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At least 397 records · Page 22Linked to original sources
PubMed2001

Recovery of infectious human parainfluenza type 2 virus from cDNA clones and properties of the defective virus without V-specific cysteine-rich domain.

A full-length cDNA clone was constructed from the genome of the human parainfluenza type 2 virus (hPIV2). First, Vero cells were infected with recombinant vaccinia virus expressing T7 RNA polymerase, and then the plasmid encoding the antigenome sequence was transfected into Vero cells together with polymerase unit plasmids, NP, P, and L, which were under control of the T7 polymerase promoter. Subsequently, the transfected cells were cocultured with fresh Vero cells. Rescue of recombinant hPIV2 (rPIV2) from cDNA clone was demonstrated by finding the introduced genetic tag. As an application of reverse genetics, we introduced one nucleotide change (UCU to ACU) to immediate downstream of the RNA-editing site of the V gene in the full-length hPIV2 cDNA and were able to obtain infectious viruses [rPIV2V(-)] from the cDNA. The rPIV2V(-) possessed a defective V protein that did not have the unique cysteine-rich domain in its carboxyl terminus (the V-protein-specific domain). The rPIV2V(-) showed no growth in CV-1 and FL cells. Replication of the rPIV2V(-) in these cells, however, was partially recovered by adding anti-interferon (IFN)-beta antibody into the culture medium, showing that the rPIV2V(-) is highly sensitive against IFN and that no growth of rPIV2V(-) in CV-1 and FL cells is mainly due to its hypersensitivity to endogenously produced IFN. These findings indicate that the V-protein-specific domain of hPIV2 is related to IFN resistance. On the other hand, the rPIV2V(-) efficiently replicated in Vero cells, which are known as a IFN-non-producers. However, the virus yields of rPIV2V(-) in Vero cells were 10- to100-fold lower than those of control rPIV2, although syntheses of the viral-specific proteins and their mRNAs in rPIV2V(-)-infected Vero cells were augmented up to 48 p.i. in comparison with those of rPIV2. Furthermore, the rPIV2V(-) virions showed anomalous in size as compared with rPIV2 virions. These results suggest that the V protein plays an important role in the hPIV2 assembly, maturation, and morphogenesis.

Animals↗
PubMed1991

From linked marker to gene.

An area of increasing importance in human genetics research is the strategy of 'reverse genetics' or 'positional cloning', whereby a gene associated with a genetic disease is isolated on the basis of its approximate chromosomal position. This article and the accompanying centrepage diagram describe the main features of this strategy and outline the techniques involved; they are designed to be used for teaching purposes and as a general reference for those from other fields of genetics research.

Chromosome Mapping↗
PubMed2004

Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes.

Human metapneumovirus (HMPV) is a recently recognized causative agent of respiratory tract disease in individuals of all ages and especially young infants. HMPV remains poorly characterized and has been reported to replicate inefficiently in vitro. Complete consensus sequences were recently determined for two isolates representing the two proposed HMPV genetic subgroups. We have developed a reverse genetic system to produce one of these isolates, CAN97-83, entirely from cDNA. We also recovered a version, rHMPV-GFP, in which the enhanced green fluorescent protein (GFP) was expressed from a transcription cassette inserted as the first gene, leaving the 41-nt leader region and first 16 nt of the N gene undisturbed. The ability to monitor GFP expression in living cells greatly facilitated the initial recovery of this slow-growing virus. In addition, the ability to express a foreign gene from an engineered transcription cassette confirmed the identification of the HMPV transcription signals and identified the F gene-end signal as being highly efficient for transcription termination. The ability to recover virus containing a foreign insert in this position indicated that the viral promoter is contained within the 3'-terminal 57 nt of the genome. Recombinant HMPV replicated in vitro as efficiently as biologically derived HMPV, whereas the kinetics and final yield of rHMPV-GFP were reduced several-fold. Conditions for trypsin treatment were investigated, providing for improved virus yields. Another version of HMPV, rHMPV+G1F23, was recovered that contained a second copy of the G gene and two extra copies of F in promoter-proximal positions in the order G1-F2-F3. Thus, this recombinant genome would encode 11 mRNAs rather than eight and would be 17.3 kb long, 30% longer than that of the natural virus. Nonetheless, the rHMPV+G1F23 virus replicated in vitro with an efficiency that was only modestly reduced compared to rHMPV and was essentially the same as rHMPV-GFP. Northern blot analysis showed that the increased number and promoter-proximal location of the added copies of the F and G genes resulted in a more than 6- and 14-fold increase in the expression of F and G mRNA, respectively, and sequence analysis confirmed the intactness of the added genes in recovered virus. Thus, it should be feasible to construct an HMPV vaccine virus containing extra copies of the G and F putative protective antigen genes to increase antigen expression or to provide representation of additional antigenic lineages or subgroups of HMPV.

