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At least 1,009 records · Page 56Linked to original sources
PubMed1993

Overexpression of a truncated growth hormone receptor in the sex-linked dwarf chicken: evidence for a splice mutation.

Sex-linked dwarfism in chickens is a form of GH resistance that resembles the Laron syndrome in humans. The dwarfism found in chickens is due to a mutant gene (dw) carried on the sex chromosome. The homozygous dwarf (dwdw) chicken is characterized by reductions in stature and plasma insulin-like growth factor-I (IGF-I) levels. Despite the absence of hepatic GH-binding activity, Southern blot analysis shows that there is no gross structural change in the gene for the GH receptor (GHR) in this strain of dwdw chicken. GH-dependent IGF-I production can be restored in cultured dwdw hepatocytes after transfection and transient expression of a chicken GHR (cGHR) cDNA, indicating that other factors that participate in GH-mediated IGF-I synthesis are intact. Northern blot analysis of liver, muscle, fat, and pituitary RNA from normal (DwDw) chickens shows a major transcript of 4.3 kilobases (kb) and three minor transcripts (0.8, 1.7, and 3.2 kb), which correspond to the cGHR. In contrast, the 0.8-kb transcript is the major cGHR transcript expressed in these tissues from dwdw chickens. Northern blot analysis with domain-specific probes shows that the 0.8-kb transcript in DwDw and dwdw liver contains only a small portion of the extracellular domain of the cGHR. A cDNA clone encoding this transcript has been isolated from a liver library prepared from a normal chicken.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Oocyte-specific expression of growth/differentiation factor-9.

Growth/differentiation factor-9 (GDF-9) is a previously described member of the transforming growth factor-beta superfamily expressed specifically in the ovary in adult mice. Using in situ hybridization methods, we have localized the expression of GDF-9 messenger RNA (mRNA) exclusively to oocytes. GDF-9 mRNA was detected in oocytes at all stages of follicular development, except in primordial follicles, in both neonatal and adult ovaries. GDF-9 mRNA continued to be expressed in oocytes after ovulation, but disappeared by 1.5 days after fertilization. Based on Western analysis of ovarian extracts using antibodies raised against recombinant GDF-9 protein, GDF-9 mRNA expressed by oocytes appears to be translated. A human homolog of GDF-9 was isolated from a complementary DNA library prepared from adult ovary mRNA. The predicted human protein is 90% identical to murine GDF-9 in the mature portion of the molecule. These results are significant because no other growth factor-like molecules have been shown to be expressed specifically by oocytes and, together with results of previous studies, suggest that ovarian development and function are regulated by factors produced by both oocytes and support cells of the ovary.

Amino Acid Sequence↗
PubMed1987

Cloning of cDNAs for two beta-tubulin isotypes expressed in murine T cell lymphoma, L5178Y and analysis of their translation products.

Three beta-tubulin isoforms, M beta Ib, M beta Ia and M beta II, were detected by isoelectric focusing gel electrophoresis (IEF) in extracts of cultured cells of a mouse T cell lymphoma, L5178Y. To investigate the origin(s) of the isoforms, we have isolated cDNA clones encoding beta-tubulin from a cDNA library prepared from poly (A)+ RNA of L5178Y cells. Seventeen cDNA clones carrying the entire coding sequences of beta-tubulin were isolated and classified into two distinct isotypes, represented by two clones designated pMT27 and pMT49, according to the results of restriction mapping. On the basis of the nucleotide sequences of the two cDNAs, pMT27 and pMT49 were identified as mouse beta-tubulin isotypes 3 and 5, respectively. By using in vitro translation products of hybrid-selected mRNAs and of the SP6 in vitro transcripts of the cDNAs, polypeptides encoded by the two cDNA clones were analyzed by IEF. We found that pMT27 and pMT49 encode M beta Ib and M beta Ia, respectively. In addition, M beta II was detected in translation products of mRNA specifically hybridized to pMT49, but not in those of the in vitro transcript of pMT49 DNA. These results suggest that M beta II is the translation product of mRNA whose 3'-untranslated region is highly homologous to that of pMT49.

Animals↗
PubMed2004

Porcine (Sus scrofa) cellular FLICE-like inhibitory protein (cFLIP): molecular cloning and comparison with the human and murine cFLIP.

