Temperature-sensitive mutants of Dictyostelium discoideum

Three classes of temperature-sensitive mutants of the cellular slime mold Dictyostelium discoideum have been isolated. One class contains strains able to grow at 22 C but not at 27 C. Cells of these strains can develop into sorocarps at both temperatures. Another class contains strains which can grow at both temperatures but can only develop at the lower temperature. The third class contains strains unable to grow or develop at 27 C. Those strains whose development is temperature-sensitive appear to carry mutations which affect the cells only during the period of aggregation before the construction of a multicellular sorocarp. When pairs of growth-temperature-sensitive (GTS) strains develop in mixed aggregates, temperature-resistant (TR) cells are formed at a frequency of about 10(-4). These TR cells transmit the phenotype in a relatively stable hereditary fashion. However, temperature-sensitive segregants can be isolated from TR strains even after six clonal passages. Mixed incubation of pairs of morphologically aberrant GTS strains was found to give rise to TR progeny which develop normally. These progeny clones independently segregate morphologically aberrant strains and temperature-sensitive strains. The results indicate that several temperature-sensitive and morphological mutations are recessive and nonidentical.     

Motility mutants of Dictyostelium discoideum

We describe six motility mutants of Dictyostelium discoideum in this report. They were identified among a group of temperature-sensitive growth (Tsg) mutants that had been previously isolated using an enrichment for phagocytosis-defective cells. The Tsg mutants were screened for their ability to produce tracks on gold-coated cover slips, and several strains were found that were temperature-sensitive for migration in this assay. Analysis of spontaneous Tsg+ revertants of 10 migration-defective strains identified six strains that co-reverted the Tsg and track formation phenotypes. Characterization of these six strains indicated that they were defective at restrictive temperature in track formation, phagocytosis of bacteria, and pseudopodial and filopodial activity, while retaining normal rates of oxygen consumption and viability. Because they had lost this group of motile capabilities, these strains were designated motility mutants. The Tsg+ revertants of these mutants, which coordinately recovered all of the motile activities, were found at frequencies consistent with single genetic events. Analysis of the motility mutants and their revertants suggests a relationship between the motility mutations in some of these strains and genes affecting axenic growth.     

Induction of fruiting in two aggregateless mutants of Dictyostelium discoideum

Five general groups of morphogenetically aberrant mutants of Dictyostelium discoideum were isolated. Each group of mutants was characterized either by the absence of any fruiting structures or by the formation of abnormal fructifications. Among these developmental mutants were two aggregateless isolates, Agg⁻-1 and Agg⁻-2, that could be induced to form normal sorocarps under certain conditions. Sorocarps of the normal D. discoideum type were formed when growing myxamoebae from either of these mutants were allowed to come in contact with myxamoebae of the other mutants, wild-type D. discoideum, D. purpureum, or D. mucoroides. No sorocarps were formed when myxamoebae of Agg⁻-1 and Agg⁻-2 were paired. These two aggregateless mutants, while incapable of aggregating or fruiting when cultivated singly with Escherichia coli B/r on a glucose-salts medium, formed normal fruiting structures after being freed of what appeared to be a product of bacterial growth. The spores produced by Agg⁻-1 and Agg⁻-2 myxamoebae again gave rise to aggregateless clones of the original parental types.     

Production and specificity of monoclonal antibodies against calmodulin from Dictyostelium discoideum

Monoclonal antibodies were raised against calmodulin purified from Dictyostelium discoideum. To increase its antigenicity, the calmodulin was conjugated to keyhole limpet hemocyanin; mice were immunized with the conjugate. Hybridomas producing antibodies against calmodulin were identified by screening culture supernatants with calmodulin coupled to bovine serum albumin. The specificity of antibodies from hybridoma culture supernatants was tested by Western blot of Dictyostelium cell lysates. For the purpose, methods were developed that permitted sensitive detection of calmodulin bound to membranes. The key elements of the blotting protocol were used of PVDF membrane, transfer conducted in phosphate buffer, and glutaraldehyde fixation after transfer. These methods permitted detection of as little as 0.1 ng of calmodulin spotted directly onto the membrane, or 10 ng transferred from an SDS polyacrylamide gel. Ten calmodulin-specific antibodies were identified; most of these reacted preferentially with the calcium-containing form of Dictyostelium calmodulin. Several of the monoclonal antibodies cross-reacted with calmodulin from bovine brain.     

Thymidine-requiring mutants of Dictyostelium discoideum.

Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.     

