Yeast communities associated with artisanal mezcal fermentations from Agave salmiana.

The aims of this work were to characterize the fermentation process of mezcal from San Luis Potosi, México and identify the yeasts present in the fermentation using molecular culture-dependent methods (RFLP of the 5.8S-ITS and sequencing of the D1/D2 domain) and also by using a culture-independent method (DGGE). The alcoholic fermentations of two separate musts obtained from Agave salmiana were analyzed. Sugar, ethanol and major volatile compounds concentrations were higher in the first fermentation, which shows the importance of having a quality standard for raw materials, particularly in the concentration of fructans, in order to produce fermented Agave salmiana must with similar characteristics. One hundred ninety-two (192) different yeast colonies were identified, from those present on WL agar plates, by RFLP analysis of the ITS1-5.8S- ITS2 from the rRNA gene, with restriction endonucleases, HhaI, HaeIII and HinfI. The identified yeasts were: Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia kluyveri, Zygosaccharomyces bailii, Clavispora lusitaniae, Torulaspora delbrueckii, Candida ethanolica and Saccharomyces exiguus. These identifications were confirmed by sequencing the D1-D2 region of the 26S rRNA gene. With the PCR-DGGE method, bands corresponding to S. cerevisiae, K. marxianus and T. delbrueckii were clearly detected, confirming the results obtained with classic techniques.

Variaciones hematológicas postoperatorias endermolipectomía / Postoperative hematological variations in dermolipectomy

La tétrada deformante de la pared abdominal está constituida por la obesidad, la distensión abdominal, la gravidez y la diástasis muscular. Nos planteamos como objetivo de nuestro trabajo el demostrar la variación que sufren el hematocrito y la hemoglobina con relación al porcentaje del peso corporal total que representan los colgajos dermograsos extirpados en una dermolipectomía abdominal, tomando como parámetro los valores obtenidos a las 24 horas de la cirugía y a los7 días de postoperatorio. Diseñamos un estudio prospectivo observacional en el que analizamos 93 pacientes operados entre el 1 de agosto del2007 y el 31 de diciembre de 2008. Las variables analizadas fueron las modificaciones sufridas por el hematocrito y la hemoglobina en relación al tanto por ciento de peso corporal total que representan los colgajos extirpados. El promedio de descenso del hematocrito a las 24 horas de la intervención fue del 6,19 % y el de la hemoglobina a las 24 horas de la intervención fue de1,9 gr/l; los valores a los 7 días de postoperatorio fueron de 3,84% y 1,25 gr/l respectivamente. Como conclusión, destacamos la necesidad de comprender la importancia de una correcta preparación prequirúrgica de los pacientes que se van a someter a una dermolipectomía abdominal, para evitar complicaciones en el postoperatorio inmediato y tardío, mejorando así su selección para disminuirla morbilidad de esta intervención quirúrgica (AU)

The deforming tetrad of the abdominal wall is formed byobesity, abdominal distension, gravidity and muscle diastases. Our objective is to show the variation of the hematocrit and hemoglobin in relation to the percentage of the total bodymass that represents the fatty skin folds extirpated in a dermolipectomy, having as parameter the one obtained 24 hours after surgery and at 7 postoperative day. We design an observational prospective study on 93 patients who underwent an abdominal dermolipectomy between august 1st 2007 and december 31st 2008.Analyzed variables were hematocrit and hemoglobin variations related to the percentage of total body mass that represents the skin folds extirpated. The average decrease of the hematocrit 24 hours after surgery was 6,19% and the hemoglobin 1,9gr/l; 7 days later were 3,84% and 1,25gr/l respectively. As a conclusion, we remark the importance of a correct presurgical care of the patients to avoid immediate and distant postoperative complications in abdominal dermolipectomy, improving selection to diminish morbidity (AU)

[Varicella outbreak in a village in Uruguay]. / Estudio de un brote de varicela en un pueblo del Uruguay.

