We have dissected the autonomously replicating sequence ARS121 using site-directed in vitro mutagenesis. Three domains important for origin function were identified; one of these is essential and contains an 11-base-pair sequence resembling the canonical ARS core consensus; the second region, deletion of which affects the efficiency of the origin, is located 3' to the T-rich strand of the essential sequence and encompasses several elements with near matches to the ARS core consensus; the third region, containing two OBF1 DNA-binding sites and located 5' to the essential sequence, also affects the efficiency of the ARS. Here we demonstrate that a synthetic OBF1 DNA-binding site can substitute for the entire third domain in origin function. A dimer of the synthetic binding site, fused to a truncated origin containing only domains one and two, restored the origin activity to the levels of the wild-type ARS. The stimulation of origin function by the synthetic binding site was relatively orientation independent and could occur at distances as far as 1 kilobase upstream to the essential domain. Based on these results we conclude that the OBF1 DNA-binding site is an enhancer of DNA replication. We suggest that the DNA-binding site and the OBF1 protein are involved in the regulation of the activation of nuclear origins of replication in Saccharomyces cerevisiae.Replication of eukaryotic nuclear DNA is initiated at the start of the S phase and proceeds by multiple initiations along a chromosome, generating multiple replicons. However, the regulation and the mechanism of genomic DNA replication are not understood. Saccharomyces cerevisiae provides a convenient model system for studying DNA
Small, acid-soluble spore proteins SASP-oa, SASP-,, and SASP--y as well as a SASP-j-lacZ gene fusion product were found only within the forespore compartment of sporulating Bacillus subtilis cells by using immunoelectron microscopy. The aJ$-type SASP were associated almost exclusively with the forespore nucleoid, while SASP-y was somewhat excluded from the nucleoid. These different locations of oda,-type and -y-type small, acid-soluble spore proteins within the forespore are consistent with the different roles for these two types of proteins in spore resistance to UV light.Approximately 10% of the protein of dormant spores of Bacillus ,subtilis is composed of a group of small, acidsoluble spore proteins (SASP) (18). The SASP are synthesized in parallel during sporulation within the developing forespore and are rapidly degraded during spore germination, thus providing amino acids for protein synthesis during this period of development. Three proteins, termed SASP-a, SASP-1, and SASP-y, make up approximately 75% of the SASP pool. SASP-a and SASP-3 are almost identical in primary sequence, and there are a number of minor SASP with primary sequences similar to those of SASP-cx and SASP-P. In contrast, SASP-,y has a more different primary sequence, and there is only a single y-type SASP (18).Other than its role in providing amino acids for protein synthesis during spore germination, SASP--y appears to have no function in spores (6). However, the a/,B-type SASP also play a key role in the resistance of spores to UV light, as they are intimately involved in the modification of the UV photochemistry of spore DNA which is essential for spore UV resistance (11,12,14). While the. mechanism whereby a/p-type SASP affect spore DNA photochemistry is not known, it seems likely that it involves direct SASP-DNA interactions, and it is known that SASP are localiied within the spore core, the site of spore DNA (18). SASP can also bind to DNA in vitro (18). However, this binding is weak, and attempts to isolate spore DNA or spore nucleoids with significant associated SASP have failed (17). One study using intact Bacillus megaterium spores and UV-induced protein-DNA cross-linking did provide evidence that significant ao/-type SASP was associated with spore DNA in vivo (15). However, from this study it was not possible to determine what percentage of these SASP were DNA associated.Because of the limitations of these previous techniques, we decided to attempt to localize various SASP within B. subtilis spores and forespores by using immunoelectron microscopy. However, initial attempts to localize SASP in dormant spores by using this technique were unsuccessful, since the cross-linking agents used in fixation (paraformaldehyde with or without glutaraldehyde) did not penetrate dormant spores sufficiently to prevent SASP movement during subsequent steps. The lack of penetration of dormant spores by cross-linking agents is not surprising in light of * Corresponding author. what is known of dormant-spore permeability (5) as well as the resi...
This study provides useful baseline data for guiding future disease control programs.
We previously identified a protein activity from Saccharomyces cerevisiae, OBF1, that bound specifically to a DNA element present in autonomously replicating sequences ARS120 and ARS121 (S. Eisenberg C. Civalier, and B. K. Tye, Proc. Natl. Acad. Sci. USA 85:743-746, 1988). OBF1 has now been purified to near homogeneity by conventional protein and DNA affinity chromatography. Electrophoresis of the purified protein in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two polypeptides. The major protein band had a relative molecular size of 123 kilodaltons, and the minor protein band, which constituted only a small fraction of total protein, had a molecular size of 127 kilodaltons. Both polypeptides cochromatographed with the specific ARS120 DNA-binding activity and formed a stable protein-DNA complex, isolatable by sedimentation through sucrose gradients. Using antibodies, we have shown that both polypeptides are associated with the isolated protein-DNA complexes. The ARS DNA-binding activity had a Stokes radius of 54 A (5.4 nm) and a sedimentation coefficient of 4.28S, as determined by gel filtration and sedimentation through glycerol gradients, respectively. These physical parameters, together with the denatured molecular size values, suggested that the proteins exist in solution as asymmetric monomers. Since both polypeptides recognized identical sequences and had similar physical properties, they are probably related. In addition to binding to ARS120, we found that purified OBF1 bounds with equal affinity to ARS121 and with 5-and 10-fold-lower affinity to ARS) and HMRE, respectively. Furthermore, in the accompanying paper (S. S. Walker, S. C. Francesconi, B. K. Tye, and S. Eisenberg, Mol. Cell. Biol. 9:2914-2921, 1989), we demonstrate the existence of a high, direct correlation between the ability of purified OBFI to bind to ARS121 and optimal in vivo ARS121 activity as an origin of replication. These findings, taken together, suggest a role for OBF1 in ARS function, presumably at the level of initiation of DNA replication at the ARS.The replication of eucaryotic chromosomal DNA proceeds by numerous initiations along a chromosome, generating multiple replicons. It is believed that in Sacc haromyces cerevisiae the initiation sites at replicons are specific and correspond to the autonomously replicating sequences (ARSs). ARSs were isolated as DNA fragments that confer on plasmids bearing them the ability to replicate autonomously in yeast cells (29). Both in vitro (6,13,16) and in vivo (3,11,19,23)
Francisella tularensis subsp. holarctica is widely disseminated in North America and the boreal and temperate regions of the Eurasian continent. Comparative genomic analyses identified a 1.59-kb genomic deletion specific to F. tularensis subsp. holarctica isolates from Spain and France. Phylogenetic analysis of strains carrying this deletion by multiple-locus variable-number tandem repeat analysis showed that the strains comprise a highly related set of genotypes, implying that these strains were recently introduced or recently emerged by clonal expansion in France and the Iberian Peninsula.
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.