The chromosomal DNA of Streptomyces griseus 2247 (a derivative of strain IFO3237) was digested with several restriction endonucleases and analyzed by pulsed-field gel electrophoresis (PFGE). Digestion with AseI and DraI gave 15 and 9 fragments, respectively, the total sizes of which were 7.8 Mb. All the AseI and DraI fragments were aligned on a linear chromosome map by using linking plasmids and cosmids. PFGE analysis of the intact chromosome also showed a linear DNA band of about 8 Mb. Detailed physical maps of both terminal regions were constructed; they revealed the presence of a 24-kb terminal inverted repeat on each end. PFGE analysis with and without proteinase K treatment suggested that each end of the chromosome carries a protein molecule.Streptomyces species are gram-positive soil bacteria with a high level of GϩC base composition (70 to 74%) in their DNA (4). They display a complex life cycle which culminates in spore formation (3) and involves the production of a large number of secondary metabolites such as enzyme inhibitors, herbicides, and over 70% of naturally occurring antibiotics (2). These characteristics make this genus an attractive research subject from both academic and industrial points of view.Streptomyces griseus is one of the physiologically best-studied species among streptomycetes. Studies of various S. griseus strains without genealogical relationship revealed that this species sporulates well in liquid culture (19) and produces the classical antibiotic streptomycin. It also produces A-factor, an autoregulating hormone essential for both sporulation and streptomycin production (16,20). A preliminary attempt to construct a genetic map of the S. griseus NRRL3851 chromosome (33, 34) was not followed up because of its genetic instability, which is an obstacle to genetic studies of this physiologically interesting Streptomyces species.Recently, macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) allowed the construction of physical maps of bacterial chromosomes. Thus, it was shown that the chromosomes of Streptomyces lividans and Streptomyces coelicolor A3 (2) have a linear topology (5, 28, 29). Moreover, several reports revealed a wide variety of bacterial chromosome structures (15). For example, Borrelia burgdorferi has a linear chromosome (9), Agrobacterium tumefaciens contains a linear and a circular chromosome (1), and Rhodobacter sphaeroides has two circular chromosomes (38). Therefore, the long-standing belief that bacteria have one circular chromosome has been disproved: linear chromosomes are not a monopoly of eukaryotes.To characterize physically the S. griseus chromosome, we made use of PFGE analysis of macrorestriction fragments of the chromosome and the alignment of these fragments by hybridization with linking plasmids and cosmids. Here, we describe a linear chromosome map of S. griseus 2247 and the analysis of its terminal structure. MATERIALS AND METHODSBacterial strains, plasmids, and media. S. griseus strains were obtained from the IFO (Institute for Fer...

The aggregation propensity of humanized antibody after heat treatment is evaluated in the presence of six buffer species. The comparison under equivalent pH showed high aggregation propensity on phosphate and citrate buffer. In contrast, 2-(N-Morpholino) ethane sulfonate (MES), 3-(N-Morpholino) propane sulfonate (MOPS), acetate and imidazole buffer showed lower aggregation propensity than the above two buffers. Meanwhile, unfolding temperature evaluated by differential scanning calorimetry measurement was not altered among these buffer species. The light scattering analysis suggested that heat-denatured intermediate was aggregated slightly on MES and acetate buffer. Therefore, it was found that the different aggregation propensity among buffer species was caused from the aggregation propensity of heat-denatured intermediate rather than the unfolding temperature. Furthermore, it was revealed that the aggregation dependency on buffer species is accounted for by the specific molecular interaction between buffer and IgG, rather than the ionic strength. On the contrary, on the analyses of unfolding and aggregation propensity by molecular dissection of IgG into Fab and Fc fragments, aggregation propensity of Fc fragment on MES, acetate and phosphate buffer was almost the same as whole IgG. From the above results, it was suggested that the specific interaction between buffer molecule and Fc domain of IgG was involved in the aggregation propensity of heat-denatured IgG.

We have recently constructed a physical map of the Streptomyces griseus 2247 genome using the restriction enzymes AseI and DraI, which revealed that this strain carries a 7.8 Mb linear chromosome. Based on this map, precise macrorestriction fragment and cosmid maps were constructed for both ends of the chromosome, which localized the afsA gene 150 Kb from the left end. Two afsA- mutants were found to have suffered chromosomal deletions that removed the afsA locus. The sizes of the deletions were 20 and 130 Kb at the right end and 180 and 350 kb at the left end, respectively. Hybridization experiments using cosmids carrying a deletion endpoint indicated that the ends of the chromosome in the mutants were fused to form a circular chromosome.

Suppression of IgG1 aggregation can be attributed to the interaction between Arg and hydrophobic residues; on the other hand, facilitation of aggregation and degradation is presumably due to the interaction between Arg and some acidic residues, which could be competitively inhibited by simultaneously adding either Asp or Glu.

To examine the effect of the conformational stability of the CH2 domain on aggregation and peptide cleavage of a humanized IgG1, we carried out size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of incubated sample solutions. By comparing the residual percentage of monomer after incubation at 60 and 80°C at various pH levels, we found that aggregation and peptide cleavage of the humanized IgG1 occurred during long incubation at 60°C under acidic conditions. Next, we confirmed cleavage of the Asp272-Pro273 peptide bond in the CH2 domain. Comparison of the cleavage rates of the IgG1 monomer and a peptide containing the same Asp-Pro sequence revealed that the conformational stability of the CH2 domain retards cleavage of the Asp272-Pro273 peptide bond at 60°C and pH 4.0. The finding of aggregation and peptide cleavage of the humanized IgG1 after long incubation at 60°C under acidic conditions was supported by another finding: there were lower unfolding temperatures of the CH2 domain at pH 4.0 and 5.0. We conclude that the conformational stability of the CH2 domain is closely related to aggregation and peptide cleavage of the humanized IgG1 under acidic conditions. We also found that the 2-[N-morpholino] ethane sulfonate buffer inhibits aggregation of the IgG1 at pH 4.0-5.0 and 7.0-8.0.