Biomedical subjects

W D Park

Publications and source records attributed to W D Park.

At least 19 recordsLinked to original sources

PubMed2003

Molecular markers linked to the blast resistance gene Pi-z in rice for use in marker-assisted selection.

Rice blast, caused by the fungal pathogen Pyricularia grisea, is a serious disease affecting rice-growing regions around the world. Current methods for identification of blast-resistant germplasm and progeny typically utilize phenotypic screening. However, phenotypic screens are influenced by environmental conditions and the presence of one resistance gene can sometimes phenotypically mask other genes conferring resistance to the same blast race. Pi-z is a dominant gene located on the short arm of chromosome 6 that confers complete resistance to five races of blast. Using sequence data found in public databases and degenerate primer pairs based on the P-loop, nucleotide binding sites and kinase domain motifs of previously cloned resistance genes, we have developed PCR-based DNA markers that cosegregate with the gene. These markers are polymorphic in a wide range of germplasm, including the narrow crosses characteristic of applied rice-breeding programs. They can now be used as a low cost, high-throughput alternative to conventional phenotypic screening for direct detection of blast resistance genes, allowing rapid introgression of genes into susceptible varieties as well as the incorporation of multiple genes into individual lines for more-durable blast resistance.

Chromosome Mapping↗

PubMed2003

Viscosity of rice flour: a rheological and biological study.

Rice breeders frequently use rapid visco analysis (RVA) as in index of rice quality. Potentially, viscosity curves could also be used to predict the sensory properties of a sample of rice or the processing properties of rice when used as an ingredient. The aim of this study is to determine the contribution of the main components of rice flour-starch and protein polymers and lipids-to the viscosity curve, accounting for biological and rheological contributions, and interactions with water. By deconstructing the rice flour, resistance to shear is generally the primary factor that affects rheological processes in the RVA, often masking the physical processes of cooking. Thus, higher concentrations of water reveal more about the true biological and physical processes of the transition from a powder to paste. Proteins contribute to peak height, offset thixotropy, and contribute to the final viscosity. Starch-lipid complexes can mask differences in the molecular structures of amylose and amylopectin, and removing lipids alters the structure of the paste significantly, which consequently alters viscosity curves.

Amylose↗

PubMed2001

Niemann-Pick C variant detection by altered sphingolipid trafficking and correlation with mutations within a specific domain of NPC1.

Niemann-Pick disease type C (NPC) is a fatal, autosomal recessive lipidosis characterized by lysosomal accumulation of unesterified cholesterol and multiple neurological symptoms, such as vertical supranuclear ophthalmoplegia, progressive ataxia, and dementia. More than 90% of cases of NPC are due to a defect in Niemann-Pick C1 (NPC1), a late endosomal, integral membrane protein that plays a role in cholesterol transport or homeostasis. Biochemical diagnosis of NPC has relied on the use of patient skin fibroblasts in an assay to demonstrate delayed low-density lipoprotein (LDL)-derived cholesterol esterification and a cytological technique-filipin staining-to demonstrate the intracellular accumulation of cholesterol. A small percentage of patients, referred to as "NPC variants," present with clinical symptoms of NPC but show near-normal results of these biochemical tests, making laboratory confirmation of NPC disease problematic. Here, we demonstrate that NPC-variant fibroblast samples can be detected as sphingolipid storage disease cells, using a fluorescent sphingolipid analog, BODIPY-lactosylceramide. This lipid accumulated in endosomes/lysosomes in variant cells preincubated with LDL cholesterol but targeted to the Golgi complex in normal cells under these conditions. The reproducibility of this technique was validated in a blinded study. In addition, we performed mutation analysis of the NPC1 gene in NPC variant and "classical" NPC cell samples and found a high incidence of specific mutations within the cysteine-rich region of NPC1 in variants. We also found that 5 of the 12 variant cell samples had no apparent defect in NPC1 but were otherwise indistinguishable from other variant cells. This is a surprising result, since, in general, approximately 90% of patients with NPC possess defects in NPC1. Our findings should be useful for the detection of NPC variants and also may provide significant new insight regarding NPC1 genotype/phenotype correlations.

Alleles↗

PubMed1999

Transcript accumulation and utilization of alternate and non-consensus splice sites in rice granule-bound starch synthase are temperature-sensitive and controlled by a single-nucleotide polymorphism.

