Le Gene Research Articles

Mapping and Functional Analysis of LE Gene in a Lethal Etiolated Rice Mutant at Seedling Stage

An EMS-induced mutant le (lethal etiolated) obtained from a rice variety Zhongjian 100, was characterized by lethal etiolated phenotypes with significantly lower contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids, as well as significantly decreased the number of chloroplast grana and irregular and less-stacked grana lamella. The root length, root surface area and root volume of le plants were markedly reduced relative to the wild type. The mutant le showed significantly lower catalase activity and total protein content, significantly higher peroxidase activity,. Using the map-based cloning method, LE gene was mapped to an interval of 48 kb between markers RM16107 and RM16110 on rice chromosome 3. A mutation (from T to C) at the nucleotide position 692 bp of LOC_Os03g59640 (ChlD) occurred, resulting in a change from leucine to proline. By crossing HM133 (a pale green mutant with a single-base substitution of A for G on the exon 10 of ChlD subunit) with a heterozygous line of le (LEle), two plant lines heterozygous at the LE locus and HM133 locus were obtained. Among 15 transgenic plants, 3 complementation lines displayed normal leaf color with significant higher total chlorophyll and chlorophyll a and b contents. The ChlD subunit of the mutant was unable to interact with the ChlI subunit, as demonstrated by a yeast two-hybrid assay. The mutation in le led to lethal etiolated phenotype, but no such phenomenon was observed in the mutants. The interaction between the ChlD and ChlI subunits, chlorophyll synthesis, and chloroplast development may all be impacted by the mutation, which could ultimately result in plant death. The mutation in the AAA+ domain of ChlD blocked the interaction of ChlDle with ChlI, leading to the loss of function of ChlD and hindering chlorophyll synthesis and chloroplast development, and ultimately causing lethal etiolated phenotype of the mutant.

A novel genotyping method for rapid identification of the Le gene to select patients for diagnosis with CA19-9

BackgroundThe antigenic determinant of CA19-9 is synthesized by the α1,3/4fucosyltransferase encoded by the Le gene in the Lewis blood group system. Accordingly, a diagnosis with CA19-9 is not appropriate forLe-negative patients who possess the Le gene-mutated le alleles homozygously. MethodsA Le gene-specific PCR was undertaken to determine c59T>G by using a set of tag-sense and biotin-labeled anti-sense primers and a peptide nucleic acid-le-clamp which bound to G59 in the le alleles. Following mixing with streptavidin-coatedbluelatex beads, the PCR products were developed on a strip on which the complementary tag oligonucleotide to theLe gene-specific amplicon was immobilized. ResultsWhen the PCR products were developed on the strip, a clear line was rapidly observed in Le-positive but not in Le-negative individuals. In contrast, a significant number of cancer patients with Lewis-negative phenotype were found to possess CA19-9, while they were specifically genotyped asLe/-. No contradictory results were observed in cancer patients (n = 315) with respect to their Lewis genotypes and CA19-9 levels. Conclusionsc59T>G occurred commonly in the le alleles could be specifically and rapidly identified by the present method. This method appeared to be relevant forselecting cancer patientsto bediagnosed with CA19-9.

Quantitative trait loci for stem strength properties and lodging in two pea biparental mapping populations

AbstractLodging is the major factor that determines whether pea (Pisum sativum L.) can be mechanically harvested. The aim of this experiment was to investigate the relationship between stem mechanical parameters (diameter, stem wall thickness, stiffness [rigidity], and maximum self‐weight moment [stem strength]) and lodging in pea. Three years of data on stem mechanical parameters and 2 yr of data on lodging resistance were collected on the ‘Carneval’ × ‘MP1401’ pea cultivars and Wt10245 × Wt11238 lines. Mechanical parameters were measured separately for three stem sections: lower, middle, and upper. Nine quantitative trait loci (QTL) associated with stem mechanical parameters were identified in the Carneval × MP1401 genetic map. Eight QTL associated with stem mechanical parameters were identified in the Wt10245 × Wt11238 genetic map. These results confirmed QTL for lodging resistance found previously, especially in regards to located in chr5LG3 close to the A004 marker. Quantitative trait loci for stem diameter and stem strength of the upper and lower stem colocated with the Rfs marker in chr5LG3. A plant height QTL was detected close to AB141 marker, 2 cM (3.3 Mbp) from the Le gene. These results enable identification of pea genomic regions associated with agriculturally important traits and provide a foundation for further research to improve lodging resistance in pea.

