A simplified protocol for fast plasmid DNA sequencing

Zimmermann, Jürgen; Voß, H.; Schwager, Christian; Stegemann, J.; Erfle, Holger; Stucky, K.; Kristensen, Tom; Ansorge, W.

doi:10.1093/nar/18.4.1067

Cited by 84 publications

(41 citation statements)

References 2 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The resulting fragments were cloned in the multiple cloning site of plasmid pBluescript, and the resulting plasmids transformed to E. coli DH5␣ cells. Dideoxy sequencing reactions were done using T7 DNA polymerase, with either 5Ј-end-labeled primers or with unlabeled primers and fluorescein-labeled ATP (17,18). Nucleotide sequencing was done with the automated laser fluorescent DNA sequencer (Amersham Pharmacia Biotech).…”

Section: Methodsmentioning

confidence: 99%

Hydrophobic Amino Acid Residues in the Acceptor Binding Site Are Main Determinants for Reaction Mechanism and Specificity of Cyclodextrin-glycosyltransferase

Veen¹,

Leemhuis²,

Kralj³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Cyclodextrin-glycosyltransferases (CGTases) (EC 2.4.1.19) preferably catalyze transglycosylation reactions with glucosyl residues as acceptor, whereas the homologous ␣-amylases catalyze hydrolysis reactions using water as acceptor. This difference in reaction specificity is most likely caused by the acceptor binding site. To investigate this in detail we altered the acceptor site residues Lys-232, Phe-183, Phe-259, and Glu-264 of Bacillus circulans strain 251 CGTase using site-directed mutagenesis. Lys-232 is of general importance for catalysis, which appears to result mainly from stabilization of the conformation of the loop containing the catalytic nucleophile Asp-229 and His-233, a residue that has been implied in transition state stabilization. Glu-264 contributes to the disproportionation reaction only, where it is involved in initial binding of the (maltose) acceptor. Phe-183 and Phe-259 play important and distinct roles in the transglycosylation reactions catalyzed by CGTase. Mutation of Phe-183 affects especially the cyclization and coupling reactions, whereas Phe-259 is most important for the cyclization and disproportionation reactions. Moreover, the hydrophobisity of Phe-183 and Phe-259 limits the hydrolyzing activity of the enzyme. Hydrolysis can be enhanced by making these residues more polar, which concomitantly results in a lower transglycosylation activity. A double mutant was constructed that yielded an enzyme preferring hydrolysis over cyclization (15:1), whereas the wild type favors cyclization over hydrolysis (90:1).Cyclodextrin-glycosyltransferases (CGTase) 1 (EC 2.4.1.19) belong to the ␣ -amylase family (glycosyl hydrolase family 13)(1), an important group of starch-converting enzymes. Catalysis in the ␣ -amylase family proceeds via a covalently linked intermediate (2), which basically divides the reaction in two steps. In the first step the donor substrate (starch or oligosaccharide) is processed, yielding the covalent intermediate (donor reaction). In the second step the acceptor reacts with this intermediate, resulting in product formation (acceptor reaction). Whereas ␣ -amylases usually catalyze a hydrolysis reaction using water as acceptor, CGTases mainly catalyze transglycosylation reactions in which the acceptor is either the non-reducing end glucose of another oligosaccharide (disproportionation) or the non-reducing end glucose of the covalently linked oligosaccharide intermediate, resulting in formation of a cyclodextrin (cyclization). Also the reverse of the cyclization, in which a cyclodextrin is cleaved and transferred to an accepting oligosaccharide (coupling), is catalyzed by CGTase (3). The main determinants that cause the difference in reaction specificity between ␣ -amylases and CGTases are thus likely to be found at the acceptor binding sites.The crystal structures of the Bacillus circulans strain 251 CGTase in complex with an acarbose-derived maltononaose inhibitor (4, 5) and a maltononaose substrate (2) have revealed the nature of the acceptor site. In the maltononaose s...

show abstract

Section: Methodsmentioning

confidence: 99%

Hydrophobic Amino Acid Residues in the Acceptor Binding Site Are Main Determinants for Reaction Mechanism and Specificity of Cyclodextrin-glycosyltransferase

