Analysis of the promoters for the two immunity genes present in the ColE3-CA38 plasmid using two new promoter probe vectors.

We have constructed two new promoter probe vectors which carry a polylinker derived from plasmid pUC19 proximal to the 5' end of a promoter-less galactokinase gene. Using these two vectors we have demonstrated that the ColE3imm gene and the ColE8imm gene present on the ColE3-CA38 plasmid have their own promoters, independent of the SOS promoter of the colicin E3 structural gene. The activity of two terminators, one located proximal to the 5' end of the ColE8imm gene, the other located proximal to the 5' end of the lys gene, were shown by a comparison of the galactokinase activity conferred by several of the recombinant plasmids.     

Hierarchical order of critical residues on the immunity-determining region of the Im7 protein which confer specific immunity to its cognate colicin.

The directed mutagenesis study of the Im7 protein of colicin E7 revealed that three residues, D31, D35, and E39, located in the loop 1 and helix 2 regions of the protein were critical for initiating the complex formation with its cognate colicin E7. Interestingly, the importance of these three critical residues in conferring specific immunity to its own colicin was exhibited in a hierarchical order, respectively. Moreover, we found that existence of the three critical residues was common among the DNase-type Im proteins. Most likely the three residues of the DNase-type immunity proteins are critical for initiating the unique protein-protein interactions with their cognate colicin. In addition, replacement of the helix 2 of Im7 by the corresponding region of Im8 produced a phenotype of the mutant protein very similar to that of Im8. This result suggests that the DNase-type Im proteins indeed share a "homologous-structural framework" and evolution of the Im proteins may be engendered by minor amino acid changes in this specific immunity-determining region without causing structural alteration of the proteins.     

A facile analytical method for the identification of protease gene profiles from Bacillus thuringiensis strains

Five pairs of degenerate universal primers have been designed to identify the general protease gene profiles from some distinct Bacillus thuringiensis strains. Based on the PCR amplification patterns and DNA sequences of the cloned fragments, it was noted that the protease gene profiles of the three distinct strains of B. thuringiensis subsp. kurstaki HD73, tenebrionis and israelensis T14001 are varied. Seven protease genes, neutral protease B (nprB), intracellular serine protease A (ispA), extracellular serine protease (vpr), envelope-associated protease (prtH), neutral protease F (nprF), thermostable alkaline serine protease and alkaline serine protease (aprS), with known functions were identified from three distinct B. thuringiensis strains. In addition, five DNA sequences with unknown functions were also identified by this facile analytical method. However, based on the alignment of the derived protein sequences with the protein domain database, it suggested that at least one of these unknown genes, yunA, might be highly protease-related. Thus, the proposed PCR-mediated amplification design could be a facile method for identifying the protease gene profiles as well as for detecting novel protease genes of the B. thuringiensis strains.     

The zinc ion in the HNH motif of the endonuclease domain of colicin E7 is not required for DNA binding but is essential for DNA hydrolysis

The HNH motif was originally identified in the subfamily of HNH homing endonucleases, which initiate the process of the insertion of mobile genetic elements into specific sites. Several bacteria toxins, including colicin E7 (ColE7), also contain the 30 amino acid HNH motif in their nuclease domains. In this work, we found that the nuclease domain of ColE7 (nuclease-ColE7) purified from Escherichia coli contains a one-to-one stoichiometry of zinc ion and that this zinc-containing enzyme hydrolyzes DNA without externally added divalent metal ions. The apo-enzyme, in which the indigenous zinc ion was removed from nuclease-ColE7, had no DNase activity. Several divalent metal ions, including Ni²⁺, Mg²⁺, Co²⁺, Mn²⁺, Ca²⁺, Sr²⁺, Cu²⁺ and Zn²⁺, re-activated the DNase activity of the apo-enzyme to various degrees, however higher concentrations of zinc ion inhibited this DNase activity. Two charged residues located at positions close to the zinc-binding site were mutated to alanine. The single-site mutants, R538A and E542A, showed reduced DNase activity, whereas the double-point mutant, R538A + E542A, had no observable DNase activity. A gel retardation assay further demonstrated that the nuclease-ColE7 hydrolyzed DNA in the presence of zinc ions, but only bound to DNA in the absence of zinc ions. These results demonstrate that the zinc ion in the HNH motif of nuclease-ColE7 is not required for DNA binding, but is essential for DNA hydrolysis, suggesting that the zinc ion not only stabilizes the folding of the enzyme, but is also likely to be involved in DNA hydrolysis.     

