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Biomedical subjects

B W Sobral

Publications and source records attributed to B W Sobral.

At least 19 recordsLinked to original sources
PubMed2006

Plant pathogen forensics: capabilities, needs, and recommendations.

A biological attack on U.S. crops, rangelands, or forests could reduce yield and quality, erode consumer confidence, affect economic health and the environment, and possibly impact human nutrition and international relations. Preparedness for a crop bioterror event requires a strong national security plan that includes steps for microbial forensics and criminal attribution. However, U.S. crop producers, consultants, and agricultural scientists have traditionally focused primarily on strategies for prevention and management of diseases introduced naturally or unintentionally rather than on responding appropriately to an intentional pathogen introduction. We assess currently available information, technologies, and resources that were developed originally to ensure plant health but also could be utilized for postintroduction plant pathogen forensics. Recommendations for prioritization of efforts and resource expenditures needed to enhance our plant pathogen forensics capabilities are presented.

Bioterrorism↗
PubMed2001

The Arabidopsis Information Resource (TAIR): a comprehensive database and web-based information retrieval, analysis, and visualization system for a model plant.

Arabidopsis thaliana, a small annual plant belonging to the mustard family, is the subject of study by an estimated 7000 researchers around the world. In addition to the large body of genetic, physiological and biochemical data gathered for this plant, it will be the first higher plant genome to be completely sequenced, with completion expected at the end of the year 2000. The sequencing effort has been coordinated by an international collaboration, the Arabidopsis Genome Initiative (AGI). The rationale for intensive investigation of Arabidopsis is that it is an excellent model for higher plants. In order to maximize use of the knowledge gained about this plant, there is a need for a comprehensive database and information retrieval and analysis system that will provide user-friendly access to Arabidopsis information. This paper describes the initial steps we have taken toward realizing these goals in a project called The Arabidopsis Information Resource (TAIR) (www.arabidopsis.org).

Arabidopsis↗
PubMed2001

Bioinformatics for rice resources.

The distinguishing feature of the 'new biology' is that it is information intensive. Not only does it demand access to and assimilation of vast data sets accumulated by engineered laboratory processes, but it also demands a previously unimaginable level of data integration across data types and sources. There are various information resources available for rice. In addition, there are various information resources that are not focused on rice but that contain rice data. The challenge for rice researchers and breeders is to access this wealth of data meaningfully. This challenge will grow significantly as international efforts aimed at sequencing the entire rice genome come into full swing. Only through concerted efforts in bioinformatics will the power of these public data be brought to bear on the needs of rice researchers and breeders worldwide. These efforts will need to focus on two large but distinct areas: (1) development of an effective bioinformatics infrastructure (hardware systems, software systems, and software engineers and support staff) and (2) computational biology research in visualization and analysis of very large, complex data sets, such as those that will be developed using high-throughput expression technologies, large-scale insertional mutagenesis, and biochemical profiling of various types. In the midst of the large flow of high-throughput data that the international rice genome sequencing efforts will produce, it is also imperative that integration of those data with unique germplasm data held in trust by the CGIAR be a part of the informatics infrastructure. This paper will focus on the state of rice information resources, the needs of the rice community, and some proposed bioinformatics activities to support these needs.

Algorithms↗
PubMed2000

Comparative analysis of expressed sequences in Phytophthora sojae.

Phytophthora sojae (Kaufmann and Gerdemann) is an oomycete that causes stem and root rot on soybean (Glycine max L. Merr) plants. We have constructed three cDNA libraries using mRNA isolated from axenically grown mycelium and zoospores and from tissue isolated from plant hypocotyls 48 h after inoculation with zoospores. A total of 3,035 expressed sequence tags (ESTs) were generated from the three cDNA libraries, representing an estimated 2,189 cDNA transcripts. The ESTs were classified according to putative function based on similarity to known proteins, and were analyzed for redundancy within and among the three source libraries. Distinct expression patterns were observed for each library. By analysis of the percentage G+C content of the ESTs, we estimate that two-thirds of the ESTs from the infected plant library are derived from P. sojae cDNA transcripts. The ESTs originating from this study were also compared with a collection of Phytophthora infestans ESTs and with all other non-human ESTs to assess the similarity of the P. sojae sequences to existing EST data. This collection of cDNA libraries, ESTs, and accompanying annotation will provide a new resource for studies on oomycetes and on soybean responses to pathogen challenge.

DNA, Complementary↗
PubMed1999

Discrimination of Rhizobium tropici and R. leguminosarum strains by PCR-specific amplification of 16S-23S rDNA spacer region fragments and denaturing gradient gel electrophoresis (DGGE).

