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PubMed1999

Anthelmintic resistance: past, present and future.

Anthelmintic resistance continues to increase in geographic range, in the number of species affected and the range of drugs involved. Several aspects of resistance have emerged as important issues. They include lack of genetic reversion, presence of side resistance and lack of universality. Furthermore, resistant isolates recovered from the field may have different characteristics to those selected in pen passage. Research into anthelmintic resistance has not progressed far beyond the stage of descriptive research. Some progress has been made in developing control strategies and in diagnosing resistance, especially in the development and adoption of in-vitro tests. However, these still need improvements in their ability to detect resistance to closantel and avermectin/milbemycin anthelmintics. Less progress into understanding the basis of resistance has occurred. Research priorities include improvement of diagnostic tests and the development of molecular tests, particularly for resistance to levamisole and the avermectin/milbemycins. Resistance itself, as a selectable marker for genetic transfection in parasites, is a potential tool for investigating parasite biology.

Animals↗
PubMed1997

Authentic and chimeric full-length genomic cDNA clones of bovine viral diarrhea virus that yield infectious transcripts.

Bovine viral diarrhea virus (BVDV) is the most insidious and devastating viral pathogen of cattle in the United States. Disease control approaches must be based on detailed knowledge of virus biology. To develop reverse-genetic systems to study the molecular biology of the virus, we first constructed a plasmid containing the entire genome of BVDV cloned as cDNA. Subsequently, we showed that infectious BVDV was produced by cells transfected with uncapped RNA transcribed in vitro from the cDNA clone. This result defined functional 5' and 3' termini in viral genomic RNA and established the biological importance of the proposed internal ribosome entry site element in the 5' untranslated region of the viral genome. BVDV rescued from the infectious cDNA clone has an in vitro phenotype similar to that of the wild-type parent, the National Animal Disease Laboratory strain of BVDV. A deletion of a single codon in the full-length genomic BVDV cDNA clone, encoding glutamic acid at position 1600, gave rise to sequence-tagged virus easily identified by restriction fragment length polymorphism analysis of reverse transcription-PCR amplicons. Suitability of the molecular clone of BVDV for genomic manipulations was shown by substitution of the major envelope glycoprotein E2/gp53 with that of the Singer strain, giving rise to a chimeric virus. The predicted change in antigenic structure of the chimeric virus could be readily identified with strain-specific monoclonal antibodies by neutralization and immunofluorescence assays. Immediate applications of this system include development of safe and effective live vaccine strains possessing predetermined defined attenuating mutations.

Animals↗
PubMed2005

Adult scl+/+ murine hemangioblasts persist in allogeneic mutant blastocysts but fail to rescue the scl-/- phenotype.

Isolated and expanded scl (+) adult murine progenitors show a strong endothelial and hematopoietic differentiation potential and have been considered to be the adult equivalent of the hemangioblast. These unique cells may provide effective therapeutic approaches to tissue damage resulting from hypoxemia or chronic ischemia. Here, we study the fate of adult scl (+/+) during development and their ability to reverse genetic defects in scl expression. scl (+/+) adult stem cells (clone RM26) did not persist during embryonic development after injection into blastocysts of allogeneic wild-type mice on day E 3.5. However, GFP(+)-marked scl (+/+) cells were detected in all possible genotypes from allogeneic scl (+/+) intercrosses (scl (+/+), scl (+/-), scl (-/-) on day E 9.5 after the cloned cells were injected into scl-mutant blastocysts on day E 3.5. Nevertheless, there was no indication of phenotypic rescue of the mutant blastocysts despite the continued presence of scl (+/+) RM26 cells in the allogeneic embryonic environment. The results show that differentiated stem cells providing a defective gene may exert effects during development when there is a reparative demand, but they are not capable of reversing the effects of a mutant phenotype during embryonic development. These effects should be considered when evaluating the efficacy of stem cells for therapeutic reversal of inborn errors of development.

Animals↗
PubMed2006

Complete genome analysis of RFLP 184 isolates of porcine reproductive and respiratory syndrome virus.

