Inheritance of Autogamy and the Killer Trait in Euplotes minuta

SYNOPSIS. Eight interfertile strains of Euplotes minuta collected from two localities in the Mediterranean Sea are shown to comprise a single breeding system of multiple mating types. One strain regularly passes through autogamy and, in crosses to non-autogamous strains, preliminary evidence for the genic control of autogamy was obtained. The cytogenetic events of conjugation and autogamy are briefly described. Three strains are "killers." Killer cells liberate a substance into the surrounding medium which has a lethal effect on cells of other strains called "sensitives." This substance, "euplotein," is not produced by sensitives. The inheritance of the killer trait is apparently under the control of a cytoplasmic factor but present attempts to identify the factor microscopically were without success. The discussion stresses the potential usefulness of this species for studies of protozoan genetics and development.     

Metabolism of alpha-factor by a mating type cells of Saccharomyces cerevisiae.

When a mating type cells of Saccharomyces cerevisiae are exposed to the mating pheromone alpha-factor in liquid cultures, there is a time-dependent loss of alpha-factor activity from the culture fluid. This loss of biological activity can be directly correlated with the proteolysis of the pheromone by a mating type cells. The metabolism of alpha-factor by a mating type cells may be measured by using either in vitro 125I-labeled or in vivo 35S-labeled pheromone. Addition of chloroquine to growing cultures of a mating type cells at concentrations which cause no detectable alterations in cell growth produces a potentiation of alpha-factor mediated cell cycle arrest. This potentiation of alpha-factor activity is directly correlated with the inhibition of alpha-factor proteolysis. Thus, while proteolytic digestion of alpha-factor appears to be related to the mechanism whereby a mating type cells "detoxify" alpha-factor and recover from cell cycle arrest, proteolysis of the mating factor is not necessary for alpha-factor mediated cell cycle arrest.     

Differentiation of Reproductive Cells in Volvox carteri*

SYNOPSIS. The life cycle of Volvox carteri was studied in axenic culture using the NB-3 and the NB-7 strains isolated from Nebraska. Vegetative colonies of both strains contain 8–12 asexual reproductive cells (gonidia) which divide to form daughter colonies. During daughter colony formation, the reproductive cells of the daughters are delimited at an early stage of cleavage. Gonidia are delimited at the division from 16 to 32 cells, but eggs and male initial cells are not differentiated until the division of the 32-celled stage. In all instances the reproductive cells are the products of unequal cleavages. Male and female colonies are formed in separate clones. Female colonies contain approximately 20 eggs. Male colonies have approximately 50 male initial cells, each of which forms a sperm bundle containing 64 or 128 sperm. Sperm bundles penetrate female colonies and fertilize the eggs. Zygote formation, zygote germination, and the development of gone colonies is described. Sexual type was inherited in a 1:1 ratio. Male colonies appear spontaneously in the male strain, but female colonies were formed in the female strain only in the presence of a substance produced by colonies from male cultures. This female inducing substance is produced in male cultures primarily, if not exclusively, by male colonies rather than by vegetative colonies. The female inducing substance is heat labile and non-dialyzable. Activity is destroyed by Pronase, but not by trypsin, chymotrypsin or ribonuclease. Gonidia appear to be most susceptible to female induction during the early stages of their expansion prior to cleavage.     

Growth and differentiation of wild type amoebae of Physarum polycephalum in liquid culture

A method for growing wild type amoebal strains of Physarum polycephalum in two-membered liquid culture is presented. The medium is a simple buffered salts solution. We found that a minimal level of divalent cation was required for growth. All amoebal strains tried to date have grown under our conditions in stationary culture. Growth under gyratory conditions was only successful at 60 rpm or less and consistent growth required a period of adaptation over several transfers. Differentiation of two apogamic strains, CL and CH1, were compared. Contrary to the results seen on agar plates, the time of onset for the first committed amoebae was identical for both strains in liquid culture. Attempts to demonstrate mating between two genetically compatible amoebal strains grown together in liquid culture were not successful.     