With the aim of detecting Rhizobium species directly in the environment, specific PCR primers for Rh. tropici and Rh. leguminosarum were designed on the basis of sequence analysis of 16S-23S rDNA spacer regions of several Rh. tropici, Rh. leguminosarum and Agrobacterium rhizogenes strains. Primer specificity was checked by comparison with available rDNA spacer sequences in databases, and by PCR using DNA from target and reference strains. Sequence polymorphisms of rDNA spacer fragments among strains of the same species were detected by denaturing gradient gel electrophoresis (DGGE). The specific PCR primers designed in this study could be applied to evaluate the diversity of Rh. tropici and Rh. leguminosarum by analysing the polymorphisms of 16S-23S spacer rDNA amplified from either whole-cell or soil-extracted DNA.

DNA Primers↗
PubMed1999

Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily.

The nucleotide binding site (NBS) is a characteristic domain of many plant resistance gene products. An increasing number of NBS-encoding sequences are being identified through gene cloning, PCR amplification with degenerate primers, and genome sequencing projects. The NBS domain was analyzed from 14 known plant resistance genes and more than 400 homologs, representing 26 genera of monocotyledonous, dicotyle-donous and one coniferous species. Two distinct groups of diverse sequences were identified, indicating divergence during evolution and an ancient origin for these sequences. One group was comprised of sequences encoding an N-terminal domain with Toll/Interleukin-1 receptor homology (TIR), including the known resistance genes, N, M, L6, RPP1 and RPP5. Surprisingly, this group was entirely absent from monocot species in searches of both random genomic sequences and large collections of ESTs. A second group contained monocot and dicot sequences, including the known resistance genes, RPS2, RPM1, I2, Mi, Dm3, Pi-B, Xa1, RPP8, RPS5 and Prf. Amino acid signatures in the conserved motifs comprising the NBS domain clearly distinguished these two groups. The Arabidopsis genome is estimated to contain approximately 200 genes that encode related NBS motifs; TIR sequences were more abundant and outnumber non-TIR sequences threefold. The Arabidopsis NBS sequences currently in the databases are located in approximately 21 genomic clusters and 14 isolated loci. NBS-encoding sequences may be more prevalent in rice. The wide distribution of these sequences in the plant kingdom and their prevalence in the Arabidopsis and rice genomes indicate that they are ancient, diverse and common in plants. Sequence inferences suggest that these genes encode a novel class of nucleotide-binding proteins.

Amino Acid Motifs↗
PubMed1997

Comparative mapping of Andropogoneae: Saccharum L. (sugarcane) and its relation to sorghum and maize.

Comparative genetic maps of Papuan Saccharum officinarum L. (2n = 80) and S. robustum (2n = 80) were constructed by using single-dose DNA markers (SDMs). SDM-framework maps of S. officinarum and S. robustum were compared with genetic maps of sorghum and maize by way of anchor restriction fragment length polymorphism probes. The resulting comparisons showed striking colinearity between the sorghum and Saccharum genomes. There were no differences in marker order between S. officinarum and sorghum. Furthermore, there were no alterations in SDM order between S. officinarum and S. robustum. The S. officinarum and S. robustum maps also were compared with the map of the polysomic octoploid S. spontaneum 'SES 208' (2n = 64, x = 8), thus permitting relations to homology groups ("chromosomes") of S. spontaneum to be studied. Investigation of transmission genetics in S. officinarum and S. robustum confirmed preliminary results that showed incomplete polysomy in these species. Because of incomplete polysomy, multiple-dose markers could not be mapped for lack of a genetic model for their segregation. To coalesce S. officinarum and S. robustum linkage groups into homology groups (composed of homologous pairing partners), they were compared with sorghum (2n = 20), which functioned as a synthetic diploid. Groupings suggested by comparative mapping were found to be highly concordant with groupings based on highly polymorphic restriction fragment length polymorphism probes detecting multiple SDMs. The resulting comparative maps serve as bridges to allow information from one Andropogoneae to be used by another, for breeding, ecology, evolution, and molecular biology.

Journal Article↗
PubMed1995

Genetic variability of Paecilomyces fumosoroseus isolates revealed by molecular markers.

