ABSTRACT
Robot-assisted radical prostatectomy causes discomfort in the immediate postoperative period. This randomised controlled trial investigated if intrathecal bupivacaine/morphine, in addition to general anaesthesia, could be beneficial for the postoperative quality of recovery. One hundred and fifty-five patients were randomly allocated to an intervention group that received intrathecal 12.5 mg bupivacaine/300 µg morphine (20% dose reduction in patients > 75 years) or a control group receiving a subcutaneous sham injection and an intravenous loading dose of 0.1 mg.kg-1 morphine. Both groups received standardised general anaesthesia and the same postoperative analgesic regimen. The primary outcome was a decrease in the Quality of Recovery-15 (QoR-15) questionnaire score on postoperative day 1. The intervention group (n = 76) had less reduction in QoR-15 on postoperative day 1; median (IQR [range]) 10% (1-8 [-60% to 50%]) vs. 13% (5-24 [-6% to 50%]), p = 0.019, and used less morphine during the admission; 2 mg (1-7 [0-41 mg]) vs. 15 mg (12-20 [8-61 mg]), p < 0.001. Furthermore, they perceived lower pain scores during exertion; numeric rating scale (NRS) 3 (1-6 [0-9]) vs. 5 (3-7 [0-9]), p = 0.001; less bladder spasms (NRS 1 (0-2 [0-10]) vs. 2 (0-5 [0-10]), p = 0.001 and less sedation; NRS 2 (0-3 [0-10]) vs. 3 (2-6 [0-10]), p = 0.005. Moreover, the intervention group used less rescue medication. Pruritus was more severe in the intervention group; NRS 4 (1-7 [0-10]) vs. 0 (0-1 [0-10]), p = 0.000. We conclude that despite a modest increase in the incidence of pruritus, multimodal pain management with intrathecal bupivacaine/morphine remains a viable option for robot-assisted radical prostatectomy.
Subject(s)
Anesthesia, Spinal/methods , Anesthetics, Local , Bupivacaine , Injections, Spinal , Morphine , Prostatectomy/methods , Robotic Surgical Procedures/methods , Aged , Anesthesia Recovery Period , Anesthesia, General , Anesthetics, Local/administration & dosage , Bupivacaine/administration & dosage , Humans , Male , Middle Aged , Pain Measurement , Postoperative Complications/epidemiology , Postoperative Period , Pruritus/chemically induced , Pruritus/epidemiology , Surveys and Questionnaires , Treatment OutcomeABSTRACT
OBJECTIVE: Congenital heart defects (CHD) are still missed frequently in prenatal screening programs, which can result in severe morbidity or even death. The aim of this study was to evaluate the quality of fetal heart images, obtained during the second-trimester standard anomaly scan (SAS) in cases of CHD, to explore factors associated with a missed prenatal diagnosis. METHODS: In this case-control study, all cases of a fetus born with isolated severe CHD in the Northwestern region of The Netherlands, between 2015 and 2016, were extracted from the PRECOR registry. Severe CHD was defined as need for surgical repair in the first year postpartum. Each cardiac view (four-chamber view (4CV), three-vessel (3V) view and left and right ventricular outflow tract (LVOT, RVOT) views) obtained during the SAS was scored for technical correctness on a scale of 0 to 5 by two fetal echocardiography experts, blinded to the diagnosis of CHD and whether it was detected prenatally. Quality parameters of the cardiac examination were compared between cases in which CHD was detected and those in which it was missed on the SAS. Regression analysis was used to assess the association of sonographer experience and of screening-center experience with the cardiac examination quality score. RESULTS: A total of 114 cases of isolated severe CHD at birth were analyzed, of which 58 (50.9%) were missed and 56 (49.1%) were detected on the SAS. The defects comprised transposition of the great arteries (17%), aortic coarctation (16%), tetralogy of Fallot (10%), atrioventricular septal defect (6%), aortic valve stenosis (5%), ventricular septal defect (18%) and other defects (28%). No differences were found in fetal position, obstetric history, maternal age or body mass index (BMI) or gestational age at examination between missed and detected cases. Ninety-two cases had available cardiac images from the SAS. Compared with the detected group, the missed group had significantly lower cardiac examination quality scores (adequate score (≥ 12) in 32% vs 64%; P = 0.002), rate of proper use of magnification (58% vs 84%; P = 0.01) and quality scores for each individual cardiac plane (4CV (2.7 vs 3.9; P < 0.001), 3V view (3.0 vs 3.8; P = 0.02), LVOT view (1.9 vs 3.3; P < 0.001) and RVOT view (1.9 vs 3.3; P < 0.001)). In 49% of missed cases, the lack of detection was due to poor adaptational skills resulting in inadequate images in which the CHD was not clearly visible; in 31%, the images showed an abnormality (mainly septal defects and aortic arch anomalies) which had not been recognized at the time of the scan; and, in 20%, the cardiac planes had been obtained properly but showed normal anatomy. Multivariate regression analysis showed that the volume of SAS performed per year by each sonographer was associated significantly with quality score of the cardiac examination. CONCLUSIONS: A lack of adaptational skills when performing the SAS, as opposed to circumstantial factors such as BMI or fetal position, appears to play an important role in failure to detect CHD prenatally. The quality of the cardiac views was inadequate significantly more often in undetected compared with detected cases. Despite adequate quality of the images, CHD was not recognized in 31% of cases. A high volume of SAS performed by each sonographer in a large ultrasound center contributes significantly to prenatal detection. In 20% of undetected cases, CHD was not visible even though the quality of the images was good. © 2019 Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.
Subject(s)
Clinical Competence/statistics & numerical data , Fetal Heart/diagnostic imaging , Heart Defects, Congenital/diagnostic imaging , Missed Diagnosis/statistics & numerical data , Ultrasonography, Prenatal/statistics & numerical data , Case-Control Studies , Female , Fetal Heart/embryology , Heart Defects, Congenital/embryology , Heart Defects, Congenital/epidemiology , Humans , Netherlands/epidemiology , Pregnancy , Pregnancy Trimester, Second , RegistriesABSTRACT
OBJECTIVE: The aim of our study is to explore whether the cerebral growth is delayed in fetuses with congenital heart defects (CHD) in the second and early third trimester. METHODS: A prospective cohort study was conducted in 77 CHD cases, with 75 healthy controls. 3D cerebral volume acquisition was performed sequentially. The volumes of the fetal hemicerebrum and extracerebral fluid were compared by linear regression analysis, and the Sylvian fissure was measured. RESULTS: Between 19 and 32 weeks of gestation, 158 measurements in cases and 183 measurements in controls were performed (mean 2.2/subject). The volume growth of the hemicerebrum (R2 = 0.95 vs. 0.95; p = 0.9) and the extracerebral fluid (R2 = 0.84 vs. 0.82, p = 0.9) were similar. Fetuses with abnormal oxygen delivery to the brain have a slightly smaller brain at 20 weeks of gestation (p = 0.02), but this difference disappeared with advancing gestation. CHD cases demonstrated a slightly shallower Sylvian fissure (mean ratio 0.146 vs. 0.153; p = 0.004). CONCLUSIONS: Our study shows no differences in cerebral growth, studied in an unselected cohort, with successive cases of isolated CHD. Even in the severest CHD cases, cerebral size is similar in the early third trimester. The cause and meaning of a shallower Sylvian fissure is unclear; possibly, it is a marker for delayed cerebral maturation or it might be an expression of decreasing amount of extracerebral fluid.
