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Abstract: Phytoestrogens such as isoflavonoids and lignans have been postulated as breast cancer protective constituents in soy and whole-grain cereals. We investigated the ability of isoflavones (IFs) and flaxseed to modulate spontaneous mammary tumor development in female heterozygous Tg.NK (MMTV/c-neu) mice. Two different exposure protocols were applied, either from 4 wk of age onward (postweaning) or during gestation and lactation (perinatal). In the postweaning exposure study, mice were fed IFs or flaxseed in a high-fat diet. In addition, flaxseed in a low-fat diet was tested. Postweaning exposure to IFs and flaxseed tended to accelerate the onset of mammary adenocarcinoma development, although tumor burden at necropsy was not changed significantly. Perinatal IF exposure resulted in enhanced mammary gland differentiation, but palpable mammary tumor onset was not affected. However, tumor burden at necropsy in the perinatal exposure study was significantly increased in the medium- and high-IF dose groups. Comparison of both exposure scenarios revealed a strongly accelerated onset of tumor growth after perinatal high-fat diet exposure compared with the low-fat diet. This study shows that breast cancer-modulating effects of phytoestrogens are dependent both on the background diet and on the timing of exposure in the life cycle. [ABSTRACT FROM AUTHOR]

Abstract Background A validated instrument to assess the effects of chronic cough on health status in patients with chronic obstructive pulmonary disease (COPD) is currently not available. The Leicester Cough Questionnaire (LCQ) is a cough-specific health status questionnaire which is originally validated for a population of general patients presenting with chronic cough. We examined the psychometric performance of the LCQ in patients with COPD and chronic productive cough. Methods Concurrent validity, internal consistency, reproducibility and responsiveness were determined. The St. George's Respiratory Questionnaire (SGRQ) and the Short Form-36 (SF-36) were used as external criteria. Questionnaires were completed at the start of the study. After 2 and 12 weeks the LCQ was repeated, together with a global rating of change. Results In total 54 patients were included. Concurrent validity analysis showed significant correlations between corresponding domains of the LCQ and the SGRQ (rs -0.31 to -0.60). Corresponding domains of the LCQ and the SF-36 showed weaker correlations (rs 0.04 to 0.41). Internal consistency was adequate for two of the three domains (Cronbach's α 0.74 - 0.86). Test-retest reliability in stable patients was high (intraclass correlation coefficients 0.79 - 0.93). The mean difference after two weeks was 0.73 (± 1.75). Responsiveness analysis indicated that the LCQ was able to detect changes after 12 weeks. Conclusion The LCQ is a valid, reliable, responsive instrument to measure health status in COPD patients with chronic productive cough. Trial Registration ClinicalTrials.gov: NCT01071161

The effect of ethylene tetramerisation ligand structures on 1-octene selectivity is well studied. However, by-product formation is less understood. In this work, a range of PNP ligand structures are correlated with the full product selectivity and with catalyst activity. As steric bulk on the N-substituent increases, the product selectivity shifts from >10 % to < 3% of both C6 cyclics and C16+ by-products. 1-Octene peaks at ca. 70%. Thereafter, only 1-hexene increases. Similar selectivity changes were observed for ortho-Ph-substituted PNP ligands. The C10-14 selectivity was less affected by the ligand structure. The ligand effect on the changes in selectivity is explained mechanistically. Lastly, an increase in ligand steric bulk was found to improve catalyst activity and reduce polymer formation by an order of magnitude. It is proposed that steric bulk promotes formation of cationic catalytic species which are responsible for selective ethylene oligomerisation., (© 2020 Wiley-VCH GmbH.)

A preliminary report is presented by an international project team working on developing a model for a structured and holistic approach to companioning parishioners in the journey of formation in the Christian life. A holistic model involves working in three domains: positive psychology, spirituality, and personal and social ethics. Structure is provided by utilizing four self-assessment instruments to inform the work the pastor and the parishioner do together.

Aim: The purpose of this article is to present the results of a private cardiac surgical practice. This information could also serve as a hermeneutical text for new wisdom., Methods: A personal database of 1 750 consecutive patients who had had coronary artery bypass graft (CABG) surgery was statistically analysed. Mortality and major morbidity figures were compared with large registries. Risk factors for postoperative death were determined., Results: Over a period of 12 years, 1 344 (76.8%) males and 406 (23.2%) females were operated on. The observed mortality rate was 3.03% and the expected mortality rate (EuroSCORE) was 3.87%. After stepwise logistic regression, independent risk factors for death were urgency (intra-aortic balloon pump), renal impairment (chronic kidney disease, stage III), re-operation and an additional procedure. Apart from the 53 deaths, another 91 patients had major complications., Conclusion: Mortality and morbidity rates compared favourably with other international registries. Mortality was related to co-morbidities. This outcome contributes to a hermeneutical understanding focusing on new spiritual wisdom and meaning for the surgeon.