Isolation of germination mutants of Dictyostelium discoideum

A simple method to separate spores from amoebae of Dictyostelium discoideum has been devized and used to isolate spore germination mutants. A subclass of these mutants is temperature sensitive for germination and growth.     

Selection of chemotaxis mutants of Dictyostelium discoideum

A method has been developed for the efficient selection of chemotaxis mutants of Dictyostelium discoideum. Mutants defective in the chemotactic response to folate could be enriched up to 30-fold in one round of selection using a chamber in which a compartment that contained the chemoattractant was separated by a sandwich of four nitrocellulose filters from a compartment that contained buffer. Mutagenized cells were placed in the center of the filter layer and exposed to the attractant gradient built up between the compartments for a period of 3-4 h. While wild-type cells moved through the filters in a wave towards the compartment that contained attractant, mutant cells remained in the filter to which they were applied. After several repetitions of the selection procedure, mutants defective in chemotaxis made up 10% of the total cell population retained in that filter. Mutants exhibiting three types of alterations were collected: motility mutants with either reduced speed of movement, or altered rates of turning; a single mutant defective in production of the attractant-degrading enzyme, folate deaminase; and mutants with normal motility but reduced chemotactic responsiveness. One mutant showed drastically reduced sensitivity in folate-induced cGMP production. Morphogenetic alterations of mutants defective in folate chemotaxis are described.     

Carbohydrate and other epitopes of the contact site A glycoprotein of Dictyostelium discoideum as characterized by monoclonal antibodies

A series of monoclonal antibodies against a developmentally regulated protein of Dictyostelium discoideum, the contact site A glycoprotein, were used in immunoblots to label proteins of cells harvested at three stages of development: during the growth phase, at the aggregation competent stage, and at the slug stage. The antibodies fell into two groups according to their reactivity with partially or fully deglycosylated forms of the 80 kDa glycoprotein. Group A antibodies reacted not only with a 66 kDa, but also with a 53 kDa product of tunicamycin-treated wild-type cells, and they reacted with a 68 kDa component produced by HL220, a mutant that carries a specific defect in glycosylation. The 68 kDa product of the mutant was not completely unglycosylated. Like the 80 kDa glycoprotein of the wild type, which carried sulfate at carbohydrate residues, the mutant product was sulfated. In the presence of tunicamycin, the mutant produced a 53 kDa component indistinguishable from that of the wild type, which represents, most likely, the non-N-glycosylated protein portion of the contact site A glycoprotein. The group A antibodies showed almost no cross-reactivity with other proteins of the developmental stages tested, in accord with their postulated specificity for the protein moiety of the contact site A molecule. Group B antibodies did not react with the 53 kDa product of tunicamycin-treated cells, nor with the 68 kDa component of mutant HL220. These antibodies were of varying specificity. Some of them were almost as specific as group A antibodies, others cross-reacted with many proteins, particularly of the slug stage. Competition or non-competition between various group B antibodies for binding to the contact site A glycoprotein allowed sub-classification of these antibodies. According to two criteria, group B antibodies were characterized as anti-carbohydrate antibodies: (1) some of these antibodies were blocked by N-acetylglucosamine; (2) none of them reacted with the 68 kDa product or any other protein of mutant HL220. These results indicate that the 80 kDa glycoprotein carries two types of carbohydrate: type 1 carbohydrate that is sulfated and present on the 68 kDa product of mutant HL220, and type 2 carbohydrate that reacts with group B antibodies and is present on the 66 kDa product of tunicamycin-treated wild-type cells. Type 2 carbohydrate moieties are also present on many glycoproteins that are enriched in the prespore area of the slugs.(ABSTRACT TRUNCATED AT 400 WORDS)     

鉴定单克隆抗体识别蛋白质抗原表位方法的建立

为了建立鉴定治疗性单克隆抗体识别蛋白质抗原表位的方法,选择程序死亡受体-1(PD-1)作为目的蛋白。基于丙氨酸扫描策略,建立了定点突变技术和哺乳动物细胞表达系统相结合的抗原突变体快速表达方法,确定了真核表达元件扩增和细胞转染表达的条件。共表达了150个PD-1蛋白突变体,鉴定了这些突变体与抗PD-1抗体帕博利珠单抗的结合能力。根据蛋白突变体与抗体的结合力并结合蛋白结构分析确定了帕博利珠单抗的抗原表位,与已报道的基于晶体结构的抗原表位高度一致,表明本方法操作简单、准确性高,可用于治疗性单克隆抗体的抗原表位作图。     

Elucidation of the antigenic determinant in lipotropin for two monoclonal antibodies

Two monoclonal antibodies were found to give enhanced affinity for β-lipotropin when mixed, as evidenced by competitive radioimmunoassay. Both monoclonals were found to react with a pentapeptide Ala-Glu-Leu-Glu-Tyr, which is a sequence of high local hydrophilicity within the N-terminal section of β-lipotropin.     