Two full-length genomes of recently emerged virulent isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were sequenced and compared to other PRRSV strains. The results revealed that these two isolates (named MN184), of North American lineage, represented the shortest PRRSV genomes sequenced to date with a nucleotide length of 15019 bases. Genetic analysis demonstrated that the two isolates were not identical and shared approximately 87 and 59% nucleotide identity with prototype North American strain VR-2332 and European strain Lelystad, respectively. Three quite variable regions were identified, corresponding to putative nsp1beta, putative nsp2 and ORF5. Nsp2, the most variable region, shared only 66-70% amino acid similarity to other sequenced North American-like PRRSV nsp2 proteins. Further study revealed that the nsp2 protein of the MN184 isolates contained three discontinuous deletions when compared to strain VR-2332 nsp2 protein, with the sizes of 111, 1, and 19 amino acids corresponding to strain VR-2332 positions 324-434, 486 and 505-523, respectively. The results suggest that targeted manipulation of PRRSV through nsp2 modification by reverse genetics may yield promising vectors for vaccine development, as has been recently demonstrated [Han, J., Faaberg, K.S., Wang, Y., Liu, H., 2005. Non-structural protein 2 mutants of PRRSV strain VR-2332 infectious clone based on deletions seen in RFLP184 isolates are viable. In: PRRS International Symposium Proceedings, vol. 8, Saint Louis, MO].

Amino Acid Sequence↗
PubMed1998

The role of the Aspergillus fumigatus areA gene in invasive pulmonary aspergillosis.

The areA gene of Aspergillus nidulans is a positive-acting transcriptional factor required for the expression of genes involved in the utilization of a broad range of nitrogen sources other than ammonium and glutamine. We have investigated the role in pathogenesis of the corresponding gene (AfareA) of Aspergillus fumigatus, a causative agent of invasive pulmonary aspergillosis. Stable and unstable AfareA- strains were constructed and tested for altered virulence in mice on the basis of host survival, mixed infection experiments and genetic reversion studies. These showed that the AfareA gene contributes to, but is not essential for, virulence. Strains carrying extragenic mutations that partially suppress the AfareA- phenotype for growth on different nitrogen sources were tested for altered virulence in mixed infection studies. One of these was found to be more virulent than the parental AfareA- strain.

Animals↗
PubMed2002

Building a hair: tip growth in Arabidopsis thaliana root hairs.

The Arabidopsis thaliana root hair is used as a model for studying tip growth in plants. We review recent advances, made using physiological and genetic approaches, which give rise to different, yet compatible, current views of the establishment and maintenance of tip growth in epidermal cells. For example, an active calcium influx channel localized at the tip of Arabidopsis root hairs has been identified by patch-clamp measurements. Actin has been visualized in vivo in Arabidopsis root hairs by using a green-fluorescent-protein-talin reporter and shown to form a dense mesh in the apex of the growing tip. The kojak gene, which encodes a protein similar to the catalytic subunit of cellulose synthase, is needed in the first stages of hair growth. A role for LRX1, a leucine-rich repeat extensin, in determining the morphology of the cell wall of root hairs has been established using reverse genetics. The new information can be integrated into a general and more advanced view of how these specialized plant cells grow.

Arabidopsis↗
PubMed2006

Spider egg case core fibers: trimeric complexes assembled from TuSp1, ECP-1, and ECP-2.

