Biomedical subjects

B U Lee

Publications and source records attributed to B U Lee.

3 recordsLinked to original sources

PubMed1996

Characterization of rco-1 of Neurospora crassa, a pleiotropic gene affecting growth and development that encodes a homolog of Tup1 of Saccharomyces cerevisiae.

The filamentous fungus Neurospora crassa undergoes a well-defined developmental program, conidiation, that culminates in the production of numerous asexual spores, conidia. Several cloned genes, including con-10, are expressed during conidiation but not during mycelial growth. Using a previously described selection strategy, we isolated mutants that express con-10 during mycelial growth. Selection was based on expression of an integrated DNA fragment containing the con-10 promoter-regulatory region followed by the initial segment of the con-10 open reading frame fused in frame with the bacterial hygromycin B phosphotransferase structural gene (con10'-'hph). Resistance to hygromycin results from mutational alterations that allow mycelial expression of the con-10'-'hph gene fusion. A set of drug-resistant mutants were isolated; several of these had abnormal conidiation phenotypes and were trans-acting, i.e., they allowed mycelial expression of the endogenous con-10 gene. Four of these had alterations at a single locus, designated rco-1 (regulation of conidiation). Strains with the rco-1 mutant alleles were aconidial, female sterile, had reduced growth rates, and formed hyphae that coiled in a counterclockwise direction, opposite that of the wild type. The four rco-1 mutants had distinct conidiation morphologies, suggesting that conidiation was blocked at different stages. Wild-type rco-1 was cloned by a novel procedure employing heterokaryon-assisted transformation and ligation-mediated PCR. The predicted RCO1 polypeptide is a homolog of Tup1 of Saccharomyces cerevisiae, a multidomain protein that mediates transcriptional repression of genes concerned with a variety of processes. Like tup1 mutants, null mutants of rco-1 are viable and pleiotropic. A promoter element was identified that could be responsible for RCO1-mediated vegetative repression of con-10 and other conidiation genes.

Amino Acid Sequence↗

PubMed1995

A tactile sensory system of Myxococcus xanthus involves an extracellular NAD(P)(+)-containing protein.

CsgA is a cell surface protein that plays an essential role in tactile responses during Myxococcus xanthus fruiting body formation by producing the morphogenic C-signal. The primary amino acid sequence of CsgA exhibits homology with members of the short-chain alcohol dehydrogenase (SCAD) family and several lines of evidence suggest that NAD(P)+ binding is essential for biological activity. First, the predicted CsgA secondary structure based on the 3 alpha/20 beta-hydroxysteroid dehydrogenase crystal structure suggests that the amino-terminal portion of the protein contains an NAD(P)+ binding pocket. Second, strains with csgA alleles encoding amino acid substitutions T6A and R10A in the NAD(P)+ binding pocket failed to develop. Third, exogenous MalE-CsgA rescues csgA development, whereas MalE-CsgA with the amino acid substitution CsgA T6A does not. Finally, csgA spore yield increased approximately 20% when containing 100 nM of MalE-CsgA was supplemented with 10 microM of NAD+ or NADP+. Conversely, 10 microM of NADH or NADPH delayed development for approximately 24 hr and depressed spore levels approximately 10%. Together, these results argue that NAD(P)+ binding is critical for C-signaling. S135 and K155 are conserved amino acids in the catalytic domain of SCAD members. Strains with csgA alleles encoding the amino acid substitutions S135T or K155R failed to develop. Furthermore, a MalE-CsgA protein containing CsgA S135T was not able to restore development to csgA cells. In conclusion, amino acids conserved in the coenzyme binding pocket and catalytic site are essential for C-signaling.

Amino Acid Sequence↗

PubMed1992

csgA expression entrains Myxococcus xanthus development.

The development cycle of the myxobacterium Myxococcus xanthus consists of three partially overlapping morphological stages referred to as rippling, fruiting body formation, and sporulation, all of which are absent in csgA null mutants. The CsgA gene product is an extracellular protein, referred to as the C signal, which is essential for developmental cell-cell interactions. csgA expression increases throughout development, reaching its peak during sporulation. CsgA was made limiting for development by constructing nested deletions upstream from the csgA gene, which resulted in reduced csgA expression. Successively larger deletions resulted in termination of development at earlier and earlier stages, with rippling requiring approximately 20% maximum csgA expression, fruiting body formation requiring approximately 30% expression, and sporulation requiring 82% expression. Conversely, artificial induction of csgA also induced development provided nutrients were limiting. These results suggest that steady increases in CsgA over the course of development entrain the natural sequence of morphological events. The csgA upstream region appears to process information concerning the levels of nutrients, peptidoglycan components, and the B signal. In the absence of nutrients, a region extending 400 bp upstream from the start site of transcription was necessary for development and maximal csgA expression. In the presence of low levels of nutrients, a region extending approximately 930 bp upstream was essential for the same tasks. It appears that the upstream region extending from -400 to -930 stimulates csgA expression in the presence of excess carbon, nitrogen, and phosphate, thereby allowing development to go to completion.

Base Sequence↗