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Sökning: L773:0378 1119

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1.
  • Carlsson, Peter, et al. (författare)
  • Bacillus subtilis citM, the structural gene for dihydrolipoamide transsuccinylase: cloning and expression in Escherichia coli
  • 1987
  • Ingår i: Gene. - : Elsevier BV. - 1879-0038 .- 0378-1119. ; 61:2, s. 217-224
  • Tidskriftsartikel (refereegranskat)abstract
    • The 2-oxoglutarate dehydrogenase multienzyme complex is composed of three different subenzymes: 2-oxoglutarate dehydrogenase (E1o), dihydrolipoamide transsuccinylase (E2o), and dihydrolipoamide dehydrogenase (E3). Bacillus subtilis E1o and E2o are encoded by the citK and citM genes, respectively. A 3.4-kb BamHI DNA fragment containing citK and citM markers was isolated from a library of B. subtilis DNA in Escherichia coli. Functional E2o was expressed from the cloned DNA both in B. subtilis and E. coli. E2o had an apparent Mr of 60000 when expressed in E. coli. The B. subtilis E2o component complemented an E. coli E2o-defective mutant in vivo and in vitro. It is concluded that functional B. subtilis E2o can be produced in E. coli and can interact with E. coli and E1o and E3 to form an active chimeric enzyme complex.
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2.
  • Dueñas, M., et al. (författare)
  • A point mutation in a murine immunoglobulin V-region strongly influences the antibody yield in Escherichia coli
  • 1995
  • Ingår i: Gene. - 0378-1119. ; 158:1, s. 61-66
  • Tidskriftsartikel (refereegranskat)abstract
    • Recombinant DNA technology has made it possible to produce specific Fab and scFv antibody (Ab) fragments in prokaryotic host cells. Using vectors designed for periplasmic expression of encoded Ab fragments, we have been studying how the sequence and genetic localization of the light chain (L-chain) variable region gene of a mouse Ab (CB-Nm.1) determined the level of Ab production. The variable region was shown to belong to the VKV family and contained a previously unreported Ile72. Nine different Ab constructions were tested in monocistronic (scFv) or dicistronic (Fab) operons for their ability to affect the synthesis level of the L-chain. When the gene coding for the L-chain was located downstream from the Fd fragment gene, the substitution of codons encoding Ile by a codon encoding Thr was found to be crucial for any expression of the L-chain fragment. This was, however, not accompanied by an increase in L-chain-specific mRNA, neither was there any change in the size of the mRNA. The fact that the unmutated L-chain protein was produced from cells transformed with certain other constructions indicated that the protein as such was not incompatible with the prokaryotic environment. Together, this suggested that the translation process was involved in the restricted production of the L-chain. Thus, surprisingly small substitutions significantly affected the expression level, a fact that will have important implications on the library size expressed in prokaryotic hosts, including phagedisplayed Ab libraries.
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3.
  • Furebring, Christina, et al. (författare)
  • Evaluation of novel control elements by construction of eukaryotic expression vectors
  • 1997
  • Ingår i: Gene. - 0378-1119. ; 188:2, s. 191-198
  • Tidskriftsartikel (refereegranskat)abstract
    • A novel mammalian eukaryotic expression vector for the production of immunoglobulin heavy chain (IgH) genes has been designed. This expression vector contains the variable heavy chain (VH) promoter, the IgH intron enhancer (μE) and the IgH 3' enhancer (3'E). This construct, designated pTIF-1, was stably transfected into the myeloma cell line J558L. A fivefold increase in the expression level of a rearranged IgH gene was observed when using the pTIF-1 vector containing the 3'E compared to an expression vector lacking this enhancer. Interestingly, this positive effect on the expression level of the 3' enhancer appears to be position independent. The introduction of two recently identified Ig control elements, HS3 and HS4, to the vector cassette did not further elevate the expression level in the cell line tested. The pTIF-1 vector can be used for expression of any antibody specificity, using PCR amplification of the VDJ region of interest. Furthermore, the constant region can easily be exchanged, which further facilitates studies to dissect different effector functions of IgH constant genes.
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4.
