A method to study the rapid phosphorylation-related modulation of neutral trehalase activity by temperature shifts in yeast.

Heat shock enhanced the synthesis of neutral trehalase in growing cells of Saccharomyces cerevisiae, as detected by immunological methods. The activity of the enzyme was measured in extracts obtained by two methods: cells were either harvested by filtration and subsequent disruption with glass beads at 0-4 degrees C or immediately frozen with liquid nitrogen in the presence of Triton X-100, followed by thawing at 30 degrees C. The first procedure yielded artificially high activities of neutral trehalase in heat-shocked cells due to rapid (less than 1 min) activation during handling at 4 degrees C before homogenization. Activity of the enzyme in these homogenates decreased 75-90% upon a treatment with alkaline phosphatase, indicating that activation was due to phosphorylation. The second procedure yielded low trehalase activities for heat-shock treated cells, much higher activities for cells shifted back for some seconds to 27 degrees C, and very low activities again for cells shifted from 27 to 40 degrees C for a second time. Thus, permeabilization of cells following rapid freezing in Triton X-100 is a method of choice to study post-translational modulation of the neutral trehalase of S. cerevisiae by phosphorylation and dephosphorylation.

The ontogeny of diversification at the immunoglobulin heavy chain locus in Xenopus.

Since the larval and adult antibody responses are distinct and restricted in the clawed toad Xenopus, it offers a near ideal model for studying the ontogeny of antibody repertoires and the mechanisms involved. Immunoglobulin heavy chain (IgH) cDNA clones and B cell IgH DNA clones from various larval and adult libraries have been analysed in isogenic Xenopus. Some features are similar in adults and tadpoles, while others differ and explain the particularities observed previously at the protein level. Among the similarities we found are: (i) the mode of rearrangements (there are approximately 50% abortive events in B cells from both stages), (ii) VH family usage (10 of 11 known VH families are expressed proportionally to the number of VH elements per family), and (iii) JH usage (of the eight to nine Xenopus JH elements, two are used in approximately 70% of the VH regions in both stages of development). We found that there is relatively higher membrane exon expression in tadpoles compared with adults; and that most of the differences come from the diversification of CDR3 through DH usage and N diversification. Unlike in mammals, Xenopus DH elements are used with a remarkable flexibility with inversion, fusions and usage in different reading frames, but tadpoles show a strong bias for the usage of only a few DH elements and of a preferred reading frame. There is N diversification, which further increases CDR3 heterogeneity, in adult Xenopus but virtually none in tadpoles. These observations can account for the fact that larval antibody responses are less heterogeneous than those of adults.

Genetic basis of the antibody repertoire in Xenopus: analysis of the Vh diversity.

The Xenopus IgH locus includes various variable (VH) families, several putative diversity (DH) and at least seven joining (JH) elements, but--although structurally very similar to the mammalian locus--it contributes to a restricted antibody repertoire. The largest three VH families contain 15-30 VH elements which are interspersed at the VHI-VHII and VHII-VHIII boundaries. Twenty-nine genomic and eight expressed VH regions have been sequenced. Each VH family has distinct promoter elements with up to three octamers (ATGCCTAAAT) in either orientation. The incidence of pseudogenes ranges from less than 15% in VHI and VHII to approximately 50% in VHIII, consistent with their relative expression. CDR1 and CDR2 show low overall diversity with nucleotide divergence limited to parts of the CDRs. Randomly selectedly VH elements share CDR1 and CDR2, in some cases also with expressed VH regions. Thus, the complexity of VH elements is not maximal. Patterns of sequence similarities or identities indicate recombination or gene conversion events; sets of direct and inverted repeats flank the sites of, or lie within FR or CDR sequences where these genetic events may occur. Restricted antibody diversity in Xenopus seems therefore to be at least partially related to low complexity of VH elements, frequence of pseudogenes and expression regulated by specific promoter elements; diversity may potentially be increased by (non)homologous recombination events.

Saccharomyces cerevisiae Ras/cAMP pathway controls post-diauxic shift element-dependent transcription through the zinc finger protein Gis1.

