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PubMed1998

Unveiling the mechanism of action and regulation of the steroidogenic acute regulatory protein.

Stimulation of steroid-producing cells of the gonads and adrenals with trophic hormone (LH, and ACTH, respectively) produces a marked increase in steroid hormone synthesis within minutes. The rate-limiting step in this acute steroidogenic response is the transport of cholesterol from the outer to the inner mitochondrial membrane, where the first committed step in steroid synthesis is performed by the side-chain cleavage enzyme system (P450scc), resulting in the production of pregnenolone. This process of cholesterol translocation is blocked by inhibitors of protein synthesis (i.e. cycloheximide) indicating that the effect of trophic hormones, acting through the intermediacy of cAMP, most likely involves the de novo synthesis of a protein that is rapidly inactivated. The recently identified steroidogenic acute regulatory protein (StAR) appears to be the most likely candidate for the labile protein: (1) StAR is synthesized in response to cAMP and the StAR preprotein disappears rapidly in the presence of inhibitors of protein synthesis; (2) StAR has an N-terminal targeting sequence that directs the protein to the mitochondria; and (3) StAR protein is expressed almost exclusively in steroid-producing cells, its presence is correlated with steroid hormone production, and lack of functional StAR causes the autosomal recessive disease congenital lipoid adrenal hyperplasia (lipoid CAH), characterized by markedly impaired gonadal and adrenal steroid hormone synthesis. We have demonstrated that StAR is a target for serine phosphorylation mediated by protein kinase A (PKA), a process that is essential to maximizing StAR activity. StAR import by mitochondria is not essential to its steroidogenesis enhancing activity, and more likely, represents a means of rapidly inactivating StAR. Truncation mutations and site-directed mutations in StAR demonstrated that the C-terminus of the protein contains the functionally important domains. Further, we have demonstrated potent steroidogenic activity of recombinant StAR protein on isolated mitochondria from bovine corpus luteum using protein that lacks the mitochondrial targeting sequence. These observations confirm that StAR import is not essential for its steroidogenic activity and suggest that StAR acts directly on the outer mitochondrial membrane in the absence of intermediary cytosolic factors. More recently, we have found that StAR functions as a cholesterol transfer protein that does not require a protein receptor or co-factor, suggesting that StAR acts directly on lipids of the outer mitochondrial membrane to promote cholesterol translocation.

Animals↗

PubMed2006

Identification of a tyrosine-phosphorylated CCCTC-binding nuclear factor in capacitated mouse spermatozoa.

The molecular basis of mammalian sperm capacitation, either in vivo in the female reproductive tract, or in vitro, is poorly understood. It is well known that sperm capacitation is associated with an increase in tyrosine phosphorylation of a subset of proteins. We resolved the phosphoproteins in the cell lysate of mouse sperm after capacitation by 2-DE. One tyrosine-phosphorylated 130-kDa spot was trypsin-digested, and six oligopeptide sequences were established from the MS data. These were confirmed in a CCCTC-binding nuclear factor (CTCF), a widely expressed and highly conserved protein. Further, both an anti-phosphotyrosine antibody and an anti-CTCF antibody showed immunoreactivity to a 130-kDa component in the immunoprecipitates obtained after incubation of the cell lysate from the capacitated sperm using another anti-CTCF antibody. The data support the presence of a tyrosine-phosphorylated CTCF in the capacitated sperm. Immunolocalization of the CTCF revealed fluorescent staining in the acrosome region in both capacitated and incapacitated sperm. The electrophoretic mobility shift assay, using a CTCF target sequence 5'-GGCGGCGCCGCTAGGGGTCTCTCT-3' found in the promoter of the amyloid beta-protein precursor, manifested that, relative to CTCF in the incapacitated sperm, the tyrosine-phosphorylated protein in the capacitated sperm had stronger affinity to the CTCF target sequence.

Amino Acid Sequence↗

PubMed1993

Mutational specificity of oxidative DNA damage.

