Species-specific aggregation factor in sponges. IV. Inactivation of the aggregation factor by mucoid cells from another species.

The role of protein synthesis in the cell union was investigated in conjugation of Blepharisma intermedium. In order to avoid possible complications due to the occurrence of other processes in conjugation, the homotypic cell union, in which conjugation is arrested at the stage of cell union without further changes, was used. Such unions were induced by treating cells of one mating type with the gamone of the other mating type for about 2 h. The induction of cell union was regularly accompanied by increased protein synthesis, which started 5 min after the beginning of the gamone treatment and continued for about 2 h. When protein synthesis was inhibited by cycloheximide, cell union was also inhibited. The extent of the two inhibitions were closely correlated. We concluded that gamone induces proteins and that protein synthesis is essential for cell union. Proteins synthesized in gamone-treated and non-treated cells were also separated and compared. Consideration of these results leads to a hypothesis that most of the gamone-induced proteins are membrane proteins normally synthesized, though in lesser amount, in non-conjugating cells and that cells gain the capacity to unite when these proteins are accumulated at a restricted area on the cell surface by another gamone-controlled mechanism.     

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.     

Cell surface control in Blepharisma intermedium (Protozoa ciliata). Blocking of gamone II-induced homotypic pairing by different membrane interacting ligands

PHA, Con-A, or anti-tubulin antibodies inhibit homotypic pair formation, in B. intermedium mating type-I cells in the presence of suboptimal concentrations of gamone II. The inhibition is dependent on the dose of gamone added; the structural conformation and the relative concentration of the inhibitor; and the time of addition of the inhibitor. The block can be selectively prevented by competitive inhibitors of each ligand. The receptors for the inhibitors are distinctive and there is no cross-reaction between the ligands. It is concluded that ligand binding and subsequent receptor-ligand aggregation must induce a change within the cell-surface membrane, which distorts the distribution and/or affects an optimal conformational aspect of a specific membrane-receptor system for the gamone, a prerequisite for cell pair formation.     

DNA methylation in vegetative and conjugating cells of a protozoan ciliate: Blepharisma japonicum

We report here the presence of N⁶-methyladenine (MeAde) in the macronuclear DNA (maDNA) of Blepharisma japonicum vegetative cells. We have further investigated the relationship between DNA methylation and cell union in cells activated for conjugation. Such activation was induced by treating cells of mating type I with complementary gamone 2. We found a reduction of about 24% of MeAde content in gamone-treated cells ready for cell union. First indications of the presence and reduction of MeAde content came from electrophoresis of maDNA digested by appropriate restriction endonucleases. Chromatographic determination of the amount of methylated base by HPLC substantiated these observations. In vegetative cells, 1.576 ± 0.02% of total adenine was found to be methylated as opposed to 1.193 ± 0.04% in activated cells. The HPLC analysis of maDNA also revealed a peak with a retention time corresponding to that of 5-hydroxymethyluracil, already found in some species of dinoflagellates. In that gamone treatment is correlated with a differential gene expression (indicated by a differential RNA and protein synthesis), our results suggest that there is a relationship between macronuclear genome activation and demethylation of maDNA. This is the first report of a correlation between gene activation and adenine demethylation in a eukaryotic organism.     

Behavioural changes induced by the conjugation-inducing pheromones,gamone 1 and 2, in the ciliate Blepharisma japonicum

Preconjugant interactions between complementary mating-type cells in ciliates occur before sexual reproduction. The interactions include retardation of swimming behaviour, courtship dancing, chemoattraction, nuclear activation, cell division, or cell agglutination, depending on ciliate species. In Blepharisma japonicum, chemoattraction of mating-type I by mating-type II has been reported previously. It has been shown that chemoattraction here is caused by a conjugation-inducing substance called gamone 2 secreted by mating-type II cells. In this study, we show that mating-type II cells accumulate near the site where gamone 1 secreted by mating-type I cells is present at a high concentration. We also show that the behaviour of individual cells changes when exposed to the complementary mating-type gamone; cells begin to rotate and swim slowly, thus shortening their minimum path length (final displacement of a cell from its origin). These results suggest that gamones 1 and 2 induce behavioural changes in type II and I cells, respectively, and that gamone-stimulated cells may accumulate at the site with the highest activity of the complementary gamone, after repetition of swimming changes in the gradient of gamone concentration. This reciprocal induction of the changes in behaviour may increase the probability of sexual encounters for conjugation.     

