Polymerase chain reaction (PCR) is one of the most important laboratory techniques used in molecular biology, genetics and molecular diagnostics. The success of a PCR-based method largely depends on the correct nucleic acid sequence analysis in silico prior to a wet-bench experiment. Here, we report the development of an online Java-based software for virtual PCR on linear or circular DNA templates and multiple primer or probe search from large or small databases. Primer or probe sensitivity and specificity are predicted by searching a database to find sequences with an optimal number of mismatches, similarity and stability. The software determines primer location, orientation, efficiency of binding and calculates primer melting temperatures for standard and degenerate oligonucleotides. The software is suitable for batch file processing, which is essential for automation when working with large amounts of data. The online Java software is available for download at http://primerdigital.com/tools/pcr.html. Accession numbers for the sequences resulting from this study: EU140956 EU177767 EU867815 EU882730 FJ975775-FJ975780 HM481419 HM481420 KC686837-KC686839 KM262797.
To study subdomain organization of the potato virus X (PVX) movement protein (MP) encoded by the first gene in the triple gene block (TGB), we mutated the 25-kDa TGBp1 protein. The N-terminal deletion of the helicase motifs I, IA, and II resulted in loss of the ATPase activity and RNA binding. A frameshift mutation truncating the C-terminal motifs V and VI gave rise to increase of the TGBp1 ATPase activity and had little effect on RNA binding in vitro. Fusions of the green fluorescent protein with 25-kDa MP and its derivative lacking motifs V-VI exhibited similar fluorescence patterns in epidermal cells of Nicotiana benthamiana leaves. Cell-to-cell movement of the 25K-deficient PVX genome was not complemented by the TGBp1 of Plantago asiatica mosaic potexvirus (PlAMV) but was efficiently complemented by a chimeric TGBp1 consisting of the N-terminal part of PlAMV protein (motifs I-IV) and the PVX-specific C-terminal part (motifs V-VI). These results suggest that NTP hydrolysis, RNA binding, and targeting to the specific cellular compartment(s) are associated with the N-terminal domain of the TGBp1 including the helicase motifs I-IV and that the C-terminal domain is involved in specific interactions with other virus proteins.
One of the key mechanisms linking cell signaling and control of gene expression is reversible phosphorylation of transcription factors. FOXC2 is a forkhead transcription factor that is mutated in the human vascular disease lymphedema-distichiasis and plays an essential role in lymphatic vascular development. However, the mechanisms regulating FOXC2 transcriptional activity are not well understood. We report here that FOXC2 is phosphorylated on eight evolutionarily conserved proline-directed serine/threonine residues. Loss of phosphorylation at these sites triggers substantial changes in the FOXC2 transcriptional program. Through genome-wide location analysis in lymphatic endothelial cells, we demonstrate that the changes are due to selective inhibition of FOXC2 recruitment to chromatin. The extent of the inhibition varied between individual binding sites, suggesting a novel rheostat-like mechanism by which expression of specific genes can be differentially regulated by FOXC2 phosphorylation. Furthermore, unlike the wild-type protein, the phosphorylation-deficient mutant of FOXC2 failed to induce vascular remodeling in vivo. Collectively, our results point to the pivotal role of phosphorylation in the regulation of FOXC2-mediated transcription in lymphatic endothelial cells and underscore the importance of FOXC2 phosphorylation in vascular development. Forkhead box (Fox) proteins are a family of transcription factors (TFs) that play an important role in development, cell cycle regulation, and other key biological processes (1). In mammals, more than 40 forkhead family members have been identified, all sharing the evolutionarily conserved forkhead DNA binding domain. Despite the similarity in their DNA-binding domains, different members of the forkhead family have evolved distinct functional roles. The forkhead transcription factor FOXC2 was first demonstrated to play a role in the morphogenesis of the cardiovascular system during embryonic development (2, 3). Subsequent studies revealed that FOXC2 is also implicated in lymphatic vascular development and disease. Mutations in FOXC2 cause lymphedema-distichiasis (LD; OMIM 153400) characterized by lymphedema and double rows of eyelashes (4). In both humans and mice, FOXC2 is highly expressed in the developing lymphatic vessels, as well as in the adult lymphatic valves (5, 6). The critical role of FOXC2 in lymphatic vascular development has been underscored by the demonstration of abnormal lymphatic patterning and failure to form lymphatic valves in Foxc2-deficient mice (5,7,8). LD patients develop similar defects characterized by lymph and venous reflux, indicating failure or absence of lymphatic and venous valves (9, 10). On a mechanistic level, FOXC2 genomic binding sites are enriched in NFATC1 consensus sequences, and the two transcription factors cooperate in vivo during lymphatic vascular morphogenesis (8).Phosphorylation of transcription factors represents a rapid and reversible mechanism for dynamic regulation of transcriptional networks (11). Most of the Fox...
