The chromosomal constitution of wine strains of Saccharomyces cerevisiae

A general procedure is described for determining the chromosomal constitution of industrial strains of Saccharomyces cerevisiae based on analysis of segregation frequencies for input markers among random spore progeny of industrial-laboratory strain hybrids. The multiply auxotrophic haploid testers used carried a dominant erythromycin-resistance marker, allowing hybrids to be selected in mass matings with spores produced by the wild-type industrial strains. Analysis of a number of independent crosses between the haploid testers and an unselected population of spores of each wine strain distinguished between disomic, trisomic and tetrasomic chromosomal complements in the parents. Possible explanations for a significant class of aberrant segregation frequencies are discussed. Results of the analysis indicate that UCD Enology 522 (Montrachet) is diploid and possibly trisomic for chromosome VII; 522X is diploid; UCD Enology 505 (California Champagne) is disomic for chromosome XVI, trisomic for chromosomes I, II, III, VI, VIII, IX, X, XII, XV, tetrasomic for chromosomes IV, XI, XIII, XIV and either trisomic or tetrasomic for chromosomes V and VII; and that UCD Enology 595 (Pasteur Champagne) is disomic for chromosomes I, II, III, IX, XVI, trisomic for chromosomes IV, VI, X, XII, XIV, XV, tetrasomic for chromosomes V, VIII, XI, XIII and either disomic or tetrasomic for chromosome VII.     

Common industrial sake yeast strains have three copies of the AQY1-ARR3 region of chromosome XVI in their genomes

Genomic analysis of industrial yeast strains is important for exploitation of their potential. We analysed the genomic structure of the most widely used sake yeast strain, Kyokai no. 7 (K7), by DNA microarray. Since the analysis suggested that the copy number of the AQY1-ARR3 region in the right arm of chromosome XVI was amplified, we performed Southern blot analysis using the AQY1 gene as a probe. The probe hybridized to three bands in the widely used sake strains derived from K7, but only to one band of 1.4 kb in the laboratory strains. Since the extra two bands were not observed in old sake strains, or in other industrial strains, the amplification of this region appeared to be specific for the widely used sake strains. The copy number of the AQY1-ARR3 region appeared to have increased by chromosomal translocation, since chromosomal Southern blot analysis revealed that the AQY1 probe hybridized to chromosomes IV and XIII, in addition to chromosome XVI, in which AQY1 of the laboratory strain is encoded. The chromosomal translocation was also confirmed by PCR analysis using primers that amplify the region containing the breakpoint. Cloning and sequencing of cosmids that encode the AQY1-ARR3 region revealed that this region is flanked by TG(1-3) repeats on the centromere-proximal side in chromosomes IV and XIII, suggesting that amplification of this region occurred by homologous recombination through TG(1-3) repeats. These results demonstrate the genomic characteristics of the modern widely used sake strains that discriminate them from other strains.     

Cloning, sequencing and application of the LEU2 gene from the sour dough yeast Candida milleri

We have cloned by complementation in Saccharomyces cerevisiae and sequenced a LEU2 gene from the sour dough yeast Candida milleri CBS 8195 and studied its chromosomal location. The LEU2 coding sequence was 1092 nt long encoding a putative beta-isopropylmalate dehydrogenase protein of 363 amino acids. The nucleotide sequence in the coding region had 71.6% identity to S. cerevisiae LEU2 sequence. On the protein level, the identity of C. milleri Leu2p to S. cerevisiae Leu2p was 84.1%. The CmLEU2 DNA probe hybridized to one to three chromosomal bands and two or three BamHI restriction fragments in C. milleri but did not give any signal to chromosomes or restriction fragments of C. albicans, S. cerevisiae, S. exiguus or Torulaspora delbrueckii. Using CmLEU2 probe for DNA hybridization makes it easy to quickly identify C. milleri among other sour dough yeasts.     

