Origins of cultivars of Chrysanthemum—Evidence from the chloroplast genome and nuclear LFY gene

The origins of cultivated chrysanthemums have attracted considerable attention, but they remain poorly known. Here, we reconstructed the phylogeny of representative well‐known cultivars and wild species of the genus Chrysanthemum using chloroplast genomes and the nuclear LEAFY gene. Our results suggest that geographic and ecological factors may determine the opportunities for wild species to be involved in the origin of the cultivars. The wild species C. indicum, C. zawadskii, C. dichrum, C. nankingense, C. argyrophyllum, and C. vestitum were likely directly or indirectly involved as paternal species of most of the chrysanthemum cultivars examined in this study. Yet, the maternal species is supported to be a lineage of an extinct wild Chrysanthemum species and its subsequent cultivars, as all accessions of chrysanthemum cultivars sampled formed a strongly supported clade, distinct from all other species of Chrysanthemum in the plastome tree. Thus, the cultivated chrysanthemums originated from multiple hybridizations involving several paternal species rather than only two or a few wild species, with an extinct species and its subsequent cultivars serving as the maternal parents. This finding is consistent with Chrysanthemum having high rates of hybridization and gene flow, which has been demonstrated within previous studies; nevertheless, it is important to unravel the role of an extinct wild Chrysanthemum species as the ultimate maternal parent species for all the chrysanthemum cultivars. Our results also suggest that C. vestitum from Tianzhu and Funiu Mountains in Anhui and Henan Provinces of China represent two distinct cryptic species.     

The structure and function of the chloroplast photosynthetic membrane — a model for the domain organization

Recent work on the domain organization of the thylakoid is reviewed and a model for the thylakoid of higher plants is presented. According to this model the thylakoid membrane is divided into three main domains: the stroma lamellae, the grana margins and the grana core (partitions). These have different biochemical compositions and have specialized functions. Linear electron transport occurs in the grana while cyclic electron transport is restricted to the stroma lamellae. This model is based on the following results and considerations. (1) There is no good candidate for a long-range mobile redox carrier between PS II in the grana and PS I in the stroma lamellae. The lateral diffusion of plastoquinone and plastocyanin is severely restricted by macromolecular crowding in the membrane and the lumen respectively. (2) There is an excess of 14±18% chlorophyll associated with PS I over that of PS II. This excess is assumed to be localized in the stroma lamellae where PS I drives cyclic electron transport. (3) For several plant species, the stroma lamellae account for 20±3% of the thylakoid membrane and the grana (including the appressed regions, margins and end membranes) for the remaining 80%. The amount of stroma lamellae (20%) corresponds to the excess (14–18%) of chlorophyll associated with PS I. (4) The model predicts a quantum requirement of about 10 quanta per oxygen molecule evolved, which is in good agreement with experimentally observed values. (5) There are at least two pools of each of the following components: PS I, PS II, cytochrome bf complex, plastocyanin, ATP synthase and plastoquinone. One pool is in the grana and the other in the stroma compartments. So far, it has been demonstrated that the PS I, PS II and cytochrome bf complexes each differ in their respective pools.Abbreviations PS I and PS II Photosystem I and II - P 700 reaction center of PS I - LHC II light-harvesting complex II     

Function of 3′ non-coding sequences and stop codon usage in expression of the chloroplast psaB gene in Chlamydomonas reinhardtii