Subject(s)
Cerebrum/embryology , Fetal Development , Heart Defects, Congenital/physiopathology , Brain/diagnostic imaging , Brain/embryology , Cerebrospinal Fluid/diagnostic imaging , Cerebrum/diagnostic imaging , Cohort Studies , Female , Heart Defects, Congenital/diagnostic imaging , Heart Defects, Congenital/embryology , Humans , Linear Models , Pregnancy , Pregnancy Trimester, Second , Pregnancy Trimester, ThirdABSTRACT
OBJECTIVES: Severe right ventricular outflow tract obstruction (RVOTO) is a potential complication in recipient twins of twin-to-twin transfusion syndrome (TTTS) that requires postnatal follow-up or treatment. We aimed to evaluate pregnancy characteristics of neonates with RVOTO from complicated monochorionic twin pregnancies, determine the incidence of RVOTO in TTTS cases and construct a prediction model for its development. METHODS: This was an observational cohort study of all complicated monochorionic twin pregnancies with a postnatal diagnosis of RVOTO examined at our center. Cases were referred for evaluation of the need for fetal therapy or intervention because of TTTS, selective intrauterine growth restriction (sIUGR) or multiple congenital malformations in one of the twins. Ultrasound data were retrieved from our monochorionic twin database. Among liveborn TTTS recipients treated prenatally with laser therapy, those with RVOTO were compared with those without RVOTO (controls). We describe four additional cases with RVOTO that were not TTTS recipients. RESULTS: A total of 485 twin pregnancies received laser therapy for TTTS during the study period. RVOTO was diagnosed in 3% (11/368) of liveborn TTTS recipients, of whom two showed mild Ebstein's anomaly. Before laser therapy, pericardial effusion was seen in 45% (5/11) of RVOTO cases (P < 0.01) and abnormal A-wave in the ductus venosus (DV) in 73% (8/11) (P = 0.03), significantly higher proportions than in controls. Mean gestational age at laser therapy was 17 + 3 weeks in RVOTO cases compared with 20 + 3 weeks in controls (P = 0.03). A prediction model for RVOTO was constructed incorporating these three significant variables. One TTTS donor had RVOTO after the development of transient hydrops following laser therapy. Three larger twins in pregnancies complicated by sIUGR developed RVOTO, the onset of which was detectable early in the second trimester. CONCLUSIONS: RVOTO occurs in TTTS recipient twins but can also develop in TTTS donors and larger twins of pregnancies complicated by sIUGR. Abnormal flow in the DV, pericardial effusion and early gestational age at onset of TTTS are predictors of RVOTO in TTTS recipients, which suggests increased vulnerability to hemodynamic imbalances in the fetal heart in early pregnancy. These findings could guide diagnostic follow-up protocols after TTTS treatment. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.
Subject(s)
Fetofetal Transfusion/diagnostic imaging , Prenatal Diagnosis , Twins , Ventricular Outflow Obstruction/epidemiology , Child, Preschool , Cohort Studies , Female , Fetofetal Transfusion/complications , Follow-Up Studies , Humans , Incidence , Infant , Male , Netherlands/epidemiology , Pregnancy , ROC Curve , Sensitivity and Specificity , Ventricular Outflow Obstruction/diagnosis , Ventricular Outflow Obstruction/etiologyABSTRACT
AIMS: Saccharomyces cerevisiae does not express any xylose-specific transporters. To enhance the xylose uptake of S. cerevisiae, directed evolution of the Gal2 transporter was performed. METHODS AND RESULTS: Three rounds of error-prone PCR were used to generate mutants with improved xylose-transport characteristics. After developing a fast and reliable high-throughput screening assay based on flow cytometry, eight mutants were obtained showing an improved uptake of xylose compared to wild-type Gal2 out of 41 200 single yeast cells. Gal2 variant 2·1 harbouring five amino acid substitutions showed an increased affinity towards xylose with a faster overall sugar metabolism of glucose and xylose. Another Gal2 variant 3·1 carrying an additional amino acid substitution revealed an impaired growth on glucose but not on xylose. CONCLUSIONS: Random mutagenesis of the S. cerevisiae Gal2 led to an increased xylose uptake capacity and decreased glucose affinity, allowing improved co-consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: Random mutagenesis is a powerful tool to evolve sugar transporters like Gal2 towards co-consumption of new substrates. Using a high-throughput screening system based on flow-through cytometry, various mutants were identified with improved xylose-transport characteristics. The Gal2 variants in this work are a promising starting point for further engineering to improve xylose uptake from mixed sugars in biomass.