To reveal the molecular regulation mechanism of selective follicular atresia in porcine ovaries, we isolated the porcine cDNA encoding cellular FLICE-like inhibitory protein (cFLIP), which inhibits death receptor-mediated apoptosis signal transduction. Two alternative splicing isoforms of cFLIP, porcine cellular FLIP-short form (pcFLIPS, 642 bp and 214-aa) and -long form (pcFLIPL, 1446 bp and 482-aa), were identified from a cDNA library prepared from follicular granulosa cells of pig ovaries. pcFLIPS and pcFLIPL indicated high identities with human and murine cFLIP, and both of them contain two tandem specific amino acid regions (death effector domain: DED) in their N-terminal, suggesting that pcFLIPS and pcFLIPL inhibit the death receptor-mediated apoptosis signal by binding to other pro-apoptotic factors mediated by DED. pcFLIPS contains a short C-terminal region, while pcFLIPL has a caspase-like domain in the C-terminal region. The reverse transcription-polymerase chain reaction analysis revealed that both pcFLIPS and pcFLIPL mRNAs were highly expressed in granulosa cells of healthy follicles, suggesting that these cFLIPs play important roles in the regulation mechanism of apoptosis in ovarian follicular granulosa cells. The present data will contribute to understanding of the physiological roles of cFLIPs in the apoptosis regulation in porcine tissues.

Amino Acid Sequence↗
PubMed1996

Cloning and sequence analysis of a cDNA encoding salmon (Onchorhynchus keta) liver transglutaminase.

We isolated cDNA clones encoding a transglutaminase (TGase: EC 2.3.2.13) from a salmon (Onchorhynchus keta) cDNA library prepared from the liver. In the cDNA sequence combined, an open reading frame coding for a protein of 680 aa was found. The deduced sequence showed a considerable similarity (62.4%) to that of red sea bream TGase. By comparison of sequence similarity to other TGases, the structure of salmon TGase was like tissue type TGases, rather than membrane-associated type or plasma type TGases. As a structural feature of salmon TGase, 3 aa residues were substituted in the 25 aa sequence around the active site Cys residue, which is conserved among several tissue type TGases. The critical residues thought to form the catalytic-center triad (Cys272, His331, and Asp301) were found in the highly conserved region, but the region surrounding Tyr511, which corresponds to the residue participates in hydrogen-bond interactions of active center domain, was less similar to other TGases, except for red sea bream TGase. These finding suggests that the overall structure of fish TGase resembles tissue-type TGases, but has some unique structure.

Amino Acid Sequence↗
PubMed2000

Pyrithiamine resistance gene (ptrA) of Aspergillus oryzae: cloning, characterization and application as a dominant selectable marker for transformation.

A pyrithiamine (PT) resistance gene (ptrA) was cloned from a genomic DNA library prepared from a PT resistant mutant of Aspergillus oryzae. It conferred high resistance to PT on an A. oryzae industrial strain as well as A. nidulans. Nucleotide sequence analysis showed that the ptrA gene contained one intron (58-bp) and encodes 327 amino acid (aa) residues. Additionally, the deduced aa sequence has 72% and 63% identity to Fusarium solani sti35 encoding a stress-inducible protein and Saccharomyces cerevisiae THI4 encoding an enzyme involved in thiamine biosynthesis, respectively, indicating that ptrA is a mutated allele of a gene belonging to the THI4 family. The mutation point was identified in the conserved motif in 5'-flanking region of these three THI4 homologous genes (ptrA, sti35, and THI4). The introduction of the ptrA gene allowed an A. oryzae industrial strain to grow on the minimum medium containing PT (0.1 mg/l) on which an untransformed strain did not grow. This result indicates that the ptrA is applicable as a dominant selectable marker for transformation of A. oryzae.

Amino Acid Sequence↗
PubMed2006

Cloning and expression of 1,2-alpha-mannosidase gene (fmanIB) from filamentous fungus Aspergillus oryzae: in vivo visualization of the FmanIBp-GFP fusion protein.

1,2-alpha-Mannosidase catalyzes the specific cleavage of 1,2-alpha-mannose residues from protein-linked N-glycan. In this study, a novel DNA sequence homologous to the authentic 1,2-alpha-mannosidase was cloned from a cDNA library prepared from solid-state cultured Aspergillus oryzae. The fmanIB cDNA consisted of 1530 nucleotides and encoded a protein of 510 amino acids in which all consensus motifs of the class I alpha-mannosidase were conserved. Expression of the full length of 1,2-alpha-mannosidase cDNA by the Aspergillus host, though it has rarely been done with other filamentous-fungal mannosidase, was successful with fmanIB and caused an increase in both intracellular and extracellular mannosidase activity. The expressed protein (FmanIBp) specifically hydrolyzed 1,2-alpha-mannobiose with maximal activity at a pH of 5.5 and a temperature of 45 degrees C. With Man(9)GlcNAc(2) as the substrate, Man(5)GlcNAc(2) finally accumulated while hydrolysis of the 1,2-alpha-mannose residue of the middle branch was rate-limiting. To examine the intracellular localization of the enzyme, a chimeric protein of FmanIBp with green fluorescent protein was constructed. It showed a dotted fluorescence pattern in the mycelia of Aspergillus, indicative of the localization in intracellular vesicles. Based on these enzymatic and microscopic results, we estimated that FmanIBp is a fungal substitute for the mammalian Golgi 1,2-alpha-mannosidase isozyme IB. This and our previous report on the presence of another ER-type mannosidase in A. oryzae (Yoshida et al., 2000) support the notion that the filamentous fungus has similar steps of N-linked glycochain trimming to those in mammalian cells.