Paecilomyces fumosoroseus (Deuteromycotina:Hyphomycetes) is a fungus that is potentially useful for the bio-control of economically important agricultural pests, such as whitefly (Bemisia tabaci). Arbitrarily primed PCR and PCR with tRNA consensus primers were used to analyze genetic variability among 27 P. fumosoroseus isolates, 15 of which came from the same host, B. tabaci, one P. lilacinus isolate, used as an outgroup, 9 previously unidentified Paecilomyces isolates. Fifteen 10-mer oligonucleotide primers of arbitrary sequence revealed 322 scorable binary characters. Principal coordinates and cluster analysis of characters showed that most of the P. fumosoroseus and Paecilomyces sp. isolates were in three phenetic groups with > 65% internal similarity. Two of the three arbitrary phenetic groups were closely related (76% similarity) with the third group quite different (only 14% similarity) from the first two. The phenetic groups did not correlate with geographical origin or host species. Genetic variability of isolates infecting whitefly in Florida was detected. Isolates from B. tabaci were represented in two of the three groups, and different genotypes were identified even when they were isolated from an epizootic population in India and Pakistan. There was no evidence of host-specific selection of genotypes, as has been shown in other entomopathogenic fungi. Three isolates morphologically classified as P. fumosoroseus were clustered in a phenetic group which displayed only 14% similarity to the other isolates of this species. Seven isolated that presented problems for morphological classification were found to be similar or, in three cases, identical to other P. fumosoroseus isolates that dit not present problems for morphological classification.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

DNA markers for differentiating isolates of Paecilomyces lilacinus.

Paecilomyces lilacinus is an agent for the potential biological control of soil nematodes. Arbitrarily primed PCR was used to fingerprint the genomes of 28 isolates of this fungus. Most (72%) of the isolates originated from soil of different regions of Brazil. Fourteen 10-mer oligonucleotide primers of arbitrary sequence revealed 293 scorable binary characters. Distinct genotypes were obtained for each isolate. Cluster analysis showed a high level of variability among these genotypes. The similarity among pairwise comparisons of the isolates varied from 84.3% to 7.6%, with a mean of 63.5%. No clearly defined phenetic groups were identified by cluster or multivariate analyses. No correlation with geographical origin or host was detected. In addition, PCR with four pairs of consensus tRNA gene primers was performed on a subsample of 12 P. lilacinus isolates, three P. farinosus isolates, two P. fumosoroseus isolates, and one isolate of P. amoenoroseus. An inferred phylogeny based on 112 binary characters obtained by tRNA-PCR showed a monophyletic group which contained most of the P. lilacinus isolates. In contrast, three isolates of P. farinosus were not in a monophyletic group under the inferred phylogeny. These results suggest that tRNA fingerprinting could provide a valuable tool which could be used to develop the molecular taxonomy of Paecilomyces, as morphological characteristics of asexual structures cannot entirely resolve species.

Base Sequence↗
PubMed1995

tRNA intergenic spacers reveal polymorphisms diagnostic for Xanthomonas albilineans.

A PCR-based detection system was developed for Xanthomonas albilineans, a pathogen of sugarcane, and other related xanthomonads, using the conserved sequence of two adjacent tRNA genes and the variable length and sequence of the spacer region between them. An appropriate region was identified as follows: tRNA genes with the same anticodon from a wide variety of bacteria were aligned and the most frequent base at each position was chosen to derive primers that would anneal to the gene in either orientation. Pairs of such primers were screened against various Xanthomonas species and members of related genera using PCR at low to moderate annealing stringency. A subset of these pairs of tRNA consensus primers gave one or more PCR products which generally displayed interspecific length variability. The primer pair 5'-3' tRNA(ala) and 3'-5' tRNA(ile) showed interspecific length polymorphisms between X. albilineans and all other Xanthomonas species examined. These PCR products were cloned and sequenced from four isolates of X. albilineans and four isolates from different pathovars of X. campestris, and the spacer length variation confirmed. Specific tRNA gene primers were derived from the tRNA gene sequences. These primers yielded a PCR product of a characteristic length within most Xanthomonas species and pathovars tested. When a primer that projected from tRNA(ala) into the 3' end of the variable intergenic spacer was used with a tRNA(ile)-specific primer, PCR was a very sensitive diagnostic test for X. albilineans-infected sugarcane and gave no product or only a faint product with other species of bacteria. The specificity of this PCR-based detection system was further enhanced by a nested PCR reaction that took advantage of the fact that the tRNA(ala)-tRNA(ile) region was found to be embedded in a 16S rRNA-23S rRNA gene spacer. By amplifying the region between the 16S rRNA gene and tRNA(ile) or between the tRNA(ala) and the 23S rRNA gene, the subsequent nested PCR product was shown to be X. albilineans-specific.