Veen¹,

Leemhuis²,

Kralj³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…12 Small fragments were sequenced manually using T7-polymerase sequencing kit (Sequenase, Pharmacia, Uppsala, Sweden).…”

Section: Sequence Analysismentioning

confidence: 99%

A comparison of genomic structures and expression patterns of two closely related flanking genes in a critical lung cancer region at 3p21.3

et al. 1999

View full text Add to dashboard Cite

In the search for a tumour suppressor gene in the 3p21.3 region we isolated two genes, RBM5 and RBM6. Gene RBM5 maps to the region which is homozygously deleted in the small cell lung cancer cell line GLC20; RBM6 crosses the telomeric breakpoint of this deletion. Sequence comparison revealed that at the amino acid level both genes show 30% identity. They contain two zinc finger motifs, a bipartite nuclear signal and two RNA binding motifs, suggesting that the proteins for which RBM5 and RBM6 are coding have a DNA/RNA binding function and are located in the nucleus. Northern and Southern analysis did not reveal any abnormalities. By SSCP analysis of 16 lung cancer cell lines we found only in RBM5 a single presumably neutral mutation. By RT-PCR we demonstrated the existence of two alternative splice variants of RBM6, one including and one excluding exon 5, in both normal lung tissue and lung cancer cell lines. Exclusion of exon 5 results in a frameshift which would cause a truncated protein of 520 amino acids instead of 1123 amino acids. In normal lung tissue, the relative amount of the shorter transcript was much greater than that in the lung tumour cell lines, which raises the question whether some tumour suppressor function may be attributed to the derived shorter protein.

show abstract

“…Sequences of putstive genes were then aligned with updated protein On the cosmid. To the order of the EcoRI fragments, primers were databases, The deduced amino acid sequences designed and sequences obtained with the entire reactions were prepared using Tip20 were also analysed using the GeneQuiz automated preference values (Table 1) were calculated using a cosmid as a DNA for sequence search program (Casari et al, 1995 (Zimmerman et al, 1990) and T7 CodonFrequency). The lower the value, the DNA polymerase-and with the modifications greater the probability that the ORF represents necessary for using internal labelling with a functional gene.…”

Section: Sequencing Strategies Atid Metliodsmentioning

confidence: 99%

Sequencing and Analysis of a 35·4 kb Region on the Left Arm of Chromosome IV from Saccharomyces cerevisiae Reveal 23 Open Reading Frames

Eide

Sander

Prydz

1996

Yeast

View full text Add to dashboard Cite

The complete DNA sequence of cosmid clone 31A5 containing a 35452 bp segment from the left arm of chromosome IV from Saccharomyces cerevisiae, was determined from an ordered set of subclones in combination with primer walking on the cosmid. The sequence contains 23 open reading frames (ORFs) of more than 100 amino acid residues and the tRNA-Val2a gene. Five ORFs corresponded to the known yeast genes SNQZ, SESl, GCVI, RPL2B and RPS18A. The DNA sequence for RPSl8A is interrupted by an intron. One O R F corresponded to a part of the yeast gene HEX2 at the end of the cosmid insert. Four ORFs encoded putative proteins which showed strong homologies to other previously known proteins, three of yeast origin and one of non-yeast origin. Two ORFs were classified as having borderline homologies: one had similarity to two protein families and another to two protein products of unknown function from other species. The remaining 11 ORFs bore no significant similarity to any published protein. The complete DNA sequence has been submitted to the EMBL data library, Accession Number X95966.

show abstract

A simplified protocol for fast plasmid DNA sequencing

Cited by 84 publications

References 2 publications

Hydrophobic Amino Acid Residues in the Acceptor Binding Site Are Main Determinants for Reaction Mechanism and Specificity of Cyclodextrin-glycosyltransferase

Hydrophobic Amino Acid Residues in the Acceptor Binding Site Are Main Determinants for Reaction Mechanism and Specificity of Cyclodextrin-glycosyltransferase

A comparison of genomic structures and expression patterns of two closely related flanking genes in a critical lung cancer region at 3p21.3

Sequencing and Analysis of a 35·4 kb Region on the Left Arm of Chromosome IV from Saccharomyces cerevisiae Reveal 23 Open Reading Frames

Contact Info

Product

Resources

About