scholarly journals Molecular Cloning of a Novel Mouse Testis-specific Spermatogenic Cell Apoptosis Inhibitor Gene mTSARG7 as a Candidate Oncogene

2005 ◽  
Vol 37 (6) ◽  
pp. 396-405 ◽  
Author(s):  
Xiao-Jun Tan ◽  
Xiao-Wei Xing ◽  
Lu-Yun Li ◽  
Zhao-Di Wu ◽  
Chang-Gao Zhong ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mrna Expression ◽  
Expressed Sequence Tag ◽  
Mouse Testis ◽  
Control Group ◽  
Amino Acid Residues ◽  
Apoptosis Inhibitor ◽  
Expressed Sequence ◽  
Candidate Oncogene

Abstract A novel mouse gene, mTSARG7 (GenBank accession No. AY489184), with a full cDNA length of 2279 bp and containing 12 exons and 11 introns, was cloned from a mouse expressed sequence tag (GenBank accession No. BE644543) that was significantly up-regulated in cryptorchidism. The gene was located in mouse chromosome 8A1.3 and encoded a protein containing 403 amino acid residues that was a new member of the acyltransferase family because the sequence contained the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The mTSARG7 protein and AU041707 protein shared 83.9% identity in 402 amino acid residues. Expression of the mTSARG7 gene was restricted to the mouse testis. The results of the in situ hybridization analysis revealed that the mTSARG7 mRNA was expressed in mouse spermatogonia and spermatocytes. Subcellular localization studies showed that the EGFP-tagged mTSARG7 protein was localized in the cytoplasm of GC-1 spg cells. The mTSARG7 mRNA expression was initiated in the mouse testis in the second week after birth, and the expression level increased steadily with spermatogenesis and sexual maturation of the mouse. The results of the heat stress experiment showed that the mTSARG7 mRNA expression gradually decreased as the heating duration increased. The pcDNA3.1 Hygro(–)/mTSARG7 plasmid was constructed and introduced into GC-1 spg cells by liposome transfection. The mTSARG7 can accelerate GC-1 spg cells, causing them to traverse the S-phase and enter the G2-phase, compared with the control group where this did not occur as there was no transfection of mTSARG7. In conclusion, our results suggest that this gene may play an important role in spermatogenesis and the development of cryptorchid testes, and is a testis-specific apoptosis candidate oncogene.

Identification of a Novel Gene SRG4 Expressed at Specific Stages of Mouse Spermatogenesis

2004 ◽  
Vol 36 (5) ◽  
pp. 351-359 ◽  
Author(s):  
Xiao-Wei Xing ◽  
Lu-Yun Li ◽  
Gang Liu ◽  
Jun-Jiang Fu ◽  
Xiao-Jun Tan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Mouse Testis ◽  
Amino Acid Residues ◽  
Complex Process ◽  
Testis Cdna Library ◽  
New Gene ◽  
Testis Cdna ◽  
Gene 4

Abstract Spermatogenesis is a complex process. Two spermatocytes expression sequence tags (ESTs) BG101130 and BG100990 were found. Their putative amino acid sequences have high homology with rat Spag4 (sperm antigen 4). By electrical hybridization, a novel cDNA encoding polypeptide of 348 amino acid residues was identified from a mouse testis cDNA library. The new gene was designated as SRG4 (Spermatogenesis related gene 4) (GenBank accession No. AY307077). Results of Northern blot and RTPCR revealed that SRG4 expressed specifically in mouse testis. Changes of SRG4 expression in mouse different development stages were observed by RT-PCR. The SRG4 mRNA was hardly detected in 2 weeks postpartum, and expressed abundantly from 3 weeks later, reaching top lever at 4–5 weeks, while slightly down in aging mouse testis. Results of in situ hybridization showed that SRG4 gene expressed abundantly in spermatocytes, round spermatids. This indicated SRG4 gene may play an important role in mouse meiotic divisions of spermatocytes.

