Isolation Of Homologs Research Articles

Separation of Molar Weight-Distributed Polyethylene Glycols by Reversed-Phase Chromatography—II. Preparative Isolation of Pure Single Homologs

The isolation of single homologs of polyethylene glycol by preparative reversed-phase chromatography is investigated. A thermodynamic model developed accurately previously describes the retention times of individual homologs as function of their size, temperature, and mobile phase composition under linear, diluted conditions. The model is extended to predict limiting retention times for linear gradient operation in preparative applications. Isocratic and gradient-based separations are studied under strongly overloaded conditions. Baseline separation of homologs up to 3000 g/mol is demonstrated. Quantitative production of pure single homologs up to molar weights of 1000 g/mol was performed using an automated setup.

Identification and Characterization of Transcription Factors Regulating Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus.

Biosynthesis of the therapeutically valuable terpenoid indole alkaloids (TIAs), in the medicinal plant Catharanthus roseus, is one of the most elaborate and complex metabolic processes. Although genomic and transcriptomic resources have significantly accelerated gene discovery in the TIA pathway, relatively few genes of transcription factors (TFs) have been identified and characterized thus far. Systematic identification of TFs and elucidation of their functions are crucial for understanding TIA pathway regulation. The successful discovery of TFs in the TIA pathway has relied mostly on three different approaches, (1) identification of cis-regulatory motifs (CRMs) present in the pathway gene promoters as they often provide clues on potential TFs that bind to the promoters, (2) co-expression analysis, based on the assumption that TFs regulating a metabolic or developmental pathway exhibit similar spatiotemporal expression as the pathway genes, and (3) isolation of homologs of TFs known to regulate structurally similar or diverse specialized metabolites in different plant species. TFs regulating TIA pathway have been isolated using either an individual or a combination of the three approaches. Here we describe transcriptome-based coexpression analysis and cis-element determination to identify TFs in C. roseus. In addition, we describe the protocols for generation of transgenic hairy roots, Agrobacterium infiltration of flowers, and electrophoretic mobility shift assay (EMSA). The methods described here are useful for the identification and characterization of potential TFs involved in the regulation of special metabolism in other medicinal plants.

Polyphenol Oxidases in Crops: Biochemical, Physiological and Genetic Aspects.

Enzymatic browning is a colour reaction occurring in plants, including cereals, fruit and horticultural crops, due to oxidation during postharvest processing and storage. This has a negative impact on the colour, flavour, nutritional properties and shelf life of food products. Browning is usually caused by polyphenol oxidases (PPOs), following cell damage caused by senescence, wounding and the attack of pests and pathogens. Several studies indicated that PPOs play a role in plant immunity, and emerging evidence suggested that PPOs might also be involved in other physiological processes. Genomic investigations ultimately led to the isolation of PPO homologs in several crops, which will be possibly characterized at the functional level in the near future. Here, focusing on the botanic families of Poaceae and Solanaceae, we provide an overview on available scientific literature on PPOs, resulting in useful information on biochemical, physiological and genetic aspects.

Characterization of the homologs of flerovium with crown ether based extraction chromatography resins: studies in hydrochloric acid

Eichrom’s Pb resin, a crown-ether-based extraction chromatography resin, was characterized for separation of the flerovium (Fl) homologs, Pb and Sn. Batch uptake of Pb(II) and Sn(IV) radionuclides was determined from an HNO3 matrix. Pb(II) was strongly retained on the resin at all HNO3 concentrations, while Sn(IV) showed no uptake. Extraction kinetics for Pb(II) were examined and show suitable uptake on the second time scale. Separation methods for the isolation of individual homologs, Pb(II) and Sn(IV), have been established using 2 mL pre-packed vacuum flow Pb resin columns.

Flower Development and Perianth Identity Candidate Genes in the Basal Angiosperm Aristolochia fimbriata (Piperales: Aristolochiaceae).