A software method has been developed which uses ellipse fitting to analyse electron diffraction patterns from polycrystalline materials. The method, which requires minimal user input, can determine the pattern centre and the diameter of diffraction rings with sub-pixel precision. This enables accurate crystallographic information to be obtained in a rapid and consistent manner. Since the method fits ellipses, it can detect, quantify and correct any elliptical distortion introduced by the imaging system. Distortion information derived from polycrystalline patterns as a function of camera length can be subsequently recalled and applied to single crystal patterns, resulting in improved precision and accuracy. The method has been implemented as a plugin for the DigitalMicrograph software by Gatan, and is a freely available via the internet., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Doping of graphene via low energy ion implantation could open possibilities for fabrication of nanometer-scale patterned graphene-based devices as well as for graphene functionalization compatible with large-scale integrated semiconductor technology. Using advanced electron microscopy/spectroscopy methods, we show for the first time directly that graphene can be doped with B and N via ion implantation and that the retention is in good agreement with predictions from calculation-based literature values. Atomic resolution high-angle dark field imaging (HAADF) combined with single-atom electron energy loss (EEL) spectroscopy reveals that for sufficiently low implantation energies ions are predominantly substitutionally incorporated into the graphene lattice with a very small fraction residing in defect-related sites.

To obtain insight into the genome-wide transcriptional response of heterologous carotenoid production in Saccharomyces cerevisiae, the transcriptome of two different S. cerevisiae strains overexpressing carotenogenic genes from the yeast Xanthophyllomyces dendrorhous grown in carbon-limited chemostat cultures was analysed. The strains exhibited different absolute carotenoid levels as well as different intermediate profiles. These discrepancies were further sustained by the difference of the transcriptional response exhibited by the two strains. Transcriptome analysis of the strain producing high carotenoid levels resulted in specific induction of genes involved in pleiotropic drug resistance (PDR). These genes encode ABC-type and major facilitator transporters which are reported to be involved in secretion of toxic compounds out of cells. β-Carotene was found to be secreted when sunflower oil was added to the medium of S. cerevisiae cells producing high levels of carotenoids, which was not observed when added to X. dendrorhous cells. Deletion of pdr10, one of the induced ABC transporters, decreased the transformation efficiency of a plasmid containing carotenogenic genes. The few transformants that were obtained had decreased growth rates and lower carotenoid production levels compared to a pdr5 deletion and a reference strain transformed with the same genes. Our results suggest that production of high amounts of carotenoids in S. cerevisiae leads to membrane stress, in which Pdr10 might play an important role, and a cellular response to secrete carotenoids out of the cell., (Copyright © 2010 John Wiley & Sons, Ltd.)

Highly dense hexagonally ordered two-dimensional arrays of Si nanocrystals embedded in SiO(2) nanodots were fabricated on a silicon substrate by using a self-assembled porous anodic alumina thin film as a masking layer through which electrochemical oxidation of the Si substrate and ultralow energy Si implantation took place. After removal of the alumina film and high temperature annealing of the samples, hexagonally ordered Si nanocrystals embedded within SiO(2) nanodots were obtained, having sizes in the few tens of nanometer range. The fabricated ordered structures show significant potential for applications either in basic physics experiments or as building blocks for nanoelectronic and nanophotonic devices.

Mating of Blakeslea trispora and other molds of the order Mucorales requires the interaction of mycelia of opposite sex, (+) and (-), leading to the development of specialized structures and to an enhanced accumulation of beta-carotene. Industry obtains beta-carotene by co-cultivating appropriate strains of Blakeslea ("mated cultures"). Gene transcription in single and mated cultures was assayed by cDNA-AFLP, a technique to observe the differential expression of subsets of mRNA fragments. Overexpression in mated cultures is about ten times more frequent than underexpression. We obtained and sequenced fragments of 97 candidate genes that appeared to be overexpressed during mating and confirmed four of them by reverse transcription and real-time PCR. Comparisons with gene sequences from other organisms suggest functions in carotene biosynthesis (4 genes), energy metabolism (8), cell wall synthesis (1), transfer of acetyl groups (1), and regulatory processes (10). Sodium acetate inhibited sexual overexpression in about two-thirds of the candidate genes and acted as a signal with broad effects on the metabolism and the morphology of mated cultures. Our work offers new materials for the study of carotene biosynthesis and its regulation and for the improvement of carotene production with Mucorales.

