| 1. |
- Carlsson, Peter, et al.
(författare)
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Bacillus subtilis citM, the structural gene for dihydrolipoamide transsuccinylase: cloning and expression in Escherichia coli
- 1987
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Ingår i: Gene. - : Elsevier BV. - 1879-0038 .- 0378-1119. ; 61:2, s. 217-224
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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)
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A point mutation in a murine immunoglobulin V-region strongly influences the antibody yield in Escherichia coli
- 1995
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Ingår i: Gene. - 0378-1119. ; 158:1, s. 61-66
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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)
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Evaluation of novel control elements by construction of eukaryotic expression vectors
- 1997
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Ingår i: Gene. - 0378-1119. ; 188:2, s. 191-198
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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)
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Exploiting sequence space : Shuffling in vivo formed complementarity determining regions into a master framework
- 1998
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Ingår i: Gene. - 0378-1119. ; 215:2, s. 471-476
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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)
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Comparison of the complete sequence of the str operon in Salmonella typhimurium and Escherichia coli
- 1992
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Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 120:1, s. 93-98
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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)
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Fusidic acid-resistant mutants define three regions in elongation factor G of Salmonella typhimurium
- 1994
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Ingår i: Gene. - : Elsevier BV. - 0378-1119 .- 1879-0038. ; 143:1, s. 9-55
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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)
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A cluster of genes encoding major isozymes of lignin peroxidase and manganese peroxidase from the white-rot fungus Trametes versicolor
- 1996
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Ingår i: Gene. - : Elsevier BV. - 1879-0038 .- 0378-1119. ; 170:1, s. 31-38
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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)
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The alpha1-microglobulin/bikunin gene : characterization in mouse and evolution
- 1999
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Ingår i: Gene. - 0378-1119. ; 234:2, s. 36-329
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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)
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Gene expression analysis by signature pyrosequencing
- 2002
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Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 289:1-2, s. 31-39
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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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| 11. |
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| 12. |
- Altman, S, et al.
(författare)
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Catalysis by the RNA subunit of RNase P
- 1989
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Ingår i: Gene. - : Elsevier BV. - 0378-1119 .- 1879-0038. ; 82:1, s. 63-64
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Forskningsöversikt (refereegranskat)abstract
- RNase P, an enzyme that contains both RNA and protein components, cleaves tRNA precursors to generate mature 5' termini. The catalytic activity of RNase P resides in the RNA component, with the protein cofactor affecting the rate of the cleavage reaction. The reaction is also influenced by the nature of the tRNA substrate.
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| 13. |
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| 14. |
- Andersson, T., et al.
(författare)
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Shotgun sequencing and microarray analysis of RDA transcripts
- 2003
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Ingår i: Gene. - : Elsevier BV. - 0378-1119 .- 1879-0038. ; 310, s. 39-47
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Tidskriftsartikel (refereegranskat)abstract
- Monitoring of differential gene expression is an important step towards understanding of gene function. We describe a comparison of the representational difference analysis (RDA) subtraction process with corresponding microarray analysis. The subtraction steps are followed in a quantitative manner using a shotgun cloning and sequencing procedure that includes over 1900 gene sequences. In parallel, the enriched transcripts are spotted onto microarrays facilitating large scale hybridization analysis of the representations and the difference products. We show by the shotgun procedure that there is a high diversity of gene fragments represented in the iterative RDA products (92-67% singletons) with a low number of shared sequences (<9%) between subsequent subtraction cycles. A non redundant set of 1141 RDA clones were immobilized on glass slides and the majority of these clones (97%) gave repeated good fluorescent signals in a subsequent hybridization of the labelled and amplified original cDNA. We observed only a low number of false positives (<2%) and a more than twofold differential expression for 32% (363) of the immobilized RDA clones. In conclusion, we show that by random sequencing of the difference products we obtained an accurate transcript profile of the individual steps and that large-scale confirmation of the obtained transcripts can be achieved by microarray analysis.
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| 15. |
- Angerth, Tomas, et al.