Amino Acid Sequence↗
PubMed1999

Expression of a major piroplasm surface protein of Theileria sergenti in sporozoite stage.

A 32 kilodalton major piroplasm surface protein (MPSP) is expressed abundantly on the surface of intraerythrocytic piroplasms of Theileria sergenti and is considered to be a candidate antigen for vaccine development against piroplasmosis. In this study, transcripts of MPSP gene were detected in an expression cDNA library prepared from T. sergenti-infected tick salivary glands. Expression of MPSP in the sporozoite stage was also confirmed by immunoblot analysis. Its expression at the sporozoite and intraerythrocytic stages gives scope for possible induction of protective immunity being targeted at both stages by immunization with recombinant MPSP.

Animals↗
PubMed2007

Breast cancer-associated gene 3 (BCA3) is a novel Rac1-interacting protein.

UNLABELLED: BCA3 was identified in a yeast two-hybrid screen as a novel Rac1-interacting partner in osteoclasts. BCA3 binds directly to Rac and, in vivo, binds GTP-Rac but not GDP-Rac. Perinuclear co-localization of BCA3 and Rac1 is observed in CSF-1-treated osteoclasts. Overexpression of BCA3 attenuates CSF-1-induced cell spreading. We conclude that BCA3 regulates CSF-1-dependent Rac activation. INTRODUCTION: Rac1, a ubiquitously expressed GTPase, is a mediator of colony-stimulating factor 1 (CSF-1)-dependent actin remodeling in osteoclasts. Because the role of Rac in osteoclasts has not been fully defined, we undertook a yeast two-hybrid screen to identify Rac-interacting partners in these cells. MATERIALS AND METHODS: A yeast two-hybrid screen was undertaken using a cDNA library prepared from osteoclast-like cells as prey and either native Rac1 or constitutively active Rac1 (Q61L) as bait. Radiolabeled breast cancer-associated gene 3 (BCA3) protein constructs were generated in vitro using rabbit reticulate lysates and used in vitro binding assays with Rac1. In vivo binding was assessed using myc-tagged Rac1(Q61L) and HA-tagged BCA3. PBD pull-down assays were used to determine if GTP-loaded Rac1 preferentially bound BCA3. Co-localization of Rac1 and BCA3 in osteoclasts was assessed using confocal immunofluorescence. The functional significance of the BCA3-Rac1 interaction was assessed by examining the effect of overexpressing BCA3 in RAW 264.7 cells on the subsequent spreading response to CSF-1. RESULTS: One of three positive clones from the wildtype Rac1 screen and all three positive clones from the Rac1(Q61L) screen encoded the same protein, BCA3. BCA3 expression in osteoclasts was confirmed by RT-PCR and immunocytochemistry. BCA3 bound directly to Rac1 in vitro. Deletional analysis indicated that amino acids 76-125 in BCA3 are important for its ability to bind Rac. In vivo association of the two proteins was shown by co-immunoprecipitation of BCA3 and Rac1. Only GTP-bound-Rac but not GDP-bound Rac could interact with BCA3 in vivo. Confocal immunocytochemistry showed perinuclear co-localization of BCA3 and Rac1 in CSF-1-treated neonatal rat osteoclasts but not in resting osteoclasts. Overexpression of BCA3 markedly attenuated the spreading response to CSF-1 in RAW 264.7 cells. CONCLUSIONS: These data establish that BCA3 is a novel Rac1-interacting protein and suggest that it may influence the ability of Rac1 to remodel the actin cytoskeleton.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Duocalins: engineered ligand-binding proteins with dual specificity derived from the lipocalin fold.

