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Biomedical subjects

L L Domier

Publications and source records attributed to L L Domier.

At least 19 recordsLinked to original sources

A genetic linkage map of the soybean cyst nematode Heterodera glycines.

A genetic linkage map of the soybean cyst nematode (SCN) Heterodera glycines was constructed using a population of F2 individuals obtained from matings between two highly inbred SCN lines, TN16 and TN20. The AFLP fingerprinting technique was used to genotype 63 F2 progeny with two restriction enzyme combinations (EcoRI/MseI and PstI/TaqI) and 38 primer combinations. The same F2 population was also genotyped for Hg-cm-1 (H. glycines chorismate mutase-1), a putative virulence gene, using real-time quantitative PCR. Some of the markers were found to be distributed non-randomly. Even so, of the 230 markers analyzed, 131 could be mapped onto ten linkage groups at a minimum LOD of 3.0, for a total map distance of 539 cM. The Hg-cm-1 locus mapped to linkage group III together with 16 other markers. The size of the H. glycines genome was estimated to be in the range of 630-743 cM, indicating that the current map represents 73-86% of the genome, with a marker density of one per 4.5 cM, and a physical/genetic distance ratio of between 124 kb/cM and 147 kb/cM. This genetic map will be of great assistance in mapping H. glycines markers to genes of interest, such as nematode virulence genes and genes that control aspects of nematode parasitism.

Animals↗

Variability and transmission by Aphis glycines of North American and Asian Soybean mosaic virus isolates.

The variability of North American and Asian strains and isolates of Soybean mosaic virus was investigated. First, polymerase chain reaction (PCR) products representing the coat protein (CP)-coding regions of 38 SMVs were analyzed for restriction fragment length polymorphisms (RFLP). Second, the nucleotide and predicted amino acid sequence variability of the P1-coding region of 18 SMVs and the helper component/protease (HC/Pro) and CP-coding regions of 25 SMVs were assessed. The CP nucleotide and predicted amino acid sequences were the most similar and predicted phylogenetic relationships similar to those obtained from RFLP analysis. Neither RFLP nor sequence analyses of the CP-coding regions grouped the SMVs by geographical origin. The P1 and HC/Pro sequences were more variable and separated the North American and Asian SMV isolates into two groups similar to previously reported differences in pathogenic diversity of the two sets of SMV isolates. The P1 region was the most informative of the three regions analyzed. To assess the biological relevance of the sequence differences in the HC/Pro and CP coding regions, the transmissibility of 14 SMV isolates by Aphis glycines was tested. All field isolates of SMV were transmitted efficiently by A. glycines, but the laboratory isolates analyzed were transmitted poorly. The amino acid sequences from most, but not all, of the poorly transmitted isolates contained mutations in the aphid transmission-associated DAG and/or KLSC amino acid sequence motifs of CP and HC/Pro, respectively.

Animals↗

Effect of individual Sumai 3 chromosomes on resistance to scab spread within spikes and deoxynivalenol accumulation within kernels in wheat.

Two sets of substitution lines were developed by crossing individual monosomic lines of Chinese Spring (recipient) with scab (Fusarium graminearum) resistant cultivar Sumai 3 (donor) and then using the monosomics as the recurrent male parent for four backcrosses (without selfing after each backcross). The disomic substitution lines were separated from selfed BC4F2 plants. Chromosome specific SSR markers were analyzed for polymorphism between Sumai 3 and Chinese Spring. Polymorphic markers were used to identify substitution lines for specific chromosomes. Based on the specific SSR markers, chromosome substitutions occurred in thirty-six lines, and six lines segregated alleles from the two parents or were homozygous for the allele from Chinese Spring. These substitution lines were used to evaluate Type II (spread within the head) and Type V (deoxynivalenol accumulation within kernels) scab resistance. The objective was to use the substitution lines to evaluate the effect of individual chromosomes of Sumai 3 on Type 11 and Type V scab resistance in the greenhouse. Significant differences in Type II scab resistance and deoxynivalenol (DON) levels among different Chinese Spring (Sumai 3) substitution lines were detected. Positive chromosome substitution effects on Type II scab resistance were found on chromosomes 2B, 3B. 6B, and 7A from Sumai 3. Chromosomes 3B and 7A also reduced DON accumulation within the kernels, while chromosomes IB, 2D, and 4D from Sumai 3 increased DON concentration. Chromosome 7A from Sumai 3 had the largest effect on resistance to scab spread and DON accumulation. Additional research is in progress on the scab resistance conferred by chromosome 7A.

