Biochemical and molecular characterization of a rice glutelin allele for the GluA-1 gene

The rice ( Oryza sativa L.) mutant of glu4a, lacking the glutelin alpha-2 subunit while the alpha-1 subunit increased (alpha-1H/alpha-2L), was used in this study. Two-dimensional electrophoresis analysis revealed that the mutant lacked the polypeptide pI6.71/alpha-2 encoded by glu4 while forming a new polypeptide of pI6.50/alpha-1. Experiments were conducted to identify the relationships between the mutated polypeptides of the mutant and to illustrate the mutation mechanism of the allele. Peptide mapping and amino-acid sequence analyses revealed that the newly formed glu4a encoded polypeptide pI6.50/alpha-1 of high homology with the deleted pI6.71/alpha-2 polypeptide which was encoded by glu4 (GluA-1). The nucleotide sequence revealed that the iso-electric point variation of the pI6.50/alpha-1 polypeptide was caused by a point mutation with nucleotide replacement at the variable region of the gene. These results suggested the possibility of altering glutelin quality by using single gene mutation.     

Inheritance of alleles for glutelin alpha-2 subunit genes in rice and identification of their corresponding cDNA clone

Rice glutelins consist of acidic (alpha) and basic (beta) subunits which are further separated into three polypeptide components assigned as alpha-1, alpha-2, and alpha-3 subunit components and beta-1, beta-2 and beta-3 subunit components. Nine rice mutant lines with a decreased amount of the glutelin alpha-2 subunit component (alpha-2L) were obtained by screening about 6,800 potential mutant lines derived from the fertilized egg treatment with N-methyl-N-nitrosourea (MNU) using the SDS-PAGE method. The mutants were classified into three types of the increased alpha-1 subunit (alpha-1H/alpha-2L), the decreased beta-2 subunit (beta-2L/alpha-2L) and the increased alpha-3 subunit (alpha-3H/alpha-2L) represented by EM278, CM1707 and EM659, respectively. Iso-electric focus (IEF) analysis revealed that all of the mutants had an extremely low amount of a polypeptide with a 6.71 pI value, whereas a polypeptide with either a 6.50 pI value or with a 6.90 pI value increased significantly in alpha-1H/alpha-2L mutants or in alpha-3H/alpha-2L mutants, respectively. The beta-2L/alpha-2L mutants had a decreased amount of a basic polypeptide with a 8.74 pI value. Genetic analysis revealed that the three types of mutants were controlled by a single incomplete dominant gene respectively, and the three are alleles. The gene was temporarily named glu4, which was found to be located on chromosome 1 linked with the eg and spl6 genes. Two-dimensional electrophoresis analysis revealed that the glu4 encoded polypeptides of pI 6.71/alpha-2 and pI 8.74/beta-2. Amino acid sequence analysis suggested that the mutated acidic polypeptide was the product of a GluA subfamily gene. Northern and RT-PCR analyses revealed that glu4 corresponded to the GluA-1 gene.     

Mutants lacking glutelin subunits in rice: mapping and combination of mutated glutelin genes

Nine mutant lines lacking glutelin subunits were selected from M₂ seeds of about 10000 M₁ plants mutagenized with gamma rays or EMS and from 1400 mutant lines selected originally for morphological characters. There were three types of mutants, one line lacking the largest subunit among four minor bands of glutelin acidic subunits (Type 1), five lines lacking the second largest subunit band (Type 2), and three lines lacking the third largest subunit band (Type 3). Mutants lacking the smallest subunit band were not found. Type 1 lacked 2 of the 10 spots of glutelin acidic subunits separated by two-dimensional electrophoresis and 1 of the 11 spots of the 57-kDa glutelin precursor. Type 2 lacked 2 spots of acidic subunits and 1 spot of the 57-kDa glutelin precursor, and had low amounts of 1 of the 8 spots of glutelin basic subunits. Type 3 mutants lacked each of 1 spot of the acidic subunits and glutelin precursor and had low amount of 1 spot of the basic subunits. Genetic analysis of the mutated genes showed that these mutant characters were controlled by single recessive genes named glu-1, glu-2, and glu-3, respectively. Mutated genes of different lines of the same type were found to be at the same locus. RFLP analysis of F₂ plants between the mutant lines and cv `Kasalath' indicated that glu-1 is on chromosome 2, glu-2 on chromosome 10, and glu-3 on chromosome 1. These mutant genes were combined by crossing, and a line lacking the 3 minor bands of the glutelin acidic subunits was developed. However, the total glutelin content of this line was not remarkably reduced, showing a only 13% decrease. Received: 1 April 1996 / Accepted: 14 June 1996     

