Molecular characterization of spontaneous and induced mutations in the three homoeologous waxy genes of Japanese barnyard millet [Echinochloa esculenta (A. Braun) H. Scholz]

Japanese barnyard millet is an important food source in East Asian countries. However its crumbly texture limits desirability and consumption. Controlling amylose level in the endosperm is important to improve the eating quality of the millet. Because it is well known that the waxy gene determines the amylose level in the endosperm, we conducted a molecular analysis of the gene. Segregation analysis revealed that wild-type cultivars had three functional genes while low-amylose cultivars had one. We determined complete sequences of the three homoeologous waxy structural genes, EeWx1, EeWx2 and EeWx3, in a wild-type cultivar. These sequences showed high homology in the exon regions (97 %), and lower homology in the introns (82 %). Two spontaneous mutations were characterized in the low-amylose cultivars. In addition, one induced mutation was found in the fully waxy cultivar, Chojuromochi. Spontaneous mutations are deletions of whole and terminal regions in the EeWx2 and EeWx3 alleles, respectively. The induced mutation is a single-base deletion that led to a premature termination codon in EeWx1. These findings led us to develop useful markers for selecting low-amylose and waxy lines in millet.     

The molecular basis of mutations at the Waxy locus from Amaranthus caudatus L.: evolution of the waxy phenotype in three species of grain amaranth

Polymorphisms at the Waxy locus of Amaranthus caudatus L. collected from a wide range of regions were used to investigate genetic diversity and mutation sites. A comparison of the Waxy locus revealed a very high level of sequence conservation. This result clearly showed low environmental and evolutionary variability in the Waxy gene. We also performed screening to confirm the mutation sites in the coding sequences of all accessions. The results indicate that one insertion in the coding region of Waxy genes was responsible for the change in perisperm starch leading to the waxy phenotype in all accessions of this species, and thus that a single mutation event altered the regulation of the Waxy gene during the domestication of this crop. In addition, phylogenetic analysis showed that waxy phenotypes within each of three species, A. caudatus, A. cruentus and A. hypochondriacus, originated separately or differentiated from nonwaxy phenotypes of each species through a single mutational event (i.e., a frame shift or base substitution). We also compared obvious structural features of the coding sequence of waxy and nonwaxy phenotypes with those of low-amylose phenotypes in A. caudatus. The Waxy coding sequences of low-amylose phenotypes do not show polymorphisms and are identical with those of waxy phenotypes. This could mean that there is another gene that encodes a key enzyme responsible for amylose synthesis as the elementary quantity in tissues other than perisperm in A. caudatus.     

Molecular characteristics of two new <Emphasis Type="Italic">waxy</Emphasis> mutations in China waxy maize

The waxy gene mutation causes waxy maize grain to have a sticky quality. China has numerous waxy maize landraces and is thought to be the place of origin of waxy maize. The most abundant waxy maize resources in China are located in the Yunnan province and its surrounding areas. We collected 57 waxy maize landraces from Yunnan province and cloned and sequenced the waxy gene from its fourth to eighth exon. Two new waxy gene mutations, named wx-Cin4 and wx-124, were identified. The wx-Cin4 mutation is a 466-bp retrotransposon inserted into exon six. The wx-124 mutation is a 116-bp miniature inverted-repeat transposable element inserted into exon seven. This is the first time a 124-type mutation has been found in a maize waxy gene. The discovery of the two specific waxy mutations from landraces collected in Yunnan province provides new evidence supporting the hypothesis that China is the origin area for waxy maize.     

A 25 bp-long insertional mutation in the BmVarp gene causes the waxy translucent skin of the silkworm,Bombyx mori

In Bombyx mori, there are more than 35 mutant strains whose larval skin color is transparent. The waxy translucent strain ow is one of the oily mutants which lack accumulation of uric acid in the epidermis. Here we performed positional cloning of the ow gene using the Bombyx draft genome sequence. For fine structure mapping, we succeeded to narrow the ow linked region to approximately 150 kb, and identified the ow candidate gene by annotation analysis and DNA sequencing. The complete cDNA sequences of the ow gene from wild-type strains were 3501 bp-long and potentially encoded a protein of 920 amino acids. We found a 25 bp-long insertion in this gene in the ow mutant strain, resulting in a frame-shift mutation and generation of a premature stop codon. A BLAST search revealed that this protein had high homology to Varp, a recently identified protein containing a vacuolar sorting protein 9 domain and ankyrin repeats, and we termed the silkworm protein BmVarp. Varp has been shown to regulate endosome dynamics, suggesting that BmVarp may play an important role in the incorporation and/or accumulation of uric acid in the epidermis.     