Chromosomes, Plant↗

Sequence requirements for translation initiation of Rhopalosiphum padi virus ORF2.

Rhopalosiphum padi virus (RhPV) is an aphid-infecting virus with a 10-kb ssRNA genome that contains two large open reading frames (ORFs). ORF1 and ORF2 encode the nonstructural and structural polyproteins, respectively. Both ORFs are preceded by noncoding regions of 500 nt that could function as internal ribosome entry segments (IRESes). The sequence for ORF2 lacks an obvious initiation codon, but an out-of-frame AUG codon is present that could translate ORF2 through a +1 frameshift. To investigate the mechanisms of translation initiation of ORF2, a series of point and deletion mutations were constructed and transcribed and translated in vitro. A bicistronic plasmid containing two copies of the RhPV intergenic region translated both ORFs efficiently, indicating that the region functioned as an IRES in vitro. Deletion analysis showed that the region required for activity of the IRES extended from 178 nt upstream and 6 nt downstream of the 5' border of ORF2. Changes in the out-of-frame AUG codon had little effect on translation initiation, but mutations that included the first and second codons of ORF2 or that disrupted a putative pseudoknot structure near the 3' end of the IRES failed to initiate protein synthesis. Sequence analysis of the in vitro synthesized proteins showed that the first amino acid of the polyprotein corresponded to the second codon of ORF2. These results show that in vitro the noncoding region upstream of RhPV ORF2 functions as an IRES that contains a pseudoknot that directs translation initiation at a non-AUG codon. If the in vitro synthesized proteins have not been processed by an aminopeptidase, translation is initiated at the second (GCA) codon of ORF2.

5' Untranslated Regions↗

Oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial virus-F protein induces a systemic immune response.

Respiratory syncytial virus (RSV) is one of the most important pathogens of infancy and early childhood. Here a fruit-based edible subunit vaccine against RSV was developed by expressing the RSV fusion (F) protein gene in transgenic tomato plants. The F-gene was expressed in ripening tomato fruit under the control of the fruit-specific E8 promoter. Oral immunization of mice with ripe transgenic tomato fruits led to the induction of both serum and mucosal RSV-F specific antibodies. The ratio of immunoglobulin subclasses produced in response to immunization suggested that a type 1 T-helper cell immune response was preferentially induced. Serum antibodies showed an increased titer when the immunized mice were exposed to inactivated RSV antigen.

Administration, Oral↗

Phylogenetic position of the North American isolate of Pasteuria that parasitizes the soybean cyst nematode, Heterodera glycines, as inferred from 16S rDNA sequence analysis.

A 1341 bp sequence of the 16S rDNA of an undescribed species of Pasteuria that parasitizes the soybean cyst nematode, Heterodera glycines, was determined and then compared with a homologous sequence of Pasteuria ramosa, a parasite of cladoceran water fleas of the family Daphnidae. The two Pasteuria sequences, which diverged from each other by a dissimilarity index of 7%, also were compared with the 16S rDNA sequences of 30 other bacterial species to determine the phylogenetic position of the genus Pasteuria among the Gram-positive eubacteria. Phylogenetic analyses using maximum-likelihood, maximum-parsimony and neighbour-joining methods showed that the Heterodera glycines-infecting Pasteuria and its sister species, P. ramosa, form a distinct line of descent within the Alicyclobacillus group of the Bacillaceae. These results are consistent with the view that the genus Pasteuria is a deeply rooted member of the Clostridium-Bacillus-Streptococcus branch of the Gram-positive eubacteria, neither related to the actinomycetes nor closely related to true endospore-forming bacteria.

Animals↗

Combined AFLP and RFLP mapping in two hexaploid oat recombinant inbred populations.