Cell-to-cell contact by locally differentiated surfaces in conjugation of Blepharisma

Local functional differences of the cell surface were investigated in the formation of cell unions in conjugation of Blepharisma intermedium. When cells of this ciliate are treated by the gamone (the conjugation-inducing substance) of the complementary mating type, they first unite by cilia and then more intimately by the direct contact of cell bodies. The ciliary union was found to be formed by a specific contact between two morphologically distinguishable surfaces, the adoral zone of membranelles (AZM) of one cell and the anterior extension of the undulating membrane (extUM) of the other. The AZM gains the capacity to unite first and the extUM next. The extUM of gamone-treated cells sticks not only to the AZM but also to glass and some other artificial substrata under some conditions. These stickings are inhibited by cycloheximide which also inhibits the ciliary union. The role of specific contact between AZM and extUM in the cell recognition is discussed.     

Gamone Production in Large-Scale Cultures of Euplotes octocarinatus1

We report a method that allows us to grow and maintain the freshwater ciliate Euplotes octocarinatus in large quantities. Frequent exchange of culture fluid proved more effective than aeration in obtaining high cell densities (4200 cells/ml) and reasonable doubling times in large-scale cultures. For harvesting gamone 1, the cell density was raised to 10,000 cells/ml. Under these conditions, the cells continued to produce and secrete gamone; they were slightly starved, but they no longer divided. Cell-free fluid with a steady and relatively high yield of gamone was obtained from two such cultures over a period of five months. We isolated gamone 1 also from cell homogenates and compared it with secreted gamone 1, but found no differences in the gamones from these two sources.     

Purification and characterization of a pheromone that induces sexual cell division in the unicellular green alga Closterium ehrenbergii

Closterium ehrenbergii is a unicellular charophycean alga consisting of two sexes: mating type plus (mt⁺) and minus (mt^–). The sexual reproductive process consists of five steps: formation of sexual pairs, cell division of each member of a pair, formation of conjugation papillae, release of protoplasts from gametangial cells, and fusion of protoplasts to form a zygote. The second step, called sexual cell division (SCD), produces two gametangial cells from one vegetative mother cell. The SCD of mt⁺ cell is mediated by a diffusible sex pheromone, named SCD-inducing pheromone (SCD-IP). This pheromone is released from mt^– cells in the light, and the presence of mt⁺ cells stimulates its secretion from mt^– cells. SCD-IP was purified by sequential column-chromatographic fractionation from culture medium in which both mating type cells had been co-cultured. Purified SCD-IP is a glycoprotein with an apparent molecular mass of 20 kDa. The molecular mass of the SCD-IP was estimated to be 18 kDa by mass spectrometry. Amino-terminal and two internal amino acid sequences of the pheromone revealed significant similarity to another Closterium pheromone, protoplast release-inducing protein (PR-IP) inducer of Closterium peracerosum-strigosum-littorale complex (C. pslc). These two pheromones induced different morphological reactions in each Closterium species. Based on these results, the diversity of sex pheromones is discussed.     

