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Query: UNIPROT:P06889 (Mol)
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1. The yield of fragment C was only modestly affected by Mut phenotype, and the site and type of integration event. 2. Fragment C accumulation was closely correlated with gene dosage and maximal expression levels required high gene copy number. 3. Yields were greatly increased in controlled fermenters, compared to shake-flasks, owing to the high cell density achieved and to an increased efficiency of induction (2.5- to 10-fold). 4. In fermenter inductions of a 14-copy strain, fragment C accumulated to 27% of total protein, giving an estimated yield of 12 g/L. 5. Considerable clonal variation in the level of expression occurred with transplacement transformants, and this was owing to a diversity of different integration events and to differences in gene copy number. 6. These multicopy transplacement events occur by in vivo circularization of transforming DNA fragments followed by repeated single-crossover integration. This is presumably a general phenomenon, such that it should be possible to obtain multicopy integrants from all P. pastoris transplacement transformations.
Methods Mol Biol 1998
PMID:Expression of tetanus toxin fragment C. 968 Jun 41

When maintained under continuous selection with the folate inhibitor, methotrexate, cultured Aedes albopicfus mosquito cells amplify an 200 kb region of DNA containing the dihydrofolate reductase gene. To determine whether the amplicon contained additional coding regions, Southern blots of cosmid clones containing amplicon DNA were probed separately with reverse-transcribed mRNA from methotrexate-sensitive and methotrexate-resistant cells. Cosmid pWED118 contained five EcoRI fragments (A, B, C, F, G) ranging in size from 2 to 5 kb that hybridized with cDNA from resistant cells. Of these, fragments B and F also hybridized to probe representing mRNA from sensitive cells, and all but fragment G hybridized to repetitive DNA from wild-type cells. Fragment G, which appeared to encode a low copy number gene in wild-type cells that subsequently became part of the dihydrofolate reductase amplicon in methotrexate-resistant cells, hybridized strongly to a 7 kb band and more weakly to bands measuring 9 and 3 kb on Northern blots containing RNA from resistant cells. Fragment G contained a 1203 bp open reading frame, encoding 401 amino acids homologous to synaptic vesicle protein SV2, a member of a transmembrane transporter family expressed in neural and endocrine cells. The region of homology included the six N-terminal transmembrane domains, an internal cytoplasmic loop, a seventh transmembrane domain, and most of an intravesicular loop. This partial sequence, which appears to correspond to a truncated gene generated during formation of the dihydrofolate reductase amplicon, provides a useful basis for more extensive characterization of an important gene family that may be the target of novel insecticides.
Insect Mol Biol 1998 Nov
PMID:The mosquito dihydrofolate reductase amplicon contains a truncated synaptic vesicle protein gene. 972 69

Solanum tuberosum L. DNA sequences containing simple sequence repeat (SSR) motifs were extracted from the EMBL database, cDNA and selectively enriched small-insert DNA libraries. Enrichment was achieved using either triplex affinity capture or single-strand hybridisation selection. One hundred and twelve primer pairs which successfully amplified products of the correct size from potato DNA were ultimately designed and synthesised. Ninety-eight of these revealed length polymorphisms in a panel of four diploid and two tetraploid clones, in agreement with the high information content of this class of markers which has been found in other species. All of the markers were assigned a quality score of 1-5 based on their potential usefulness. Eighty-nine loci from 65 of the primer pairs were located on two genetic linkage maps of potato by segregation analysis of the amplified alleles. Fifty-two of the SSRs were clearly single locus. The maps were aligned using 23 SSR primer pairs and 13 RFLP loci mapped in both populations. The markers described constitute a class which should replace Restriction Fragment Length Polymorphisms (RFLP) as the markers of choice for future genetic studies in potato. The sequences of the primers, together with other information on these markers are provided.
Mol Gen Genet 1998 Aug
PMID:Isolation, characterisation and mapping of simple sequence repeat loci in potato. 974 66