Cloning and characterization of human liver cytosolic β-glycosidase

2001 ◽  
Vol 356 (3) ◽  
pp. 907-910 ◽  
Author(s):  
Michelle de GRAAF ◽  
Irene C. van VEEN ◽  
Ida H. van der MEULEN-MUILEMAN ◽  
Winald R. GERRITSEN ◽  
Herbert M. PINEDO ◽  
...  
Keyword(s):  
Amino Acid ◽  
Human Liver ◽  
Expressed Sequence Tag ◽  
Eukaryotic Expression ◽  
Glycoside Hydrolases ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Mammalian Liver ◽  
Cloned Cdna ◽  
Expressed Sequence

Cytosolic β-glucosidase (EC 3.2.1.21) from mammalian liver is a member of the family 1 glycoside hydrolases and is known for its ability to hydrolyse a range of β-d-glycosides, including β-d-glucoside and β-d-galactoside. We therefore refer to this enzyme as cytosolic β-glycosidase. We cloned the cDNA encoding the human cytosolic β-glycosidase by performing PCR on cDNA prepared from total human liver RNA. Specific primers were based on human expressed sequence tags found in the expressed sequence tag database. The cloned cDNA contained 1407nt with an open reading frame encoding 469 amino acid residues. Amino acid sequence analysis indicates that human cytosolic β-glycosidase is most closely related to lactase phlorizin hydrolase and klotho protein. The enzyme was characterized by using cell lysates of COS-7 cells transfected with a eukaryotic expression vector containing the cDNA. The biochemical, kinetic and inhibition properties of the cloned enzyme were found to be identical with those reported for the enzyme purified from human liver.

scholarly journals Beta-centractin: characterization and distribution of a new member of the centractin family of actin-related proteins.

1994 ◽  
Vol 5 (12) ◽  
pp. 1301-1310 ◽  
Author(s):  
S W Clark ◽  
O Staub ◽  
I B Clark ◽  
E L Holzbaur ◽  
B M Paschal ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cdna Clone ◽  
Expressed Sequence Tag ◽  
Full Length ◽  
Northern Analysis ◽  
Related Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Full Length Cdna ◽  
Dynactin Complex ◽  
Expressed Sequence

An examination of human-expressed sequence tags indicated the existence of an isoform of centractin, an actin-related protein localized to microtubule-associated structures. Using one of these tags, we isolated and determined the nucleotide sequence of a full-length cDNA clone. The protein encoded represents the first example of multiple isoforms of an actin-related protein in a single organism. Northern analysis using centractin-specific probes revealed three species of mRNA in HeLa cells that could encode centractin isoforms. One mRNA encodes the previously-identified centractin (now referred to as alpha-centractin). The full-length cDNA clone isolated using the expressed sequence tag encodes a new member of the centractin family, beta-centractin. A probe specific for alpha-centractin hybridized to the third species of mRNA observed (referred to as gamma-centractin). Comparisons of Northern blots of human tissues indicated that alpha-centractin and beta-centractin mRNAs are equally distributed in all populations of mRNA examined, whereas the expression of gamma-centractin appears to be tissue specific. The amino acid sequence of beta-centractin, deduced from the cDNA, indicates a 91% identity with alpha-centractin, increasing to 96% similarity when conservative amino acid changes are taken into account. As antibodies previously raised against alpha-centractin reacted only poorly with beta-centractin, new antibodies were produced and combined with two-dimensional gel electrophoresis to discriminate the two isoforms. Using this system, the subcellular distribution of the alpha- and beta-isoforms were determined. Both isoforms were found predominantly in the cytosolic fraction as a part of a previously identified 20S complex (referred to as the dynactin complex) with no evidence for a free pool of either isoform. The isoforms were found in a constant ratio of approximately 15:1 (alpha:beta) in the dynactin complex.

scholarly journals How [FeFe]-Hydrogenase Facilitates Bidirectional Proton Transfer

2019 ◽  
Author(s):  
Moritz Senger ◽  
Viktor Eichmann ◽  
Konstantin Laun ◽  
Jifu Duan ◽  
Florian Wittkamp ◽  
...  
Keyword(s):  
Amino Acid ◽  
Proton Transfer ◽  
Active Site ◽  
Molecular Hydrogen ◽  
H2 Production ◽  
Amino Acid Residues ◽  
Difference Spectroscopy ◽  
Dynamic Changes ◽  
In Situ Ir

