Purification and characterization of new mating pheromones of the ciliate Euplotes raikovi

Cell union in mating pairs in the ciliate Euplotes raikovi is controlled by a system of multiple mating types which are inherited with alleles codominant at the genetic locus mat and expressed via diffusible mating pheromones. The mating pheromones Er-2, Er-3, and Er-11 were purified from cells homozygous for the mat-2, mat-3, and mat-11 alleles, respectively. These pheromones are proteins of similar Mr (11,000-12,000) and acidity (pI 3.7-4.0) and are active at a concentration that varies from 2.9 X 10(-12) to 1.2 X 10(-11) M. Data on amino acid composition revealed that an unusually high amount of cysteine (12-15.7%) and poor contents of basic amino acids are common to every pheromone. On the basis of this uniformity in the main biochemical traits, which also holds for the previously purified pheromone Er-1, it was concluded that E. raikovi mating pheromones are members of a family of proteins structurally diversified from each other to varying extents.     

Identification and initial characterization of an autocrine pheromone receptor in the protozoan ciliate Euplotes raikovi

The polypeptide pheromone Er-1, purified from the ciliate Euplotes raikovi of mating type I and genotype mat-1/mat-1, was iodinated with 125I-Bolton-Hunter reagent to a sp act of 0.45-0.73 mu Ci/microgram of protein. This preparation of 125I-Er-1 bound specifically to high affinity binding sites on the same cells of mating type I. Binding of 125I-Er-1 occurred with an apparent Kd of 4.63 +/- 0.12 X 10(-9) M in cells in early stationary phase. It was estimated that these cells carry a total number of approximately 5 X 10(7) sites/cell, with a site density that falls in the range of 1,600-1,700/microns 2 of cell surface. Unlabeled Er-1, other homologous pheromones such as Er-2 and Er-10, antibodies specific for Er-1, and human IL-2 were shown to act as effective inhibitors of specific binding of 125I-Er-1 to mating type I cells. The "autocrine" nature of the identified specific high affinity binding sites for Er-1 was further substantiated by cross-linking experiments. These experiments revealed that mating type-I cell membranes contain one protein entity of Mr = 28,000 that is capable of reacting specifically with the homodimeric native form of Er-1.     

Purification, characterization, and amino acid sequence of the mating pheromone Er-10 of the ciliate Euplotes raikovi

The mating pheromone Er-10 from mat-10 homozygous Euplotes raikovi was purified by a three-step purification procedure with an overall yield of 62%. It was identified as a protein of molecular weight 8000 having an isoelectic point of 3.9. Its complete primary structure was determined by automated Edman degradation of the whole protein after performic acid oxidation and of peptides generated by cyanogen bromide and Staphylococcus aureus V8 protease. The proposed sequence is Asp1-Leu-Cys-Glu-Gln-Ser-Ala-Leu-Gln-Cys10-Asn-Glu-Gln-Gly-Cys-His -Asn-Phe-Cys- Ser20-Pro-Glu-Asp-Lys-Pro-Gly-Cys-Leu-Gly-Met30-Val-Trp-Asn- Pro-Glu-Leu-Cys- Pro38. The calculated molecular weight of 4191.7, which is in good agreement with the value of m/z 4190.7 obtained by fission fragment ionization mass spectrometry, suggests that the native structure is a dimer with three intrachain disulfide bonds in each subunit. The amino acid sequence is 43% identical with that of the E. raikovi mating pheromone Er-1, with the identities concentrated in the amino-terminal half. The half-cystine locations are conserved, but Er-10 is two residues shorter than Er-1. Prediction of the secondary structure suggests that Er-10 may also contain a helical structure at the amino terminus. These results indicate that the mating pheromones of E. raikovi form a homologous family.     

Isolation of the mating-inducing factor of the ciliate Euplotes

Numerous strains of different mating types of the marine ciliate Euplotes raikovi have been found to be autonomous excreters into the surrounding medium of specific mating-inducing factors (gamones) (Luporini, P et al., J exp zool 226 (1983) 1 [9]). The gamone from the mating type represented by strain 13 has been isolated and identified as a glycoprotein with a molecular weight (MW) of about 12 kD and a pI of 4. It has been termed euplomone r 13. At a concentration of 3 × 10⁻¹² M, euplomone r 13 specifically induces cells of a complementary mating type to unite in conjugation within 2 h.     