Effect of Carrier and Administration on Luteinizing Hormone Release By Gonadotropin Releasing Hormone

Ovariectomized ewes in groups of four were administered either saline (I) or 50 μg of gonadotropin releasing hormone (II to VII). Methods of administration and carriers were: II, intramuscular saline; III, intravenous saline; IV, subcutaneous saline; V, subcutaneous gelatin; VI, subcutaneous carboxymethylcellulose; and VII, subcutaneous gelatin capsule. Only subcutaneous administration of gonadotropin releasing hormone in the gelatin capsule (VII) prolonged the interval from treatment to luteinizing hormone peak; however, there were fewer ewes with peak luteinizing hormone concentations equal to or greater than 100 ng/ml in VII than II, IV, V, and VI. Ewes in V, VI, and VII had luteinizing hormone surges of longer duration than II and III. Ewes in VII had the longest luteinizing hormone surge, which was 7.5 h, even though luteinizing hormone concentrations did not return to pretreatment concentrations by 8 h posttreatment in three of the four ewes.     

Genomic reorganization between two sibling yeast species,Saccharomyces bayanus and Saccharomyces cerevisiae

Genomic comparison of two sibling yeast species, Saccharomyces bayanus and Saccharomyces cerevisiae, was performed by Southern blot analysis with various S. cerevisiae gene probes following electrophoretic karyotyping. Fifteen genes on chromosome IV of S. cerevisiae were examined and classified into two groups. Gene probes of CEN4 and TRP1, as well as six other genes located on the left arm of the chromosome hybridized to a 1100-kb chromosome of S. bayanus that is smaller than chromosome IV of S. cerevisiae. On the other hand, probes of seven genes located on the right arm of chromosome IV hybridized to a 1350-kb chromosome that is homeologous to chromosome IV, judging from its size. Two genes located on the left arm of chromosome II hybridized to the 1350-kb chromosome, while four genes on the right arm hybridized to the 1100-kb chromosome. These pieces of evidence indicate that chromosomes II and IV of S. cerevisiae are rearranged into 1350-kb and 1100-kb chromosomes in S. bayanus. Furthermore, it is suggested that chromosome XV is rearranged into two chromosomes (800 and 850 kb in size) in S. bayanus. The translocation points of chromosomes II and IV were delimited using S. cerevisiae prime clone membranes. The results indicated that the translocation points are located close to the FUR4 locus on chromosome II and close to the RAD57 locus on chromosome IV.     

Co-existence of two types of chromosome in the bottom fermenting yeast,Saccharomyces pastorianus

The bottom fermenting yeasts in our collection were classified as Saccharomyces pastorianus on the basis of their DNA relatedness. The genomic organization of bottom fermenting yeast was analysed by Southern hybridization using eleven genes on chromosome IV, six genes on chromosome II and five genes on chromosome XV of S. cerevisiae as probes. Gene probes constructed from S. cerevisiae chromosomes II and IV hybridized strongly to the 820-kb chromosome and the 1500-kb chromosome of the bottom fermenting yeast, respectively. Five gene probes constructed from segments of chromosome XV hybridized strongly to the 1050-kb and the 1000-kb chromosomes. These chromosomes are thought to be S. cerevisiae-type chromosomes. In addition, these probes also hybridized weakly to the 1100-kb, 1350-kb, 850-kb and 700-kb chromosome. Gene probes constructed from segments including the left arm to TRP1 of chromosome IV and the right arm of chromosome II hybridized to the 1100-kb chromosome of S. pastorianus. Gene probes constructed using the right arm of chromosome IV and the left arm of chromosome II hybridized to the 1350-kb chromosome of S. pastorianus. These results suggested that the 1100-kb and 1350-kb chromosomes were generated by reciprocal translocation between chromosome II and IV in S. pastorianus. Three gene probes constructed using the right arm of chromosome XV hybridized weakly to the 850-kb chromosome, and two gene probes from the left arm hybridized weakly to the 700-kb chromosome. These results suggested that chromosome XV of S. cerevisiae was rearranged into the 850-kb and 700-kb chromosomes in S. pastorianus. These weak hybridization patterns were identical to those obtained with S. bayanus. Therefore, two types of chromosome co-exist independently in bottom fermenting yeast: one set which originated from S. bayanus and another set from S. cerevisiae. This result supports the hypothesis that S. pastorianus is a hybrid of S. cerevisiae and S. bayanus. © 1998 John Wiley & Sons, Ltd.     