Hydrogenases are metalloenzymes that catalyse the interconversion of protons and molecular hydrogen, H2. [FeFe]-hydrogenases show particularly high rates of hydrogen turnover and have inspired numerous compounds for biomimetic H2 production. Two decades of research on the active site cofactor of [FeFe]-hydrogenases have put forward multiple models of the catalytic proceedings. In comparison, understanding of the catalytic proton transfer is poor. We were able to identify the amino acid residues forming a proton transfer pathway between active site cofactor and bulk solvent; however, the exact mechanism of catalytic proton transfer remained inconclusive. Here, we employ in situ IR difference spectroscopy on the [FeFe]-hydrogenase from Chlamydomonas reinhardtii evaluating dynamic changes in the hydrogen-bonding network upon catalytic proton transfer. Our analysis allows for a direct, molecular unique assignment to individual amino acid residues. We found that transient protonation changes of arginine and glutamic acid residues facilitate bidirectional proton transfer in [FeFe]-hydrogenases.<br>

scholarly journals Molecular cytogenetic identification of a wheat – Thinopyrum ponticum substitution line with stripe rust resistance

Genome ◽  
2017 ◽  
Vol 60 (10) ◽  
pp. 860-867 ◽  
Author(s):  
Chen Zhu ◽  
Yanzhen Wang ◽  
Chunhuan Chen ◽  
Changyou Wang ◽  
Aicen Zhang ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Stripe Rust ◽  
Expressed Sequence Tag ◽  
Wheat Breeding ◽  
Substitution Line ◽  
Stripe Rust Resistance ◽  
Fish Analysis ◽  
Thinopyrum Ponticum ◽  
Expressed Sequence

Thinopyrum ponticum (Th. ponticum) (2n = 10x = 70) is an important breeding material with excellent resistance and stress tolerance. In this study, we characterized the derivative line CH1113-B13-1-1-2-1 (CH1113-B13) through cytological, morphological, genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), expressed sequence tag (EST), and PCR-based landmark unique gene (PLUG) marker analysis. The GISH analysis revealed that CH1113-B13 contained 20 pairs of common wheat chromosomes and one pair of JSt genomic chromosomes. Linkage analysis of Th. ponticum using seven EST and seven PLUG markers indicated that the pair of alien chromosomes belonged to the seventh homeologous group. Nulli-tetrasomic and FISH analysis revealed that wheat 7B chromosomes were absent in CH1113-B13; thus, CH1113-B13 was identified as a 7JSt (7B) substitution line. Finally, adult-stage CH1113-B13 exhibited immunity to wheat stripe rust. This substitution line is therefore a promising germplasm resource for wheat breeding.

scholarly journals Human diadenosine 5′,5′′′-P1,P4-tetraphosphate pyrophosphohydrolase is a member of the MutT family of nucleotide pyrophosphatases

1995 ◽  
Vol 311 (3) ◽  
pp. 717-721 ◽  
Author(s):  
N M H Thorne ◽  
S Hankin ◽  
M C Wilkinson ◽  
C Nuñez ◽  
R Barraclough ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Expressed Sequence Tag ◽  
Sequence Motif ◽  
Diadenosine Tetraphosphate ◽  
Expressed Sequence

The cDNA and derived amino acid sequence of human diadenosine 5′,5‴-P1,P4-tetraphosphate pyrophosphohydrolase have been determined with the aid of the GenBank Expressed Sequence Tag database. This enzyme possesses a modification of the MutT sequence motif found in certain nucleotide pyrophosphatases. It is unrelated to the enzymes of diadenosine tetraphosphate catabolism found in prokaryotes and fungi.

Analysis of expressed sequence tags from the ectoparasitic nematode Xiphinema index

Nematology ◽  
2005 ◽  
Vol 7 (1) ◽  
pp. 95-104 ◽  
Author(s):  
Cleber Furlanetto ◽  
Linda Cardle ◽  
Derek J.F. Brown ◽  
John T. Jones
Keyword(s):  
Expressed Sequence Tag ◽  
In Situ Hybridisation ◽  
Cdna Libraries ◽  
Small Scale ◽  
Xiphinema Index ◽  
Genes Encoding ◽  
Predicted Signal Peptide ◽  
Basal Bulb ◽  
Expressed Sequence

AbstractWe report the results of a small-scale expressed sequence tag project performed on the ectoparasitic nematode Xiphinema index. Approximately 1400 genes were sequenced, 70% from a cDNA library generated from dissected basal bulbs (containing the pharyngeal gland cells) and 30% from cDNA libraries generated from whole mixed stage nematodes. A large portion of the bulb library (48%) was composed of proteins with no matches in the database. Further analysis of these genes revealed that a total of 51 contigs were present, half of which encoded novel secreted proteins. By contrast, the whole nematode library contained more housekeeping and nematode-specific genes. Only one of the novel genes from the whole nematode library had a predicted signal peptide at the N-terminus. Genes encoding transthyretin-like proteins were abundant in the bulb library and in situ hybridisation confirmed that one of these is expressed in the basal bulb. Genes encoding a variety of proteases, which were shown using in situ hybridisation to be expressed in the gut, were also identified.