In this paper we describe our studies on the mutagenic consequences of oxidative DNA damage introduced by radiation-induced OH radicals (.OH) and by exposure to singlet oxygen (1O2), released by thermo-dissociation of the endoperoxide 3,3'-(1,4-naphthalidene) dipropionate (NDPO2). We have made use of M13mp10 bacteriophage and pUC18 plasmid DNA, containing a 144 base pair (bp) insert in the lacZ alpha gene. This 144 bp insert was used as a mutational target sequence. When dilute aqueous solutions of double-stranded (ds) M13mp10 (plus 144 bp insert) were gamma-irradiated in the presence of oxygen (O2; 100% .OH) or nitrous oxide (N2O; 90% .OH, 10% .H), very specific mutation spectra were found. Mainly bp substitutions were observed, of which C/G to G/C transversions are the predominant type. Moreover, the mutations are for the most part concentrated into two mutational hot spots: a minor and major one. Differences between the oxic (O2) and anoxic (N2O) mutation spectra could also be observed. Under N2O-1 bp deletions were detected, which are absent in the presence of O2, and in the anoxic spectrum more C/G to A/T transversions are present. To investigate whether these differences were due to the small amount of H radicals, which are formed under N2O, ds M13mp10 (plus 144 bp insert) was exposed to gamma-rays in phosphate buffer under nitrogen (55% .H, 45% .OH). Under these conditions a remarkable shift was observed from C/G-->G/C to C/G-->A/T transversions, while the mutations were far more scattered along the 144 bp sequence and no -1 bp deletions were detected. These results strongly suggest that H radicals do not cause -1 bp deletions, but may be responsible for the observed C/G to A/T transversions. The kind of bp substitution not only appeared to be dependent on the type of the water radicals, but also appeared to be strongly influenced by the replicon in which the target sequence is incorporated. When an oxygenated solution of pUC18 plasmid DNA (plus 144 bp insert) is irradiated, mainly C/G to A/T transversions were found at the same major hot spot instead of C/G to G/C transversions when the 144 bp sequence is part of M13mp10 DNA. Finally, in agreement with the observation that 1O2 reacts preferentially with guanine in DNA, a guanine is involved in most of the mutations scored after exposure of single-stranded (ss) M13mp10 DNA to NDPO2-generated 1O2.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1996

The molecular basis of Boston-type craniosynostosis: the Pro148-->His mutation in the N-terminal arm of the MSX2 homeodomain stabilizes DNA binding without altering nucleotide sequence preferences.

Craniosynostosis, Boston type is an autosomal dominant disorder that results in the premature fusion of calvarial bones and ensuing abnormalities in skull shape. We showed previously that this disorder is tightly linked to the Msx2 homeobox gene on the long arm of chromosome 5, and that affected individuals bear a mutated copy of Msx2. In addition, transgenic mice in which either mutant or wild-type mouse Msx2 is overexpressed in the developing skull also exhibit craniosynostosis. That both mutant and wild-type Msx2 elicit craniosynostosis in transgenic mice and that the Boston type mutation is dominant led us to hypothesize that the mutation might enhance the normal function of Msx2. The mutation is located in position 7 of the N-terminal arm of the homeodomain, a region implicated in both target sequence recognition and protein-protein interactions. Here we test the hypothesis that the Pro148-->His mutation alters the DNA binding properties of Msx2. Using gel shift and binding site selection analyses, we show that the mutation enhances the affinity of Msx2 for a set of known Msx2 target sequences but has little or no effect on the site specificity of Msx2 binding. The enhancement of Msx2 binding is due largely if not entirely to an increased stability of the mutant Msx2-DNA complex. These data provide a molecular-level explanation of how the Pro148-->His mutation enhances Msx2 function and thus leads to the dominant craniosynostosis phenotype.

Animals↗

PubMed2001

Clinical protocol. A phase 1 open-label clinical trial of the safety and tolerability of single escalating doses of autologous CD4 T cells transduced with VRX496 in HIV-positive subjects.