新的水稻谷蛋白α—1亚基缺失突变体

从水稻受精卵MNU处理后代中获得4个谷蛋白α－1亚基缺失突变品系。SDS－PAGE和IEF分析表明这些突变体在共同缺失1条pI6.82多肽的同时,或形成新的多肽,或其他多肽表现量增加,这些突变体是由结构基因控制的,IEF分析同时显示2条多肽pI6.82和pI8.58源自同一条谷蛋白前驱体。这4个突变体对于改良水稻谷蛋白品质、研究谷蛋白生物合成遗传调控机制以及揭示谷蛋白基因功能是不可多得的研究材料。     

水稻种子贮藏谷蛋白的微细异质性

利用灵敏的等聚焦与SDS－PAGE合的双向电泳分析方法,从水稻（Oryza sativa L.)种子贮藏谷蛋白中至少可以分离为13条酸性和19条碱性多 肽,依据谷蛋白多肽的表现量推测,水稻谷蛋白主要由约6个主效基因控制,肽图谱与N－端氨基酸序列分析可清晰将谷蛋白酸性多肽分为两组,此两组恰好与谷蛋白GluA和GluB两个cDNA克隆组相吻合。     

Association analysis of the glutelin synthesis genes <Emphasis Type="Italic">GluA</Emphasis> and <Emphasis Type="Italic">GluB1</Emphasis> in a <Emphasis Type="Italic">Japonica</Emphasis> rice collection

Glutelin is the most significant seed storage protein and is regarded as an important nutrient quality trait in rice. Research on the genetic basis of the glutelin content distinction in rice will provide more choices for the diets of people with kidney disease and diabetes. The GluA and GluB1 genes play important roles in the process of glutelin synthesis. In this study, 128 Japonica rice accessions with wide geographic distributions were collected to construct the association panel. Among all the 128 accessions, both sequences of the GluA and GluB1 genes were obtained, and nucleotide polymorphisms were detected. A total of 46 SNPs and eight InDels, six SNPs and four InDels were found in the GluA and GluB1 gene sequences, respectively. Eight haplotypes and two haplotypes were classified based on the SNPs in the coding region of the GluA and GluB1 genes, respectively. Moreover, the association of the polymorphic sites in the two genes with glutelin content in the tested population was estimated. The results revealed that five SNPs in the GluA gene, one SNP and one InDel in the GluB1 gene were associated with glutelin content at a significant level (P < 0.01). Corresponding markers were also designed to check the alleles of GluA and GluB1 genes. These results suggested that polymorphisms in the GluA and GluB1 genes in rice could be utilized in molecular marker-assisted selection to improve the nutrient quality of rice breeding programmes.     

The correlation between expression and localization of a foreign gene product in rice endosperm

Glucagon-like peptide 1 (GLP-1) is a 30 amino acid peptide hormone involved in insulin stimulation that is dependent upon blood glucose levels. We have previously reported that when this short peptide gene was directly expressed under the control of a glutelin promoter and its signal peptide, it was not accumulated in transgenic rice seed due to gene silencing. However, when the modified GLP-1 (mGLP-1) gene was enlarged to 5xmGLP-1 (mGLPx5) by tandem repeat, no silencing was observed. The mGLPx5 peptide could be accumulated in rice seed and its localization was mainly limited to the endoplasmic reticulum (ER). We also investigated alternative cellular localization sites that would increase accumulation. The relationship between the expression level and localization was examined by attaching the chitinase signal peptide to mGLPx5 to direct it into the intercellular space (apoplast), or by expression as a fusion protein with glutelin by insertion into a variable region of the acidic subunit, thus directing the peptide to protein body II (PB II). Attachment of the KDEL ER retention signal to the 6xmGLP-1 (mGLPx6) or its fusion to the C-terminus of the 13 kDa prolamin directed the peptide to the ER or PB I, respectively. Unexpectedly, these results indicated that mGLPx5 without any signal except for the glutelin signal peptide was accumulated to the greatest extent in rice endosperm. It can thus be concluded that the ER is a suitable intracellular organelle for accumulation of mGLPx5 peptide.     