Screening of Israeli Holstein-Friesian cattle for restriction fragment length polymorphisms using homologous and heterologous deoxyribonucleic acid probes

Genomic DNA of Israeli Holstein-Friesian dairy cattle were screened with a battery of 17 cloned or subcloned DNA probes in an attempt to document restriction fragment length polymorphisms at a number of genetic loci. Restriction fragment length polymorphisms were observed at the chymosin, oxytocin-neurophysin I, lutropin beta, keratin III, keratin VI, keratin VII, prolactin, and dihydrofolate reductase loci. Use of certain genomic DNA fragments as probes produced hybridization patterns indicative of satellite DNA at the respective loci. Means for distinguishing hybridizations to coding sequences for unique genes from those to satellite DNA were developed. Results of this study are discussed in terms of strategy for the systematic development of large numbers of bovine genomic polymorphisms.     

Characterization of multiple forms of lactophorin isolated from bovine milk whey

A glycoprotein that reacted to the antisoluble glycoprotein of bovine milk fat globule membrane was purified from the proteose-peptone of whey and designated lactophorin. Lactophorin was separated into seven components. Lactophorin and the seven components were rich in aspartic acid, threonine, serine, glutamic acid, leucine, and lysine. The content of threonine, glycine, isoleucine, lysine, and arginine varied in each component. The ratio of fucose, mannose, galactose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid of lactophorin, which contains about 18% saccharide, were 1, 6.6, 10.3, 5.5, 9.7, and 11.6, respectively, while the respective ratio of the seven components were 1, 5 to 6, 7 to 9, 3 to 4, 6 to 8, and 4 to 12. Sialic acid content varied in each component. Protein-carbohydrate linkage was N- and o-glucoside linkage. Lactophorin consisted of seven polypeptides (I to VII) with apparent molecular weights 17,000 to 67,000. Bands I, II, VI, and VII were glycoprotein. Bands VI and VII were major and had antigenicity to anti-soluble glycoprotein, while bands I to V were minor polypeptides. Component 1 consisted of only one polypeptide (VII), whereas the components 2 to 7 contained two major (VI, VII, or both) and several minor polypeptides. The sedimentation pattern of each component was a single and almost symmetrical peak. Sedimentation coefficient was 3.79 to 5.64 S and also varied in lactophorin. The results indicate that lactophorin has multiple forms.     

Character impact odorants of the apple cultivars Elstar and Cox Orange

Extracts obtained from Elstar and Cox-Orange apples (Malus var. domestica Borkh.) were screened for potent odorants by aroma extract dilution analysis (AEDA). (E)-beta-Damascenone (I), (E)-beta-damascone (II), hexanal (III), (Z)-3-hexenal (IV), (Z)-2-nonenal (V), linalool (VI), butyl acetate (VII), 2-methylbutyl acetate (VIII) and hexyl acetate (IX) were identified in Elstar and I, III-V, ethyl butanoate (X), ethyl 2-methylbutanoate (XI), 1-octen-3-one (XII), (E)-2-octenal (XIII), methional (XIV), (E)-2-nonenal (XV), eugenol (XVI) and wine lactone (XVII) in Cox Orange. The release of odorants by the cultivars was analysed by gas chromatography-olfactometry of headspace samples. Esters X and XI showed the highest odour activities in the air above Elstar apples and ester X, acetaldehyde (XVIII), 2-methylbutanol (XIX) and ethyl methylpropanoate (XX) in that of Cox Orange. Preparation of a homogenate changed the headspace composition of potent odorants. Hexyl acetate (IX) became the key odorant of Elstar and aldehydes III and IV those of Cox Orange. Quantification and calculation of odour activity values indicated I, IV, V, VII, VIII and IX as the character impact odorants of the fruit flesh of the two cultivars. Elstar contained more I, V, VII and IX and Cox Orange more IV than the other cultivar. The concentrations of VIII were similar.     