Identification and Characterization of LARGE EMBRYO, a New Gene Controlling Embryo Size in Rice (Oryza sativa L.)

BackgroundAlthough embryo accounts for only 2–3% of the total weight of a rice grain, it is a good source of various nutrients for human health. Because enlarged embryo size causes increase of the amount of nutrients and bioactive compounds stored within rice grain, giant embryo mutants of rice (Oryza sativa L.) are excellent genetic resources for improving the nutritional value of rice grains.ResultsThree giant embryo mutants, including large embryo (le), giant embryo (ge) and super-giant embryo (ges), with variable embryo size were used in this study. We investigated whether genes controlling embryo size in these mutants (le, ge and ges) were allelic to each other. Although ge and ges was allelic to GIANT EMBRY (GE), le was not allelic to ge and ges in allelism test. The GE gene carried a unique nucleotide substitution in each of the two mutants (ge and ges), resulting in non-synonymous mutations in exon 2 of GE in both mutants. However, the GE gene of the le mutant did not carry any mutation, suggesting that the enlarged embryo phenotype of le was governed by another gene. Using map-based cloning, we mapped the LE gene to the short arm of chromosome 3. The le mutant showed mild enlargement in embryo size, which resulted from an increase in the size of scutellar parenchyma cells. The LE encodes a C3HC4-type RING finger protein and was expressed to relatively high levels in seeds at a late developmental stage. Knockdown of LE expression using RNA interference increased the embryo size of rice grains, confirming the role of LE in determining the embryo size.ConclusionOverall, we identified a new gene controlling embryo size in rice. Phenotypic and molecular characterization results suggest that the le mutant will serve as a valuable resource for developing new rice cultivars with large embryos and nutrient-dense grains.

Regulation of the gibberellin pathway by auxin and DELLA proteins.

The synthesis and deactivation of bioactive gibberellins (GA) are regulated by auxin and by GA signalling. The effect of GA on its own pathway is mediated by DELLA proteins. Like auxin, the DELLAs promote GA synthesis and inhibit its deactivation. Here, we investigate the relationships between auxin and DELLA regulation of the GA pathway in stems, using a pea double mutant that is deficient in DELLA proteins. In general terms our results demonstrate that auxin and DELLAs independently regulate the GA pathway, contrary to some previous suggestions. The extent to which DELLA regulation was able to counteract the effects of auxin regulation varied from gene to gene. For Mendel's LE gene (PsGA3ox1) no counteraction was observed. However, for another synthesis gene, a GA 20-oxidase, the effect of auxin was weak and in WT plants appeared to be completely over-ridden by DELLA regulation. For a key GA deactivation (2-oxidase) gene, PsGA2ox1, the up-regulation induced by auxin deficiency was reduced to some extent by DELLA regulation. A second pea 2-oxidase gene, PsGA2ox2, was up-regulated by auxin, in a DELLA-independent manner. In Arabidopsis also, one 2-oxidase gene was down-regulated by auxin while another was up-regulated. Monitoring the metabolism pattern of GA(20) showed that in Arabidopsis, as in pea, auxin can promote the accumulation of bioactive GA.

Smoking and serum CA19‐9 levels according to Lewis and secretor genotypes

CA19-9, a marker for cancers of biliary tract, pancreas and colorectum, is not synthesized in those with no enzyme activity genotype (le/le) of Lewis (Le) gene. No enzyme activity genotype (se/se) of secretor (Se) gene is known to have an association with high serum CA19-9 levels. There are also variations in serum CA19-9 levels independent of the genotypes. This study aimed to examine the associations of serum CA19-9 levels with smoking, alcohol drinking and body mass index (BMI; kg/m(2)), after the adjustments of Le and Se genotypes. Subjects were 486 health check-up examinees (158 males and 328 females) aged from 39 to 90 years in Hokkaido, Japan. Genotyping was conducted for 3 polymorphisms; Le T59G (59T for Le allele and 59G for le allele), Se A385T (385A for Se allele and 385T for sej allele), and Se pseudogene (se5 allele). The genotypes of Le and Se were deterministic factors of serum CA19-9. Those with Le/Le & se/se had the highest mean, while CA19-9 was not detected or very low in those with le/le. Although no associations were observed with alcohol drinking and BMI, a significant association was observed with smoking. Among those with Le/Le, the geometric mean of CA19-9 was significantly lower for current smokers than for noncurrent smokers (p = 0.011 in 4-way ANOVA with age, sex and Se genotype). When hemoglobin A1c was further adjusted, the association became stronger (p = 0.0027). In addition to polymorphic variations, some components of cigarette smoke may influence the production or destruction of CA19-9.

