Search PubMed⌕ Search

Biomedical subjects

R W Hammond

Publications and source records attributed to R W Hammond.

At least 19 recordsLinked to original sources

Predicted ATP-binding cassette systems in the phytopathogenic mollicute Spiroplasma kunkelii.

Spiroplasma kunkelii is a cell wall-free, helical, and motile mycoplasma-like organism that causes corn stunt disease in maize. The bacterium has a compact genome with a gene set approaching the minimal complement necessary for cellular life and pathogenesis. A set of 21 ATP-binding cassette (ABC) domains was identified during the annotation of a draft S. kunkelii genome sequence. These 21 ABC domains are present in 18 predicted proteins, and are components of 16 functional systems, which account for 5% of the protein coding capacity of the S. kunkelii genome. Of the 16 systems, 11 are membrane-bound transporters, and two are cytosolic systems involved in DNA repair and the oxidative stress response; the genes for the remaining three hypothetical systems harbor nonsense and/or frameshift mutations, so their functional status is doubtful. Assembly of the 11 multicomponent transporters, and comparisons with other known systems permitted functional predictions for the S. kunkelii ABC transporter systems. These transporters convey a wide variety of substrates, and are critical for nutrient uptake, multidrug resistance, and perhaps virulence. Our findings provide a framework for functional characterization of the ABC systems in S. kunkelii.

ATP-Binding Cassette Transporters↗

The complete nucleotide sequence, genome organization, and specific detection of Beet mosaic virus.

Beet mosaic virus (BtMV) was identified almost five decades ago but has not been fully characterized at the molecular level. In this study, we have determined for the first time the complete nucleotide sequence of BtMV genomic RNA and have developed a specific molecular means for its diagnosis. The viral genome of BtMV comprises 9591 nucleotides, excluding the 3' terminal poly (A) sequence, and contains a single open reading frame (ORF) that begins at nt 166 and terminates at nt 9423, encoding a single polyprotein of 3086 amino acid residues. A 3' untranslated region of 168 nucleotides follows the ORF. The deduced genome organization is typical for a member of the family Potyviridae and includes 10 proteins: P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb and coat protein (CP). Nine putative protease cleavage sites were predicted computationally and by analogy with genome arrangements of other potyviruses. Conserved sequence motifs of homologous proteins of other potyviruses were found in corresponding positions of BtMV. BtMV is a distinct species of the genus Potyvirus with the most closely related species being Peanut mottle virus ( approximately 55% amino acid identity). Based on the nucleotide sequence obtained, we have developed a virus-specific RT-PCR assay for accurate diagnosis and differentiation of BtMV.

Amino Acid Sequence↗

The complete nucleotide sequence of isolate BYMV-GDD of Bean yellow mosaic virus, and comparison to other potyviruses.

The complete nucleotide sequence of Bean yellow mosaic virus (BYMV) gladiolus isolate GDD was determined and compared to broad bean isolates BYMV-MB4 and BYMV-S. The BYMV-GDD genome (9528 nt) was more similar to BYMV-MB4 (9532 nt) than to BYMV-S (9547 nt), which has "atypical" symptom expression and host range. The greatest variability occurred in the 5' untranslated region, P1 protein, and NIa-VPg protein, the N-terminal two thirds of HC-Pro, and the C-terminal one third of P3. Each of these regions has been correlated with symptom or host differences between isolates of other potyviruses, and may contribute to the "atypical" nature of BYMV-S.

5' Untranslated Regions↗

Gene content and organization of an 85-kb DNA segment from the genome of the phytopathogenic mollicute Spiroplasma kunkelii.

Spiroplasma kunkelii, the causative agent of corn stunt disease in maize (Zea maysL.), is a helical, cell wall-less prokaryote assigned to the class Mollicutes. As part of a project to sequence the entire S. kunkelii genome, we analyzed an 85-kb DNA segment from the pathogenic strain CR2-3x. This genome segment contains 101 ORFs and two tRNA genes. The majority of the ORFs code for predicted proteins that can be assigned to respective clusters of orthologous groups (COGs). These COGs cover diverse functional categories including genetic information storage and processing, cellular processes, and metabolism. The most notable gene cluster in this genome segment is a super-operon capable of encoding 24 ribosomal proteins. The organization of genes in this operon reflects the unique evolutionary position of the spiroplasma. Gene duplications, domain rearrangements, and frameshift mutations in the segment are interpreted as indicators of phase variation in the spiroplasma. To our knowledge, this is the first analysis of a large genome segment from a plant pathogenic spiroplasma.