Development of PCR markers to detect the glb1 and Lgc1 mutations for the production of low easy-to-digest protein rice varieties

Limiting the ingestion of protein is the fundamental idea in the diet therapy for patients with chronic renal failure. Two mutations involved in the content of major rice storage proteins useful for developing low easy-to-digest protein rice variety have been isolated. The glb1 mutation causes the deficiency of α-globulin, and the Lgc1 mutation reduces the glutelin content. By combining the glb1 and the Lgc1 mutations, it is possible to reduce the easy-to-digest protein content by approximately 50%. The Lgc1 mutation has been shown to be caused by a 3.5-kb deletion between the glutelin structural genes, GluB4 and GluB5, while the molecular basis of glb1 mutation has been less understood. PCR analysis of the glb1 mutation revealed a 62.8-kb deletion, including the structural gene of α-globulin. Based on these lines of information, we generated PCR markers that make it possible to detect the glb1 and Lgc1 mutations. Using those PCR markers, we genotyped F₂ plants segregating for the glb1 mutation and the Lgc1 mutation and confirmed the consistency of genotype and phenotype. Because the PCR marker sets can distinguish heterozygotes, they will be very useful in developing new varieties of low easy-to-digest protein rice.     

Expansion and contraction of the DUP240 multigene family in Saccharomyces cerevisiae populations

The influence of duplicated sequences on chromosomal stability is poorly understood. To characterize chromosomal rearrangements involving duplicated sequences, we compared the organization of tandem repeats of the DUP240 gene family in 15 Saccharomyces cerevisiae strains of various origins. The DUP240 gene family consists of 10 members of unknown function in the reference strain S288C. Five DUP240 paralogs on chromosome I and two on chromosome VII are arranged as tandem repeats that are highly polymorphic in copy number and sequence. We characterized DNA sequences that are likely involved in homologous or nonhomologous recombination events and are responsible for intra- and interchromosomal rearrangements that cause the creation and disappearance of DUP240 paralogs. The tandemly repeated DUP240 genes seem to be privileged sites of gene birth and death.     

Parental somatic and germ-line mosaicism for a multiexon deletion with unusual endpoints in a type III collagen (COL3A1) allele produces Ehlers-Danlos syndrome type IV in the heterozygous offspring.

Ehlers-Danlos syndrome (EDS) type IV is a dominantly inherited disorder that results from mutations in the type III collagen gene (COL3A1). We studied the structure of the COL3A1 gene of an individual with EDS type IV and that of her phenotypically normal parents. The proband was heterozygous for a 2-kb deletion in COL3A1, while her father was mosaic for the same deletion in somatic and germ cells. In fibroblasts from the father, approximately two-fifths of the COL3A1 alleles carried the deletion, but only 10% of the COL3A1 alleles in white blood cells were of the mutant species. The deletion in the mutant allele extended from intron 7 into intron 11. There was a 12-bp direct repeat in intron 7 and intron 11, the latter about 60 bp 5' to the junction. At the breakpoint there was a duplication of 10 bp from intron 11 separated by an insertion of 4 bp contained within the duplicated sequence. The father was mosaic for the deletion so that the gene rearrangement occurred during his early embryonic development prior to lineage allocation. These findings suggest that at least some of the deletions seen in human genes may occur during replication, rather than as a consequence of meiotic crossing-over, and that they thus have a risk for recurrence when observed de novo.     