Aristolochia fimbriata (Aristolochiaceae: Piperales) exhibits highly synorganized flowers with a single convoluted structure forming a petaloid perianth that surrounds the gynostemium, putatively formed by the congenital fusion between stamens and the upper portion of the carpels. Here we present the flower development and morphology of A. fimbriata, together with the expression of the key regulatory genes that participate in flower development, particularly those likely controlling perianth identity. A. fimbriata is a member of the magnoliids, and thus gene expression detected for all ABCE MADS-box genes in this taxon, can also help to elucidate patterns of gene expression prior the independent duplications of these genes in eudicots and monocots. Using both floral development and anatomy in combination with the isolation of MADS-box gene homologs, gene phylogenetic analyses and expression studies (both by reverse transcription PCR and in situ hybridization), we present hypotheses on floral organ identity genes involved in the formation of this bizarre flower. We found that most MADS-box genes were expressed in vegetative and reproductive tissues with the exception of AfimSEP2, AfimAGL6, and AfimSTK transcripts that are only found in flowers and capsules but are not detected in leaves. Two genes show ubiquitous expression; AfimFUL that is found in all floral organs at all developmental stages as well as in leaves and capsules, and AfimAG that has low expression in leaves and is found in all floral organs at all stages with a considerable reduction of expression in the limb of anthetic flowers. Our results indicate that expression of AfimFUL is indicative of pleiotropic roles and not of a perianth identity specific function. On the other hand, expression of B-class genes, AfimAP3 and AfimPI, suggests their conserved role in stamen identity and corroborates that the perianth is sepal and not petal-derived. Our data also postulates an AGL6 ortholog as a candidate gene for sepal identity in the Aristolochiaceae and provides testable hypothesis for a modified ABCE model in synorganized magnoliid flowers.

Isolation, expression and evolution of FERTILIZATION INDEPENDENT ENDOSPERM homologs in Podostemaceae.

Podostemaceae is an interesting family of angiosperms with unusual development and morphology. Among these, double fertilization, a defining feature of angiosperms is invariably missing in the family. Consequently, embryo development in the seeds takes place without endosperm. In recent years, the role of polycomb genes has garnered much interest because of their crucial role in seed development. Some of these genes have been reported from many unrelated species, underlining their high conservation. Thus, it becomes exciting to know the role of these genes in podostemads, which are devoid of double fertilization and endosperm. Here, we report the isolation, characterization and expression patterns of homologs of Fertilization Independent Endosperm (FIE) in two species of Podostemaceae, Zeylanidium olivaceum and Polypleurum stylosum. FIE like homologs could be identified in Z. olivaceum (ZoFIE) and P. stylosum (PsFIE). The predicted amino acid sequence of FIE homologs showed similarity to other homologs, containing the conserved seven WD40 repeats. Expression studies revealed that ZoFIE and PsFIE transcripts were present in the vegetative tissue (thallus in Podostemaceae) and the seedlings, similar to the model plants. However, the ZoFIE and PsFIE expression disappeared in the flowering stages. This unique pattern of expression suggests that in the absence of double fertilization and endosperm the expression of FIS complex genes perhaps is obliterated in Podostemaceae.

Production and isolation of homologs of flerovium and element 115 at the Lawrence Livermore National Laboratory Center for Accelerator Mass Spectrometry

New procedures have been developed to isolate no-carrier-added (NCA) radionuclides of the homologs and pseudo-homologs of flerovium (Hg, Sn) and element 115 (Sb), produced by 12–15 MeV proton irradiation of foil stacks with the tandem Van-de-Graaff accelerator at the Lawrence Livermore National Laboratory Center for Accelerator Mass Spectrometry (CAMS) facility. The separation of 113Sn from natIn foil was performed with anion-exchange chromatography from hydrochloric and nitric acid matrices. A cation-exchange chromatography method based on hydrochloric and mixed hydrochloric/hydroiodic acids was used to separate 124Sb from natSn foil. A procedure using Eichrom TEVA resin was developed to separate 197Hg from Au foil. These results demonstrate the suitability of using the CAMS facility to produce NCA radioisotopes for studies of transactinide homologs.

The Role of Atonal Factors in Mechanosensory Cell Specification and Function.

Atonal genes are basic helix-loop-helix transcription factors that were first identified as regulating the formation of mechanoreceptors and photoreceptors in Drosophila. Isolation of vertebrate homologs of atonal genes has shown these transcription factors to play diverse roles in the development of neurons and their progenitors, gut epithelial cells, and mechanosensory cells in the inner ear and skin. In this article, we review the molecular function and regulation of atonal genes and their targets, with particular emphasis on the function of Atoh1 in the development, survival, and function of hair cells of the inner ear. We discuss cell-extrinsic signals that induce Atoh1 expression and the transcriptional networks that regulate its expression during development. Finally, we discuss recent work showing how identification of Atoh1 target genes in the cerebellum, spinal cord, and gut can be used to propose candidate Atoh1 targets in tissues such as the inner ear where cell numbers and biochemical material are limiting.

