Luis Rodríguez

Cloning and sequence analysis of the invertase geneINV1 from the yeastPichia anomala

A genomic library from the yeastPichia anomala has been constructed and employed to clone the gene encoding the sucrose-hydrolysing enzyme invertase by complementation of a sucrose non-fermenting mutant ofSaccharomyces cerevisiae. The cloned gene,INV1, was sequenced and found to encode a polypeptide of 550 amino acids which contained a 22 amino-acid signal sequence and ten potential glycosylation sites. The amino-acid sequence shows significant identity with other yeast invertases and also withKluyveromyces marxianus inulinase, a yeast β-fructofuranosidase which has a different substrate specificity. The nucleotide sequences of the 5′ and 3′ non-coding regions were found to contain several consensus motifs probably involved in the initiation and termination of gene transcription.

Synthesis of Saccharomyces cerevisiae invertase by Schizosaccharomyces pombe.

In order to gain information on the ability of the glycosylation system of Schizosaccharomyces pombe to process heterologous glycoproteins, the expression of Saccharomyces cerevisiae invertase in the former yeast was studied. Sc. pombe cells are able to produce enzymatically active invertase from the S. cerevisiae SUC2 gene introduced by transformation and the enzyme is glycosylated and secreted into the cell wall. However, Sc. pombe transformants do not glycosylate the heterologous enzyme as their own invertase since it is not bound by the lectin from Bandeiraea simplicifolia seeds, which indicates the absence of terminal galactose residues. Moreover, the electrophoretic mobility of the heterologous invertase is similar to that of the large enzyme from S. cerevisiae, both in its native form and after being deglycosylated with Endo H. These results suggest that the polypeptide chain of S. cerevisiae invertase is the primary factor for the glycosylation in Sc. pombe cells.

Cloning and Characterization of a Candida albicans Gene Homologous to Fructose-1,6-Bisphosphatase Genes

By sequencing of the DNA adjacent to the Candida albicans SEC61 gene, an open reading frame encoding a polypeptide of 331 amino acids was found. The predicted protein showed a strong homology with the fructose-1,6-bisphosphatase [FbPase] from other organisms, and conserved regions included the catalytic motif found in all known FbPases. Although the cloned gene did not complement the growth failure of a Saccharomyces cerevisiae fbp1 mutant in media with gluconeogenic carbon sources, it was transcribed in the transformants in a fashion that indicates a partial repression by glucose. A similar control on the transcription of this gene and on FbPase activity was found in wild-type C. albicans, where the cloned gene (CaFBP1) was shown to be localized in a single chromosomal locus in the genome.

High-resolution optical imaging of the core of the globular cluster M15 with FastCam

We present high-resolution I-band imaging of the core of the globular cluster M15 obtained at the 2.5-m Nordic Optical Telescope with FastCam, a low readout noise L3CCD-based instrument. Short exposure times (30ms) were used to record 200 000 images (512×512 pixels each) over a period of 2 h and 43 min. The lucky imaging technique was then applied to generate a final image of the cluster centre with full width at half-maximum ∼0.1 arcsec and 13 × 13 arcsec^2 field of view. We obtained a catalogue of objects in this region with a limiting magnitude of I = 19.5. I-band photometry and astrometry are reported for 1181 stars. This is the deepest I-band observation of the M15 core at this spatial resolution. Simulations show that crowding is limiting the completeness of the catalogue. At shorter wavelengths, a similar number of objects have been reported using Hubble Space Telescope (HST)/Wide Field Planetary Camera observations of the same field. The cross-match with the available HST catalogues allowed us to produce colour–magnitude diagrams where we identify new blue straggler star candidates and previously known stars of this class.

The sequence of a 15 769 bp segment of Pichia anomala identifies the SEC61 and FBP1 genes and five new open reading frames

We have determined the sequence of a 15 769 bp DNA segment of Pichia anomala. The sequence contains seven complete open reading frames (ORFs) longer than 100 amino acids and a putative tRNA gene. Two of the ORFs code for the well‐characterized genes SEC61 (which codes for the core subunit of the ER translocation complex) and FBP1 (encoding fructose‐1,6‐bisphosphatase). A gene coding for a protein similar to S. cerevisiae YDL054c was found between the two genes. These three genes show a different organization (intermingled triples) in three yeast species: Saccharomyces cerevisiae, Candida albicans and P. anomala. Two out of the four remaining ORFs show weak homology with different proteins from other species and the other two show non‐significant similarity with previously sequenced genes. The nucleotide sequence has been submitted to the EMBL database under Accession No. AJ306295. Copyright

Functional conservation of cytosolic proteins required for endosomal vesicle fusion

Recent studies suggest that intracellular membrane traffic relies upon families of related proteins which confer specificity to individual transport reactions but which operate in tandem with a ubiquitous fusogenic complex containing the N‐ethylmaleimide‐sensitive fusion protein (NSF). The extent to which components of this process are functionally conserved is apparent from the finding that yeast Sec18 protein (Sec18p) can substitute for mammalian NSF in intra‐Golgi transport reactions. Here we report that yeast cytosol can support mammalian endosomal vesicle fusion, demonstrating conservation of cytosolic components required for this reaction. Furthermore, under conditions in which the fusion reaction is NSF‐dependent we show that yeast Sec18p can functionally substitute for NSF, showing that the yeast protein is capable of catalysing at least two distinct mammalian membrane fusion events. In addition we exploit the complex pattern of sensitivity of the mammalian reaction to N‐ethylmaleimide (NEM), coupled with the use of yeast cytosol, to dissect a number of factors required for fusion. We reveal at least three novel NEM‐sensitive activities. One of these can be restored by yeast cytosol suggesting that it is functionally conserved.

Effect of anticalmodulin drugs on the action of yeast α factor pheromone

Saccharomyces cerevisiae α factor pheromone arrest growth of cells of the a mating type (MAT a) at the G1 phase of the cell cycle. When treatment of MAT a cells with α factor was carried out in the presence of anticalmodulin drugs, trifluoperazine or chlorpromazine, the extent of cell growth arrest induced by α factor was reduced or even became undetectable. These results lend support to the hypothesis that calmodulin plays a role as mediator in the action of α factor on MAT a cells.

Asymptotic representations for hypergeometric-Bessel type function and fractional integrals

The paper is devoted to the study of asymptotic relations for the function λγ,σ(β)(z)=β/Γ(γ+1-1/β) ∫1∞ (tβ - 1)γ-1/βtσ e-ztdt generalising Tricomi confluent hypergeometric function and modified Bessel function of the third kind. The full asymptotic representations for λγ,σ(β)(z) at zero and infinity are established. Applications are given to obtain full asymptotic expansions near zero and infinity for the Liouville fractional integral (Iα_f)(x)=1/Γ(α)∫x∞ f(t)dt/(t-x)1-α (x > 0; α ∈ C, Re(α) > 0) and for the Erdelyi-Kober-type fractional intergral (Iα_;β,ηf)(x)= βxβη/Γ(α) ∫x∞(tβ(1-α-η)-1/ f(t)dt)/(tβ-xβ )1-α) (x >0; α ∈C, Re(α) >0) with β > 0 and η ∈ C of power-exponential function f(t), and for three other fractional integrals.