A comprehensive density functional theory (DFT) study analysing the bulk and various low Miller index surfaces of Hägg Fe carbide (Fe(5)C(2)), considered to be the active phase in Fe-catalysed Fischer-Tropsch synthesis (FTS), has been carried out. The DFT determined bulk structure of Hägg Fe carbide (Fe(5)C(2)) is found to be in good agreement with reported monoclinic (C 2/c) XRD data, independently of whether a monoclinic (C 2/c) or triclinic ([Formula: see text]) bulk structure is used as input for calculations. Attention is focused on the construction of a surface energy stability trend with subsequent correlation with particular surface properties. It is found that a (010) Miller index plane results in the most stable surface (2.468 J m(-2)), while a (101) surface is the least stable (3.281 J m(-2)). The systematic comparison of calculated surface energies with surface properties such as the number of dangling bonds and surface atom density (within a broken bond model), as well as unrelaxed surface energies, relative ruggedness of surfaces, degree of surface relaxation upon optimization, total spin density changes of surfaces compared to the bulk, etc, result in only an approximate correlation with the surface stability trend in selected cases. From the results it is concluded that the relative surface energies fall in a narrow range and that a large number of additional surfaces may be defined, e.g. from higher Miller index planes, sharing similar surface energy values. The results serve to demonstrate the rich complexity and diverse nature of the Fe carbide phase responsible for FTS, effectively laying the foundation for further fundamental studies.

To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially beta-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product beta-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of beta-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of beta-carotene by S. cerevisiae.

The fungus Aspergillus niger is an industrial producer of pectin-degrading enzymes. The recent solving of the genomic sequence of A. niger allowed an inventory of the entire genome of the fungus for potential carbohydrate-degrading enzymes. By applying bioinformatics tools, 12 new genes, putatively encoding family 28 glycoside hydrolases, were identified. Seven of the newly discovered genes form a new gene group, which we show to encode exoacting pectinolytic glycoside hydrolases. This group includes four exo-polygalacturonan hydrolases (PGAX, PGXA, PGXB and PGXC) and three putative exo-rhamnogalacturonan hydrolases (RGXA, RGXB and RGXC). Biochemical identification using polygalacturonic acid and xylogalacturonan as substrates demonstrated that indeed PGXB and PGXC act as exo-polygalacturonases, whereas PGXA acts as an exo-xylogalacturonan hydrolase. The expression levels of all 21 genes were assessed by microarray analysis. The results from the present study demonstrate that exo-acting glycoside hydrolases play a prominent role in pectin degradation.

The electrochemistry of the salts, [emim]2[UBr6] and [emim]2[UO2Br4] ([emim] = 1-ethyl-3-methylimidazolium), has been investigated in both a basic and an acidic bromoaluminate(III) ionic liquid. In the basic ionic liquid, the hexabromo salt undergoes a one-electron reversible reduction process at a stationary glassy carbon disc electrode, while the tetrabromodioxo salt was reduced to a uranium(IV) species by an irreversible two-electron process with the simultaneous transfer of oxide to the ionic liquid. On the other hand, dissolution of either of the salts in an acidic bromoaluminate(III) ionic liquid resulted in the formation of the same electroactive species. The solid state structures of the uranium chloride salts, [emim]2[UCl6] and [emim]2[UO2Cl4], have previously been reported, but have now been re-evaluated using a new statistical model developed in our group, to determine the presence or absence of weak hydrogen bonding interactions in the crystalline state.

