Molecular analysis of Francisella tularensis subspecies tularensis and holarctica.

Rapid methods are needed for public health and military applications to specifically identify Francisella tularensis, the causative agent of tularemia in humans. A comparative analysis of the capabilities of multiple technologies was performed using a well-defined set of organisms to determine which approach would provide the most information in the shortest time. High-resolution molecular techniques, including pulsed-field gel electrophoresis, amplified fragment length polymorphism, and ribotyping, provided subspecies level identification within approximately 24 hours after obtaining an isolate, whereas multilocus variable number tandem repeat analysis with 8 or 25 targets provided strain level discrimination within about 12 hours. In contrast, Raman spectroscopy provided species level identification in 10 minutes but could not differentiate between subspecies tularensis and holarctica.

The multifunctional protein OBF1 is phosphorylated at serine and threonine residues in Saccharomyces cerevisiae.

We have purified a DNA replication enhancer-binding protein, OBF1, from yeast cells grown in a medium containing 32P-labeled orthophosphate. The purified 32P-labeled protein comigrated on polyacrylamide gels with OBF1 bands identified by immunoblotting with anti-OBF1 antibodies. Furthermore, trypsin treatment of the 32P-labeled OBF1 revealed several phosphorylated peptides, suggesting that OBF1 is multiply phosphorylated in vivo. Incubation of phosphorylated peptides with calf intestinal phosphatase liberated the radiolabel as free phosphate, indicating a phosphoester linkage. Acid hydrolysis of the tryptic peptides revealed 32P-label label comigrating with phosphoserine; some of it, however, was also identified as phosphothreonine. Using anti-OBF1 antibodies, we cloned the OBF1 gene from a lambda gt11 yeast expression library. The DNA sequence of the isolated gene and its over-expression in yeast indicated that OBF1 is identical to ABF-1 and BAF1 proteins, believed to have a role in transcriptional repression and activation. Therefore, we suggest that OBF1 is a multifunctional protein, acting in transcription and replication, and that these activities are regulated by phosphorylation.

A DNA replication enhancer in Saccharomyces cerevisiae.

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.

Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences.

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 ARS1 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 purify OBF1 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 OBF1 protein and its DNA-binding site are important for the function of an autonomously replicating sequence in Saccharomyces cerevisiae.

The autonomously replicating sequence ARS121 was cloned as a 480-base-pair (bp) long DNA fragment that confers on plasmids autonomous replication in Saccharomyces cerevisiae. This fragment contains two OBF1-binding sites (sites I and II) of different affinities, as identified by a gel mobility shift assay and footprint analysis. Nucleotide substitutions (16 to 18 bp) within either of the two sites obliterated detectable in vitro OBF1 binding to the mutagenized site. Linker substitution (6 bp) mutations within the high-affinity site I showed effects similar to those of the complete substitution, whereas DNA mutagenized outside the binding site bound OBF1 normally. We also tested the mitotic stability of centromeric plasmids bearing wild-type and mutagenized copies of ARS121. Both deletion of the sites and the extensive base alterations within either of the two OBF1-binding sites reduced the percentage of plasmid-containing cells in the population from about 88% to 50 to 63% under selective growth and from about 46% to 15 to 20% after 10 to 12 generations of nonselective growth. Furthermore, linker (6 bp) substitutions within site I, the high-affinity binding site, showed similar deficiencies in plasmid stability. In contrast, plasmids containing linker substitutions in sequences contiguous to site I displayed wild-type stability. In addition, plasmid copy number analysis indicated that the instability probably resulted not from nondisjunction during mitosis but rather from inefficient plasmid replication. The results strongly support the notion that the OBF1-binding sites and the OBF1 protein are important for normal ARS function as an origin of replication.

Immunoelectron microscopic localization of small, acid-soluble spore proteins in sporulating cells of Bacillus subtilis.

Small, acid-soluble spore proteins SASP-alpha, SASP-beta, and SASP-gamma as well as a SASP-beta-lacZ gene fusion product were found only within the forespore compartment of sporulating Bacillus subtilis cells by using immunoelectron microscopy. The alpha/beta-type SASP were associated almost exclusively with the forespore nucleoid, while SASP-gamma was somewhat excluded from the nucleoid. These different locations of alpha/beta-type and gamma-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.