Here we show that emb-30 is required for metaphase-to-anaphase transitions during meiosis and mitosis in Caenorhabditis elegans. Germline-specific emb-30 mutant alleles block the meiotic divisions. Mutant oocytes, fertilized by wild-type sperm, set up a meiotic spindle but do not progress to anaphase I. As a result, polar bodies are not produced, pronuclei fail to form, and cytokinesis does not occur. Severe-reduction-of-function emb-30 alleles (class I alleles) result in zygotic sterility and lead to germline and somatic defects that are consistent with an essential role in promoting the metaphase-to-anaphase transition during mitosis. Analysis of the vulval cell lineages in these emb-30(class I) mutant animals suggests that mitosis is lengthened and eventually arrested when maternally contributed emb-30 becomes limiting. By further reducing maternal emb-30 function contributed to class I mutant animals, we show that emb-30 is required for the metaphase-toanaphase transition in many, if not all, cells. Metaphase arrest in emb-30 mutants is not due to activation of the spindle assembly checkpoint but rather reflects an essential emb-30 requirement for M-phase progression. A reduction in emb-30 activity can suppress the lethality and sterility caused by a null mutation in mdf-1, a component of the spindle assembly checkpoint machinery. This result suggests that delaying anaphase onset can bypass the spindle checkpoint requirement for normal development. Positional cloning established that emb-30 encodes the likely C. elegans orthologue of APC4/Lid1, a component of the anaphase-promoting complex/cyclosome, required for the metaphase-to-anaphase transition. Thus, the anaphase-promoting complex/cyclosome is likely to be required for all metaphase-to-anaphase transitions in a multicellular organism.
Arylamine N-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote Salmonella typhimurium. NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it. nat genes were cloned fromMycobacterium smegmatis and Mycobacterium tuberculosis, and expressed in Escherichia coli andM. smegmatis. The induced M. smegmatis NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from S. typhimurium recognizes NAT fromM. smegmatis and cross-reacts with recombinant NAT fromM. tuberculosis. Overexpression of mycobacterialnat genes in E. coli results in predominantly insoluble recombinant protein; however, with M. smegmatisas the host using the vector pACE-1, NAT proteins from M. tuberculosis and M. smegmatis are soluble. M. smegmatis transformants induced to express the M. tuberculosis nat gene in culture demonstrated a threefold higher resistance to isoniazid. We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.
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