A novel protein,sperm head and tail associated protein (SHTAP), interacts with cysteine‐rich secretory protein 2 (CRISP2) during spermatogenesis in the mouse

Background information. CRISP2 (cysteine‐rich secretory protein 2) is a sperm acrosome and tail protein with the ability to regulate Ca²⁺ flow through ryanodine receptors. Based on these properties, CRISP2 has a potential role in fertilization through the regulation of ion signalling in the acrosome reaction and sperm motility. The purpose of the present study was to determine the expression, subcellular localization and the role in spermatogenesis of a novel CRISP2‐binding partner, which we have designated SHTAP (sperm head and tail associated protein). Results. Using yeast two‐hybrid screens of an adult testis expression library, we identified SHTAP as a novel mouse CRISP2‐binding partner. Sequence analysis of all Shtap cDNA clones revealed that the mouse Shtap gene is embedded within a gene encoding the unrelated protein NSUN4 (NOL1/NOP2/Sun domain family member 4). Five orthologues of the Shtap gene have been annotated in public databases. SHTAP and its orthologues showed no significant sequence similarity to any known protein or functional motifs, including NSUN4. Using an SHTAP antiserum, multiple SHTAP isoforms (～20–87 kDa) were detected in the testis, sperm, and various somatic tissues. Interestingly, only the ～26 kDa isoform of SHTAP was able to interact with CRISP2. Furthermore, yeast two‐hybrid assays showed that both the CAP (CRISP/antigen 5/pathogenesis related‐1) and CRISP domains of CRISP2 were required for maximal binding to SHTAP. SHTAP protein was localized to the peri‐acrosomal region of round spermatids, and the head and tail of the elongated spermatids and sperm tail where it co‐localized with CRISP2. During sperm capacitation, SHTAP and the SHTAP—CRISP2 complex appeared to be redistributed within the head. Conclusions. The present study is the first report of the identification, annotation and expression analysis of the mouse Shtap gene. The redistribution observed during sperm capacitation raises the possibility that SHTAP and the SHTAP—CRISP2 complex play a role in the attainment of sperm functional competence.     

High Molecular Weight Glutenin Subunit Composition of Indian Wheats and Their Relationships with Dough Strength

One hundred and seventy two wheat varieties including twenty-five durum wheat cultivars were evaluated for high molecular weight glutenin subunit (HMW-GS) composition using SDS-PAGE. The relationship between HMW-GS and sedimentation tests for dough strength was studied. Three alleles were present at the Glu-A1 locus, eight at Glu-B1 and two at Glu-D1 in bread wheat. The data indicated the prevalence of the Glu-A1b allele (63.5%) at the Glu-A1 and Glu-D1a (71.4%) at Glu-D1 loci. Three alleles, namely Glu-B1b (30.61%), Glu-B1c (25.85%) and Glu-B1i (34.00%) represented about 90% of the alleles at Glu-B1 locus. The combination of Glu-A1b, Glu-B1i and Glu-D1d alleles exhibited highest dough strength as measured by sedimentation value in comparison to other combinations (p<0.001). However, this combination was present only in 7% of the samples evaluated. In durum wheat, the null allele (Glu-A1c) was observed more frequently (76%) than the Glu-A1b allele (24%). Glu-B1f and Glu-B1e alleles represented equally (32% each). Protein subunits 13+16 and 6+8 were found correlated positively (p<0.05) with improved dough strength as compared to subunit 20 in durum wheat. This information can be a valuable reference for designing breeding programme for the improvement of bread and pasta making quality of bread and durum wheats, respectively in India.     

Characterization of Low Molecular Weight Glutenin Subunit Gene Representing Glu-B3 Locus of Indian Wheat Variety NP4

Low molecular weight (LMW) glutenin subunits represent major part (30%) of storage proteins in wheat endosperm and determine the quality of dough. Despite their importance few LMW glutenin genes have been characterized so far and none from Indian wheat variety. In the present investigation PCR technique was employed to characterize LMW-GS gene representing Glu-B3 locus from Indian bread wheat cultivar NP4. The deduced protein sequence coded by Glu-B3 locus of LMW-GS gene from NP4 showed the presence of regular structure of the repetitive domain with varying numbers of glutamine (Q) residues and the presence of 1^st cysteine residue within the repetitive domain at 40^th position in mature polypeptide. Such structure might increase and stabilize the gluten polymer through intermolecular interactions of the large numbers of glutamine side chains and cysteine residues for intermolecular disulphide bond formation leading to stronger dough quality of NP4. Moreover, Glu-B3 specific primers could also be used for identifying 1BL/1RS translocation in addition to amplifying LMW glutenin genes. There was no amplification in 1B/1R translocation lines as short arm of wheat was replaced by short arm of rye chromosome in these lines. Such information can be useful in wheat improvement for dough properties for better chapati and bread quality.     