A combined RFLP and AFLP map was constructed for hexaploid oat (Avena spp.). The segregation of AFLP markers was scored in two hexaploid oat recombinant inbred line (RIL) populations, the 'Kanota' x 'Ogle' RFLP population, and a population derived from 'Clintland64' and 'IL86-5698', barley yellow dwarf virus (BYDV)-sensitive and BYDV-tolerant lines, respectively. More than 300 AFLP markers were scored in each population, of which 97 could be scored in both populations. AFLP markers were linked to RFLP markers in 32 of 36 'Kanota' x 'Ogle' RFLP linkage groups. The addition of the AFLP markers to the 'Kanota' x 'Ogle' RFLP data set combined markers from four pairs of linkage groups and increased the size of the map from 1402 cM to 2351 cM. Thirty linkage groups were observed in the 'Clintland64' x 'IL86-5698' population, two of which could be consolidated by comparing the maps from both populations. The AFLP and RFLP markers showed very similar distributions in the 'Kanota' x 'Ogle' population with a tendency of each type of marker to cluster with markers of the same type. The placement of a set of AFLP markers on the 'Kanota' x 'Ogle' linkage map will enrich the RFLP map and allow others to relate AFLP markers for agronomically important genes to the reference 'Kanota' x 'Ogle' linkage map.

Avena↗

Nucleotide sequence analysis shows that Rhopalosiphum padi virus is a member of a novel group of insect-infecting RNA viruses.

Rhopalosiphum padi virus (RhPV) is an aphid virus that has been considered a member of the Picornaviridae based on physicochemical properties. The 10,011-nt polyadenylated RNA genome of RhPV was completely sequenced. Analysis of the sequence revealed the presence of two open reading frames (ORFs). The predicted amino acid sequence of ORF1, representing the first 6600 nt of the RhPV genome, showed significant similarity to the nonstructural proteins of several plant and animal RNA viruses. Direct sequence analysis of the RhPV capsid proteins showed that ORF2, which represents the last 2900 nt, encodes the three structural proteins (28, 29, and 30 kDa). The predicted amino acid sequence of ORF2 is very similar to the corresponding regions of Drosophila C virus, Plautia stali intestine virus, and to a partial sequence from the 3' end of the cricket paralysis virus genome. The site of initiation of protein synthesis for ORF2 could not be determined from the amino acid and nucleotide sequences. ORF1 is preceded by 579 nt of noncoding RNA and the two ORFs are separated by more than 500 nt of noncoding RNA. Like picornaviruses, these regions may function to facilitate the cap-independent initiation of translation of the two ORFs. These data suggest that RhPV, Drosophila C virus, Plautia stali intestine virus, and probably cricket paralysis virus are members of a unique group of small RNA viruses that infect primarily insects.

Amino Acid Sequence↗

Identification and map location of TTR1, a single locus in Arabidopsis thaliana that confers tolerance to tobacco ringspot nepovirus.

The interaction between Arabidopsis and the nepovirus tobacco ringspot virus (TRSV) was characterized. Of 97 Arabidopsis lines tested, all were susceptible when inoculated with TRSV grape strain. Even though there was systemic spread of the virs, there was a large degree of variation in symptoms as the most sensitive lines died 10 days after inoculation, while the most tolerant lines either were symptomless or developed only mild symptoms. Four lines were selected for further study based on their differential reactions to TRSV. Infected plants of line Col-0 and Col-0 gl1 flowered and produced seeds like noninfected plants, while those of lines Estland and H55 died before producing seeds. Symptoms appeared on sensitive plants approximately 5 to 6 days after inoculation. Serological studies indicated that in mechanically inoculated seedlings, the virus, as measured by coat protein accumulation, developed at essentially the same rates and to the same levels in each of the four lines, demonstrating that differences in symptom development were not due to a suppression of virus accumulation. Two additional TRSV strains gave similar results when inoculated on the four lines. Genetic studies with these four Arabidopsis lines revealed segregation of a single incompletely dominant locus controlling tolerance to TRSV grape strain. We have designated this locus TTR1. By using SSLP and CAPS markers, TTR1 was mapped to chromosome V near the nga129 marker. Seed transmission frequency of TRSV for Col-0 and Col-0 gl1 was over 95% and their progeny from crosses all had seed transmission frequencies of over 83%, which made it possible to evaluate the segregation of TTR1 in F2 progeny from infected F1 plants without inoculating F2 plants. Seed transmission of TRSV will be further exploited to streamline selection of individuals for fine mapping the TTR1 gene. The identification of tolerant and sensitive interactions between TRSV and A. thaliana lines provides a model system for genetic and molecular analysis of plant tolerance to virus infection.

Arabidopsis↗

The complete nucleotide sequence of apple mosaic virus RNA-3.