Spider silk proteins are well-known for their extraordinary mechanical properties, displaying remarkable strength and toughness. In this study, matrix-assisted laser desorption ionization (MALDI) tandem time-of-flight (TOF) mass spectrometry (MS/MS) and reverse genetics were used to isolate a new cDNA sequence that encodes for a protein assembled into egg case silk from the black widow spider, Latrodectus hesperus. Analysis of the primary sequence of this protein reveals approximately 52% identity to the egg case protein 1 (ECP-1) fibroin-like family member. On the basis of the similarity in the primary sequence and expression pattern, we have named this factor egg case protein 2 (ECP-2). Alignments of ECP-1 and ECP-2 demonstrate highly conserved N termini, with 16 Cys residues found within the first 153 amino acids. Traditional ensemble repeats found within reported fibroins were poorly represented in the primary sequence of ECP-2, but scattered blocks of polyalanine were present, along with a C terminus rich in GA repeats. Reverse transcription quantitative PCR analysis showed that ECP-2 is predominantly expressed in the tubuliform gland. Relative to ECP-1, ECP-2 mRNA levels were determined to be >2-fold higher. MALDI MS/MS analysis of peptide fragments generated from the large-diameter core fiber after enzymatic digestion and acid hydrolysis demonstrated the presence of a fiber that is trimeric in nature, containing tubuliform spidroin 1 (TuSp1), ECP-1, and ECP-2. We also report an additional primary sequence for TuSp1, demonstrating that TuSp1 contains two Cys residues within a nonrepetitive N-terminal region. In combination with the distinctive protein architectures of ECP-1 and ECP-2, along with their co-localization with TuSp1 in the core fiber, our findings suggest that ECP-1 and ECP-2 play important structural roles in the egg case silk fiber.

Amino Acid Motifs↗
PubMed2004

Characterization of CD8+ T cell repertoire diversity and persistence in the influenza A virus model of localized, transient infection.

Influenza virus infection of C57BL/6 mice provides a well-characterized model for the study of acute CD8(+) T cell responses and for the analysis of memory in the absence of antigen persistence. The advent of tetramer reagents and intracellular cytokine staining, coupled with techniques such as single cell RT-PCR and influenza reverse genetics, has enabled the detailed molecular dissection of different epitope-specific primary, memory and secondary immune CD8(+) T cell responses. The approach offers novel insights into the factors determining the selection of immune repertoires, and their functional consequences for CD8(+) T cell-mediated immunity.

Animals↗
PubMed2006

A pentatricopeptide repeat protein facilitates the trans-splicing of the maize chloroplast rps12 pre-mRNA.

The pentatricopeptide repeat (PPR) is a degenerate 35-amino acid repeat motif that is widely distributed among eukaryotes. Genetic, biochemical, and bioinformatic data suggest that many PPR proteins influence specific posttranscriptional steps in mitochondrial or chloroplast gene expression and that they may typically bind RNA. However, biological functions have been determined for only a few PPR proteins, and with few exceptions, substrate RNAs are unknown. To gain insight into the functions and substrates of the PPR protein family, we characterized the maize (Zea mays) nuclear gene ppr4, which encodes a chloroplast-targeted protein harboring both a PPR tract and an RNA recognition motif. Microarray analysis of RNA that coimmunoprecipitates with PPR4 showed that PPR4 is associated in vivo with the first intron of the plastid rps12 pre-mRNA, a group II intron that is transcribed in segments and spliced in trans. ppr4 mutants were recovered through a reverse-genetic screen and shown to be defective for rps12 trans-splicing. The observations that PPR4 is associated in vivo with rps12-intron 1 and that it is also required for its splicing demonstrate that PPR4 is an rps12 trans-splicing factor. These findings add trans-splicing to the list of RNA-related functions associated with PPR proteins and suggest that plastid group II trans-splicing is performed by different machineries in vascular plants and algae.

Alleles↗
PubMed1994

Mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation.