(författare)
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Cloning and structural analysis of a gene encoding a mouse mastocytoma proteoglycan core protein : Analysis of its evolutionary relation to three cross hybridizing regions in the mouse genome
- 1990
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Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 93:2, s. 235-240
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Tidskriftsartikel (refereegranskat)abstract
- Serglycin (SGC) is a Ser-Gly-repeat-containing protein, used as proteoglycan core protein in the parietal yolk sac and in mast cell, where glycosaminoglycan side chains are attached to the serine residues of the repeat region. In this article, the structure of the gene SGC encoding mouse SGC is reported. The gene is divivided into three exons, which are all contained within a region of approximately 13 kb. Nucleotide (nt) sequence analysis was carried out on a region of 1.2 kb upstream from the first exon. The region containing the two promoters (active in parietal yolk sac and in mast cells, respectively) was analyzed for the presence of recognition sites for known DNA-binding proteins. A number of sequence closely related to known recognition sites were found in both promoters, and one consensus octamer-binding site could be identified in the putative yolk-sac promoter. Multiple regions in the mouse genome hybridizing with DNA fragments covering the Ser-Gly repeat region have previously been described, and it has been sugggested that these loci may represent other proteoglycan core proteins. Analysis of nt sequence was carried out on three out of the more than 15 of three regions present in the mouse genome. However, none of the clones analyzed was found to have any open reading frame in the region of cross-hybridization which possibly could code for a SGC protein. Instead, one of the clones was found to contain an exon encoding a highly basic protein, unrelated to SGC protein. Instead, one of the clones was found to contain an exon a highly basic protein, unrelated to SGC. Hence, no evidence ws found for a multigene family of Ser-Gly-repeat-containing proteoglycan-encoding genes.
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| 16. |
- Borang, S., et al.
(författare)
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Monitoring of the subtraction process in solid-phase representational difference analysis : characterization of a candidate drug
- 2001
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Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 271:2, s. 183-192
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we have applied and evaluated a modified cDNA representational difference analysis (RDA) protocol based on magnetic bead technology to study the molecular effects of a candidate drug (N,N'-diacetyl-L-cystine, DiNAC) in a model for atherosclerosis. Alterations in a gene expression profile induced by DiNAC were investigated in a human monocytic cell line (THP-1) differentiated into macrophage-like cells by lipopolysaccharide and further exposed to DiNAC. Three rounds of subtraction have been performed and the difference products from the second and third rounds have been characterized in detail by analysis of over 1000 gene sequences. Two protocols for analysis of the subtraction products have been evaluated, a shotgun approach and size selection of both distinct fragments and band-patterned smear. We demonstrate that in order to obtain a representative view of the most abundant gene fragments, the shotgun procedure is preferred. The obtained sequences were analyzed against the UniGene and Expressed Gene Anatomy Database (EGAD) databases and the results were visualized and analyzed with the ExProView software enabling rapid pair-wise comparison and identification of individual genes or functional groups of genes with altered expression levels. The identified differentially expressed gene sequences were comprised of both genes with known involvement in atherosclerosis or cholesterol biosynthesis and genes previously not implicated in these processes. The applicability of a solid-phase shotgun RDA protocol, combined with virtual chip monitoring, results in new starting points for characterization of novel candidate drugs.
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| 17. |
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| 18. |
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| 19. |
- Brodin, B., et al.
(författare)
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Cloning and characterization of spliced fusion transcript variants of synovial sarcoma : SYT/SSX4, SYT/SSX4v, and SYT/SSX2v. Possible regulatory role of the fusion gene product in wild type SYT expression
- 2001
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Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 268:02-jan, s. 173-182
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Tidskriftsartikel (refereegranskat)abstract
- The synovial sarcoma translocation t(X;18)(p11.2; q11.2) results in the fusion of the SYT gene on chromosome 18 to exon 5 of either SSX1 or SSX2 genes on chromosome X. We recently reported that the SSX4 gene is also involved in such a translocation. In the present investigation we cloned and sequenced the full-length cDNA of SYT/SSX1, SYT/SSX2 and SYT/SSX4 from synovial sarcoma tissues. We isolated a novel fusion transcript type Variant involving the fusion of SYT with exon 6 of the SSX4 gene (SYT/SSX4v). The SYT/SSX4 and SYT/SSX2 open reading frame also differed from previously reported SYT/SSX sequences by an in-frame addition of 93bp exon located in the junction between exon 7 and 8 of the SYT. This exon is identical to that reported for the murine SYT but has not been previously found in the human transcript. Two SYT transcripts, with and without the 93 bp exon, were co-expressed in mouse NIH3T3 cells, human malignant cells and human testis tissue, but not in human normal fibroblasts. Stable transfection of an SYT/SSX4 expression vector into human and murine cell lines correlated with a down-regulation of SYT transcripts. This was also observed in a synovial sarcoma tumor expressing SYT/SSX4. This suggests that the SYT/SSX fusion gene may regulate SYT expression from the normal allele and as such alter the normal function of SYT.
