Evolution and structure of 5S rDNA loci in allotetraploid Nicotiana tabacum and its putative parental species

Nicotiana tabacum (tobacco) is an allotetraploid derived from ancestors of the modern diploids, N. sylvestris and N. tomentosiformis. We identified and characterized two distinct families of 5S ribosomal DNA (rDNA) in N. tabacum; one family had an average 431 bp unit length and the other a 646 bp unit length. In the diploid species, N. sylvestris and N. tomentosiformis, the 5S rDNA unit lengths are 431 bp and 644 bp respectively. The non-coding spacer sequence of the short unit in tobacco had high sequence homology to the spacer of N. sylvestris5S rDNA, while the longer spacer of tobacco had high homology with the 5S spacer of N. tomentosiformis. This suggests that the two 5S families in tobacco have their origin in the diploid ancestors. The longer spacer sequence had a GC rich sub-region (called the T-genome sub-region) that was absent in the short spacer. Pulsed field gel analysis and fluorescent in situ hybridization to tobacco metaphase chromosomes showed that the two families of 5S rDNA units are spatially separate at two chromosomal loci, on chromosomes S8 (short family) and T8 (long family). The repeat copy number at each chromosomal locus showed heterogeneity between different tobacco cultivars, with a tendency for a decrease in the copy number of one family to be compensated by an increase in the copy number of the second family. Sequence analysis reveals there is as much diversity in 5S family units within the diploid species as there is within the T and S-genome 5S family units respectively, suggesting 5S diversification within each family had occurred before tobacco speciation. There is no evidence of interlocus homogenization of the two 5S families in tobacco. This is therefore substantially different to 18-26S rDNA where interlocus gene conversion has substantially influenced most sequences of S and T genome origin; possible reasons are discussed.     

The origin of tobacco's T genome is traced to a particular lineage within Nicotiana tomentosiformis (Solanaceae)

Nicotiana tabacum (tobacco) is a natural allotetraploid. The maternal genome donor is not controversial and is probably derived from an ancestor of N. sylvestris. The paternal, T-genome donor has been less clear, with N. tomentosiformis, N. otophora, or an introgression hybrid proposed. Here we provide evidence that the T genome of N. tabacum is derived from a particular lineage of N. tomentosiformis. We show that the repetitive sequences of geminiviral origin, GRD53 and GRD3, are present in the genomes of N. tabacum cultivars, a tobacco cell suspension culture TBY-2, and N. tomentosiformis ac. NIC 479/84. Surprisingly, they are not present in another three varieties of N. tomentosiformis. A detailed cytogenetic analysis also revealed that N. tomentosiformis ac. NIC 479/84 most closely resembles the N. tabacum T genome in the location of other tandem repetitive sequences. Thus, tobacco formed after divergence within N. tomentosiformis, and the spectrum of potential donors of the paternal genome can be narrowed to a genotype of N. tomentosiformis characterized by the presence of GRD53 and GRD3 repeats. It is clear that future paternity studies in tobacco should use N. tomentosiformis ac. NIC 479/84 rather than any other accession.     

Amino acids do not suppress proteolysis in premature neonates

To determine whether increased amino acid availability can reduce proteolysis in premature neonates and to assess the capacity of infants born prematurely to acutely increase the irreversible catabolism of the essential amino acids leucine (via oxidation) and phenylalanine (via hydroxylation to form tyrosine), leucine and phenylalanine kinetics were measured under basal conditions and in response to a graded infusion of intravenous amino acids (1.2 and 2.4 g. kg(-1). day(-1)) in clinically stable premature (approximately 32 wk gestation) infants in the 1st wk of life. In contrast to the dose-dependent suppression of proteolysis seen in healthy full-term neonates, the endogenous rates of appearance of leucine and phenylalanine (reflecting proteolysis) were unchanged in response to amino acids (297 +/- 21, 283 +/- 19, and 284 +/- 31 micromol. kg(-1). h(-1) for leucine and 92 +/- 6, 92 +/- 4, and 84 +/- 7 micromol. kg(-1). h(-1) for phenylalanine). Similar to full-term neonates, leucine oxidation (40 +/- 5, 65 +/- 6, and 99 +/- 7 micromol. kg(-1). h(-1)) and phenylalanine hydroxylation (12 +/- 1, 16 +/- 1, and 20 +/- 2 micromol. kg(-1). h(-1)) increased in a stepwise fashion in response to graded amino acids. This capacity to increase phenylalanine hydroxylation may be crucial to meet tyrosine needs when exogenous supply is limited. Finally, to determine whether amino acids stimulate glucose production in premature neonates, glucose rate of appearance was measured during each study period. In response to amino acid infusion, rates of endogenous glucose production were unchanged (and near zero).     