Cloning of Two New cry Genes from Bacillus thuringiensis subsp. wuhanensis Strain

With PCR products as probes, we have cloned two new cry-type genes from Bacillus thuringiensis subsp. wuhanensis. The deduced amino acid sequence of the first clone is 77.3% identical to Cry1Ga1. The deduced protein sequence of the second clone is 69.8–78.7% identical to that of Cry1B group. The nomenclature assignment of these two clones is, therefore, named Cry1Gb1 and Cry1Bd1, respectively. The Cry1Bd1 is toxic to Plutella xylostella larvae, and the Cry1Gb1 is toxic to Pieris rapae larvae. Received: 2 August 1999 / Accepted: 18 October 1999     

DNA methylation profiling in Barrett's esophagus and esophageal adenocarcinoma reveals unique methylation signatures and molecular subclasses

Barrett's esophagus (BE) is a metaplastic process whereby the normal stratified, squamous esophageal epithelium is replaced by specialized intestinal epithelium. Barrett's is the only accepted precursor lesion for esophageal adenocarcinoma (EAC), a solid tumor that is rapidly increasing in incidence in western countries. BE evolves into EAC through intermediate steps that involve increasing degrees of dysplasia. Current histologic criteria are quite subjective and the clinical behavior of BE is highly variable and difficult to predict using these standards. It is widely believed that molecular alterations present in BE and EAC will provide more precise prognostic and predictive markers for these conditions than the current clinical and histologic features in use. In order to further define molecular alterations that can classify unique groups of BE and EAC, we utilized methylation microarrays to compare the global gene methylation status of a collection of normal squamous, BE, BE + high-grade dysplasia (HGD), and EAC cases. We found distinct global methylation signatures, as well as differential methylation of specific genes, that discriminated these histological groups. We also noted high and low methylation epigenotypes among the BE and EAC cases. Additional validation of those CpG sites that distinguished BE from BE + HGD and EAC may lead to the discovery of useful biomarkers with potential clinical applications in the diagnosis and prognosis of BE and EAC.     

Immunomodulatory compounds from Pestalotiopsis leucothës, an endophytic fungus from Tripterygium wilfordii

The immunomodulatory effects of three compounds designated BS, GS, and YS produced by Pestalotiopsis leucothës, an endophytic fungus isolated from Tripterygium wilfordii, were evaluated. The 50% inhibition concentration (IC₅₀) value of BS in the proliferative assay with various stimulating agents such as phytohemagglutinin-M (PHA-M), phorbol myristate acetate (PMA)/ionomycin, mixed lymphocyte reaction (MLR) and poke weed mitogen (PWM) was 0.35, 1.6, 0.8 and 5.4 μg/ml, respectively. In addition, BS significantly inhibited the production of cytokines such as interleukin (IL)-1β, IL-2, interferon (IFN)-γ and tumor necrosis factor (TNF)-α, by peripheral blood mononuclear cells (PBMNC) and soluble IL-2 receptor expression at concentrations greater than 1 μg/ml. Inhibition of PHA stimulated PBMNC proliferation and IL-2 and sIL-2R production by BS indicates that it is a T-cell specific immunosuppressant. However, BS also moderately inhibited immunoglobulin (Ig) G and M at concentrations greater than 1 μg/ml suggesting that it also has B cell immunosuppressive effects. YS was 10% less active than BS in all assay systems. In contrast, GS exhibited both suppression and enhancement of PBMNC proliferation in the presence of various stimulants. However, GS inhibited PWM stimulated PBMNC proliferation and IL-4 and IgG and IgM production at concentrations above 1 μg/ml. All three fungal compounds altered the percentage of T-lymphocyte subpopulations only at high concentrations. Cell viability was not affected at the immunosuppressive concentrations of these compounds. In conclusion, work from our laboratory has identified three potentially potent immunomodulatory compounds from P. leucothës. These compounds have variable effects on T- and B-cells and monocytes. They may partially explain the immunosuppressive activity of T. wilfordii. In addition, they may represent a new source of immunomodulatory compounds for the treatment of human immune mediated diseases.     

Identification of an essential cleavage site in ColE7 required for import and killing of cells

Colicin E7 (ColE7), a nuclease toxin released from Escherichia coli, kills susceptible bacteria under environmental stress. Nuclease colicins are processed during translocation with only the cytotoxic nuclease domains traversing the inner membrane to cleave tRNA, rRNA, or DNA in the cytoplasm of target cells. In this study, we show that the E. coli periplasmic extract cleaves ColE7 between Lys(446) and Arg(447) in the presence or absence of its inhibitor Im7 protein. Several residues near cleavage sites were mutated, but only mutants of Arg(447) completely lost in vivo cell-killing activity. Both the full-length and the nuclease domain of Arg(447) mutants retained their nuclease activities, indicating that failure to kill cells was not a consequence of damage to the endonuclease activity of the enzyme. Moreover, the R447E ColE7 mutant was not cleaved at its 447 site by periplasmic extracts or transported into the cytoplasm of target cells. Collectively, these results suggest that ColE7 is cleaved at Arg(447) during translocation and that cleavage is an essential step for ColE7 import into the cytoplasm of target cells and its cell-killing activity. Conserved basic residues aligned with Arg(447) have also been found in other nuclease colicins, implying that the processing at this position may be common to other colicins during translocation.