Morphological and Mutational Analysis of Mating in Ustilago hordei

Martinez-Espinoza, A. D., Gerhardt, S. A., and Sherwood, J. E. 1993. Morphological and mutational analysis of mating in Ustilago hordei. Experimental Mycology 17, 200-214. Ustilago hordei is a basidiomycete that causes covered smut on barley. Mating in U. hordei, which is controlled by a single locus with two alleles, results in the conversion of haploid, nonpathogenic yeast-like sporidia to dikaryotic, pathogenic mycelia. When sporidia of the opposite mating type were mixed and placed on water agar, both cell types produced conjugation tubes within 2 h at 21°C. Growth of conjugation tubes was directed toward the tip of tubes arising from cells of the opposite mating type. These tubes fused and the dikaryotic mycelium emerged from the conjugation bridge. Sporidia separated by a dialysis membrane were still capable of inducing conjugation tube formation by cells of the opposite mating type, indicating the involvement of diffusible small-molecular-weight mating factors (pheromones). Numerous nutritional and environmental variables were examined in order to optimize conjugation tube induction. Twenty-six mutants that fail to form dikaryotic mycelium have been isolated and characterized. These mutants were arranged into classes based on their ability to form conjugation tubes, the ability to induce conjugation tube formation by opposite mating-type cells, and cell morphology. These mutants provide an indication of the genetic complexity involved in this critical phase of the U. hordei life cycle.     

Characterization of a renal tubular epithelial cell line which secretes the autologous target antigen of autoimmune experimental interstitial nephritis

Proximal tubular epithelial cells from mice which develop autoimmune interstitial nephritis were found to express the nephritogenic target antigen, 3M-1. Anti-3M-1 mAbs (alpha 3M-1-Ab) were used to positively select for 3M-1-secreting tubular epithelium and, after stabilization in culture, this new cell line (MCT) was examined for the production of several moieties important to either immune interactions or to the development of extracellular matrix. Alkaline phosphatase-staining MCT cells also express epithelial growth factor receptors with a Kd of 0.87 nM and an epithelial growth factor receptor constant (Ro) of 2.1 X 10(4) receptors/cell. MCT culture supernatants contain greater amounts of laminin, and types IV and V procollagens compared to types I and III procollagens, and growing MCT cells on type I collagen matrix causes them to preferentially secrete even more type IV and V procollagen. The 30,000-Mr 3M-1 antigen could be immunoprecipitated from biosynthetically labeled MCT cell supernatants with alpha 3M-1-Ab. An identical-sized moiety was isolated by immunoaffinity chromatography from collagenase-solubilized mouse kidney tubular basement membranes. The 3M-1 antigen can be found on the MCT cell surface by radioimmunoassay, or deposited in a linear array in the extracellular matrix surrounding the MCT cells in culture by immunofluorescence. Mature messenger RNA species for both class I and class II major histocompatibility complex (MHC) molecules were detected by Northern hybridization, and their corresponding cell surface gene products were detected by cytofluorography of MCT cells stained with haplotype-specific antibodies. Both the cell surface 3M-1 and the small amounts of detected class II MHC molecules appear to be biologically functional, as MCT cells can support the proliferation of 3M-1-specific, class II MHC-restricted helper T cells in culture. These findings suggest that MCT cells provide all the necessary biological parameters for interfacing both as the target of a nephritogenic immune response, and as a potential source for new extracellular matrix which develops as a fibrogenic response to interstitial nephritis.     

SEX PHEROMONES THAT INDUCE SEXUAL CELL DIVISION IN THE CLOSTERIUM PERACEROSUM–STRIGOSUM–LITTORALE COMPLEX (CHAROPHYTA)1

Sexual cell division (SCD) that produces two gametangial cells from one vegetative mother cell is the first step observed morphologically in the sexual reproduction in the Closterium peracerosum–strigosum– littorale complex. SCD‐inducing activities specific for each mating‐type cells were detected in the medium in which both mating type cells has been cocultured. Mating‐type minus (mt ^? ) cells released SCD‐inducing substance specific for mating‐type plus (mt ⁺ ) cells and were designated as SCD‐ inducing pheromone (IP)‐minus, whereas mt ^? specific substances released from mt ⁺ cells were designated as SCD‐IP‐plus. Culture medium was subjected to gel filtration, and then SCD‐IP‐plus and SCD‐IP‐minus chemical were found to have the molecular masses of 90–100 kDa and 10–20 kDa, respectively. It was evident that light was imperative for this type of signaling. Gametangial cells of both mating types were obtained from vegetative cells by treatment with SCD‐IPs. Gametangial mt ⁺ cells showed high competency for conjugation with vegetative mt ^? cells, whereas gametangial mt ^? cells showed low competency for conjugation with vegetative mt ⁺ cells. These results indicate that SCD in both mating type cells is induced by high molecular weight sex pheromones and that the roles of gametangial cells in the process of conjugation differ by sex.     