UNLABELLED: A varicella outbreak occurred in a Uruguayan village that introduced the varicella vaccine in 1999 with currently high vaccination rates. AIM: To investigate the cause of the outbreak, vaccine effectiveness, and to describe the measures adopted. MATERIAL AND METHODS: Cases that occurred in the kindergarten and schools in the village were investigated. Vaccination cards were examined, history of chickenpox and clinical characteristic of the current episode were obtained and the outcome of the measures was evaluated. An estimate was made of the vaccine's effectiveness. RESULTS: 37 cases of varicella were reported, 14 occurring in previously vaccinated children, in a one total population of 313 children. The global effectiveness of the vaccine was 80%, and 100% for severe cases. A shift of cases towards older ages was demonstrated; vaccinated children had a trend of less fever and lower number of lesions. Immunization of healthy unvaccinated children, mainly adolescents stopped the outbreak. COMMENTS: The vaccine proved to be highly effectiveness. In an outbreak situation, immunization policies should consider "catch up" vaccination in non-immunized adolescents without a previous history of varicella.

Estudio de un brote de varicela en un pueblo del Uruguay / Varicella outbreak in a village in Uruguay

A varicella outbreak occurred in a Uruguayan village that introduced the varicella vaccine in 1999 with currently high vaccination rates. Aim: To investigate the cause of the outbreak, vaccine effectiveness, and to describe the measures adopted. Material and Methods: Cases that occurred in the kindergarten and schools in the village were investigated. Vaccination cards were examined, history of chickenpox and clinical characteristic of the current episode were obtained and the outcome of the measures was evaluated. An estimate was made of the vaccine's effectiveness. Results: 37 cases of varicella were reported, 14 occurring in previously vaccinated children, in a one total population of 313 children. The global effectiveness of the vaccine was 80 percent, and 100 percent for severe cases. A shift of cases towards older ages was demonstrated; vaccinated children had a trend of less fever and lower number of lesions. Immunization of healthy unvaccinated children, mainly adolescents stopped the outbreak. Comments: The vaccine proved to be highly effectiveness. In an outbreak situation, immunization policies should consider "catch up" vaccination in non-immunized adolescents without a previous history of varicella.

Un brote de varicela acaeció en un pueblo uruguayo que había introducido la vacunación anti-varicela en 1999 y tenía altas coberturas de vacunación. Objetivo: Investigar las causas del brote, la efectividad de la vacunación y evaluar las medidas adoptadas. Material y Métodos: Se investigó los casos ocurridos enjardines de infantes y colegios. Se revisó el carné de vacunación de cada niño además de averiguar por historia previa de varicela, las características clínicas de los casos y el resultado de las medidas adoptadas. Se hizo una estimación de la efectividad de la vacuna. Resultados: Se presentaron 37 casos de varicela, 14 de los cuales habían recibido la vacuna, en una población total de 313 niños. La efectividad global de la vacuna fue de 80 por ciento, y de 100 por ciento para los casos graves. Se constató un desplazamiento de la enfermedad hacia edades mayores; además, los casos en vacunados tuvieron una tendencia a presentar menos fiebre y un número menor de lesiones. La vacunación de aquellos que no habían tenido la varicela y no estaban vacunados antes, detuvo el brote epidémico. Comentarios: Se demostró la efectividad de la vacuna. La política de vacunación debiera evaluar si es necesario proceder a la vacunación "de rescate" en adolescentes no vacunados y que no exhiben el antecedente de haber padecido la varicela.

Embryonic skulls of titanosaur sauropod dinosaurs.

Little is known about the cranial anatomy of the taxonomically diverse and geographically widespread titanosaurs, a paucity that has hindered inferences about the genealogical history and evolutionary development of the latest sauropod dinosaurs. Newly discovered fossil eggs containing embryonic remains from the Late Cretaceous of Argentina provide the first articulated skulls of titanosaur dinosaurs. The nearly complete fetal skulls shed light on the evolution of some of the most notable cranial features of sauropod dinosaurs, including the retraction of the external nares, the forward rotation of the braincase, and the abbreviation of the infraorbital region.