The complete nucleotide sequence of apple mosaic ilarvirus (ApMV) RNA-3 has been determined from cloned viral cDNAs. The 5' terminus of RNA-3 was determined by direct RNA sequencing, while the 3' end was determined by polyadenylation of genomic RNA and sub-cloning using oligo dT. ApMV RNA-3 is 2056 bases in length and encodes at least two open reading frames. It is similar in size and genome organization to the RNA-3 of other members of the Bromoviridae, which includes ilarviruses. The CP gene is in the 3' half of the molecule, and another large open reading frame is upstream of the CP gene and can potentially encode a protein of 32,400 daltons. This peptide is the same size and shows limited sequence homology to an open reading frame located at the 5' end of RNA 3 in tobacco streak and prune dwarf ilarviruses and alfalfa mosaic virus, which is postulated to be the viral movement protein. The nucleic acid sequence was not homologous to tobacco streak virus, prune dwarf virus, alfalfa mosaic virus or other members of the Bromoviridae. The 5'-non-coding region of ApMV RNA-3 contains a 15 base palindromic sequence which encloses a sequence resembling the ICR-2 regions of eukaryotic tRNA gene promoters.

Amino Acid Sequence↗

Expression of multiple eukaryotic genes from a single promoter in Nicotiana.

We engineered an expression unit composed of three eukaryotic genes driven by a single plant-active promoter and demonstrated functional expression in planta. The individual genes were linked as translational fusions to produce a polyprotein using spacer sequences encoding specific heptapeptide cleavage recognition sites for NIa protease of tobacco vein mottling virus (TVMV). The NIa gene itself was included as the second gene of the multi-gene unit. The first and third genes, obtained from the TR region of pTi15955, encoded enzymatic functions associated with the mannityl opine biosynthetic pathway. The mannityl opine conjugase gene (mas2) was the first unit of the construct and provided the native plant-active promoter and 5' untranslated regulatory sequence. The third gene (mas1), encoding the mannityl opine reductase, furnished the native 3' untranslated region. Cis-processing of the polyprotein by the NIa protease domain was demonstrated in vitro using rabbit reticulocyte lysate and wheat germ cell-free translation systems. Tobacco plant cells transformed with the multi-gene unit produced detectable levels of mannopine, mannopinic acid, and their biosynthetic intermediates, deoxyfructosyl-glutamate and deoxyfructosyl-glutamine. This indicates that the polygene construct results in a set of functional enzymatic activities that constitute a complete metabolic pathway.

Animals↗

Polymorphism of genes coding for nuclear 18S rRNA indicates genetic distinctiveness of anastomosis group 10 from other groups in the Rhizoctonia solani species complex.

DNA polymorphism in the 18S nuclear rRNA gene region was investigated by using 11 restriction endonucleases for 161 isolates of 25 intraspecific groups (ISGs) representing 11 reported anastomosis groups (AGs) of Rhizoctonia solani. A PCR-based restriction mapping method in which enzymatically amplified DNA fragments and subfragments were digested with one or two restriction enzymes was employed. Four types of DNA restriction maps of this region were constructed for these 25 ISGs. Map type I of the 18S rDNA region was represented by isolates of a majority of R. solani ISGs. Map types II and III, represented by ISG 2E and 9 isolates and 5C isolates, respectively, differed from map I by the absence of one (map type II) or two (map type III) restriction sites. Map type IV, represented by ISG 10A and B (or AG 10) isolates, showed significant restriction site variations, with five enzymes in this region compared with those of the remaining ISGs or AGs. Ten of the 25 restriction sites in the 18S rRNA gene region were informative and selected for analysis. Previously reported restriction maps of the 5.8S rRNA gene region, including the internal transcribed spacers, were aligned with each other, and 12 informative restriction sites were identified. These data were used alone and in combination to evaluate group relationships. Analyses derived from these data sets by maximum parsimony and likelihood methods showed that AG 10 isolates were distinct and distantly related to the majority isolates of the other AGs of this species complex.

Base Sequence↗

Detection of the readthrough protein of barley yellow dwarf virus.

The single open reading frame (ORF) 5 encoding the 50-kDa protein of barley yellow dwarf virus PAV-IL (BYDV-PAV-IL) was expressed in bacteria, purified, and used as an immunogen/antigen to produce/screen antibodies specific to the 50-kDa protein. Two monoclonal antibodies (MAb PAV-IL-22 kDa and MAb PAV-IL-50 kDa) raised against BYDV-PAV-IL could specifically detect the presence of the 72-kDa readthrough protein in extracts from the BYDV-infected leaf tissue. The results suggest that ORF 5 (50-kDa protein) is translated by readthrough of ORF 3 (22-kDa coat protein) to produce the 72-kDa protein. The readthrough protein is thought to be a structural protein on the external surface of BYDV.