  • Jirholt, P., et al. (författare)
  • Exploiting sequence space : Shuffling in vivo formed complementarity determining regions into a master framework
  • 1998
  • Ingår i: Gene. - 0378-1119. ; 215:2, s. 471-476
  • Tidskriftsartikel (refereegranskat)abstract
    • A novel approach in molecular design is presented, where in vivo formed complementarity determining regions (CDR) from antibody genes were shuffled into a specific framework region. A synthetic gene library of soluble VH-fragments was created and the complexity of the library was determined by sequencing. The synthetic genes were diverse and contained random combinations of CDR from different germlines. All CDR were randomised in one step and by using in vivo formed CDR, the length, sequence and combination were varied simultaneously.
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5.
  • Johanson, Urban, et al. (författare)
  • Comparison of the complete sequence of the str operon in Salmonella typhimurium and Escherichia coli
  • 1992
  • Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 120:1, s. 93-98
  • Tidskriftsartikel (refereegranskat)abstract
    • The nucleotide (nt) sequences of the str operon in Escherichia coli K-12 and Salmonella typhimurium LT2 were completed and compared at the nt and amino acid (aa) level. The order of conservation at the nt and aa level is rpsL greater than tufA greater than rpsG greater than f usA. A striking difference is that the rpsG-encoded ribosomal protein, S7, in E. coli K-12 is 23 aa longer than in S. typhimurium. The very low (0.18) codon adaptation index of this part of the E. coli K-12-encoding gene and the unusual stop codon (UGA) suggest that this is a relatively recent extension. A trend towards a higher G+C content in fusA (gene encoding elongation factor (EF)-G) and tufA (gene encoding EF-Tu) in S. typhimurium is noted. In fusA, nt substitutions at all three positions in a codon occur at a much higher frequency than expected from the number of nt substitutions in the gene, assuming they are random and independent events. An analysis of substitutions in this and other genes suggests that the triple substitutions in fusA, and some other genes, are the result of the sequential accumulation of individual mutations, probably driven by selection pressure for particular codons or aa.
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6.
  • Johanson, U, et al. (författare)
  • Fusidic acid-resistant mutants define three regions in elongation factor G of Salmonella typhimurium
  • 1994
  • Ingår i: Gene. - : Elsevier BV. - 0378-1119 .- 1879-0038. ; 143:1, s. 9-55
  • Tidskriftsartikel (refereegranskat)abstract
    • We have sequenced fusA, the gene coding for elongation factor G (EF-G), in 18 different mutants of Salmonella typhimurium selected as fusidic acid resistant (FuR). In addition, we have sequenced two previously described FuR mutants from Escherichia coli. In all cases, the resistance is due to a mutation in one of three separate regions in fusA. The three clusters of mutant sites superimpose on regions that are well conserved, suggesting that they are of a more general functional importance. To further classify the mutants, we have measured the minimal inhibitory concentration (MIC) for Fu and for two other antibiotics which interfere with translocation on the ribosome, kanamycin (Km) and spectinomycin (Sp). The levels of resistance to Fu for each of the mutants are significantly higher than in the wild type (wt), and vary by about one order of magnitude between the highest and the lowest. Most of the mutants are also more resistant to Km than the wt, although the level of resistance is low and the variation small. In contrast, about half of the mutants are more sensitive to Sp than the wt, with only one being more resistant. Only three of the twenty mutants behave like the wt with respect to the non-selected phenotypes, KmR and SpR.
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7.