Base Sequence↗

Phylogeny of isolates of Prunus necrotic ringspot virus from the Ilarvirus Ringtest and identification of group-specific features.

Isolates of Prunus necrotic ringspot virus (PNRSV) were examined to establish the level of naturally occurring sequence variation in the coat protein (CP) gene and to identify group-specific genome features that may prove valuable for the generation of diagnostic reagents. Phylogenetic analysis of a 452 bp sequence of 68 virus isolates, 20 obtained from the European Union Ilarvirus Ringtest held in October 1998, confirmed the clustering of the isolates into three distinct groups. Although no correlation was found between the sequence and host or geographic origin, there was a general trend for severe isolates to cluster into one group. Group-specific features have been identified for discrimination between virus strains.

Base Sequence↗

Genetic variability of genomic RNA 2 of four tobacco rattle tobravirus isolates from potato fields in the Northwestern United States.

Sequence analysis of RNA 2 of four Tobacco rattle virus (TRV) isolates collected from potato fields in Oregon (OR2, Umt1), Washington (BM), and Colorado (Cot2) revealed significant homologies to the ORY isolate from North America. Phylogenetic analysis based on a comparison of nucleotide (nt) and amino acid (aa) sequences with other members of the genus Tobravirus indicates that the North American isolates cluster as a distinct group. All of the RNAs are predicted to contain open reading frames (ORFs) potentially encoding the coat protein (CP, ORF 2a) and 37.6 kDa (ORF 2b) ORFs. In addition, they all contain a region of similarity to the 3' terminus of RNA 1 of ORY, including a truncated portion of the 16 kDa cistron from the 3' end of RNA 1. Three of the isolates, which are nematode transmissible, OR2, BM, and Cot2, also contain a third putative ORF (ORF 2c) which encodes a protein of 33.6 kDa. The fourth isolate, Umt1, which is not nematode transmissible, is the most divergent of the isolates as it encodes a truncated version of ORF 2c. The ORF 2c deletion in Umt1 may contribute to its inability to be transmitted by the vector. The results reported in this article indicate again that the TRV genome is flexible. Interestingly, although both isolates Umt1 and Cot2 were mechanically transmitted to tobacco from potato, only Umt1 exhibits the deletion in RNA 2. TRV Isolate Umt1, therefore, appears to be another example of rapid adaptation of the TRV genome to non-field conditions.

Animals↗

Molecular characterization of the genome of Maize rayado fino virus, the type member of the genus Marafivirus.

The complete nucleotide sequence of the single-stranded RNA genome of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus, is 6305 nucleotides (nts) in length and contains two putative open reading frames (ORFs). The largest ORF (nt 97-6180) encodes a polyprotein of 224 kDa with sequence similarities at its N-terminus to the replication-associated proteins of other viruses with positive-strand RNA genomes and to the papainlike protease domain found in tymoviruses. The C-terminus of the 224-kDa ORF also encodes the MRFV capsid protein. A smaller, overlapping ORF (nt 302-1561) encodes a putative protein of 43 kDa with unknown function but with limited sequence similarities to putative movement proteins of tymoviruses. The nucleotide sequence and proposed genome expression strategy of MRFV is most closely related to that of oat blue dwarf virus (OBDV). Unlike OBDV, MRFV RNA does not appear to contain a poly(A) tail, and it encodes a putative second overlapping open reading frame.

Amino Acid Sequence↗

Differential transport of DNA by a rectified Brownian motion device.

An interdigitated electrode array (IDEA) device has been designed and used to transport DNA based on a Brownian ratchet mechanism. This migration is produced by the periodic formation of an asymmetric sawtooth electric field in the device. Oligonucleotides of 25, 50, and 100 bases in length were tested using two different array geometries. DNA transport as a function of DNA size, electric field frequency, and array geometry is shown to be in qualitative agreement with theory. Such a device could provide for DNA separations over a broad size range, and can be readily scaled as a component in a microfabricated DNA analysis system.

Animals↗

Development and evaluation of a complementation-dependent gene delivery system based on cucumber mosaic virus.

