SOMATIC CELL INCOMPATIBILITY IN DIDYMIUM IRIDIS: LOCUS IDENTIFICATION AND FUNCTION

Somatic incompatibility in two strains of the myxomycete Didymium iridis is controlled by at least 13 loci: seven fusion loci and six clear-zone loci. Details on correlating loci of the Hon 1 strain with Pan 1 loci are given and a unified nomenclature, applicable to both strains, has been developed from data presented in this paper. Although fusion loci generally prevent fusion between different plasmodial incompatibility phenotypes, studies on individual loci have shown that a limited transient fusion may occasionally take place. Thus, the differences between the fusion and clear-zone loci are not as distinct as once thought. However, the 13 somatic incompatibility loci are still easily designated as either fusion or clear-zone loci, and no locus has thus far been found with true intermediate function. Studies on individual locus function are also discussed.     

CONTROL OF PLASMODIAL FUSION IN A PANAMANIAN ISOLATE OF DIDYMIUM IRIDIS

Plasmodial fusion in a Panamanian isolate (Pan. 1) of Didymium iridis is controlled by at least six fusion loci. Only plasmodia with identical phenotypes for these six loci are capable of fusion. No evidence was obtained in this study to indicate that any one fusion locus more strongly affects the fusion process than any of the other loci. Control of plasmodial fusion in the Pan. 1 strain of D. iridis is similar to that found earlier for a Honduran strain (Hon. 1).     

CYTOPHOTOMETRIC MEASUREMENT OF NUCLEAR DNA IN SEVEN HETEROTHALLIC ISOLATES OF DIDYMIUM IRIDIS,A MYXOMYCETE

Absorption cytophotometry was used to measure nuclear Feulgen-DNA content of myxamoebae and Plasmodia in seven heterothallic isolates of Didymium iridis. Measurements of myxamoebal nuclei from clones of four isolates (Hon 1, Pan 1, Pan 2, and CR 5) gave a mean DNA value of 0.34, whereas the nuclei of Plasmodia which develop from each of the four intraisolate crosses had a mean value of 0.63. These values correspond to the 2C haploid level in myxamoebae and the 4C diploid level in Plasmodia. DNA values in two additional isolates (Pan 3 and CR 2) are much higher than the mean for the other five. Accordingly, it is proposed that these may be polyploid. The question of polyploidy in D. iridis and in other myxomycetes is evaluated. The seventh isolate, Ky 1, is taxonomically very close to D. nigripes and was not included in calculations of mean values for D. iridis.     

NUCLEAR DNA CONTENT OF MYXAMOEBAE AND PLASMODIA IN SIX NON-HETEROTHALLIC ISOLATES OF A MYXOMYCETE,DIDYMIUM IRIDIS

The nuclear DNA content of six non-heterothallic isolates of the myxomycete Didymium iridis was measured by combining the Feulgen reaction with absorption microspectrophotometry. This allowed us to distinguish between homothallic (sexual) and apogamic (non-sexual) isolates. Four of the isolates studied, Panamanian 4 and 5, California 1, and Missouri 1 are homothallic. Moreover, the average DNA content of the myxamoebal and plasmodial nuclei (0.32 and 0.61 respectively) does not differ significantly from the calculated haploid and diploid values for heterothallic isolates of D. iridis (0.34 and 0.63). Hence, it is concluded that in each of these isolates the myxamoebae are haploid and the plasmodia diploid. In two of the isolates investigated, Georgia 1 and Hawaii 1, the DNA content of the myxamoebal and plasmodial nuclei did not differ significantly. Therefore, in both of these isolates the plasmodia appear to develop apogamically. In addition the mean DNA values recorded for the Ha-1 isolate suggest that it is aneuploid.     

HETEROTHALLISM AND HOMOTHALLISM: A STUDY OF 27 ISOLATES OF DIDYMIUM IRIDIS,A TRUE SLIME MOLD

Single pore studies of 27 isolates of Didymium iridis from several geographical locations revealed that 11 are heterothallic, displaying a one-locus, multiple allelic mating system. Collectively, 9 of the 11 possess a total of 12 mating types in one multiple allelic series. Each of the remaining two isolates has a pair of mating types which are either incompatible with the 12 or they have not yet been tested against them. Sixteen isolates proved to be homothallic.     

