Molecular cloning of the cDNA for ligatin

1989 ◽  
Vol 93 (2) ◽  
pp. 227-232
Author(s):  
E.R. Jakoi ◽  
A.L. Brown ◽  
Y.S. Ho ◽  
R. Snyderman
Keyword(s):  
Consensus Sequence ◽  
Blot Hybridization ◽  
Rabbit Antiserum ◽  
Acceptor Site ◽  
Protein Fusion ◽  
Reading Frame ◽  
Pair Sequence ◽  
Partial Cdna ◽  
Partial Cdna Clone ◽  
Trafficking Receptor

We describe the first isolation and sequence of a partial cDNA clone encoding ligatin, a trafficking receptor for phosphoglycoproteins. The clone was isolated from a human U937 promonocyte lambda gt11 cDNA library using rabbit antiserum to rat ileal ligatin. RNA blot hybridization revealed that the intact receptor transcript in human cells is 2.4 kilobases (kb). DNA sequencing together with expression of protein fusion products in Escherichia coli demonstrated that the cloned segment begins with a 1.2 kb open reading frame potentially encoding a 7.5 × 10(3) Mr section of the 10 × 10(3) Mr receptor followed by a 3′ tail of 948 bases. The 225 bases of coding sequence correspond to the carboxyl region of ligatin and contain a potential acceptor site for asparagine-linked glycosylation. Neither a poly(A) sequence nor polyadenylation signal was found at the 3′ end of the clone. In the 3′ untranslated region there is a paired consensus sequence (TGAGnnnTTTTTCA) that is analogous to a conserved 12 base-pair sequence present in clusters in several growth-controlled mRNAs, including those for c-fos and beta-actin. The identity of this clone as ligatin was confirmed immunologically using antisera to an encoded fusion protein and three independent regions of its derived sequence. In addition, one of these antibodies produced a punctate immunofluorescence pattern within the cytosol of U937 cells in a similar fashion to anti-ligatin serum.

Cloning and characterization of an electrogenic Na/HCO3− cotransporter from the squid giant fiber lobe

2007 ◽  
Vol 292 (6) ◽  
pp. C2032-C2045 ◽  
Author(s):  
Peter M. Piermarini ◽  
Inyeong Choi ◽  
Walter F. Boron
Keyword(s):  
Giant Axon ◽  
Predicted Amino Acid Sequence ◽  
Giant Fiber ◽  
Reading Frame ◽  
Excitable Membranes ◽  
Current Voltage ◽  
Partial Cdna ◽  
Partial Cdna Clone ◽  
Current Voltage Relationship

The squid giant axon is a classic model system for understanding both excitable membranes and ion transport. To date, a Na+-driven Cl-HCO3− exchanger, sqNDCBE—related to the SLC4 superfamily and cloned from giant fiber lobe cDNA—is the only HCO3−-transporting protein cloned and characterized from a squid. The goal of our study was to clone and characterize another SLC4-like cDNA. We used degenerate PCR to obtain a partial cDNA clone (squid fiber clone 3, SF3), which we extended in both the 5′ and 3′ directions to obtain the full-length open-reading frame. The predicted amino-acid sequence of SF3 is similar to sqNDCBE, and a phylogenetic analysis of the membrane domains indicates that SF3 clusters with electroneutral Na+-coupled SLC4 transporters. However, when we measure pHi and membrane potential—or use two-electrode voltage clamping to measure currents—on Xenopus oocytes expressing SF3, the oocytes exhibit the characteristics of an electrogenic Na/HCO3− cotransporter, NBCe. That is, exposure to extracellular CO2/HCO3− not only causes a fall in pHi, followed by a robust recovery, but also causes a rapid hyperpolarization. The current-voltage relationship is also characteristic of an electrogenic NBC. The pHi recovery and current require HCO3− and Na+, and are blocked by DIDS. Furthermore, neither K+ nor Li+ can fully replace Na+ in supporting the pHi recovery. Extracellular Cl− is not necessary for the transporter to operate. Therefore, SF3 is an NBCe, representing the first NBCe characterized from an invertebrate.

scholarly journals Identification and Initial Characterization of the Murine Gammaherpesvirus 68 Gene M3, Encoding an Abundantly Secreted Protein

1999 ◽  
Vol 73 (5) ◽  
pp. 4524-4529 ◽  
Author(s):  
Victor van Berkel ◽  
Karen Preiter ◽  
Herbert W. Virgin ◽  
Samuel H. Speck
Keyword(s):  
Secreted Protein ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Murine Gammaherpesvirus ◽  
Amino Terminal ◽  
Partial Cdna ◽  
Rapid Amplification ◽  
Gammaherpesvirus 68 ◽  
Partial Cdna Clone ◽  
Murine Gammaherpesvirus 68

ABSTRACT Several viruses, including members of the gammaherpesvirus family, encode proteins that are secreted into the extracellular environment. We have identified an abundant 44-kDa secreted protein that is present in the supernatant of fibroblasts infected with murine gammaherpesvirus 68 (γHV68; also referred to as MHV-68) but not in that of uninfected fibroblasts. Sequence analysis of the amino terminus and of internal peptides revealed that this protein is encoded by the γHV68 M3 open reading frame (ORF). The amino-terminal sequence of the secreted protein starts at residue 25 of the M3 ORF, consistent with the first 24 residues functioning as a signal peptide. Northern blot analysis revealed a single abundant ∼1.4-kb early-late lytic transcript encoded by the M3 ORF. Analysis of a partial cDNA clone and subsequent analyses of products of rapid amplification of cDNA ends coupled with S1 nuclease protection assays demonstrate that the M3 protein is encoded by an unspliced, polyadenylated mRNA initiating at bp 7294 and terminating at bp 6007 of the γHV68 genome. The 3′ end of the M3 transcript maps 9 bp downstream of a consensus polyadenylation signal. Thus, the predicted M3 ORF is a functional gene that encodes an abundant secreted protein which is a candidate for interacting with host cellular receptors or cytokines.

scholarly journals First Report of Cucumber mosaic virus in Desert Rose in Taiwan

Plant Disease ◽  
2012 ◽  
Vol 96 (4) ◽  
pp. 593-593 ◽  
Author(s):  
Y. K. Chen ◽  
Y. S. Chang ◽  
Y. W. Lin ◽  
M. Y. Wu
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Chenopodium Quinoa ◽  
Rabbit Antiserum ◽  
Serological Tests ◽  
Reading Frame ◽  
First Report ◽  
Reverse Transcription Pcr ◽  
Wilt Virus ◽  
Line Patterns

Desert rose (Adenium obesum (Forssk.) Roem. & Schult, family Apocynaceae) is native to southeastern Africa, and is a perennial potted ornamental with colorful flowers that are popular in Taiwan. Symptoms of mosaic and chlorotic ringspots and line patterns on leaves were observed in July 2010, on all eight plants in a private garden in Potzu, Chiayi, Taiwan. Spherical virus particles with a diameter of approximately 28 nm were observed in crude sap prepared from symptomatic leaves. Virus culture was established by successive local lesion isolation in Chenopodium quinoa and was maintained in the systemic host Nicotiana tabacum van Hicks. The virus was mechanically transmissible to indicator plants and induced symptoms similar to those incited by Cucumber mosaic virus (CMV). Observed symptoms included local lesions on inoculated leaves of C. amaranticolor and systemic mosaic in Cucumis sativus, Lycopersicon esculentum, N. benthamiana, N. glutinosa, and N. rustica. On N. tabacum, necrotic ringspots developed on inoculated leaves followed by systemic mosaic. Serological tests using ELISA assays and western blotting indicated that the virus reacted positively to a rabbit antiserum prepared to CMV (4). Amplicons of an expected size (1.1 kb) were obtained in reverse transcription-PCR with primers specific to the 3′-half of CMV RNA 3 (3) using total RNA extracted from infected desert rose and N. tabacum. The amplified cDNA fragment was cloned and sequenced (GenBank Accession No. AB667971). Nucleotide sequences of the coat protein open reading frame (CP ORF) (657 nt) had 92 to 96% and 76 to 77% sequence identity to those of CMV in subgroups I (GenBank Accession Nos. NC_001440, D00385, M57602, D28780, and AB008777) and II (GenBank Accession Nos. L15336, AF127976, AF198103, and M21464), respectively. Desert roses infected by Tomato spotted wilt virus (TSWV) (1) and CMV (2) have been reported previously. In spite of the plants showing mosaic symptoms similar to that caused by CMV (2) and chlorotic ringspots and line patterns caused by TSWV (1), only CMV was detected in and isolated from these infected desert roses. However, the possibility of mixed infection of CMV and other viruses were not excluded in this research. To our knowledge, this is the first report of CMV infection in desert rose plants occurring in Taiwan. References: (1) S. Adkins and C. A. Baker. Plant Dis. 89:526, 2005. (2) C. A. Baker et al. Plant Dis. 87:1007, 2003. (3) Y. K. Chen et al. Arch. Virol. 146:1631, 2001. (4) Y. K. Chen and C. C. Yang. Plant Dis. 89:529, 2005.

Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells

1993 ◽  
Vol 13 (8) ◽  
pp. 5085-5098
Author(s):  
A M Carothers ◽  
G Urlaub ◽  
D Grunberger ◽  
L A Chasin
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Consensus Sequence ◽  
Donor Site ◽  
Chinese Hamster ◽  
Base Substitution ◽  
Acceptor Site ◽  
Rnase Protection ◽  
Single Base

Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a splice site beyond the consensus sequence.

scholarly journals Isolation and characterization of a vinculin cDNA from chick-embryo fibroblasts

1987 ◽  
Vol 245 (2) ◽  
pp. 595-603 ◽  
Author(s):  
G J Price ◽  
P Jones ◽  
M D Davison ◽  
B Patel ◽  
I C Eperon ◽  
...  
Keyword(s):  
Chick Embryo ◽  
Fusion Protein ◽  
Tyrosine Kinases ◽  
Consensus Sequence ◽  
Coli Strain ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Internal Repeat ◽  
Isolation And Characterization ◽  
Embryo Fibroblasts

A chick-embryo fibroblast lambda gt11 cDNA library was screened with affinity-purified antibodies to chick gizzard vinculin. One recombinant was purified to homogeneity and the fusion protein expressed in Escherichia coli strain C600. The fusion protein was unstable, but polypeptides that reacted with vinculin antibodies, but not non-immune immunoglobulin, were detected by Western blotting. The recombinant contained a single EcoRI fragment of 2891 bp with a single open reading frame. The deduced protein sequence could be aligned with that of six CNBr-cleavage peptides and two tryptic peptides derived from chicken gizzard vinculin. AUG-247 has tentatively been identified as the initiation codon, as it is contained within the consensus sequence for initiation sites of higher eukaryotes. The cDNA lacks 3′ sequence and encodes 74% of the vinculin sequence, presuming the molecular mass of vinculin to be 130,000 Da. Analysis of the deduced sequence showed no homologies with other protein sequences, but it does display a triple internal repeat of 112 amino acid residues covering residues 259-589. The sequences surrounding the seven tyrosine residues in the available sequence were aligned with the tyrosine autophosphorylation consensus sequence found in protein tyrosine kinases. Tyr-822 showed a good match to this consensus, and may represent one of the two major sites of tyrosine phosphorylation by pp60v-sre. Northern blots showed that the 2.89 kb vinculin cDNA hybridized to one size of mRNA (approx. 7 kb) in chick-embryo fibroblasts, chick smooth muscle and chick skeletal muscle. Southern blots revealed multiple hybridizing bands in genomic DNA.

Chromosomal Assignment of HepG2 3′-Directed Partial cDNA Sequences by Southern Blot Hybridization Using Monochromosomal Hybrid Cell Panels

Genomics ◽  
1994 ◽  
Vol 22 (1) ◽  
pp. 127-136 ◽  
Author(s):  
Atsushi Fukushima ◽  
Kousaku Okubo ◽  
Hidehiko Sugino ◽  
Naohiro Hori ◽  
Ryo Matoba ◽  
...  
Keyword(s):  
Southern Blot ◽  
Hybrid Cell ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Chromosomal Assignment ◽  
Cdna Sequences ◽  
Partial Cdna ◽  
Monochromosomal Hybrid

Isolation of a cDNA clone complementary to sequences for a 34-kilodalton protein which is a pp60v-src substrate

1984 ◽  
Vol 4 (9) ◽  
pp. 1935-1938
Author(s):  
H G Tomasiewicz ◽  
R Cook-Deegan ◽  
D M Chikaraishi
Keyword(s):  
Cdna Clone ◽  
Chicken Embryo ◽  
Kinase Activity ◽  
Chicken Embryo Fibroblast ◽  
Src Kinase ◽  
Cell Protein ◽  
Fibroblast Cells ◽  
Chicken Cell ◽  
Partial Cdna ◽  
Partial Cdna Clone

We have isolated a partial cDNA clone containing sequences complementary to a mRNA encoding a 34- to 36-kilodalton normal chicken cell protein which is a substrate for pp60v-src kinase activity. Using this 34-kilodalton cDNA clone as a probe, we determined that the size of the 34-kilodalton mRNA was 1,100 nucleotides and the level of the 34-kilodalton RNA was the same in various tissues of mature chickens but was significantly higher in chicken embryo fibroblast cells.

Cortisol alters carbonic anhydrase-mediated renal sulfate secretion

2003 ◽  
Vol 285 (6) ◽  
pp. R1430-R1438 ◽  
Author(s):  
Ryan M. Pelis ◽  
James E. Goldmeyer ◽  
Joseph Crivello ◽  
J. Larry Renfro
Keyword(s):  
Carbonic Anhydrase ◽  
Proximal Tubule ◽  
Plasma Membranes ◽  
Sequence Similarity ◽  
Renal Proximal Tubule ◽  
Protein Abundance ◽  
High Sequence Similarity ◽  
Partial Cdna ◽  
Partial Cdna Clone ◽  
Low Cortisol

Active transepithelial sulfate secretion rate by winter flounder renal proximal tubule epithelium in primary culture (fPTC) is dependent on intracellular carbonic anhydrase (CA) and enhanced by cortisol. To further evaluate this relationship, a partial cDNA clone (327 bp) of carbonic anhydrase II (CAII) with high sequence similarity to CAII from numerous species including fish, chicken, and human was obtained from fPTCs. The majority of CA activity and CAII protein was present in the cytosol of fPTCs; however, significant amounts of both (in addition to SDS-resistant CA activity, i.e., CAIV-like isoform) were present in concentrated plasma membranes. CAII from concentrated membranes migrated differently than purified CAII on nondenaturing PAGE gels, suggesting that CAII associates with another membrane component. Treatment of fPTCs with the cell-soluble CA inhibitor methazolamide (100 μM) caused a 58% reduction in active transepithelial SO42- secretion. fPTCs that were previously cultured under high-cortisol concentrations, when subjected to 5 days of low physiological levels of cortisol, had decreased CA activity (28%), CAII protein abundance (65%), and net active SO42- secretion (28%), with no effect on epithelial differentiation. Methazolamide and low-cortisol treatment in combination inhibited net active SO42- secretion 56%, which was not different than the effect of methazolamide treatment alone. These data indicate that cortisol directly increases renal CA activity, CAII protein abundance, and CA-dependent SO42- secretion in the marine teleost renal proximal tubule.

scholarly journals Effect of Intragenic Rearrangement and Changes in the 3′ Consensus Sequence on NSP1 Expression and Rotavirus Replication

2001 ◽  
Vol 75 (5) ◽  
pp. 2076-2086 ◽  
Author(s):  
John T. Patton ◽  
Zenobia Taraporewala ◽  
Dayue Chen ◽  
Vladimir Chizhikov ◽  
Melinda Jones ◽  
...  
Keyword(s):  
Cell Culture ◽  
Consensus Sequence ◽  
Active Role ◽  
Serial Passage ◽  
Selective Advantage ◽  
Wild Type ◽  
Reading Frame ◽  
Group A Rotaviruses ◽  
Group A ◽  
Dsrna Genome

ABSTRACT The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3′ termini ended with 5′-UGAACC-3′ instead of the 3′ consensus sequence 5′-UGACC-3′, which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3′ consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3′ consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3′ end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3′ consensus sequence.

scholarly journals Isolation of a cDNA clone complementary to sequences for a 34-kilodalton protein which is a pp60v-src substrate.

1984 ◽  
Vol 4 (9) ◽  
pp. 1935-1938 ◽  
Author(s):  
H G Tomasiewicz ◽  
R Cook-Deegan ◽  
D M Chikaraishi
Keyword(s):  
Cdna Clone ◽  
Chicken Embryo ◽  
Kinase Activity ◽  
Chicken Embryo Fibroblast ◽  
Src Kinase ◽  
Cell Protein ◽  
Fibroblast Cells ◽  
Chicken Cell ◽  
Partial Cdna ◽  
Partial Cdna Clone

We have isolated a partial cDNA clone containing sequences complementary to a mRNA encoding a 34- to 36-kilodalton normal chicken cell protein which is a substrate for pp60v-src kinase activity. Using this 34-kilodalton cDNA clone as a probe, we determined that the size of the 34-kilodalton mRNA was 1,100 nucleotides and the level of the 34-kilodalton RNA was the same in various tissues of mature chickens but was significantly higher in chicken embryo fibroblast cells.

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