AFLPs mark different genomic regions compared with RFLPs: a case study in tomato.

A set of three tomato chromosome 7 introgression lines (ILs) containing overlapping segments of Lycopersicon pennellii DNA was screened with a set of 10 EcoRI-MseI and 10 PstI-MseI AFLP primer combinations. A large number of markers were identified that mapped to one of the four regions of chromosome 7 defined by the set of three ILs. Because many of the identified markers have known map positions in three tomato reference maps, their location on the tomato genome could be verified. It was demonstrated that the three chromosome 7 ILs carried a chromosome 10 region harbouring a cluster of six AFLP markers that had not been detected before using RFLPs. The causes and implications of this observation are discussed.     

Carrying capacity models should not use fixed prey density thresholds: a plea for using more tools of behavioural ecology

Earlier studies have developed models of carrying capacity to predict the number of animals a certain area can support. These models assume that resources are not renewed after consumption ('standing stock' models), and that the initial number of prey and the rate of prey consumption determine the time a population of foragers can live in an area. Within such areas, foragers give up feeding at a sub-site or patch when intake rates no longer cover energy expenditure. To improve the success rate of the models' predictions, we here change the existing rate-maximising models into fitness-maximising models, and include dynamics in the availability of patches. These new (conceptual) models show that the approaches used so far may over- as well as underestimate carrying capacity. We review empirical studies that have aimed to estimate carrying capacity, and discuss how concepts have been confused. We make explicit suggestions on how to proceed in predicting carrying capacities in future studies.     

Reconstitution and use of highly active human CDK1:Cyclin‐B:CKS1 complexes

As dividing cells transition into mitosis, hundreds of proteins are phosphorylated by a complex of cyclin‐dependent kinase 1 (CDK1) and Cyclin‐B, often at multiple sites. CDK1:Cyclin‐B phosphorylation patterns alter conformations, interaction partners, and enzymatic activities of target proteins and need to be recapitulated in vitro for the structural and functional characterization of the mitotic protein machinery. This requires a pure and active recombinant kinase complex. The kinase activity of CDK1 critically depends on the phosphorylation of a Threonine residue in its activation loop by a CDK1‐activating kinase (CAK). We developed protocols to activate CDK1:Cyclin‐B either in vitro with purified CAKs or in insect cells through CDK‐CAK co‐expression. To boost kinase processivity, we reconstituted a ternary complex consisting of CDK1, Cyclin‐B, and CKS1. In this work, we provide and compare detailed protocols to obtain and use highly active CDK1:Cyclin‐B (CC) and CDK1:Cyclin‐B:CKS1 (CCC).     

Increased risk of atherosclerosis by elevated plasma levels of phospholipid transfer protein

Plasma phospholipid transfer protein (PLTP) is thought to be involved in the remodeling of high density lipoproteins (HDL), which are atheroprotective. It is also involved in the metabolism of very low density lipoproteins (VLDL). Hence, PLTP is thought to be an important factor in lipoprotein metabolism and the development of atherosclerosis. We have overexpressed PLTP in mice heterozygous for the low density lipoprotein (LDL) receptor, a model for atherosclerosis. We show that increased PLTP activity results in a dose-dependent decrease in HDL, and a moderate stimulation of VLDL secretion (相似文献   

Structural and resting state functional connectivity of the subthalamic nucleus: identification of motor STN parts and the hyperdirect pathway

Deep brain stimulation (DBS) for Parkinson's disease often alleviates the motor symptoms, but causes cognitive and emotional side effects in a substantial number of cases. Identification of the motor part of the subthalamic nucleus (STN) as part of the presurgical workup could minimize these adverse effects. In this study, we assessed the STN's connectivity to motor, associative, and limbic brain areas, based on structural and functional connectivity analysis of volunteer data. For the structural connectivity, we used streamline counts derived from HARDI fiber tracking. The resulting tracks supported the existence of the so-called "hyperdirect" pathway in humans. Furthermore, we determined the connectivity of each STN voxel with the motor cortical areas. Functional connectivity was calculated based on functional MRI, as the correlation of the signal within a given brain voxel with the signal in the STN. Also, the signal per STN voxel was explained in terms of the correlation with motor or limbic brain seed ROI areas. Both right and left STN ROIs appeared to be structurally and functionally connected to brain areas that are part of the motor, associative, and limbic circuit. Furthermore, this study enabled us to assess the level of segregation of the STN motor part, which is relevant for the planning of STN DBS procedures.     

