Cloning and characterization of a novel human SPRYD4 gene encoding a putative SPRY domain-containing protein.

We report here the cloning and characterization of a novel human SPRYD4 gene which encodes a SPRY domain containing protein. The SPRYD4 gene is isolated from the human brain cDNA library, and mapped to 12q13.2 by searching the UCSC genomic database. The SPRYD4 cDNA is 1201 base pairs in length and contains an open reading frame encoding 207 amino acids. The SPRYD4 gene consists of two exons and encodes a putative protein with a SPRY domain ranging from 86 to 203 amino acids. The RT-PCR analysis reveals that SPRYD4 is ubiquitously expressed in 18 human tissues. However, it is strongly expressed in kidney, bladder, brain, thymus and stomach, while weakly expressed liver, testis, uterus, spleen and lung. Subcellular localization demonstrates that SPRYD4 protein is localized in the nuclear when overexpressed in COS-7 cell.     

Root-specific expression of a Zea mays gene encoding a novel glycine-rich protein,zmGRP3

The isolation and characterization of a cDNA clone from Zea mays coding for a novel glycine-rich protein (GRP) is described. The corresponding 1.4 kb mRNA accumulates exclusively in roots (primary, lateral seminal and crown roots) of young maize seedlings, following developmentally specific patterns. In agreement with previously described GRPs from other plant species the derived protein sequence exhibits a hydrophobic domain at the N-terminal region followed by repeated glycine-rich motifs. Genomic Southern analysis indicates that the zmGRP3 gene is present in the maize genome as one or two copies or at a low copy number.     

Identification and characterization of Veph, a novel gene encoding a PH domain-containing protein expressed in the developing central nervous system of vertebrates

We isolated Veph, a novel gene encoding a pleckstrin homology (PH) domain-containing protein from a mouse. Veph was strongly expressed in the embryonic brain, and its expression level gradually decreased in later stages. In situ hybridization analysis of sectioned embryo brains revealed that Veph was expressed exclusively in the ventricular zone. We then isolated a zebrafish orthologue of Veph (zVeph). As observed in the mouse gene, zVeph was expressed in the ventricular zone of developing brain and spinal cord. Blockage of zVeph expression by injection of zVeph-specific morpholino antisense oligo into zebrafish fertilized eggs resulted in a defect in the midbrain-hindbrain boundary and otic vesicle formation, suggesting the important function of zVeph in central nervous system (CNS) development. On the other hand, homozygous knockout mice of Veph showed no significant defect in the CNS, pointing to possible different functions of Veph between the zebrafish and mouse.     

Mfsd2a encodes a novel major facilitator superfamily domain-containing protein highly induced in brown adipose tissue during fasting and adaptive thermogenesis

This study describes the identification of Mfsd2a (major facilitator superfamily domain-containing protein 2a), a novel mammalian major facilitator superfamily domain-containing protein, and an additional closely related protein, Mfsd2b. Most intron/exon junctions are conserved between the two genes, suggesting that they are derived from a common ancestor. Mfsd2a and Mfsd2b share a 12 transmembrane alpha-helical domain structure that bears greatest similarity to that of the bacterial Na(+)/melibiose symporters. Confocal microscopy demonstrated that Mfsd2a localizes to the endoplasmic reticulum. Mfsd2a is expressed in many tissues and is highly induced in liver and BAT (brown adipose tissue) during fasting. Mfsd2a displays an oscillatory expression profile in BAT and liver, consistent with a circadian rhythm. Although the basal level of Mfsd2a expression is relatively low in mouse BAT, it is greatly induced during cold-induced thermogenesis and after treatment with betaAR (beta-adrenergic receptor) agonists. This induction is totally abolished in beta-less (betaAR-deficient) mice. These findings indicate that Mfsd2a is greatly up-regulated in BAT during thermogenesis and that its induction is controlled by the betaAR signalling pathway. The observed induction of Mfsd2a expression in cultured BAT cells by dibutyryl-cAMP is in agreement with this conclusion. The present study suggests that Mfsd2a plays a role in adaptive thermogenesis.     

Molecular characterization of a novel soybean gene encoding a neutral PR-5 protein induced by high-salt stress