Phenotypic suppression of morphogenetic mutants of Dictyostelium discoideum.

The effects of cAMP pulses on the capacity of 15 aggregateless mutants to differentiate and construct fruiting bodies are compared to those obtained when mutant cells are starved with wild-type amoebae. Mutant strains are classified into three main groups depending upon the degree to which their phenotypic defects can be corrected. These data extend studies published earlier [Darmon, M., Brachet, P., and Pereira da Silva, L. (1975). Chemotactic signals induce cell differentiation in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA72, 3163–3166; Pereira da Silva, L., Darmon, M., Brachet, P., Klein, C., and Barrand, P. (1975). Induction of cell differentiation by the chemotactic signal in Dictyostelium discoideum. In “Proceedings of the Tenth FEBS Meeting,” pp. 269–276]. (1) Only one mutant was unresponsive both to cAMP pulses and to the presence of wild-type amoebae and did not display any of the properties of differentiated cells. (2) Following treatment with cAMP pulses, 11 mutants developed certain properties of aggregation-competent amoebae. They increased their levels of cellular phosphodiesterase, showed an enhanced chemotactic sensitivity to cAMP, and established specific cell contacts. None of these amoebae could differentiate further. They did co-aggregate to some extent with wild-type cells, but failed to differentiate into spores. Rather, mutant cells were excluded from the pseudoplasmodium during the process of morphogenesis of the fruiting body. (3) In contrast, the aggregateless phenotype of three mutants was fully corrected by both cAMP pulses and the presence of wild-type cells. These findings are discussed on the basis of a relationship between the chemotactic signal and cell differentiation.     

Defective intercellular cohesion in glycosylation mutants of Dictyostelium discoideum

In order to identify the biological roles of protein-linked oligosaccharides, we have isolated mutants by a selection for amoebae with temperature-sensitive defects in glycan assembly and processing. Of these, 75% were also temperature sensitive for development [Boose and Henderson, 1986]. Two such mutants with distinct developmental phenotypes and glycosylation patterns are described. Mutant HT7 cannot complete aggregation at the restrictive temperature and is defective in expression of EDTA-resistant cohesion. The biochemical defect appears to be early in glycan processing. A revertant of HT7 has recovered aggregation capability, EDTA-resistant cohesion, and reverted almost totally to wild-type glycosylation. Mutant HT15 aggregates at the restrictive temperature but then disperses into a cell lawn. It is less deficient in EDTA-resistant cohesion than HT7 and has a different glycosylation profile. These results provide strong support for a role of protein N-linked oligosaccharides in aggregation-stage intercellular cohesion.     

Fusion-inhibiting monoclonal antibodies and their relevant antigens in relation to sexual process of Dictyostelium discoideum

Sexual cell fusion occurs between NC4 and HM1, the heterothallic strains in Dictyostelium discoideum. Cells of these strains are fusion incompetent when cultured on agar plates in the light and become fusion competent upon cultivation in a liquid medium in darkness. Two cell-surface components, gp70 and gp138, have been identified and characterized as being relevant to sexual cell fusion. Both are glycoproteins, and the former is detected only in fusion-competent HM1 cells, while the latter is detected both in fusion-competent HM1 and fusion-competent NC4 cells. We therefore suspect gp 70 to be responsible for cell recognition and gp138, for membrane fusion. Therefore, NC4 cells are expected to possess specific surface molecule(s) that can be recognized by HM1 cells. In the present study, we raised monoclonal antibodies (mAbs) against membrane fractions of NC4 cells and selected fusion-inhibiting mAbs to identify novel molecules related to sexual cell fusion in D. discoideum. Out of the five mAbs we obtained three, DE1, GG6, and HH9, were characterized. DE1 recognized antigens that specifically existed in fusion-competent NC4 cells but not in fusion-incompetent NC4 or HM1 cells. GG6 recognized cell-surface proteins with approximate molecular weights of 125 and 32 kDa in both fusion-competent NC4 and fusion-competent HM1 cells. In addition GG6 also recognised other proteins commonly present in fusion-incompetent cells. The 125 kDa protein appeared to be the same as gp138. The epitope recognized by HH9 was sodium dodecyl sulfate (SDS)-sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)