The proposed study is a Phase I trial to evaluate a HIV-based lentiviral vector carrying an antisense sequence targeted to HIV in the treatment of HIV infection. The primary objective of this Phase I study is to determine the safety and tolerability of treatment with autologous CD4+ T cells modified (transduced) ex vivo with VRX496 when administered to HIV-infected patients. VRX496 is a completely gutted lentiviral vector and does not code for any viral proteins. The viral vector contains an antisense sequence targeted to the HIV envelope (env) gene. VRX496 directly interferes with wild-type HIV (wt-HIV) expression via anti-env antisense expression in vector transduced CD4 cells that become infected with wt-HIV. Expression of the anti-HIV antisense env from a HIV vector transcript would target wt-HIV RNA and destroy it, and hence, decrease productive HIV replication from CD4 T cells. The clinical goal for this treatment approach is to decrease viral loads and promote CD4 T cell survival in vivo. Data from in vitro studies suggest that HIV vectors such as VRX496 could potentially reduce viral loads in HIV-infected individuals and thus could delay the onset to AIDS while promoting CD4 T cell survival and providing the immune system with a better chance to control the infection. Additionally, preliminary results from experiments in SCID mice (mice with transplanted human immune cells) indicate that the human cells transduced with VRX496 and implanted into the SCID mice do not elicit any overt adverse effects. HIV-infected patients (CD4 T cell count of >200/mm3, discontinued from HAART therapy) will undergo leukoapheresis with subsequent CD4 T cell isolation. Patient CD4 T cells will be transduced ex vivo with the vector, expanded for 8-11 days, and then the modified cells will be reintroduced into the patient. Each subject will receive a single intravenous injection infused over 30 minutes; subjects will be examined 24, 48, and 72 hours post-injection and weekly for 4 weeks. Patients will receive one of four different ascending doses (1 x 10(9), 3 x 10(9), 1 x 10(10), and 3 X 10(10) cells/patient). Doses will be administered to four independent, sequential subject cohorts of 3 patients. Groups will be administered escalating doses at 6-week intervals after safety has been demonstrated in the previous group. Follow-up examinations will be conducted 1, 3, and 6 months post-injection. Long term follow-up including RCR testing will be performed.

Adolescent↗

PubMed1994

In vitro selection of antisense oligonucleotides targeted to a hairpin structure.

Antisense oligonucleotides are widely used to selectively prevent pre-RNA splicing, mRNA translation or cDNA synthesis from a retroviral RNA template. However, intramolecular folding of the RNA chain can sequester the target sequence into a stable structure. Consequently, the antisense effect can be greatly reduced or even abolished. Hydrogen donor and acceptor sites are still available on nucleic acid bases involved in secondary structures. However, the rational design of antisense sequences able to recognize the three dimensional array of these sites is not available. We used an in vitro selection procedure to fish out aptastrucs, i.e., oligomers able ("apte") to bind to a structure. A population of randomly synthesized oligonucleotides was mixed with the structure of interest and oligodeoxynucleotide sequences bound to the target were selected and amplified. The selection involves the destruction of the unbound candidates by a restriction enzyme. This procedure can be used both for RNA and DNA target structures and does not require the purification of the bound oligonucleotides at each cycle of selection. Several cycles of selection-amplification, followed by cloning and sequencing, allowed us to identify three oligonucleotides able to form a complex with a DNA hairpin. Due to the sequence of the selected candidates, these aptastruc-hairpin complexes involve very likely non-canonical interactions between the two partners.

DNA↗

PubMed1998

Integrin receptor-targeted transfer peptides for efficient delivery of antisense oligodeoxynucleotides.

Integrin receptor-targeted transfer of oligodeoxynucleotides (ODNs) by small synthetic peptides was used for improving delivery of antisense ODNs. An 18-mer phosphodiester bond containing ODN complementary to c-myb-encoded mRNA was complexed with several transfer peptides, containing as their parts two modules: (a) an RGD-motif as targeting sequence for integrin receptor and (b) nucleocapsid protein (NCp) 7 of HIV-1 or NCp7-derived peptides for complex formation with the ODNs. The amount of antisense ODN required for the inhibition of proliferation of human myeloid cell line HL-60 in vitro can be more than 50-fold reduced by complexing with transfer peptides. The efficiency of antisense delivery was increased by multimerization of the targeting sequence for the integrin receptor. Competition with integrin peptide abolished the effect, indicating that the integrin receptor is indeed responsible for the reaction.