Identification and Properties of the Genes Encoding Microcin E492 and Its Immunity Protein

The gene coding for the immunity protein (mceB) and the structural gene of microcin E492 (mceA), a low-molecular-weight channel-forming bacteriocin produced by a strain of Klebsiella pneumoniae, have been characterized. The microcin gene codes for a precursor protein of either 99 or 103 amino acids. Protein sequencing of the N-terminal region of microcin E492 unequivocally identified this gene as the microcin structural gene and indicated that this microcin is synthesized as a precursor protein that is cleaved at either amino acid 15 or 19, at a site resembling the double-glycine motif. The gene encoding the 95-amino-acid immunity protein (mceB) was identified by cloning the DNA segment that encodes only this polypeptide into an expression vector and demonstrating the acquisition of immunity to microcin E492. As expected, the immunity protein was found to be associated with the inner membrane. Analysis of the DNA sequence indicates that these genes belong to the same family as microcin 24, and they do not share structural motifs with any other known channel-forming bacteriocin. The organization of the microcin- and immunity protein-encoding genes suggests that they are coordinately expressed.     

Molecular characterisation of the Wx-B1 allelic variants identified in cultivated emmer wheat and comparison with those of durum wheat

Emmer wheat is a neglected crop that could be used in the breeding of modern durum wheat for quality, one important aspect of which is the starch composition that is related to the waxy proteins. A collection of 87 accessions of Spanish emmer wheat was analysed for waxy protein composition by SDS?CPAGE. No polymorphism was found for the Wx-A1 gene. However, for the Wx-B1 gene, three alleles were detected, two of them new. The whole gene sequence of these alleles was amplified by PCR in three fragments, which were digested with several endonucleases to determine internal differences in the sequence. These variants were also compared with the Wx alleles present in durum wheat. Differences in size and restriction sites were detected. DNA sequence analysis confirmed that the alleles found in emmer wheat are different from those in durum wheat. The first data suggested that these alleles showed a different influence on the amylose content of these lines. The variation found could be used to enlarge the gene pool of durum and emmer wheat, and design new materials with different amylose content.     

Deletion commonly found in Waxy gene of Japanese and Korean cultivars of Job’s tears (Coix lacryma-jobi L.)

We isolated the entire sequence of the coding region of Waxy gene of a non-waxy accession of Job??s tears (Coix lacryma-jobi) by PCR-based methods. We also compared the entire sequences of the gene between two non-waxy accessions and three waxy cultivars and found a 275-bp deletion in the coding region (exons 10?C11) of this gene specific to waxy cultivars. We showed by PCR genotyping that this deletion is commonly found in Japanese and Korean cultivars and confirmed that this deletion resulted in lack of Wx protein. We also confirmed that this polymorphism of the gene co-segregates with phenotypes in endosperm and pollen. These results suggest that this PCR-based marker will be useful in breeding of Job??s tears and that genetic information obtained in other grass species will be also useful in genetics and breeding of Job??s tears.     

Origin and evolution of the waxy phenotype in Amaranthus hypochondriacus: evidence from the genetic diversity in the Waxy locus

The existence of polymorphism in the Waxy locus in a large gene pool of 53 strains with various waxy phenotypes from samples of Amaranthus hypochondriacus collected from different regions was investigated in an origin-and-evolution study. First, we screened all strains for a mutation point (G–A polymorphism in exon 6) by using PCR–RFLP and/or direct sequence analysis. The results showed that the nonsense mutation in the coding region (exon 6) of the Waxy gene was responsible for the change in perisperm starch, leading to a waxy phenotype in all strains. Second, phylogenetic analysis, which was based on the Waxy variation, indicated diverse waxy types occurring separately and independently in certain domesticated regions in Mexico. Finally, we designated nine molecular types by comparing obvious structural variations in the coding region of the Waxy gene. Among the molecular types, A. hypochondriacus contained Type III in three subtypes with the waxy phenotype, with evolutionary routes that could originate from Type II in accordance with G–A polymorphism. In addition, these types had the same mutation points by which the Waxy gene was converted into the waxy phenotype. Therefore, the present results showed that the nonsense mutation is a unique event in the evolution of waxy phenotypes in this crop. This study will provide useful information for understanding the evolutionary process of the waxy phenotype.     