The carboxy terminus of the hemagglutinin (HA) of influenza A viruses contains three cysteine residues which are highly conserved among HA subtypes. It has previously been shown for the H2, H3, and H7 subtypes of HA that these cysteine residues are modified by the covalent attachment of palmitic acid. In order to study the role of the acylated cysteines in the formation of infectious influenza viruses, we introduced mutations into the HA of influenza A/WSN/33 virus (H1 subtype) by reverse-genetics techniques. We found that the cysteine at position 563 of the cytoplasmic tail is required for infectious-particle formation. The cysteine at position 560 can be changed to alanine or tyrosine to yield virus strains that are attenuated in cell cultures. The change from cysteine at position 553 to serine or alanine does not significantly alter the phenotype of the virus. The requirement for a cysteine at position 563 suggests a functional role for palmitylation of the cytoplasmic tail. This interpretation is further supported by experiments in which two or more of the cysteine residues were mutated, eliminating potential palmitylation sites. None of these double or triple mutations resulted in infectious virus. Selection of revertants of the attenuated cysteine-to-tyrosine mutant (mutation at position 560) always resulted in reversion to cysteine rather than to other amino acids. Although our data indicate a biological role for the conserved cysteine residues in the cytoplasmic tail of the HA of influenza viruses, we cannot exclude the possibility that structural constraints in the cytoplasmic tail of the HA--rather than altered palmitylation--are the determining factors for infectious-particle formation.

Amino Acid Sequence↗
PubMed2000

Sequence determination and molecular analysis of two strains of bovine parainfluenza virus type 3 that are attenuated for primates.

The Kansas/15626/84 (Ka) and Shipping Fever (SF) strains of bovine parainfluenza virus type 3 (BPIV3) replicate less efficiently than human PIV3 (HPIV3) in the upper and lower respiratory tract of rhesus monkeys, and BPIV3 Ka is also highly attenuated in humans and is in clinical trials as a candidate vaccine against HPIV3. To initiate an investigation of the genetic basis of the observed attenuation phenotype of BPIV3 in primates, the complete genomic sequences of Ka and SF genomes were determined and compared to those of BPIV3 strain 910N and two HPIV3 strains, JS and Wash/47885/57. There is a high degree of identity between the five PIV3 viruses in their 55 nucleotide (nt) leader (83.6%) and 44 nt trailer (93.2%) sequences. The five viruses display amino acid sequence identity ranging from 58.6% for the phosphoprotein to 89.7% for the matrix protein. Interestingly, the majority of amino acid residues found to be variable at a given position in a five-way protein alignment are nonetheless identical within the viruses of either host species (BPIV3 or HPIV3). These host-specific residues might be products of distinct selective pressures on BPIV3 and HPIV3 during evolution in their respective hosts. These host-specific sequences likely include ones which are responsible for the host range differences, such as the efficient growth of BPIV3 in bovines compared to its restricted growth in primates. It should now be possible using the techniques of reverse genetics to import sequences from BPIV3 into HPIV3 and identify those nt or protein sequences which attenuate HPIV3 for primates. This information should be useful in understanding virus-host interactions and in the development of vaccines to protect against HPIV3-induced disease.

Animals↗
PubMed2004

National Science Foundation-sponsored workshop report. Draft plan for soybean genomics.

Recent efforts to coordinate and define a research strategy for soybean (Glycine max) genomics began with the establishment of a Soybean Genetics Executive Committee, which will serve as a communication focal point between the soybean research community and granting agencies. Secondly, a workshop was held to define a strategy to incorporate existing tools into a framework for advancing soybean genomics research. This workshop identified and ranked research priorities essential to making more informed decisions as to how to proceed with large scale sequencing and other genomics efforts. Most critical among these was the need to finalize a physical map and to obtain a better understanding of genome microstructure. Addressing these research needs will require pilot work on new technologies to demonstrate an ability to discriminate between recently duplicated regions in the soybean genome and pilot projects to analyze an adequate amount of random genome sequence to identify and catalog common repeats. The development of additional markers, reverse genetics tools, and bioinformatics is also necessary. Successful implementation of these goals will require close coordination among various working groups.

Computational Biology↗
PubMed2003

Binding of sulfonylurea by AtMRP5, an Arabidopsis multidrug resistance-related protein that functions in salt tolerance.