Single mating type-specific genes and their 3′ UTRs control mating and fertility in Cochliobolus heterostrophus

To determine the number of proteins required for mating type (MAT) locus-regulated control of mating in Cochliobolus heterostrophus, MAT fragments of various sizes were expressed in MAT deletion strains. As little as 1.5?kb of MAT sequence, encoding a single unique protein in each mating type (MAT-1 and MAT-2), conferred mating ability, although an additional 160?bp of 3^′ UTR was needed for production of ascospores. No other mating type-specific genes involved in mating identity or fertility were found. Thus, although homologs of the C. heterostrophus MAT-1 and MAT-2 genes exist in the filamentous ascomycetes Neurospora crassa and Podospora anserina, C. heterostrophus does not appear to have mating type-specific homologs of two additional genes required by both N. crassa and P.?anserina for successful sexual reproduction. Three genes were identified in the common DNA flanking the MAT locus: a gene encoding a GTPase-activating protein and an ORF of unknown function lie 5^′ while a β-glucosidase encoding gene lies found 3^′. None of these genes appears to be involving in the mating process.     

A New Mating Compatibility Locus in PHYSARUM POLYCEPHALUM

The rate and extent of plasmodium formation were studied in mating tests involving pairs of largely isogenic amoebal strains compatible for mating-type (mt) alleles. A systematic variability was observed: plasmodia formed either rapidly and extensively or slowly and inefficiently. Plasmodium formation was found to be 10³- to 10⁴-fold more extensive in "rapid" crosses than in "slow" crosses. A genetic analysis revealed that the variability reflects the influence of a multiallelic compatibility locus that determines mating efficiency. This compatibility locus (designated matB), together with the original mating type locus, mt (in this work designated matA), constitute a tetrapolar mating specificity system in Physarum polycephalum.     

Genetics of CHLAMYDOMONAS REINHARDTII Diploids. II. the Effects of Diploidy and Aneuploidy on the Transmission of Non-Mendelian Markers

The transmission of two non-Mendelian drug resistance markers has been studied in crosses of Chlamydomonas reinhardtii involving diploids and aneuploids with different mating type genotypes. Under normal laboratory conditions for gametogenesis, mating and zygote maturation, the transmission pattern of the non-Mendelian markers sr-u-1 (resistance to streptomycin) and spr-u-1-27-3 (resistance to spectinomycin) is primarily determined by the mating type genotypes of the parental cells. Our results confirm and expand an earlier observation suggesting that an apparent codominant function of the female (mt⁺) allele in regulating chloroplast gene transmission in meiosis appears to be distinct and separate from its recessive function in regulating mating behavior. The chloroplast DNA complement (as indexed by the number of extranuclear DNA-containing bodies) may exert a secondary effect on the transmission of these markers. Within a mating type group (mt⁺/mt^- or mt^-/mt^-) a cell line with more chloroplast DNA tended to transmit its non-Mendelian markers more frequently than a cell line with less chloroplast DNA.     

Identification of a new mating group and reproductive isolation in the <Emphasis Type="Italic">Closterium peracerosum–strigosum–littorale</Emphasis> complex

Reproductive isolation is essential for the process of speciation. In order to understand speciation, it is necessary to compare one mating group with other phylogenetically related but reproductively isolated groups. The Closterium peracerosum–strigosum–littorale (C. psl.) complex is a unicellular isogamous zygnematophycean alga, which is believed to share a close phylogenetic relationship with the land plants. In this study, we identified a new mating group, named group G, of C. psl. complex and compared its physiological and biochemical characteristics with the mating group I-E, which was closely related to the mating group G. Zygospores are typically formed as a result of conjugation between mating-type plus (mt⁺) and mating-type minus (mt^?) cells in the same mating group during sexual reproduction. Crossing experiments revealed mating groups G and I-E were reproductively isolated from each other, but the release of lone protoplasts from mt^? cells of mating group G was induced in the presence of mt⁺ cells of mating group I-E. In fact, the sex pheromone, protoplast-release-inducing protein of mating group I-E induced the release of protoplasts from mt^? cells of mating group G. When mt⁺ and mt^? cells of both mating groups I-E and G were co-cultured (multiple-choice matings), the zygospore formation of mating group G, but not that of mating group I-E, was inhibited. Based on these results, we propose a possible mechanism of reproductive isolation between the two mating groups and suggest the presence of sexual interference between mating group G and mating group I-E.     