The rate of mRNA decay is an important step in the control of gene expression in prokaryotes, eukaryotes and cellular organelles. Factors that determine the rate of mRNA decay in chloroplasts are not well understood. Chloroplast mRNAs typically contain an inverted repeat sequence within the 3 untranslated region that can potentially fold into a stem-loop structure. These stem-loop structures have been suggested to stabilize the mRNA by preventing degradation by exonuclease activity, although such a function in vivo has not been clearly established. Secondary structures within the translation reading frame may also determine the inherent stability of an mRNA. To test the function of the inverted repeat structures in chloroplast mRNA stability mutants were constructed in the psaB gene that eliminated the 3 flanking sequences of psaB or extended the open reading frame into the 3 inverted repeat. The mutant psaB genes were introduced into the chloroplast genome of Chlamydomonas reinhardtii. Mutants lacking the 3 stem-loop exhibited a 75% reduction in the level of psaB mRNA. The accumulation of photosystem I complexes was also decreased by a corresponding amount indicating that the mRNA level is limiting to PsaB protein synthesis. Pulse-chase labeling of the mRNA showed that the decay rate of the psaB mRNA was significantly increased demonstrating that the stem-loop structure is required for psaB mRNA stability. When the translation reading frame was extended into the 3 inverted repeat the mRNA level was reduced to only 2% of wild-type indicating that ribosome interaction with stem-loop structures destabilizes chloroplast mRNAs. The non-photosynthetic phenotype of the mutant with an extended reading frame allowed us to test whether infrequently used stop codons (UAG and UGA) can terminate translation in vivo. Both UAG and UGA are able to effectively terminate PsaB synthesis although UGA is never used in any of the Chlamydomonas chloroplast genes that have been sequenced.     

BioGPS and GXD: mouse gene expression data—the benefits and challenges of data integration

Mouse gene expression data are complex and voluminous. To maximize the utility of these data, they must be made readily accessible through databases, and those resources need to place the expression data in the larger biological context. Here we describe two community resources that approach these problems in different but complementary ways: BioGPS and the Mouse Gene Expression Database (GXD). BioGPS connects its large and homogeneous microarray gene expression reference data sets via plugins with a heterogeneous collection of external gene centric resources, thus casting a wide but loose net. GXD acquires different types of expression data from many sources and integrates these data tightly with other types of data in the Mouse Genome Informatics (MGI) resource, with a strong emphasis on consistency checks and manual curation. We describe and contrast the “loose” and “tight” data integration strategies employed by BioGPS and GXD, respectively, and discuss the challenges and benefits of data integration. BioGPS is freely available at http://biogps.org. GXD is freely available through the MGI web site (www.informatics.jax.org) or directly at www.informatics.jax.org/expression.shtml.     

Effects of site-directed mutation on the function of the chloroplast ATP synthase ε subunit

The previous work in our lab showed that the spinach chloroplast ATP synthase ε mutant with 3 amino acid residues deleted from the N-terminus had much lower ability to inhibit ATP hydrolysis and block proton leakage in comparison to a mutant with 1 or 2 residues deleted from the N-terminus. The present study aimed at determining whether there is special importance in the structure and function of the N-terminal third residue of the chloroplast ε subunit. The leucine residue at the N-terminal third site (Leu3) of the spinach chloroplast ε subunit was replaced with Ile, Phe, Thr, Arg, Glu or Pro by site-directed mutagenesis, forming mutants εL3I, εL3F, εL3T, εL3R, εL3E and εL3P, respectively. These ε variants all showed lower abilities to inhibit ATP hydrolysis and to block proton leakage, as compared to the wild type ε subunit (εWT). The abilities of mutants εL3I and εL3F to restore the ATP synthesis activity of reconstituted membranes were higher than those of εWT, but the abilities of the other ε variants were lower than that of εWT. These results indicate that the hydrophobic and neutral characteristics of Leu3 of the chloroplast ε subunit are very important for its ability to inhibit ATP hydrolysis and block proton leakage, and for the ATP synthesis ability of ATP synthase.     

B lymphocyte differentiation and the control of IgM μ chain expression

The biosynthesis of IgM μ polypeptides was studied in isolated populations of normal B lymphocytes and in various IgM-producing cell lines. Membrane and secretory μ were found to be distinct polypeptide species, with separate biosynthetic intermediates from the translation stage onwards. Various B cell populations express different portions of the two biosynthetic μ pathways. Normal, resting small B lymphocytes do not secrete detectable μ and lack the later intermediate forms of secretory μ. However, they apparently possess, and translate, secretory μ mRNA, and show earlier secretory μ intermediate protein forms. Resting B cells thus exert posttranslational control over secretory μ expression. Since the later intermediate forms of secretory μ, which are lacking in small B cells, are due to carbohydrate modifications of the μ chain, it is suggested that the carbohydrate portion may be involved in regulating the expression of the secretory μ glycoprotein. In contrast to small B cells, highly differentiated IgM-secreting cells control the expression of membrane μ by a pretranslational mechanism.     