Rapid isolation of gene homologs across taxa: Efficient identification and isolation of gene orthologs from non-model organism genomes, a technical report

BackgroundTremendous progress has been made in the field of evo-devo through comparisons of related genes from diverse taxa. While the vast number of species in nature precludes a complete analysis of the molecular evolution of even one single gene family, this would not be necessary to understand fundamental mechanisms underlying gene evolution if experiments could be designed to systematically sample representative points along the path of established phylogenies to trace changes in regulatory and coding gene sequence. This isolation of homologous genes from phylogenetically diverse, representative species can be challenging, especially if the gene is under weak selective pressure and evolving rapidly.ResultsHere we present an approach - Rapid Isolation of Gene Homologs across Taxa (RIGHT) - to efficiently isolate specific members of gene families. RIGHT is based upon modification and a combination of degenerate polymerase chain reaction (PCR) and gene-specific amplified fragment length polymorphism (AFLP). It allows targeted isolation of specific gene family members from any organism, only requiring genomic DNA. We describe this approach and how we used it to isolate members of several different gene families from diverse arthropods spanning millions of years of evolution.ConclusionsRIGHT facilitates systematic isolation of one gene from large gene families. It allows for efficient gene isolation without whole genome sequencing, RNA extraction, or culturing of non-model organisms. RIGHT will be a generally useful method for isolation of orthologs from both distant and closely related species, increasing sample size and facilitating the tracking of molecular evolution of gene families and regulatory networks across the tree of life.

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits. Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ. Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.

Underexpression of the plant NOTCHLESS gene, encoding a WD-repeat protein, causes pleitropic phenotype during plant development

WD-repeat proteins are involved in a breadth of cellular processes. While the WD-repeat protein encoding gene NOTCHLESS has been involved in the regulation of the Notch signaling pathway in Drosophila, its yeast homolog Rsa4p was shown to participate in 60S ribosomal subunit biogenesis. The plant homolog ScNLE was previously characterized in Solanum chacoense (ScNLE) as being involved in seed development. However, expression data and reduced size of ScNLE underexpressing plants suggested in addition a role during shoot development. We here report the detailed phenotypic characterization of ScNLE underexpressing plants during shoot development. ScNLE was shown to be expressed in actively dividing cells of the shoot apex. Consistent with this, ScNLE underexpression caused pleiotropic defects such as a reduction in aerial organ size, a reduction in some organ numbers, delayed flowering, and an increase in stomatal index. Analysis of adaxial epidermal cells revealed that both cell number and cell size were reduced in mature leaves of ScNLE underexpressing lines. Two-hybrid screens with the Nle domain and the WD-repeat domain of ScNLE allowed the isolation of homologs of yeast MIDASIN and NSA2 genes, the products of which are involved in 60S ribosomal subunit biogenesis in yeast. A ScNLE-GFP chimeric protein was localized in both the cytoplasm and nucleus. These data altogether suggest that ScNLE likely plays a role in 60S ribosomal subunit biogenesis, which is essential for proper cellular growth and proliferation during plant development.

高等植物Pre-rRNA processing factor 相同遺伝子の単離と特徴付け

高等植物Pre-rRNA processing factor 相同遺伝子の単離と特徴付け

Isolation of acetolactate synthase homologs in common sunflower

A common sunflower population from Howard, SD (HSD) was previously determined to be cross-resistant to imazethapyr and chlorimuron-ethyl, both acetolactate synthase–inhibiting (ALS) herbicides. Experiments were conducted to determine if target-site polymorphisms could act as a mechanism of ALS-inhibitor herbicide resistance in the HSD common sunflower. Approximately 1,600 nucleotides were amplified by polymerase chain reaction and sequenced from putativeALSgene(s) in common sunflower and Jerusalem artichoke. In sunflower, two different amplification products were detected that differed by a nine-basepair deletion. This suggested the presence of at least two ALS genes in common sunflower that could contribute to the herbicide resistance phenotype. In addition, an Ala205to Val205substitution was observed in several clones from resistant common sunflower (amino acid position is relative to the full-length mouse-ear cress ALS protein). Previously documented mutations at this position in other species indicated that it might play a vital role in conferring resistance to one or more ALS-inhibitor herbicides.

Novel purification method for mammalian seminal plasma phospholipid-binding proteins reveals the presence of a novel member of this family of protein in stallion seminal fluid.

