Nap Hal, an Indian Landrace of Wheat, Contains Unique Genes for Better Biscuit Making Quality

Nap Hal, an Indian landrace of wheat, exhibited unique characteristics suitable for biscuit making quality. Double null trait at Glu-D1 locus in Nap Hal was associated with reduced gluten strength as reflected in low sedimentation volume (16.0 ml, 6 g test), lower Farinograph peak time (2.0 min) and lower tolerance (higher break down 140 BU) to mixing. PCR analysis of puroindoline genes showed the presence of both pinA and glycine type pinB associated with soft grain texture of wheat. This is the first report that Nap Hal contains unique combination of soft grain characteristics determined by puroindolines as well as low gluten strength determined by null alleles at Glu-D1 locus. The combination of both soft grain characteristics and weak gluten is useful in developing varieties suitable for biscuit making where the use of chemical improvers can be reduced.     

Recurrent nonsense mutation in exon 7 of the phenylalanine hydroxylase gene

Summary A new mutation (CGA to TGA) in codon 261 of exon 7 of the phenylalanine hydroxylase gene transforms Arg²⁶¹ to a stop codon in two unrelated patients of German and Turkish origin. The different ethnic backgrounds and the different polymorphic characteristics of the two mutant alleles suggest an independent origin of the mutation. This is the second defect detected in codon 261 of the phenylalanine hydroxylase gene, a codon that thus appears to be a mutation hot spot.     

Development of Transgenic Mice Containing an Introduced Stop Codon on the Human Methylmalonyl-CoA Mutase Locus

The mutation R403stop was found in an individual with mut⁰ methylmalonic aciduria (MMA) which resulted from a single base change of C→T in exon 6 of the methylmalonyl-CoA mutase gene (producing a TGA stop codon). In order to accurately model the human MMA disorder we introduced this mutation onto the human methylmalonyl-CoA mutase locus of a bacterial artificial chromosome. A mouse model was developed using this construct.The transgene was found to be intact in the mouse model, with 7 copies integrated at a single site in chromosome 3. The phenotype of the hemizygous mouse was unchanged until crossed against a methylmalonyl-CoA mutase knockout mouse. Pups with no endogenous mouse methylmalonyl-CoA mutase and one copy of the transgene became ill and died within 24 hours. This severe phenotype could be partially rescued by the addition of a transgene carrying two copies of the normal human methylmalonyl-CoA mutase locus. The “humanized” mice were smaller than control litter mates and had high levels of methylmalonic acid in their blood and tissues.This new transgenic MMA stop codon model mimics (at both the phenotypic and genotypic levels) the key features of the human MMA disorder. It will allow the trialing of pharmacological and, cell and gene therapies for the treatment of MMA and other human metabolic disorders caused by stop codon mutations.     

Two Novel Mutations on Exon 8 and Intron 65 of COL7A1 Gene in Two Chinese Brothers Result in Recessive Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa is an inherited bullous dermatosis caused by the COL7A1 gene mutation in autosomal dominant or recessive mode. COL7A1 gene encodes type VII collagen – the main component of the anchoring fibrils at the dermal–epidermal junction. Besides the 730 mutations reported, we identified two novel COL7A1 gene mutations in a Chinese family, which caused recessive dystrophic epidermolysis bullosa (RDEB). The diagnosis was established histopathologically and ultrastructurally. After genomic DNA extraction from the peripheral blood sample of all subjects (5 pedigree members and 136 unrelated control individuals), COL7A1 gene screening was performed by polymerase chain reaction amplification and direct DNA sequencing of the whole coding exons and flanking intronic regions. Genetic analysis of the COL7A1 gene in affected individuals revealed compound heterozygotes with identical novel mutations. The maternal mutation is a 2-bp deletion at exon 8 (c.1006_1007delCA), leading to a subsequent reading frame-shift and producing a premature termination codon located 48 amino acids downstream in exon 9 (p.Q336EfsX48), consequently resulting in the truncation of 2561 amino acids downstream. This was only present in two affected brothers, but not in the other unaffected family members. The paternal mutation is a 1-bp deletion occurring at the first base of intron 65 (c.IVS5568+1delG) that deductively changes the strongly conserved GT dinucleotide at the 5′ donor splice site, results in subsequent reading-through into intron 65, and creates a stop codon immediately following the amino acids encoded by exon 65 (GTAA→TAA). This is predicted to produce a truncated protein lacking of 1089 C-terminal amino acids downstream. The latter mutation was found in all family members except one of the two unaffected sisters. Both mutations were observed concurrently only in the two affected brothers. Neither mutation was discovered in 136 unrelated Chinese control individuals. This study reveals novel disease-causing mutations in the COL7A1 gene.     

