Isolation of nitrogen deregulated mutants of erythromycin biosynthesis

Summary Ammonium represses erythromycin synthesis bySaccharopolyspora erythraea and insensitive mutants to this effect were isolated. Six mutants were selected and one of them produces 50% more antibiotic than the wild type in 100 mM NH₄Cl as nitrogen source. Glutamine synthetase and alanine dehydrogenase levels in the mutants were determined and no differences with wild type strain were observed.     

Acridine-linked oligonucleotide probes for the short dAT-rich motifs in the 3'-untranslated region of cytokine genes

We have investigated the use of oligonucleotide probes for identifying cDNA clones containing the short dAT-rich motifs found in the 3'-untranslated region of cytokine genes. To obtain sufficiently stable duplexes between the octameric probes used to identify genes containing the sequence dTATTTATT and its complement, it was necessary to couple an intercalating agent, an acridine derivative (acr), to the 5'-positions of the probes. The resulting octamers 5'-acr-dAATAAATA and, particularly, 5'-acr-dTATTTATT were successfully used to distinguish the complementary sequences in cDNA from internal, single point mismatched sequences. Southern blot analyses of plasmids containing IL-1 beta and IL-8 gave positive results with the 3' degenerate probe, 5'-acr-dTATTTATTN, clearly showing that the very short probe approach can be used in this type of analysis. Subsequently, in slot blot analyses we found that, even without the degenerate nucleotide, N, plasmids bearing cytokine sequences with at least 7 contiguous matched nucleotides could be unambiguously identified with 5'-acr-dTATTTATT. Unfortunately, because of the ubiquity of these dAT-rich sequences in bacterial DNA, it was not possible to use these probes for direct colony screening. In contrast to the results obtained with DNA, at the RNA level, with IL-1 beta mRNA bound to nitrocellulose, the hybrid formed with 5'-acr-dAATAAATA was very unstable, even in 1M LiCl solution at 2 degrees C; however, in the same salt solution the slightly longer acridine-coupled probes 5'-acr-dAATAAATAGGG and 5'-acr-dAAAGAACAA remained hybridized to their complementary sequences up to about 18 degrees C.     

Knowledge-based simulation of DNA metabolism: prediction of enzyme action

We have developed a knowledge-based simulation of DNA metabolismthat accurately predicts the actions of enzymes on DNA undera large number of environmental conditions. Previous simulationsof enzyme systems rely predominantly on mathematical models.We use a frame-based representation to model enzymes, substratesand conditions. Interactions between these objects are expressedusing production rules and an underlying truth maintenance system.The system performs rapid inference and can explain its reasoning.A graphical interface provides access to all elements of thesimulation, including object representations and explanationgraphs. Predicting enzyme action is the first step in the developmentof a large knowledge base to envision the metabolic pathwaysof DNA replication and repair. Received on February 1, 1990; accepted on October 2, 1990     

Cloning, sequencing and mutational analysis of the cytochrome c552 gene (cycB) from Bradyrhizobium japonicum strain 110

We report the cloning and nucleotide sequence analysis of the cytochrome c552 gene (cycB) of Bradyrhizobium japonicum strain 110. The gene was identified with help of an oligonucleotide that was designed on the basis of the amino acid sequence determined for purified cytochrome c552 of B. japonicum strain CC705. The cycB gene product has an N-terminal 23-amino acid signal peptide that is missing in the mature cytochrome c552 protein. A B. japonicum cycB insertion mutant was constructed which had no observable phenotypic defects in denitrification and symbiotic nitrogen fixation. Thus, the function of c552 remains unknown.     

Multiple xylanases of Cellulomona fimi are encoded by distinct genes

Cellulomonas fimi genomic DNA encoding xylanase activity has been cloned and expressed in Escherichia coli. As judged by DNA hybridization and restriction analysis, twelve xylanase-positive clones carried a minimum of four different xylanase (xyn) genes. The encoded enzymes were devoid of cellulase activity but three of the four bound to Avicel.     

The role of the epidermal growth factor-1 and hydrophobic stack domains of human factor IX in binding to endothelial cells.

To determine the function and specificity in factor IX of the first epidermal growth factor (EGF)-like domain and the eight-amino acid hydrophobic stack encoded by exon C (residues 39-46), these domains were replaced by the corresponding polypeptide regions of factor X and chimeric proteins were produced in human embryo kidney cells. Both chimeras were activated by factor XIa at a rate similar to plasma factor IX and exhibited calcium-dependent fluorescence quenching similar to plasma factor IX. Both chimeras competed equally for binding to the endothelial cell receptor. Our findings make it unlikely that the first EGF-like domain or the hydrophobic stack of factor IX are responsible for the specific binding of factor IX to its endothelial cell receptor.