Identification of fluorescent U(V) and U(VI) microparticles in a multispecies biofilm by confocal laser scanning microscopy and fluorescence spectroscopy

Fluorescent uranium(V) and uranium(VI) particles were observed for the first time in vivo by a combined laser fluorescence spectroscopy and confocal laser scanning microscopy approach in a living multispecies biofilm grown on biotite plates. These particles ranged between 1 and 7 um in width and up to 20 microm in length and were located at the bottom and at the edges of biofilms colonies. Analysis of amplified 16S rRNA fragments and fluorescence in situ hybridization were used to characterize the biofilm communities. Laser fluorescence spectroscopy was used to identify these particles. The particles showed either a characteristic fluorescence spectrum in the wavelength range of 415-475 nm, indicative for uranium(V), or in the range of 480-560 nm, which is typical for uranium(VI). Particles of uranium(V) as well as uranium(VI) were simultaneously observed in the biofilms. These uranium particles were attributed for uranium(VI) to biologically mediated precipitation and for uranium(V) to redox processes taking place within the biofilm. The detection of uranium(V) in a multispecies biofilm was interpreted as a short-lived intermediate of the uranium(VI) to uranium(IV) redox reaction. Its presence clearly documents that the uranium(VI) reduction is not a two electron step but that only one electron is involved.     

Sequence Analysis of 203 Kilobases from Saccharomyces cerevisiae Chromosome VII

The nucleotide sequences of five major regions from chromosome VII of Saccharomyces cerevisiae have been determined and analysed. These regions represent 203 kilobases corresponding to approximately one-fifth of the complete yeast chromosome VII. Two fragments originate from the left arm of this chromosome. The first one of about 15·8 kb starts approximately 75 kb from the left telomere and is bordered by the SKI8 chromosomal marker. The second fragment covers the 72·6 kb region between the chromosomal markers CYH2 and ALG2. On the right chromosomal arm three regions, a 70·6 kb region between the MSB2 and the KSS1 chromosomal markers and two smaller regions dominated by the KRE11 marker and another one in the vicinity of the SER2 marker were sequenced. We found a total of 114 open reading frames (ORFs), 13 of which were completely overlapping with larger ORFs running in the opposite direction. A total of 44 yeast genes, the physiological functions of which are known, could be precisely mapped on this chromosome. Of the remaining 57 ORFs, 26 shared sequence homologies with known genes, among which were 13 other S. cerevisiae genes and five genes from other organisms. No homology with any sequence in the databases could be found for 31 ORFs. Furthermore, five Ty elements were found, one of which may not be functional due to a frame shift in its Ty1B amino acid sequence. The five chromosomal regions harboured five potential ARS elements and one sigma element together with eight tRNA genes and two snRNAs, one of which is encoded by an intron of a protein-coding gene. © 1997 by John Wiley & Sons, Ltd.     

Molecular identification of yeast species associated with 'Hamei'--a traditional starter used for rice wine production in Manipur, India