Amino Acid Sequence↗

PubMed2000

Nuclear localization of enzymatically active green fluorescent protein-CTP:phosphocholine cytidylyltransferase alpha fusion protein is independent of cell cycle conditions and cell types.

To address the recent controversy about the subcellular localization of CTP:phosphocholine cytidylyltransferase alpha (CTalpha), this study was designed to visualize green fluorescent protein (GFP). CTalpha fusion proteins directly and continuously under different conditions of cell cycling and in various cell lines. The GFP. CTalpha fusion proteins were enzymatically active and capable of rescuing mutant cells with a temperature-sensitive CT. The expressed GFP.CTalpha fusion protein was localized to the nucleus in all cell lines and required the N-terminal nuclear targeting sequence. Serum depletion/replenishment did not cause shuttling of CTalpha between the nucleus and cytoplasm. Moreover, the subcellular localization of CTalpha was examined continuously through all stages of the cell cycle in synchronized cells. No shuttling of CTalpha between the nucleus and cytoplasm was observed at any stage of the cell cycle. Stimulation of cells with oleate had no effect on the localization of CTalpha. The GFP.CTalpha lacking the nuclear targeting sequence stayed exclusively in the cytoplasm. Regardless of their localization, the GFP.CTalpha fusion proteins were equally active for phosphatidylcholine synthesis and mutant rescue. We conclude that the nuclear localization of CTalpha is a biological event independent of cell cycle in most mammalian cells and is unrelated to activation of phosphatidylcholine synthesis.

Animals↗

PubMed1990

A method for difference cloning: gene amplification following subtractive hybridization.

We describe a procedure for genomic difference cloning, a method for isolating sequences present in one genomic DNA population ("tester") that is absent in another ("driver"). By subtractive hybridization, a large excess of driver is used to remove sequences common to a biotinylated tester, enriching the "target" sequences that are unique to the tester. After repeated subtractive hybridization cycles, tester is separated from driver by avidin/biotin affinity chromatography, and single-stranded target is amplified by the polymerase chain reaction, rendering it double-stranded and clonable. We model two situations: the gain of sequences that result from infection with a pathogen and the loss of sequences that result from a large hemizygous deletion. We obtain 100- to 700-fold enrichment of target sequences.

Bacteriophage lambda↗

PubMed1988

Both immunoglobulin promoter and enhancer sequences are targets for suppression in myeloma-fibroblast hybrid cells.

When immunoglobulin (Ig)-producing B cells are fused with fibroblastic cells, expression of Igs is suppressed by a mechanism that selectively abolishes transcription of Ig genes. The suppression is also maintained in proliferating hybrids. We have used gene transfer followed by cell fusion to study this phenomenon further. Here we report that expression of a rearranged Ig heavy chain gene, stably integrated into a myeloma genome, is completely suppressed upon fusion with fibroblasts by a mechanism that is equally active on the endogenous myeloma lambda light chain gene. To define regulatory sequences within the Ig transcriptional unit that are involved in this down-regulation, we examined the transcriptional contributions of the IgH chain gene enhancer and the kappa light chain gene promoter individually by linking them to a heterologous reporter gene. Mouse myeloma cells were stably transformed with such test constructs and subsequently fused with mouse fibroblasts. To avoid any significant loss of chromosomes, hybrid cells were isolated shortly after fusion by fluorescence-activated cell sorting, and proliferating hybrids were harvested within 2-3 weeks. On the basis of RNase protection mapping of cytoplasmic RNA, and of nuclear run-on assays we showed that both the kappa light chain promoter and the IgH chain enhancer contain regulatory information that is made redundant or is suppressed in the hybrid environment.

Animals↗

PubMed2026

KIT and FLT3-ITD mutations do not predict outcomes in pediatric core-binding factor acute myeloid leukemia: findings from the C-HUANAN-AML-15 multicenter cohort study.