Ultralow energy Ar+ and O+ ion beam irradiation of low density polyethylene has been carried out under controlled dose and monoenergetic conditions. XPS of Ar+-treated surfaces exposed to ambient atmosphere show that the bombardment of 50 eV Ar+ ions at a total dose of 10(16) cm(-2) gives rise to very reactive surfaces with oxygen incorporation at about 50% of the species present in the upper surface layer. Using pure O+ beam irradiation, comparatively low O incorporation is achieved without exposure to atmosphere (approximately 13% O in the upper surface). However, if the surface is activated by Ar+ pretreatment, then large oxygen contents can be achieved under subsequent O+ irradiation (up to 48% O). The results show that for very low energy (20 eV) oxygen ions there is a dose threshold of about 5 x 10(15) cm(-2) before surface oxygen incorporation is observed. It appears that, for both Ar+ and O+ ions in this regime, the degree of surface modification is only very weakly dependent on the ion energy. The results suggest that in the nonequilibrium plasma treatment of polymers, where the ion flux is typically 10(18) m(-2) s(-1), low energy ions (<50 eV) may be responsible for surface chemical modification.

As part of the international evaluation program coordinated by ILSI/HESI, the potential of DNA repair deficient Xpa-/- mice and the double knockout Xpa-/-.p53+/- mice for short term carcinogenicity assays was evaluated. For comparison also wild-type C57BL/6 mice (WT) were included in these studies. Four test compounds were administered to groups of 15 male and 15 female Xpa-/- mice, Xpa-/-.p53+/- mice and WT mice for 39 weeks. The model compounds investigated were haloperidol, reserpine (nongenotoxic rodent carcinogens, putative human noncarcinogens), phenacetin (genotoxic rodent carcinogen, suspected human carcinogen), and D-mannitol (noncarcinogen in rodents and humans). The test compounds were administered as admixture to rodent diet at levels up to 25 mg/kg diet for haloperidol, 7.5 mg/kg diet for reserpine, 0.75% for phenacetin, and 10% for D-mannitol. These levels included the maximum tolerable dose (MTD). Survival was not affected with any of the test compounds. Haloperidol, reserpine and D-mannitol were negative in the carcinogenicity assay with Xpa-/- and Xpa-/-.p53+/- mice, showing low and comparable tumor incidences in controls and high-dose animals. The results obtained with phenacetin may be designated equivocal in Xpa-/-.p53+/- mice, based on the occurrence of a single rare tumor in the target organ (kidney) accompanied by a low incidence of hyperplastic renal lesions and a high incidence of karyomegaly. These results are in agreement with the currently known carcinogenic potential of the 4 test compounds in humans.

In the yeast Saccharomyces cerevisiae three dominant flocculation genes, FLO1, FLO5 and FLO8 have been described. Until now only the FLO1 gene, which is located at chromosome I, has been cloned and sequenced. FLO5 and FLO8 were previously localized at chromosomes I and VIII respectively (Vezinhet, F., Blondin, B. and Barre, P. (1991). Mapping of the FLO5 gene of Saccharomyces cerevisiae by transfer of a chromosome during cytoduction. Biotechnol. Lett. 13, 47-52; Yamashita, I. and Fukui, S. (1983). Mating signals control expression of both starch fermentation genes and a novel flocculation gene FLO8 in the yeast Saccharomyces. Agric. Biol. Chem. 47, 2889-2896). This was not in agreement with our results. Here, we report the location of FLO5 and FLO8 on chromosomes VIII and I respectively. By induced chromosome loss and genetic mapping, the FLO5 gene was localized at the right end of chromosome VIII approximately 34 cM centromere distal of PET3. This is part of the region that is present both at chromosome I and chromosome VIII. The location of FLO5 in this area of chromosome VIII made it necessary to re-evaluate the localization of FLO8, which was previously thought to occur in this region. Both genetic and physical mapping showed that FLO8 is allelic to FLO1. Hence, there are only two known dominant flocculation genes, FLO1 and FLO5. Analysis of the nucleotide sequence of chromosome VIII of a non-flocculent strain revealed an open reading frame encoding a putative protein that is approximately 96% identical to the Flo1 protein. This suggests that both dominant flocculation genes encode similar, cell wall-associated, proteins with the same function in the flocculation mechanism.

Northern analysis showed that DNA from the flocculation gene FLO1 hybridized to mRNA molecules of 4.8 kb. This transcript was specific for the FLO1 gene at the right end of chromosome I since disruption of this gene resulted in the disappearance of the transcript. We further found an absolute correlation between flocculation and the presence of transcripts hybridizing to FLO1 DNA, both in various flocculent and non-flocculent strains and in cells from the non-flocculating and flocculating stages of growth. In all cases transcripts were present in flocculating and absent from non-flocculating cultures. From these results we conclude that the FLO1 gene is transcriptionally regulated. Mutations in TUP1 or SSN6 cause flocculation. Several transcripts hybridizing to FLO1 DNA were present in the mutants but not in the corresponding wild-type strains. Disruption of the FLO1 gene in the tup1 and ssn6 strains showed that one of the transcripts corresponded to the FLO1 gene. Disruption of FLO1 did not abolish flocculation completely but only reduced it, indicating that at least two flocculation genes, including FLO1, are activated or derepressed by mutations in the TUP1/SSN6 regulatory cascade.