To extend our understanding of amplicon structure in methotrexate-resistant Mtx-5011-256 Aedes albopictus mosquito cells, we examined a series of cosmids containing genomic DNA corresponding to the unique 3'-end of the Type 1 dihydrofolate reductase amplicon. Cosmid pWED118 contained five EcoRI fragments ranging from 2 to 5 kb (A, B, C, F, G) that hybridized to cDNA from methotrexate-resistant cells. Of these, fragments B and F hybridized weakly to first-strand cDNA from sensitive cells and shared considerable sequence identity. Fragment G occurred twice in the map of pWED118; one copy mapped within a 10 kb BssHII core fragment from the Type I amplicon and a second copy mapped downstream in the 48 kb BssHII core fragment. Hybridization signals among fragments contained in overlapping cosmids suggested that a branch point defining two or more subtypes of the Type I amplicon occurs within or near the 10 kb BssHII genomic DNA fragment. A 1.8 kb sequence common to fragments B and F included an approximately 0.4 kb region that shared sequence similarities with a LINE element from Aedes aegypti and with a repeated sequence from Anopheles gambiae. In addition, these elements shared amino acid similarity to a reverse transcriptase from the nematode, Caenorhabditis elegans. Shared sequence between Aedes and Anopheles elements supports the hypothesis that an ancestral LINE-like element was active in mosquito genomes prior to the divergence of the subfamilies Culicinae and Anophelinae. The presence of homologies to LINE-like elements near a branch point in the dihydrofolate reductase amplicon is consistent with a possible role of repeated sequences in amplicon shortening.
Insect Biochem Mol Biol 1998 Aug
PMID:Similarities to a LINE element shared by Anopheline and Culicine mosquitos map to the distal end of dihydrofolate reductase amplicons in Aedes albopictus mosquito cells. 975 71

Carboxyflavins were found to be potent selective inhibitors of Taq DNA polymerase in a polymerase chain reaction. The inhibitions were dose-dependent, and complete inhibitions were observed at the concentration of 3.0 microM. Carboxyflavins were much less, or not sensitive to the DNA polymerases tested such as calf thymus DNA polymerase alpha, rat DNA polymerase beta, human immunodeficiency virus type 1 reverse transcriptase, the Klenow Fragment of E. coli DNA polymerase I and T4 DNA polymerase. To our knowledge, there is no other report of an agent that selectively inhibits only a thermophilic polymerase. Interestingly, the carboxyflavins were able to prevent DNA synthesis in the murine lymphoid leukemia cell line L1210 in vitro; almost complete inhibitory levels were achieved in the range of less than 10 microM.
Int J Mol Med 1998 Sep
PMID:Carboxyflavins, novel inhibitors of Taq DNA polymerase. 985 99

The 395-residue proteolytic fragment E3, which comprises the two most C-terminal LG modules of the mouse laminin alpha1 chain, was previously shown to contain major binding sites for heparin, alpha-dystroglycan and sulfatides. The same fragment (alpha1LG4-5) and its individual alpha1LG4 and alpha1LG5 modules have now been obtained by recombinant production in mammalian cells. These fragments were apparently folded into a native form, as shown by circular dichroism, electron microscopy and immunological assays. Fragment alpha1LG4-5 bound about five- to tenfold better to heparin, alpha-dystroglycan and sulfatides than E3. These binding activities could be exclusively localized to the alpha1LG4 module. Side-chain modifications and proteolysis demonstrated that Lys and Arg residues in the C-terminal region of alpha1LG4 are essential for heparin binding. This was confirmed by 14 single to triple point mutations, which identified three non-contiguous basic regions (positions 2766-2770, 2791-2793, 2819-2820) as contributing to both heparin and sulfatide binding. Two of these regions were also recognized by monoclonal antibodies which have previously been shown to inhibit heparin binding. The same three regions and a few additional basic residues also make major contributions to the binding of the cellular receptor alpha-dystroglycan, indicating a larger binding epitope. The data are also consistent with previous findings that heparin competes for alpha-dystroglycan binding.
J Mol Biol 1999 Mar 26
PMID:Analysis of heparin, alpha-dystroglycan and sulfatide binding to the G domain of the laminin alpha1 chain by site-directed mutagenesis. 1008 Aug 89