QUANTITATIVE MICROSPECTROPHOTOMETRY OF NUCLEAR DNA IN SELFING STRAINS OF THE MYXOMYCETE DIDYMIUM IRIDIS

Genetic and cytochemical investigations of the origin, development, nuclear activity, and ploidy level of Plasmodia obtained from selfed clones S-2 and B₁P-33 of the heterothallic myxomycete, Didymium iridis, are presented. To demonstrate that selfing did not result from contamination of the clones, or mutations at the mating-type locus, crosses were made between F₁ clones and clones of known mating types. The data were inconsistent with these two possibilities. DNA was quantified by Feulgen-DNA microspectrophotometry. All cellular phases studied (logarithmic amoebae, swarmers, and encysted amoebae) appear to be haploid, with the nuclear DNA being in the replicated (2C) state. The plasmodia are in all cases diploid; however, the data indicate that the selfed Plasmodia are in an extended G₁ condition. The nuclear DNA content of these is therefore 2C, whereas that of the cross Plasmodium is 4C. Sporangial nuclei exhibit DNA in diploid replicated (4C) category.     

FURTHER STUDIES IN MULTIPLE ALLELOMORPH HETEROTHALLISM IN THE MYXOMYCETE DIDYMIUM IRIDIS

Analysis of an isolate of Didymium iridis from Costa Rica indicates that it possesses a pair of incompatibility factors. These factors have been shown to be allelic with those of a Honduras and a Panama isolate. Data presented in this paper show that, among them, the 3 isolates possess 6 incompatibility alleles at 1 locus.     

MULTIPLE ALLELES AT THE INCOMPATIBILITY LOCUS IN THE MYXOMYCETE DIDYMIUM IRIDIS

Collins , O'Neil Ray . (Queens Coll., New York City.) Multiple alleles at the incompatibility locus in the Myxomycete Didymium iridis. Amer. Jour. Bot. 50(5): 477–480. 1963.—Working with 2 isolates of Didymium iridis, from Honduras and Panama, respectively, it was shown that each isolate possesses a pair of incompatibility factors. On the basis of results from inter-isolate crosses, one pair was designated A¹ A² and the other A³ A⁴ to indicate that these factors actually constitute a series of multiple alleles at a single locus.     

COMPARATIVE MEASUREMENTS OF NUCLEAR DNA IN A HETEROTHALLIC AND A SELF-FERTILE ISOLATE OF THE MYXOMYCETE,DIDYMIUM IRIDIS

Comparative measurements were made of the nuclear Feulgen-DNA content of a heterothallic and a self-fertile isolate of the myxomycete Didymium iridis. Plasmodial nuclei of both isolates contain the diploid amount of DNA. The replicated diploid (4C) values for the heterothallic and the self-fertile isolates are 5.66 and 5.95, respectively. Myxamoebae, however, are quite dissimilar in their nuclear DNA content. Those of the heterothallic isolates, Honduran 1–2 (A¹) and Panamanian 2–4 (A⁷), have mean values of 3.81 and 3.69, whereas myxamoebae of the self-fertile Philippine-1 isolate were found to have a mean value of 6.07. Myxamoebae of the Ph-1 isolate are, therefore, at the same ploidy level as the Ph-1 Plasmodium. Mean DNA values for Ph-1 sporangial nuclei were in category 4C. Measurement of the DNA content of mitotic metaphases in sporangia at T = 6 hr confirmed that the mean DNA content of both Ph-1 myxamoebae and plasmodial nuclei is equivalent to 4C. It is concluded that nuclear phase alternance is lacking in the Ph-1 isolate and that the Plasmodium of this isolate develops by apogamy.     

GENETICS OF STERILITY IN HYBRIDS BETWEEN TWO SUBSPECIES OF DROSOPHILA

Hybrids between D. pseudoobscura bogotana and D. pseudoobscura pseudoobscura are fertile except for males produced in one of the two reciprocal crosses. As there is no premating isolation between these subspecies, nonreciprocal male sterility represents the first step in speciation. Genetic analysis reveals two causes of hybrid F₁ sterility: a maternal effect and incompatibilities between chromosomes within males. The maternal effect appears to play the greatest role in hybrid sterility. The X chromosome has the largest effect on fertility of any chromosome, a ubiquitous result in analyses of hybrid sterility and inviability in Drosophila. This effect is entirely attributable to a region comprising less than 30% of the X chromosome. These results are compared to those from a similar study of D. pseudoobscura-D. persimilis hybrids, an older and more reproductively isolated species pair in the same lineage. Such comparisons may allow one to identify the genetic changes characterizing the early versus late stages of speciation.     