In Manipur state of North-Eastern India, wine from glutinous rice using traditional solid state starter called 'Hamei' is particularly interesting because of its unique flavour. A total of 163 yeast isolates were obtained from fifty four 'Hamei' samples collected from household rice wine preparations in tribal villages of Manipur. Molecular identification of yeast species was carried out by analysis of the restriction digestion pattern generated from PCR amplified internal transcribed spacer region along with 5.8S rRNA gene (ITS1-5.8S-ITS2). Seventeen different restriction profiles were obtained from the size of PCR products and the restriction analysis with three endonucleases (Hae III, Cfo I and Hinf I). Nine groups were identified as S. cerevisiae, Pichia anomala, Trichosporon sp., Candida tropicalis, Pichia guilliermondi, Candida parapsilosis, Torulaspora delbrueckii, Pichia fabianii and Candida montana by comparing this ITS-RFLP profile with type strains of common wine yeasts, published data and insilico analysis of ITS sequence data available in CBS yeast database. ITS-RFLP profile of eight groups was not matching with available database of 288 common wine yeast species. The most frequent yeast species associated with 'Hamei' were S. cerevisiae (32.5%), P. anomala (41.7%) and Trichosporon sp. (8%). The identity of major groups was confirmed by additional restriction digestion of ITS region with Hind III, EcoRI, Dde I and Msp I. The genetic diversity of industrially important S. cerevisiae group was investigated using Pulsed Field Gel Electrophoresis (PFGE). Although most of the 53 strains of S. cerevisiae examined were exhibited a common species specific pattern, a distinct degree of chromosomal length polymorphism and variable number of chromosomal DNA fragments were observed with in the species. Cluster analysis showed seven major karyotypes (K1-K7) with more than 83% similarity. The karyotype pattern K1 was the most frequent (67.9%) among the strains from different samples. Other karyotypes K2-K7 were very unique with less than 80% similarity. Finally using mitochondrial DNA restriction analysis (mt-DNA RFLP), S. cerevisiae strains belonging to the major karyotype K1 were distinctly differentiated with highly polymorphic bands by Hinf I and Hae III endonucleases.     

In situ bioreduction of technetium and uranium in a nitrate-contaminated aquifer

The potential to stimulate an indigenous microbial community to reduce a mixture of U(VI) and Tc(VII) in the presence of high (120 mM) initial NO3- co-contamination was evaluated in a shallow unconfined aquifer using a series of single-well, push-pull tests. In the absence of added electron donor, NO3-, Tc(VII), and U(VI) reduction was not detectable. However, in the presence of added ethanol, glucose, or acetate to serve as electron donor, rapid NO3- utilization was observed. The accumulation of NO2-, the absence of detectable NH4+ accumulation, and the production of N2O during in situ acetylene-block experiments suggest that NO3- was being consumed via denitrification. Tc(VII) reduction occurred concurrently with NO3- reduction, but U(VI) reduction was not observed until two or more donor additions resulted in iron-reducing conditions, as detected by the production of Fe(II). Reoxidation/remobilization of U(IV) was also observed in tests conducted with high (approximately 120 mM) but not low (approximately 1 mM) initial NO3- concentrations and not during acetylene-block experiments conducted with high initial NO3-. These results suggest that NO3(-)-dependent microbial U(IV) oxidation may inhibit or reverse U(VI) reduction and decrease the stability of U(IV) in this environment. Changes in viable biomass, community composition, metabolic status, and respiratory state of organisms harvested from down-well microbial samplers deployed during these tests were consistent with the conclusions that electron donor additions resulted in microbial growth, the creation of anaerobic conditions, and an increase in activity of metal-reducing organisms (e.g., Geobacter). The results demonstrate that it is possible to stimulate the simultaneous bioreduction of U(VI) and Tc(VII) mixtures commonly found with NO3- co-contamination at radioactive waste sites.     

几种调控酒精代谢物质解酒效果的比较研究

目的:研究几种调控酒精代谢物质在解酒方面的效果差异比较。方法:取NIH小鼠若干只,按自配产品随机分组,通过解酒实验、防醉实验和攀附实验观察各调控酒精代谢物质的解酒效果差异。结果:解酒、防醉效果由高至低依次为产品V>产品VI>产品II>产品III>产品VII>产品VIII>产品IV>产品I,抗攀附功能障碍能力由高至低依次为产品II>产品III>产品V>产品VI>产品VII>产品VIII>产品I>产品IV。结论:解酒配方中加入酸类物质的、糖酸比值较大的和加入葛根素或茶多酚的,解酒、防醉和纠正运动失调的作用效果更为明显。其中茶多酚的解酒、防醉效果比葛根素明显,而葛根素的纠正运动失调作用效果比茶多酚明显。     