A homeotic mutation influences the wing vibration patterns during mating in males of the silkworm moth Bombyx mori

An abnormality in the wing vibration pattern in males of the E^Nc homeotic mutant of Bombyx mori was investigated. The wild-type (+/+) males show a switching of the rhythmic wing vibrations from a sequential pattern to an intermittent pattern during mating, whereas the E^Nc mutants show a sequential pattern both before and during mating. Wing motions in +/+ males became small during mating, but those in +/E^Nc males did not. Ablation of the head ganglia of +/+ and +/E^Nc males during mating caused no change in the motor patterns of wing vibrations. Ablation or cooling of the posterior abdomen in the +/+ males during mating caused sequential wing vibrations, suggesting that the change in wing vibrations is induced by signals from the posterior abdomen. The pterothoracic ganglion in the +/E^Nc males is separated into two ganglia, in contrast to the complete ganglionic fusion in the +/+ males. The neurons in the pterothoracic ganglion stained from abdominal nerve cords are homologus in +/+ and +/E^Nc males, but many of these in +/E^Nc males are elongated along the anteroposterior axis. These results suggest that the wing vibration pattern is restricted by genetic factors through reconstruction of the thoracic nervous system during metamorphosis.     

Mating systems and predictors of relative reproductive success in a Cutthroat Trout subspecies of conservation concern

Mating systems and patterns of reproductive success in fishes play an important role in ecology and evolution. While information on the reproductive ecology of many anadromous salmonids (Oncorhynchus spp.) is well detailed, there is less information for nonanadromous species including the Yellowstone Cutthroat Trout (O. clarkii bouvieri), a subspecies of recreational angling importance and conservation concern. Using data from a parentage‐based tagging study, we described the genetic mating system of a migratory population of Yellowstone Cutthroat Trout, tested for evidence of sexual selection, and identified predictors of mating and reproductive success. The standardized variance in mating success (i.e., opportunity for sexual selection) was significantly greater for males relative to females, and while the relationship between mating success and reproductive success (i.e., Bateman gradient) was significantly positive for both sexes, a greater proportion of reproductive success was explained by mating success for males (r ² = 0.80) than females (r ² = 0.59). Overall, the population displayed a polygynandrous mating system, whereby both sexes experienced variation in mating success due to multiple mating, and sexual selection was variable across sexes. Tests for evidence of sexual selection indicated the interaction between mating success and total length best‐predicted relative reproductive success. We failed to detect a signal of inbreeding avoidance among breeding adults, but the group of parents that produced progeny were on average slightly less related than adults that did not produce progeny. Lastly, we estimated the effective number of breeders (N _b) and effective population size (N _e) and identified while N _b was lower than N _e, both are sufficiently high to suggest Yellowstone Cutthroat Trout in Burns Creek represent a genetically stable and diverse population.     

Control of cell type in yeast by the mating type locus: The α1-α2 hypothesis

We have extended the genetic analysis of four mutants carrying defective MATα alleles in order to determine how the mating type locus controls yeast cell types: a, a, and $\text{a}\text{α}$. First, we have mapped the defect in the mutant VC73 to the mating type locus by diploid and tetraploid segregation analysis. Second, we have determined that the mutations in these strains define two complementation groups, MATα1 and MATα2. The MATα1 gene is proposed to be a positive regulator of α mating functions. The MATα2 gene product is proposed to have two roles, as a negative regulator of a-specific mating functions and as a regulator of $\text{a}\text{α}$ cell functions (required for sporulation, for inhibition of mating and other processes). This view of MATα leads to the prediction that matα1^?matα2^? mutants should have the mating ability of an a cell and that matα1^?matα2^?/MATα strains should mate as α and be unable to sporulate. Such double mutants have been constructed and behave as predicted. We therefore propose that a-specific mating functions in MATa cells are constitutively expressed due to the absence of the MATα2 gene product and that α-specific mating functions are not expressed due to the absence of the MATα1 gene product.     