Animals↗
PubMed1993

Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1.

Expression vectors based on DNA or plus-stranded RNA viruses are being developed as vaccine carriers directed against various pathogens. Less is known about the use of negative-stranded RNA viruses, whose genomes have been refractory to direct genetic manipulation. Using a recently described reverse genetics method, we investigated whether influenza virus is able to present antigenic structures from other infectious agents. We engineered a chimeric influenza virus which expresses a 12-amino-acid peptide derived from the V3 loop of gp120 of human immunodeficiency virus type 1 (HIV-1) MN. This peptide was inserted into the loop of antigenic site B of the influenza A/WSN/33 virus hemagglutinin (HA). The resulting chimeric virus was recognized by specific anti-V3 peptide antibodies and a human anti-gp120 monoclonal antibody in both hemagglutination inhibition and neutralization assays. Mice immunized with the chimeric influenza virus produced anti-HIV antibodies which were able to bind to synthetic V3 peptide, to precipitate gp120, and to neutralize MN virus in human T-cell culture system. In addition, the chimeric virus was also capable of inducing cytotoxic T cells which specifically recognize the HIV sequence. These results suggest that influenza virus can be used as an expression vector for inducing both B- and T-cell-mediated immunity against other infectious agents.

Amino Acid Sequence↗
PubMed2000

Genetic analysis of essential hypertension in Japanese populations.

The reverse genetic approach, which examines genetic factors underlying the root of pathogenesis first, is a powerful tool to clarify the genetic cause of essential hypertension. Using the rat cross model, studies of the genetically hypertensive inbred rat model indicated several candidate loci on the rat chromosome responsible for blood pressure, but failed to identify the exact causal gene. Moreover, it was not certain that the rat data really reflect the human case. Thus, we shifted our focus to human genetics and carried out case control studies using the candidate gene approach. We mainly focused on gene components of the renin-angiotensin system as candidates, finding that angiotensinogen gene polymorphisms are genetic predisposing factors for hypertension. However, the results obtained from case-control studies using Japanese subjects were not consistent, suggesting that there was a problem in control sampling. In our recent study, we recruited more than 5,000 residents of an urban community as a general population and examined the association between genetic factors and their health status. Our results indicate that angiotensin-converting enzyme gene polymorphism is a male-specific genetic risk for essential hypertension. In light of our previous investigations, we present a discussion concerning the design of future studies of the genetics of hypertension.

Angiotensinogen↗
PubMed2006

Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb.