Granule-bound starch synthase (GBSS), a product of the waxy gene in rice (Oryza sativa L.), is necessary for the synthesis of amylose in the endosperm. In an extended pedigree of 89 rice cultivars, we have previously shown that all cultivars with more than 18% amylose had the sequence AGGTATA at the leader intron 5' splice site, while all cultivars with a lower proportion of amylose had the sequence AGTTATA. This single-nucleotide polymorphism reduces the efficiency of GBSS pre-mRNA processing. It also results in alternate splicing at multiple sites, some of which have non-consensus sequences. Here we demonstrate that this same G-to-T polymorphism is also associated with differential sensitivity to temperature during the period of grain development. Cultivars with the sequence AGTTATA have a substantial increase in accumulation of mature GBSS transcripts at 18 degrees C compared to 25 or 32 degrees C. The selection of leader intron 5' splice sites is also affected by temperature in these cultivars. A 5' splice site -93 upstream from that used in high-amylose varieties predominates at 18 degrees C. At higher temperatures there is increased utilization of a 5' splice site at -I and a non-consensus site at +1. Potential implications of differential 5' splice site selection and associated differences in 3' splice site selection on transcript stability and translational efficiency are discussed.

Alternative Splicing↗

PubMed1998

Use of alternate splice sites in granule-bound starch synthase mRNA from low-amylose rice varieties.

The rice Waxy gene encodes a granule-bound starch synthase (GBSS) necessary for the synthesis of amylose in endosperm tissue. We have previously shown that a CT microsatellite near the transcriptional start site of the GBSS gene can distinguish 7 alleles that accounted for more than 80% of the variation in apparent amylose content in an extended pedigree of 89 US rice cultivars (Oryza sativa L.). Furthermore, all the cultivars with 18% or less amylose were shown to have the sequence AGTTATA at the putative leader intron 5' splice site, while all cultivars with a higher proportion of amylose had AGGTATA. Here we demonstrate that this single-base mutation reduces the efficiency of GBSS pre-mRNA processing and results in alternate splicing at three cryptic sites. The predominant 5' splice site in CT18 low-amylose varieties is 93 bp upstream of the splice site used in intermediate and high amylose varieties and is immediately 5' to the CT microsatellite that we previously demonstrated to be tightly correlated with amylose content. Use of the leader intron 5' splice site at either -93 or -1 in conjunction with the predominant 3' splice site results in formation of a small open reading frame 38 bp upstream of the normal ATG and out of frame with it. This open reading frame is not produced when any of the 5' leader intron splice sites are used in conjunction with an alternate 3' splice site five bases further downstream which was observed in all rice varieties tested.

Alternative Splicing↗

PubMed1997

Epistasis for three grain yield components in rice (Oryza sativa L.).

The genetic basis for three grain yield components of rice, 1000 kernel weight (KW), grain number per panicle (GN), and grain weight per panicle (GWP), was investigated using restriction fragment length polymorphism markers and F4 progency testing from a cross between rice subspecies japonica (cultivar Lemont from USA) and indica (cv. Tequing from China). Following identification of 19 QTL affecting these traits, we investigated the role of epistasis in genetic control of these phenotypes. Among 63 markers distributed throughout the genome that appeared to be involved in 79 highly significant (P < 0.001) interactions, most (46 or 73%) did not appear to have "main" effects on the relevant traits, but influenced the trait(s) predominantly through interactions. These results indicate that epistasis is an important genetic basis for complex traits such as yield components, especially traits of low heritability such as GN and GWP. The identification of epistatic loci is an important step toward resolution of discrepancies between quantitative trait loci mapping and classical genetic dogma, contributes to better understanding of the persistence of quantitative genetic variation in populations, and impels reconsideration of optimal mapping methodology and marker-assisted breeding strategies for improvement of complex traits.

Adaptation, Physiological↗

PubMed1997

Genetics of hybrid sterility and hybrid breakdown in an intersubspecific rice (Oryza sativa L.) population.