The centromere of Kluyveromyces lactis was delimited to a region of approximately 280 bp, encompassing KlCDEI, II, and III. Removal of 6 bp from the right side of KlCDEIII plus flanking sequences abolished centromere function, and removal of 5 bp of KlCDEI and flanking sequences resulted in strongly reduced centromere function. Deletions of 20-80 bp from KlCDEII resulted in a decrease in plasmid stability, indicating that KlCDEII must have a certain length for proper centromere function. Centromeres of K. lactis do not function in Saccharomyces cerevisiae and vice versa. Adapting the length of KlCDEII to that of ScCDEII did not improve KlCEN function in S. cerevisiae, while doubling the ScCDEII length did not improve ScCEN function in K. lactis. Thus the difference in CDEII length is not in itself responsible for the species specificity of the centromeres from each of the two species of budding yeast. A chimeric K. lactis centromere with ScCDEIII instead of KlCDEIII was no longer functional in K. lactis, but did improve plasmid stability in S. cerevisiae, although to a much lower level than a wild-type ScCEN. This indicates that the exact CDEIII sequence is important, and suggests that the flanking AT-rich CDEII has to conform to specific sequence requirements.

The location and sequence of the PDA1 gene, encoding the E1 alpha subunit of the pyruvate dehydrogenase (PDH) complex from Saccharomyces cerevisiae, were determined. The PDA1 gene was located on a 6.2 kb fragment of chromosome V, approximately 18 kb centromere distal to RAD3. Consistent with this, the PDA1 gene was genetically mapped at 4 cM from RAD3. A part of the 6.2 kb fragment of chromosome V was sequenced. The nucleotide sequence contained the PDA1 open reading frame and the entire putative promoter. Computer analysis revealed a putative GCN4 binding motif in the PDA1 promoter. The presence of transcriptional elements was experimentally determined by deletion analysis. To this end, ExoIII deletions were constructed in the 5' to 3' direction of the PDA1 promoter and effects on transcription were determined by Northern analysis. Transcription was unaffected upon deletion to position -190 relative to the ATG start codon. Deletions from position -148 and beyond, however, reduced promoter activity at least 40-fold. Apparently the 42 bp between nucleotides -190 and -148 contain an element essential for transcription. Inactivation of the PDA1 promoter could not be attributed to deletions of a recognizable TATA element or any known yeast regulatory motifs. The possible role of the CCCTT sequence present in the 42 bp region and also in the promoters of the other genes encoding subunits of the PDH complex is discussed.

Expression of the PDA1 gene encoding the E1 alpha subunit of the pyruvate dehydrogenase complex (PDH complex) and activity of the complex were investigated in cells grown under several conditions. Comparable amounts of PDA1 mRNA and E1 alpha subunit were detected in cells from batch and chemostat cultures grown on various carbon sources, showing constitutive expression of PDA1 at the transcriptional and translational levels. Induction of the regulatory GCN4 mechanism upon histidine starvation, using the anti-metabolite 3-amino-1,2,4-triazole, increased the levels of PDA1 mRNA by approximately 40%. However, a corresponding increase of E1 alpha concentration or activity of the PDH complex could not be detected. Hence, expression of the PDA1 gene is only regulated to a small extent, if at all, by the GCN4 mechanism. Contrary to the constant levels of PDA1 mRNA and E1 alpha subunit in both batch and chemostat cultures, the specific activity of the PDH complex varied with the culture conditions. The activity of the PDH complex in chemostat cultures was approximately two-threefold higher than in batch cultures grown on the same carbon sources. Overproduction of the E1 alpha subunit in batch cultures resulted in a two-threefold increase in the activity of the PDH complex. Taken together, these results indicate that the activity of the PDH complex is mainly regulated by post-translational modification of the E1 alpha subunit. Expression of PDA1 and activity of the PDH complex were also detected in cultures grown under conditions where no physiological significance of the PDH complex was expected, i.e. during anaerobic growth on glucose or aerobic growth on ethanol. Apparently, the switch from oxidative growth to fermentation occurs without much effect on the PDH complex. These observations suggest that the PDH complex has an alternative function besides sugar catabolism.