基于lolB和toxR基因的水产品中霍乱弧菌双重PCR检测方法

选择lolB和toxR两种基因序列设计2对特异性引物,建立一种针对霍乱弧菌所有生物型菌株的双重PCR检测方法,扩增目的片段大小分别为519bp和779bp。结果表明:在同步扩增中,仅霍乱弧菌模板可同时扩增出2种基因片段,4株对照菌模板无任何扩增条带;敏感性检测结果显示,该双重PCR最低能检测3.42×103CFU/mL菌落数的霍乱弧菌。所建立的基于lolB和toxR两种基因的双重PCR检测方法特异性强、敏感性高、方法简单、用时短,可用于霍乱弧菌检测。     

Reduction of U(VI) Incorporated in the Structure of Hematite

U(VI) doped hematite was synthesized and exposed to two different organic reductants with E(0) of 0.23 and 0.70 V. A combination of HAADF-TEM and EXAFS provided evidence that uranium was incorporated in hematite in uranate, likely octahedral coordination. XPS indicated that structurally incorporated U(VI) was reduced to U(V), whereas non-incorporated U(VI) was reduced to U(IV). Specifically, the experiments indicate that U(V) was the dominant oxidation state of uranium in hematite around Eh -0.24 to -0.28 V and pH 7.7-8.6 for at least up to 5 weeks of reaction time. U(V), but not U(IV), was also detected in hematite at Eh +0.21 V (pH 7.1-7.3). The results support the hypothesis, based on previous experimental and theoretical work, that the stability field of U(V) is widened relative to U(IV) and U(VI) in uranate coordination environments where the coordination number of U is less than 8.     

Possible gene interchange between plasmid and chromosome in yeast

Genomic DNAs isolated from 420 yeast strains stocked in the Department of Fermentation Technology, Hiroshima University (HUT) were screened for the presence of a plasmid sequence both as plasmid or in the chromosome. Five DNA samples gave rise to a positive hybridization signal when 32P-labelled Zygosaccharomyces plasmid pSR1 was used as a probe. Two among these contain hybridizing sequences as plasmids while the other three apparently were chromosomal. Two chromosomal DNA segments of HUT 7195 (Zygosaccharomyces spp.) which hybridized with pSR1 probe were cloned and sequenced. Both DNAs hybridized with a plasmid sequence covering the P gene of pSR1. One of the two segments contains a large open reading frame which can encode 410 amino acid residues. The deduced amino acid sequence is closely related with that of the P gene of pSR1. The present finding suggests that there was an interchange(s) of a gene between yeast plasmid(s) and chromosomes.     

The electrophoretic karyotype of two strains of Candida albicans by transverse alternate field electrophoresis reveals higher number of chromosomes ranging from 1 to 3·5 Mb

The advent of the powerful electrophoretic technique, pulsed field gel electrophoresis, first developed on the yeast Saccharomyces cerevisiæ, has brought a vital impulse to the genetic study on the opportunistic pathogen Candida albicans. We report here on sizing and numbering of Candida chromosomes using transverse alternate field electrophoresis. Our results indicate the occurrence of nine to ten electrophoretic bands (depending on type of Candida strain), that range in approximate size from 1 to 3·5 Mbp, and may account for a higher overall chromosome number, because at least two of these bands appear to be doublets. This number of bands, with smaller size, is considerably higher than previously reported.     

