Evolutionary history of tall fescue morphotypes inferred from molecular phylogenetics of the <Emphasis Type="Italic">Lolium</Emphasis>-<Emphasis Type="Italic">Festuca</Emphasis>species complex

Background  

The agriculturally important pasture grass tall fescue (Festuca arundinacea Schreb. syn. Lolium arundinaceum (Schreb.) Darbysh.) is an outbreeding allohexaploid, that may be more accurately described as a species complex consisting of three major (Continental, Mediterranean and rhizomatous) morphotypes. Observation of hybrid infertility in some crossing combinations between morphotypes suggests the possibility of independent origins from different diploid progenitors. This study aims to clarify the evolutionary relationships between each tall fescue morphotype through phylogenetic analysis using two low-copy nuclear genes (encoding plastid acetyl-CoA carboxylase [Acc1] and centroradialis [CEN]), the nuclear ribosomal DNA internal transcribed spacer (rDNA ITS) and the chloroplast DNA (cpDNA) genome-located matK gene. Other taxa within the closely related Lolium-Festuca species complex were also included in the study, to increase understanding of evolutionary processes in a taxonomic group characterised by multiple inter-specific hybridisation events.     

Comparison of homoeolocus organisation in paired BAC clones from white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.) and microcolinearity with model legume species

Background  

White clover (Trifolium repens L.) is an outbreeding allotetraploid species and an important forage legume in temperate grassland agriculture. Comparison of sub-genome architecture and study of nucleotide sequence diversity within allopolyploids provides insight into evolutionary divergence mechanisms, and is also necessary for the development of whole-genome sequencing strategies. This study aimed to evaluate the degree of divergence between the O and P' sub-genomes of white clover through sequencing of BAC clones containing paired homoeoloci. The microsyntenic relationships between the genomes of white clover and the model legumes Lotus japonicus and Medicago truncatula as well as Arabidopsis thaliana were also characterised.     

PDA: Pooled DNA analyzer

Background  

Association mapping using abundant single nucleotide polymorphisms is a powerful tool for identifying disease susceptibility genes for complex traits and exploring possible genetic diversity. Genotyping large numbers of SNPs individually is performed routinely but is cost prohibitive for large-scale genetic studies. DNA pooling is a reliable and cost-saving alternative genotyping method. However, no software has been developed for complete pooled-DNA analyses, including data standardization, allele frequency estimation, and single/multipoint DNA pooling association tests. This motivated the development of the software, 'PDA' (Pooled DNA Analyzer), to analyze pooled DNA data.     

Reversible depression of oxygen consumption in isolated liver mitochondria during hibernation

The biochemical mechanisms by which hibernators cool as they enter torpor are not fully understood. In order to examine whether rates of substrate oxidation vary as a function of hibernation, liver mitochondria were isolated from telemetered ground squirrels (Spermophilus lateralis) in five phases of their annual hibernation cycle: summer active, and torpid, interbout aroused, entrance, and arousing hibernators. Rates of state 3 and state 4 respiration were measured in vitro at 25 degrees C. Relative to mitochondria from summer-active animals, rates of state 3 respiration were significantly depressed in mitochondria from torpid animals yet fully restored during interbout arousals. These findings indicate that a depression of ADP-dependent respiration in liver mitochondria occurs during torpor and is reversed during the interbout arousals to euthermia. Because this inhibition was determined to be temporally independent of entrance and arousal, it is unlikely that active suppression of state 3 respiration causes entrance into torpor by facilitating metabolic depression. In contrast to the observed depression of state 3 respiration in torpid animals, state 4 respiration did not differ significantly among any of the five groups, suggesting that alterations in proton leak are not contributing appreciably to downregulation of respiration in hibernation.     

