SEXUAL DIFFERENTIATION OF GRIFFITHSIA (CERAMIALES, RHODOPHYTA): NUCLEAR PLOIDY LEVEL OF MIXED-PHASE PLANTS IN G. JAPONICA1

Mixed-phase plants of Griffithsia japonica Okamura spontaneously occurred in a laboratory culture. Four female plants produced tetrasporangia and spermatangia in addition to their normal female reproductive structures (bisexual/mixed-phase plants), and four male plants produced tetrasporangia as well as spermatangia (male/mixed-phase plants). To determine the nuclear ploidy level of these mixed-phase plants, relative nuclear sizes of male, female, tetrasporangial, and mixed-phase plants were measured using a microscopic image analysis system. Haploid gametophytes could be distinguished from diploid tetrasporophytes by relative nuclear sizes, with the later having nuclei twice the size of the former. Relative nuclear sizes of the mixed-phase plants were similar to those of the haploid plants. Thus, the mixed-phase plants were determined to be haploid. Haploid mixed-phase plants of G. japonica have a potential to produce male, female and tetrasporangial reproductive structures. Sex determination models are discussed to explain "haploid" mixed-phase phenomena in red algae .     

Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana

Four mutants that show the delayed leaf senescence phenotype were isolated from Arabidopsis thaliana . Genetic analyses revealed that they are all monogenic recessive mutations and fall into three complementation groups, identifying three genetic loci controlling leaf senescence in Arabidopsis . Mutations in these loci cause delay in all senescence parameters examined, including chlorophyll content, photochemical efficiency of photosystem II, relative amount of the large subunit of Rubisco, and RNase and peroxidase activity. Delay of the senescence symptoms was observed during both age-dependent in planta senescence and dark-induced artificial senescence in all of the mutant plants. The results indicate that the three genes defined by the mutations are key genetic elements controlling functional leaf senescence and provide decisive genetic evidence that leaf senescence is a genetically programmed phenomenon controlled by several monogenic loci in Arabidopsis . The results further suggest that the three genes function at a common step of age-dependent and dark-induced senescence processes. It is further shown that one of the mutations is allelic to ein2-1 , an ethylene-insensitive mutation, confirming the role of ethylene signal transduction pathway in leaf senescence of Arabidopsis .     

Ginkgolide B production in cultured cells derived from Ginkgo biloba L. leaves

Callus cultures and cell suspension cultures derived from Ginkgo biloba L. leaves produced ginkgolidc B. In cell suspension cultures, the production reached a maximum by the 13th day of subculture and followed by a sharp decrease. The medium of Murashige and Skoog induced the highest ginkgolide B content in cultures while the medium of Schenk and Hildebrandt promoted cell growth. For the maximal production of ginkgolide B, cells were cultured in Murashige and Skoog medium modified to contain 1.0 mg/l of -naphthaleneacetic acid, 0.1 mg/1 of kinetin, 30 g/1 sucrose and 1.25 mM potassium phosphate with a molar ratio of ammonium to nitrate ions of 1 3.Abbreviations B5 Gamborg et al (1968) medium - GKB Ginkgolide B - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic aicd - SH Schenk and Hildebrandt (1972) medium     

Cloning and chromosomal assignment of the bovine interleukin-2 receptor alpha (IL-2Rα) gene

The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number U24226.     

New taxol assay method using tubulin-assembly stimulation

Summary A novel taxol determination method which involves the tubulin-assembly stimulation is described. The tubulin-assembly was monitored by turbidity change at 350nm. In a limited range of taxol concentration (0 to 24 M), taxol stimulated tubulin-assembly linearly. And this linear relation was observed from 20min to 30min after the reaction started. Bioactive derivatives of taxol, such as cephalomanin and 7-epi-10-deacetyltaxol also stimulated the tubulin-assembly. However, baccatin III, which was known as less active taxol derivative did not stimulate tubulin assembly. This result showed that the stimulation of tubulin assembly has a relationship with the antimiotic activity. This assay method have several advantages. 1) Time required for the measurement is relatively short. 2) Multiple samples can be measured simultaneously. 3) It can remove interference of less active taxane compounds more selectively than immuno-assay. Consequently, this method can be used to determine taxol concentration in biological samples. Especially, this method can be used for large scale selection of cell line and primary screening of new antimiotic compounds.     

The binding of isolated mesophyll cells from barley leaves to hyphae of Pyrenophora teres

Live mesophyll cells from barley leaves were isolated and used to investigate contact phenomena between these cells and hyphae of the barley pathogen Pyrenophora teres. When incubated with colonies of P. teres, the isolated cells became attached to the hyphae. The binding of the cells may have been mediated by an affinity between cellulose in the mesophyll cell wall and extracellular material produced by the fungal hyphae. Different fungi exhibited a wide range of affinities for the mesophyll cells. It is suggested that isolated mesophyll cells may be a useful tool in the study of host-pathogen interfaces.     