Evolution and differentiation of MSHR gene in different species

Coat color offers some prospects for evolutionary studies due to its large amount of presumably adaptive coat color variation and conserved genetic mechanisms of generating different coat colors in different species. Melanocyte-stimulating hormone receptor (MSHR) gene is responsible for intraspecific and interspecific color variation in mammals and birds. A total number of 206 MSHR gene sequences belonging to 84 species, 58 genera, and 20 families were analyzed to investigate its evolution and differentiation in different species. Most of the species have 954 bp and stop codon TGA. Species in Callithrix and Callimico have a stop codon mutation from TGA to TGG and elongate 81 bp with TAG as stop codon. Species in Phasianidae, Fringillidae, and Lemuridae also use TAG as stop codon. The Sus scrofa had an insertion of AACCAGACC encoding Asn-Gln-Thr from 85 to 93 bp. In Bovidae, a brown strain of cow with 966 bp due to the 12-bp duplication of GGCATTGCCCGG from 670 to 681 bp encoding for Gly-Ile-Ala-Arg was found. Teiidae has the smallest number of total mutations (6), silent mutations (3), nonsynonymous mutations (3), average number of nucleotide differences (1.519), synonymous nucleotide diversity (pi(s) = 0.0030), and nonsynonymous nucleotide diversity (pi(a) = 0.0029), and Hominidae, Lemuridae, Canidae, and Teiidae have higher ratio of pi(a)/pi(s) (0.537-0.973). The reconstructed phylogenetic tree of MSHR gene of families is basically consistent with the taxonomy of National Center for Biotechnology Information.     

Puroindolines: the molecular genetic basis of wheat grain hardness

The variation in grain hardness is the single most important trait that determines end-use quality of wheat. Grain texture classification is based primarily on either the resistance of kernels to crushing or the particle size distribution of ground grain or flour. Recently, the molecular genetic basis of grain hardness has become known, and it is the focus of this review. The puroindoline proteins a and b form the molecular basis of wheat grain hardness or texture. When both puroindolines are in their `functional' wild state, grain texture is soft. When either one of the puroindolines is absent or altered by mutation, then the result is hard texture. In the case of durum wheat which lacks puroindolines, the texture is very hard. Puroindolines represent the molecular-genetic basis of the Hardness locus on chromosome 5DS and the soft (Ha) and hard (ha) alleles present in hexaploid bread wheat varieties. To date, seven discrete hardness alleles have been described for wheat. All involve puroindoline a or b and have been designated Pina-D1b and Pinb-D1b through Pinb-D1g. A direct role of a related protein, grain softness protein (as currently defined), in wheat grain texture has yet to be demonstrated.     

The soybean retroelement SIRE1 uses stop codon suppression to express its envelope-like protein

The soybean SIRE1 family of Ty1/copia retrotransposons encodes an envelope-like gene (env-like). We analysed the DNA sequences of nine SIRE1 insertions and observed that the gag/pol and env-like genes are in the same reading frame and separated by a single UAG stop codon. The six nucleotides immediately downstream of the stop codon conform to a degenerate nucleotide motif, CARYYA, which is sufficient to facilitate stop codon suppression in tobacco mosaic virus. In vivo stop codon suppression assays indicate that SIRE1 sequences confer leakiness to the UAG stop codon at an efficiency of 5%. These data suggest that SIRE1 retro-elements use translational suppression to express their envelope-like protein; this is in contrast with all characterized retroviruses, which express the envelope protein from a spliced genomic messenger RNA.     

Identification of a novel splicing mutation and 1-bp deletion in the 17α-hydroxylase gene of Japanese patients with 17α-hydroxylase deficiency

We report studies of two unrelated Japanese patients with 17α-hydroxylase deficiency caused by mutations of the 17α-hydroxylase (CYP17) gene. We amplified all eight exons of the CYP17 gene, including the exon-intron boundaries, by the polymerase chain reaction and determined their nucleotide sequences. Patient 1 had novel, compound heterozygous mutations of the CYP17 gene. One mutant allele had a guanine to thymine transversion at position +5 in the splice donor site of intron 2. This splice-site mutation caused exon 2 skipping, as shown by in vitro minigene expression analysis of an allelic construct, resulting in a frameshift and introducing a premature stop codon (TAG) 60 bp downstream from the exon 1-3 boundary. The other allele had a missense mutation of His (CAC) to Leu (CTC) at codon 373 in exon 6. These two mutations abolished the 17α-hydroxylase and 17,20-lyase activities. Restriction fragment length polymorphism (RFLP) analysis with a mismatch oligonucleotide showed that the patient’s mother and brother carried the splice-site mutation, but not the missense mutation. Patient 2 was homozygous for a novel 1-bp deletion (cytosine) at codon 131 in exon 2. This 1-bp deletion produces a frameshift in translation and introduces a premature stop codon (TAG) proximal to the highly conserved heme iron-binding cysteine at codon 442 in microsomal cytochrome P450 steroid 17α-hydroxylase (P450c17). RFLP analysis showed that the mother was heterozygous for the mutation. Received: 15 November 1997 / Accepted: 15 March 1998     

