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Title / MacVector


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Graphical Sequence Editing ... of a pool of at least 40 DNA damage-inducible genes whose functions are involved in DNA replication, DNA repair, mutagenesis, and control of the cell cycle (3, 5, 8, 11). This regulon is controlled by two key proteins, RecA and LexA, the positive and negative regulators, respectively (1, 12, 15, 22). Under normal condi- tions LexA binds specifically to an operator region located within the promoters of the SOS genes, repressing their ex- pression (8, 22). When DNA is damaged or replication is stalled, RecA protein forms a nucleofilament with the resulting single-stranded DNA, achieving its activated conformation (RecA*). RecA* mediates the autocatalytic cleavage of LexA between residues Ala 84 and Gly 85 , resulting in two parts, the N-terminal DNA binding domain and the C-terminal domain, which is involved in dimerization of the repressor (13, 14). In this process, the catalytic site formed by Ser 119 and Lys 156 is responsible for the hydrolysis of the Ala-Gly bond through a biochemical reaction characteristic of serine proteases (16). As a consequence of the cleavage, LexA no longer binds DNA, the SOS genes are derepressed, and their products participate in repairing the DNA lesions to guarantee cell survival. This process is known as the SOS response and was first suggested in the 1970s by Miroslav Radman (19). Once DNA is repaired, RecA* loses its active conformation, LexA autohydrolysis ceases, and the repressor accumulates again, inhibiting the expression of the SOS genes. The lexA gene seems to be widespread in the Bacteria do- main. However, lexA is not found in the fully sequenced ge- nomes of several bacterial species, such as Aquifex aeolicus, moniae, Campylobacter jejuni , Helicobacter pylori, and Porphy- romonas gingivali s. Moreover, it appears that a lexA -like gene does not exist in the Archaea domain, as lexA homologues are absent in the 19 archaeal genomes currently available (4). Recently, the three-dimensional structure of the E. coli LexA protein has been solved (16). In E. coli , LexA binds specifically to a DNA motif known as the SOS-box or LexA- box (22). Comparative analysis of the 31 E. coli LexA binding sites shows a 16-bp consensus sequence that responds to the motif CTGN 10 CAG (5). Besides the E. coli SOS-box, two more LexA-boxes have been identified so far in bacteria. The directed repeat GTTCN 7 GTTC is recognized by LexA protein of members of the alpha-class Proteobacteria , such as Rhodobacter sphaeroides and Rhizobium etli (6, 21). In gram-positive bacteria such as Bacillus subtilis and Mycobacterium tuberculosis, the consensus sequence CGAACRNRYGTTYG is the target for this repressor (2, 24). Dehalococcoides ethenogenes is an anaerobic bacterium ca- pable of dechlorinating tetrachloroethene, one of the most common groundwater contaminants, to ethene (17). D. ethenogenes is rather enigmatic taxonomically. Its cell wall composition is unlike that of gram-positive or gram-negative bacteria, instead more closely resembling the cell wall of archaea (17). However, on the basis of 16S rRNA gene sequence compari- sons, D. ethenogenes has been assigned to the green nonsulfur bacteria, a division of physiologically diverse species with rel- atively few cultured representatives (10). The unusual phylogenetic position, structural properties, and metabolic capabil- ities of D. ethenogenes make it an interesting organism for further biological characterization. Here, we report the cloning of the key regulatory gene lexA from D. ethenogenes , purifica- tion of the protein, and characterization of its recognition site. Prior to this report, there were no data available pertaining to the motif recognized by LexA in this bacterial division. Finally, we report a genomic analysis of the composition of the D. ethenogenes LexA network. Identi fi cation and cloning of D. ethenogenes lexA gene. The D. ethenogenes strain 195 genome sequence is currently unas- sembled, but contigs are available for performing BLAST anal- ysis at the Institute for Genomic Research and at the National Center of Biotechnology Information ( fi ). The Escherichia coli LexA amino acid sequence was used to query the D. ethenogenes genome with the TBLASTN pro- gram, revealing a protein homologue of 212 amino acids. Al- though D. ethenogenes is not a gram-positive organism, its LexA protein is highly homologous to the LexA proteins be- longing to several members of this bacterial phylum, such as Bacillus halodurans , Staphylococcus aureus, and Clostridium perfringens . Speci fi cally, D. ethenogenes LexA protein shows the highest level of identity with Bacillus halodurans LexA (40%), while the D. ethenogenes repressor is only 31% identical to E. coli LexA (Fig. 1). Furthermore, D. ethenogenes LexA contains all of the conserved residues involved in repressor autocleav- age (Ala 88 , Gly 89 , Ser 331 , and Lys 170 ) as deduced from CLUSTAL W alignment of different LexA proteins (Fig. 1). It is important to note that nine codons downstream of the pro- posed initiation site for the 212-amino-acid open reading frame there is a 203-amino-acid protein starting with methio- nine. However, this truncated version of D. ethenogenes LexA would lack several N-terminal residues likely to be essential to the LexA structure, since it would lack important residues belonging to the fi rst ␣ -helix characterized by nuclear magnetic resonance spectroscopy