Development of two sequence-specific PCR markers linked to the le gene that reduces pod shattering in narrow-leafed Lupin (Lupinus angustifolius L.)

Wild types of narrow-leaf lupin (Lupinus angustifolius L.) have seed pods that shatter upon maturity, leading to the loss of their seeds before or during the harvest process. Two recessive genes have been incorporated into domesticated cultivars of this species to maximize harvest-ability of the produce. One of these genes is called lentus (le). Two microsatellite - anchored fragment length polymorphism (MFLP) candidate markers were identified as closely linked to the le gene in a recombinant inbred line (RIL) population derived from a domesticated x wild type cross. The candidate MFLP markers were isolated from the gel, re-amplified by PCR, cloned and sequenced. The MFLP polymorphisms were converted into sequence-specific PCR-based markers. Linkage analysis by MapManager indicated that one of the markers, LeM1, was 2.6 centiMorgans (cM) and the other, LeM2, was 1.3 cM from the gene, with both being on the same side. The correlation between the marker genotype and the plant phenotype for the le gene is 95% for the Australian cultivars, and approximately 36% on wild types tested. These markers may be useful in marker assisted selection for the le gene when introgressing wild material into lupin breeding programs.

Hormone interactions and regulation of Unifoliata, PsPK2, PsPIN1 and LE gene expression in pea (Pisum sativum) shoot tips.

The Unifoliata (Uni) gene plays a major role in development of the compound leaf in pea, but its regulation is unknown. In this study, we examined the effects of plant hormones on the expression of Uni, PsPK2 (the gene for a pea homolog of Arabidopsis PID, a regulator of PIN1 targeting), PsPIN1 (the major gene for a putative auxin efflux carrier) and LE (a gibberellin biosynthesis gene, GA3ox), and also examined mutual hormonal regulation of these genes, in pea shoot tips, including a number of mutants. The Uni promoter possessed putative auxin and gibberellin response elements. The PsPIN1 mRNA levels were increased in afila, which replaces leaflets with branched tendrils; and reduced in tendrilless, which replaces tendrils with leaflets, compared with the wild type (WT). In contrast, mRNA levels of LE were increased in uni and tendrilless and decreased in afila compared with the WT. Uni, PsPK2 and PsPIN1 are positively regulated by gibberellin and auxin, and were induced to higher levels by simultaneous application of auxin and gibberellin. Auxin induction of Uni, PsPK2 and PsPIN1 did not require de novo protein synthesis. LE was positively regulated by auxin and cytokinin. In conclusion, these results support the hypothesis that auxin and gibberellin positively regulate Uni, which controls pea compound leaf development. Also, Uni, PsPIN1, PsPK2 and LE are expressed differentially in the leaf mutants, suggesting that mutual regulation by auxin and gibberellin promotes compound leaf development.

Identification of internal sequences in the 3' leader region of human respiratory syncytial virus that enhance transcription and confer replication processivity.

Previous studies of respiratory syncytial virus have shown that the 44-nucleotide (nt) leader (Le) region is sufficient to initiate RNA replication, producing antigenome RNA, and that the Le and adjoining gene start (GS) signal of the first gene are sufficient to initiate transcription, producing mRNA. A cis-acting element necessary for both transcription and replication was mapped within the first 11 nt at the 3' end of Le. In the present study the remainder of the Le region was mapped to identify sequences important for transcription and replication. A series of minigenomes with mutant Le sequences was generated, and their ability to direct transcription and replication was determined by Northern blot analysis, which examined full-length antigenome and mRNA, and by primer extension analysis, which examined antigenome and mRNA initiation. With regard to transcription, nt 36 to 43, located immediately upstream of the GS signal, were found to be necessary for optimal levels of mRNA synthesis, although the GS signal in conjunction with the 3'-terminal region of Le was sufficient to direct accurate mRNA synthesis initiation. With regard to replication, the first 15 nt of Le were found to be sufficient to direct initiation of antigenome synthesis, but nt 16 to 34 were required in addition for efficient encapsidation and production of full-length antigenome. Analysis of transcripts produced from di- and tricistronic minigenomes indicated that a significant proportion of abortive replicases continue RNA synthesis to the end of the first gene and then continue in a transcription mode along the remainder of the genome.