Isolation and amino acid sequence of a mating pheromone produced by mating type α cells of Saccharomyces exiguus

A peptide, termed α^se pheromone, was isolated as a mating pheromone from culture filtrate of mating type a cells of Saccharomyces exiguus. The peptide showed both agglutinability-inducing activity to a cells of S. cerevisiae and shmoo-inducing action to a cells of S. cerevisiae, S. kluyveri and S. exiguus. The amino acid sequence of α^se pheromone was determined as H-Trp-His-Trp-Leu-Arg-Leu-Ser-Tyr-Gly-Gln-Pro-Ile-Tyr-OH by mass spectrometry, sequence analysis and enzymatic digestion.     

Light Affects Flagellar Agglutinability in Chlamydomonas eugametos by Modification of the Agglutinin Molecules

The effect of light on the sexual competence of a light-sensitive mating type minus strain (mt⁻) of Chlamydomonas eugametos obtained by crossing a light-sensitive mating type plus strain (mt⁺) with a light-insensitive mt⁻ strain is described. As previously demonstrated for the mt⁺ parent, this study of one of the mt⁻ offspring shows that (a) a light-sensitive mechanism affects flagellar agglutinability in a rapid process that does not require protein synthesis; (b) only the activity of the flagellar agglutinins (glycoproteins responsible for agglutination) is susceptible to light while agglutinins on the cell body surface are not affected by light. We further demonstrate that (a) membrane vesicles naturally released from nonagglutinable dark gametes remain inactive. Extracts of these vesicles also remain inactive even though they contain agglutinin-like components; (b) inactive mt⁻ agglutinin is present in extracts of flagella from nonagglutinable dark gametes by comparison of its chromatographic, electrophoretic, and immunogenic properties with those of active agglutinin. When purified of all other flagellar proteins, it remains inactive; (c) a monoclonal antibody directed against the sexual agglutination site of the mt⁻ agglutintin discriminates between active and inactive agglutinins when present in a native state on the flagellar surface, but is unable to discriminate between them when they are denatured in sodium dodecyl sulfate-electrophoresis gels and blotted onto nitrocellulose. Taken collectively these observations suggest that light activation involves the chemical modification of the agglutinins in situ on the flagellar surface.     

Mating behavior in mutant strains of Drosophila melanogaster at different population densities

The effects of mutations and genetic background on the mating activity of males and receptivity of females Drosophila melanogaster have been studied at different population densities. Population density, as well as its combinations with other factors, significantly affects mating behavior of D. melanogaster. There are two distinct trends in the effect of this factor on mating behavior: the maximum larval overpopulation may cause either a significant suppression of the behaviors studied or an increase in their expressivity. The mating behaviors of w ^a and cn mutants against a certain genetic background changed similarly in response to varying population density.     

Sexual differentiation in Mucor: Trisporic acid response mutants and mutants blocked in zygospore development

Spores of a minus strain of Mucor mucedo (Bref.) were treated with 1-methyl-[3-nitro]-1-nitro-soguanidine and mutants were isolated either by testing for zygophore induction with externally supplied trisporic acids (TA) or by mating with wild type plus colonies. Mutants were found defective (Tar^?) or temperature-sensitive (Tar-Ts) in their reaction towards trisporic acids, blocked or temperature-sensitive in their mating with plus strain (Mat^? or Mat-Ts) or temperature-sensitive in zygospore development (Zyg-Ts). The inability to react against externally supplied trisporic acids was not necessarily coupled with an inability to mate with plus strain (phenotype Tar^? Mat⁺). This indicated that the diffusion and uptake of trisporic acids is not a necessary prerequisite to the sexual interaction of Mucor mating types.