Acute intermittent porphyria caused by a single base insertion of C in exon 15 of the porphobilinogen deaminase gene that results in a frame shift and premature stopping of translation

A single base insertion of C in exon 15 of the porphobilinogen deaminase (PBG-D) gene was observed in a patient with acute intermittent porphyria (AIP) by polymerase chain reaction (PCR)-direct sequencing analysis. The insertion locates between positions -22 and -21 from the translation termination codon TAA, causes a frame shift, and results in a stop codon located 4 codons downstream from the insertion (premature stopping of translation). The mutation generates an MspI recognition site, which can be used, in turn, to detect the mutant allele. Analysis of the cDNA fragments amplified by PCR revealed the existence of the abnormal PBG-D mRNA from the mutant allele in the patient.     

Marker–trait association analysis of kernel hardness and related agronomic traits in a core collection of wheat lines

Identification of marker–trait associations is the first step towards marker-assisted selection in plant breeding. Here we assess genetic diversity and population structure of 94 diverse wheat elite lines and use genome-wide association mapping to identify marker–trait associations for five important traits: kernel hardness (KHA), thousand-kernel weight, grain protein content, test weight (TWT), and plant height (PHT). The 94 accessions employed in this study were grouped into three subpopulations based on the first three principal components, which accounted for 51.5 % of the variations. A mixed linear model was used to detect marker–trait associations incorporating covariance of population structure and relative kinship. A total of six marker loci was significantly associated with KHA, TWT, and PHT after the correction of false discovery rate (α _c  = 0.05). The gene pinB was found to be highly associated with KHA, and is reported to be a major determinant of KHA together with the gene pinA at the Ha locus on chromosome 5D. Marker XwPt-7187 on chromosome 2A was also significantly associated with KHA, two Diversity Arrays Technology markers XwPt-1250 and XwPt-4628 with TWT, and marker Xgwm512 with PHT, making the first report of marker–trait associations in these genomic regions.     

Evolution of Prokaryotic Genes by Shift of Stop Codons

De novo origin of coding sequence remains an obscure issue in molecular evolution. One of the possible paths for addition (subtraction) of DNA segments to (from) a gene is stop codon shift. Single nucleotide substitutions can destroy the existing stop codon, leading to uninterrupted translation up to the next stop codon in the gene’s reading frame, or create a premature stop codon via a nonsense mutation. Furthermore, short indels-caused frameshifts near gene’s end may lead to premature stop codons or to translation past the existing stop codon. Here, we describe the evolution of the length of coding sequence of prokaryotic genes by change of positions of stop codons. We observed cases of addition of regions of 3′UTR to genes due to mutations at the existing stop codon, and cases of subtraction of C-terminal coding segments due to nonsense mutations upstream of the stop codon. Many of the observed stop codon shifts cannot be attributed to sequencing errors or rare deleterious variants segregating within bacterial populations. The additions of regions of 3′UTR tend to occur in those genes in which they are facilitated by nearby downstream in-frame triplets which may serve as new stop codons. Conversely, subtractions of coding sequence often give rise to in-frame stop codons located nearby. The amino acid composition of the added region is significantly biased, compared to the overall amino acid composition of the genes. Our results show that in prokaryotes, shift of stop codon is an underappreciated contributor to functional evolution of gene length.     

