Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the ability of our cloned murine beta-casein locus to direct the exogenous gene expression in the milk of transgenic mice, the human t-PA variant mammary gland expression vector under the control of murine beta-casein gene regulatory elements was constructed, in which the human t-PA variant signal-pro peptide sequence was replaced with murine beta-casein signal peptide sequence and the human t-PA variant mature peptide cDNA was inserted into the second exon of beta-casein gene. The fusion gene was microinjected in the fertilized mice eggs. A total of 285 embryos were microinjected and transferred into 13 surrogate mother mice. Twelve positive transgenic mice were identified through PCR and Southern blot analysis among 42 new born mice. Human t-PA variant was expressed in the milk of 7 transgenic mice, the highest expression level attained to 3.6593 micrograms/ml. The results demonstrated that the murine beta-casein gene regulatory elements can direct the human t-PA variant gene successfully express in the milk of transgenic mice. It lays great foundation for the research on the beta-casein knock-in mice mammary gland bioreactor model construction.
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PMID:[Murine beta-casein gene sequences direct a human tissue plasminogen activator variant expressed in the milk of transgenic mice]. 1269 95

The sequences of the cDNA variant lumbrokinase and the genomic DNA of beta-casein gene expression regulatory elements have been submitted to the GenBank(R), DDBJ, EMBL and GSDB Nucleotide Sequence Databases under the accession numbers AY327442C and AY311384 respectively. In order to identify whether the product of a genetically modified or newly isolated eukaryotic gene has biological activity, the gene of interest is usually subcloned into a mammalian expression vector and then expressed in an in vitro system such as in tissue culture. In the present study an efficient in vivo system has been developed by employing a mammary-gland-specific vector and expressing the targeted protein in the lactating-goat mammary glands. In this system, the synthesized lumbrokinase cDNA variant (LK-m) and the tissue-type plasminogen activator (tPA) cDNA were selected as genes of interest and cloned downstream of the goat beta-casein regulatory sequence. The LK-m- and tPA-expressing plasmids were prepared to high purity and portions (100-800 microg) injected into lactating-goat mammary-gland tissues. High-level expression of the LK-m and tPA was detected, by a fibrin-agarose plate assay, as fibrin-lysis activity. A dynamic study showed that the specific expression starts immediately after injection, generally reaches peak in 6-9 h, persists for 20-24 h at peak and the expression lasts for 4 days with gradual decline in the amounts expressed. The potential use of this system as bioreactor for the production of biological proteins in place of transgenic animals is implicated from this study.
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PMID:A convenient method for the identification and expression of eukaryotic genes. 1515 42

A murine beta-casein gene targeting vector was constructed using the cloned genomic sequence. The short arm was 2.7 kb including mouse beta-casein gene 5' flanking sequence, exon1, intron1 and partial exon2. The long arm is a 3.4 kb fragment including partial intron2, exon3 approximately 7, intron3 approximately 6 and partial intron7. The human t-PA mutant cDNA was subcloned in the exon2 and fused with the mice beta-casein signal peptide sequence. The positive selective marker neo was placed in the middle of intron2. A tk negative selective marker was just outside the short arm. TC-1 ES cells were cultured and amplified on G418 resistant feeder layer. The linearized targeting construct DNAs of 45 microg were introduced into 2 x 10(7) ES cells by electroporation. Totally 192 ES clones were picked up after cultured in G418 and Gancyclovir for 7 days. The colonies were amplified and subjected to genomic DNA preparation. The genomic DNAs were digested with EcoR I and used for Southern blot analysis. A probe inside the 5' homologous arm was used for hybridization. A 9.8 kb band was found in wild type, but the band was shift down from 9.8 kb to 6.6 kb in the beta-casein gene targeted allele because a new EcoR I site was introduced into the exon2 along with the human t-PA mutant gene. There were 9.8 kb and 6.6 kb bands in targeted ES cells. One clone of targeted ES cells with correct homologous recombination events was obtained among 78 analyzed clones. It lays foundation for gene targeted mice making.
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PMID:[Study on a human tissue-type plasminogen activator mutant cDNA knocked-in the beta-casein gene site of murine ES cells]. 1549 Aug 72

We have expanded our recent on-line LC-MS platform for large peptide analysis to combine collision-induced dissociation (CID), electron-transfer dissociation (ETD), and CID of an isolated charge-reduced (CRCID) species derived from ETD to determine sites of phosphorylation and glycosylation modifications, as well as the sequence of large peptide fragments (i.e., 2000-10,000 Da) from complex proteins, such as beta-casein, epidermal growth factor receptor (EGFR), and tissue plasminogen activator (t-PA) at the low femtomol level. The incorporation of an additional CID activation step for a charge-reduced species, isolated from ETD fragment ions, improved ETD fragmentation when precursor ions with high m/z (approximately >1000) were automatically selected for fragmentation. Specifically, the identification of the exact phosphorylation sites was strengthened by the extensive coverage of the peptide sequence with a near-continuous product ion series. The identification of N-linked glycosylation sites in EGFR and an O-linked glycosylation site in t-PA were also improved through the enhanced identification of the peptide backbone sequence of the glycosylated precursors. The new strategy is a good starting survey scan to characterize enzymatic peptide mixtures over a broad range of masses using LC-MS with data-dependent acquisition, as the three activation steps can provide complementary information to each other. In general, large peptides can be extensively characterized by the ETD and CRCID steps, including sites of modification from the generated, near-continuous product ion series, supplemented by the CID-MS2 step. At the same time, small peptides (e.g., <or=2+ ions), which lack extensive ETD or CRCID fragmentation, can be characterized by the CID-MS2 step. A more targeted approach can then be followed in subsequent LC-MS runs to obtain additional information, if needed. Overall, the recently introduced ETD not only provides useful structural information, but also enhances the confidence of all assignments. The sensitivity of this new approach on the chromatographic time scale is similar to the previous Extended Range Proteomic Analysis (ERPA) using CID-MS2 and CID-MS3. The new LC-MS platform can be anticipated to be a useful approach for the comprehensive characterization of complex proteins.
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PMID:On-line LC-MS approach combining collision-induced dissociation (CID), electron-transfer dissociation (ETD), and CID of an isolated charge-reduced species for the trace-level characterization of proteins with post-translational modifications. 1790 Jan 80