The carboxy-terminal tail of the Ste2 receptor is involved in activation of the G protein in the Saccharomyces cerevisiae alpha-pheromone response pathway.

The Ste2 gene encodes the yeast alpha-pheromone receptor that belongs to the superfamily of seven-transmembrane G protein-coupled receptors. Binding of pheromone induces activation of the heterotrimeric G protein triggering growth arrest in G1 phase and induction of genes required for mating. By random PCR-mediated mutagenesis we isolated mutant 8L4, which presents a substitution of an asparagine residue by serine at position 388 of the alpha-factor receptor. The 8L4 mutant strain shows phenotypic defects such as: reduction in growth arrest after pheromone treatment, diminished activation of the Fus1 gene, and impaired mating competence. The asparagine residue lies in the second half of the intracellular protruding C-terminal tail of the receptor, and its replacement by serine affects interaction with both the G(alpha) and Gbeta subunits. Since expression of the receptor as well as its kinetic parameters, i.e., ligand affinity and receptor number, are unaffected in the mutant strain, we propose that association of the C-terminal tail of the receptor with G(alpha) and Gbeta subunits is required for proper activation of the heterotrimeric G protein. Besides its described role in downregulation and in formation of preactivation complex, the results here shown indicate that the C-terminal tail of the receptor plays an active role in transmitting the stimulus of mating pheromone to the heterotrimeric G protein.

The KlGpa1 gene encodes a G-protein alpha subunit that is a positive control element in the mating pathway of the budding yeast Kluyveromyces lactis.

The cloning of the gene encoding the KlGpa1p subunit was achieved by standard PCR techniques and by screening a Kluyveromyces lactis genomic library using the PCR product as a probe. The full-length open reading frame spans 1,344 nucleotides including the stop codon. The deduced primary structure of the protein (447 amino acid residues) strongly resembles that of Gpa1p, the G-protein alpha subunit from Saccharomyces cerevisiae involved in the mating pheromone response pathway. Nevertheless, unlike disruption of Gpa1 from S. cerevisiae, disruption of KlGpa1 rendered viable cells with a reduced capacity to mate. Expression of a plasmidic KlGpa1 copy in a DeltaKlgpa1 mutant restores full mating competence; hence we conclude that KlGpa1p plays a positive role in the mating pathway. Overexpression of the constitutive subunit KlGpa1p(K(364)) (GTP bound) does not induce constitutive mating; instead it partially blocks wild-type mating and is unable to reverse the sterile phenotype of DeltaKlgpa1 mutant cells. K. lactis expresses a second Galpha subunit, KlGpa2p, which is involved in regulating cyclic AMP levels upon glucose stimulation. This subunit does not rescue DeltaKlgpa1 cells from sterility; instead, overproduction of KlGpa2p slightly reduces the mating of wild-type cells, suggesting cross talk within the pheromone response pathway mediated by KlGpa1p and glucose metabolism mediated by KlGpa2p. The DeltaKlgpa1 DeltaKlgpa2 double mutant, although viable, showed the mating deficiency observed in the single DeltaKlgpa1 mutant.

The Leu-132 of the Ste4(Gbeta) subunit is essential for proper coupling of the G protein with the Ste2 alpha factor receptor during the mating pheromone response in yeast.