We have investigated the chromatin structure of Kluyveromyces lactis centromeres in isolated nuclei of K. lactis and Saccharomyces cerevisiae by using micrococcal nuclease and DNAse I digestion. The protected region found in K. lactis is approximately 270 bp long and encompasses the centromeric DNA elements, KlCDEI, KlCDEII, and KlCDEIII, but not KlCDE0. Halving KlCDEII to 82 bp impaired centromere function and led to a smaller protected structure (210 bp). Likewise, deletion of 5 bp from KlCDEI plus adjacent flanking sequences resulted in a smaller protected region and a decrease in centromere function. The chromatin structures of KlCEN2 and KlCEN4 present on plasmids were found to be similar to the structures of the corresponding centromeres in their chromosomal context. A different protection pattern of KlCEN2 was detected in S. cerevisiae, suggesting that KlCEN2 is not properly recognized by at least one of the centromere binding proteins of S. cerevisiae. The difference is mainly found at the KlCDEIII side of the structure. This suggests that one of the components of the ScCBF3-complex is not able to bind to KlCDEIII, which could explain the species specificity of K. lactis and S. cerevisiae centromeres.

The nucleotide sequence was determined of a 2.1 kb DNA fragment located at approximately 35 kb to the right of the centromere of chromosome VI from Saccharomyces cerevisiae. Analysis revealed the presence of a tRNA(GLy) gene, part of a delta element and a remarkable palindromic sequence. The longest open reading frame found encodes a putative protein of 195 amino acids. Although the fragment was isolated by hybridization to a human diacylglycerol kinase cDNA, no evidence was obtained for the presence of a gene encoding diacylglycerol kinase.

The cloned part of the flocculation gene FLO1 of Saccharomyces cerevisiae (Teunissen, A.W.R.H., van den Berg, J.A. and Steensma, H.Y. (1993). Physical localization of the flocculation gene FLO1 on chromosome I of Saccharomyces cerevisiae, Yeast, in press) has been sequenced. The sequence contains a large open reading frame of 2685 bp. The amino acid sequence of the putative protein reveals a serine- and threonine-rich C-terminus (46%), the presence of repeated sequences and a possible secretion signal at the N-terminus. Although the sequence is not complete (we assume the missing fragment consists of repeat units), these data strongly suggest that the protein is located in the cell wall, and thus may be directly involved in the flocculation process.

The nucleotide sequences of five of the six centromeres of the yeast Kluyveromyces lactis were determined. Mutual comparison of these sequences led to the following consensus: a short highly conserved box (5'-ATCACGTGA-3') flanked by an AT-rich (+/- 90%) stretch of +/- 160 bp followed by another conserved box (5'-TNNTTTATGTTTCCGAAAATTAATAT-3'). These three elements were named KlCDEI, KlCDEII, and KlCDEIII respectively, by analogy with the situation in Saccharomyces cerevisiae. In addition, a second 100 bp AT-rich (+/- 90%) element, named KlCDE0, was found +/- 150 bp upstream of KlCDEI. The sequences of both KlCDEI and KlCDEIII are highly conserved between K. lactis and S. cerevisiae; however, centromeres of K. lactis do not function in S. cerevisiae and vice versa. The most obvious differences between the centromeres of the two yeast species are the length of the AT-rich CDEII, which is 161-164 bp in K. lactis versus 78-86 bp in S. cerevisiae and the presence in K. lactis of KlCDE0, which is not found in S. cerevisiae.

The genetics of flocculation in the yeast Saccharomyces cerevisiae are poorly understood despite the importance of this property for strains used in industry. To be able to study the regulation of flocculation in yeast, one of the genes involved, FLO1, has been partially cloned. The identity of the gene was confirmed by the non-flocculent phenotype of cells in which the C-terminal part of the gene had been replaced by the URA3 gene. Southern blots and genetic crosses showed that the URA3 gene had integrated at the expected position on chromosome I. A region of approximately 2 kb in the middle of the FLO1 gene was consistently deleted during propagation in Escherichia coli and could not be isolated. Plasmids containing the incomplete gene, however, were still able to cause weak flocculation in a non-flocculent strain. The 3' end of the FLO1 gene was localized at approximately 24 kb from the right end of chromosome I, 20 kb centromere-proximal to PHO11. Most of the newly isolated chromosome I sequences also hybridized to chromosome VIII DNA, thus extending the homology between the right end of chromosome I and chromosome VIII to approximately 28 kb.