Conversion of lysine 91 to methionine or glutamic acid in human choriogonadotropin alpha results in the loss of cAMP inducibility.

Human choriogonadotropin (hCG) is a heterodimeric glycoprotein hormone. The alpha subunit comprises 92 amino acids, of which 6 are Lys residues (Morgan, F.G., Birken, S., and Canfield, R.E. (1975) J. Biol. Chem. 250, 5247-5258). Our photoaffinity-labeling studies indicate that several of these Lys residues make contact with the lutropin receptor and are covalently cross-linked to the receptor. Lys-91 of the alpha subunit is of interest because deletion of the two alpha C-terminal residues, Lys-91 and Ser-92, results in a significant reduction in the bioactivity of lutropin and thyrotropin (Cheng, K.-W., Glazer, A.N., and Pierce, J.G. (1973) J. Biol. Chem. 248, 7930-7937). To determine the importance of Lys-alpha 91, we substituted it with Arg, Met, or Glu. The resulting mutant alpha cDNA constructs were co-transfected into CHO cells with the wild type hCG beta cDNA construct. Secreted hCG dimers were assayed for binding to receptors on porcine granulosa cells and stimulation of cAMP synthesis in a murine Leydig tumor cell line. The natural hCG, wild type hCG, and all mutant hCGs recognized the receptor, although with somewhat divergent affinities. However, there was a striking difference in the ability of cAMP induction. The natural hCG, wild type hCG, and Lys-91----Arg mutant hCG induced cAMP synthesis, whereas the Lys-91----Met and Lys-91----Glu mutants did not. These results demonstrate that Lys-91 is important for receptor modulation in the stimulation of cAMP synthesis.     

The Calvin cycle enzyme pentose-5-phosphate 3-epimerase is encoded within the cfx operons of the chemoautotroph Alcaligenes eutrophus.

Several genes (cfx genes) encoding Calvin cycle enzymes in Alcaligenes eutrophus are organized in two highly homologous operons comprising at least 11 kb. One cfx operon is located on the chromosome; the other is located on megaplasmid pHG1 of the organism (B. Bowien, U. Windh?vel, J.-G. Yoo, R. Bednarski, and B. Kusian, FEMS Microbiol. Rev. 87:445-450, 1990). Corresponding regions of about 2.7 kb from within the operons were sequenced. Three open reading frames, designated cfxX (954 bp), cfxY (765 bp), and cfxE (726 bp), were detected at equivalent positions in the two sequences. The nucleotide identity of the sequences amounted to 94%. Heterologous expression of the subcloned pHG1-encoded open reading frames in Escherichia coli suggested that they were functional genes. The observed sizes of the gene products CfxX (35 kDa), CfxY (27 kDa), and CfxE (25.5 kDa) closely corresponded to the values calculated on the basis of the sequence information. E. coli clones harboring the cfxE gene showed up to about 19-fold-higher activities of pentose-5-phosphate 3-epimerase (PPE; EC 5.1.3.1) than did reference clones, suggesting that cfxE encodes PPE, another Calvin cycle enzyme. These data agree with the finding that in A. eutrophus, PPE activity is significantly enhanced under autotrophic growth conditions which lead to a derepression of the cfx operons. No functions could be assigned to CfxX and CfxY.     

Induction of ovulation and oocyte maturation of amphibian (Rana dybowskii) ovarian follicles by protein kinase C activation in vitro.

We previously reported that protein kinase C (PKC) activation induced meiotic maturation (germinal vesicle breakdown, GVBD) of Rana dybowskii follicular oocytes cultured in vitro without hormone treatment. The experiments reported here were carried out to establish whether ovarian follicles ovulated in response to PKC activation during culture. A phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was used for PKC activation. TPA addition (10 microM) to cultured ovarian fragments induced ovulation and maturation of the oocytes similar to that seen following addition of frog pituitary homogenate (FPH, 0.05 pituitary/ml) or progesterone (0.5 microgram/ml). Such changes were not observed when ovarian fragments were treated with inactive phorbol ester. The time course of TPA-induced ovulation was similar to that produced by FPH-stimulated ovulation. Both TPA- and FPH-stimulated ovulation and maturation were blocked by treatment with cycloheximide, forskolin (an adenylate cyclase stimulator), and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7; a PKC inactivator). FPH treatment markedly increased progesterone levels in the medium during ovarian fragment culture whereas TPA treatment failed to elevate progesterone levels. Thus, TPA treatment mimics FPH and progesterone in inducing ovulation and meiotic maturation in cultured amphibian ovarian fragments. The data strongly suggest that PKC plays an important role in regulating ovulation as well as in modulating amphibian oocyte maturation during follicular differentiation.