Base Sequence↗
PubMed1993

A genetic linkage map of Saccharum spontaneum L. 'SES 208'.

The arbitrarily primed polymerase chain reaction was used to detect single-dose polymorphisms that, in turn, were used to generate a linkage map of a polyploid relative of cultivated sugarcane, Saccharum spontaneum 'SES 208' (2n = 64). The mapping population was composed of 88 progeny from a cross between SES 208 and a diploidized haploid derived from SES 208 by anther culture, ADP 85-0068. This cross allowed direct analysis of meiosis in SES 208 and gametic segregation ratios to be observed. One hundred twenty-seven 10-mer oligonucleotide primers of arbitrary sequence were selected from a pool of 420 primers used to screen the mapping parents. Three hundred thirty-six of the 420 primers amplified 4,540 loci or 13.5 loci per primer. The selected 127 primers revealed 2,160 loci of which 279 were present in SES 208 and absent in ADP 85-0068 and easily scored. Two hundred and eight (74.6%) of these 279 polymorphisms were single-dose polymorphisms (i.e., they displayed 1:1 segregation, chi 2 at 98% confidence level). Linkage analysis (theta = 0.25, LOD = 9.0 for two-point analysis, then theta = 0.25, LOD = 6.0 for multipoint analysis) of single-dose polymorphisms placed them into 42 linkage groups containing at least 2 markers. These single-dose markers span 1,500 contiguous centimorgans (cM) with 32 markers remaining unlinked (15.4%). From this 208-marker map we estimated the genome size of SES 208 to be 2,500 cM. The map has a predicted coverage of 85.1% at 30 cM, meaning that any new marker placed has an 85.1% chance of being within 30 cM of an existing marker. Furthermore, we show that SES 208 behaves like an autopolyploid because (i) the ratio of single-dose markers to higher dose markers fit the assumption of auto-octaploidy and (ii) the absence of repulsion phase linkages. This is the first genetic map constructed directly on a polyploid species for which no diploid relatives are known.

Base Sequence↗
PubMed1993

Physical map of the genome of Rhizobium meliloti 1021.

A physical map of the genome of Rhizobium meliloti 1021 is presented. The physical sizes of the three replicons in this genome had previously been determined and are as follows: the chromosome, 3.4 Mb; pSym-b, 1.7 Mb; and pSym-a, 1.4 Mb. The physical maps for this GC-rich genome contain AT-rich restriction sites for SwaI (5'-TAAATTTA-3'), PacI (5'-TTAATTAA-3'), PmeI (5'-GTTTAAAC-3'), and, for pSym-b, SpeI (5'-ACTAGT-3'). In addition, the endonuclease I-CeuI cleaved the 23S rRNA genes in this genome, and perhaps in most eubacterial genomes. I-CeuI digestion and polymerase chain reaction amplification of rrn regions were used to determine that there are at least three rrn loci in R. meliloti, all of which are located on the chromosome. The orientation of the rrn loci was determined by Southern blotting with probes from rrn sequences located 5' and 3' to the I-CeuI site. The rrn loci are clustered in one part of the chromosome and are oriented so that transcription will occur away from a single point in the circle, as observed for the origin of replication in the Escherichia coli and Salmonella typhimurium chromosomes. Fifteen genes that had been tagged by Tn5 insertion were localized to fragments on the chromosome physical map by using the IS50 as a probe in Southern blots. In addition, glt and gap were placed on the physical map by using Southern hybridization with cloned genes. The fortuitous occurrence of SpecI site in Tn5-233 was used to physically map 10 genetically mapped Tn5-233 integrations on pSym-b and to anchor the physical map to the genetic map. Finally, we demonstrate the usefulness of the map by localizing a total of 12 previously unmapped transposon insertions in the genome. This is the first physical map of the genome of a multireplicon member of the family Rhizobiaceae as well as the first physical map of a Rhizobium chromosome.

Base Sequence↗
PubMed1992

Analysis of large DNA from soybean (Glycine max L. Merr.) by pulsed-field gel electrophoresis.

The technique of pulsed-field gel electrophoresis (PFE) has been used to study chromosomal regions and entire genomes of several organisms. Techniques are presented for the isolation of high molecular weight DNA from embedded soybean protoplasts and the conditions for separating large DNA fragments using PFE. Digestion was detected by Southern hybridization using single copy nodulin clones. These data are being used to generate a physical map of the nodulin region(s) of the soybean genome.

DNA↗
PubMed1991

Electrophoretic separation of the three Rhizobium meliloti replicons.