Assessment of allelic variation in serpin gene (Srp5B) in Indian wheats

Serpins (Serine proteinase inhibitors) are the family of proteins involved in the inhibition of proteinases from endogenous and other sources and thus have role in disease and pest resistance. In addition, serpins have the properties influencing grain quality traits. The genes coding for serpin proteins are present on the long arm of the 5B chromosome. In the present investigation, allelic variations in the serpin genes encoded by Srp5Bb were assessed using CAP-PCR among the 300 wheat varieties developed in India during the last 100?years. Eighty percent of the varieties exhibited wild type (Srp5Ba) and 20% mutated (Srp5Bb) form of the allele. Moreover, the frequency of Srp5Bb allele decreased among the recently released varieties as compared to older varieties. Only six out of 50 varieties used as checks in national trials showed the presence of Srp5Bb allele. There was no relationship between Srp5B allelic types and grain hardness. The information is useful to further investigate the role of Srp5B alleles in disease resistance and imparting grain quality.     

Transfer of grain softness from 5U-5A wheat-Aegilops triuncialis substitution line to bread wheat through induced homeologous pairing

Journal of Plant Biochemistry and Biotechnology - Bread wheat sustains genes for grain softness on “Ha” locus of short arm of 5D chromosome. Pina-D1 and Pinb-D1 alleles of...     

Pleiotropic behaviour of a cyanophage AS-1-resistant mutant of Anacystis nidulans

Summary A cyanophage AS-1-resistant mutant strain of Anacystis nidulans exhibited a slow rate of nutrient uptake compared to the wild type. The increased Cu⁺⁺ sensitivity of the mutant could be correlated with higher rates of Cu⁺⁺ uptake. The results are discussed in the light of alterations in the proteins involved in permeability of the outer membrane.     

Biochemical Basis and Molecular Genetics of Processing and Nutritional Quality Traits of Wheat

Wheat is unique in the sense that large numbers of end-use products such as chapati, bread, biscuits, noodles and pasta products are made from it. This is possible because of visco-elastic property imparted to the dough by its gluten proteins. Grain texture, gluten constituents and starch composition in the endosperm are major determinants of end-product quality. Each end-use product has its own specific requirements. Therefore, to expedite the breeding programme for the development of product-specific varieties, understanding the grain components at biochemical/molecular level and their relationship with quality, is needed. Impressive advances have been made in understanding the subunit composition of glutenins, gliadins and starch at biochemical and molecular levels. Molecular markers for these components have been identified, and are being used in breeding for the improvement of wheat quality. Wheat is also deficient of certain micronutrients such as Fe and Zn and lysine content. There is increasing concern globally for the improvement of the nutritional quality of wheat for micronutrient content, and protein and starch quality. Transgenic approach will be helpful in manipulating metabolic pathways for enhancing micronutrient bioavailability and increased lysine content. In this review, information on recent developments that have taken place in understanding the biochemical basis and molecular genetics of processing and nutritional quality of wheat grain and its future implications, has been provided.     

New Frame Shift Mutation in Puroindoline B in Indian Wheat Cultivars Hyb65 and NI5439

Puroindoline genes pinA and pinB are the main components of the 15 kD friabilin protein reported to be associated with kernel softness. However, grain hardness of Hyb65 and NI5439, the two Indian wheat varieties, could not be explained based on the earlier identified alleles in puroindolines in wheat. Hyb65 and NI5439 are hard but based on the earlier identified allelic forms of puroindolines both the varieties could have been soft. In this investigation, puroindolines (a and b) from Hyb65 and NI5439 were characterised to understand their role in determining grain hardness. The sequence of puroindoline genes from both the varieties indicated that there was no mutation in pinA. However, there was frame shift mutation in pinB generated by insertion of a guanine residue 126 bp downstream from the start codon in both the varieties. This created new hardness allele of pinB designated as pinb-D1h. Frame shift also culminated into stop codon (TGA) 231 bp downstream from the start codon terminating protein synthesis at 77^th amino acid position. Five more stop codons (4TGA & 1TAG) were also created to the downstream positions of the first stop codon because of frame shift. There was additional point mutation in NI5439 (transition from A to G) resulting into change of amino acid residue from thymine to arginine at 205^th nucleotide position. Thus single nucleotide change in pinB resulted into truncated pin B and consequently the harder texture.