Subject(s)
Monosaccharide Transport Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/enzymology , Xylose/metabolism , Biological Transport , Directed Molecular Evolution , Glucose/metabolism , High-Throughput Screening Assays , Monosaccharide Transport Proteins/metabolism , Mutagenesis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
OBJECTIVE: To evaluate the effect of fetal anemia and intrauterine transfusion on ventricular shortening fraction. METHODS: The end-diastolic and end-systolic transverse dimensions of the left and right ventricles were obtained using M-mode ultrasonography. The shortening fractions of both ventricles were calculated at three time points: before, immediately after and one day after intrauterine transfusion. The blood volume given at intrauterine transfusion was expressed as a percentage of estimated fetoplacental blood volume. RESULTS: Complete measurements were obtained from 49 transfusions in 23 fetuses. Intrauterine transfusion was performed at a median gestational age of 31 (range, 19-35) weeks. Median hemoglobin concentration before and after intrauterine transfusion was 7.9 (range, 2.7-13.7) g/dL and 14.3 (range, 12.7-16.1) g/dL, respectively. Both left and right ventricular shortening fractions differed significantly between the three time points. Left ventricular shortening fraction decreased immediately after transfusion in 43 (88%) of the 49 procedures. Right ventricular shortening fraction decreased immediately after transfusion in 42 (86%) of the 49 procedures. At the first intrauterine transfusion, there was only a weak correlation between the decrease in shortening fraction of both ventricles and the transfused volume (left: R(2) = 0.15; P = 0.20/right: R(2) = 0.005; P = 0.81). CONCLUSION: Transfusion significantly decreases the shortening fraction of both ventricles of the fetal heart. There is, however, little correlation between the decrease in shortening fraction and the volume of red cells given at intrauterine transfusion. Published by John Wiley & Sons, Ltd.
Subject(s)
Anemia/physiopathology , Blood Transfusion, Intrauterine , Fetal Diseases/physiopathology , Fetal Heart/physiology , Ultrasonography, Prenatal/methods , Ventricular Function/physiology , Adult , Anemia/therapy , Blood Volume/physiology , Erythrocyte Count , Female , Fetal Diseases/therapy , Fetal Heart/diagnostic imaging , Gestational Age , Heart Ventricles/diagnostic imaging , Heart Ventricles/embryology , Humans , Myocardial Contraction/physiology , PregnancyABSTRACT
Yeast cells growing in the presence of glucose or a related rapidly-fermented sugar differ strongly in a variety of physiological properties compared to cells growing in the absence of glucose. Part of these differences appear to be caused by the protein kinase A (PKA) and related signal transduction pathways. Addition of glucose to cells previously deprived of glucose triggers cAMP accumulation, which is apparently mediated by the Gpr1-Gpa2 G-protein coupled receptor system. However, the resulting effect on PKA-controlled properties is only transient when there is no complete growth medium present. When an essential nutrient is lacking, the cells arrest in the stationary phase G0. At the same time they acquire all characteristics of cells with low PKA activity, even if there is ample glucose present. When the essential nutrient is added again, a similar PKA-dependent protein phosphorylation cascade is triggered as observed after addition of glucose to glucose-deprived cells, but which is not cAMP-mediated. Because the pathway involved requires a fermentable carbon source and a complete growth medium, at least for its sustained activation, it has been called "fermentable growth medium (FGM)-induced pathway."
ABSTRACT
In frozen dough applications a prefermentation period during the preparation of the dough is unavoidable and might also be important to obtain bread with a good texture. A major disadvantage of the prefermentation period is that it is associated with a rapid loss of the freeze resistance of the yeast cells. A major goal for the development of new baker's yeast strains for use in frozen dough applications is the availability of strains that maintain a better freeze resistance during the prefermentation period. We have isolated mutants that retain a better stress resistance during the initiation of fermentation. Some of these showed the same growth rate and fermentation capacity as the wild type cells. These mutants are called 'fil', for deficient infermentation induced loss of stress resistance. First we used laboratory strains and heat stress treatment, given shortly after the initiation of fermentation, as the selection protocol. The first two mutants isolated in this way were affected in the glucose-activation mechanism of the Ras-cAMP pathway. The fil1 mutant had a partially inactivating point mutation in CYR1, the gene encoding adenylate cyclase, while fil2 contained a nonsense mutation in GPR1. GPR1 encodes a member of the G-protein coupled receptor family which acts as a putative glucose receptor for activation of the Ras-cAMP pathway. In a next step we isolated fil mutants directly in industrial strains using repetitive freeze treatment of doughs as selection protocol. Surviving yeast strains were tested individually for maintenance of fermentation capacity after freeze treatment in laboratory conditions and also for the best performing strains in frozen doughs prepared with yeast cultivated on a pilot scale. The most promising mutant, AT25, displayed under all conditions a better maintenance of gassing power during freeze-storage. It was not affected in other commercially important properties and will now be characterised extensively at the biochemical and molecular level.