The Saccharomyces cerevisiae protein kinase Rim15 was identified previously as a component of the Ras/cAMP pathway acting immediately downstream of cAMP-dependent protein kinase (cAPK) to control a broad range of adaptations in response to nutrient limitation. Here, we show that the zinc finger protein Gis1 acts as a dosage-dependent suppressor of the rim15Delta defect in nutrient limitation-induced transcriptional derepression of SSA3. Loss of Gis1 results in a defect in transcriptional derepression upon nutrient limitation of various genes that are negatively regulated by the Ras/cAMP pathway (e.g. SSA3, HSP12 and HSP26). Tests of epistasis as well as transcriptional analyses of Gis1-dependent expression indicate that Gis1 acts in this pathway downstream of Rim15 to mediate transcription from the previously identified post-diauxic shift (PDS) element. Accordingly, deletion of GIS1 partially suppresses, and overexpression of GIS1 exacerbates the growth defect of mutant cells that are compromised for cAPK activity. Moreover, PDS element-driven expression, which is negatively regulated by the Ras/cAMP pathway and which is induced upon nutrient limitation, is almost entirely dependent on the presence of Gis1.

Evolution of immunoglobulin light chain genes: analysis of Xenopus IgL isotypes and their contribution to antibody diversity.

The amphibian Xenopus laevis expresses several types of immunoglobulin light chain (IgL). cDNA clones for two IgL isotypes, C sigma 1 and C sigma 2, were analysed. C sigma is expressed in spleen and mitogen-stimulated B cells, like another Xenopus IgL type, termed C rho. C sigma shares less than 33% residues with C rho or with CL regions of shark, chicken and mammals. This suggests that C sigma diverged from a common ancestor of CL regions before or at the emergence of amphibians. Two families of VL elements, V sigma 1 and V sigma 2 are associated with C sigma (but not with C rho). They rearrange to their own set of JL elements, J sigma 1 and J sigma 2, which are poorly related to other J elements of the Ig gene family. The Xenopus genome contains a few V sigma 2 and multiple V sigma 1 elements (comparable with mammalian V kappa), but only two C sigma genes. Thus, the organization and expression of Xenopus IgL loci are apparently similar to mammalian IgL loci but different from shark and chicken IgL loci. Only a few VL elements are expressed, since cDNA clones show extensive sharing of CDR1 and CDR2 sequences; some clones differ only in CDR3. Rearranging VL and JL elements increases CDR3 diversity in both V sigma families, but abortive rearrangements are frequent in V sigma 1 regions. The very poor heterogeneity of expressed VL elements therefore appears to limit antibody diversity in Xenopus.

Cloning and disruption of a gene required for growth on acetate but not on ethanol: the acetyl-coenzyme A synthetase gene of Saccharomyces cerevisiae.

A DNA fragment of Saccharomyces cerevisiae with high homology to the acetyl-coenzyme A (acetyl-CoA) synthetase genes of Aspergillus nidulans and Neurospora crassa has been cloned, sequenced and mapped to chromosome I. It contains an open reading frame of 2139 nucleotides, encoding a predicted gene product of 79.2 kDa. In contrast to its ascomycete homologs, there are no introns in the coding sequence. The first ATG codon of the open reading frame is in an unusual context for a translational start site, while the next ATG, 24 codons downstream, is in a more conventional context. Possible implications of two alternative translational start sites for the cellular localization of the enzyme are discussed. A stable mutant of this gene, obtained by the gene disruption technique, had the same low basal activity of acetyl-CoA synthetase as wild-type cells when grown on glucose but completely lacked the strong increase in activity upon entering the stationary phase, providing direct proof that the gene encodes an inducible acetyl-CoA synthetase (ACS1) of yeast. As expected, the mutant was unable to grow on acetate as sole carbon source. Nevertheless, it showed normal induction of isocitrate lyase on acetate media, indicating that activity of acetyl-CoA synthetase is dispensable for induction of the glyoxylate cycle in S. cerevisiae. Surprisingly, disruption of the ACS1 gene did not affect growth on media containing ethanol as the sole carbon source, demonstrating that there are alternative pathways leading to acetyl-CoA under these conditions.

Disruption of TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae, causes accumulation of trehalose-6-phosphate and loss of trehalose-6-phosphate phosphatase activity.