Anticalins comprise a novel class of receptor proteins with predetermined ligand specificities which were engineered using the lipocalin fold. Attractive features of these artificial ligand-binding proteins include their small size and monomeric nature, being composed of a single polypeptide chain. Here we report the construction of a functional fusion protein from two independent anticalins, a so-called duocalin. The gene for the fusion protein was assembled from nucleotide sequences encoding an anticalin with fluorescein specificity on the one hand and an anticalin with digoxigenin specificity on the other. Both engineered lipocalins were previously selected from a random library prepared on the basis of the bilin-binding protein, a natural lipocalin abundant in insects. The corresponding fusion protein was expressed in a secretable form in E. coli cells and isolated from the periplasmic fraction using the Strep-tag method. The major fraction of the purified protein appeared to possess the proper pattern of altogether four disulphide bonds. The ligand-binding behaviour of the fusion protein was investigated both by solid phase ELISA and in fluorescence titration experiments. Our results demonstrate that the novel fusion protein has retained both ligand specificities. Up to now, dimerized ligand-binding proteins were mostly derived from recombinant antibody fragments. Compared with those constructs the duocalins, either with bispecific or with bivalent target recognition properties, should provide useful reagents for various purposes in biotechnology.

Carrier Proteins↗
PubMedDate not supplied

Changes in phenylalanine ammonia-lyase activity and gene expression during storage of asparagus spears.

A cDNA clone coding phenylalanine ammonia-lyase (PAL) was isolated from a cDNA library prepared from asparagus spears (Asparagus officinalis L. cv. Welcome) using the reverse transcription-polymerase chain reaction (RT-PCR). The partial cDNA clone encoded an mRNA of 527 bp and the derived amino acid sequence was found highly homologous to PAL from rice, maize and barley. Northern blot analysis showed an increase of pAS-PAL mRNA until 24 h at 20 degrees C, which coincided well with PAL activity and fiber development, suggesting that the increase is a response to the wounding associated with harvest.

Amino Acid Sequence↗
PubMed2000

Taenia solium: molecular cloning and serologic evaluation of 14- and 18-kDa related, diagnostic antigens.

We are attempting to design a simpler assay based on synthetic or recombinant antigens to replace the labor-intensive enzyme-linked immunoelectrotransfer blot (EITB-C), which is currently used to diagnose Taenia solium cysticercosis. From the lentil lectin-bound fraction of cyst glycoproteins (the LLGP fraction used in the EITB-C), we previously identified and purified 2 related polypeptides of 14- and 18-kDa that demonstrated diagnostic usefulness. Using degenerate oligonucleotide primers corresponding to amino acid sequences of these polypeptides and a cDNA library prepared from T. solium cysticerci, we amplified cDNA clones that represent the 14- and 18-kDa polypeptides. These clones share sequence homology at the nucleotide and amino acid levels. Synthetic polypeptides that represented the full-length, mature proteins (sTS14 and sTS18) were assessed for serologic potential using an ELISA. sTS14, but not sTS18, demonstrated utility as a diagnostic antigen. sTS14 was recognized by antibodies in a majority of the sera from patients with cysticercosis and none of the sera from persons with other helminth infections or uninfected human sera. Furthermore, polyclonal antibodies to sTS14 reacted with 6 discrete proteins present in the LLGP cyst fraction, suggesting that TS14 is a subunit of other previously described antigens used for diagnosing cysticercosis.

Amino Acid Sequence↗
PubMed2024

Reflective Evaluation of Next-Generation Sequencing Data during Early Phase Detection of the Delta Variant.

During the SARS-CoV-2 pandemic, next-generation sequencing (NGS) technologies like the Ion Torrent S5 and Illumina MiSeq, alongside advanced software, improved genomic surveillance in South Africa. This study analysed anonymized samples from the Eastern Cape using Genome Detective and NextClade, showing Ion Torrent S5 and Illumina MiSeq success rates of 96% and 94%, respectively. The study focused on genomic coverage (above 80%) and mutation detection (below 100), with the Ion Torrent S5 achieving 99% coverage compared to Illumina MiSeq's 80%, likely due to different primers used in amplification. The Ion Torrent S5 was more effective in sequencing varied viral loads, whereas Illumina MiSeq had difficulties with lower loads. Both platforms were adept at identifying clades, successfully differentiating between Beta (<45%) and Delta variants (<30%), despite minor discrepancies in assignments due to Illumina MiSeq's lower coverage, leading to a failure rate of up to 6%. Manual library preparation showed similar sample processing and clade identification capabilities for both platforms. However, differences in sequencing duration (3.5 vs. 36 hours), automation level, genomic coverage (80% vs. 99%), and viral load compatibility were noted, highlighting each platform's unique advantages and challenges in SARS-CoV-2 genomic surveillance. In conclusion, the Illumina MiSeq and Ion Torrent S5 platforms are both efficacious in executing whole-genome sequencing (WGS) via amplicons, facilitating precise, accurate, and high-throughput examinations of SARS-CoV-2 viral genomes. However, it is important to note the existence of disparities in the quality of data produced by each platform. Each system offers unique benefits and limitations, rendering them viable choices for the genomic surveillance of SARS-CoV-2.