Mating Type Linked Mutations Which Disrupt the Uniparental Transmission of Chloroplast Genes in Chlamydomonas

In Chlamydomonas reinhardtii, chloroplast genomes are normally transmitted by the mating type plus (mt+) parent and mitochondrial genomes by the mating type minus (mt-) parent. In this paper we describe three new nuclear mutations, designated mat-3-1 to -3, which are tightly linked to the mt+ allele and permit high transmission of chloroplast genomes from the mt- parent, but have no effect on transmission of mitochondrial genomes. We also show that mat-1, reported by others to be a nuclear mutation linked to mt- which promotes transmission of chloroplast genomes by the mt- parent, is probably a vegetative diploid since it contains both mt+ and mt- alleles. Vegetative diploids behave as if they are mt- with respect to mating, but possess a level of chloroplast gene transmission intermediate between that of haploid mt- and mt+ stocks.     

中国禾本科植物内生真菌研究4. Epichloё yangzii的人工杂交

作者首先研究Epichloё yangzii在植株上的人工杂交,明确了供试菌株的交配型。然后将分离自无子座鹅观草属植物的23株"Neotyphodium属"真菌孢子分别与E.yangzii的子座杂交,其中发现有21株与E.yangzii(mat-1,mat-2)杂交不亲和,有2株与E.yangzii(mat-1)杂交亲和。利用tubB基因片段对8株"Neotyphodium属"真菌菌株进行系统发育分析,结果表明与E.yangzii杂交亲和的2个菌株和E.yangzii聚为一枝,而其它6个菌株形成独立的分枝,进一步证实了这2个菌株是有时在宿主植物上不形成子座的E.yangzii。这说明了在宿主植物上的人工杂交是区别有时不产子座的E.yangzii和Neotyphodium属真菌的有效手段。     

Interaction of alpha-agglutinin and a-agglutinin, Saccharomyces cerevisiae sexual cell adhesion molecules

alpha-Agglutinin and a-agglutinin are complementary cell adhesion glycoproteins active during mating in the yeast Saccharomyces cerevisiae. They bind with high affinity and high specificity: cells of opposite mating types are irreversibly bound by a few pairs of agglutinins. Equilibrium and surface plasmon resonance kinetic analyses showed that the purified binding region of alpha-agglutinin interacted similarly with purified a-agglutinin and with a-agglutinin expressed on cell surfaces. At 20 degrees C, the K(D) for the interaction was 2 x 10(-9) to 5 x 10(-9) M. This high affinity was a result of a very low dissociation rate ( approximately 2.6 x 10(-4) s(-1)) coupled with a low association rate (= 5 x 10(4) M(-1) s(-1)). Circular-dichroism spectroscopy showed that binding of the proteins was accompanied by measurable changes in secondary structure. Furthermore, when binding was assessed at 10 degrees C, the association kinetics were sigmoidal, with a very low initial rate. An induced-fit model of binding with substantial apposition of hydrophobic surfaces on the two ligands can explain the observed affinity, kinetics, and specificity and the conformational effects of the binding reaction.     

Cloning the Mating Types of the Heterothallic Fungus Podospora Anserina: Developmental Features of Haploid Transformants Carrying Both Mating Types

DNAs that encode the mating-type functions (mat+ and mat-) of the filamentous fungus Podospora anserina were cloned with the use of the mating-type A probe from Neurospora crassa. Cloning the full mat information was ascertained through gene replacement experiments. Molecular and functional analyses of haploid transformants carrying both mating types lead to several striking conclusions. Mat+ mat- strains are dual maters. However, the resident mat information is dominant to the mat information added by transformation with respect to fruiting body development and ascus production. Moreover, when dual mating mat+ mat- strains are crossed to mat+ or mat- testers, there is strong selection, after fertilization, that leads to the loss from the mat+ mat- nucleus of the mat information that matches that of the tester. Finally, the mat locus contains at least two domains, one sufficient for fertilization, the other necessary for sporulation.     

Developmental analysis of the cell recognition mechanism in the ciliate Euplotes raikovi.