Spontaneous Selfing (Intraclonal Conjugation) in the Ciliate Euplotes patella Syngen 2 Takes Place without a Change in Mating Type Expression

ABSTRACT. Cells of Euplotes patella syngen 2 have been found to excrete mating-type-specific gamones (mating-inducing factors) into the surrounding medium. In strains of E. patella syngen 2, intraclonal conjugation (selfing) sometimes occurred spontaneously. Cell-free fluids of clonal cultures in which spontaneous selfing occurred were not effective in inducing homotypic pair formation in other cultures of the same mating type as the culture from which the cell-free fluids were derived. This indicates that selfing cultures produce only those gamones expected of their own mating types. This result suggests that spontaneous selling in E. patella syngen 2 is not due to change of mating type expression. The possible mechanisms for spontaneous selfing in E. patella syngen 2 are discussed.     

Role of the pheromone-inducible surface protein Asc10 in mating aggregate formation and conjugal transfer of the Enterococcus faecalis plasmid pCF10.

The high transfer frequency of pheromone-inducible conjugative plasmids of Enterococcus faecalis in liquid culture is due in part to the formation of mating aggregates. These aggregates result from the interaction of two surface components, aggregation substance (AS), which is plasmid encoded, and the chromosomally encoded binding substance (BS). In the accompanying paper (S.-M. Kao, S. B. Olmsted, A. S. Viksnins, J.C. Gallo, G. M. Dunny, J. Bacteriol, 173:7650-7664, 1991), the sequence of the prgB gene encoding the AS molecule (Asc10) produced by pheromone-induced cells carrying plasmid pCF10 is presented. Here we report the results of genetic and immunological experiments which define the role of Asc10 in aggregation and plasmid transfer. These data indicate expression of AS on the surface of an E. faecalis cell and its binding to BS expressed on a second cell are required for the formation of a mating pair and the efficient transfer of pCF10 in liquid matings. However, the orientation of the receptors was not critical for transfer; ie., AS expressed on recipient cells could facilitate plasmid transfer via binding to BS on the donor. Our results suggest that additional (as yet unidentified) products are involved in forming the channel that ultimately serves to transfer the DNA, with AS-BS binding serving primarily to generate the initial attachment between cells. The putative prgC gene product, identified by DNA sequencing (data presented in the accompanying paper), could be involved in transfer events occurring subsequent to aggregation.     

Isolation of a Peptidyl Factor, α Substance-IA,Inducing Sexual Agglutinability in Saccharomyces cerevisiae

After a eleven-steps purification, a peptidyl factor named α substance-Ia was isolated in pure form from a culture filtrate of α type cells of the heterothallic Saccharomyces cerevisiae. The substance induced the sexual agglutinability in a haploid cells belonging to the opposite mating type at concentrations from 0.4 to 0.8 ng/ml.     

Organizational behavior of human umbilical vein endothelial cells

Culture conditions that favor rapid multiplication of human umbilical vein endothelial cells (HUV-EC) also support long-term serial propagation of the cells. This is routinely achieved when HUV-EC are grown in Medium 199 (M-199) supplemented with fetal bovine serum (FBS) and endothelial cell growth factor (ECGF), on a human fibronectin (HFN) matrix. The HUV-EC can shift from a proliferative to an organized state when the in vitro conditions are changed from those favoring low density proliferation to those supporting high density survival. When ECGF and HFN are omitted, cultures fail to achieve confluence beyond the first or second passage: the preconfluent cultures organize into tubular structures after 4-6 wk. Some tubes become grossly visible and float in the culture medium, remaining tethered to the plastic dish at either end of the tube. On an ultrastructural level, the tubes consist of cells, held together by junctional complexes, arranged so as to form a lumen. The smallest lumens are formed by one cell folding over to form a junction with itself. The cells contain Weibel-Palade bodies and factor VIII-related antigen. The lumens contain granular, fibrillar and amorphous debris. Predigesting the HFN matrix with trypsin (10 min, 37 degrees C) or plasmin significantly accelerates tube formation. Thrombin and plasminogen activator had no apparent effect. Disruption of the largest tubes with trypsin/EDTA permits the cells to revert to a proliferative state if plated on HFN, in M-199, FBS, and ECGF. These observations indicate that culture conditions that do not favor proliferation permit attainment of a state of nonterminal differentiation (organization) by the endothelial cell. Furthermore, proteolytic modification of the HFN matrix may play an important role in endothelial organization.     