Recently, a new member of the ABC transporter superfamily of Arabidopsis, AtMRP5, was identified and characterized. In the present work, we found that AtMRP5 can bind specifically to sulfonurea when it is expressed in HEK293 cells. We also present evidence for a new role of AtMRP5 in the salt stress response of Arabidopsis. We used reverse genetics to identify an Arabidopsis mutant (atmrp5-2) in which the AtMRP5 gene was disrupted by transferred DNA insertion. In root-bending assays using Murashige and Skoog medium supplemented with 100 mm NaCl, root growth of atmrp5-2 was substantially inhibited in contrast to the almost normal growth of wild-type seedlings. This hypersensitive response of the atmrp5-2 mutant was not observed during mannitol treatment. The root growth of the wild-type plant grown in Murashige and Skoog medium supplemented with the MRP inhibitor glibenclamide and NaCl was inhibited to a very similar extent as the root growth of atmrp5-2 grown in NaCl alone. The Na(+)-dependent reduction of root growth of the wild-type plant in the presence of glibenclamide was partially restored by diazoxide, a known K+ channel opener that reverses the inhibitory effects of sulfonylureas in animal cells. Moreover, the atmrp5-2 mutant was defective in 86Rb+ uptake. When seedlings were treated with 100 mm NaCl, atmrp5-2 seedlings accumulated less K+ and more Na+ than those of the wild type. These observations suggest that AtMRP5 is a putative sulfonylurea receptor that is involved in K+ homeostasis and, thus, also participates in the NaCl stress response.

Arabidopsis↗
PubMed1994

Functions involved in bacteriophage P2-induced host cell lysis and identification of a new tail gene.

Successful completion of the bacteriophage P2 lytic cycle requires phage-induced lysis of its Escherichia coli host, a process that is poorly understood. Genetic analysis of lysis-deficient mutants defined a single locus, gene K, which lies within the largest late transcription unit of P2 and maps between head gene L and tail gene R. We determined and analyzed the DNA sequence of a ca. 2.1-kb EcoRV fragment that spans the entire region from L to R, thus completing the sequence of this operon. This region contains all of the functions necessary for host cell lysis. Sequence analysis revealed five open reading frames, initially designated orf19 through orf23. All of the existing lysis mutants--ts60, am12, am76, and am218--were located in orf21, which must therefore correspond to gene K. The K gene product has extensive amino acid sequence similarity to the product of gene R of bacteriophage lambda, and its exhibits endolysin function. Site-directed mutagenesis and reverse genetics were used to create P2 amber mutants in each of the four other newly identified open reading frames. Both orf19 (gene X) and orf20 (gene Y) encode essential functions, whereas orf22 (lysA) and orf23 (lysB) are nonessential. Gene Y encodes a polypeptide with striking similarities to the family of holin proteins exemplified by gpS of phage lambda, and the Yam mutant displayed the expected properties of a holin mutant. The gene products of lysA and lysB, although nonessential, appear to play a role in the correct timing of lysis, since a lysA amber mutant caused slightly accelerated lysis and a lysB amber mutant slightly delayed lysis of nonpermissive strains. Gene X must encode a tail protein, since lysates from nonpermissive cells infected with the X amber mutant were complemented in vitro by similar lysates of cells infected with P2 head mutants but not with tail mutants.

Amino Acid Sequence↗
PubMed2004

Recovery of human metapneumovirus genetic lineages a and B from cloned cDNA.

Human metapneumovirus (hMPV) is a newly discovered pathogen associated with respiratory tract illness, primarily in young children, immunocompromised individuals, and the elderly. The genomic sequence of the prototype hMPV isolate NL/1/00 without the terminal leader and trailer sequences has been reported previously. Here we describe the leader and trailer sequences of two hMPV isolates, NL/1/00 and NL/1/99, representing the two main genetic lineages of hMPV. Minigenome constructs in which the green fluorescent protein or chloramphenicol acetyltransferase genes are flanked by the viral genomic ends derived from both hMPV lineages and transcribed using a T7 RNA polymerase promoter-terminator cassette were generated. Cotransfection of minigenome constructs with plasmids expressing the polymerase complex components L, P, N, and M2.1 in 293T or baby hamster kidney cells resulted in expression of the reporter genes. When the minigenome was replaced by a sense or antisense full-length cDNA copy of the NL/1/00 or NL/1/99 viral genomes, recombinant virus was recovered from transfected cells. Viral titers up to 10(7.2) and 10(5.7) 50% tissue culture infective dose/ml were achieved with the sense and antisense plasmids, respectively. The recombinant viruses replicated with kinetics similar to those of the parental viruses in Vero cells. This reverse genetics system provides an important new tool for applied and fundamental research.