Euplotes raikovi, like other ciliates, passes through a postconjugal immaturity, operatively identified by an apparent cell inability to form mating pairs under experimental conditions that are the same as those used for inducing mating at maturity. In cells homozygous for the gene mat-2, which controls the pheromone Er-2, Er-2 mRNA synthesis and mature Er-2 secretion were shown to start from the very beginning of the life cycle and continue throughout immaturity, although to extents estimated to be 5- to 10-fold lower than at maturity. In addition, experiments of 125I-Er-2 binding and crosslinking provided evidence that autocrine pheromone-binding sites, showing values of the dissociation constant of the order of 10(-9) M, are on the surface of immature cells. The number of these sites per cell was estimated to increase from less than 10(6) per cell of 5-7 fissions of age, to about 16 x 10(6) at maturity. These results were taken to suggest that a pheromone-receptor production is stimulated during immaturity by autocrine pheromone binding to cells and that this production might be essential for the development of a pheromone-receptor density high enough to transform the cell from "immature" to "adult," that is competent to respond as well to pheromones of conspecific, genetically different cells.     

A quantitative analysis of DNA extraction and purification from compost

We quantified both DNA and humic acid concentrations during the extraction and purification of DNA from compost. The DNA extraction method consisted of bead-beating with SDS for cell lysis, poly(ethylene glycol)-8000 precipitation for preliminary DNA purification, and chromatography on a 10-ml Sephadex G-200 column for final DNA purification. Direct microscopic observation of pre- and post-lysis samples revealed that 95.3+/-2.3% of native cells was lysed. Sixty-three percent of the original DNA was lost during purification, resulting in a final DNA yield of 18.2+/-3.8 microg DNA/g of wet compost. The humic acid content was reduced by 97% during the purification steps resulting in a final humic acid concentration of 27+/-4.7 ng humic acid/microl. The purified DNA fragments were up to 14 kbp in size and were sufficiently free of contaminants to allow both restriction enzyme digestion by four different enzymes and PCR amplification of 16S rDNA.     

Expression, purification, and characterization of the recombinant kringle 1 domain from tissue-type plasminogen activator.

A novel fusion protein expression plasmid that allows ready purification and subsequent facile release of the target molecule has been constructed and employed to express in Escherichia coli and purify the tissue plasminogen activator kringle 1 domain ([K1tPA] residues C92-C173). The resulting plasmid encodes the tight lysine-binding kringle (K)1 domain of human plasminogen ([K1HPg]) followed by a peptide (PfXa) containing a factor Xa-sensitive bond, downstream of which [K1tPA] was inserted. The recombinant (r) [K1HPg]PfXa[K1tPA] fusion polypeptide was purified from various cell fractions in one step by Sepharose-lysine affinity chromatography. After cleavage with fXa, the mixture was repassaged over Sepharose-lysine, whereupon the r-[K1tPA]-containing polypeptide passed unretarded through the column. A homogeneous preparation of this material was then obtained after a simple step employing fast protein liquid chromatography. The purified r-[K1tPA], which contained the amino acid sequence SNAS[K1tPA]S, provided an amino-terminal amino acid sequence, through at least 20 amino acid residues, that was identical to that predicted from the cDNA sequence. The molecular mass of r-SNAS[K1tPA]S, determined by electrospray mass spectrometry, was 9621.9 +/- 4.0 (expected molecular mass, 9623.65). 1H-NMR spectroscopy and thermal stability studies of r-SNAS[K1tPA]S revealed that the purified material was properly folded and similar to other isolated kringle domains. Additionally, employment of this methodology revealed that only a very weak interaction between epsilon-aminocaproic acid and the isolated r-[K1tPA] domain occurred.     

Use of claydite-immobilized oil-oxidizing microbial cells for purification of water from oil

Oil-oxidizing bacteria were isolated from oil-polluted soil and water samples and identified as Acinetobacter calcoaceticus K-4, Nocardia vaceinii K-8, Rhodococcus erythropolis EK-1, and Mycobacterium sp. K-2. It was found that immobilization of the bacteria on an expanded clay aggregate accelerated their growth and consumption of hydrocarbon substrates. It was also found that water polluted with 100 mg/l oil could be purified with Rhodococcus erythropolis EK-1 and Nocardia vaceinii K-8 cells immobilized in this way. The dependence of the degree of water purification on its flow rate, aeration, and availability of nitrogen and phosphorus sources was determined. The efficiency of water purification from oil by immobilized Rhodococcus erythropolis EK-1 cells at high flow rates (of up to 0.68 l/h), low aeration (of 0.1 l/l per min) and an intermittent supply of 0.01% diammonium phosphate reached 99.5-99.8%.     