Association of Lewis and Secretor gene polymorphisms and Helicobacter pylori seropositivity among Japanese-Brazilians.

Secretor ( Se) and Lewis ( Le) genes are involved in the synthesis of Lewis b (Le(b)) and type I antigens throughout the body, especially in the epithelial cells of gastric mucosa. Helicobacter pylori can attach to the gastric epithelial cells with the blood group antigen-binding adhesin, which binds to Le(b) or H type I carbohydrate structures. In a previous study, a marked association between H. pylori seropositivity and polymorphism of the Se and Le genes was observed among Japanese outpatients of a gastroenterology clinic. The present work aims to investigate the associations between Se and Le gene polymorphisms and H. pylori infection among Japanese-Brazilians. The subjects consisted of 942 healthy volunteer Japanese-Brazilians, who were tested for the presence of anti- H. pylori IgG antibodies and genotyped for Se and Le polymorphisms. The sex-age-adjusted odds ratios (aORs) for H. pylori seropositivity were 0.99 for the Sese genotype relative to the SeSe genotype (95% confidence interval [CI], 0.73-1.33), and 1.03 for sese relative to SeSe (95% CI, 0.71-1.48). On the other hand, the aOR for the subjects with the le allele ( Lele or lele) relative to the LeLe genotype was 1.48 (95% CI, 1.07-1.79). When the Se and Le genotypes were analyzed in combination according to risk group, no statistically significant association was observed. These results are inconsistent with previous work and may have been modulated by an external factor or some other unidentified factor. Japanese-Brazilians are genotypically the same as Japanese, but their lifestyle is adapted to that of Brazil. Further investigations are necessary to clarify this influence on susceptibility to H. pylori infection.

The Roles of the NANA and LEPIDA Genes in Regulating the Stem Growth in Arabidopsis thaliana

Genetic, physiological, and morphological studies of dwarf mutants of Arabidopsis thaliana (L.) Heynh. from the collection of the Department of Genetics and Breeding, Moscow State University, showed that the NA and LE genes are involved in regulating elongation of internode cells and sensitivity to various hormones. The na mutation suppressed stem growth only in the presence of the active LE gene. The absence of the LE activity (in the lele homozygote) restored stem growth of the na mutant to the level characteristic of the le-2 mutant, and a decrease in LE activity (in LEle heterozygote) almost completely suppressed the na phenotype. Phenotypic analysis of homozygous double mutants and heterozygotes obtained by crossing the na and le-2 mutants showed that the recessive le-2 allele has an epistatic effect on the semidominant na allele and that the genes possibly control consecutive steps of one biochemical pathway or one morphogenetic process. A hypothetical scheme was proposed for the interaction of the NA and LE gene products.

Smoking and polymorphisms of fucosyltransferase gene Le affect success of H. pylori eradication with lansoprazole, amoxicillin, and clarithromycin.

Identification of factors influencing success of Helicobacter pylori (HP) eradication is important for clinical practice. We have prospectively conducted an HP eradication study in the Aichi Cancer Center with a total of 142 patients available for analysis. The overall success rate was 61.3% (95% confidence interval 52.7-69.3%). Smoking during the medication for eradication significantly decreased the success rate (42.9%), whereas smoking cessation during the treatment was associated with a similar rate as for non-smokers (66.7%). We also examined links between an eradication outcome and polymorphisms of Le, Se, IL1A, IL1B, IL1RN and MPO genes, but with one exception none showed any association. The non-functional le allele of Le polymorphisms, leading to decreased expression of Le(b) antigen to which HP attaches with adhesin, showed a beneficial effect for success. Although further clarification is necessary, our study indicated that smoking cessation and Le gene polymorphisms may affect the success rate of HP eradication.