Although core-binding factor acute myeloid leukemia (CBF-AML) is generally considered a favorable-risk subtype in children, disease relapse remains a significant concern. The prognostic relevance of co-occurring mutations, particularly KIT and FLT3-ITD, remains debatable, and treatment intensity may modulate their impact. This multicenter analysis included 289 children (< 14 years) with newly diagnosed CBF-AML enrolled in the C-HUANAN-AML-15 study (2015-2023). KIT and FLT3-ITD mutations were identified via cytogenetic analysis and targeted sequencing. Measurable residual disease (MRD) was evaluated by multiparameter flow cytometry (MFC) and quantitative polymerase chain reaction (PCR) following induction chemotherapy. Survival analyses were performed using Kaplan-Meier and Cox regression methods. This multicenter analysis included 289 children (< 14 years) with newly diagnosed CBF-AML enrolled in the C-HUANAN-AML-15 study (2015-2023). KIT and FLT3-ITD mutations were identified via cytogenetic analysis and targeted sequencing. Measurable residual disease (MRD) was evaluated by multiparameter flow cytometry (MFC) and quantitative polymerase chain reaction (PCR) following induction chemotherapy. Survival analyses were performed using Kaplan-Meier and Cox regression methods. KIT mutations were detected in 103 patients (35.6%), predominantly involving exon 17 (69.9%), and were associated with extramedullary disease, sex chromosome loss, and trisomy 22. No significant differences in 5-year event-free survival (EFS), overall survival (OS), or cumulative incidence of relapse (CIR) were observed between patients with and without KIT mutations. FLT3-ITD mutations (5.5% of patients) did not adversely affect outcomes. Neither mutation independently predicted survival. MRD positivity (MFC-MRD ≥ 0.1%) after the second induction cycle strongly predicted inferior EFS and OS and higher CIR, with corresponding results observed for molecular MRD and parallel findings for PCR-based MRD. In this large multicenter cohort, KIT and FLT3-ITD mutations did not adversely affect the prognosis of pediatric CBF-AML treated according to the C-HUANAN-AML-15 protocol. MRD after induction was the most powerful predictor of relapse and survival, underscoring its importance for risk stratification in future pediatric AML trials.

Humans↗

PubMed2002

The DNA architectural protein HMGB1 displays two distinct modes of action that promote enhanceosome assembly.

HMGB1 (also called HMG-1) is a DNA-bending protein that augments the affinity of diverse regulatory proteins for their DNA sites. Previous studies have argued for a specific interaction between HMGB1 and target proteins, which leads to cooperative binding of the complex to DNA. Here we propose a different model that emerged from studying how HMGB1 stimulates enhanceosome formation by the Epstein-Barr viral activator Rta on a target gene, BHLF-1. HMGB1 stimulates binding of individual Rta dimers to multiple sites in the enhancer. DNase I and hydroxyl radical footprinting, electrophoretic mobility shift assays, and immobilized template assays failed to reveal stable binding of HMGB1 within the complex. Furthermore, mutational analysis failed to identify a specific HMGB1 target sequence. The effect of HMGB1 on Rta could be reproduced by individual HMG domains, yeast HMO1, or bacterial HU. These results, combined with the effects of single-amino-acid substitutions within the DNA-binding surface of HMGB1 domain A, argue for a mechanism whereby DNA-binding and bending by HMGB1 stimulate Rta-DNA complex formation in the absence of direct interaction with Rta or a specific HMGB1 target sequence. The data contrast with our analysis of HMGB1 action on another BHLF-1 regulatory protein called ZEBRA. We discuss the two distinct modes of HMGB1 action on a single regulatory region and propose how HMGB1 can function in diverse contexts.

Animals↗

PubMed1993

C/G to A/T transversions represent the main type of mutation induced by gamma-irradiation in double-stranded M13mp10 DNA in a nitrogen-saturated solution.