scholarly journals Coexpression of Corticotropin-Releasing Hormone and Urotensin I Precursor Genes in the Caudal Neurosecretory System of the Euryhaline Flounder (Platichthys flesus): A Possible Shared Role in Peripheral Regulation

Endocrinology ◽  
2004 ◽  
Vol 145 (12) ◽  
pp. 5786-5797 ◽  
Author(s):  
Weiqun Lu ◽  
Louise Dow ◽  
Sarah Gumusgoz ◽  
Matthew J. Brierley ◽  
Justin M. Warne ◽  
...  
Keyword(s):  
Amino Acid ◽  
Adaptive Plasticity ◽  
Neurosecretory System ◽  
Amino Acid Residues ◽  
Major Site ◽  
Caudal Neurosecretory System ◽  
Genes Encoding ◽  
Releasing Factors ◽  
Circulating Levels

Abstract CRH and urotensin I (UI) are neuroendocrine peptides that belong to the superfamily of corticotropin-releasing factors. In mammals, these peptides regulate the stress response and other central nervous system functions, whereas in fish an involvement for UI in osmoregulation has also been suggested. We have identified, characterized, and localized the genes encoding these peptides in a unique fish neuroendocrine organ, the caudal neurosecretory system (CNSS). The CRH and UI precursors, isolated from a European flounder CNSS library, consist of 168 and 147 amino acid residues, respectively, with an overall homology of approximately 50%. Both precursors contain a signal peptide, a divergent cryptic region and a 41-amino acid mature peptide with cleavage and amidation sites. Genomic organization showed that whole CRH and UI coding sequences are contained in a single exon. Northern blot analysis and quantitative PCR of a range of tissues confirmed the CNSS as a major site of expression of both CRH and UI and thus serves as a likely source of circulating peptides. In situ hybridization demonstrated that CRH and UI colocalize to the same cells of the CNSS. Our findings suggest that, in euryhaline fish, the CNSS is a major site of production of CRH and probably contributes to the high circulating levels observed in response to specific environmental challenges. Furthermore, the localization of CRH and UI within the same cell population suggests an early, possibly shared role for these peptides in controlling stress-mediated adaptive plasticity.

scholarly journals Expression and distribution of synaptotagmin isoforms in the zebrafish retina

2020 ◽  
Author(s):  
Diane Henry ◽  
Christina Joselevitch ◽  
Gary G. Matthews ◽  
Lonnie P. Wollmuth
Keyword(s):  
Amino Acid ◽  
Large Family ◽  
Amino Acid Residues ◽  
Ribbon Synapses ◽  
The Family ◽  
Class 1 ◽  
The Central Nervous System ◽  
Asynchronous Release ◽  
The Brain

ABSTRACTSynaptotagmins belong to a large family of proteins. While various synaptotagmins have been implicated as Ca2+ sensors for vesicle replenishment and release at conventional synapses, their roles at retinal ribbon synapses remain incompletely understood. Zebrafish is a widely used experimental model for retinal research. We therefore investigated the homology between human, rat, mouse, and zebrafish synaptotagmins 1 to 10 using a bioinformatics approach. We also characterized the expression and distribution of various synaptotagmin (syt) genes in the zebrafish retina using RT-PCR and in situ hybridization, focusing on the family members whose products likely underlie Ca2+-dependent exocytosis in the central nervous system (synaptotagmins 1, 2, 5 and 7). We find that most zebrafish synaptotagmins are well conserved and can be grouped in the same classes as mammalian synaptotagmins, based on crucial amino acid residues needed for coordinating Ca2+ binding and determining phospholipid binding affinity. The only exception is synaptotagmin 1b, which lacks 34 amino acid residues in the C2B domain and is therefore unlikely to bind Ca2+ there. Additionally, the products of zebrafish syt5a and syt5b genes share identity with mammalian class 1 and 5 synaptotagmins. Zebrafish syt1, syt2, syt5 and syt7 paralogues are found in the zebrafish brain, eye, and retina, excepting syt1b, which is only present in the brain. The complementary expression pattern of the remaining paralogues in the retina suggests that syt1a and syt5a may underlie synchronous release and syt7a and syt7b may mediate asynchronous release or other Ca2+ dependent processes in different types of retinal neurons.

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