Increased interest in using zebrafish as a model organism has led to a resurgence of fin regeneration studies. This has allowed for the identification of a large number of gene families, including signaling molecules and transcription factors, which are expressed during regeneration. However, in cases where no specific inhibitor is available for the gene product of interest, determination of a functional role for these genes has been difficult. Here we demonstrate that in vivo electroporation of morpholino oligonucleotides is a feasible approach for protein knock-down during fin regeneration. Morpholino oligonucleotides against fgfr1 and msxb were utilized and knock-down of both proteins resulted in reduced fin outgrowth. Importantly, Fgfr1 knock-down phenocopied outgrowth inhibition obtained with an Fgfr1 inhibitor. Furthermore, this method provided direct evidence for a functional role for msxb in caudal fin regeneration. Finally, knock-down of Fgfr1, but not Msxb, affected the blastemal expression of msxc, suggesting this technique can be used to determine epistasis in genetic pathways affecting regeneration. Thus, this convenient reverse genetic approach allows researchers to quickly (1) assess the function of genes known to be expressed during fin regeneration, (2) screen genes for functional relevance during fin regeneration, and (3) assign genes to the molecular pathways underlying fin regeneration.

Animals↗
PubMed1988

Molecular genetics in muscular dystrophy research: revolutionary progress.

The contribution of "reverse genetic" strategies to neuromuscular disease research is evident in the progression of breakthroughs that have recently culminated in the cloning of the Duchenne muscular dystrophy (DMD) cDNA. The resultant improvements in our understanding of the genetic basis of Becker muscular dystrophy (BMD) and DMD serve as models for similar investigation of other heritable disorders. These genetic advances have outpaced concurrent work on the molecular pathogenesis of the dystrophic process, with the curious result that inferences about the DMD protein's amino acid sequence have preceded any information about its function or intracellular localization. In recognition that this foundation sets the stage for the rapid elucidation of the disease's pathogenesis, we review the experimental basis of such advances, with reference to relevant progress in basic myology, pathology, and molecular biology. We conclude with a view towards the ultimate clinical implications of these experimental breakthroughs.

Animals↗
PubMed1991

The Marfan syndrome locus: confirmation of assignment to chromosome 15 and identification of tightly linked markers at 15q15-q21.3.

The Marfan syndrome is a common autosomal dominant disorder of connective tissue. Despite many years of intensive investigation, the primary genetic defect has not yet been identified. Reverse genetic methods, targeted at mapping this disease gene, have resulted in an initial report of linkage of the genetic locus for the Marfan phenotype in Finnish families to two polymorphic markers on chromosome 15. We have investigated four large multiplex American families with classic Marfan syndrome using standard genetic linkage methods. Our data confirm the assignment of the Marfan syndrome gene to chromosome 15, but establish a more centromeric location (defined by markers D15S25 and D15S1) as the most probable site for the genetic defect (lod score = 12.1, theta = 0.00). These data should facilitate identification and characterization of the Marfan syndrome gene and, in selected families, have immediate application to diagnosis of equivocal cases or prenatal counseling.

Chromosome Mapping↗
PubMed2004

Genetic and genomic approaches to reward and addiction.

Drug addiction is recognized as a mental disease affecting the brain's natural reward system. Drugs of abuse strongly activate reward structures in the brain and induce lasting changes in behavior that reflect changes in neuron physiology and biochemistry. With the ultimate goal of developing therapeutic interventions, it is of interest to determine the molecular and cellular components of motivation and reward, and identify those gene products that contribute to the process of drug addiction. Our laboratory has chosen three general genetic approaches to examine reward and addiction: reverse genetics to assess the role of candidate genes in drug responsiveness, forward genetics to discover novel regulators of dopamine transmission, and gene expression profiling to define gene sets in different brain structures that contribute to the molecular and neurobiological basis of reward.

Animals↗
PubMed2006

An arginine-to-proline mutation in a domain with undefined functions within the helicase protein (Nsp13) is lethal to the coronavirus infectious bronchitis virus in cultured cells.

