The bialaphos resistance gene, bar, was used as a selectable marker to isolate the bialaphos production genes (bap) from the Sfreptomyces uiridochromogenes genome. The S. uiridochromogenes bar gene was cloned on overlapping restriction fragments using pIJ680 and pIJ702 in the bialaphos-sensitive host, S. Zittidans. Although the restriction endonuclease cleavage map of these fragments was not similar to the bap cluster of S. hygroscopicus, the presence and location of bar and four other bap genes as well as a gene required for the transcriptional activation of the cluster (brpA) was demonstrated by heterologous cloning experiments using a series of previously characterized bialaphos-nonproducing S. hygroscopicus mutants. Since recombination-deficient mutants of streptomycetes have not been isolated, restored function provided by cloned homologous DNA results from both recombination (marker rescue) and complementation in frans. In contrast to our previously reported homologous cloning experiments where we were able to define the position of mutant alleles by recombination, in these heterologous cloning experiments we observed little if any recombination between plasmid-cloned genes and the chromosome. As a result, this approach allowed us to define the location and orientation of functional genes using a genetic complementation test. The organization of the clustered S. uiridochromogenes bup genes was indistinguishable from the corresponding S. hygroscopicus mutant alleles. The fact that the S. uiridochrornogenes transcriptional regulatory gene, brpA, functioned in S. hygroscopicus implied that some transcriptional regulatory signals may also be interchangeable. In these two Streptomyces species, which have considerable nucleotide sequence divergence, the complex biochemical and genetic organization of the bialaphos biosynthetic pathway is conserved.
a b s t r a c tLeucine-rich repeat and fibronectin type III domain-containing (LRFN) family proteins are thought to be neuronal-specific proteins that play essential roles in neurite outgrowth and synapse formation. Here, we focused on expression and function of LRFN4, the fourth member of the LRFN family, in non-neural tissues. We found that LRFN4 was expressed in a wide variety of cancer and leukemia cell lines. We also found that expression of LRFN4 in the monocytic cell line THP-1 and in primary monocytes was upregulated following macrophage differentiation. Furthermore, we demonstrated that LRFN4 signaling regulated both the transendothelial migration of THP-1 cells and the elongation of THP-1 cells via actin cytoskeleton reorganization. Our data indicate that LRFN4 signaling plays an important role in the migration of monocytes/macrophages.
Weinactivated the bialaphos (BA) resistance gene {bar) of a BAproducer, Streptomyces hygroscopicus, by the gene replacement technique. The resulting BA-sensitive mutant (Bar") was able to produce little BAbut considerable amount of an intermediate demethylphosphinothricin (DMPT).The Bar" mutant was still able to convert the N-acetyl derivative (AcDMPT) of DMPTto BA. Introduction of normal bar containing plasmid restored both BAresistance and BAbiosynthesis to levels as high as the parental BAproducer. By contrast, introducing a multi copy glutamine synthetase gene (glnA) into the Bar" mutant restored BAresistance but not BA production. Thus, the bar gene plays a crucial role in both self-defense and a step of BAbiosynthesis in the BA-producing S. hygroscopicus.
Wehave isolated and studied the genes involved in the alanylation step in the biosynthesis of a herbicide, bialaphos which is produced by Streptomyces hygroscopicus. Three bialaphos-nonproducing mutants, NP60, NP61 and NP62, isolated from S. hygroscopicus by treatment with AT-methyl-N'-nitro-TV-nitrosoguanidine were defective for the alanylation step and were not restored to productivity by any locus of the gene cluster previously cloned. Three plasmids were isolated using NP60, NP61 and NP62 as recipients. The genes which restored productivity to NP61 and NP62hybridized to the contiguous region of the bialaphos biosynthetic gene cluster. The gene cluster involved in the bialaphos production was about 35 kb long. The gene which restored productivity to NP60 did not hybridize to the bialaphos biosynthetic gene cluster. VM3and VM4, putative alanylation blocked mutants, were derived from a bialaphos producer by gene replacement of an unidentified region of the biosynthetic gene cluster with an in vitro altered DNAsequence. The genes which restored productivity to VM3and VM4were located between the genes which code for phosphinomethylmalic acid synthase and demethylphosphinothricin acetyltransferase in the cluster. These results suggest that multiple genes are involved in the alanylation step.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.