F1 hybrid sterility and "hybrid breakdown" of F2 and later generations in rice (Oryza sativa L.) are common and genetically complicated. We used a restriction fragment length polymorphism linkage map and F4 progeny testing to investigate hybrid sterility and hybrid breakdown in a cross between "widely compatible" O. sativa ssp. japonica cultivar Lemont from the Southern U.S. and ssp. indica cultivar Teqing from China. Our results implicate different genetic mechanisms in hybrid sterility and hybrid breakdown, respectively. Hybrid sterility appeared to be due to recombination within a number of putative differentiated "supergenes" in the rice genome, which may reflect cryptic structural rearrangements. The cytoplasmic genome had a large effect on fertility of both male and female gametes in the F1 hybrids. There appeared to be a pair of complementary genes that behaved like "wide compatibility" genes. This pair of genes and the "gamete eliminator" (S1) or "egg killer" (S-5) may influence the phenotypic effects of presumed supergenes in hybrids. Hybrid breakdown appeared to be largely due to incompatibilities between indica and japonica alleles at many unlinked epistatic loci in the genome. These proposed mechanisms may partly account for the complicated nature of postreproductive barriers in rice.

Chromosome Mapping↗

PubMed1997

Comparative evaluation of within-cultivar variation of rice (Oryza sativa L.) using microsatellite and RFLP markers.

The objective of this study was to determine an efficient way of detecting within-cultivar variation in rice varieties obtained from national and international germplasm collections. Seventy-one rice cultivars were evaluated for within-cultivar variation using a combination of phenotypic, RFLP, and microsatellite or simple sequence length polymorphism (SSLP). Variation between individuals within and accession and between duplicate accessions within a cultivar was detected even in cultivars that had been purified by phenotypic evaluation. Landrace cultivars were more heterogeneous and displayed a larger number of both RFLP and SSLP alleles than did modern cultivars. Microsatellite markers detected a greater number of alleles and were able to discriminate between even closely related individuals more efficiently than RFLPs. Some microsatellite markers were more informative than others for assessing genetic diversity. Single markers revealed 5.6-61.1% of the total variation detected by the 10 SSLP markers. Some marker combinations were complementary, providing more information than others. Several combinations of 4 SSLP markers detected as much as 94% of the total within-cultivar variation detected by the 10 SSLP markers. These results suggest that the use of four well-chosen microsatellites would be an efficient method for evaluating the heterogeneity of rice accessions.

Gene Frequency↗

PubMed1995

Sink- and vascular-associated sucrose synthase functions are encoded by different gene classes in potato.

Two differentially regulated classes of sucrose synthase genes, Sus3 and Sus4, were identified in potato. They cannot be classified as Sus1 and Sus2 types based on sequence homology and appear to have evolved after the divergence of the major families of dicotyledonous plants but before the divergence of tomato and potato. The potato sucrose synthase clones Sus3-65 and Sus4-16 share an 87% nucleotide identity in the coding regions, and both are interrupted by 13 introns, including a long leader intron. Potato Sus3 genes are expressed at the highest levels in stems and roots and appear to provide the vascular function of sucrose synthase. In contrast, Sus4 genes are expressed primarily in the storage and vascular tissue of tubers and appear to facilitate sink function. The genes are differentially regulated in root tips, with Sus3 expressed at high levels in the cell division zone and Sus4 expressed at high levels in the meristem and cap.

Amino Acid Sequence↗

PubMed1995

High-level tuber expression and sucrose inducibility of a potato Sus4 sucrose synthase gene require 5' and 3' flanking sequences and the leader intron.

The 3.6 kb of 5' flanking sequence, leader intron, and 0.7 kb of 3' sequence from the potato sucrose synthase gene Sus4-16 are sufficient to direct high-level expression in developing tubers, in basal tissues of axillary buds and shoots, and in meristems and caps of roots, and to confer sucrose inducibility in leaves. By examining a series of deletion and substitution constructs in transgenic potato plants, we found that this pattern of expression requires 5' flanking sequences both upstream and downstream of position -1500 and that sequences between positions -1500 and -267 are essential for sucrose induction. Replacement of the native 3' sequence with the nopaline synthase 3' sequence resulted in the loss of sucrose inducibility and of expression in basal tissues of axillary buds. A general decrease in expression in other tissues was also observed. Removal of the 1612-bp leader intron also had a dramatic effect on both the pattern and level of expression.

Base Sequence↗

PubMed1995

A potato Sus3 sucrose synthase gene contains a context-dependent 3' element and a leader intron with both positive and negative tissue-specific effects.