Genetic manipulation of the RNA genomes by reverse genetics is a powerful tool to study the molecular biology and pathogenesis of RNA viruses. During construction of an infectious clone from a Vero cell-adapted coronavirus infectious bronchitis virus (IBV), we found that a G-C point mutation at nucleotide position 15526, causing Arg-to-Pro mutation at amino acid position 132 of the helicase protein, is lethal to the infectivity of IBV on Vero cells. When the in vitro-synthesized full-length transcripts containing this mutation were introduced into Vero cells, no infectious virus was rescued. Upon correction of the mutation, infectious virus was recovered. Further characterization of the in vitro-synthesized full-length transcripts containing the G15526C mutation demonstrated that this mutation may block the transcription of subgenomic RNAs. Substitution mutation of the Arg132 residue to a positively charged amino acid Lys affected neither the infectivity of the in vitro-synthesized transcripts nor the growth properties of the rescued virus. However, mutation of the Arg132 residue to Leu, a conserved residue in other coronaviruses at the same position, reduced the recovery rate of the in vitro-synthesized transcripts. The recovered mutant virus showed much smaller-sized plaques. On the contrary, a G-C and a G-A point mutations at nucleotide positions 4330 and 9230, respectively, causing Glu-Gln and Gly-Glu mutations in or near the catalytic centers of the papain-like (Nsp3) and 3C-like (Nsp5) proteinases, did not show detectable detrimental effect on the rescue of infectious viruses and the infectivity of the rescued viruses.

Amino Acid Sequence↗
PubMed2003

The transformation of the model organism: a decade of developmental genetics.

The past decade has seen the development of powerful techniques to dissect the molecular processes that regulate development. New tools have been used to reveal the basis of cell polarity, morphogen gradients and regulation of signaling in developing animals. Cell biology and developmental biology have become closely intertwined, and many genes that had been thought of as regulators of general cell biological (housekeeping) functions have been shown to act as specific developmental regulators. Vertebrate developmental genetics is now flourishing, with forward and reverse genetics in both zebrafish and the mouse providing new dimensions to our understanding of development.

Animals↗
PubMed1997

DHR3, an ecdysone-inducible early-late gene encoding a Drosophila nuclear receptor, is required for embryogenesis.

Response to the steroid hormone ecdysone in Drosophila is controlled by genetic regulatory hierarchies that include eight members of the nuclear receptor protein family. The DHR3 gene, located within the 46F early-late ecdysone-inducible chromosome puff, encodes an orphan nuclear receptor that recently has been shown to exert both positive and negative regulatory effects in the ecdysone-induced genetic hierarchies at metamorphosis. We used a reverse genetics approach to identify 11 DHR3 mutants from a pool of lethal mutations in the 46F region on the second chromosome. Two DHR3 mutations result in amino acid substitutions within the conserved DNA binding domain. Analysis of DHR3 mutants reveals that DHR3 function is required to complete embryogenesis. All DHR3 alleles examined result in nervous system defects in the embryo.

Amino Acid Sequence↗
PubMed2001

Phenotypic characterization of three phylogenetically conserved stem-loop motifs in the mengovirus 3' untranslated region.

An alignment of cardiovirus sequences led to the prediction of three conserved stem-loops in the 3' untranslated region (UTR) of mengovirus. Deletions of each stem were engineered in mengovirus cDNAs and also in mengovirus replicons, in which part of the viral capsid sequences were replaced with the firefly luciferase gene. The effect of deletion on RNA infectivity and plaque phenotype was evaluated after transfection of viral transcripts into HeLa cells or by luciferase assays of cellular extracts after transfection with RNA replicons. Stem I (mengovirus bases 7666 to 7687) was found to be dispensable for viral growth or exponential luciferase expression. Deletion of stem III (bases 7711 to 7721) was lethal to the virus, and the replicons were incapable of RNA synthesis. Deletion of stem II (DeltaII; bases 7692 to 7705) produced an intermediate phenotype, in that replicons had marginal RNA synthesis activity but transfection with genomic RNA usually failed to produce plaques after normal incubation times (31 h, 37 degrees C). In a few of the DeltaII transfections, however, plaques were observed after long incubation, especially if the cells received large amounts of RNA (3 microg per 3 x 10(6) cells). Viruses from two DeltaII-derived plaques were isolated and amplified. Their RNAs were converted into cDNA, sequenced, and mapped for genotype. Each maintained the DeltaII deletion and, in addition, had one or two reversion mutations, which were characterized by reverse genetics as responsible for the phenotypes. One reversion caused an amino acid change in the polymerase (3D(pol)), and the other was localized to the 3' UTR, upstream of stem I.