防风悬浮培养中的体细胞胚发生

将防风（Ｓａｐｏｓｈｎｉｋｏｖｉａ ｄｉｖａｒｉｃａｔａ （Ｔｕｒｃｚ．） Ｓｃｈｉｓｃｈｋ．）试管苗幼根接种在含０．５ ｍ ｇ／Ｌ ２,４－Ｄ 的ＭＳ培养基上诱导出愈伤组织,继而在含１０％ 椰子乳和０．５ ｍ ｇ／Ｌ ２,４－Ｄ的ＭＳ液体培养基中振荡分散建立了细胞悬浮物。至胚性细胞团产生后转至无激素培养基中形成体细胞胚。观察了从单细胞直到球形胚发生的显微及超微结构的变化。在胚性细胞团阶段细胞内出现周质内质网,它的出现可能与细胞团向原胚过渡时的特殊代谢相联系。从单细胞至原胚各阶段细胞内均含有圆球体,到原胚时数量大增,而且其中央电子致密的基质变小变浅,周边部分扩大并更加透明,暗示圆球体在胚胎发生早期起着贮藏蛋白或脂蛋白的作用,后来消耗于原胚的建成而衍生为脂滴。球形胚阶段,在很多细胞的液泡中出现团块状的液泡蛋白。此外还观察到,悬浮培养时单细胞的聚集能促进体细胞胚的发生     

GENETICAL AND CYTOLOGICAL EVIDENCE FOR CHROMOSOMAL ELIMINATION IN A TRUE SLIME MOLD,DIDYMIUM IRIDIS

In crosses involving a polyploid myxamoebal clone. CR 2-25*, F₁ plasmodia and myxamoebae display a variety of unexpected ploidy levels as indicated by nuclear DNA measurements. Genetic analyses of the F₁ generations reveal either complete elimination of certain genetic markers or greatly skewed segregation ratios. On the basis of these two kinds of evidence, it is assumed that chromosome elimination occurs at some stage (or stages) following karyogamy between parental nuclei. The possible significance of polyploidy in relation to myxomycete speciation and evolution is discussed.     

REGENERATION AND SEXUAL DIFFERENTIATION OF GRIFFITHSIA JAPONICA (CERAMIACEAE,RHODOPHYTA) THROUGH SOMATIC CELL FUSION1

Hybrid cells were obtained from somatic cell fusion among male, female, and tetrasporangial plants in Griffithsia japonica Okamura by a wound-healing process. Isolated fusion cells regenerated new mature plants with mixed reproductive structures. The plants regenerated from hybrid cells between male and female plants developed into 1) spermatangiate, 2) carpogonial, 3) bisexual with spermatangia and carpogonial branches, 4) mixed-phase with spermatangia and tetrasporangia, or 5) bisexual/mixed-phase plants with spermatangia, carpogonial branches, and tetrasporangia. About 70% of the plants regenerated from hybrid cells between male and female plants produced tetrasporangia that were always formed with spermatangia on a single cell. Some of those tetrasporangia released tetraspores, six of which gave rise to mature plants. The plants regenerated from hybrid cells between male and tetrasporangial plants developed into spermatangiate, tetrasporangiate, or mixed-phase plants with spermatangia and tetrasporangia. The plants regenerated from hybrid cells between female and tetrasporangial plants developed into carpogonial, tetrasporangiate, or mixed-phase plants with carpogonial branches and tetrasporangia. All types of reproductive structures we re functional.     

CONTROLLED PRODUCTION OF PROLIFERATING SOMATIC CELL HYBRIDS

The techniques described permit the controlled production of large numbers of proliferating somatic cell hybrids in a relatively short period of time. Sendai virus is used to promote cell hybridization. β-propriolactone is employed as the inactivating agent of Sendai virus since it produces complete loss of viral infectivity while preserving viral fusion capacity. Cells are fused in monolayer, instead of in suspension, since fixing cells in two dimensions permits one to control cell contacts during the fusion event through the expedient of varying multiplicities of the parental cells and the total cell density. Under the conditions described, a several hundred fold increase in the number of hybrid clones obtained is seen as compared to the controls.     