In order to identify amino acid residues of Ste4p involved in receptor recognition and/or receptor-G protein coupling, we employed random in vitro mutagenesis and a genetic screening to isolate mutant Ste4p subunits with altered pheromone response. We generated a plasmid library containing randomly mutagenized Ste4 ORFs, followed by phenotypic selection of ste4p mutants by altered alpha pheromone response in yeast cells. Subsequently, we analyzed mutant ste4-10 which has a replacement of the almost universally conserved leucine 132 by phenylalanine. This residue lies in the first blade of the beta propeller structure proposed by crystallographic analysis. By overexpression experiments we found that mutant ste4p subunit triggers the mating pathway at wild type levels in both wild type and receptorless strains. When expressed in a ste4 background, however, the mutant G protein is activated inefficiently by mating pheromone in both a and alpha cells. The mutant ste4-10p was tested in the two-hybrid system and found to be defective in its interaction with the Gpa1p, but has a normal association with the C-termini end of the Ste2p receptor. These observations strongly suggest that the Leu-132 of the Ste4p subunit is essential for efficient activation of the G protein by the pheromone-stimulated receptor and that this domain could be an important point for physical interaction between the Gbeta and the Galpha subunits.

A Saccharomyces cerevisiae mutant lacking a K+/H+ exchanger.

The KHA1 gene corresponding to the open reading frame YJL094c (2.62 kb) encoding a putative K+/H+ antiporter (873 amino acids) in Saccharomyces cerevisiae was disrupted by homologous recombination. The core protein is similar to the putative Na+/H+ antiporters from Enterococcus hirae (NAPA gene) and Lactococcus lactis (LLUPP gene) and the putative K+/H+ exchanger from Escherichia coli (KEFC gene). Disruption of the KHA1 gene resulted in an increased K+ accumulation and net influx without a significant difference in efflux, as well as an increased growth rate, smaller cells, and twice the cell yield per glucose used. Flow cytometry analysis showed an increase of the DNA duplication rate in the mutant. Kinetic studies of 86Rb+ uptake showed the same saturable system for wild-type and disruptant strains. Mutant cells also produced a greater acidification of the medium coincident with an internal pH alkalinization and showed a higher oxygen consumption velocity. We speculate that higher K+ accumulation and increased osmotic pressure accelerate the cell cycle and metabolic activity.

Two unusual amino acid substitutions in cytochrome b of the colorless alga Polytomella spp.: correlation with the atypical spectral properties of the bH heme.

The dithionite-reduced spectra of the purified bc1 complexes from the colorless alga Polytomella spp. and the closely related green alga Chlamydomonas reinhardtii were compared. The spectrum of the bc1 complex from C. reinhardtii showed a profile similar to those of the bc1 complexes from other species. In contrast, the bc1 complex from Polytomella spp. exhibits a double-peak spectrum in the alpha-band region, where the absorption bands of cytochrome c1 and cytochrome b are completely resolved. To further understand the molecular basis of these spectroscopic differences, the mitochondrial gene encoding cytochrome b of Polytomella spp. was cloned, sequenced, and compared with that of C. reinhardtii. The Polytomella spp. cytochrome b gene is 1113 bp long and does not contain introns. The deduced protein sequence exhibits 56% identity and 68% similarity with the cytochrome b of C. reinhardtii, and in a phylogenetic analysis it clearly affiliated with the b-type cytochromes of C. reinhardtii and C. smithii. A comparison of the primary sequences of the Polytomella spp. cytochrome b with other b-type cytochromes, and its analysis based on the structure featuring eight transmembrane stretches, allowed the identification of a tyrosine in position 114, which substitutes for a tryptophan present in all mitochondrial b-type cytochromes sequenced to date. In addition, the primary sequence of the cytochrome b from Polytomella spp. has a serine at position 36, instead of a nonpolar residue (alanine or leucine) found in all other species. In the proposed model for cytochrome b, both residues Tyr114 and Ser36 are in close proximity to the high-potential bH heme. The above data suggest that the polar residues Y114 and S36, each one by itself or in combination, may interact with heme bH of Polytomella spp. and, thus, may be responsible for the unique spectroscopic characteristics of cytochrome b.

Use of pulsed-field gel electrophoresis typing to study an outbreak of infection due to Serratia marcescens in a neonatal intensive care unit.