To examine which sequences are involved in regulating the potato sucrose synthase gene Sus3-65, we examined a series of deletion and substitution constructs in transgenic potato and tobacco plants. In a construct containing 3.9 kb of 5' flanking region, substitution of the native 3' sequence with the nopaline synthase 3' sequence and deletion of the leader intron did not significantly affect expression in vegetative tissues. However, in a construct containing only 320 bp of 5' flanking region, these changes had marked effects. Replacing the native 3' sequences with nopaline synthase 3' sequences caused a six- to 20-fold increase in expression in vascular tissue, and removing the leader intron almost completely abolished expression in potato plants. Surprisingly, removal of the leader intron from either the full-length construct or a construct containing only 320 bp of 5' flanking sequence reduced expression in vascular tissue of tobacco anthers at later stages of development but increased expression in pollen by more than 100-fold.

Base Sequence↗

PubMed1994

Nuclear protein factors binding to a class I patatin promoter region are tuber-specific and sucrose-inducible.

Genes encoding patatin, the major storage protein of the potato tuber, are generally divided into two classes, class I and class II. The expression of the class I patatin genes is normally tuber-specific, but can be induced in leaves by high concentrations of sucrose. By employing electrophoretic mobility shift assays (EMSA), we have identified nuclear protein factors that interact specifically with the proximal portion of the class I patatin promoter that is required for tuber-specific and sucrose-inducible expression. The factors were detected in nuclear extracts prepared from potato tubers and sucrose-induced leaves, but not in extracts from leaves of normal potato plants. Four putative transcription factor-binding sites were localized using DNase I footprinting. Competitive EMSA was employed to show that the same protein factor binds to at least two of the sites (boxes D and M). Interestingly, these two binding sites are highly homologous to light-responsive elements present in genes for the ribulose-1,5-bisphosphate carboxylase small subunit.

Base Sequence↗

PubMed1991

The expression of class I patatin gene fusions in transgenic potato varies with both gene and cultivar.

Patatin is a family of glycoproteins that contributes about 40% of the total soluble protein in tubers of potato (Solanum tuberosum L.). The protein is encoded by a multigene family of 50-70 genes which have been divided into classes I and II on the basis of sequence homology. The promoters of two class I genes, PS20 and PS3/27, were transcriptionally fused to beta-glucuronidase and transformed into the potato cultivars Désirée and Maris Bard. Examination of the expression levels in large populations of microtubers indicated that the PS20 promoter produced beta-glucuronidase activities 5-fold lower in Désirée than Maris Bard whereas the PS3/27 promoter showed similar levels in both cultivars. Furthermore, the relative expression levels from the two promoters were reversed in the two cultivars. The beta-glucuronidase enzyme activity was correlated with the mRNA level but not the copy number of the introduced gene. The implications for the use of patatin promoters in the genetic modification of tubers is discussed.

Carboxylic Ester Hydrolases↗

PubMed1991

Genetic and physical mapping of the patatin genes in potato and tomato.

Genes for the major storage protein of potato, patatin, have been mapped genetically and physically in both the potato and tomato genomes. In potato, all patatin genes detected by the cDNA clone pGM01 map to a single locus at the end of the long arm of chromosome 8. By means of pulsed field gel electrophoresis (PFGE) it was possible further to delimit this locus, containing 10-15 copies of the gene, to a maximum size of 1.4 million base pairs. Hybridizations with class-specific clones suggest that the locus is at least partially divided into domains containing the two major types of patatin genes, class I and II. In tomato, patatin-homologous sequences were found to reside at the orthologous locus at the end of chromosome 8. The approximately three copies in tomato were localized by PFGE to a single fragment of 300 kilobases. Whereas the class II-specific 5' promoter sequences reside in tomato at the same locus as the coding sequences, the single class I-specific copy of the 5' promoter sequences was localized on chromosome 3 with no coding sequence attached to it. A clone from this chromosome 3 locus of tomato was isolated and by restriction fragment length polymorphism mapping it could be further shown that a similar class I-specific sequence also exists on chromosome 3 of potato. As in tomato, this copy on chromosome 3 is not linked to a coding sequence for patatin. The results are discussed with respect to genome evolution and PFGE analysis of complex gene families.