Expression of seasonality in Merinos d'Arles ewes of different genotypes at the MT1 melatonin receptor gene

Spontaneous ovulatory activity (SOA) in spring has been used to study the out-of-season breeding ability of Merinos d'Arles (MA) ewes. Within this breed, an association was found between more intense seasonality and genotype -/- at a MnlI restriction site (allele - for its absence v. + for its presence) in Exon II of the MT1 receptor gene. This study was designed to ascertain whether this association results in a direct effect of the MT1 genotype on the expression of seasonality in MA ewes. In the first year of the study, genotyping of 314 MA ewes at locus MnlI was carried out and resulted in frequencies of 43.0%, 44.9% and 12.1% for genotypes +/+, +/- and -/-, respectively. The SOA of these ewes was determined in early April of two consecutive years by assaying plasma progesterone concentrations in two blood samples taken 9 days apart. Groups of 30 ewes of each homozygous genotype (+/+ and -/-) were identified from this population and their SOA was followed by taking blood samples at regular intervals between January and mid-April of the second and third year of the study. In the second year, groups of ewes were managed together on rangelands, whereas in the third year each group was split into two subgroups given differential feed levels. The results clearly showed that genotype had no significant effect on SOA during the 2- to 3-month period preceding the introduction of rams for spring mating. In the second year of the study, in which the experimental procedure allowed a fair comparison of the fertility of ewes in spring mating, fertility was similar for both genotypes. The reciprocity of the association was not demonstrated and the MnlI polymorphic site could not be used as a genetic marker of selection for out-of-season breeding ability, at least not in the MA breed. The percentage of cycling ewes significantly decreased between January and April, and older ewes (5 or 6 years old depending on the year of the study) were more cyclic than younger ones (2 and 3 years old, respectively). The differential feeding level of ewes from early February did not significantly affect their SOA during the time period studied in the third year of the study.     

Detection of white spot syndrome virus (WSSV) of shrimp by means of monoclonal antibodies (MAbs) specific to an envelope protein (28 kDa)

The vp28 gene encoding an envelope protein (28 kDa) of white spot syndrome virus (WSSV) was amplified from WSSV-infected tiger shrimp that originated from Malaysia. Recombinant VP28 protein (r-28) was expressed in Escherichia coli and used as an antigen for preparation of monoclonal antibodies (MAbs). Three murine MAbs (6F6, 6H4 and 9C10) that were screened by r-28 antigen-based enzyme-linked immunosorbent assay (ELISA) were also able to recognize viral VP28 protein as well as r-28 on Western blot. Three non-overlapping epitopes of VP28 protein were determined using the MAbs in competitive ELISA; thus, an antigen-capture ELISA (Ac-ELISA) was developed by virtue of these MAbs. Ac-ELISA can differentiate WSSV-infected shrimp from uninfected shrimp and was further confirmed by a polymerase chain reaction (PCR) and Western blot. Approximately 400 pg of purified WSSV sample and 20 pg of r-28 could be detected by Ac-ELISA, which is comparable in sensitivity to PCR assay but more sensitive than Western blot in the detection of purified virus. Hemolymph and tissue homogenate samples collected from a shrimp farm in Malaysia during December 2000 and July 2001 were also detected by Ac-ELISA and PCR with corroborating results.     

Isolation and partial characterization of gamone 1 of Euplotes octocarinatus

A polypeptide, termed gamone 1, was isolated and purified to homogeneity from culture filtrates of mating type VII of the freshwater ciliate Euplotes octocarinatus. The gamone induces intraclonal conjugation in cells of certain other mating types. The isolation and purification of the gamone was carried out by a combination of two chromatographic steps. The purified gamone was found to be still effective in a concentration of approx. 10⁻¹⁵M.     

