https://www.cbs.dtu.dk/services/SignalP-4.1/paper-3.0.pdf

Improved Prediction of Signal Peptides: SignalP 3.0

DNA base composition was determined by reversed-phase high-performance liquid chromatography (HPLC). DNA was hydrolysed into nucleosides with nuclease P1 and bacterial alkaline phosphatase. The mixture of nucleosides was applied to HPLC without any further purification. One determination by chromatography needed 2 μg of hydrolysed nucleosides and took only 8 min. The relative standard error of nucleoside analysis was less than 1%. The system described here gives a direct and precise method for determining DNA base composition.

Determination of DNA base composition by reversed-phase high-performance liquid chromatography

Preparation of transforming deoxyribonucleic acid by phenol treatment

Protein phosphorylation and regulation of adaptive responses in bacteria.

DnaK and other members of the 70-kilodalton heat-shock protein (hsp70) family promote protein folding, interaction, and translocation, both constitutively and in response to stress, by binding to unfolded polypeptide segments. These proteins have two functional units: a substrate-binding portion binds the polypeptide, and an adenosine triphosphatase portion facilitates substrate exchange. The crystal structure of a peptide complex with the substrate-binding unit of DnaK has now been determined at 2.0 angstroms resolution. The structure consists of a beta-sandwich subdomain followed by alpha-helical segments. The peptide is bound to DnaK in an extended conformation through a channel defined by loops from the beta sandwich. An alpha-helical domain stabilizes the complex, but does not contact the peptide directly. This domain is rotated in the molecules of a second crystal lattice, which suggests a model of conformation-dependent substrate binding that features a latch mechanism for maintaining long lifetime complexes.

/pdf/structural-analysis-of-substrate-binding-by-the-molecular-2a1dx17hgt.pdf

Structural Analysis of Substrate Binding by the Molecular Chaperone DnaK

We show that a collection of 93 E. coli mutations which map between thr and leu and which block phage lambda DNA replication define two closely linked cistrons. Work published in the accompanying paper shows that these mutations also affect host DNA replication, so we designate them dnaJ and dnaK; the gene order is thr--dnaK--dnaJ--leu. Demonstration of two cistrons was possible with the isolation of lambda transducing phages carrying one or the other or both of the dna genes. These phages were employed in phage vs bacterial complementation studies which unambiguously show that dnaK and dnaJ are different cistrons.

Genetic analysis of two genes, dnaJ and dnaK , necessary for Escherichia coli and bacteriophage lambda DNA replication

Bacillus subtilis Marburg is nonpermissive for the multiplication of bacteriophages SP10 and ϕNR2. A permissive mutant was derived from the Marburg strain, and the genetic determinants of nonpermissiveness were analyzed by PBS1 transduction. The simultaneous presence of two genes as mutant alleles, nonA and nonB, was necessary for permissiveness. The gene nonA is linked very closely to rfm (cotransfer: 95%); nonB is located between dal and purB (cotransfer of nonB and purB6: 48%). The genetic determinant of host-specific restriction intrinsic to the Marburg strain (hsrM) was found to be identical or very closely linked to nonB. The segregation of nonB and hsrM has never been observed in the course of transduction analysis. The mutation, hsrM1, diminishes the restriction activity, but not the host-controlled modification.

Mapping of genes determining nonpermissiveness and host-specific restriction to bacteriophages in Bacillus subtilis Marburg.

The nucleotide sequence of phoR, the positive and negative regulatory gene for alkaline phosphatase and phosphodiesterase formation in Bacillus subtilis, was determined. The sequence data predicted an open reading frame of 1,740 base pairs (579 amino acids) which overlaps the 5 base pairs of the preceding phoP coding sequence. The deduced amino acid sequence was significantly homologous with that of the Escherichia coli phoR gene product, which is the sensory element for the pho regulon.

Nucleotide sequence of the Bacillus subtilis phoR gene.

Two DNA fragments which complement the alkaline phosphatase-negative mutation phoP of Bacillus subtilis were cloned from a B. subtilis chromosome with the prophage vector phi CM (a derivative of phi 105). One of the fragments contained the regulatory gene phoR in addition to phoP. Nucleotide sequence analysis of the phoP region revealed that the phoP gene product consists of 241-amino-acid residues and that the sequence of these amino acids is extensively homologous with the sequence of the phoB gene product. This protein is the positive regulator for the phosphate regulon in Escherichia coli. It therefore appears that phoP is a regulatory gene for alkaline phosphatase synthesis in B. subtilis.

Hiuga Saito

Papers

Preparation of transforming deoxyribonucleic acid by phenol treatment

Genetic analysis of two genes, dnaJ and dnaK , necessary for Escherichia coli and bacteriophage lambda DNA replication

Mapping of genes determining nonpermissiveness and host-specific restriction to bacteriophages in Bacillus subtilis Marburg.

Nucleotide sequence of the Bacillus subtilis phoR gene.

Cloning and nucleotide sequence of phoP, the regulatory gene for alkaline phosphatase and phosphodiesterase in Bacillus subtilis.