Auxin-Gibberellin Interactions in Pea: Integrating the Old with the New

Recent findings on auxin-gibberellin interactions in pea are reviewed, and related to those from studies conducted in the 1950s and 1960s. It is now clear that in elongating internodes, auxin maintains the level of the bioactive gibberellin, GA1, by promoting GA1 biosynthesis and by inhibiting GA1 deactivation. These effects are mediated by changes in expression of key GA biosynthesis and deactivation genes. In particular, auxin promotes the step GA20 to GA1, catalyzed by a GA 3-oxidase encoded by Mendel’s LE gene. We have used the traditional system of excised stem segments, in which auxin strongly promotes elongation, to investigate the importance for growth of auxin-induced GA1. After excision, the level of GA1 in wild-type (LE) stem segments rapidly drops, but the auxin indole-3-acetic acid (IAA) prevents this decrease. The growth response to IAA was greater in internode segments from LE plants than in segments from the le-1 mutant, in which the step GA20 to GA1 is impaired. These results indicate that, at least in excised segments, auxin partly promotes elongation by increasing the content of GA1. We also confirm that excised (light-grown) segments require exogenous auxin in order to respond to GA. On the other hand, decapitated internodes typically respond strongly to GA1 application, despite being auxin-deficient. Finally, unlike the maintenance of GA1 content by auxin, other known relationships among the growth-promoting hormones auxin, brassinosteroids, and GA do not appear to involve large changes in hormone level.

Control of gibberellin levels and gene expression during de-etiolation in pea.

Gibberellin A(1) (GA(1)) levels drop significantly in wild-type pea (Pisum sativum) plants within 4 h of exposure to red, blue, or far-red light. This response is controlled by phytochrome A (phyA) (and not phyB) and a blue light receptor. GA(8) levels are increased in response to 4 h of red light, whereas the levels of GA(19), GA(20), and GA(29) do not vary substantially. Red light appears to control GA(1) levels by down-regulating the expression of Mendel's LE (PsGA3ox1) gene that controls the conversion of GA(20) to GA(1), and by up-regulating PsGA2ox2, which codes for a GA 2-oxidase that converts GA(1) to GA(8). This occurs within 0.5 to 1 h of exposure to red light. Similar responses occur in blue light. The major GA 20-oxidase gene expressed in shoots, PsGA20ox1, does not show substantial light regulation, but does show up-regulation after 4 h of red light, probably as a result of feedback regulation. Expression of PsGA3ox1 shows a similar feedback response, whereas PsGA2ox2 shows a feed-forward response. These results add to our understanding of how light reduces shoot elongation during de-etiolation.

Control of Gibberellin Levels and Gene Expression during De-Etiolation in Pea

Gibberellin A1 (GA1) levels drop significantly in wild-type pea (Pisum sativum) plants within 4 h of exposure to red, blue, or far-red light. This response is controlled by phytochrome A (phyA) (and not phyB) and a blue light receptor. GA8 levels are increased in response to 4 h of red light, whereas the levels of GA19, GA20, and GA29 do not vary substantially. Red light appears to control GA1 levels by down-regulating the expression of Mendel's LE (PsGA3ox1) gene that controls the conversion of GA20 to GA1, and by up-regulating PsGA2ox2, which codes for a GA 2-oxidase that converts GA1 to GA8. This occurs within 0.5 to 1 h of exposure to red light. Similar responses occur in blue light. The major GA 20-oxidase gene expressed in shoots, PsGA20ox1, does not show substantial light regulation, but does show up-regulation after 4 h of red light, probably as a result of feedback regulation. Expression of PsGA3ox1 shows a similar feedback response, whereasPsGA2ox2 shows a feed-forward response. These results add to our understanding of how light reduces shoot elongation during de-etiolation.

Lewis and Secretor Gene Effects on Lewis Antigen and Postnatal Development of Lewis Blood Type

Lewis (Le) and Secretor (Se) gene effects on postnatal change in Lewis antigens are described based on the quantitative analysis of Lewis antigens in blood. Genuine Lewis blood types were determined by Le and Se genotyping in DNA samples obtained from umbilical cord blood and infant blood. Lewis^a (Le^a) and Lewis^b (Le^b) antigen levels were measured by a time-resolved fluoroimmunoassay. We found that Lewis phenotypes in infants over 9 months of age agreed with the genuine types. In cord blood, the dosage effect of the Se gene on antigen levels was observed, but the Le gene effect was not observed. Both Se and Le gene effects were observed in newborn babies’ blood 3–6 days after birth.