主要谷物水分测定方法比较研究

对大米、小麦和玉米水分测定方法进行了比较研究.水分测定方法包括:(Ⅰ)GB/T 5497 105℃恒重法(烘箱干燥105℃,3 h)、(Ⅱ)GB/T 5497定温定时烘干法(烘箱干燥130℃,40 min)、(Ⅲ)ISO712粉状物测定(烘箱干燥130℃,90 min)、(Ⅳ)ISO712常规法(烘箱干燥130℃,2 h)、(Ⅴ)GB/T 10362粉碎玉米水分测定(烘箱干燥130℃,4 h)、(Ⅵ)GB/T 10362整粒玉米水分测定(烘箱干燥130℃,38 h)和(Ⅶ)M105/72(烘箱干燥105℃,72 h).在小麦和大米水分测定及同一类型的粮食水分测定中,方法(Ⅳ)测定值要大于方法(Ⅰ)、(Ⅱ)、(Ⅲ);其标准偏差要小于这三种方法,精确度较高,方法(Ⅰ)、(Ⅱ)、(Ⅲ)测定值与方法(Ⅳ)测定值的相关系数较高.玉米水分测定中,方法(Ⅴ)测定值和精确度大于方法(Ⅰ)、(Ⅱ),这两种方法的测定结果与方法(Ⅴ)的相关系数较高.     

Characterization of adenylyl cyclases in cultured human granulosa cells

Granulosa cells play an essential role in follicular development and formation of corpora lutea. Many functions of granulosa-lutein cells are controlled by activation of G protein-coupled receptors and the formation of cyclic AMP (cAMP) by adenylyl cyclase. There are at least nine mammalian adenylyl cyclase isoenzymes, which show different sensitivities towards other signalling systems. The aim of this study was to identify the types of adenylyl cyclase present in human granulosa cells and to investigate its functional regulation by G proteins, calcium and the protein kinase C and A pathways. Granulosa cells were obtained from women undergoing IVF. The cells were maintained in primary culture and they consistently expressed mRNA coding for adenylyl cyclase I, III, VI, VII and IX. The signals for adenylyl cyclase V and VIII were more variable among patients and there was no signal for adenylyl cyclase II. The expression of multiple adenylyl cyclase proteins was confirmed by immunochemistry with subtype-specific antibodies. The formation of cAMP in cultured cells was stimulated many times by hCG (EC(50) value 4.2 iu ml(-1)) and by prostaglandin E(2) (PGE(2); EC(50) = 0.75 micromol l(-1)) in a concentration-dependent manner, thus confirming the presence of receptors coupled positively to G(s). The diterpene forskolin, which stimulates all isoforms of adenylyl cyclase except for adenylyl cyclase IX, increased cAMP formation to higher levels than hCG or PGE(2). The strong stimulation by forskolin indicates that adenylyl cyclase IX is unlikely to be the major source of cyclase activity in these cells. Basal and forskolin- or PGE(2)-stimulated adenylyl cyclase activity was amplified 1.5-2.0 times by phorbol-12,13-dibutyrate, indicating that protein kinase C-sensitive enzymes (for example, adenylyl cyclase types IV, V, VI or VII) may be active in the cells. In contrast, hCG-stimulated activity was inhibited (76 +/- 6%) by phorbol ester. Stimulation of G(i) with the alpha-adrenoceptor agonist clonidine inhibited hCG-induced cyclase activity. This finding indicates that adenylyl cyclase II and IV subtypes, which are stimulated by betagamma subunits released from G(i), are not predominant. Increases in intracellular free calcium concentrations by the ionophore A23187, the calcium-ATPase inhibitor thapsigargin or by fluprostenol, a selective prostanoid FP receptor agonist, which is known to open calcium channels in granulosa cells, or removal of calcium by EGTA, had no significant effects on basal or forskolin-stimulated formation of cAMP. These results indicate that subtypes adenylyl cyclase I, III and VIII, which are activated by calcium, and adenylyl cyclase V and VI, which are inhibited by calcium, are not dominant isoforms in granulosa-lutein cells. The protein kinase A inhibitor H89 had no effects on formation of cAMP; this finding rules out the involvement of adenylyl cyclase V and VI subtypes, which are subjected to negative feedback by protein kinase A. These results indicate that adenylyl cyclase VII is the dominant functional isoenzyme in human granulosa-lutein cells.