To get more insight into the possible mutagenic consequences of DNA damage induced by radiation-generated H radicals (.H), a nitrogen-saturated solution of double-stranded (ds) M13mp10 DNA in phosphate buffer was irradiated with gamma-rays. Under these conditions 55% of the DNA-damaging species consists of H radicals and 45% of OH radicals (.OH). The mutations were investigated in a 144-bp mutational target sequence inserted into the lacZ alpha gene. A very specific mutation spectrum was obtained with respect to the type of mutations. Twenty out of the 28 radiation-induced mutations were C/G to A/T transversions; the remaining 8 mutations were 4 C/G to G/C transversions, 2 C/G to T/A transitions, one T/A to A/T transversion and only one -1 bp deletion. The mutations were rather randomly distributed along the 144-bp mutation target sequence with no clear mutational hot spots. When these results are compared with those we have obtained previously after irradiation of ds M13mp10 DNA under O2 (100% .OH) or N2O (90% .OH; 10% .H) (Hoebee et al., 1988, 1989), the data strongly suggest that H radicals may be responsible for the observed C/G to A/T transversions but not for -1 bp deletions.

Bacteriophage M13↗

PubMed1997

Transcriptional activity and constitutive nuclear localization of the ETS protein Elf-1.

Elf-1 is a lymphoid-specific transcription factor that belongs to the ETS protein family. It can bind to DNA target sequences within a variety of cytokine genes. We demonstrate that Elf-1 is constitutively localized in the nucleus which is dependent on the presence of amino acids 86-265. Analysis of Gal4-Elf-1 fusion proteins revealed that the N-terminal 86 amino acids of Elf-1 contain a transcriptional activation domain, the activity of which is attenuated by an internal repression domain. Furthermore, Elf-1 interacts specifically with the E74 target sequence and can stimulate transcription driven by the E74 site independent of mitogenic signaling. Thus, Elf-1 is able to stimulate gene transcription which may be required for the development and activity of lymphocytes.

Animals↗

PubMed1986

Molecular assembly of tobacco mosaic virus in vitro.

TMV assembly starts with a specific interaction between the assembly origin on the RNA and a disk aggregate of coat protein. The assembly origin is located in the 30K protein cistron for common and tomato strains of TMV and in the coat protein cistron for cowpea strain of TMV and for CGMMV. All the assembly origins have three essential structures: a long base-paired hairpin loop structure; a target sequence, GAPuGUUG, at the top of the hairpin loop structure; and a tract where every third base is a purine. The protein aggregate responsible for the initiation of TMV assembly is a 20S disk, a two-layered aggregate of 34 protein subunits. The two layers of a disk open apart onto the central hole and this structure may be critical for the disk to interact with the assembly origin on the RNA. The target sequence may bind specifically to this structure. Although only a low concentration of 20S disks exists in the usual assembly condition, one disk is enough to initiate TMV assembly. TMV elongation proceeds in two directions. Elongation to the 5'-end proceeds rapidly by preferential incorporation of protein subunits (or A protein) and in 5-7 min gives rise to 260 nm intermediate particles whose 5'-end is coated. A model of elongation toward the 5'-end is shown in Fig. 15. Protected RNAs from nuclease digestion during the assembly reaction produce a banding pattern on gels by electrophoresis. The banding pattern reflects features of the RNA rather than protein that are used in the assembly reaction, since the pattern was the same for assembly between TMV-RNA and CGMMV protein subunits as for assembly between TMV-RNA and TMV protein containing 20S aggregates. The 20S aggregate in the assembly solution has a helical structure with 39 protein subunits rather than the disk structure. Rapid addition of 20S helical aggregates to the top of the growing rod seems to be impossible because of its topological complexity. Elongation toward the 3'-end does not start for at least the first 4 min after initiation. It probably cannot begin until the 5'-end RNA tail disappears into the intermediate rod. Elongation toward the 3'-end favors 20S aggregates as the protein source and gives rise to the full-length rods in about 30 min after the initiation. There are no topological difficulties in adding 20S helical aggregates to the protruding RNA tail.(ABSTRACT TRUNCATED AT 400 WORDS)

Genes, Viral↗

PubMed2002

Characterization of the small antisense CI RNA that regulates bacteriophage P4 immunity.