Structural analyses of the permease like protein SoxT: A member of the sulfur compound metabolizing sox operon

One of the oldest known gene clusters that are involved in biological oxidation processes is the sox operon. This operon is present in different microbial species. In the present study an attempt has been made to analyze the probable structural role of SoxT protein from Pseudaminobacter salicylatoxidans. This protein has been predicted to be a permease-like protein. A comparative model of the protein has been made and analyzed. The possible membrane spanning region of the protein has been detected by structural bioinformatics approach. The inducer of the sulfur oxidation process has been predicted. And thereby the plausible mechanism of the transport of the sulfur anion inside the bacterial cell has been elucidated. Since this is the first study regarding the structural aspect of the protein this study may shed light on the theory of the yet unknown molecular mechanism of the sulfur oxidation process by sox operon.     

Differential regulation of waxy gene expression in rice endosperm

Summary In order to examine the effects of different alleles on the gene expression at the waxy locus, the Wx gene product which controls the synthesis of amylose was isolated from endosperm starch of rice plants and analysed by electrophoretic techniques. The major protein bound to starch granules was absent in most of waxy strains and increased with the number of Wx alleles in triploid endosperms, suggesting that the major protein is the Wx gene product. In addition to wx alleles which result in the absence or drastic reduction of the Wx gene product and amylose, differentiation of Wx alleles seemed to have occurred among nonwaxy rice strains. At least two Wx alleles with different efficiencies in the production of the major protein as well as amylose were detected. These alleles are discussed in relation to regulation of the gene expression.     

Bacteriophage-coded thymidylate synthetase. Evidence that the T4 enzyme is a capsid protein

It has previously been shown that T4 bacteriophage-coded dihydrofolate reductase is a capsid protein, specifically an element of the tail plate. This paper presents evidence that thymidylate synthetase is also a structural protein. Antiserum prepared against purified T4 thymidylate synthetase neutralizes T4 infectivity. Evidence is presented that structural thymidylate synthetase is the target of the antiphage component of the serum.The td gene in T4 codes for thymidylate synthetase. We have crossed the td gene from phage T6 into T4 and eliminated other T6 genetic material from the hybrid phage by extensive backcrossing. The hybrid phage, T4td^T6, is inactivated at 60 °C significantly more rapidly than the parent phage, T4D. Thus, the td gene is a determinant of a physical property of the virion, providing direct confirmation that thymidylate synthetase is a capsid protein. At present the role of the virion-bound enzyme is unknown.     

Diverse origins of waxy foxtail millet crops in East and Southeast Asia mediated by multiple transposable element insertions

The naturally occurring waxy and low-amylose variants of foxtail millet and other cereals, like rice and barley, originated in East and Southeast Asia under human selection for sticky foods. Mutations in the GBSS1 gene for granule-bound starch synthase 1 are known to be associated with these traits. We have analyzed the gene in foxtail millet, and found that, in this species, these traits were originated by multiple independent insertions of transposable elements and by subsequent secondary insertions into these elements or deletion of parts of the elements. The structural analysis of transposable elements inserted in the GBSS1 gene revealed that the non-waxy was converted to the low-amylose phenotype once, while shifts from non-waxy to waxy occurred three times, from low amylose to waxy once and from waxy to low amylose once. The present results, and the geographical distribution of different waxy molecular types, strongly suggest that these types originated independently and were dispersed into their current distribution areas. The patterns of GBSS1 variation revealed here suggest that foxtail millet may serve as a key to solving the mystery of the origin of waxy-type cereals in Asia. The GBSS1 gene in foxtail millet provides a new example of the evolution of a gene involved in the processes of domestication and its post-domestication fate under the influence of human selection. Electronic Supplementary Material Supplementary material is available for this article at     

Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2

Mutant plants defective in the assimilation of nitrate can be selected by their resistance to the herbicide chlorate. In Arabidopsis thaliana, mutations at any one of nine distinct loci confer chlorate resistance. Only one of the CHL genes, CHL3, has been shown genetically to be a nitrate reductase (NR) structural gene (NIA2) even though two NR genes (NIA1 and NIA2) have been cloned from the Arabidopsis genome. Plants in which the NIA2 gene has been deleted retain only 10% of the wildtype shoot NR activity and grow normally with nitrate as the sole nitrogen source. Using mutagenized seeds from the NIA2 deletion mutant and a modified chlorate selection protocol, we have identified the first mutation in the NIA1 NR structural gene. nia1, nia2 double mutants have only 0.5% of wild-type shoot NR activity and display very poor growth on media with nitrate as the only form of nitrogen. The nial-1 mutation is a single nucleotide substitution that converts an alanine to a threonine in a highly conserved region of the molybdenum cofactor-binding domain of the NR protein. These results show that the NIA1 gene encodes a functional NR protein that contributes to the assimilation of nitrate in Arabidopsis.     