Hypertonic induction of aquaporin-5 expression through an ERK-dependent pathway

Aquaporin-5 (AQP5) is a water channel protein expressed in lung, salivary gland, and lacrimal gland epithelia. Each of these sites may experience fluctuations in surface liquid osmolarity; however, osmotic regulation of AQP5 expression has not been reported. This study demonstrates that AQP5 is induced by hypertonic stress and that induction requires activation of extracellular signal-regulated kinase (ERK). Incubation of mouse lung epithelial cells (MLE-15) in hypertonic medium produced a dose-dependent increase in AQP5 expression; AQP5 protein peaked by 24 h and returned to baseline levels within hours of returning cells to isotonic medium. AQP5 induction was observed only with relatively impermeable solutes, suggesting an osmotic pressure gradient is required for induction. ERK was selectively activated in MLE-15 cells by hypertonic stress, and inhibition of ERK activation with two distinct mitogen-activated extracellular regulated kinase kinase (MEK) inhibitors, U0126 and PD98059, blocked AQP5 induction. AQP5 induction was also observed in the lung, salivary, and lacrimal glands of hyperosmolar rats, suggesting potential physiologic relevance for osmotic regulation of AQP5 expression. This report provides the first example of hypertonic induction of an extrarenal aquaporin, as well as the first association between mitogen-activated protein kinase signaling and aquaporin expression.     

Snowdrop lectin (GNA) has no acute toxic effects on a beneficial insect predator,the 2-spot ladybird (Adalia bipunctata L.)

Two-spot ladybird (Adalia bipunctata L.) larvae were fed on aphids (Myzus persicae (Sulz.)) which had been loaded with snowdrop lectin (Galanthus nivalis agglutinin; GNA) by feeding on artificial diet containing the protein. Treatment with GNA significantly decreased the growth of aphids. No acute toxicity of GNA-containing aphids towards the ladybird larvae was observed, although there were small effects on development. When fed a fixed number of aphids, larvae exposed to GNA spent longer in the 4th instar, taking 6 extra days to reach pupation; however, retardation of development was not observed in ladybird larvae fed equal weights of aphids. Ladybird larvae fed GNA-containing aphids were found to be 8-15% smaller than controls, but ate a significantly greater number of aphids (approx. 40% to pupation). GNA was shown to be present on the microvilli of the midgut brush border membrane and within gut epithelial cells in ladybird larvae fed on GNA-dosed aphids, although disruption of the brush border was not observed. It is hypothesised that GNA does not have significant direct toxic or adverse effects on developing ladybird larvae, but that the effects observed may be due to the fact that the aphids fed on GNA are compromised and are thus a suboptimal food.     

In Depth Characterization of Repetitive DNA in 23 Plant Genomes Reveals Sources of Genome Size Variation in the Legume Tribe Fabeae

The differential accumulation and elimination of repetitive DNA are key drivers of genome size variation in flowering plants, yet there have been few studies which have analysed how different types of repeats in related species contribute to genome size evolution within a phylogenetic context. This question is addressed here by conducting large-scale comparative analysis of repeats in 23 species from four genera of the monophyletic legume tribe Fabeae, representing a 7.6-fold variation in genome size. Phylogenetic analysis and genome size reconstruction revealed that this diversity arose from genome size expansions and contractions in different lineages during the evolution of Fabeae. Employing a combination of low-pass genome sequencing with novel bioinformatic approaches resulted in identification and quantification of repeats making up 55–83% of the investigated genomes. In turn, this enabled an analysis of how each major repeat type contributed to the genome size variation encountered. Differential accumulation of repetitive DNA was found to account for 85% of the genome size differences between the species, and most (57%) of this variation was found to be driven by a single lineage of Ty3/gypsy LTR-retrotransposons, the Ogre elements. Although the amounts of several other lineages of LTR-retrotransposons and the total amount of satellite DNA were also positively correlated with genome size, their contributions to genome size variation were much smaller (up to 6%). Repeat analysis within a phylogenetic framework also revealed profound differences in the extent of sequence conservation between different repeat types across Fabeae. In addition to these findings, the study has provided a proof of concept for the approach combining recent developments in sequencing and bioinformatics to perform comparative analyses of repetitive DNAs in a large number of non-model species without the need to assemble their genomes.     