The megaplasmids and the chromosome from the bacterium Rhizobium meliloti 1021 were separated in preparative quantities by using transverse alternating-field gel electrophoresis. The genetic content of each electrophoretically separated band was determined by Southern hybridization with replicon-specific probes and by comparison with Agrobacterium tumefaciens transconjugants harboring either pSym-a or pSym-b megaplasmids. Pulsed-field gel electrophoresis analyses of PacI (5'-TTAATTAA-3') and SwaI (5'-ATTTAAAT-3') digests of the whole genome and of the separated replicons were used to calculate genome sizes in two R. meliloti strains. In these strains, PacI digestion yielded only four fragments for the entire genome. The sizes of the PacI fragments from R. meliloti 1021 in megabase pairs (Mb) were 3.32 +/- 0.30, 1.42 +/- 0.13, 1.21 +/- 0.10, and 0.55 +/- 0.08, for a total genome size of 6.50 +/- 0.61 Mb. Southern hybridization with replicon-specific probes assigned one PacI fragment to the chromosome of R. meliloti 1021, one to pRme1021a, and two to pRme1021b. PacI digestion of A. tumefaciens pTi-cured, pSym transconjugants confirmed these assignments. In agreement with PacI data, the addition of the six SwaI fragments from R. meliloti 1021 gave a genome size of 6.54 +/- 0.43 Mb. pRme1021a was calculated to be 1.42 +/- 0.13 Mb, 1.34 +/- 0.09 Mb, and 1.38 +/- 0.12 Mb on the basis of PacI digestion, SwaI digestion, and the migration of uncut pRme1021a, respectively. pRme1021b was calculated to be 1.76 +/- 0.18 Mb, 1.65 +/- 0.10 Mb, and 1.74 +/- 0.13 Mb on the basis of PacI digestion, SwaI digestion, and the migration of uncut pRme1021B, respectively. The R. meliloti 1021 chromosome was calculated to be 3.32 +/- 0.30 Mb, 3.55 +/- 0.24 Mb, and 3.26 +/- 0.46 Mb on the basis of PacI data, SwaI data, and the migration of uncut chromosome, respectively.

Base Sequence↗
PubMed1991

The genomes of the family Rhizobiaceae: size, stability, and rarely cutting restriction endonucleases.

The lack of high-resolution genetic or physical maps for the family Rhizobiaceae limits our understanding of this agronomically important bacterial family. On the basis of statistical analyses of DNA sequences of the Rhizobiaceae and direct evaluation by pulsed-field agarose gel electrophoresis (PFE), five restriction endonucleases with AT-rich target sites were identified as the most rarely cutting: AseI (5'-ATTAAT-3'), DraI (5'-TTTAAA-3'), SpeI (5'-ACTAGT-3'), SspI (5'-AATAAT-3'), and XbaI (5'-TCTAGA-3'). We computed the sizes of the genomes of Bradyrhizobium japonicum USDA 424 and Rhizobium meliloti 1021 by adding the sizes of DNA fragments generated by SpeI digests. The genome sizes of R. meliloti 1021 and B. japonicum USDA 424 were 5,379 +/- 282.5 kb and 6,195 +/- 192.4 kb, respectively. We also compared the organization of the genomes of free-living and bacteroid forms of B. japonicum. No differences between the PFE-resolved genomic fingerprints of free-living and mature (35 days after inoculation) bacteroids of B. japonicum USDA 123 and USDA 122 were observed. Also, B. japonicum USDA 123 genomic fingerprints were unchanged after passage through nodules and after maintenance on a rich growth medium for 100 generations. We conclude that large-scale DNA rearrangements are not seen in mature bacteroids or during free-living growth on rich growth media under laboratory conditions.

Base Sequence↗
PubMed1990

Genome analysis of Bradyrhizobium japonicum serocluster 123 field isolates by using field inversion gel electrophoresis.

The genomes of 11 Bradyrhizobium japonicum serocluster 123 field isolates were analyzed by using field inversion gel electrophoresis. Genomic fingerprints produced by digestion of intact genomic DNA in agarose plugs with the rare-cutting restriction enzymes AseI, DraI, SpeI, and XbaI showed that the isolates were genetically diverse. Few (30 to 50%) isolates exhibited the same fingerprint as the USDA serogroup strain with which they are antigenically related. Southern hybridization with a nifHD gene probe to the blotted field inversion electrophoresis gels provided further evidence of the relatedness between members of serogroups 123 and 127.

Colony Count, Microbial↗