The First Structure of a Mycobacteriophage,the Mycobacterium abscessus subsp. bolletii Phage Araucaria

The unique characteristics of the waxy mycobacterial cell wall raise questions about specific structural features of their bacteriophages. No structure of any mycobacteriophage is available, although ∼3,500 have been described to date. To fill this gap, we embarked in a genomic and structural study of a bacteriophage from Mycobacterium abscessus subsp. bolletii, a member of the Mycobacterium abscessus group. This opportunistic pathogen is responsible for respiratory tract infections in patients with lung disorders, particularly cystic fibrosis. M. abscessus subsp. bolletii was isolated from respiratory tract specimens, and bacteriophages were observed in the cultures. We report here the genome annotation and characterization of the M. abscessus subsp. bolletii prophage Araucaria, as well as the first single-particle electron microscopy reconstruction of the whole virion. Araucaria belongs to Siphoviridae and possesses a 64-kb genome containing 89 open reading frames (ORFs), among which 27 could be annotated with certainty. Although its capsid and connector share close similarity with those of several phages from Gram-negative (Gram⁻) or Gram⁺ bacteria, its most distinctive characteristic is the helical tail decorated by radial spikes, possibly host adhesion devices, according to which the phage name was chosen. Its host adsorption device, at the tail tip, assembles features observed in phages binding to protein receptors, such as phage SPP1. All together, these results suggest that Araucaria may infect its mycobacterial host using a mechanism involving adhesion to cell wall saccharides and protein, a feature that remains to be further explored.     

Mutation loci and intragenic selection marker of the granule-bound starch synthase gene in waxy maize

Four pairs of specific PCR primers have been designed on the basis of the sequence of the granule-bound starch synthase gene (GBSS; dominant non-waxy gene Wx) and used to amplify its homologous sequence from thirteen waxy and two non-waxy inbred lines. Results from electrophoresis indicated that the recessive waxy gene was wx, derived from the dominant non-waxy gene Wx by mutation at its 3′ end. The sequence of the mutated 3′ end was amplified by the TAIL-PCR technique. Sequence alignment showed that the mutation of the wx gene was caused by transposition of the aldehyde dehydrogenase gene rf2. Two pairs of specific primers were designed on the basis of the sequence difference between the dominant gene Wx and its mutated recessive allele wx and used as intragenic selection markers to identify individual plants of genotypes WxWx, Wxwx, and wxwx by PCR amplification from the segregating population of the F₂ generation crossed between waxy and non-waxy inbred lines. Iodine solution staining and starch component assay showed that all the 35 F₂ plants identified as genotype WxWx produced non-waxy kernels of the F₃ generation and that all 33 F₂ plants identified as genotype wxwx produced waxy kernels of the F₃ generation. This result can be used to improve the selection efficiency of waxy maize breeding and for selection of other single genes and major polygenes.     

Isolation of the transposable element hupfer from the entomopathogenic fungus Beauveria bassiana by insertion mutagenesis of the nitrate reductase structural gene

A transposable element has been isolated from the entomopathogenic fungus Beauveria bassiana by trapping it in the nitrate reductase structural gene, which has been cloned from this species. The element had inserted in the first exon of the nia gene and appeared to have duplicated the sequence TA at the site of insertion. It was 3336?bp long with 30-bp imperfect, inverted, terminal repeats. The element, called hupfer, contained an open reading frame encoding a 321-amino acid protein similar to the IS630- or mariner-Tc1-like transposases, and a residual sequence of about 2?kb which was not significantly similar to any published sequence. There are fewer than five copies of this transposable element present per genome in the fungus.     

Assembly of the mitochondrial membrane system: Sequence analysis of a yeast mitochondrial ATPase gene containing the oli-2 and oli-4 loci

The mitochondrial DNA (mtDNA) segments of several ρ^? mutants carrying the oli-2, oli-4 and pho-1 loci have been sequenced. The segments contain a common structural gene sequence that has been identified to include all three genetic markers. The gene codes for a protein with a molecular weight of 28,257. This new gene is located between 61.5 and 62.6 units on the wild-type map of Saccharomyces cerevisiae and is transcribed from the same DNA strand as most other yeast mitochondrial genes sequenced to date. The amino acid composition and sequence deduced from the DNA sequence indicate that the protein is very hydrophobic, with three long domains (>30 residues) consisting of nonpolar amino acids. Based on its molecular weight, the gene product is tentatively proposed to be either subunit 3 or 6 of the oligomycin-sensitive ATPase.