GENETIC CONTROL OF MITOCHONDRIAL THYMIDINE KINASE IN HUMAN-MOUSE AND MONKEY-MOUSE SOMATIC CELL HYBRIDS

Distinctive thymidine (dT) kinase molecular forms are present in mouse, human, and monkey mitochondria. Disk polyacrylamide gel electrophoresis (disk PAGE) analyses have shown that the mitochondrial-specific dT kinases differ from cytosol dT kinases in relative electrophoretic mobilities (Rm). Furthermore, the mouse mitochondrial dT kinase differs in Rm value from primate mitochondrial dT kinases. The mouse and primate cytosol dT kinases can also be distinguished. Disk PAGE analyses have been carried out on the cytosol and mitochondrial dT kinases of human-mouse (WIL-8) and monkey-mouse (mK·CV^III) somatic cell hybrids in order to learn whether the mitochondria of the hybrid cells contained murine mitochondrial-specific, primate mitochondrial-specific, or both dT kinases. WIL-8 cells were derived from cytosol dT kinase-negative, mitochondrial dT kinase-positive mouse fibro blasts and from cytosol dT kinase-positive, mitochondrial dT kinase-positive human embryonic lung cells; they contained mostly mouse chromosomes and a few human chromosomes, including the determinant for human cytosol dT kinase. The mK·CV^III cells were derived from cytosol dT kinase-negative, mitochondrial dT kinase-positive mouse kidney cells and from cytosol dT kinase-positive, mitochondrial dT kinase-positive monkey kidney cells; they contained mostly mouse chromosomes and a few monkey chromosomes, including the determinant for monkey cytosol dT kinase. Disk PAGE analyses demonstrated that the mitochondria of human-mouse and monkey-mouse somatic cell hybrids contained the mouse-specific mitochondrial dT kinase but not the human- or monkey-specific mitochondrial dT kinase. These findings suggest that primate cytosol and mitochondrial thymidine kinase genes are coded on different chromosomes.     

海南岛两个虫媒病毒分离物的初步鉴定

本文报告,1985年夏从海南岛采集的6种蚊子中,分离到4株乙型脑炎病毒和38株非乙脑病毒,其中两株(HN8和HN99)为甲组虫媒病毒。二者抗原性非常相近,对乳小白鼠脑内、腹腔和皮下接种部有较强的致病力,能在多种组织培养细胞上增殖并产生病变,能在鸡胚纤维母细胞上形成空斑,能凝集多种动物红细胞,血凝pH范围为5.75～6.4,最适pH为5.75或6.0,血凝素在4～37℃均较稳定,血凝素对福尔马林敏感。病毒呈球形,有包膜,直径60～65nm,属RNA病毒,对热、酸、乙醚和紫外线均敏感。血清学鉴定表明这两株病毒只与甲组虫媒病毒抗体有反应,尤其与MAY病毒抗原性关系较密切。同年从当地人、畜血清中检查了HN99病毒抗体,其阳性率为:健康人14％,猪23％,山羊35％。另外还与北京的M4株病毒作了初步比较,并对新毒株及我国其它有关毒株的分类学地位提出了初步看法。     

PROTOPLAST FUSION: A NOVEL APPROACH TO ORGANELLE GENETICS IN HIGHER PLANTS

In the majority of higher plants there is maternal inheritance of cytoplasmic organelles and, as a consequence, there are few opportunities for the study of the effects on plant phenotype of having cytoplasm initially containing organelles of both parents. Now the availability of somatic plant protoplasts, which can be fused together and suitably cultured to produce somatic hybrid plants, is enabling the effects of such hybrid cytoplasms to be investigated in higher plants exhibiting maternal inheritance. A very wide range of cytoplasmic genetic diversity, including mitochondrial and chloroplast recombinants, can be produced by such somatic hybridizations, and a theoretical model is presented to show the origins of this wide range of cytoplasmic diversity. Cybrids produced by such protoplast fusions have been shown to be of importance in plant breeding especially in relation to transfer of cytoplasmic male sterility and herbicide resistance. Protoplast fusion, including the fusion of gametic and somatic protoplasts, is also enabling the study of the inheritance of cytoplasmic controlled traits in higher plants.