ABSTRACT
Phosphatidylinositol (PI) and its phosphorylated derivatives known as phosphoinositides (PIPs), are essential regulators of cell signaling and membrane trafficking, cytoskeletal dynamics, and nuclear functions. Disruption of PI metabolism is associated with disorders such as immune dysfunction, cardiovascular disease, and cancer; therefore, there is currently great interest in studying PIPs and their metabolic enzymes. Here, we describe a method for the separation of fluorescent PI and its seven fluorescent phosphorylated derivatives by CE-LIF. The CE method utilizes a Tris buffer and sodium deoxycholate in the presence of 30% 1-propanol and 5% of a dynamic coating reagent, EOTrol low reverse (EOTrol LR). It is simple, fast, highly sensitive, and it offers LODs in the order of 1.5 amol. The effect of cations such as lithium, sodium, potassium, cesium, barium, manganese, zinc, magnesium, calcium, spermine, and gentamicin were evaluated. Calcium and magnesium provided the best selectivity and resolution for the separation of the analytes while magnesium offered the best data reproducibility. The developed CE method would be useful in the studies of enzymatic activity in the PI and PIPs metabolic pathways using CE-based in vitro and CE cell-based assays, and/or for drug screening.
Subject(s)
Electrophoresis, Capillary/methods , Fluorescent Dyes/analysis , Phosphatidylinositols/analysis , Calcium/chemistry , Cations, Divalent , Hydrogen-Ion Concentration , Magnesium/chemistry , Phosphatidylinositols/isolation & purification , Phosphorylation , Reproducibility of Results , TemperatureABSTRACT
Capillary electrophoresis (CE) is a high-resolution technique for the separation of complex biological and chemical mixtures. CE continues to emerge as a powerful tool in the determination of lipids. Here we review the analytical potential of CE for the determination of a wide range of lipids. The different classes of lipids are introduced, and the different modes of CE and optimization methods for the separation of lipids are described. The advantages and disadvantages of the different modes of CE compared to traditional methods like gas chromatography (GC) and liquid chromatography (LC) in the determination of lipids are discussed. Finally, the potential of CE in the determination of lipids in the future is illustrated.
Subject(s)
Electrophoresis, Capillary/methods , Lipids/analysis , Capillary Electrochromatography , Chromatography, Micellar Electrokinetic Capillary , Electrophoresis, Microchip , Lipids/chemistry , Lipids/classificationABSTRACT
The Alu Ya-lineage is a group of related, short interspersed elements (SINEs) found in primates. This lineage includes subfamilies Ya1-Ya5, Ya5a2 and others. Some of these subfamilies are still actively mobilizing in the human genome. We have analyzed 2482 elements that reside in the human genome draft sequence and focused our analyses on the 2318 human autosomal Ya Alu elements. A total of 1470 autosomal loci were subjected to polymerase chain reaction (PCR)-based assays that allow analysis of individual Ya-lineage Alu elements. About 22% (313/1452) of the Ya-lineage Alu elements were polymorphic for the insertion presence on human autosomes. Less than 0.01% (5/1452) of the Ya-lineage loci analyzed displayed insertions in orthologous loci in non-human primate genomes. DNA sequence analysis of the orthologous inserts showed that the orthologous loci contained older pre-existing Y, Sc or Sq Alu subfamily elements that were the result of parallel forward insertions or involved in gene conversion events in the human lineage. This study is the largest analysis of a group of "young", evolutionarily related human subfamilies. The size, evolutionary age and variable allele insertion frequencies of several of these subfamilies makes members of the Ya-lineage useful tools for human population studies and primate phylogenetics.
Subject(s)
Alu Elements , Evolution, Molecular , Genome, Human , Sequence Analysis, DNA , Animals , Cell Line , Chromosomes, Human , Databases, Nucleic Acid , Gene Conversion , Humans , Molecular Sequence Data , Polymorphism, GeneticABSTRACT
The preTa subfamily of long interspersed elements (LINEs) is characterized by a three base-pair "ACG" sequence in the 3' untranslated region, contains approximately 400 members in the human genome, and has low level of nucleotide divergence with an estimated average age of 2.34 million years old suggesting that expansion of the L1 preTa subfamily occurred just after the divergence of humans and African apes. We have identified 362 preTa L1 elements from the draft human genomic sequence, investigated the genomic characteristics of preTa L1 insertions, and screened individual elements across diverse human populations and various non-human primate species using polymerase chain reaction (PCR) assays to determine the phylogenetic origin and levels of human genomic diversity associated with the L1 elements. All of the preTa L1 elements analyzed by PCR were absent from the orthologous positions in non-human primate genomes with 33 (14%) of the L1 elements being polymorphic with respect to insertion presence or absence in the human genome. The newly identified L1 insertion polymorphisms will prove useful as identical by descent genetic markers for the study of human population genetics. We provide evidence that preTa L1 elements show an integration site preference for genomic regions with low GC content. Computational analysis of the preTa L1 elements revealed that 29% of the elements amenable to complete sequence analysis have apparently escaped 5' truncation and are essentially full-length (approximately 6kb). In all, 29 have two intact open reading frames and may be capable of retrotransposition.