Subject(s)
Fermentation , Freezing , Saccharomyces cerevisiae/metabolism , Mutation , Trehalose/metabolismABSTRACT
Using repetitive freezing and thawing, different mutant industrial Saccharomyces cerevisiae strains with increased freeze resistance have been isolated. To get a better insight in the mechanisms responsible for this elevated resistance and to give us the opportunity to modify other strains so that they become more suitable for use in frozen dough preparations, we applied the microarray technology in order to identify genes that are differentially expressed in a freeze-resistant mutant when compared to a freeze-sensitive industrial yeast strain.
Subject(s)
Fermentation , Genes, Fungal , Saccharomyces cerevisiae/genetics , Freezing , Saccharomyces cerevisiae/metabolismABSTRACT
In the yeast Saccharomyces cerevisiae the accumulation of cAMP is controlled by an elaborate pathway. Only two triggers of the Ras adenylate cyclase pathway are known. Intracellular acidification induces a Ras-mediated long-lasting cAMP increase. Addition of glucose to cells grown on a non-fermentable carbon source or to stationary-phase cells triggers a transient burst in the intracellular cAMP level. This glucose-induced cAMP signal is dependent on the G alpha-protein Gpa2. We show that the G-protein coupled receptor (GPCR) Gpr1 interacts with Gpa2 and is required for stimulation of cAMP synthesis by glucose. Gpr1 displays sequence homology to GPCRs of higher organisms. The absence of Gpr1 is rescued by the constitutively activated Gpa2Val-132 allele. In addition, we isolated a mutant allele of GPR1, named fil2, in a screen for mutants deficient in glucose-induced loss of heat resistance, which is consistent with its lack of glucose-induced cAMP activation. Apparently, Gpr1 together with Gpa2 constitute a glucose-sensing system for activation of the cAMP pathway. Deletion of Gpr1 and/or Gpa2 affected cAPK-controlled features (levels of trehalose, glycogen, heat resistance, expression of STRE-controlled genes and ribosomal protein genes) specifically during the transition to growth on glucose. Hence, an alternative glucose-sensing system must signal glucose availability for the Sch9-dependent pathway during growth on glucose. This appears to be the first example of a GPCR system activated by a nutrient in eukaryotic cells. Hence, a subfamily of GPCRs might be involved in nutrient sensing.
Subject(s)
Cyclic AMP/metabolism , GTP-Binding Protein alpha Subunits , Glucose/metabolism , Heterotrimeric GTP-Binding Proteins , Receptors, Cell Surface/metabolism , Receptors, G-Protein-Coupled , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Blotting, Northern , Cyclic AMP-Dependent Protein Kinases/metabolism , Fungal Proteins/metabolism , GTP-Binding Proteins/metabolism , Gene Expression Regulation, Fungal , Genes, Fungal , Molecular Sequence Data , Mutation , Receptors, Cell Surface/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Signal TransductionABSTRACT
Adenylate cyclase activity in Saccharomyces cerevisiae is dependent on Ras proteins. Both addition of glucose to glucose-deprived (derepressed) cells and intracellular acidification trigger an increase in the cAMP level in vivo. We show that intracellular acidification, but not glucose, causes an increase in the GTP/GDP ratio on the Ras proteins independent of Cdc25 and Sdc25. Deletion of the GTPase-activating proteins Ira1 and Ira2, or expression of the RAS2(val19) allele, causes an enhanced GTP/GDP basal ratio and abolishes the intracellular acidification-induced increase. In the ira1Delta ira2Delta strain, intracellular acidification still triggers a cAMP increase. Glucose also did not cause an increase in the GTP/GDP ratio in a strain with reduced feedback inhibition of cAMP synthesis. Further investigation indicated that feedback inhibition by cAPK on cAMP synthesis acts independently of changes in the GTP/GDP ratio on Ras. Stimulation by glucose was dependent on the Galpha-protein Gpa2, whose deletion confers the typical phenotype associated with a reduced cAMP level: higher heat resistance, a higher level of trehalose and glycogen and elevated expression of STRE-controlled genes. However, the typical fluctuation in these characteristics during diauxic growth on glucose was still present. Overexpression of Ras2(val19) inhibited both the acidification- and glucose-induced cAMP increase even in a protein kinase A-attenuated strain. Our results suggest that intracellular acidification stimulates cAMP synthesis in vivo at least through activation of the Ras proteins, while glucose acts through the Gpa2 protein. Interaction of Ras2(val19) with adenylate cyclase apparently prevents its activation by both agonists.