To engineer cucumber mosaic virus (CMV-Ix) into a gene vector, genome component RNA 3 of the virus was modified and split into two sub-components, RNA 3A and RNA 3B. In RNA 3A, the open reading frame of the movement protein (MP) was replaced by a reporter gene encoding the green fluorescent protein (GFP), to monitor virus replication and movement. In RNA 3B, the coat protein (CP) gene was eliminated and a multiple cloning site (MCS) was created for foreign gene insertion. Each sub-component alone is defective and relies on its companion sub-component to restore full RNA 3 function. The vector system was evaluated for its ability to deliver and express the bacterial beta-glucuronidase (GUS) gene and a modified bean yellow mosaic virus coat protein (BYMV-CP) gene in Nicotiana benthamiana plants. Results showed that the engineered virus was able to move from cell to cell in the inoculated leaf and enter the minor veins of the inoculated leaf. Foreign gene expression was detected in the inoculated leaves. However, intermolecular recombination between RNA 3A and 3B occurred frequently, preventing efficient systemic expression of the foreign gene(s). Modifications and further evaluations are being undertaken to improve the gene delivery system.

Capsid↗

Both point mutation and RNA recombination contribute to the sequence diversity of citrus viroid III.

Field-grown citrus trees often harbor complex mixtures of 4-5 different viroid species, and the presence of citrus viroid III (CVd-III) has been shown to reduce the rate of tree growth without inducing disease. To more fully define the structure of its quasi-species, we have examined nine citrus viroid complexes for the presence of previously undescribed sequence variants of CVd-III. Analysis of 86 full-length cDNAs generated from these nine viroid complexes by RT-PCR revealed the presence of 20 new CVd-III variants. Chain lengths ranged from 293-297 nucleotides, and sequence changes were confined largely to the lower portions of the central conserved region and variable domain. The previously described variants CVd-IIIa (297 nt) and CVd-IIIb (294 nt) were clearly predominant, but phylogenetic analysis indicated that certain isolates may contain representatives of two additional fitness peaks. At least one group of CVd-III variants appears to have arisen as a result of RNA recombination. Populations recovered from diseased/declining trees were the most diverse, but even dwarfing isolates originating from old line Shamouti trees showed considerable variability.

Base Sequence↗

Characterization of a tomato protein kinase gene induced by infection by Potato spindle tuber viroid.

Viroids--covalently closed, circular RNA molecules in the size range of 250 to 450 nucleotides-are the smallest known infectious agents and cause a number of diseases of crop plants. Viroids do not encode proteins and replicate within the nucleus without a helper virus. In many cases, viroid infection results in symptoms of stunting, epinasty, and vein clearing. In our study of the molecular basis of the response of tomato cv. Rutgers to infection by Potato spindle tuber viroid (PSTVd), we have identified a specific protein kinase gene, pkv, that is transcriptionally activated in plants infected with either the intermediate or severe strain of PSTVd, at a lower level in plants inoculated with a mild strain, and not detectable in mock-inoculated plants. A full-length copy of the gene encoding the 55-kDa PKV (protein kinase viroid)-induced protein has been isolated and sequence analysis revealed significant homologies to cyclic nucleotide-dependent protein kinases. Although the sequence motifs in the catalytic domain suggest that it is a serine/threonine protein kinase, the recombinant PKV protein autophosphorylates in vitro on serine and tyrosine residues, suggesting that it is a putative member of the class of dual-specificity protein kinases.

Amino Acid Sequence↗

DNA transport by a micromachined Brownian ratchet device.

We have micromachined a silicon-chip device that transports DNA with a Brownian ratchet that rectifies the Brownian motion of microscopic particles. Transport properties for a DNA 50-mer agree with theoretical predictions, and the DNA diffusion constant agrees with previous experiments. This type of micromachine could provide a generic pump or separation component for DNA or other charged species as part of a microscale lab-on-a-chip. A device with reduced feature size could produce a size-based separation of DNA molecules, with applications including the detection of single-nucleotide polymorphisms.

DNA↗

Differentiation of closely related but biologically distinct cherry isolates of Prunus necrotic ringspot virus by polymerase chain reaction.