Short 5'-flanking regions of the Amy gene of Drosophila kikkawai affect amylase gene expression and respond to food environments

Evolution of the duplicated genes and regulation in gene expression is of great interest, especially in terms of adaptation. Molecular population genetic and evolutionary studies on the duplicated amylase genes of Drosophila species have suggested that their 5'-flanking (cis-regulatory) regions play an important role in evolution of these genes. For better understanding of evolution of the duplicated amylase genes and gene expression, we studied functional significance of the Amy1 gene of Drosophila kikkawai using in vitro deletion mutagenesis followed by P-element-mediated germline transformation. We found that a 1.6-kb of the 5'-flanking region can produce strikingly higher level of larval amylase activity on starch food compared with that on glucose food. We found two cis-regulatory elements, which increase larval amylase activity on starch food. We also found a larval cis-regulatory element, which responds to the food difference. This food-response element is necessary for the function of the element increasing larval activity on starch food. A 5-bp deletion in a putative GRE caused high amylase activity, indicating a cis-regulatory element decreasing amylase activity. These cis-regulatory elements identified in the 5'-flanking region could be the targets of natural selection.     

Two types of FtsH protease subunits are required for chloroplast biogenesis and Photosystem II repair in Arabidopsis

FtsH protease is important in chloroplast biogenesis and thylakoid maintenance. Although bacteria contain only one essential FTSH gene, multiple genes exist in cyanobacteria and higher plants. However, the functional significance of FTSH multiplication in plants is unclear. We hypothesized that some FTSH genes may be redundant. To test this hypothesis, we generated double mutant combinations among the different FTSH genes in Arabidopsis thaliana. A double mutant of ftsh1 and ftsh8 showed no obvious phenotypic alterations, and disruption of either FTSH1 or FTSH5 enhanced the phenotype of the ftsh2 mutant. Unexpectedly, new phenotypes were recovered from crosses between ftsh2 and ftsh8 and between ftsh5 and ftsh1, including albinism, heterotrophy, disruption of flowering, and severely reduced male fertility. These results suggest that the duplicated genes, FTSH1 and FTSH5 (subunit type A) and FTSH2 and FTSH8 (subunit type B), are redundant. Furthermore, they reveal that the presence of two types of subunits is essential for complex formation, photosystem II repair, and chloroplast biogenesis.     

Complementation of bacteriophage induction and recombination defects in Escherichia coli RecA(-) mutants by expression of the cloned T4 bacteriophage uvsX gene

Previous workers reported that the T4 bacteriophage UvsX protein could promote neither RecA-LexA-mediated DNA repair nor induction of lysogenized bacteriophage, only recombination. Reexamination of these phenotypes demonstrated that, in contrast to these prior studies, when this gene was cloned into a medium but not a low-copy-number vector, it stimulated both a high frequency of spontaneous induction and mitomycin C-stimulated bacteriophage induction in a strain containing a recA13 mutation, but not a recA1 defect. The gene when cloned into a low- or medium- copy-number vector also promoted a low frequency of recombination of two duplicated genes in Escherichia coli in a strain with a complete recA gene deletion. These results suggest that a narrow concentration range of T4 UvsX protein is required to promote both high-frequency spontaneous and mitomycin C-stimulated bacteriophage induction in a recA13 gene mutant, but it facilitates recombination of duplicated genes at only a very low frequency in E. coli RecA(-) mutants with a complete recA deletion. These results also suggest that the different UvsX phenotypes are affected differentially by the concentration of UvsX protein present.     