The 25K movement protein (MP) of potato virus X (PVX) is encoded by the 5'-proximal gene of three overlapping MP genes forming a 'triple gene block'. The PVX 25K MP (putative NTPase-helicase) has been synthesized in Escherichia coli as a recombinant containing a six-histidine tag at the amino terminus. The His-tagged 25K protein was purified in a onecolumn Ni-chelate affinity chromatography procedure. In the absence of any other viral factors, this protein had obvious Mg2+-dependent ATPase activity, which was stimulated slightly (1.7-1.9-fold) by various polynucleotides. Like other viral proteins possessing ATPase-helicase motifs and many plant viral movement proteins, the PVX 25K MP was able to bind nucleic acids in vitro. The RNA binding activity of the 25K MP was pronounced only at very low salt concentrations and was independent of its ATPase activity.
The parechoviruses differ in many biological properties from other picornaviruses, and their replication strategy is largely unknown. In order to identify the viral RNA replication complex in human parechovirus type 1 (HPEV-1)-infected cells, we located viral protein and RNA in correlation to virus-induced membrane alterations. Structural changes in the infected cells included a disintegrated Golgi apparatus and disorganized, dilated endoplasmic reticulum (ER) which had lost its ribosomes. Viral plus-strand RNA, located by electron microscopic (EM) in situ hybridization, and the viral protein 2C, located by EM immunocytochemistry were found on clusters of small vesicles. Nascent viral RNA, visualized by 5-bromo-UTP incorporation, localized to compartments which were immunocytochemically found to contain the viral protein 2C and the trans-Golgi marker 1,4-galactosyltransferase. Protein 2C was immunodetected additionally on altered ER membranes which displayed a complex network-like structure devoid of cytoskeletal elements and with no apparent involvement in viral RNA replication. This protein also exhibited membrane binding properties in an in vitro assay. Our data suggest that the HPEV-1 replication complex is built up from vesicles carrying a Golgi marker and forming a structure different from that of replication complexes induced by other picornaviruses.Parechoviruses belong to a recently established picornavirus genus (35) which contains two human pathogens, human parechovirus type 1 (HPEV-1) and HPEV-2, and Ljungan virus, which has been isolated from rodents. HPEV-1 infections are common and usually occur during the first years of life (46). The virus causes mostly gastrointestinal and respiratory symptoms, but it has also been associated with central nervous system infections. When the parechoviruses were first isolated, they were classified in the enterovirus genus as echoviruses 22 and 23. During their original characterization, exceptional growth properties, which were compared to those of enteroviruses, were observed. These included difficulties in adaptation of the viruses to cultures of monkey kidney cells and restriction of the cytopathic effect to peripheral parts of the cell monolayer (57).Sequence analysis of HPEV-1 revealed that the virus was genetically distant from other picornaviruses, and assignment to an independent group of picornaviruses was suggested (20). Although the organization of the parechovirus genome is similar to that of other picornaviruses, HPEV-1 exhibits some distinct differences in molecular and biological properties (45, 47), such as lack of the maturation cleavage of the capsid protein precursor VP0 to VP2 and VP4 polypeptides (47), inability to cause host cell shut-off (12), and resistance to guanidine hydrochloride (49). As shown for poliovirus (PV), the target of guanidine is the nonstructural protein 2C (34), which is a key protein in the formation and function of the PV replication complex (2,8,14,33,34,50).PV protein 2C, an ATPase (33), and particularly its precurso...
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