A family of bull seminal plasma (BSP) phospholipid-binding proteins (BSP proteins), potentiate heparin- and HDL-induced capacitation. The homologous proteins have been purified from stallion and boar seminal plasma, and detected in low concentrations in other mammalian seminal plasma. In this study, we developed a new isolation method for mammalian seminal plasma choline phospholipid-binding proteins wherein they are present in low concentrations. The method is based on the interaction of this family of proteins with egg yolk low-density lipoprotein fraction (LDF). In order to demonstrate the feasibility of the method, we incubated LDF with alcohol precipitates of bull, boar, and stallion seminal plasma. LDF were re-isolated by ultracentrifugation along with bound proteins. LDF with associated proteins were dialyzed, lyophilized, and delipidated. BSP homologous proteins were finally purified by p-aminophenyl phosphorylcholine (PPC)-agarose and/or gelatin-agarose chromatographies, and analyzed by SDS-PAGE. With this new protocol, phospholipid-binding proteins of bull, boar, and stallion seminal plasma were recovered almost 100%. A new 12 kDa stallion seminal plasma protein of the same family was also isolated and partially sequenced. The radio-immunoassay (RIA) data showed that 10 mg of LDF can bind all BSP proteins present in 120 mg of alcohol precipitated BSP proteins. These results confirm the efficiency of the method and that the LDF step could be used for the isolation of all BSP proteins homologs from different mammalian species.

The Arabidopsis HKT1 gene homolog mediates inward Na(+) currents in xenopus laevis oocytes and Na(+) uptake in Saccharomyces cerevisiae.

The Na(+)-K(+) co-transporter HKT1, first isolated from wheat, mediates high-affinity K(+) uptake. The function of HKT1 in plants, however, remains to be elucidated, and the isolation of HKT1 homologs from Arabidopsis would further studies of the roles of HKT1 genes in plants. We report here the isolation of a cDNA homologous to HKT1 from Arabidopsis (AtHKT1) and the characterization of its mode of ion transport in heterologous systems. The deduced amino acid sequence of AtHKT1 is 41% identical to that of HKT1, and the hydropathy profiles are very similar. AtHKT1 is expressed in roots and, to a lesser extent, in other tissues. Interestingly, we found that the ion transport properties of AtHKT1 are significantly different from the wheat counterpart. As detected by electrophysiological measurements, AtHKT1 functioned as a selective Na(+) uptake transporter in Xenopus laevis oocytes, and the presence of external K(+) did not affect the AtHKT1-mediated ion conductance (unlike that of HKT1). When expressed in Saccharomyces cerevisiae, AtHKT1 inhibited growth of the yeast in a medium containing high levels of Na(+), which correlates to the large inward Na(+) currents found in the oocytes. Furthermore, in contrast to HKT1, AtHKT1 did not complement the growth of yeast cells deficient in K(+) uptake when cultured in K(+)-limiting medium. However, expression of AtHKT1 did rescue Escherichia coli mutants carrying deletions in K(+) transporters. The rescue was associated with a less than 2-fold stimulation of K(+) uptake into K(+)-depleted cells. These data demonstrate that AtHKT1 differs in its transport properties from the wheat HKT1, and that AtHKT1 can mediate Na(+) and, to a small degree, K(+) transport in heterologous expression systems.

Vertebrate Slit, a Secreted Ligand for the Transmembrane Protein Roundabout, Is a Repellent for Olfactory Bulb Axons

The olfactory bulb plays a central role in olfactory information processing through its connections with both peripheral and cortical structures. Axons projecting from the olfactory bulb to the telencephalon are guided by a repulsive activity in the septum. The molecular nature of the repellent is not known. We report here the isolation of vertebrate homologs of the Drosophila slit gene and show that Slit protein binds to the transmembrane protein Roundabout (Robo). Slit is expressed in the septum whereas Robo is expressed in the olfactory bulb. Functionally, Slit acts as a chemorepellent for olfactory bulb axons. These results establish a ligand–receptor relationship between two molecules important for neural development, suggest a role for Slit in olfactory bulb axon guidance, and reveal the existence of a new family of axon guidance molecules.

Isolation and characterization of mammalian homologs of the Drosophila gene glial cells missing.

The glial cells missing (gcm) gene in Drosophila encodes a transcription factor that determines the choice between glial and neuronal fates. We report here the isolation of two mammalian gcm homologs, Gcm1 and Gcm2, and the characterization of their expression patterns during embryonic development. Although Gcm2 is expressed in neural tissues at a low level, the major sites of expression for both of the mammalian genes are nonneural, suggesting that the functions of the mammalian homologs have diverged and diversified. However, when expressed ectopically, Gcm1 can substitute functionally for Drosophila gcm by transforming presumptive neurons into glia. Thus, certain biochemical properties, although not the specificity of the tissue in which the gene is expressed, have been conserved through the evolution of the Gcm gene family.