小麦近缘种低分子量麦谷蛋白亚基基因Glu-B3克隆及系统发育分析

发掘小麦近缘种低分子量麦谷蛋白基因, 可为小麦品质改良提供更多的基因资源。文章利用Glu-B3位点特异性标记LB1F/LB1R、LB2F/LB2R、LB3F/LB3R和 LB4F/LB4R, 对普通小麦B染色体组的7个可能供体近缘种, 即硬粒小麦(T. durum)、栽培二粒小麦(T. dicoccum)、野生二粒小麦(T. dicoccoides)、拟斯卑尔脱山羊草(Ae. speltoides)、高大山羊草(Ae. longissima)、西尔斯山羊草(Ae. searsii)和双角山羊草(Ae. bicornis)共20份材料进行PCR扩增, 克隆小麦近缘种中GluB3-1、GluB3-2、GluB3-3和GluB3-4基因的等位变异, 并对Glu-B3位点基因进行系统发育分析。共获得16个新等位变异, 其中GluB3-1基因的新等位变异1个, 命名为GluB3-16, 其推导氨基酸分子量为39.2 kDa; GluB3-3的新等位变异有3个, 分别命名为GluB3-35、GluB3-36和GluB3-37, 其推导氨基酸分子量为44.5 kDa(GluB3-36)或44.6 kDa(GluB3-35和GluB3-37); GluB3-4的新等位变异12个, 分别命名为GluB3-46、GluB3-47、GluB3-48、GluB3-49、GluB3-410、GluB3-411、GluB3-412、GluB3-413、GluB3-414、GluB3-415、GluB3-416和GluB3-417, 其推导氨基酸分子量变化在38.6(GluB3-414)~ 42.5 kDa(GluB3-413)之间; 16个新等位变异都包含单一的完整开放阅读框, 具有低分子量麦谷蛋白亚基的典型结构。文章进一步拓展了低分子量麦谷蛋白基因资源, 揭示不同Glu-B3基因的进化过程不完全相同, 为有效地利用小麦近缘种材料和转基因育种提供了新的基因资源。     

Divergence of duplicated genes in maize: evolution of contrasting targeting information for enzymes in the porphyrin pathway

The divergence of sequence and expression pattern of duplicated genes provides a means for genetic innovation to occur without sacrificing an essential function. The cpx1 and cpx2 genes of maize are a singular example of duplicated genes that have diverged by deletion and creation of protein targeting information. The cpx genes encode coproporphyrinogen III oxidase ('coprogen oxidase'), which catalyzes a step in the synthesis of chlorophyll and heme. In plants, this enzyme has been found exclusively in the plastids. The cpx1 and cpx2 genes encode almost identical, catalytically active enzymes with distinctive N-terminal peptide sequences. The cpx1 gene encodes the expected plastid transit peptide, but this region is deleted from the cpx2 gene. While the 5' regions of both messenger RNAs are highly similar, the cpx2 gene has an open-reading frame that could encode a new targeting signal. GFP fused with CPX1 localized to the plastids. In contrast, the GFP fusion with CPX2 did not target plastids and appeared to localize to mitochondria. Both cpx genes are expressed ubiquitously but, based on mutant phenotype, they seem to have discrete biological roles. Seedlings homozygous for a null mutation in the cpx1 gene completely lack chlorophyll and develop necrotic lesions in the light. However, the mutant seedlings and callus cultures will grow in tissue culture in the dark, implying that they retain a capacity to produce heme. We discuss models for the evolution of the cpx genes and possible roles of mitochondrion-localized coprogen oxidase activity in maize.     

Meiotic Recombination between Duplicated Genetic Elements in SACCHAROMYCES CEREVISIAE

We have studied the meiotic recombination behavior of strains carrying two types of duplications of an 18.6-kilobase HIS4 Bam HI fragment. The first type is a direct duplication of the HIS4 Bam HI fragment in which the repeated sequences are separated by Escherichia coli plasmid sequences. The second type, a tandem duplication, has no sequences intervening between the repeated yeast DNA. The HIS4 genes in each region were marked genetically so that recombination events between the duplicated segments could be identified. Meiotic progeny of the strains carrying the duplication were analyzed genetically and biochemically to determine the types of recombination events that had occurred. Analysis of the direct vs. tandem duplication suggests that the E. coli plasmid sequences are recombinogenic in yeast when homozygous. In both types of duplications recombination between the duplicated HIS4 regions occurs at high frequency and involves predominantly interchromosomal reciprocal exchanges (equal and unequal crossovers). The striking observation is that intrachromosomal reciprocal recombination is very rare in comparison with interchromosomal reciprocal recombination. However, intrachromosomal gene conversion occurs at about the same frequency as interchromosomal gene conversion. Reciprocal recombination events between regions on the same chromatid are the most infrequent exchanges. These data suggest that intrachromosomal reciprocal exchanges are suppressed.