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| 20. |
- Danielsen, S., et al.
(författare)
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In vitro selection of enzymatically active lipase variants from phage libraries using a mechanism-based inhibitor
- 2001
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Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 272:02-jan, s. 267-274
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Tidskriftsartikel (refereegranskat)abstract
- The 'detergent lipase' Lipolasel((R)), from Thermomyces lanuginosa was subjected to a combinatorial protein engineering/phage display approach with the aim of identifying new enzyme variants with improved characteristics in the presence of detergents. First it was demonstrated that wild-type Lipolase((R)) could be produced in Escherichia coli retaining full activity and be displayed as an active enzyme fused to coat protein 3 on E. coli phage M13. A phagemid library designed to result in approximately two to three mutations per lipase gene was then constructed. Nine amino acids located in two regions close to the active site were targeted for randomization. Selections using a mechanism-based biotinylated inhibitor showed that phages displaying Lipolase((R)) could be specifically enriched from a population of control phages. Selections on a library phage stock in the presence of inhibitor and a commercial powder detergent resulted in a step-wise increase in the proportion of active clones. Analysis of 84 active clones revealed that they all expressed lipase activity, but with lower activities than that of a wild-type Lipolase((R))-producing clone. In six of the seven most active clones a wild-type serine at position 83 had been replaced by threonine, a substitution known to alter the substrate chain length preference of Lipolase((R)) variants. Furthermore, the selection had enriched enzyme variants with a high degree of conservatism in one of the variegated regions, suggesting that this region is important for enzymatic activity and that the designed selection procedure was relevant. The selected variants contained primarily basic amino acid residues within the other variegated region. Taken together, the described results show that selection protocols based on enzymatic activity can be designed for this enzyme class which should be of importance for future protein engineering attempts.
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| 21. |
- Emahazion, Tesfai, et al.
(författare)
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Identification of 167 Polymorphisms in 88 Genes from Candidate Neurodegeneration Pathways
- 1999
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Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 238:2, s. 315-324
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Tidskriftsartikel (refereegranskat)abstract
- Catalogs of intra-gene polymorphisms are needed to facilitate wide-ranging candidate gene-based association studies in common complex diseases. With this in mind, we have scanned multiple alignments of expressed sequence tags and of genomic DNA sequences (PCR products from four to eight unrelated individuals) to find polymorphisms in 195 genes putatively involved in neurodegenerative illness (including components of oxidative stress, excitotoxicity, inflammation, apoptosis and aging). This led to the discovery of 167 polymorphisms in 88 genes. These comprised 163 single nucleotide polymorphisms, one insertion/deletion, and three other variations involving more than one base pair. The polymorphisms were distributed in the exons (87), introns (70), and gene flanking regions (10). Of the exonic polymorphisms, 17 would give rise to non-synonymous amino acid substitutions. These findings now provide a valuable resource for association studies in neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease.
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| 22. |
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| 23. |
- Garcia, C. A., et al.
(författare)
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Mutation detection by pyrosequencing : sequencing of exons 5-8 of the p53 tumor suppressor gene
- 2000
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Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 253:2, s. 249-257
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Tidskriftsartikel (refereegranskat)abstract
- The ability to sequence a large number of DNA samples rapidly and accurately for detection of all possible mutations is a critical goal for the future application of DNA sequencing in routine medical diagnostics. Pyrosequencing(TM) is a non-electrophoretic real-time DNA sequencing method that uses the luciferase-luciferin light release as the detection signal for nucleotide incorporation into target DNA. For pyrosequencing of the human p53 gene, a nested multiplex PCR method for amplification of exons 5-8 was prepared. In order to investigate the use of pyrosequencing in mutation detection, DNA samples from skin-cancer patients were used. Two forms of nucleotide dispensation strategy were used, cyclic and programmed. Bi-directional pyrosequencing was performed and the overlapping sequence data produced were assembled to determine the sequence of the gene. Reliable sequencing data were obtained with both dispensation strategies, but some advantages were obtained using the programmed nucleotide dispensation approach, such as longer and faster reads, and fewer out-of-phase problems. The accuracy of pyrosequencing for detection of p53 mutations and allele distribution was demonstrated.
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| 24. |
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| 25. |
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