Semi-constitutive expression of an Arabidopsis thaliana α-tubulin gene

In Arabidopsis tissues, the pool of tubulin protein is provided by the expression of multiple -tubulin and -tubulin genes. Previous evidence suggested that the TUA2 -tubulin gene was expressed in all organs of mature plants. We now report a more detailed analysis of TUA2 expression during plant development. Chimeric genes containing TUA2 5-flanking DNA fused to the -glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Second-generation progeny of regenerated plants were analyzed by histochemical assay to localize GUS expression. GUS activity was seen throughout plant development and in nearly all tissues. The blue product of GUS activity accumulated to the highest levels in tissues with actively dividing and elongating cells. GUS activity was not detected in a few plant tissues, suggesting that, though widely expressed, the TUA2 promoter is not constitutively active.     

Subunit structure and gene control of mouse NADP—malate dehydrogenase

The NADP-dependent malate dehydrogenase (MDH) in the supernatant fraction of mouse tissues is known to occur in two allelic forms which are electrophoretically distinguishable; each produces a single band in starch gel. We have investigated the subunit structure and synthesis of NADP—MDH through electrophoretic patterns obtained from several experimental sources. (1) Heterozygotes containing both alleles yield a five-banded pattern. The bands are in an approximate frequency of 1:4:6:4:1; the two extremes correspond to the pure types and the three intermediates are presumably hybrid enzymes. The NADP—MDH molecule therefore appears to be a tetramer. (2) In muscle heterokaryons of allophenic mice (with homozygous nuclei of each genotype within a common cytoplasm), hybrid enzymes are formed; they are not formed in other allophenic tissues. Therefore the gene at this locus codes only for monomeric subunits and the tetramer is assembled in a second step in the cytoplasm. Also, both genes must function in a nucleus when the locus is active (e.g., in F₁ uninucleated cells). (3) Dissociation in vitro of mixtures of both pure types of enzymes, followed by reassociation among fragments, leads to a three-banded pattern, even after repeated cycles. Thus the tetramer must cleave in a fixed plane, to form dimers, which reassociate, rather than in a random fashion to form monomers. The most likely interpretation is that mouse NADP—MDH is an example of the type of tetramer postulated by Monod et al. (1965) and termed isologous. The dimers are held symmetrically in the tetrameric conformation by relatively weak forces; the monomeric subunits comprising the dimer are held together by stronger forces.These investigations were supported by U.S.P.H.S. grants No. HD-01646, CA-06927, and FR-05539, and by an appropriation from the Commonwealth of Pennsylvania.     

Analysis of maternal effect mutant combinations elucidates regulation and function of the overlap of hunchback and Krüppel gene expression in the Drosophila blastoderm embryo

The metameric organisation of the Drosophila embryo is generated early during development, due to the action of maternal effect and zygotic segmentation and homeotic genes. The gap genes participate in the complex process of pattern formation by providing a link between the maternal and the zygotic gene activities. Under the influence of maternal gene products they become expressed in distinct domains along the anteroposterior axis of the embryo; negative interactions between neighboring gap genes are thought to be involved in establishing the expression domains. The gap gene activities in turn are required for the correct patterning of the pair-rule genes; little is known, however, about the underlying mechanisms. We have monitored the distribution of gap and pair-rule genes in wild-type embryos and in embryos in which the anteroposterior body pattern is greatly simplified due to combinations of maternal effect mutations (staufen exuperantia, vasa exuperantia, vasa exuperantia, bicoid oskar, bicoid oskar torsolike, vasa torso exuperantia). We show that the domains of protein distribution of the gap genes hunchback and Krüppel overlap in wild-type embryos. Based on the analysis of the maternal mutant combinations, we suggest an explanation of how this overlap is generated. Furthermore, our data show that different constellations of gap gene activities provide different input for the pair-rule genes, and thus strongly suggest that the overlap of hunchback and Krüppel in wild-type is functional in the formation of the patterns of pair-rule genes.     