Formation of nonculturable Mycobacterium tuberculosis and their regeneration

Nonculturable cells were found to occur in populations of Mycobacterium tuberculosis cells during the long post-stationary phase. These cells were small (0.6-0.8 micron) ovoid and coccoid forms with intact cell walls and negligible respiratory activity, which allows them to be regarded as dormant cells. Nonculturable cells were characterized by low viability after plating onto solid medium; a minor part of the population of these cells could be cultivated in liquid medium. Cell-free culture liquid of an exponential-phase Mycobacterium tuberculosis culture or the bacterial growth factor Rpf exerted a resuscitating effect, increasing substantially the growth capacity of the nonculturable cells in liquid medium. During resuscitation of nonculturable cells, a transition from ovoid to rodlike cell shape occurred. At early stages of resuscitation, ovoid cells formed small aggregates. The recovery of culturability was associated with the formation of rod-shaped cells in the culture. The data obtained demonstrate the in vitro formation of dormant cells of Mycobacterium tuberculosis, which do not grow on solid media but can be resuscitated in liquid medium under the effect of substance(s) secreted by actively growing cells.     

试管出菇法测定刺芹侧耳的交配系统*

在MDA或PNA培养基上,用单孢分离物在试管中进行配对,观测菌丝体上的锁状联合和子实体的形成情况,判定单孢菌株间的亲和性;研究了2个刺芹侧耳菌株PE-11、PE-12和1个糙皮侧耳菌株PO-02的交配系统,以及3个菌株间的亲缘关系;结果表明刺芹侧耳和糙皮侧耳是双因子交配系统。这是一个测定侧耳属的交配系统的新方法。     

The adhesin Hwp1 and the first daughter cell localize to the a/a portion of the conjugation bridge during Candida albicans mating

The cell wall protein Hwp1 was originally demonstrated to be expressed exclusively in hyphae of Candida albicans and cross-linked to human epithelium by mammalian transglutaminase. Hwp1 is expressed on the walls of hyphae formed by a/alpha, a/a, and alpha/alpha cells. Hence, it is expressed on hyphae independently of mating type. However, Hwp1 is selectively expressed on the wall of conjugation tubes formed by a/a cells, but not alpha/alpha cells, in the mating process. This was demonstrated in all possible crosses between four unrelated natural a/a strains and four unrelated alpha/alpha strains. In zygotes, Hwp1 is restricted to that portion of the wall of the conjugation bridge contributed by the a/a parent cell. Hwp1 staining further revealed that the first daughter bud that emerges from the conjugation bridge does so from the a/a-contributed portion. Hwp1 expression and localization during the mating process is, therefore, mating type specific, opaque phase specific, and alpha-pheromone induced. These results indicate that the mating type-specific contributions to the conjugation bridge during the mating process in C. albicans are qualitatively and functionally distinct and that the a/a portion of the bridge, which selectively contains Hwp1, bears the first daughter cell in the mating process.     

Extracellular chitinase production by wild-type B-10 and mutant M-1 strains of Serratia marcescens

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, while M-10 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused overproduction of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the production of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).     

Mechanism of α Factor Biosynthesis in Saccharomyces cerevisiae

The biosynthesis of alpha factor, a mating-type-specific regulatory oligopeptide which is secreted by Saccharomyces cerevisiae cells of alpha mating type, was studied. In batch cultures only small amounts of the peptide were synthesized during the exponential growth phase. During the stationary phase, alpha factor was produced at a constant rate and accumulated in the culture medium. Inhibition of translation in wild-type cells by cycloheximide, or in mutant strains under conditions which blocked protein or ribonucleic acid (RNA) synthesis completely inhibited the production of alpha factor. These results indicate that the factor is produced by ribosomal translation of a specific messenger RNA and not by an extraribosomal mechanism of peptide synthesis.     

Mating-type locus homozygosis, phenotypic switching and mating: a unique sequence of dependencies in Candida albicans

A small proportion of clinical strains of Candida albicans undergo white-opaque switching. Until recently it was not clear why, since most strains carry the genes differentially expressed in the unique opaque phase. The answer to this enigma lies in the mating process. The majority of C. albicans strains are heterozygous for the mating type locus MTL (a/alpha) and cannot undergo white-opaque switching. However, when these cells undergo homozygosis at the mating type locus (i.e., become a/a or alpha/alpha), they can switch, and they must switch in order to mate. Even though the newly identified stages of mating mimic those of Saccharomyces cerevisiae, the process differs in its dependency on switching, and the effects switching has on gene regulation. This unique feature of C. albicans mating appears to be intimately intertwined with its pathogenesis. The unique, newly discovered dependencies of switching on homozygosis at the MTL locus and of mating on switching are, therefore, reviewed within the context of pathogenesis.