3' Untranslated Regions↗
PubMed2001

Complete genome comparison of porcine reproductive and respiratory syndrome virus parental and attenuated strains.

Two full-length porcine reproductive and respiratory syndrome virus (PRRSV) genomes, strain VR-2332 and its cell culture passaged descendent RespPRRS vaccine strain, were compared and analyzed in order to identify possible sites of attenuation. Of the 44 nucleotide changes, 13 resulted in conservative changes and 18 produced non-conservative changes. The results suggest that key amino acids in ORF1 may contribute to the phenotype of RespPRRS, which includes increased growth rate on MA-104 cells and decreased virulence in swine. The results provide a genetic basis for future manipulation of a PRRSV reverse genetics system.

5' Untranslated Regions↗
PubMed2026

The Q653R substitution in the spike protein is associated with attenuation of a GVI-1 infectious bronchitis virus strain.

The GVI-1 genotype of infectious bronchitis virus (IBV) has become increasingly prevalent in Asia. In this study, a highly pathogenic GVI-1 strain (GVI-1-WT) was attenuated by 110 serial passages in embryonated chicken eggs, yielding an attenuated strain (GVI-1-E110). Comparative genomic analysis identified two amino acid substitutions, S523I, Q653R and a nine-amino-acid truncation in the spike (S) protein. To evaluate the contribution of the two point mutations to virulence attenuation, recombinant viruses carrying Q653R and S523I substitutions were generated using a reverse genetics system based on the GVI-1-WT strain as the backbone. Their replication and pathogenicity were assessed in embryonated eggs and specific pathogen-free chickens. The Q653R substitution was associated with reduced viral replication in embryonated chicken eggs and pathogenicity in specific pathogen-free chickens, whereas the S523I mutation alone showed a limited effect but enhanced attenuation when combined with Q653R. However, the attenuation phenotype of the recombinant viruses did not fully recapitulate that of the passaged strain GVI-1-E110, suggesting that additional mutations, including the identified truncation and mutations in replicase-associated genes outside the S protein, may also contribute to virulence attenuation. This study indicates that spike protein mutations are involved in the attenuation of GVI-1 IBV strains, and provides insights into the molecular basis of IBV attenuation during serial passage. Further studies are required to elucidate the underlying mechanisms and to evaluate their potential relevance for vaccine development.

GVI-1 genotype↗
PubMed2026

Field-isolate recombinant tick-borne encephalitis viruses define reporter-stability guidelines for antiviral testing in flaviviruses.

As arthropod-borne viruses continue to threaten populations globally, there is a pressing need for experimental systems that enable rapid antiviral discovery. Reverse-genetics platforms producing recombinant reporter orthoflaviviruses have been developed to address this gap. Here, we present two new recombinant tick-borne encephalitis viruses (TBEVrec) generated on a European-subtype Haselmühl Tiho1 isolate backbone. A reporter gene, either eGFP or Nluc, was inserted in the capsid-coding region of the genome downstream of the capsid RNA regulatory signal and separated from the complete viral polyprotein by a 2A self-cleaving peptide. TBEVrec was better rescued using the circular polymerase extension reaction (CPER) than with the infectious subgenomic amplicon (ISA) method. TBEVrec replicated efficiently in relevant human cell lines, with comparable replication to wild-type TBEV in a neuronal cell line and moderately reduced titers and RNA levels in immune-derived cell lines. Using either eGFP or Nluc, we illustrate how TBEVrec enabled high-content RNAi screening, highlighting Nucleolin and PRKD1 as potential TBEV host factors, and drug testing on a benchtop plate reader. Nanopore sequencing of the eGFP insert revealed that the reporter is excised without affecting flanking regions. Comparative analysis of eGFP and Nluc further shows that this instability is time- and cell type-dependent, and that Nluc is comparatively more stable. From these observations, we outline safeguards and design principles that are broadly applicable both to the rescue of existing constructs and to the design of future recombinant reporter virus platforms.