Serratia marcescens is a well-known cause of nosocomial infections and outbreaks, particularly in critically ill neonates and immunocompromised patients. Numerous methods have been proposed for typing. We used pulsed-field gel electrophoresis (PFGE) typing to analyze an outbreak in a neonatal intensive care unit (NICU). We included 23 patient isolates from an outbreak (March to July 1995), and 10 patient isolates from different wards during the same time period. PFGE of whole-cell DNA digested by SpeI was used as a marker of strain identity. The most common presentation of the infection was sepsis in 18 of 23 (78%) neonates. Only four different biotypes were identified; biotype A8d accounted for 84% of the strains. PFGE typing revealed two clones responsible for two different clonal strain dissemination outbreaks from March to July, with 24 patient isolates being pattern A and 4 patient isolates being pattern E. PFGE typing suggests cross transmission between patients in the NICU and other wards. The isolates from 5 other patients showed distinct PFGE patterns. Extensive investigation and cultures failed to identify any environmental or staff reservoir of S. marcescens. This is one of the first reports applying PFGE to the study of S. marcescens, and this method was a useful marker of strain identity. PFGE typing distinguished strains which appeared to be the same by biotyping.

Isolation of a gene encoding a G protein alpha subunit involved in the regulation of cAMP levels in the yeast Kluyveromyces lactis.

Using chromosomal DNA from Kluyveromyces lactis as template and oligodeoxynucleotides designed from conserved regions of various G protein alpha subunits we were able to amplify by the polymerase chain reaction two products of approximately 0.5 kb (P-1) and 0.8 kb (P-2). Sequencing showed that these two fragments share high homology with genes coding for the G alpha subunits from different sources. Using the P-1 fragment as a probe we screened a genomic library from K. lactis and we cloned a gene (KlGPA2) whose deduced amino acid sequence showed, depending on the exact alignment, 62% similarity and 38% identity with Gpa1p and 76% similarity and 63% identity with Gpa2p, the G protein alpha subunits from Saccharomyces cerevisiae. KlGPA2 is a single-copy gene and its disruption rendered viable cells with significantly reduced cAMP level, indicating that this G alpha subunit may be involved in regulating the adenylyl cyclase activity, rather than participating in the mating pheromone response pathway. KlGpa2p shares some structural similarities with members of the mammalian G alpha s family (stimulatory of adenylyl cyclase) including the absence in its N-terminus of a myristoyl-modification sequence.

The deduced primary structure of subunit I from cytochrome c oxidase suggests that the genus Polytomella shares a common mitochondrial origin with Chlamydomonas.

We cloned and sequenced the mitochondrial gene encoding subunit I of cytochrome c oxidase (coxI) of Polytomella spp., a colorless alga related to Chlamydomonas. The purpose was to explore whether homology between the two species also exists at the level of a mitochondrial enzyme. The gene is 1512 bp long and contains no introns. The translated protein sequence exhibits 73.8% identity with its Chlamydomonas reinhardtii counterpart. The data obtained support the hypothesis that the separation of the colorless alga from the Chlamydomonas lineage was a late event in evolution, that occurred after the endosymbiotic process that gave rise to mitochondria.

Separate roles for N- and C-termini of the STE4 (beta) subunit of the Saccharomyces cerevisiae G protein in the mediation of the growth arrest. Lack of growth-arresting activity of mammalian beta gamma complexes.

Mating pheromone signal transduction in Saccharomyces cerevisiae involves a G protein composed to Scg1p (Gpa1p), Ste4p and Ste18p subunits, homologous to the alpha, beta and gamma subunits of mammalian G protein. Growth arrest in G1 phase is activated by the Ste4p/Ste18p complex via a downstream pathway and it is negatively controlled by the Scg1p subunit. Here we explored whether mammalian beta or gamma subunits could functionally substitute for their yeast homologues. While no evidence was obtained for functional replacement of Ste4p and Ste18p, we found that overexpression of Ste18p potentiated the effect of hybrid proteins in which the N terminus of the Ste4p subunit was replaced by that of the mammalian beta. ste4 mutants having deletions in the N terminus showed a decreased activity in signalling to the downstream effector of the pheromone response. This defect was totally cured by overexpression of Ste18p, indicating that the truncated forms of Ste4p have retained their ability to form an active complex with Ste18p. Removal of six amino acids from the C terminus of Ste4p rendered a completely inactive subunit and this defect persisted in hybrids where the C terminus was placed by that of the beta subunit, indicating that the C terminus of Ste4p is essential to trigger the effector of the yeast pheromone response pathway.

