The formation of the digits in amniota embryos is accompanied by apoptotic cell death of the interdigital mesoderm triggered through BMP signaling. Differences in the intensity of this apoptotic process account for the establishment of the different morphological types of feet observed in amniota (i.e., free-digits, webbed digits, lobulated digits). The molecular basis accounting for the differential pattern of interdigital cell death remains uncertain since the reduction of cell death in species with webbed digits is not accompanied by a parallel reduction in the pattern of expression of bmp genes in the interdigital regions. In this study we show that the duck interdigital web mesoderm exhibits an attenuated response to both BMP-induced apoptosis and TGFbeta-induced chondrogenesis in comparison with species with free digits. The attenuated response to these signals is accompanied by a reduced pattern of expression of msx-1 and msx-2 genes. Local application of FGF in the duck interdigit expands the domain of msx-2 expression but not the domain of msx-1 expression. This change in the expression of msx-2 is followed by a parallel increase in spontaneous and exogenous BMP-induced interdigital cell death, while the chondrogenic response to TGFbetas is unchanged. The regression of AER, as deduced by the pattern of extinction of fgf-8 expression, takes place in a similar fashion in the chick and duck regardless of the differences in interdigital cell death and msx gene expression. Implantation of BMP-beads in the distal limb mesoderm induces AER regression in both the chick and duck. This finding suggests an additional role for BMPs in the physiological regression of the AER. It is proposed that the formation of webbed vs free-digit feet in amniota results from a premature differentiation of the interdigital mesoderm into connective tissue caused by a reduced expression of msx genes in the developing autopod.

Cell cycle progression in the budding yeast Saccharomyces cerevisiae is controlled by the Cdc28 protein kinase, which is sequentially activated by different sets of cyclins. Previous genetic analysis has revealed that two B-type cyclins, Clb5 and Clb6, have a positive role in DNA replication. In the present study, we show, in addition, that these cyclins negatively regulate G 1 -and G 2 -specific functions. The consequences of this negative regulation were most apparent in clb6 mutants, which had a shorter pre-Start G 1 phase as well as a shorter G 2 phase than congenic wild-type cells. As a consequence, clb6 mutants grew and proliferated more rapidly than wild-type cells. It was more difficult to assess the role of Clb5 in G 1 and G 2 by genetic analysis because of the extreme prolongation of S phase in clb5 mutants. Nevertheless, both Clb5 and Clb6 were shown to be responsible for down-regulation of the protein kinase activities associated with Cln2, a G 1 cyclin, and Clb2, a mitotic cyclin, in vivo. These observations are consistent with the observed cell cycle phase accelerations associated with the clb6 mutant and are suggestive of similar functions for Clb5. Genetic evidence suggested that the inhibition of mitotic cyclin-dependent kinase activities was dependent on and possibly mediated through the CDC6 gene product. Thus, Clb5 and Clb6 may stabilize S phase by promoting DNA replication while inhibiting other cell cycle activities.

In addition to exoglucanases (EXGs) I and II, old cultures of Saccharomyces cerevisiae secreted into the culture medium a new immunologically-related material that exhibited exoglucanase activity. The new exoglucanase (EXGII1/2) was purified from stationary-phase cultures. It turned out to be a glycoprotein whose protein portion was identical to that of the other two isoenzymes in terms of ionic properties, size, amino acid composition and NH2-terminal sequence (25 residues). Disruption of the structural gene encoding EXGs I and II resulted in a strain unable to secrete all three isoenzymes. EXGII1/2 was indistinguishable in terms of molecular weight from the single intermediate detected during the deglycosylation (mediated by endo H) of EXGII by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Thus, the new isoenzyme contains only one of the two slightly elongated mannan inner cores present in enzyme II. Two intermediates were, however, detected when the deglycosylation of EXGII was monitored by ion-exchange chromatography (high-pressure liquid chromatography). Site-directed mutagenesis indicated that the major intermediate, which eluted at about the same position as enzyme II1/2, corresponded to protein molecules carrying the oligosaccharide attached to the Asn of the second sequon, whereas the minor one carried the oligosaccharide in the first potential glycosylation site. Several lines of evidence indicate that EXGII1/2 is a biosynthetic product resulting from an imbalance between the rate of protein synthesis and the glycosylation capabilities of the glycosylation machinery.

We have detected proteolytic processing of a form of exoglucanase representative of the endoplasmic reticulum (form A). This processing did not take place when form A was obtained from protoplasts lysed in the presence of either EDTA or leupeptin, two wel-characterixed inhibitors of KEX2 endoprotease from Sacchuromyces cerevisiae. Sequencing of the amino terminus of an A-like form of enzyme secreted by a kex2 mutant indicated the presence of 4 amino acids, with a pair of basic residues (Lys-Arg) at their carboxyl side, preceding the amino terminus of the wild-type external exoglucanase.

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