Null mutations in a Nudix gene, ygdP, implicate an alarmone response in a novel suppression of hybrid jamming

Induction of the toxic LamB-LacZ protein fusion, Hyb42-1, leads to a lethal generalized protein export defect. The prlF1 suppressor causes hyperactivation of the cytoplasmic Lon protease and relieves the inducer sensitivity of Hyb42-1. Since prlF1 does not cause a detectable change in the stability or level of the hybrid protein, we conducted a suppressor screen, seeking factors genetically downstream of lon with prlF1-like phenotypes. Two independent insertions in the ygdP open reading frame relieve the toxicity of the fusion protein and share two additional properties with prlF1: cold sensitivity and the ability to suppress the temperature sensitivity of a degP null mutation. Despite these similarities, ygdP does not appear to act in the same genetic pathway as prlF1 and lon, suggesting a fundamental link between the phenotypes. We speculate that the common properties of the suppressors relate to secretion defects. The ygdP gene (also known as nudH) has been shown to encode a Nudix protein that acts as a dinucleotide oligophosphate (alarmone) hydrolase. Our results suggest that loss of ygdP function leads to the induction of an alarmone-mediated response that affects secretion. Using an epitope-tagged ygdP construct, we present evidence that this response is sensitive to secretion-related stress and is regulated by differential proteolysis of YgdP in a self-limiting manner.     

Two alanines juxtaposed to aggrecan's G1 domain alter its intracellular localization

Nascent proteins translated and processed in the endoplasmic reticulum (ER) sometimes contain intrinsic signals for ER retention or ER retrieval. These signals are usually a few amino acids in length, and if alanine modifications are made within these sequences, normal transit patterns of the nascent protein frequently change. The purpose of this study was to determine whether two alanines juxtaposed to the first globular domain of aggrecan's core protein affect its transit in Chinese hamster ovary (CHO) cells. Results show that two alanines juxtaposed to the first globular domain (G1AA) minimized secretion of the protein. However, transgenic proteins with juxtaposed glutamate-phenylalanine (G1EF) or no additional amino acids (G1) were still secreted. GFP-tagged G1AA localized in the lumen of the ER but not in the Golgi. In contrast, a portion of GFP-tagged G1EF and G1 did appear in the Golgi compartment. More importantly, unique and striking accumulations of G1EF and G1 transgenic proteins were seen in large dilated regions of the ER cisternae, reminiscent of accumulations seen in alpha1-antitrypsin deficiency disease. G1AA transgenic proteins did not form these vesicles but were diffusely distributed throughout the ER lumen. These results indicate that just two juxtaposed alanines can profoundly affect a large globular protein's intracellular localization.     

Saccharomyces cerevisiae Dap1p,a novel DNA damage response protein related to the mammalian membrane-associated progesterone receptor

The response to damage is crucial for cellular survival, and eukaryotic cells require a broad array of proteins for an intact damage response. We have found that the YPL170W (DAP1 [for damage response protein related to membrane-associated progesterone receptors]) gene is required for growth in the presence of the methylating agent methyl methanesulfonate (MMS). The DAP1 open reading frame shares homology with a broadly conserved family of membrane-associated progesterone receptors (MAPRs). Deletion of DAP1 leads to sensitivity to MMS, elongated telomeres, loss of mitochondrial function, and partial arrest in sterol synthesis. Sensitivity of dap1 strains to MMS is not due to loss of damage checkpoints. Instead, dap1 cells are arrested as unbudded cells after MMS treatment, suggesting that Dap1p is required for cell cycle progression following damage. Dap1p also directs resistance to itraconazole and fluconazole, inhibitors of sterol synthesis. We have found that dap1 cells have slightly decreased levels of ergosterol but increased levels of the ergosterol intermediates squalene and lanosterol, indicating that dap1 cells have a partial defect in sterol synthesis. This is the first evidence linking a MAPR family member to sterol regulation or the response to damage, and these functions are probably conserved in a variety of eukaryotes.     

Tetracysteine genetic tags complexed with biarsenical ligands as a tool for investigating gap junction structure and dynamics

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.     

