional to the drug concentration in the growth medium. In these conditions, thymidine substitution is higher in repetitive sequences of the DNA than in unique sequences; the percent of increase of thymidine substitution in repetitive fractions versus total DNA is inversely proportional to thymidine substitution in total DNA.

Alteration of phosphodiesterase activity after 5-bromodeoxyuridine (BUdR) treatment of Dictyostelium discoideum.

Summary Amoebae of Dictyostelium discoideum , treated with 5-bromodeoxyuridine (BUdR) during the growth phase, display altered patterns of cAMP phosphodiesterase activities, and of the specific inhibitor of the excreted enzyme. During growth, treated cells excreted 5–10 times less phosphodiesterase activity than untreated cells, while there was a 2–3-fold increase in the excretion of its specific inhibitor. During the preaggregation stage, activity of the excreted inhibitor appeared earlier and in larger amounts in treated cultures, while the excreted and cellular phosphodiesterase activities did not increase.

Expression of a spore-specific gene in Dictyostelium discoideum

The expression of a previously cloned Dictyostelium discoideum spore-specific gene (Julien et al., EMBO J. 1, 1089-1093 (1982)) was investigated in wild type and mutant strains. In vitro translation of this spore-specific mRNA gave a protein of a molecular weight consistent with the mRNA size. Expressed at a low level during vegetative growth development and in stalk cells, the accumulation of this mRNA reached high values only in spore cells.