The ability to rapidly and reliably genotype mice is an important concern. Traditional methods employ labour intensive and time consuming techniques such as test crossing, gel electrophoresis or nucleic acid hybridization. Here we show that a new molecular biology workstation, the WAVE DNA Fragment Analysis System, can easily resolve polymerase chain reaction (PCR) products that have small differences in their lengths. Analysis is fully automated and takes less than 7 min per sample. Approximately 200 samples can be analysed per day with only minutes of hands-on time after completion of the PCR. Genotyping with the WAVE DNA Fragment Analysis System is a fast and efficient method with minimal manual intervention.
Mol Cell Probes 1999 Jun
PMID:A novel technique for rapid automated genotyping of DNA polymorphisms in the mouse. 1036 50

In the rat, variation in alcohol and benzodiazepine sensitivity has been correlated with an inherited variant of the GABA(A)alpha6 receptor. Our goal was to identify polymorphisms in the human GABA(A)alpha6 receptor gene and determine whether a variant of the receptor is associated with alcoholism. The GABA(A)alpha6 receptor gene coding region was screened in 80 unrelated patients with alcoholism using single strand conformational polymorphism analysis. For rapid genotyping, a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assay was developed. A relatively abundant amino acid substitution and three synonymous DNA substitutions were detected. The synonymous variants, 35A>G, 665A>G, and 1031G>C had rare-allele frequencies of 0.25, 0.02, and 0.47, respectively. The Pro385Ser substitution is located in the second intracellular domain of the receptor adjacent to a putative phosphorylation site. Pro385Ser has rarer allele frequencies of 3.3% and 4.8% in 196 Finnish alcoholic patients and 189 controls, respectively (P = NS). A naturally occurring non-conservative Pro385Ser was detected in the GABA(A)alpha6 receptor. The variant is not associated with alcoholism.
Mol Psychiatry 2000 May
PMID:Identification of a naturally occurring Pro385-Ser385 substitution in the GABA(A) receptor alpha6 subunit gene in alcoholics and healthy volunteers. 1088 35

Terminal Restriction Fragment Length Polymorphism (T-RFLP) or Fluorescent Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (FluRFLP) have made a significant impact on the way in which PCR products amplified from mixed community DNA extracts have been assessed. Technically, these approaches are essentially the same. PCR products are generated that contain at one 5' end label, typically a fluorescent moiety, that will be detected by a DNA sequencing machine. Upon digestion using a specific restriction endonuclease, labeled and unlabeled fragments are generated. This restriction endonuclease is chosen such that following this digestion, each labeled fragment corresponds to a different sequence variant. During electrophoretic separation, the DNA sequencing machine detects only these labeled fragments and therefore detects only the sequence variants. The aim of this article is to describe the protocols and demonstrate that this profiling can be performed using different DNA sequencing machines. The analysis and applications of this approach are also discussed.
Mol Biotechnol 2000 Nov
PMID:Terminal restriction fragment length polymorphism monitoring of genes amplified directly from bacterial communities in soils and sediments. 1125 10

Activation of caspases 3 and 9 is thought to commit a cell irreversibly to apoptosis. There are, however, several documented situations (e.g., during erythroblast differentiation) in which caspases are activated and caspase substrates are cleaved with no associated apoptotic response. Why the cleavage of caspase substrates leads to cell death in certain cases but not in others is unclear. One possibility is that some caspase substrates generate antiapoptotic signals when cleaved. Here we show that RasGAP is one such protein. Caspases cleave RasGAP into a C-terminal fragment (fragment C) and an N-terminal fragment (fragment N). Fragment C expressed alone induces apoptosis, but this effect could be totally blocked by fragment N. Fragment N could also block apoptosis induced by low levels of caspase 9. As caspase activity increases, fragment N is further cleaved into fragments N1 and N2. Apoptosis induced by high levels of caspase 9 or by cisplatin was strongly potentiated by fragment N1 or N2 but not by fragment N. The present study supports a model in which RasGAP functions as a sensor of caspase activity to determine whether or not a cell should survive. When caspases are mildly activated, the partial cleavage of RasGAP protects cells from apoptosis. When caspase activity reaches levels that allow completion of RasGAP cleavage, the resulting RasGAP fragments turn into potent proapoptotic molecules.
Mol Cell Biol 2001 Aug
PMID:Antiapoptotic signaling generated by caspase-induced cleavage of RasGAP. 1146 18


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