Pyruvate dehydrogenase mutants of Saccharomyces cerevisiae were isolated by disruption of the PDA1 gene. To this end, the PDA1 gene encoding the E1 alpha subunit of the pyruvate dehydrogenase complex was replaced by the dominant Tn5ble marker. Disruption of the PDA1 gene abolished production of the E1 alpha subunit and pyruvate dehydrogenase activity. Two additional phenotypes were observed in the Pdh-mutants: (a) a reduced growth rate in glucose medium which was partially complemented by the amino acid leucine; (b) an increase in formation of petites which lack mitochondrial DNA [rho0], during growth on glucose. Both phenotypes were shown to be a result of inactivation of the PDA1 gene. Explanations for these phenotypes are discussed.

The recently described dominant yeast marker Tn5ble confers phleomycin resistance on the yeast Saccharomyces cerevisiae (Gatignol, Baron and Tiraby, 1987. Mol. Gen. Genet. 207, 342-348). Incubation in non-selective medium prior to selection is critical, however, for getting phleomycin-resistant transformants. A 6-h incubation period was found to give optimal transformation frequencies, up to 10(5) transformants/micrograms plasmid, comparable to selection for uracil prototrophy (Ura+).

We describe the identification and characterization of the BMH1 gene from the yeast Saccharomyces cerevisiae. The gene encodes a putative protein of 292 amino acids which is more than 50% identical with the bovine brain 14-3-3 protein and proteins isolated from sheep brain which are strong inhibitors of protein kinase C. Disruption mutants and strains with the BMH1 gene on multicopy plasmids have impaired growth on minimal medium with glucose as carbon source, i.e. a 30-50% increase in generation time. These observations suggest a regulatory function of the bmh1 protein. In contrast to strains with an intact or a disrupted BMH1 gene, strains with the BMH1 gene on multicopy plasmids hardly grew on media with acetate or glycerol as carbon source.

A direct selection method was used to isolate centromeres from a genomic library of the yeast Kluyveromyces lactis. The method is based on the lethality at high copy number of the ochre-suppressing tRNA gene SUP11. Five different chromosomal fragments were found that confer mitotic stability to plasmids containing a replication origin of K. lactis (KARS). In addition, KARS plasmids containing these fragments have a copy number of approximately one, and each of the five fragments hybridizes to a different chromosome of K. lactis. From these results we conclude that five of the six centromeres of K. lactis have been isolated. These centromeres do not function in S. cerevisiae.

We have developed the yeast Kluyveromyces lactis as a host organism for the production of the milk-clotting enzyme chymosin. In contrast to Saccharomyces cerevisiae, we found that this yeast is capable of the synthesis and secretion of fully active prochymosin. Various signal sequences could be used to efficiently direct the secretion of prochymosin in Kluyveromyces, but not in S. cerevisiae. We conclude that the efficient synthetic and secretory capacity of this heterologous protein is a property of the yeast Kluyveromyces. These results have led to the development of a large scale production process for chymosin.

A modification and extension of the computer program REVCUT (Blumenthal et al, Nucl. Acids Res. 10, 91-101 (1982) is described. The new program searches for restriction endonuclease recognition sites that are not coding DNA sequences of a protein of known aminoacid sequence using bit patterns. The modifications make the program more accurate and extend the range of the restriction endonucleases.

The CloDF13 cop-1(Ts) mutant expresses a temperature-dependent plasmid copy number. At 42 degrees C the mutant shows a "runaway" behavior, and cells harboring this plasmid are killed. The cop-1(Ts) mutation is a G-to-A transition that disturbs one of the two methylation sites which are located opposite in the stem-loop structure within a region involved in both the initiation of primer synthesis for DNA replication and the termination of the cloacin operon transcript. We demonstrate that the mutation results in an increased primer (RNA II) synthesis resulting from nonconditional enhanced RNA II promoter activity, which at 42 degrees C causes a decrease in the amount of active replication repressor molecules (RNA I) synthesized from the opposite strand. We found that the absence of Dam methylation abolishes the mutant phenotype and that under this condition the high mutant level of RNA II synthesis is reduced, which is accompanied by a restoration of the regulation by RNA I. The role of methylation in the regulation of plasmid replication is discussed.