Physiological response to complex environments in annual Polygonum species of contrasting ecological breadth

Individual physiological response to complex environments is a major factor in the ecological breadth of species. This study compared individual patterns of both long-term and short-term response to controlled, multifactorial environments in four annual Polygonum species that differ in field distribution (P. cespitosum, P. hydropiper, P. lapathifolium, and P. persicaria). To test long-term response, instantaneous net photosynthetic rate and stomatal conductance were measured in situ on one full-sib replicate from five inbred lineages from each of five field populations per species, raised in all possible combinations of low or high light; dry, moist, or flooded soil; and poor or rich nutrient status. Short-term plastic adjustment to changes in light level was examined by switching individual plants of the four species from one of six multifactorial growth environments to the contrasting light environment, and measuring assimilation rates 1 h after transfer. The Polygonum species differed significantly in their patterns of long-term photosynthetic response to particular resources and resource combinations. The species known to have relatively broad ecological distributions (P. persicaria and P. lapathifolium) maintained high photosynthetic performance in a variety of moisture and nutrient environments when grown in high light, while the more narrowly distributed P. hydropiper maintained such functional levels only if given both high light and ample macronutrients. P. cespitosum, a species limited to shaded habitats, maintained low photosynthetic rates across the environmental range. Complex differences among the species in instantaneous water use efficiency (WUE) reflected their highly specific and to some extent independent patterns of photosynthetic and stomatal response to the multifactorial environments. The species also differed significantly in short-term physiological adjustment to changes in light level. Plants of P. persicaria and P. cespitosum reached 78% and 98%, respectively, of their maximum photosynthetic rates 1 h after transfer from low to high light, but P. hydropiper and P. lapathifolium plants reached only c. 60% of their maximum rates. When switched from high to low light, P. persicaria and P. cespitosum plants maintained 64–76% of their maximum rates, while P. hydropiper and P. lapathifolium plants decreased photosynthetic rates sharply to less than 50% of their maximum rates. These results indicate that the latter two species will be less able to maintain effective functional levels in variable light environments, a result consistent with their distributions in the field. Received: 23 May 1997 / Accepted: 3 March 1998     

Central interaction between carotid baroreceptors and skeletal muscle receptors inhibits sympathoexcitation

To determine the potential of an inhibitoryinteraction between the carotid sinus baroreflex (CSB) and the exercisepressor reflex (EPR), both pathways were activated to producesympathoexcitation. It was hypothesized that, under conditions when thebaroreflex increased sympathetic outflow, the interaction between CSBand EPR would be inhibitory. Bilateral carotid occlusion (BCO),electrically induced muscle contraction (EMC), and passive musclestretch (PMS) were used to evoke sympathoexcitation. BCO decreasedsinus pressure 50 ± 5 mmHg, and the levels of muscletension generated by EMC and PMS were 7 ± 2 and 8 ± 1 kg,respectively. This resulted in significant increases in mean arterialpressure (MAP) of 55 ± 9, 50 ± 7, and 50 ± 6 mmHg(P = not significant, BCO vs. EMC vs. PMS) and in heart rate (HR) of 7 ± 2, 19 ± 4, and 17 ± 2 beats/min (P < 0.05, BCO vs.EMC and PMS). When BCO was combined with EMC or PMS, thereflex increase in MAP was augmented (80 ± 8 and 79 ± 10 mmHg;BCO+EMC and BCO+PMS, respectively; P < 0.05). However, summation of the individual MAPresponses was greater than the response evoked during coactivation (106 ± 11 and 103 ± 12 mmHg, respectively,P < 0.05). Because summing theindividual blood pressure responses exceeded the response duringcoactivation, the net effect was that the CSB and EPR interacted in anocclusive manner. In contrast, summation of the individual chronotropic responses was the same as the response evoked during coactivation. Moreover, there was no difference in summation of the individual MAP orHR responses when muscle afferents were activated by either EMC or PMS.In conclusion, the interaction between the CSB and the EPR in controlof MAP was occlusive when both reflexes were stimulated to evokesympathoexcitation. However, summation of the reflexchanges in HR was simply additive.