in E. coli LexA (7). The BLAST analysis returned 1 kb of DNA fl anking the lexA coding sequence, which was used to design the primers AR31 and AR34 (Table 1). These primers were employed to PCR amplify the entire D. ethenogenes lexA and its promoter region with genomic DNA obtained from a 10-liter tetrachloro- ethene-methanol-fed anaerobic enrichment culture. The re- sulting 866-bp DNA fragment was then cloned into pGEM-T (Promega). To con fi rm that no mutation was introduced dur- ing the PCR, the sequence of the insert was determined by labeling DNA samples with the fmol DNA cycle sequencing system (Promega) and analyzed on an ALF sequencer (Amer- sham Pharmacia Biotech). This plasmid, called pUA969, was used as a template for further studies. Puri fi cation of D. ethenogenes LexA protein. A glutathione S -transferase (GST)-LexA fusion protein was generated. The primers AR61 and AR34 (Table 1) were used to amplify the D. ethenogenes lexA gene from pUA969. The oligonucleotide AR61 incorporates an Eco RI restriction site upstream of the lexA translational start codon, permitting fusion of the protein in frame with the GST protein. The 650-bp PCR fragment was cloned into pGEM-T, generating plasmid pUA970. After- wards, pUA970 was digested with Eco RI and Sal I, and the resulting 0.65-kb fragment was cloned into pGEX4T1 (Amer- sham Pharmacia Biotech), creating the GST-LexA fusion. This plasmid, designated pUA971, was transformed into Escherichia coli BL21 Codon Plus cells (Stratagene) to over- produce the protein. An overnight culture of the BL21 Codon Plus strain containing plasmid pUA971 was diluted in one-half liter of Luria-Bertani (LB) medium and incubated at 37 ° C until it reached an optical density at 600 nm of 0.8. At this time isopropylthiogalactopyranoside (IPTG) was added (1 mM fi nal concentration), and the culture was incubated for 4 more hours. Afterwards, cells were collected by centrifugation for 15 min at 5,000 rpm. The bacterial pellet was resuspended in phosphate-buffered saline (PBS) (10 mM Na 2 HPO 4 , 1.7 mM KH 2 PO 4 , 137 mM NaCl, 2.7 mM KCl [pH 7.4]) containing complete Mini pro- tease inhibitor cocktail (Roche) and sonicated to break the cells. The cell lysate was centrifuged at 15,000 rpm for 30 min, and the supernatant containing the soluble GST-LexA protein fusion was incubated for 2 h at 4 ° C with glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech), previously equili- brated in PBS. The beads were washed twice with PBS – 0.1% Triton X-100 and three more times with PBS to eliminate contaminant proteins. The washed glutathione-Sepharose beads containing bound GST-LexA protein were equilibrated in 0.1 M Tris-HCl (pH 8) – 0.1 M NaCl buffer, and GST-LexA was eluted in the same buffer containing 20 mM glutathione (Sigma), resulting in about 85% pure GST-LexA (Fig. 2). The GST-LexA fusion protein has the amino acid sequence Leu-Val-Pro-Arg-Gly-Ser between GST and LexA. This motif is recognized by the thrombin protease, which cleaves the Arg-Gly peptide bond. In this case, proteolysis results in D. ethenogenes LexA protein with the peptide Gly-Ser-Pro-Glu- Phe attached to the N terminus. GST-LexA bound to glutathi- one-Sepharose was incubated at room temperature for 16 h with 50 U of thrombin protease (Amersham Pharmacia Bio- tech) in 1 ml of PBS. The liquid phase containing LexA was collected, and a portion was separated by electrophoresis on a denaturing 13% polyacrylamide gel. The purity of the D. etheno- genes LexA protein was greater than 95% (Fig. 2). Determination of D. ethenogenes LexA binding motif. Anal- ysis of the DNA contig containing D. ethenogenes lexA revealed that it was located between the rpsT and fucA genes (Fig. 3A). From the TAA translational stop codon of rpsT to the putative TTG start codon of lexA , there are 104 bp that presumably contain the LexA binding site. Hence, a PCR fragment con- taining this sequence was ampli fi ed with primers AR31 and AR33DIG (Table 1). The resulting digoxigenin-labeled probe (LexA1) was used as a target to observe whether puri fi ed D. ethenogenes LexA was able to bind its own promoter in elec- trophoretic mobility shift assay experiments carried out as pre- viously described (6). Basically, 20- ␮ l reaction mixtures containing 10 ng of digoxi- genin-labeled DNA probe and 25 ng (50 nM) of D. ethenogenes LexA were incubated in binding buffer: 10 mM HEPES NaOH (pH 8), 10 mM Tris-HCl (pH 8), 5% glycerol, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 ␮ g of bulk ... If your graphics palette disappears, you can reset it it using this utility; Wellcome Sanger Institute MacVector Free - [CORRECTED] A problem with "Select All" in Sequence and Protein windows




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* Mac Users: The Java Web Start option does not work for some users due to security settings. The recommended solution is to use the bundled Mac App from the link above. Alternatively you can try to work around this by right-clicking on the buttons and saving the "jnlp" file, then right-clicking on the saved "jnlp" file and select "Open With > Java Web Start". macOS Sierra Any suggestions??? 2. Open the CD and navigate to the Network Licensing / Installers / Client Installers folder. Istituto Italiano di Tecnologia - [UPDATED] Changed shortcut of 'Copy All Sequences To Multifasta' to [COMMAND-SHIFT-J] instead of [COMMAND-SHIFT-K] url below Please click on the link below to download the full online installer for MacVector 14



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