Cloning and expression of the 3′ portion of the T4 denV gene as a lacZ fusion gene

Polyclonal antibodies have been raised against endonuclease V from the bacteriophage T4. This rabbit serum, from which endemic E. coli antibodies have been removed, reacts with a single protein from T4-infected E. coli with a molecular weight of 16078 dalton. It was confirmed that these antibodies were directed against endonuclease V through the inhibition of the pyrimidine dimer specific nicking activity of endonuclease V in an in vitro nicking assay. A phage λgt11 T4 dC DNA library was screened for phage which produced a β-galactosidase-endonuclease V fusion protein. Immunopositive clones were detected at a frequency of 0.25 % of the plaques in the library. Restriction enzyme analyses of the DNA from 45 of these phage showed that all contained a 1.8 kb T4 EcoRI fragment which had been inserted within λgt11 in a single orientation. Western analysis of proteins which were produced from an induction of lysogens made from these phage reveals a single fusion protein band with a molecular weight slightly larger than native β-galactosidase.     

Induced expression of the IER5 gene by γ-ray irradiation and its involvement in cell cycle checkpoint control and survival

The immediate-early response gene 5 (IER5) was previously shown, using microarray analysis, to be upregulated by ionizing radiation. Here we further characterized the dose- and time-dependency of radiation-induced expression of IER5 at doses from 0.5 to 15 Gy by quantitative real-time PCR analyses in HeLa cells and human lymphoblastoid AHH-1 cells. A radiation-induced increase in the IER5 mRNA level was evident 2 h after irradiation with 2 Gy in both cell lines. In AHH-1 cells the expression reached a peak at 4 h and then quickly returned to the control level, while in HeLa cells the expression only remained increased for a short period of time at around 2 h after irradiation before returning to the control. After high-dose irradiation (10 Gy), the induction of the IER5 expression was lower and delayed in AHH-1 cells as compared with 2-Gy irradiated cells. In HeLa cells, at this dose, two peaks of increased expression were observed 2 h and 12–24 h post-irradiation, respectively. RNA interference technology was employed to silence the IER5 gene in HeLa cells. siRNA-mediated suppression of IER5 resulted in an increased proliferation of HeLa cells. Cell growth and survival analyses demonstrated that suppression of IER5 significantly increased the radioresistance of HeLa cells to radiation doses of up to 6 Gy, but barely affected the sensitivity of cells at 8 Gy. Moreover, suppression of IER5 potentiated radiation-induced arrest at the G2-M transition and led to an increase in the fraction of S phase cells. Taken together, we propose that the early radiation-induced expression of IER5 affects the radiosensitivity via disturbing radiation-induced cell cycle checkpoints.     

Cloning and expression of the α-amylase gene and oxytetracycline production in Streptomyces rimosus

An 8.4 kb Sau3AI DNA fragment containing the Streptomyces rimosus TM-55 -amylase gene (amy) was ligated to a vector pIJ702, named pCYL01, and cloned into amylase deficient mutant S. lividans M2 (amy ^–). Subcloning study showed that the amy gene was localized in 3.3 kbKpnI-PstI fragment. The molecular weight of the purified -amylases of S. lividans M2/pCYL01 and S. rimosus TM-55 were estimated to be 65.7 kDa. Different sizes of recombinant plasmids carrying the amy gene had been retransferred into the parental strain of S. rimosus TM-55. Among these S. rimosus transformants, TM-55/pCYL01, TM-55/pCYL12 and TM-55/pCYL36 showed amylase activity 1.36- to 2.05-fold at the seventh day (1.61 to 2.42 units vs 1.18 units), and oxytetracycline (OTC) production 2.00- to 2.50-fold at the ninth day (approximate 140 to 170 g ml^–1 vs 72 g ml^–1), higher than that of S. rimosus TM-55 alone, respectively. These results showed that industrial microorganisms could be improved by genetic and metabolic engineering.