5' Untranslated Regions↗
PubMed2001

Molecular genetics of nucleotide sugar interconversion pathways in plants.

Nucleotide sugar interconversion pathways represent a series of enzymatic reactions by which plants synthesize activated monosaccharides for the incorporation into cell wall material. Although biochemical aspects of these metabolic pathways are reasonably well understood, the identification and characterization of genes encoding nucleotide sugar interconversion enzymes is still in its infancy. Arabidopsis mutants defective in the activation and interconversion of specific monosaccharides have recently become available, and several genes in these pathways have been cloned and characterized. The sequence determination of the entire Arabidopsis genome offers a unique opportunity to identify candidate genes encoding nucleotide sugar interconversion enzymes via sequence comparisons to bacterial homologues. An evaluation of the Arabidopsis databases suggests that the majority of these enzymes are encoded by small gene families, and that most of these coding regions are transcribed. Although most of the putative proteins are predicted to be soluble, others contain N-terminal extensions encompassing a transmembrane domain. This suggests that some nucleotide sugar interconversion enzymes are targeted to an endomembrane system, such as the Golgi apparatus, where they may co-localize with glycosyltransferases in cell wall synthesis. The functions of the predicted coding regions can most likely be established via reverse genetic approaches and the expression of proteins in heterologous systems. The genetic characterization of nucleotide sugar interconversion enzymes has the potential to understand the regulation of these complex metabolic pathways and to permit the modification of cell wall material by changing the availability of monosaccharide precursors.

Amino Acid Sequence↗
PubMed2005

Stress induces the expression of AtNADK-1, a gene encoding a NAD(H) kinase in Arabidopsis thaliana.

A novel Arabidopsis thaliana gene (AtNADK-1) was identified based on its response to radiation and oxidative stress. Levels of AtNADK-1 mRNA increase eight-fold following exposure to ionising radiation and are enhanced three-fold by treatment with hydrogen peroxide. The gene also appears to be differentially regulated during compatible and incompatible plant-pathogen interactions in response to Pseudomonas syringae pv. tomato. The full-length AtNADK-1 cDNA encodes a 58-kDa protein that shows high sequence homology to the recently defined family of NAD(H) kinases. Recombinant AtNADK-1 utilises ATP to phosphorylate both NAD and NADH, showing a two-fold preference for NADH. Using reverse genetics, we demonstrate that AtNADK-1 deficient plants display enhanced sensitivity to gamma irradiation and to paraquat-induced oxidative stress. Our results indicate that this novel NAD(H) kinase may contribute to the maintenance of redox status in Arabidopsis thaliana.

Amino Acid Sequence↗
PubMed2006

Combinatorial engineering of a gene therapy vector: directed evolution of adeno-associated virus.

BACKGROUND: Viruses are being exploited as vectors to deliver therapeutic genetic information into target cells. The success of this approach will depend on the ability to overcome current limitations, especially in terms of safety and efficiency, through molecular engineering of the viral particles. METHODS: Here we show that in vitro directed evolution can be successfully performed to randomize the viral capsid by error prone PCR and to obtain mutants with improved phenotype. RESULTS: To demonstrate the potential of this technology we selected several adeno-associated virus (AAV) capsid variants that are less efficiently neutralized by human antibodies. These mutations can be used to generate novel vectors for the treatment of patients with pre-existing immunity to AAV. CONCLUSIONS: Our results demonstrate that combinatorial engineering overcomes the limitations of rational design approaches posed by incomplete understanding of the infectious process and at the same time offers a powerful tool to dissect basic viral biology by reverse genetics.

Amino Acid Sequence↗