Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Degradation of chloroaromatics:structure and catalytic activities of wild-type chlorocatechol 2,3-dioxygenases and modified ones

<p>To improve the efficiency and to investigate the molecular determinants that direct substrate specificity of chlorocatechol 2,3-dioxygenase CbzE(GJ31), several mutant enzymes were constructed. Loci for substitutions of amino acids were selected by sequence comparisons as well as by homology modelling of known chlorocatechol 2,3-dioxygenases (CbzE(BASF), CbzE(SK1) and CbzE(16-6A)). Activity measurements with various catechols showed that most of the modifications influenced activity only to a minor degree. The amino acid at position 154 seems to be located at a non-important position in the enzyme with minor extension into the substrate tunnel. Similarly, the change of related amino acids such as D95E and Y223F did not influence the catalysis since both residues are far away from the catalytic centre and the substrate tunnel. Even the modification of isoleucine to threonine in position 310, located at the outer substrate tunnel, showed a significant alteration of activities. Position 196 seems to be of higher relevance since the modification of valine to alanine, i.e. the reduction of the side-chain, produced much alteration. The amino acid is located at the interface of inner to outer substrate tunnel. CbzE(V196A) showed high relative k(cat) for 3-chlorocatechol. A pronounced increase in activity for 3-chlorocatechol resulted by the change from alanine to valine and from aspartic acid to glycine laying in the outer substrate tunnel at position 211 and 212 respectively.</p>

Degradation of chloroaromatics by Pseudomonas putida GJ31: assembled route for chlorobenzene degradation encoded by clusters on plasmid pKW1 and the chromosome

Pseudomonas putida GJ31 has been reported to grow on chlorobenzene using a meta-cleavage pathway with chlorocatechol 2,3-dioxygenase (CbzE) as a key enzyme. The CbzE-encoding gene was found to be localized on the 180 kb plasmid pKW1 in a cbzTEXGS cluster, which is flanked by transposases and encodes only a partial (chloro)catechol meta-cleavage pathway comprising ferredoxin reductase, chlorocatechol 2,3-dioxygenase, an unknown protein, 2-hydroxymuconic semialdehyde dehydrogenase and glutathione S-transferase. Downstream of cbzTEXGS are located cbzJ, encoding a novel type of 2-hydroxypent-2,4-dienoate hydratase, and a transposon region highly similar to Tn5501. Upstream of cbzTEXGS, traNEOFG transfer genes were found. The search for gene clusters possibly completing the (chloro)catechol metabolic pathway of GJ31 revealed the presence of two additional catabolic gene clusters on pKW1. The mhpRBCDFETP cluster encodes enzymes for the dissimilation of 2,3-dihydroxyphenylpropionate in a novel arrangement characterized by the absence of a gene encoding 3-(3-hydroxyphenyl)propionate monooxygenase and the presence of a GntR-type regulator, whereas the nahINLOMKJ cluster encodes part of the naphthalene metabolic pathway. Transcription studies supported their possible involvement in chlorobenzene degradation. The upper pathway cluster, comprising genes encoding a chlorobenzene dioxygenase and a chlorobenzene dihydrodiol dehydrogenase, was localized on the chromosome. A high level of transcription in response to chlorobenzene revealed it to be crucial for chlorobenzene degradation. The chlorobenzene degradation pathway in strain GJ31 is thus a mosaic encoded by four gene clusters.