Evidence that auxin promotes gibberellin A1 biosynthesis in pea.

In shoots of the garden pea, the bioactive gibberellin (GA1) is synthesised from GA20, and the enzyme which catalyses this step (a GA 3-oxidase -- PsGA3ox1) is encoded by Mendel's LE gene. It has been reported previously that decapitation of the shoot (excision of the apical bud) dramatically reduces the conversion of [3H]GA20 to [3H]GA1 in stems, and here we show that endogenous GA1 and PsGA3ox1 transcript levels are similarly reduced. We show also that these effects of decapitation are completely reversed by application of the auxin indole-3-acetic acid (IAA) to the 'stump' of decapitated plants. Gibberellin A20 is also converted to an inactive product, GA29, and this step is catalysed by a GA 2-oxidase, PsGA2ox1. In contrast to PsGA3ox1, PsGA2ox1 transcript levels were increased by decapitation and reduced by IAA application. Decapitation and IAA treatment did not markedly affect the level of GA1 precursors. It is suggested that in intact pea plants, auxin from the apical bud moves into the elongating internodes where it (directly or indirectly) maintains PsGA3ox1 transcript levels and, consequently, GA1 biosynthesis.

Plasma α1,3-Fucosyltransferase Deficiency in Schizophrenia

Levels of plasma α1,3-fucosyltransferase (α1,3FT) were assayed in 44 patients with schizophrenia and in 50 healthy controls. Significantly reduced enzyme activities were observed in patients (p < 0.05) and 4 unrelated patients were found, for the first time in Japan, to be deficient in the enzyme activity. Two point mutations in the coding region of the FUT6 gene encoding plasma α1,3FT that were responsible for the inactivation of the enzyme activity were detected in those patients. Genotyping of the Le gene (FUT3) in these patients demonstrated that 2 of them were also FUT3 deficient and were grouped as Lewis– individuals whereas the rest were Lewis+.

The influence of the null le-2 mutation on gibberellin levels in developing pea seeds

The Le gene of pea encodes a gibberellin 3β-hydroxylase. Heterologous expression of the le-2 allele indicated that a truncated protein was produced, confirming that le-2 is a null mutation. The Le expression product was unable to metabolise GA12, but was able to produce a small quantity of GA8 from GA29. The le-2 mutation had no effect on the levels of GA1, GA4 or GA8 in developing seeds. Measurements of mRNA levels indicate that the Le gene is only weakly expressed in young pea seeds. These results explain why mutant alleles at the Le locus have no major impact on seed development, even though 3β-hydroxylated GAs are essential for normal seed development in pea. Rather, a second 3β-hydroxylase, with a different substrate specificity, may be expressed in young seeds, resulting in a different biosynthetic pathway leading to the biologically active GAs.

Senescence-induced RNases in tomato.

A main feature of leaf senescence is the hydrolysis of macromolecules by hydrolases of various types, and redistribution of released materials. We have initiated a study for the characterization of RNases involved in nucleic acid catabolism during senescence. Using a PCR-based cloning approach we isolated from tomato two senescence-induced RNase cDNA clones. Each of these cDNAs hybridized to a senescence-induced transcript in northern analysis. One RNase cDNA was identical to the tomato LX RNase while the second corresponded to the LE RNase. Both LX and LE RNase genes had originally been demonstrated to be induced after phosphate starvation of tomato cell culture but nothing was known about their expression or function in plants. We observed that the expression of the LX and LE genes is induced in leaves during an advanced stage of senescence with the LX transcript level being much more induced than that of LE. Low-level expression of the RNase genes was observed in flowers and artificially senescing detached leaves while no expression could be detected in stems, roots, or fruits at different ripening stages. Ethylene activated the LX gene expression in detached young leaves while LE gene expression, which could be transiently induced by wounding, appeared to be activated by abscisic acid. We suggest that the LX RNase has a role in RNA catabolism in the final stage of senescence, and LE may function during wounding as a plant defense protein.