The Evolutionarily Conserved Eukaryotic Arginine Attenuator Peptide Regulates the Movement of Ribosomes That Have Translated It

Translation of the upstream open reading frame (uORF) in the 5′ leader segment of the Neurospora crassa arg-2 mRNA causes reduced initiation at a downstream start codon when arginine is plentiful. Previous examination of this translational attenuation mechanism using a primer-extension inhibition (toeprint) assay in a homologous N. crassa cell-free translation system showed that arginine causes ribosomes to stall at the uORF termination codon. This stalling apparently regulates translation by preventing trailing scanning ribosomes from reaching the downstream start codon. Here we provide evidence that neither the distance between the uORF stop codon and the downstream initiation codon nor the nature of the stop codon used to terminate translation of the uORF-encoded arginine attenuator peptide (AAP) is important for regulation. Furthermore, translation of the AAP coding region regulates synthesis of the firefly luciferase polypeptide when it is fused directly at the N terminus of that polypeptide. In this case, the elongating ribosome stalls in response to Arg soon after it translates the AAP coding region. Regulation by this eukaryotic leader peptide thus appears to be exerted through a novel mechanism of cis-acting translational control.     

Human urate oxidase gene: cloning and partial sequence analysis reveal a stop codon within the fifth exon

Using the cDNA and selected genomic probes of rat urate oxidase, we have screened the human genomic library and isolated seven clones; one clone (clone 13) contained exonic regions which correspond to the exons 5, 6, and 7 of rat urate oxidase gene. The nucleotide sequence was determined for these three exons and exon/intron junctions, and compared with the sequence from the rat gene. A mutation resulting in a stop codon TGA was found in the fifth exon of the human urate oxidase gene. Sequence analysis of the polymerase chain reaction amplified DNA, corresponding to the fifth exon of urate oxidase from DNA samples from four different individuals, confirmed the same TGA stop codon in all. This single stop codon mutation and/or other mutation(s) in this gene may be responsible for the lack of urate oxidase activity in the human.     

Impact of the six nucleotides downstream of the stop codon on translation termination

The efficiency of translation termination is influenced by local contexts surrounding stop codons. In Saccharomyces cerevisiae, upstream and downstream sequences act synergistically to influence the translation termination efficiency. By analysing derivatives of a leaky stop codon context, we initially demonstrated that at least six nucleotides after the stop codon are a key determinant of readthrough efficiency in S. cerevisiae. We then developed a combinatorial-based strategy to identify poor 3′ termination contexts. By screening a degenerate oligonucleotide library, we identified a consensus sequence –CA(A/G)N(U/C/G)A–, which promotes >5% readthrough efficiency when located downstream of a UAG stop codon. Potential base pairing between this stimulatory motif and regions close to helix 18 and 44 of the 18S rRNA provides a model for the effect of the 3′ stop codon context on translation termination.     

Studies on the bacteriophage MS2: XLI. Nature of the azure mutation

Azure (or reverse amber) mutants grow normally on wild-type Escherichia coli but not on host strains harbouring a strong UAG suppressor mutation. Three different bacteriophage MS2 azure mutants obtained by treatment with nitrous acid have been characterized at the nucleotide sequence level. The 3′-end fragment of the ³²P-labelled mutant genomes was isolated by DNA:RNA hybridization and treatment with nuclease S₁, and was analyzed by mini-fingerprinting of the RNA. It is known that the wild-type MS2 polymerase gene ends with a UAG codon, followed seven triplets further by an in-phase UAA triplet. All three azure mutants contained an A → G transition in this UAA second stop codon of the polymerase gene, resulting in a second suppressible UAG (amber) codon. Analysis of revertants demonstrated that the azure mutation can be counteracted either by a true site reversion at the second stop or by the creation of a new stop signal for the polymerase gene, either UAA (ochre) or UGA (opal), before or at the first stop, or beyond the second stop. On the basis of these results, a mechanism for the azure mutation is proposed. Silent mutations (one in the coding region, three in the untranslated 3′-terminal sequence) have also been observed in these phage stocks.     

Novel deletion mutation of TRPS1 gene in a Chinese patient of trichorhinophalangeal syndrome type I

Tricho–rhino–phalangeal syndrome (TRPS) is a rare autosomal dominant disorder. Deletion or mutation of the TRPS1 gene leads to the tricho–rhino–phalangeal syndromes type I or type III. In this article, we describe a Chinese patient affected with type I TRPS and showing prominent pilar, rhinal and phalangeal abnormalities. Mutational screening and sequence analysis of TRPS1 gene revealed a previously unidentified four-base-pair deletion of nucleotides 1783–1786 (c.1783_1786delACTT). The mutation causes a frame shift after codon 593, introducing a premature stop codon after 637 residues in the gene sequence. This deletion is an unquestionable loss-of-function mutation, deleting all the functionally important parts of the protein. Our novel discovery indicates that sparse hair and metacarpal defects of tricho–rhino–phalangeal syndromes in this patient are due to this TRPS1 mutation. And this data further supports the critical role of TRPS1 gene in hair and partial skeleton morphogenesis.