Identification of the rhesus monkey HLA-G ortholog. Mamu-G is a pseudogene.

HLA-G is a nonclassical MHC class I molecule that is primarily expressed in the placenta. To investigate whether rhesus monkeys possess an HLA-G ortholog, we cloned and sequenced MHC class I cDNAs from the rhesus placenta. We identified two rhesus MHC class I cDNAs with sequence similarity to HLA-G. Each cDNA contained premature stop codons and frameshift mutations, suggesting that it was derived from an MHC class I pseudogene. Gene trees constructed using MHC class I alleles from human and nonhuman primates revealed that the rhesus placental pseudogene alleles clustered with HLA-G orthologs from the human, chimpanzee, and gorilla. These data suggested that this rhesus MHC class I pseudogene is an HLA-G ortholog. This locus was, therefore, designated Mamu (Macaca mulatta)-G. PCR amplification of a portion of Mamu-G from the genomic DNA of five rhesus monkeys resulted in the identification of five additional Mamu-G alleles and revealed the presence of four Mamu-G alleles in one rhesus monkey, suggesting that Mamu-G had been duplicated in this individual. Furthermore, the analysis of 81 MHC class I clones isolated from a rhesus placenta cDNA library did not result in the isolation of Mamu-G cDNAs, nor the isolation of any additional HLA-G homologs, suggesting that Mamu-G was transcribed at negligible levels. Given the similarity of rhesus monkey and human placenta structure and function, these data raise interesting questions regarding the role of HLA-G in pregnancy.

Rapid evolution of human pseudoautosomal genes and their mouse homologs

Comparative studies of genes in the pseudoautosomal region (PAR) of human and mouse sex chromosomes have thus far been very limited. The only comparisons that can presently be made indicate that the PARs of humans and mice are not identical in terms of gene content. Here we describe additional comparative studies of human pseudoautosomal genes and their mouse homologs. Using a somatic cell hybrid mapping panel, we have assigned the mouse homolog of the human pseudoautosomal interleukin 3 receptor alpha subunit (IL3RA) gene to mouse Chromosome (Chr) 14. Attempts to clone the mouse homolog of the human pseudoautosomal adenine nucleotide translocase-3 (ANT3) gene resulted in the isolation of the murine homologs of the human ANT1 and ANT2 genes. The mouse Ant1 and Ant2 genes are very similar in sequence to their human homologs, and we have mapped them to mouse Chromosomes (Chrs) (8 and X respectively) that exhibit conserved synteny with the chromosomes on which the human genes are located. In contrast, the homolog of ANT3 appears to be either very divergent or absent from the mouse genome. Southern blot analysis of DNA from a variety of mammalian species shows restricted conservation of human pseudoautosomal genes, a trend that also applies to the two cloned mouse homologs of these genes and to neighboring human genes in distal Xp22.3. Our observations combined with those of other workers lead us to propose a model for the evolution of the PAR that includes both rapid sequence evolution and the incremental reduction in size of the region during mammalian evolution.

Small GTP-Binding Proteins and Membrane Biogenesis in Plants.

One of the amazing features of the cellular machinery is that all organeller and membrane proteins, as well as those destined for secretion, have an attached address label for targeting to a specific site. Following synthesis, these proteins are folded, shipped, delivered, and received at the right compartment. Their assigned functions are performed only when they are properly placed at a designated site in the cell. Membrane vesicles play an essential role in protein transport as carriers of specific proteins to intracellular compartments. This process begins immediately after perception of specific signals and involves membrane ruffling, budding and transport of ER vesicles, fusion and passage through the Golgi, and release of vesicles from trans-Golgi cisternae to target to the vacuoles and plasma membrane. The components of the vesicle-mediated protein trafficking system are not, however, well defined. It is not known what kind of biochemical principles are operative for unidirectional transport of vesicles. How are vesicles fused to the target compartment? Although much remains to be understood, many studies from yeast and mammalian systems have identified some key players in this pathway. Isolation of plant homologs of some of these proteins has confirmed that these steps are conserved in evolution and must involve well-defined reactions. We focus here on the relevance of small GTP-binding proteins in vesicle-mediated protein transport (for earlier reviews, see Balch, 1990; Bednarek and Raikhel, 1992; Pryer et al., 1992; Terryn et al., 1993; Zerial and Stenmark, 1993).