A newly identified anaerobically expressed locus, aeg-46.5, which is located at min 46.5 on Escherichia coli linkage map, was cloned and analyzed. The phenotype of this gene was studied by using a lacZ operon fusion. aeg-46.5 is induced anaerobically in the presence of nitrate in wild-type and narL cells. It is repressed by the narL gene product, as it showed derepressed anaerobic expression in narL mutant cells. We postulate that aeg-46.5 is subject to multiple regulatory systems, activation as a result of anaerobiosis, narL-independent nitrate-dependent activation, and narL-mediated repression. The regulatory region of aeg-46.5 was identified. A 304-bp DNA sequence which includes the regulatory elements was obtained, and the 5' end of aeg-46.5 mRNA was identified. It was verified that the anaerobic regulation of aeg-46.5 expression is controlled on the transcriptional level. Computer analysis predicted possible control sites for the NarL and FNR proteins. The proposed NarL site was found in a perfect-symmetry element. The aeg-46.5 regulatory elements are adjacent to, but divergent from, those of the eco gene.

Genes that are expressed under anaerobic conditions were identified by operon fusion techniques with a hybrid bacteriophage of A and Mu, XplacMuS3, which creates transcriptional fusions to lacZY. Cells were screened for anaerobic expression on XG medium. Nine strains were selected, and the insertion point of the hybrid phage in each strain was mapped on the Escherchia coli chromosome linkage map. The anaerobic and aerobic expression levels of these genes were measured by I-galactosidase assays in different medium conditions and in the presence of three regulatory mutations (fnr, narL, and rpoN). The anaerobically expressed genes (aeg) located at minute 99 (aeg-99) and 75 (aeg-75) appeared to be partially regulated byfnr, and aeg-93 is tightly regulated byfnr. aeg-60 requires a functional rpoN gene for its anaerobic expression. aeg-46.5 is repressed by narL. aeg-65A and aeg-65C are partially controlled byfnr but only in media containing nitrate or fumarate. aeg47.5 and aeg-48.5 were found to be anaerobically induced only in rich media. The effects of a narL mutation on aeg-46.5 expression were observed in all medium conditions regardless of the presence or absence of nitrate. This suggests that narL has a regulatory function in the absence of exogenously added nitrate.Escherichia coli is a facultative bacterium. It can grow aerobically by using oxygen as a terminal electron acceptor or anaerobically either by fermentation or, in the presence of an alternative electron acceptor, by respiration. E. coli has evolved control mechanisms to adapt to these different environments. One manifestation of this regulation was observed in two-dimensional protein gel analysis of the total E. coli proteins. The protein profile showed that the levels of at least 125 proteins were influenced by the choice between aerobic and anaerobic growth conditions (21, 24). Determining the global regulatory mechanisms that control gene expression in response to changes in oxygen availability is a central question in bacterial genetic regulation research.Recent studies have examined the regulation of genes that respond to anaerobic respiratory conditions. The pleiotropic regulatory protein, Fnr, was identified (25). Fnr is a positive and a negative regulator which responds to the availability of oxygen. It is required for the induction of anaerobic respiratory enzymes. For example, Fnr positively regulates the expression of nitrate reductase (narGHJI at 27 min) (6, 14), fumarate reductase (frdABCD at 94 min) (12), and dimethyl sulfoxide reductase (dmsABC at 20 min) (9). An example of negative regulation by Fnr is the repression of the aerobic respiratory enzyme NADH dehydrogenase (ndh at 22 min) under anaerobic conditions (26).The genes encoding anaerobic respiration functions are also regulated according to the availability of their substrates and other alternative electron acceptors. For instance, nitrate reductase is induced by nitrate, but fumarate reductase and dimethyl sulfoxide reductase are repressed by nitrate. The products of the nar...

The yeast transcriptional activator protein, Gcn4p from Saccharomyces cerevisiae binds to the specific sequence in the promoters of many amino acid biosynthetic genes for general control. A new random saturation mutagenesis method was developed to isolate Gcn4p derivatives with only one or two mutations in the DNA binding domain without using radioactive isotope. This will be used to identify the amino acids of Gcn4p involved in protein-protein interactions. Saturation mutagenesis in the DNA binding domain of Gcn4p was performed using spiked degenerate oligonucleotides containing randomized codon bases designed specifically for only one or two base changes in the mutagenized area. These oligonucleotides were synthesized to have two flanking restriction enzyme sites for cloning to the appropriate vector. The 3' ends were mutually primed after hybridization via the palindromic sequences of the restriction enzyme sites. These molecules were then converted to double stranded DNA upon treatment with DNA polymerase. Here, a library collection of 100,680 in an altered Gcn4p pool was generated by cloning a mixed-base oligonucleotide in the place of the sequence coding for the DNA binding domains. The quality of the library was examined by DNA sequencing and found to be in good agreement with the expected statistical values. Calculated mutation frequency was 66% of mutant nucleotide rate and actual sequencing data revealed 68% mutant nucleotide rates from the sequenced library. Thus, among 21 mutants, 16 had one point mutations and 5 had two point mutations. This approach appears to be an effective and general tool for creating proteins with one or two amino acid change(s) in their molecules.

It is a challenging subject of biomedical research to develop more efficient and sensitive assay method. New method for enhancing sensitivity and precision of conventional immunological assays is developed. The antibody binding domain of Streptococcus protein G was used to make a chain of repeated antibody binding domain. The repeat chain was mixed with antibody, and multiple number of antibody bound to the repeat chain to form multiple antibody-repeat chain complex. The cross-binding between the complexes formed supercomplex, and the supercomplex amplified signals without specificity loss and background noise increase.