MATING SYSTEM AND ASYMMETRIC HYBRIDIZATION IN A MIXED STAND OF EUROPEAN OAKS

The sessile (Quercus petraea [Matt.] Liebl.) and pedunculate (Quercus robur L.) oaks are two closely related species having a wide sympatric distribution over Europe. Under natural conditions, they frequently form mixed forests, where hybridization is suspected to occur. In this paper, two different approaches have been applied to the study of the mating system and the interspecific gene flow in a mixed stand formed by the two species. The mating systems of both species have been studied separately by means of the mixed-mating model. The relative contribution of the parental species to the progenies have been estimated with two different methods. The first uses the admixture model. The second is an extension of the mixed-mating model and subdivides the outcrossing rate into intra- and interspecific components. The two species were almost completely outcrossing. This high level of outcrossing and interspecific gene flow could play an important role in the maintenance of the genetic diversity in these long-lived forest tree species. The contribution of the sessile oak to the pedunculate oak progenies varied from 17% to 48%. In contrast, ovules of sessile oak trees appear to be preferentially fertilized by other extreme sessile genotypes. We suggest that interspecific and directional gene flow was responsible for such patterns. Pedunculate oak is considered as a pioneer species and is progressively replaced by sessile oak. Our present findings add a further genetic component to this succession scheme, suggesting that unidirectional gene flow reinforces succession between the two species.     

Cloning and characterization of theeapB andeapC genes ofCryphonectria parasitica encoding two new acid proteinases, and disruption ofeapC

Two new proteinases secreted byCryphonectria parasitica, namely EapB and EapC, have been purified. The corresponding structural genes were isolated by screening a cosmid library, and sequenced. Comparison of genomic and cDNA sequences revealed that theeapB andeapC genes contain three and two introns, respectively. The products of theeapB andeapC genes as deduced from the nucleotide sequences, are 268 and 269 residues long, respectively. N-terminal amino acid sequencing data indicates that EapC is synthesized as a zymogen, which yields a mature 206-amino acid enzyme after cleavage of the prepro sequence. Similarly, sequence alignment studies suggest that EapB is secreted as a 203-residue form which shares extensive similarities not only with EapC but also with two other acid fungal proteinases. However, they display distinct structural features; for example, no cysteine residue is found in EapC. TheeapC gene was mutated using a two-step gene replacement strategy which allowed the specific introduction of several stop codons at the beginning of theeapC coding sequence in an endothiapepsin-deficient (EapA⁺)C. parasitica strain. Although the resulting strain did not secrete EapC, it still exhibited residual extracellular proteolytic activity, which could be due to EapB.     

Self-incompatibility (S) alleles of the rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily

Stylar riboncleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae. The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs. Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus × domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases. The S-RNases of apple specifically accumulated in styles following maturation of the flower bud. Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced. The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases. The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2%. Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae. A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained. The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases. The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.     

ThehapC gene ofAspergillus nidulans is involved in the expression of CCAAT-containing promoters

The 5 regulatory region of theamdS gene ofAspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence which is required for setting the basal level ofamdS expression. Mobility shift studies have identified a factor inA. nidulans nuclear extracts which binds to this CCAAT sequence. InSaccharomyces cerevisiae theHAP3 gene encodes one component of a multisubunit complex that binds CCAAT sequences. A search of the EMBL and SwissProt databases has revealed anA. nidulans sequence with significant homology to theHAP3 gene adjacent to the previously cloned regulatory geneamdR. Sequencing of the remainder of this region has confirmed the presence of a gene, designatedhapC, with extensive homology toHAP3. The predicted amino acid sequence of HapC shows extensive identity to HAP3 in the central conserved domain, but shows little conservation in the flanking sequences. A haploid carrying ahapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially slowly on acetamide as a sole carbon and nitrogen source, indicating thathapC plays a role inamdS expression. In agreement with this notion, it has been shown that thehapC deletion results in reduced levels of expression of anamdS::lacZ reporter gene and this effect is particularly evident under conditions of carbon limitation. Nuclear extracts prepared from thehapC deletion mutant show no CCAAT binding activity to theamdS orgatA promoters, indicating thathapC may encode a component of the complex binding at this sequence.     

Implication of a repression system, homologous to those of other bacteria, in the control of arginine biosynthesis genes inStreptomyces coelicolor

As with most amino acid biosynthetic pathways in streptomycetes, enzymes of arginine biosynthesis inStreptomyces coelicolor show only slight derepression in minimal medium without, as opposed to with, exogenous arginine. However, when an arginine auxotroph was cultured in limiting arginine, ornithine carbamoyltransferase (OCT) activities rose by as much as 100-fold. The response was not due to a general starvation effect. To elucidate the repression-derepression mechanism, a DNA fragment containing the upstream region of the previously isolatedS. coelicolor argCJB cluster was cloned into a multicopy vector and transformed into wild-typeS. coelicolor; a slight transient derepression of OCT was observed in minimal medium without, though not with, added arginine, consistent with titration by the insert of a negatively acting macromolecule such as a repressor. A sub-fragment carrying the 5 end ofargC and the region immediately upstream showed specific binding, in mobility shift assays, to purified AhrC, the repressor/activator of genes of arginine metabolism inBacillus subtilis. It is therefore likely that inS. coelicolor, expression of arginine biosynthesis genes is controlled by a protein homologous to the well-characterisedB. subtilis andEscherichia coli repressors.     

A T7 promoter-specific, inducible protein expression system forBacillus subtilis

The adaptation and application of theEscherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression inBacillus subtilis is reported. The expression cassette used inBacillus subtilis was tightly regulated and T7 RNA polymerase (T7 RNAP) appeared 30 min after induction. The efficiency of T7 promoter-specific gene expression inB. subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation ofE. coli -galactosidase, as well as a 1,4--glucosidase fromThermoanaerobacter brockii inB. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The-amylase ofThermoactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10–20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibited-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation