Isolation, Characterization of the hva1 Gene from Syrian Barley Varieties and Cloning into a Binary Plasmid Vector

MUC1 mucin is a transmembrane glycoprotein that is highly expressed in various cancer cell lines and is also present in most of the glandular epithelial cells including the airway. Although the presence of numerous phosphorylation sites in its cytoplasmic domain suggests its potential role as a receptor, the unavailability of a ligand for MUC1 mucin has limited our understanding of its function. In this paper, we tried to circumvent this problem by constructing a chimeric receptor containing the cytoplasmic domain of MUC1 mucin, which can be phosphorylated on activation. To this end, we constructed a chimeric plasmid vector (pCD8/MUC1) by replacing the extracellular and transmembrane domains of human MUC1 mucin with those of human CD8. Transient transfection of the vector into COS-7 cells resulted in expression of the chimeric receptor on the surface of the COS-7 cells as judged by immunologic assays with various antibodies as well as by fluorescence-activated cell-sorting analysis. Treatment of the transfected COS-7 cells with an anti-CD8 antibody resulted in a significant increase in phosphorylation of tyrosine moieties of the chimeric receptor. This chimeric receptor will serve as a powerful tool in elucidating the signaling mechanism as well as the functional role of MUC1 mucin in the airway.

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Development of a Simvastatin Selection Marker for a Hyperthermophilic Acidophile, Sulfolobus islandicus

Applied and Environmental Microbiology ◽

10.1128/aem.06095-11 ◽

2011 ◽

Vol 78 (2) ◽

pp. 568-574 ◽

Cited By ~ 33

Author(s):

Tao Zheng ◽

Qihong Huang ◽

Changyi Zhang ◽

Jinfeng Ni ◽

Qunxin She ◽

...

Keyword(s):

Selectable Marker ◽

Gene Knockout ◽

Plasmid Vector ◽

Selection Marker ◽

Content Type ◽

Sulfolobus Islandicus ◽

Gene Coding ◽

High Level ◽

Coa Reductase

ABSTRACTWe report here a novel selectable marker for the hyperthermophilic crenarchaeonSulfolobus islandicus. The marker cassette is composed of thesac7dpromoter and thehmggene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Psac7d-hmg), which confers simvastatin resistance to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting thepyrEFgene of the expression vector pSeSD for Psac7d-hmgwith which theSulfolobusexpression plasmids pSSRlacS, pSSRAherA, and pSSRNherA were constructed. Characterization ofSulfolobustransformants carrying pSSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His6-tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers (pyrEFandhmg) was subsequently exploited for genetic analysis. AherAmerodiploid strain ofS. islandicuswas constructed usingpyrEFmarker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (ΔherA) cells generated from theherAmerodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability ofS. islandicus. To our knowledge, this is the first application of an antibiotic selectable marker in genetic study for a hyperthermophilic acidophile and in the crenarchaeal lineage.

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Molecular Characterization of a Phage-Encoded Resistance System in Lactococcus lactis

Applied and Environmental Microbiology ◽

10.1128/aem.65.5.1891-1899.1999 ◽

1999 ◽

Vol 65 (5) ◽

pp. 1891-1899 ◽

Cited By ~ 42

Author(s):

Stephen McGrath ◽

Jos F. M. L. Seegers ◽

Gerald F. Fitzgerald ◽

Douwe van Sinderen

Keyword(s):

Dna Replication ◽

Topoisomerase I ◽

Plasmid Vector ◽

Dna Interaction ◽

Open Reading Frames ◽

Phage Resistance ◽

High Copy Number Plasmid ◽

Specific Fragment ◽

Reading Frames

ABSTRACT A specific fragment of the genome of Tuc2009, a temperate lactococcal bacteriophage, was shown to contain several open reading frames, whose deduced protein products exhibited similarities to proteins known to be involved in DNA replication and modification. In this way, a putative single-stranded binding protein, replisome organizer protein, topoisomerase I, and a methylase were identified. When the genetic information coding for the putative replisome organizer protein of Tuc2009, Rep2009, was supplied on a high-copy-number plasmid vector, it was shown to confer a phage-encoded resistance (Per) phenotype on its lactococcal host UC509.9. The presence of this recombinant plasmid was shown to cause a marked reduction in Tuc2009 DNA replication, suggesting that the observed phage resistance was due to titration of a factor, or factors, required for Tuc2009 DNA replication. Further experiments delineated the phage resistance-conferring region to a 160-bp fragment rich in direct repeats. Gel retardation experiments, which indicated a protein-DNA interaction between this 160-bp fragment and the Rep2009protein, were performed. UC509.9 strains harboring plasmids with randomly mutated versions of this fragment were shown to display a variable phage resistance phenotype, depending on the position of the mutations.

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Development and characterization of an infectious cDNA clone of the virulent Bucyrus strain of Equine arteritis virus

Journal of General Virology ◽

10.1099/vir.0.82415-0 ◽

2007 ◽

Vol 88 (3) ◽

pp. 918-924 ◽

Cited By ~ 38

Author(s):

Udeni B. R. Balasuriya ◽

Eric J. Snijder ◽

Hans W. Heidner ◽

Jianqiang Zhang ◽

Jessika C. Zevenhoven-Dobbe ◽

...

Keyword(s):

Reverse Genetics ◽

Severe Disease ◽

Plasmid Vector ◽

High Titre ◽

Equine Arteritis Virus ◽

Progeny Virus ◽

Cdna Clones ◽

Virulence Determinants ◽

Severity Of The Disease

Strains of Equine arteritis virus (EAV) differ in the severity of the disease that they induce in horses. Infectious cDNA clones are potentially useful for identification of genetic determinants of EAV virulence; to date, two clones have been derived from a cell culture-adapted variant of the original (Bucyrus) isolate of EAV, and it has previously been shown that recombinant virus derived from one of these (rEAV030) is attenuated in horses. A complete cDNA copy of the genome of the virulent Bucyrus strain of EAV has now been assembled into a plasmid vector. In contrast to rEAV030, recombinant progeny virus derived from this clone caused severe disease in horses, characterized by pyrexia, oedema, leukopenia, high-titre viraemia and substantial nasal shedding of virus. The availability of infectious cDNA clones that produce recombinant viruses of different virulence to horses will facilitate characterization of the virulence determinants of EAV through reverse genetics.

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Beta thalassemia in Melanesia: association with malaria and characterization of a common variant (IVS-1 nt 5 G----C)

Blood ◽

10.1182/blood.v72.1.9.bloodjournal7219 ◽

1988 ◽

Vol 72 (1) ◽

pp. 9-14 ◽

Cited By ~ 1

Author(s):

AV Hill ◽

DK Bowden ◽

DF O'Shaughnessy ◽

DJ Weatherall ◽

JB Clegg

Keyword(s):

Oligonucleotide Probe ◽

Globin Gene ◽

Plasmid Vector ◽

New Guinea ◽

Beta Thalassemia ◽

Beta Globin ◽

Beta Globin Gene ◽

Intron 1 ◽

High Incidence

Data on the distribution of beta thalassemia among over 6,000 Melanesians reveals a major difference in the carrier rates between populations in the malarious coastal regions of New Guinea and those living in the historically malaria-free Highlands. The island of Maewo in Vanuatu has a particularly high incidence of beta + thalassemia associated with a single restriction enzyme haplotype. Direct cloning into a plasmid vector and sequence analysis demonstrate that the mutation is a G to C transversion at position 5 of intron 1 of the beta- globin gene. Oligonucleotide probe surveys indicate that this variant accounted for all cases of beta thalassemia studied from Maewo. It is also common in coastal Papua New Guinea where haplotype and oligonucleotide probe data suggest that the molecular basis of beta thalassmia is more heterogeneous.

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Development of Transgenic Papaya throughAgrobacterium-Mediated Transformation

International Journal of Genomics ◽

10.1155/2013/235487 ◽

2013 ◽

Vol 2013 ◽

pp. 1-5 ◽

Cited By ~ 6

Author(s):

Md. Abul Kalam Azad ◽

Md. Golam Rabbani ◽

Latifah Amin ◽

Nik Marzuki Sidik

Keyword(s):

Agrobacterium Tumefaciens ◽

Plant Regeneration ◽

Carica Papaya ◽

Zygotic Embryo ◽

Plasmid Vector ◽

Vector System ◽

Transgenic Papaya ◽

Selection Medium ◽

Binary Plasmid ◽

Final Selection

Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation withAgrobacterium tumefaciensLBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker andβ-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated withAgrobacterium tumefacienson regeneration medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime + 50 mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls ofCarica papayacv. Shahi showed the highest positive GUS activities compared toCarica papayacv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls ofC. papayacv. Shahi andC. papayacv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformedC. papayacv. Shahi also showed the maximum number of plant regeneration compared to that ofC. papayacv. Ranchi.

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Production of Recombinant Human Creatine Kinase (r-hCK) Isozymes by Tandem Repeat Expression of M and B Genes and Characterization of r-hCK-MB

Clinical Chemistry ◽

10.1093/clinchem/47.3.471 ◽

2001 ◽

Vol 47 (3) ◽

pp. 471-476 ◽

Cited By ~ 4

Author(s):

Yoshiko Sunahara ◽

Kohji Uchida ◽

Toshio Tanaka ◽

Hirokazu Matsukawa ◽

Manabu Inagaki ◽

...

Keyword(s):

Creatine Kinase ◽

Myocardial Injury ◽

Specific Activity ◽

Plasmid Vector ◽

Molecular Size ◽

Serum Creatine Kinase ◽

Liquid Form ◽

Control Material ◽

Creatine Kinase Mb

Abstract Background: Serum creatine kinase-MB isoenzyme (CK-MB) is widely used as a marker of myocardial injury. We prepared recombinant human CK (r-hCK) MB isoenzyme and examined its potential for use as a control material for assay of CK-MB in serum. Methods: cDNAs encoding CK-M and CK-B subunits were inserted into the same plasmid vector, followed by transformation of Escherichia coli. The resulting three types of CK isoenzymes were purified by conventional chromatography. Results: The ratio of MB to MM to BB was 50:40:10 on the basis of CK activity. Highly purified CK-MB with a specific activity of 533 U/mg was produced in a yield of 5.7 mg/g of packed cells. Purified r-hCK-MB had the isoelectric point (pI 5.3) and molecular size (46 kDa for the subunit) of native CK-MB. Its immunoreactivity in an ELISA using antibody against native heart enzyme was similar to that of cardiac CK-MB. The r-hCK-MB retained >90% activity for at least 4 months at 11 °C in a delipidated serum matrix in a liquid form at a concentration of 118 U/L. Conclusions: r-hCK-MB shows key properties of the native cardiac isoenzyme and may be useful as a control and calibrator for serum assays of CK-MB.

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Beta thalassemia in Melanesia: association with malaria and characterization of a common variant (IVS-1 nt 5 G----C)

Blood ◽

10.1182/blood.v72.1.9.9 ◽

1988 ◽

Vol 72 (1) ◽

pp. 9-14 ◽

Cited By ~ 35

Author(s):

AV Hill ◽

DK Bowden ◽

DF O'Shaughnessy ◽

DJ Weatherall ◽

JB Clegg

Keyword(s):

Oligonucleotide Probe ◽

Globin Gene ◽

Plasmid Vector ◽

New Guinea ◽

Beta Thalassemia ◽

Beta Globin ◽

Beta Globin Gene ◽

Intron 1 ◽

High Incidence

Abstract Data on the distribution of beta thalassemia among over 6,000 Melanesians reveals a major difference in the carrier rates between populations in the malarious coastal regions of New Guinea and those living in the historically malaria-free Highlands. The island of Maewo in Vanuatu has a particularly high incidence of beta + thalassemia associated with a single restriction enzyme haplotype. Direct cloning into a plasmid vector and sequence analysis demonstrate that the mutation is a G to C transversion at position 5 of intron 1 of the beta- globin gene. Oligonucleotide probe surveys indicate that this variant accounted for all cases of beta thalassemia studied from Maewo. It is also common in coastal Papua New Guinea where haplotype and oligonucleotide probe data suggest that the molecular basis of beta thalassmia is more heterogeneous.

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Mechanisms Conferring Resistance to Pro-Apoptotic Cancer Gene Therapy

Journal of Cell Death ◽

10.4137/jcd.s4686 ◽

2011 ◽

Vol 4 ◽

pp. JCD.S4686

Author(s):

Shona T. Dougherty ◽

Graeme J. Dougherty

Keyword(s):

Tumor Cell ◽

Plasmid Vector ◽

Death Domain ◽

Tumor Cell Death ◽

Surface Receptors ◽

Novel Approach ◽

Cell Clones ◽

Ligand Expression

Recently, we have described a novel approach to the treatment of cancer that employs a series of vectors that encode surface expressed chimeric proteins in which the cytoplasmic death domain of Fas is fused in-frame to the extracellular domain of one of a number of cell surface receptors that recognize and bind various ligands that are differentially expressed within the tumor microenvironment. Although the majority of tumor cells transduced with such vectors are killed in the presence of the corresponding cognate ligand, a small percentage survive and in vivo may go on to repopulate a treated tumor. In order to understand the mechanisms employed by tumors to escape the cytotoxic effects of pro-apoptotic signals triggered via Fas, we isolated a large number of 293 tumor cell clones that survive following transfection with a plasmid vector encoding Flk-1/Fas, a chimeric receptor that induces tumor cell death in the presence of the pro-angiogenic cytokine VEGF. Characterization of Flk-1/Fas-positive clones revealed that while survival can most often be attributed simply to the down-regulation of VEGF ligand expression, in cells that express both receptor and ligand, other proteins involved in the regulation of apoptosis may be targeted. Specifically, a Flk-1/Fas-positive, VEGF-positive clone was identified in which expression of APAF-1 was almost completely abrogated.

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Cloning and characterization of chromosomally encoded cephalosporinase gene of Enterobacter cloacae

Canadian Journal of Microbiology ◽

10.1139/m86-061 ◽

1986 ◽

Vol 32 (4) ◽

pp. 301-309 ◽

Cited By ~ 4

Author(s):

Sylvain Guerin ◽

François Paradis ◽

Roger Guay

Keyword(s):

Escherichia Coli ◽

Molecular Mass ◽

Restriction Fragment ◽

Initial Level ◽

Plasmid Vector ◽

Enterobacter Cloacae ◽

Deletion Mapping ◽

Genomic Fragment ◽

E Coli

The cephalosporinase gene, cpa, which codes for an inducible class I chromosomal β-lactamase in Enterobacter cloacae was cloned on a fragment of 6.05 kilobase pairs inserted into plasmid pACYC184 and transferred into Escherichia coli HB101 recipient cells. The constructed hybrid plasmid, designated pGGQ101, carried a genomic fragment which retained its parental inducibility characteristics, although its expression level in transformed E. coli cells fell to 40–65% of its initial level in E. cloacae. The localization of the cpa gene on pGGQ101 plasmid was determined by Bal31 exonuclease deletion mapping and further confirmed by subcloning HindIII–AvaI restriction fragment on pMB9 plasmid vector. Labeling with [35S]methionine of pGGQ101 specified proteins in a minicell system showed that six or seven proteins are encoded by the insert. Two proteins with apparent molecular mass of 42 000 and 39 500 daltons, respectively, most probably represent the premature and mature cephalosporinase forms.

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