Illumina MiSeq↗
PubMed1995

In situ hybridization shows Dmp1 (AG1) to be a developmentally regulated dentin-specific protein produced by mature odontoblasts.

Acidic phosphorylated proteins are prominent constituents of the extracellular matrix of bone and dentin. It has been postulated that they may have important structural and regulatory roles in the process of tissue mineralization. Studies of a cDNA library, prepared from cells of the rat incisor odontoblast-pulp complex of 3 week old Sprague-Dawley rats, led to the identification of a serine-rich acidic protein, designated AG1, which appeared to be a dentin matrix component. In order to determine which cells of the odontoblast-pulp complex were responsible for the making of AG1, in situ hybridization was carried out using digoxigenin-labeled probes. The full length AG1 cDNA was subcloned into the pBluescript vector, which contains two strong promoters, T3 and T7. The sense and antisense complementary RNA (cRNA) hybridization probes were prepared by in vitro transcription using T3 and T7 polymerases in the presence of 11-dUTP. Incisor sections were obtained from rat embryos at days 16, and 20, and newborns at days 2 and 5. No AG1 mRNA was detected in the embryonic sections, but digoxigenin labeling was evident in odontoblasts secreting mineralizing dentin at postnatal days 2 and 5. Sense probes showed no hybridization. Pulp cells, Meckel's cartilage, and alveolar bone were free of hybridization with the antisense probe. Unexpectedly, a low level of digoxigenin staining was seen in the cytoplasm of secretory ameloblasts, but not in the preameloblasts, stratum intermedium or stellate reticulum of the enamel organ. These data show that AG1 expression is regulated developmentally and is restricted to secretory stage mature odontoblasts.

Alveolar Process↗
PubMed1991

Murine embryonic spinal cord and adult testis Hox-1.4 cDNAs are identical 3' to the homeo box.

The Hox-1.4 murine homeo box containing gene is expressed in both embryonic spinal cord and adult testis where different sized Hox-1.4 transcripts are detected. In order to compare the sequences of these transcripts, Hox-1.4 cDNA clones were isolated from cDNA libraries prepared from mouse embryonic spinal cord and mouse adult testis. Sequence analysis showed these clones to be identical and extend from an internal EcoRI site at position 55 in the homeo box to a poly (A) stretch 20 nucleotides 3' to the polyadenylation signal. These data indicate that murine Hox-1.4 mRNAs expressed in embryonic spinal cord and adult testis are identical 3' to the homeo box and utilize the same polyadenylation signal.

Amino Acid Sequence↗
PubMed1992

The sea urchin erg homolog defines a highly conserved erg-specific domain.

A genomic clone, isolated from a phage library prepared from the DNA of the sea urchin Lytechinus variegatus, was shown by sequence analysis to be a homolog of the ets family genes, ERG and Fli-1. It contains an open reading frame of which the coding region begins at a consensus 3' splice site and extends for 173 amino acid residues. The first 84 amino acids are homologous with all members of the ets gene family, while the remainder of the sequence is only homologous with the human ERG and murine Fli-1 genes. This latter region, designated R, represents a highly conserved erg-specific domain.

Amino Acid Sequence↗
PubMed1992

Secondary structure analysis identifies a putative mouse protein demonstrating similarity to the repeat units found in CDC4, the G protein beta subunits and related proteins.

The predicted protein product of an anonymous clone isolated from a cDNA library prepared from 12 day post coitum (p.c) embryonic mouse heart tissue demonstrated the same segmental repeats previously identified in the cell division control protein, CDC4 and the G protein beta 1 subunit. A search of the protein database subsequently identified three other classes of protein containing the repeat. Secondary structure analyses performed on the repeat sequences revealed a high degree of conservation suggesting that the repeat motif performs a specific function in a diverse range of proteins.

Amino Acid Sequence↗
PubMed1997

Nucleotide sequence of equine MxA cDNA.

A 2.6 kb cDNA species has been isolated from a cDNA library prepared from interferon-alpha stimulated equine peripheral blood leucocytes and the nucleotide sequence determined. The cDNA has a single open reading frame potentially encoding a 660 amino acid polypeptide showing a high degree of homology with known mammalian Mx proteins, including the possession of three consensus GTP-binding motifs. The protein has a calculated pI = 6.1 and in accordance with proposed nomenclature we have designated it equine MxA.

Amino Acid Sequence↗