In the immune state bacteriophage P4 prevents expression of the replication functions by premature termination of transcription. A small RNA, the CI RNA, is the trans acting factor that regulates P4 immunity, by pairing to complementary target sequences and causing premature transcription termination. The CI RNA is matured by RNAse P and PNPase from the leader region of the same operon it regulates. In this work we better characterize this molecule. CI RNA copy number was determined to be around 500 molecules per lysogenic cell. By S(1) mapping we defined the 3'-end at 8423(+/-1); thus CI RNA is 79(+/-1) nt long. The minimum region for correct processing requires two bases upstream of the CI RNA 5'-end and the CCA sequence at the 3'-end. Computer analysis by FOLD RNA of CI RNA sequence predicts a cloverleaf-like structure formed by a double-stranded stalk, a minor and a major stem loop, and a single-stranded bulge. We analysed several cI mutations, which fall either in the single or double-stranded CI RNA regions. Base substitutions in the main loop and in the single-stranded bulge apparently did not change CI RNA structure, but affected its activity by altering the complementarity with the target sequences, whereas a mutation in the secondary stem had a disruptive effect on CI RNA secondary structure. The effects of this latter mutation were suppressed by a base substitution that restored the complementarity with the corresponding base in the stem. Base substitutions in the main stem caused only local alterations in the secondary structure of CI. However, when the substitutions concerned either G8501 or its complementary base at the bottom of the stem, CI RNA was not correctly processed.

Base Sequence↗

PubMed2003

Multiplex PCR: rapid DNA cycling in a conventional thermal cycler.

Multiplex polymerase chain reaction (PCR) is a variant of PCR in which two or more target sequences are simultaneously amplified in the same reaction. In the present study we investigated the limits to which the duration of multiplex PCR steps can be shortened using the thermal cycler Gene Amp PCR system 9600 (Perkin Elmer, Oak Brook, IL). The present multiplex PCR assay simultaneously detects five different herpes viruses (HSV-1, HSV-2, VZV, CMV, and EBV) and assesses sample suitability in a single amplification round of 40 cycles. It appears that when six target sequences are simultaneously amplified in multiplex PCR, extension time is a critical parameter. Using a PCR protocol of 0 sec at 95 degrees C, 0 sec at 60 degrees C, and 0 sec at 74 degrees C with Platinum Taq DNA polymerase (Life Technologies, Gaithersburg, MD), we were able to reduce the total cycling time of the multiplex PCR assay to as little as 55 min, without affecting the yield of PCR products or the specificity of the assay. It may be necessary to optimize each specific apparatus and template, but any such optimization would be trivial.

DNA↗

PubMed2006

TaqMan real-time PCR for detection of hepatopancreatic parvovirus from Australia.

Hepatopancreatic parvovirus is an emerging disease in crustacean aquaculture. Consequently, methods of detection are needed that enable the sensitive detection and confirmation of the virus better than currently used methods such as histology and conventional polymerase chain reaction (PCR). A TaqMan based real-time PCR assay was developed for the detection of the Australian isolate of hepatopancreatic parvovirus which is only 85% similar to its nearest known relative. The TaqMan assay was developed within the capsid protein region of the genome and is optimised to detect as little as 10 copies of the targeted sequence per PCR vial. The hepatopancreatic parvovirus primers and probe were HPV140F 5'-CTA CTC CAA TGG AAA CTT CTG AGC-3', HPV140R 5'-GTG GCG TTG GAA GGC ACT TC-3' and HPV140probe 5'-FAM TAC CGC CGC ACC GCA GCA GC TAMRA-3', respectively. The assay was specific for the hepatopancreatic parvovirus strain from Australian Penaeus merguiensis as it did not detect related crustacean and canine parvoviruses from Australia. In addition, the very low homology of the target sequence with published sequences from the Thai and Korean strains of hepatopancreatic parvovirus and other prawn viruses such as WSSV, suggested this assay would be specific for the Australian hepatopancreatic parvovirus isolate. Furthermore, it detected hepatopancreatic parvovirus in 22/22 wild-caught P. merguiensis clinical samples and 473/545 (87%) farmed P. merguiensis. This assay has the potential to be used for diagnostic purposes and in robotic applications, particularly for the detection and quantitation of low-grade infections.

Animals↗