Purification of bovine and human acrosin

Acrosin from human spermatozoa was required for studies of immunological interference with fertilization, but not detailed purification scheme was available for the human enzyme. Since human semen samples cannot be obtained cheaply or in large numbers and contain relatively small amounts of acrosin, development of purification procedures was carried out with bovine semen. Bovine acrosin had not previously been fully purified, and over 1 mg of pure acrosin was obtained from 100 mL of bovine semen, by a process of saline and Triton X-100 washes of the spermatozoa, 1 mM HCl extraction, gel filtration, and ion-exchange and affinity chromatography. The bovine acrosin had a molecular weight (MW) of 39 000 and a specific activity of 93 U/mg, measured with 0.5 mM benzoyl arginine ethyl ester. The same extraction procedure could be followed for human acrosin, but better yields were obtained in the purification if the ion-exchange step was omitted. The human acrosin had a MW of 49 000, and traces of a 38 000 MW component were sometimes observed. From 14 human semen samples, containing initially 7-10 U of acrosin activity, about 2.5 U (approximately 20 micrograms of protein) could be obtained in a pure state.     

Human alfa-fetoprotein: isolation and production of monoclonal antibodies.

Alfa-fetoprotein from human cord serum was purified in a single step by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B with a final recovery of alfa-fetoprotein of about 90% and a purification factor of 900. The purified preparation was homogeneous on SDS-PAGE and native PAGE running with a relative molecular weight of 72,000. Monoclonal antibodies against this purified preparation were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. 50% of culture wells exhibited hybrid growth and 7% of these wells contained anti-AFP secreting hybrids. Positive hybrid cells were cloned twice by the limiting dilution method and 8 clones were obtained that secreted monoclonal antibodies. Five of these cell lines (3F6H10, 3F6H4, 3F6H1, 3F6G5 and 3F6G10) were selected at random for purification and characterization purposes. All 5 cell lines secreted monoclonal antibodies of IgG1 subclass which were purified by affinity chromatography on Protein A- Sepharose CL-4B column with a final recovery of 80% and a purification factor of about 13. The purified preparations were homogeneous on SDS-PAGE, native PAGE and IEF. The monoclonal antibodies were highly specific for human alfa-fetoprotein as determined by Western blotting. The affinity constants (K) of these Mab ranged from 10(6) to 10(9) l/mol.     

Evaluation of three flocculation methods for the purification of Cryptosporidium parvum oocysts from water samples

AIMS: Evaluation of three flocculation methods for the purification of Cryptosporidium parvum oocysts from tap water. METHODS AND RESULTS: Ferric sulphate, aluminium sulphate and calcium carbonate were compared for their recovery efficiency of C. parvum oocysts from tap water. Lower mean recovery was achieved by calcium carbonate (38.8%) compared with ferric sulphate (61.5%) and aluminium sulphate (58.1%) for the recovery of 2.5 x 10(5) oocysts l(-1); 2.5 oocysts l(-1) and 1 oocyst l(-1) were adequately purified using ferric sulphate flocculation. In vitro excystation experiments showed that ferric sulphate flocculation does not markedly reduce the viability of oocysts. CONCLUSIONS: Ferric sulphate flocculation is a simple and effective tool for the purification of C. parvum oocysts from tap water. SIGNIFICANCE AND IMPACT OF THE STUDY: The high recovery rates and low impact on oocyst viability provided by ferric sulphate flocculation might be useful for the detection of Cryptosporidium oocysts in environmental water samples.     

A reinvestigation on the quaternary structure of ascorbate oxidase from Cucurbita pepo medullosa

The optical properties, copper content, catalytic activity and quaternary structure of many preparations of ascorbate oxidase purified with two different methods were examined. Fresh samples appeared identical and were characterized by optical ratios A280/A610 = 25 +/- 1 and A330/A610 = 0.8 +/- 0.05, by specific activity toward ascorbate of 3.48 +/- 0.05 mol g-1 min-1 and by a copper content of 8 +/- 0.3 mol/145 000 Mr. The enzyme is composed of two non-covalently linked subunits of slightly different molecular mass (75 000 and 72 000 respectively). These subunits cannot be further resolved by reduction of disulfide bonds. Proteolytic cleavage of the protein chains was observed during purification and storage in the absence of the protease inhibitor 6-amino caproic acid. Ascorbate oxidase exists as a monomer at neutral pH and undergoes reversible association into higher molecular weight species at slightly acid pH values. Association is not accompanied by spectroscopic or catalytic changes.