Preparations of the trehalose-6-phosphate synthase/phosphatase complex from Saccharomyces cerevisiae contain three polypeptides with molecular masses 56, 100 and 130 kDa, respectively. Recently, we have cloned the gene for the 56-kDa subunit of this complex (TPS1) and found it to be identical with CIF1, a gene essential for growth on glucose and for the activity of trehalose-6-phosphate synthase. Peptide sequencing of the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex (TPS2) revealed one sequence to be 100% identical with the deduced amino acid sequence of the upstream region of PPH3 on the right arm of chromosome IV. This sequence was used to clone an upstream region of PPH3 containing an open reading frame of 2685 nucleotides, predicted to encode a polypeptide of 102.8 kDa. The N-terminal sequence, as well as three internal amino acid sequences, obtained from peptide sequencing of the 100-kDa subunit, were identical with specific regions of the deduced amino acid sequence. Thus, the sequence cloned represents TPS2, the gene encoding the 100-kDa subunit of the trehalose-6-phosphate synthase/phosphatase complex. Interestingly, a stretch of about 500 amino acids from the first part of TPS2 was 33% identical with the entire TPS1 sequence. Disruption of TPS2 had no effect on trehalose-6-phosphate synthase activity but caused complete loss of trehalose-6-phosphate phosphatase activity, measured in vitro, and accumulation of excessive amounts of trehalose-6-phosphate instead of trehalose upon heat shock or entrance into stationary phase in vivo. These results suggest that TPS2 codes for the structural gene of the trehalose-6-phosphate phosphatase. Heat shock induced an increase in trehalose-6-phosphate phosphatase activity and this was preceded by an accumulation in TPS2 mRNA, suggesting that the trehalose-6-phosphate phosphatase is subjected to transcriptional control under heat-shock conditions.

Saccharomyces cerevisiae cAMP-dependent protein kinase controls entry into stationary phase through the Rim15p protein kinase.

The Saccharomyces cerevisiae protein kinase Rim15p was identified previously as a stimulator of meiotic gene expression. Here, we show that loss of Rim15p causes an additional pleiotropic phenotype in cells grown to stationary phase on rich medium; this phenotype includes defects in trehalose and glycogen accumulation, in transcriptional derepression of HSP12, HSP26, and SSA3, in induction of thermotolerance and starvation resistance, and in proper G1 arrest. These phenotypes are commonly associated with hyperactivity of the Ras/cAMP pathway. Tests of epistasis suggest that Rim15p may act in this pathway downstream of the cAMP-dependent protein kinase (cAPK). Accordingly, deletion of RIM15 suppresses the growth defect of a temperature-sensitive adenylate-cyclase mutant and, most importantly, renders cells independent of cAPK activity. Conversely, overexpression of RIM15 suppresses phenotypes associated with a mutation in the regulatory subunit of cAPK, exacerbates the growth defect of strains compromised for cAPK activity, and partially induces a starvation response in logarithmically growing wild-type cells. Biochemical analyses reveal that cAPK-mediated in vitro phosphorylation of Rim15p strongly inhibits its kinase activity. Taken together, these results place Rim15p immediately downstream and under negative control of cAPK and define a positive regulatory role of Rim15p for entry into both meiosis and stationary phase.

Structural analysis of the subunits of the trehalose-6-phosphate synthase/phosphatase complex in Saccharomyces cerevisiae and their function during heat shock.

Synthesis of trehalose in the yeast Saccharomyces cerevisiae is catalysed by the trehalose-6-phosphate (Tre6P) synthase/phosphatase complex, which is composed of at least three different subunits encoded by the genes TPS1, TPS2, and TSL1. Previous studies indicated that Tps1 and Tps2 carry the catalytic activities of trehalose synthesis, namely Tre6P synthase (Tps1) and Tre6P phosphatase (Tps2), while TsI1 was suggested to have regulatory functions. In this study two different approaches have been used to clarify the molecular composition of the trehalose synthase complex as well as the functional role of its potential subunits. Two-hybrid analyses of the in vivo interactions of Tps1, Tps2, TsI1, and Tps3, a protein with high homology to TsI1, revealed that both TsI1 and Tps3 can interact with Tps1 and Tps2; the latter two proteins also interact with each other. In addition, trehalose metabolism upon heat shock was analysed in a set of 16 isogenic yeast strains carrying deletions of TPS1, TPS2, TSL1, and TPS3 in all possible combinations. These results not only confirm the previously suggested roles for Tps1 and Tps2, but also provide, for the first time, evidence that TsI1 and Tps3 may share a common function with respect to regulation and/or structural stabilization of the Tre6P synthase/phosphatase complex in exponentially growing, heat-shocked cells.