In this study, we characterized a novel soybean gene encoding a neutral PR-5 protein and compared it to two acidic isoforms of soybean PR-5 protein. This gene, designated as Glycine max osmotin-like protein, b isoform (GmOLPb, accession no. AB370233), encoded a putative protein having the greatest similarity to chickpea PR-5b (89% identity). Unlike the two acidic PR-5, GmOLPa and P21, the protein had a C-terminal elongation responsible for possible vacuolar targeting and after maturation showed a calculated molecular mass of 21.9 kDa with pI 6.0. The 3D models, predicted by the homology modeling, contained four α-helixes and 16 β-strands and formed three characteristic domains. The two acidic PR-5 proteins also showed a 3D structure very similar to GmOLPb, although the electrostatic potential on molecular surface of each PR-5 was significantly different. In the study of the gene expression under conditions of high-salt stress, GmOLPb was highly induced in the leaves of the soybean, particularly in the lower part of a leaf. The expression started at 2 h after initiation of the stress and was highly induced between 18–72 h. Gene expression of P21e (protein homologous to P21) was transiently induced by high-salt stress, but took place earlier than the gene expressions of GmOLPa and GmOLPb. Such differential expression was observed also under investigation with methyl jasmonate and salicylic acid. These results suggested that each soybean PR-5 might play a distinctive role in the defensive system protecting the soybean plant against high-salt stress, particularly in the leaves of the soybean.     

Ankyrin protein kinases: a novel type of plant kinase gene whose expression is induced by osmotic stress in alfalfa

Interaction between Medicago spp. and Sinorhizobium meliloti leads to the development of a novel organ, the root nodule. A gene, Msapk1, encoding a novel type of plant protein kinase containing a N-terminal region with an ankyrin domain, was identified and shown to be expressed both in S. meliloti-infected and spontaneous nodules in alfalfa. This gene is not exclusively associated to nodulation since its expression was detected in other plant organs. Several genes coding for ankyrin protein kinases (APKs) were detected in various plants and animals. Three closest A. thaliana homologues of Msapk1 were identified in databases and two of them were shown to express differentially in various organs using gene-specific RT-PCR. In contrast, Southern analysis suggests that a single-copy gene exists in diploid M. truncatula. By screening a M. truncatula BAC library the Mtapk1 genomic region was isolated and sequenced. Two neighbouring genes showing homologies to previously identified sequences in data banks were detected in the vicinity of the Mtapk1 gene and compared to similar regions of the three Atapk genes. The distribution of exons/introns was the same for all expressed genes of both species although Mtapk1 contained larger introns. Upon osmotic stress Msapk1 expression was induced in roots of alfalfa starting from three hours up to two days of treatment. These data suggest that Msapk1, involved in alfalfa osmotic stress responses, belongs to a novel class of plant protein kinases.     

VfCPK1, a gene encoding calcium-dependent protein kinase from Vicia faba, is induced by drought and abscisic acid

Calcium, one of the most ubiquitous second messengers, has been shown to be involved in a wide variety of responses in plants. Calcium-dependent protein kinases (CDPKs) (EC 2.7.1.37) are the predominant Ca(2+)-regulated serine/threonine protein kinase in plants and play an important role in plant calcium signal transduction. CDPKs are encoded by a large multigene family in many plants, which has been showed so far; however, the precise role of each specific CDPK is still largely unknown. A novel CDPK gene designated as VfCPK1 was cloned from epidermal peels of broad bean (Vicia faba L.) leaves using the rapid amplification of cDNA ends (RACE)-PCR technique and its expression was studied in detail. The VfCPK1 cDNA is 1783 bp long and contains an open reading frame of 1482 bp encoding 493 amino acids. VfCPK1 contains all conserved regions found in CDPKs and shows a high level of sequence similarity to many other plant CDPKs. VfCPK1 was highly expressed in leaves, especially in leaf epidermal peels of broad bean in mRNA and protein levels. Expressions of VfCPK1 at both the mRNA and protein levels were increased in leaves treated with abscisic acid or subjected to drought stress. Potential roles of VfCPK1 in epidermal peels are discussed. The nucleotide sequence data reported here were deposited in the GenBank database under accession number AY753552.     

Cloning and expression analysis of the gene encoding fibrinogen-like protein A,a novel regeneration-related protein from Apostichopus japonicus

Fibrinogen-like protein A (FGLA), a member of the fibrinogen-related protein superfamily, exists in different tissues of vertebrates and invertebrates. FGLA plays crucial roles including innate immune response, blood clotting and regeneration. In this study, the fibrinogen-like protein A (fglA) was cloned from Apostichopus japonicus using rapid amplification of cDNA ends PCR techniques. The cDNA sequence of fglA is 1,524 bp with a 849 bp open reading frame encoding a polypeptide of 282 amino acids, with an N-terminal signal peptide and a conserved C-terminal domain. Bioinformatic analysis revealed that the predicted molecular weight of the whole protein is 31.9 kDa and it has an isoelectric point of 5.64. In-situ hybridization demonstrated that fglA is widely distributed in body wall, intestines, longitudinal muscles and respiratory tree. The expression levels of fglA during different regeneration stages in the body wall, intestine and respiratory trees were analyzed by real-time PCR. The expression of fglA gradually increased within 1 h in body wall, and reached a plateau before decreasing to the basal level. This indicates that fglA is associated with the regeneration of Apostichopus japonicus.