Carboxylic Ester Hydrolases↗

PubMed1988

Characterization of the lipid acyl hydrolase activity of the major potato (Solanum tuberosum) tuber protein, patatin, by cloning and abundant expression in a baculovirus vector.

Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum) tubers. This protein has been reported not only to serve as a storage protein, but also to exhibit enzymic activity. By using a baculovirus system to express protein from the patatin cDNA clone pGM01, it was unambiguously shown that the patatin coded by this DNA has lipid acyl hydrolase and acyltransferase activities. The enzyme is active with phospholipids, monoacylglycerols and p-nitrophenyl esters, moderately active with galactolipids, but is apparently inactive with di- and tri-acylglycerols.

Acyltransferases↗

PubMed1988

Molecular characterization of the patatin multigene family of potato.

Patatin is a family of glycoproteins that accounts for up to 40% of the total soluble protein in potato tubers. We isolated and characterized 25 patatin genomic clones. All of these exhibit different restriction patterns, but can be divided into two classes based on the presence (class II) or absence (class I) of a 22-bp sequence within the 5'-untranslated region. We determined the complete nucleotide sequence of the class-I clone PS20 and the 5'-flanking sequence of seven additional clones. The transcribed region of PS20 spans 3197 bp and is divided by six introns. The seven exons encode a transcript which is identical to the cDNA, cloned in pGM203 [Mignery et al., Nucl. Acids Res. 12 (1984) 7987-8000]. The 5'-flanking sequences of both class-I and class-II patatin genes are highly homologous up to bp position -87 and then diverge. This conserved region contains the CAAT and TATA homologies as well as a homology to the core enhancer sequence. Within a class there are additional large regions of homology, but differences exist that allow the patatin genes to be further divided into several discrete subclasses. S1 nuclease protection experiments with both class-I and class-II probes showed that class-I transcripts are the predominant species present in tubers. Class-II transcripts are present in tubers, but are 50-100-fold less abundant. In potato roots, class-II transcripts are the predominant species and few, if any, class-I transcripts are present.

Amino Acid Sequence↗

PubMed1987

The two classes of genes for the major potato tuber protein, patatin, are differentially expressed in tubers and roots.

The major potato tuber protein, patatin, is a family of 40kd glycoproteins that constitutes forty per cent of the soluble protein in tubers but is generally undetectable in other tissues. Fused rocket immunoelectro-phoresis was used to detect in roots patatin that is immunologically different from tuber patatin. Western blots of SDS-polyacrylamide gels show root patatin to have a different molecular weight distribution than tuber patatin isoforms, but immunoprecipitation of in vitro translation products shows the patatin precursors to be of similar molecular weight in both tissues. This suggests that post-translational processing may differ in tubers and roots. Northern blots show that tuber and root patatin mRNAs are of similar size, but tuber transcripts are about 100-fold more abundant. 5' S1 nuclease and primer extension mapping suggests the class of patatin transcripts expressed in roots (class II transcripts) to be a subset of patatin transcripts expressed in tubers (classes I and II). Class II patatin mRNAs differ from class I transcripts by the presence of a 22 nucleotide insertion just upstream of the initiation codon. These data demonstrate that expression of the patatin multigene family is differentially regulated in tubers and roots.

Base Sequence↗

PubMed1987

Use of an in vitro tuberization system to study tuber protein gene expression.

Nodal cuttings from micropropagated potato plantlets give rise to microtubers when placed on Murashige and Skoog medium containing 6% sucrose and 2.5 mg/liter kinetin and incubated in the dark at 19 degrees C. Microtubers produced from the cultivar Superior were shown to contain the same characteristic group of proteins as field-grown tubers. As with field-grown tubers, the 40,000-dalton major tuber glycoprotein, patatin, accumulated to high levels in microtubers, reaching 3.7 +/- 0.2 mg/g fresh weight after 90 d. Also in agreement with field-grown plants, stems and leaves of micropropagated plantlets did not contain detectable levels of patatin, but small amounts of an electrophoretically distinct form accumulated transiently in roots. Patatin mRNA is readily detectable in developing microtubers 15 d after transfer of the cuttings to inductive medium. Patatin mRNA was also present in roots, but as with field-grown plants, was 50- to 100-fold less abundant and could be distinguished from that in tubers by primer extension. Microtuber development and patatin accumulation were inhibited by gibberellic acid.

Gene Expression Regulation↗