  • Johansson, Tomas, et al. (författare)
  • A cluster of genes encoding major isozymes of lignin peroxidase and manganese peroxidase from the white-rot fungus Trametes versicolor
  • 1996
  • Ingår i: Gene. - : Elsevier BV. - 1879-0038 .- 0378-1119. ; 170:1, s. 31-38
  • Tidskriftsartikel (refereegranskat)abstract
    • A gene cluster from the white-rot basidiomycete Trametes (Coriolus) versicolor (Tv) PRL 572 containing three structural genes, LPGIII, LPGIV and MPGI, was characterized. The genes are arranged in the same transcriptional direction, within a 10-kb region, and found to encode quantitatively dominant isozymes of lignin peroxidase (LP) and manganese peroxidase (MP). The second gene in sequence, LPGIV, predicts a 346-amino-acid (aa) mature polypeptide (36.9 kDa, pI 4.31) which is identical with the partial aa sequence information available on the LP12 isozyme (43.1 kDa, pI 3.27). The first gene, LPGIII, encodes a 341-aa polypeptide (36.1 kDa, pI 3.93) which has not been identified at the protein level. However, the similarity to LPGIV would suggest that the predicted product is an LP-type enzyme. LPGIII andLPGIV are homologous to the tandemly arranged genes LPGII and LPGI, respectively, recently described by Jonsson and Nyman [Biochim. Biophys. Acta 1218 (1994) 408-412]. The homologous genes, LPGIII/LPGII and LPGIV/LPGI, are 99% and 96% identical in sequence, respectively, and are predicted to encode identical polypeptides, since base substitutions in the predicted exons are all synonymous. The third gene, MPGI, is different in intron-exon organization and predicted to be disrupted by five rather than six introns, as are the LP genes. The deduced polypeptide, 339 aa in size (35.9 kDa, pI 4.07), is identical with the partial aa sequence information available for isozyme MP2 (44.5 kDa, pI 3.09). The MPGI- and LPGIV-encoded polypeptides are 70% identical in sequence which suggests that MP and LP from Tv may be regarded as members of the same family within the plant peroxidase superfamily. Most importantly, this study identifies a gene encoding the MP2 isozyme, and further shows that genes encoding MP and LP can be closely linked on the chromosome and may be coordinately transcribed.
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8.
  • Lindqvist, A, et al. (författare)
  • The alpha1-microglobulin/bikunin gene : characterization in mouse and evolution
  • 1999
  • Ingår i: Gene. - 0378-1119. ; 234:2, s. 36-329
  • Tidskriftsartikel (refereegranskat)abstract
    • The 129Sv mouse gene coding for the alpha1-microglobulin/bikunin precursor has been isolated and characterized. The 11kb long gene contains ten exons, including six 5'-exons coding for alpha1-microglobulin and four 3'-exons encoding bikunin. Exon 7 also codes for the tribasic tetrapeptide RARR which connects the alpha1-microglobulin and bikunin parts. The sixth intron, which separates the alpha1-microglobulin and bikunin encoding parts, was compared in the human, mouse and a fish (plaice) gene. The size of this intron varies considerably, 6.5, 3.3 and 0.1kb in man, mouse and plaice, respectively. In all three genes, this intron contains A/T-rich regions, and retroposon elements are found in the first two genes. This indicates that this sixth intron is an unstable region and a hotspot for recombinational events, supporting the concept that the alpha1-microglobulin and bikunin parts of this gene are assembled from two ancestral genes. Finally, the nonsynonymous nucleotide substitution rate of the gene was determined by comparing coding sequences from ten vertebrate species. The results indicate that the alpha1-microglobulin part of the gene has evolved faster than the bikunin part.
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9.
  • Agaton, Charlotta, et al. (författare)
  • Gene expression analysis by signature pyrosequencing
  • 2002
  • Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 289:1-2, s. 31-39
  • Tidskriftsartikel (refereegranskat)abstract
    •  We describe a novel method for transcript profiling based on high-throughput parallel sequencing of signature tags using a non-gel-based microtiter plate format. The method relies on the identification of cDNA clones by pyrosequencing of the region corresponding to the 3'-end of the mRNA preceding the poly(A) tail. Simultaneously, the method can be used for gene discovery, since tags corresponding to unknown genes can be further characterized by extended sequencing. The protocol was validated using a model system for human atherosclerosis. Two 3'-tagged cDNA libraries, representing macrophages and foam cells, which are key components in the development of atherosclerotic plaques, were constructed using a solid phase approach. The libraries were analyzed by pyrosequencing, giving on average 25 bases. As a control, conventional expressed sequence tag (EST) sequencing using slab gel electrophoresis was performed. Homology searches were used to identify the genes corresponding to each tag. Comparisons with EST sequencing showed identical, unique matches in the majority of cases when the pyrosignature was at least 18 bases. A visualization tool was developed to facilitate differential analysis using a virtual chip format. The analysis resulted in identification of genes with possible relevance for development of atherosclerosis. The use of the method for automated massive parallel signature sequencing is discussed.
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10.
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