Antibodies, Viral↗

The nucleotide sequence of apple mosaic virus coat protein gene has no similarity with other Bromoviridae coat protein genes.

A double-stranded cDNA was synthesized from in vitro polyadenylated apple mosaic virus (ApMV) RNA 3 using oligo(dT) and sequence-specific primers, and was cloned into plasmid vectors. A set of overlapping cDNA clones was used to determine the nucleotide sequence of RNA 4. ApMV RNA 4 was found to contain an open reading frame (ORF) of 666 nucleotides, which was flanked by 5' and 3' non-translated sequences of 55 and 264 nucleotides, respectively. The ORF encoding the coat protein was identified by comparing the predicted amino acid sequence with that obtained from direct protein microsequencing of the native viral coat protein. The ORF encodes a protein with an M(r) of 25,056. The nucleotide sequence of the ApMV coat protein gene showed no similarity to those of alfalfa mosaic virus, tobacco streak virus (TSV), brome mosaic virus or cucumber mosaic virus. The predicted amino acid sequence of the amino-terminal region of the ApMV coat protein is basic, rich in cysteine residues and may contain a zinc finger motif similar to that found in TSV.

Amino Acid Sequence↗

Coat protein sequences of RMV-like strains of barley yellow dwarf virus separate them from other luteoviruses.

Illinois (IL) and Minnesota (MN) RMV-like strains of barley yellow dwarf virus (BYDV) were identified from maize displaying red leaf symptoms by enzyme-linked immunosorbent assay (ELISA) using antiserum against a New York strain (BYDV-RMV-NY). Some IL and MN strains, but not the NY strain, could be detected by ELISA with a monoclonal antibody raised against BYDV-RPV-NY. The region of the viral genome representing the coat protein gene was amplified by polymerase chain reaction, cloned and sequenced. The nucleotide sequences of the BYDV-RMV-IL and BYDV-RMV-MN coat protein genes differed at just five nucleotide positions while the BYDV-RMV-IL and BYDV-RMV-NY differed at 101 of the 591 positions. The predicted amino acid sequences of the coat proteins of RMV-like strains from IL, MN, and NY shared approximately 60% identify with those of the coat proteins of beet western yellows virus, BYDV-RPV-NY, and potato leafroll virus.

Amino Acid Sequence↗

A rapid chemiluminescent detection method for barley yellow dwarf virus.

Barley yellow dwarf virus (BYDV-PAV-IL) was detected with biotinylated in vitro transcript cDNA using a chemiluminescent substrate on nylon membranes. Signals were detected on X-ray film and quantified using either a densitometer or an ELISA plate reader. The time required for sample preparation was reduced so that the entire protocol could be completed in two days. The in vitro transcript probes could detect 1 ng of purified virus and as little as 1 microliter of sap extracts prepared from infected oat shoots.

Hordeum↗

In vitro synthesis, phosphorylation, and localization on 48 S initiation complexes of human protein synthesis initiation factor 4E.

Complementary DNA for human eukaryotic initiation factor 4E (eIF-4E) was transcribed in vitro and the transcripts used to direct protein synthesis in a cell-free reticulocyte translation system. The predominant translation product was 25 kDa, was bound to a m7GTP-Sepharose affinity column, and was specifically eluted with m7GTP. Both phosphorylated (P) and unphosphorylated (U) forms of eIF-4E were synthesized, and the P/U ratio increased as a function of incubation time in the reticulocyte lysate system. Both forms were quantitatively retained on m7GTP-Sepharose. When translation reactions were resolved on sucrose density gradients, the 35S-labeled eIF-4E sedimented predominantly at 3-4 S. However, in the presence of edeine or guanylyl imidodiphosphate, both of which cause accumulation of 48 S initiation complexes, eIF-4E was detected in the 48 S region. In the presence of sparsomycin, used to accumulate 80 S initiation complexes, no eIF-4E was observed in the 80 S region. No change in the eIF-4E distribution was caused by m7GTP. These results are consistent with a model whereby eIF-4E is transferred to the 43 S initiation complex together with mRNA and is released from the initiation complex when the 60 S ribosomal subunit joins.

Animals↗