Prunus necrotic ringspot ilarvirus (PNRSV) exists as a number of biologically distinct variants which differ in host specificity, serology, and pathology. Previous nucleotide sequence alignment and phylogenetic analysis of cloned reverse transcription-polymerase chain reaction (RT-PCR) products of several biologically distinct sweet cherry isolates revealed correlations between symptom type and the nucleotide and amino acid sequences of the 3a (putative movement protein) and 3b (coat protein) open reading frames. Based upon this analysis, RT-PCR assays have been developed that can identify isolates displaying different symptoms and serotypes. The incorporation of primers in a multiplex PCR protocol permits rapid detection and discrimination among the strains. The results of PCR amplification using type-specific primers that amplify a portion of the coat protein gene demonstrate that the primer-selection procedure developed for PNRSV constitutes a reliable method of viral strain discrimination in cherry for disease control and will also be useful for examining biological diversity within the PNRSV virus group.

DNA Restriction Enzymes↗

Effect of a pharmacist-education initiative on ketorolac use and costs in a Medicaid program.

The effects of a pharmacist-education initiative on the use and costs of ketorolac in a state Medicaid program are reviewed. An intervention letter describing changes in the manufacturer's prescribing guidelines for ketorolac and providing suggestions for interacting with physicians regarding the use of ketorolac was sent to 150 of the 301 pharmacies that participate in New Mexico's Medicaid program. The remaining 151 pharmacies served as a control group. Ketorolac claims records for three months before and after the intervention were reviewed. The mean quantity of ketorolac tablets, total days' supply, and number of prescriptions filled per pharmacy per month were calculated for both periods. The number of prescriptions not filled as a result of the intervention as well as the number that could have been avoided, the number of cases of peptic ulceration (ketorolac's major adverse effect) that would be avoided, and the associated cost savings if all the state's Medicaid pharmacies had been included in the intervention were estimated. A total of 167 pharmacies (90 intervention and 77 control) dispensed ketorolac for Medicaid patients during the study period. Ketorolac dispensing rates declined during the postintervention period in both the intervention group and the control group, but the reduction was greater in the intervention group. It was predicted that if all pharmacies were included in an intervention, 135.6 fewer prescriptions for ketorolac would be filled each year; as a result, 1.14 cases of peptic ulceration would be avoided and net Medicaid costs would be reduced by $1638. Sending educational letters to pharmacists was associated with a modest reduction in ketorolac use in a state Medicaid program; a net reduction in Medicaid costs if the intervention were extended to all pharmacies that participate in the state's Medicaid program was projected.

Analysis of Variance↗

High-speed separation of linear and supercoiled DNA by capillary electrophoresis. Buffer, entangling polymer, and electric field effects.

Capillary electrophoresis in dilute and semidilute (slightly entangled) hydroxyethyl cellulose (HEC) is shown to separate linear double-stranded DNA (ds-DNA) and supercoiled plasmid DNA in the size range 1-16 thousand base pairs in 3 min. The mobilities of linear ds-DNA fragments are stronger functions of electric field strength and buffer concentration than the mobilities of supercoiled plasmids. The effects of HEC concentration and molecular weight are similar for both forms of DNA. The behavioral differences, which are attributed to the greater stiffness of the plasmids, can be used to define conditions that maximize resolution of supercoiled and linear ds-DNA of the same or similar number of base pairs.

Buffers↗

Virulence and molecular polymorphism of Prunus necrotic ringspot virus isolates.

Prunus necrotic ringspot virus (PNRSV) occurs as numerous strains or isolates that vary widely in their pathogenic, biophysical and serological properties. Prior attempts to distinguish pathotypes based upon physical properties have not been successful; our approach was to examine the molecular properties that may distinguish these isolates. The nucleic acid sequence was determined from 1.65 kbp RT-PCR products derived from RNA 3 of seven distinct isolates of PNRSV that differ serologically and in pathology on sweet cherry. Sequence comparisons of ORF 3a (putative movement protein) and ORF 3b (coat protein) revealed single nucleotide and amino acid differences with strong correlations to serology and symptom types (pathotypes). Sequence differences between serotypes and pathotypes were also reflected in the overall phylogenetic relationships between the isolates.

Amino Acid Sequence↗

Capillary electrophoresis of supercoiled and linear DNA in dilute hydroxyethyl cellulose solution.

Capillary electrophoresis in dilute hydroxyethyl cellulose is shown to separate supercoiled DNA in the size range 2000-16,000 base pairs. The plasmids migrate more slowly than linear ds-DNA of the same sizes. Plasmid bandwidths are larger than observed for ds-DNA, allowing identification of the type of DNA by bandwidth. The differing dependence of mobility on chain length can be explained by assuming that a plasmid migrates as an elastic rod, while ds-DNA migrates as a wormlike chain.

Cellulose↗