A cryptic plasmid, pBAA1, was identified in an industrial Bacillus strain. The plasmid is 6.8 kilobases in size and is present in cells at a copy number of approximately 5 per chromosome equivalent. The p!asmid has been maintained under industrial fermentation conditions without apparent selective pressure and so is assumed to be partition proficient. The minimal replicon was localized to a 1.4-kilobase fragment which also contains the functions required for copy number control. The very low level of segregational instability of the minimal replicon suggests that it also contains functions involved in plasmid maintenance. Comparison with other plasmids indicates that pBAA1 belongs to the group of small gram-positive plasmids which replicate by a rolling cycle-type mechanism. A sequence was identified which is required for the efficient conversion of the single plus strand to the double-stranded form during plasmid replication. Deletion of this sequence resulted in a low level of segregational plasmid instability.The development of cloning vectors for use in Bacillus subtilis has relied on plasmids isolated from Staphylococcus aureus (6,7,14,15). Both structural (10,15,19,20,27) and segregational (1, 2, 10, 19, 28) instabilities were frequently observed when recombinant vectors were transformed into B. subtilis. These instabilities led to intense investigation of the mode of replication and of the stability functions of these plasmids. It has emerged that many of these plasmids replicate by a rolling cycle-type mechanism (11,12,31). The essential features of this mode of replication are (i) an origin of plus-strand synthesis, (ii) a replication protein which interacts with the plus origin to generate a nick which allows displacement synthesis of the plus strand to occur, and (iii) a signal for efficient conversion of the single strand to the double-stranded form. These features have been identified for a variety of plasmids, including pT181 (12, 16, 25) and pC194 (11, 12).Our aim is to analyze the segregational instability of plasmids in B. subtilis. It was hypothesized that the segregational instability of many S. aureus plasmids in B. subtilis is due, at least in part, to a suboptimal host-plasmid relationship. Thus, we chose to analyze the stability functions of a plasmid, pBAA1, which was resident in an industrial strain of B. subtilis. pBAA1 has been maintained under industrial fermentation conditions without apparent selection pressure. Preliminary experiments demonstrated that the copy number was low (approximately 5 per chromosome equivalent), so it was assumed that pBAA1 must encode an active partitioning function. In this paper, we report that all of the functions required for replication, copy number control, and partitioning are located on a 2.2-kilobase (kb) fragment. The DNA sequence and other evidence show that pBAA1 replicates by a rolling-circle mechanism and that some features of its replication functions are related both to 4X174 and to the gram-positive plasmids pC194, pUB110, and pFTB14.MAT...
The nucleotide sequence of a 4.39‐kb DNA fragment encoding the α‐glucosidase gene of Candida tsukubaensis is reported. The cloned gene contains a major open reading frame (ORF 1) which encodes the α‐glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa. N‐terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the α‐glucosidase precursor is proteolytically processed by removal of an N‐terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63–65 kDa and 50–52 kDa, respectively. Both subunits are secreted by the heterologous host S. cerevisiae in a glycosylated form. Coincident with its efficient expresion in the heterologus host, the C. tsukubaensisα‐glucosidase gene contains many of the canonical features of highly expressed S. cerevisiae genes. There is considerable sequence similarity between C. tsukubaensisα‐glucosidase, the rabbit sucrase‐isomaltase complex (proSI) and human lysosomal acid α‐glucosidase. The cloned DNA fragment from C. tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids. The function and identity of the polypeptide encoded by ORF 2 is not known.
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.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.