Characterization and mapping of a gene controlling shoot regeneration in tomato

The superior regeneration capacity of Lycopersicon peruvianum was introduced into the cultivated tomato Lycopersicon esculentum by backcrossing hybrid material with the tomato genotype VF11. In segregating material derived from these backcrosses, the ability to regenerate shoots on root explants cultured on a zeatin-containing medium, was highly correlated with the ability to regenerate shoots on established callus cultures. The efficient shoot-regenerating root explant system permitted us to study the genetics of this trait and to locate the genes involved, using a set of morphological markers defining all 12 tomato chromosomes. Depending on the tomato genotype, mono, -di- or trigenic ratios were observed. It is concluded that a dominant L. peruvianum allele at a locus (Rg-1) near the middle of chromosome 3 determines efficient shoot regeneration on root explants in tomato in combination with dominant alleles at one or two other loci of either L. peruvianum or L. esculentum origin. The map location of the Rg-1 locus was refined further using a number of chromosome-3-specific RFLPs. The addition of new classical and RFLP linkage data to existing literature data and subsequent processing resulted in a revised and integrated map of tomato chromosome 3. From a morphological and physiological analysis of genotypes differing in Rg phenotype, it is concluded that the genetic component associated with regeneration determines the maintenance of morphogenetic competence and not the sensitivity to hormones.     

Isolation of a 6.2 kb genomic fragment carrying the Adh1 gene of tomato and its expression in transgenic tobacco

An 11 kb Eco RI genomic fragment containing the alcohol dehydrogenase (Adh1) gene was cloned. Cross-hybridization with three Adh2 cDNA clones suggested that the entire coding region of the Adh1 gene was contained on a 6.2 kb Xba I/Hind III subfragment. Using RFLP linkage analysis, the genomic clone was mapped on chromosome 4 between the markers TG 182 and TG 65 in a position corresponding to the Adh1 locus. To further confirm the Adh1 origin of the genomic clone, tobacco plants were transformed with the 6.2 kb Xba I/Hinb III genomic subfragment. Isozyme analysis demonstrated that in transgenic tobacco plants functional tomato specific ADH-1 homodimers were synthesized as well as heterodimers composed of tobacco and tomato subunits.     

Increase of endogenous zeatin riboside by introduction of the ipt gene in wild type and the lateral suppressor mutant of tomato

We studied axillary meristem formation of the lateral suppressor (ls) mutant of tomato after elevating the endogenous cytokinin levels through introduction of the isopentenyltransferase (ipt) gene from Agrobacterium tumefaciens. Growth and development of several transformants were examined during in vitro culture. Transformants exhibited phenotypes varying in severity and were divided into four classes. A number of the ipt transformants had a normal phenotype, as non-transformed plants. Others showed a mild to severe ‘cytokinin-like’ phenotype. Transformants with a mild phenotype exhibited reduced internode length and reduced root development. Transformants with a severe phenotype showed even shorter internodes, loss of apical dominance, reduction of leaf size, production of callus at the basis of the shoots and absence of root development or development of green non-branching roots. The severity of the phenotype correlated well with the level of ipt gene expression, as measured by northern analysis. Transformants with a severe phenotype also exhibited increased levels of zeatin riboside, but zeatin levels were not elevated. The increase in endogenous zeatin riboside levels in the ls mutant did not restore axillary meristem formation, but sometimes bulbous structures were formed in the initially ‘empty’ leaf axils. Several adventitious meristems and shoots developed from below the surface of these structures. It is concluded that a reduced level of cytokinins in the ls mutant shoots is not responsible for the absence of axillary meristem formation.     

The measurement of exocytosis in plant cells

Exocytosis is of vital importance to the growth and developmentof plant cells. It is a dynamic process in which vesicles bearingpolysaccharide precursors and proteins fuse with the plasmamembrane and release their contents. Equally important, newplasma membrane is delivered by exocytosis as secretory vesiclemembrane becomes incorporated. The requirements for polysaccharides,proteins and plasma membrane are very different in differentcell types, so there must be sophisticated mechanisms for ensuringdelivery of these materials to the correct cellular locationsat the appropriate time and, particularly in the case of membrane,their recovery and recycling. Currently, little is known ofthese mechanisms in plants, but new methods for measuring exocytosisare under development, and existing techniques have alreadycontributed data of considerable relevance. Here the methodsfor measuring exocytosis are described and evaluated, with emphasison the electrophysiological measurement of capacitance as arelatively non-invasive method, and on cell-free assays becauseof their potential importance in the identification of proteinsand other factors that control exocytosis in plant cells. Key words: Exocytosis, vesicle traffic, vesicle fusion, polysaccharides, cell wall, cell plate, root cap, secretion, patch-clamping