ABSTRACT
Spin-dependent lepton-nucleon scattering data have been used to investigate the validity of the concept of quark-hadron duality for the spin asymmetry A1. Longitudinally polarized positrons were scattered off a longitudinally polarized hydrogen target for values of Q2 between 1.2 and 12 GeV2 and values of W2 between 1 and 4 GeV2. The average double-spin asymmetry in the nucleon resonance region is found to agree with that measured in deep-inelastic scattering at the same values of the Bjorken scaling variable x. This finding implies that the description of A1 in terms of quark degrees of freedom is valid also in the nucleon resonance region for values of Q2 above 1.6 GeV2.
ABSTRACT
The DNA polymerase I (PolI) from Mycobacterium tuberculosis (Mtb) was overproduced in Escherichia coli as an enzymatically active, recombinant protein with or without an N-terminal His-tag. The proteins catalysed both the DNA polymerisation of homo- and heteropolymer template-primers and the 5'-3' exonucleolytic hydrolysis of gapped and nicked substrates but lacked an associated proofreading activity. In accordance with recent predictions [Tabor, S. and Richardson, C.C. (1995) Proc. Natl. Acad. Sci. USA, 92, 6339-6343], both recombinant forms of the M. tuberculosis enzyme were unable to discriminate against dideoxynucleotide 5'-triphosphates and were thus efficiently inhibited by these chain-terminating nucleotide analogues during DNA synthesis. This unusual property might be potentially exploitable in terms of novel anti-mycobacterial drug design. A mutational analysis of 5' nuclease domain residues allowed the roles of nine invariant acidic residues to be evaluated. Acidic side chain neutralisation resulted in a > or = 20-fold reduction in activity, with the most profound reduction (> or = 10(4)-fold) being caused by neutralisation of the Asp125, Asp148 and Asp150 residues.
Subject(s)
DNA Polymerase I/chemistry , Exodeoxyribonucleases/chemistry , Mycobacterium tuberculosis/enzymology , Oligodeoxyribonucleotides/chemistry , Biopolymers , Cloning, Molecular , DNA Polymerase I/genetics , DNA Polymerase I/metabolism , Escherichia coli , Exodeoxyribonucleases/genetics , MutagenesisABSTRACT
The polA gene (encoding DNA polymerase I) from Mycobacterium tuberculosis was cloned using an internal gene segment probe generated by PCR amplification of genomic DNA [Mizrahi et al., Gene 136 (1993) 287-290]. The gene encodes a polypeptide 904 amino acids (aa) in length that shares 89% identity with a 911-aa homologue from Mycobacterium leprae. The polypeptide has all of the primary structural elements necessary for DNA polymerase and 5'-3' exonuclease activity, but lacks the motifs required for an associated 3'-5' exonuclease (proofreading) activity.
Subject(s)
DNA Polymerase I/genetics , Genes, Bacterial , Mycobacterium tuberculosis/genetics , Base Sequence , Cloning, Molecular/methods , Molecular Sequence Data , Mycobacterium tuberculosis/enzymology , Polymerase Chain Reaction , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
Internal segments of the gyrA and polA genes involved in DNA replication of Mycobacterium tuberculosis and rnhA of M. smegmatis, have been amplified by the polymerase chain reaction (PCR) using degenerate oligodeoxyribonucleotide primers based on conserved sequences. The deduced amino acid sequences were 54-66% homologous to the corresponding segments of their Escherichia coli counterparts. This method provides a useful means of cloning genes encoding DNA replication enzymes of mycobacteria.