CPER↗
PubMed2001

Cloning of the full length pig PIT1 (POU1F1) CDNA and a novel alternative PIT1 transcript, and functional studies of their encoded proteins.

PIT1 is an essential regulatory gene of growth hormone (GH), prolactin (PRL) and thyrotropin beta subunit (TSHbeta). Previously, a partial pig PIT1 cDNA and a genomic clone of the entire 3' end of the PIT1 gene was isolated, and polymorphisms at PIT1 were associated with several performance traits in the pig. In order to understand the biological function of the pig PIT1 gene and its possible application in swine genetics, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to complete the cloning of the full length cDNA for pig PIT1. The pig PIT1 cDNA and its deduced protein sequence have approximately 90% and 95% identity, respectively, with the PIT1 cDNA and protein of other mammals (human, bovine, sheep and rodents). Surprisingly, sequence comparison to other pig PIT1 sequences indicated only approximately 93% identity. Additional sequencing confirmed our sequence, and identified a new polymorphism in exon 4. Phylogenetic analysis of several mammalian PIT1 sequences indicates sequencing errors may account for the discrepancies observed in the other pig sequences reported. Several PIT1 alternative spliced forms were also identified by RT-PCR. They were the delta3PIT1 (missing entire exon 3), delta4PIT1 (missing entire exon 4) and PIT1beta (additional 26 amino acids inserted in front of exon 2) transcripts. The delta4PIT1 and PIT1beta transcripts have been found to encode functionally different proteins in rodents. The delta3PIT1 transcript is a novel isoform of PIT1. Potentially different functions between pig delta3PIT1 and PIT1 were analyzed by expressing these proteins in bacteria. The E. coli-expressed PIT1 and delta3PIT1 proteins were used with rat growth hormone (rGH) and rat prolactin (rPRL) promoter DNA in DNA mobility shift assays. The results showed that pig PIT1 can specifically bind rGH and rPRL promoter regions, but that the pig delta3PIT1 cannot, even at very high protein concentrations. Possible protein-protein interactions between delta3PIT1 and PIT1 were tested by mixing protein extracts before the gel shift assay, and the results showed that delta3PIT1 protein did not affect PIT1 binding to its target DNA. These data demonstrate the functionality of the PIT1 cDNA cloned in this study, and identify a novel delta3PIT1 transcript which encodes a protein that cannot bind rGH/rPRL target sequences.

Amino Acid Sequence↗
PubMed2007

ci21A/Asr1 expression influences glucose accumulation in potato tubers.

Asr genes are exclusively found in the genomes of higher plants. In many species, this gene family is expressed under abiotic stress conditions and during fruit ripening. The encoded proteins have nuclear localisation and consequently a transcription factor function has been suggested. Interestingly, yeast-one-hybrid experiments revealed that a grape ASR binds to the promoter of a hexose transporter gene (VvHT1). However, the role of these proteins in planta is still elusive. By using a reverse genetics approach in potato we found that modification of Asr1 expression has no incidence on the aerial phenotype of the plant but exerts a dramatic effect in tuber. Asr1 antisense potatoes displayed decreased tuber fresh weight whereas Asr1 overexpressors had a diminished number of tubers. Moreover, overexpression lines showed lower transcript levels of a plasma membrane hexose transporter and a concomitant decrease in glucose content in parenchyma cells of potato tubers. On the same hand glucose uptake rate was also reduced in one of the overexpressing lines. It thus seems likely that Asr1 is involved in the control of hexose uptake in heterotrophic organs. In addition, the transgenic plants were characterized by several other changes in steady state metabolite levels. Results presented here support a role for ci21A/Asr1 in glucose metabolism of potato tuber.

DNA Primers↗