A NADP-glutamate dehydrogenase mutant of the petit-negative yeast Kluyveromyces lactis uses the glutamine synthetase-glutamate synthase pathway for glutamate biosynthesis.

The activities of the enzymes involved in ammonium assimilation and glutamate biosynthesis were determined in wild-type and NADP-glutamate dehydrogenase (GDH) null mutant strains of Kluyveromyces lactis. The specific NADP-GDH activity from K. lactis was fivefold lower than that found in Saccharomyces cerevisiae. The glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were similar to those reported in S. cerevisiae. The NADP-GDH null mutant was obtained by transforming the uraA strain MD2/1 with a linearized integrative yeast vector harbouring a 390 bp fragment of the NADP-GDH structural gene. This mutant grew as well as the parent strain on ammonium, but showed GS and GOGAT activities higher that those found in the wild-type strain, implying that the GS-GOGAT pathway could play a leading role in glutamate biosynthesis in K. lactis. Southern blotting analysis of K. lactis chromosomes separated by contour-clamped homogeneous electric field electrophoresis, indicated that the NADP-GDH structural gene is localized on chromosome VI.

STE2/SCG1-dependent inhibition of STE4-induced growth arrest by mutant STE4 delta C6 in the yeast pheromone response pathway.

The yeast pheromone response pathway involves the activation of a heterotrimeric G protein composed by SCG1 (alpha) (also GPA1), STE4 (beta), and STE18 (gamma) subunits by the pheromone-activated receptors STE2 and STE3 in a and alpha cells, respectively. Upon exchange of bound GDP for GTP in the SCG1 subunit, the release of STE4/STE18 dimer occurs which, in turn causes activation of downstream effectors leading growth arrest and mating competence. Over-expression of STE4 also leads to growth arrest in a STE18 dependent manner. Removal of 6 amino acids from the C-terminus of STE4 rendered a subunit incapable of downstream signalling but still able to interact with STE18. This delta C6 mutant acts as a dominant negative because it blocks the growth arresting effect obtained by over-expression of STE4. The inhibitory effect of STE4 delta C6 is dependent on the presence of the SCG1 subunit in a STE2 but not ste2 background. Inhibition of the growth arresting effect of STE4 by the delta C6 mutant is not due to competition at the effector site, but rather involves an intrinsic activity of STE2 that is dependent on SCG1.

Characterization of a 5025 base pair mitochondrial DNA deletion in Kearns-Sayre syndrome.

We characterized a mitochondrial DNA deletion in a patient with Kearns-Sayre syndrome. Southern blot hybridization showed that 86 to 93% of the mitochondrial genome harbored a 5.0 kb deletion. The percentage of affected genomes is higher than in previously described cases. Direct sequencing of the breakpoint region revealed that the deletion extended 5025 bp from nt 10,050 in the tRNA Gly gene to nt 15,076 in the cytochrome b gene, thus 30% of the total mitochondrial genome was lost by this deletion. A pair of extremely short mirror sequences flanking the mitochondrial DNA breakpoints were identified. These flanking sequences differ from previously published consensus 'hot-spots', known to give rise to deletions in human mitochondrial DNA.

Molecular cloning of the plasma membrane H(+)-ATPase from Kluyveromyces lactis: a single nucleotide substitution in the gene confers ethidium bromide resistance and deficiency in K+ uptake.