Genome size evolution in relation to leaf strategy and metabolic rates revisited

BACKGROUND AND AIMS: It has been proposed that having too much DNA may carry physiological consequences for plants. The strong correlation between DNA content, cell size and cell division rate could lead to predictable morphological variation in plants, including a negative relationship with leaf mass per unit area (LMA). In addition, the possible increased demand for resources in species with high DNA content may have downstream effects on maximal metabolic efficiency, including decreased metabolic rates. METHODS: Tests were made for genome size-dependent variation in LMA and metabolic rates (mass-based photosynthetic rate and dark respiration rate) using our own measurements and data from a plant functional trait database (Glopnet). These associations were tested using two metrics of genome size: bulk DNA amount (2C DNA) and monoploid genome size (1Cx DNA). The data were analysed using an evolutionary framework that included a regression analysis and independent contrasts using a phylogenetic tree with estimates of molecular diversification times. A contribution index for the LMA data set was also calculated to determine which divergences have the greatest influence on the relationship between genome size and LMA. KEY RESULTS AND CONCLUSIONS: A significant negative association was found between bulk DNA amount and LMA in angiosperms. This was primarily a result of influential divergences that may represent early shifts in growth form. However, divergences in bulk DNA amount were positively associated with divergences in LMA, suggesting that the relationship may be indirect and mediated through other traits directly related to genome size. There was a significant negative association between genome size and metabolic rates that was driven by a basal divergence between angiosperms and gymnosperms; no significant independent contrast results were found. Therefore, it is concluded that genome size-dependent constraints acting on metabolic efficiency may not exist within seed plants.     

ATP-dependent sugar transport complexity in human erythrocytes

Human erythrocyte glucose sugar transport was examined in resealed red cell ghosts under equilibrium exchange conditions ([sugar](intracellular) = [sugar](extracellular), where brackets indicate concentration). Exchange 3-O-methylglucose (3MG) import and export are monophasic in the absence of cytoplasmic ATP but are biphasic when ATP is present. Biphasic exchange is observed as the rapid filling of a large compartment (66% cell volume) followed by the slow filling of the remaining cytoplasmic space. Biphasic exchange at 20 mM 3MG eliminates the possibility that the rapid exchange phase represents ATP-dependent 3MG binding to the glucose transport protein (GLUT1; cellular [GLUT1] of 相似文献   

Differential Sensitivities of Fast- and Slow-Cycling Cancer Cells to Inosine Monophosphate Dehydrogenase 2 Inhibition by Mycophenolic Acid

As uncontrolled cell proliferation requires nucleotide biosynthesis, inhibiting enzymes that mediate nucleotide biosynthesis constitutes a rational approach to the management of oncological diseases. In practice, however, results of this strategy are mixed and thus elucidation of the mechanisms by which cancer cells evade the effect of nucleotide biosynthesis restriction is urgently needed. Here we explored the notion that intrinsic differences in cancer cell cycle velocity are important in the resistance toward inhibition of inosine monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). In short-term experiments, MPA treatment of fast-growing cancer cells effectively elicited G0/G1 arrest and provoked apoptosis, thus inhibiting cell proliferation and colony formation. Forced expression of a mutated IMPDH2, lacking a binding site for MPA but retaining enzymatic activity, resulted in complete resistance of cancer cells to MPA. In nude mice subcutaneously engrafted with HeLa cells, MPA moderately delayed tumor formation by inhibiting cell proliferation and inducing apoptosis. Importantly, we developed a lentiviral vector–based Tet-on label-retaining system that enables to identify, isolate and functionally characterize slow-cycling or so-called label-retaining cells (LRCs) in vitro and in vivo. We surprisingly found the presence of LRCs in fast-growing tumors. LRCs were superior in colony formation, tumor initiation and resistance to MPA as compared with fast-cycling cells. Thus, the slow-cycling compartment of cancer seems predominantly responsible for resistance to MPA.     

Mitochondrial Ccs1 contains a structural disulfide bond crucial for the import of this unconventional substrate by the disulfide relay system

The copper chaperone for superoxide dismutase 1 (Ccs1) provides an important cellular function against oxidative stress. Ccs1 is present in the cytosol and in the intermembrane space (IMS) of mitochondria. Its import into the IMS depends on the Mia40/Erv1 disulfide relay system, although Ccs1 is, in contrast to typical substrates, a multidomain protein and lacks twin Cx(n)C motifs. We report on the molecular mechanism of the mitochondrial import of Saccharomyces cerevisiae Ccs1 as the first member of a novel class of unconventional substrates of the disulfide relay system. We show that the mitochondrial form of Ccs1 contains a stable disulfide bond between cysteine residues C27 and C64. In the absence of these cysteines, the levels of Ccs1 and Sod1 in mitochondria are strongly reduced. Furthermore, C64 of Ccs1 is required for formation of a Ccs1 disulfide intermediate with Mia40. We conclude that the Mia40/Erv1 disulfide relay system introduces a structural disulfide bond in Ccs1 between the cysteine residues C27 and C64, thereby promoting mitochondrial import of this unconventional substrate. Thus the disulfide relay system is able to form, in addition to double disulfide bonds in twin Cx(n)C motifs, single structural disulfide bonds in complex protein domains.