Subject(s)
Cyclic AMP/metabolism , Fungal Proteins/metabolism , GTP-Binding Protein alpha Subunits , GTP-Binding Proteins/metabolism , Glucose/metabolism , Heterotrimeric GTP-Binding Proteins , Saccharomyces cerevisiae/metabolism , Signal Transduction , ras Proteins/metabolism , Adenylyl Cyclases/metabolism , Cell Cycle Proteins/metabolism , Cyclic AMP/biosynthesis , Down-Regulation , Hydrogen-Ion Concentration , Phosphoprotein Phosphatases/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins , rap GTP-Binding Proteins , ras-GRF1ABSTRACT
The quality of brewing strains is, in large part, determined by their flocculation properties. By classical genetics, several dominant, semidominant and recessive flocculation genes have been recognized. Recent results of experiments to localize the flocculation genes FLO5 and FLO8, combined with the in silicio analysis of the available sequence data of the yeast genome, have revealed that the flocculation genes belong to a family which comprises at least four genes and three pseudogenes. All members of this gene family are located near the end of chromosomes, just like the SUC, MEL and MAL genes, which are also important for good quality baking or brewing strains. Transcription of the flocculation genes is repressed by several regulatory genes. In addition, a number of genes have been found which cause cell aggregation upon disruption or overexpression in an as yet unknown manner. In total, 33 genes have been reported that are involved in flocculation or cell aggregation.
Subject(s)
Genes, Dominant/genetics , Genes, Fungal/genetics , Membrane Proteins/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Cell Adhesion/genetics , Mannose-Binding Lectins , Molecular Sequence Data , Saccharomyces cerevisiae/cytology , TelomereABSTRACT
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.
Subject(s)
Chromosome Mapping , Fungal Proteins/genetics , Genes, Fungal , Membrane Proteins/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Flocculation , Mannose-Binding LectinsABSTRACT
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.
Subject(s)
Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Genes, Fungal , Membrane Proteins/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Transcription, Genetic , Flocculation , Mannose-Binding LectinsABSTRACT
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.
Subject(s)
Fungal Proteins/genetics , Genes, Fungal/genetics , Membrane Proteins/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Base Sequence , Molecular Sequence Data , Open Reading Frames/genetics , Repetitive Sequences, Nucleic Acid , Sequence AnalysisABSTRACT
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.
Subject(s)
Chromosomes, Fungal , Genes, Fungal , Saccharomyces cerevisiae/genetics , Chromosome Mapping , DNA, Fungal/genetics , Genetic Complementation Test , Plasmids , Restriction Mapping , Saccharomyces cerevisiae/physiologyABSTRACT
In a group of 84 adult male workers in an industrial plant, who used a common shower room, the applicability of the adhesive tape-KOH technique for routine mycologic examination was investigated. The tape technique was compared with the conventional cover slip technique in interdigital and plantar lesions. The tape technique gave more frequent positive results than the cover slip technique (74% and 57% respectively). However, examination of the tapes was more difficult than that of the cover slips. The latter method therefore appears more appropriate for routine microscopical mycologic examination.
Subject(s)
Dermatomycoses/microbiology , Fungi/isolation & purification , Adult , Bacteriological Techniques , Foot Dermatoses/microbiology , Humans , Male , Tinea Pedis/microbiologyABSTRACT
Yeast mitochondrial RNA polymerase can bind specifically to promoter-containing DNA fragments in vitro as detected by DNAse I or methidiumpropyl-EDTA. Fe(II) protection assays and gel retardation experiments. Retardation of RNA polymerase-DNA complexes was most pronounced when the promoter was located in the middle of a DNA fragment and was diminished when RNA polymerase was bound near one of the ends. This indicates that upon RNA polymerase-binding the DNA undergoes a conformational change which is most likely a bend. The degree of introduced bending correlated with the efficiency of transcription and promoter-binding in a series of promoter mutants, suggesting that bending is a functional event during promoter utilisation.