A Kluyveromyces lactis strain resistant to ethidium bromide and deficient in potassium uptake was isolated. Studies on the proton-pumping activity of the mutant strain showed that a decreased H(+)-ATPase specific activity was responsible for the observed phenotypes. The putative K. lactis PMA1 gene encoding the plasma membrane H(+)-ATPase was cloned by its ability to relieve the potassium transport defect of this mutant and by reversing its resistance to ethidium bromide. Its deduced amino acid sequence predicts a protein 899 residues long that is structurally colinear in its full length to H(+)-ATPases cloned from different yeasts, except for the presence of a variable N-terminal domain. By PCR-mediated amplification, we identified a transition from G to A that rendered the substitution of the fully conserved methionine at position 699 by isoleucine. We attribute to this amino acid change the low capacity of the mutant H(+)-ATPase to pump out protons.

Nucleotide sequence and chromosomal localization of the gene encoding the Old Yellow Enzyme from Kluyveromyces lactis.

A 6.6 kb genomic DNA fragment from the yeast Kluyveromyces lactis was isolated. Sequence analysis of this fragment revealed the presence of two incomplete open reading frames (ORFs) in one strand, one coding for the carboxyl terminus of the plasma membrane H(+)-ATPase and the other for the amino terminus of an unidentified product. In the complementary strand, a full-length ORF which encodes for a protein homologous to the yeast NADPH-dependent Old Yellow Enzyme was found. The deduced amino acid sequence of this ORF predicts a protein of 398 residues with 84% similarity in its full length to OYE1 from Saccharomyces carlsbergensis and OYE2 from Saccharomyces cerevisiae. In addition, an internal region showed considerable similarity to the bile acid-inducible polypeptide from Eubacterium sp., to the NADH oxidase from Thermoanaerobium brockii, to the trimethylamino dehydrogenase from bacterium W3A1 and to the estrogen-binding protein from Candida albicans, suggesting a functional or structural relationship between them. Inactivation of the KYE1 (Kluyveromyces Yellow Enzyme) gene by deletion of 0.6 kb fragment between positions +358 and +936 produced viable cells with a slight increase in their generation time. Haploid cells carrying the disrupted allele showed one-third of the NADPH oxidase activity, compared to wild-type cells. Southern blotting analysis of digested DNA and chromosomes separated by contour-clamped homogeneous electric field electrophoresis from K. lactis indicated that this is a single-copy gene and it is localized on chromosome II, whose molecular size has been estimated to be approximately 1.3 Mb.

A highly active ubiquinol-cytochrome c reductase (bc1 complex) from the colorless alga Polytomella spp., a close relative of Chlamydomonas. Characterization of the heme binding site of cytochrome c1.

The alga Polytomella spp. offers extraordinary advantages in the preparation of mitochondria since it lacks chloroplasts and a cell wall. In this work the mitochondrial bc1 complex from Polytomella spp. was solubilized and purified by ion exchange chromatography. The complex was found to be composed of 10 polypeptides and exhibited high rates of ubiquinol-cytochrome c oxidoreductase activity (> 300 s-1) sensitive to antimycin and myxothiazol. The molecular mass of the bc1 complex from Polytomella spp. was assayed by gel filtration and estimated to be of 256,300 Da. Therefore, this complex exhibits the unique property of behaving as a monomer. Amino-terminal sequencing of cytochrome c1 identified 7 residues, from which a deoxyoligonucleotide was designed. A second deoxyoligonucleotide was constructed based on a highly conserved region of the c1 type cytochromes. With these probes, a fragment of the cytochrome c1 gene was amplified by polymerase chain reaction and sequenced. The deduced sequence of the apoprotein exhibited a consensus binding site CXXCH. The data suggest that the cytochrome c1 from Polytomella spp. differs from other protoctists like Crithidia and Euglena, i.e. it exhibits a heme binding domain structurally related to the bovine, yeast, and Neurospora c1 type cytochromes.