Pheromone‐stimulated yeast cells and haploid gpa1 deletion mutants arrest their cell cycle in G1. Overexpression of a novel gene called MSG5 suppresses this inhibition of cell division. Loss of MSG5 function leads to a diminished adaptive response to pheromone. Genetic analysis indicates that MSG5 acts at a stage where the protein kinases STE7 and FUS3 function to transmit the pheromone‐induced signal. Since loss of MSG5 function causes an increase in FUS3 enzyme activity but not STE7 activity, we propose that MSG5 impinges on the pathway at FUS3. Sequence analysis suggests that MSG5 encodes a protein tyrosine phosphatase. This is supported by the finding that recombinant MSG5 has phosphatase activity in vitro and is able to inactivate autophosphorylated FUS3. Thus MSG5 might stimulate recovery from pheromone by regulating the phosphorylation state of FUS3.

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...

Saccharomyces cerevisiae Rrs1p is a nuclear protein that is essential for the maturation of 25 S rRNA and the 60 S ribosomal subunit assembly. In two-hybrid screening, using RRS1 as bait, we have cloned YKR081c/RPF2. Rpf2p is essential for growth and is mainly localized in the nucleolus. The amino acid sequence of Rpf2p is highly conserved in eukaryotes from yeast to human. Similar to Rrs1p, Rpf2p shows physical interaction with ribosomal protein L11 and appears to associate with preribosomal subunits fairly tightly. Northern, methionine pulse-chase, and sucrose density gradient ultracentrifugation analyses reveal that the depletion of Rpf2p results in a delayed processing of pre-rRNA, a decrease of mature 25 S rRNA, and a shortage of 60 S subunits. An analysis of processing intermediates by primer extension shows that the Rpf2p depletion leads to an accumulation of 27 SB pre-rRNA, suggesting that Rpf2p is required for the processing of 27 SB into 25 S rRNA.

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.

The nucleotide sequence of a 2.1-kilobase-pair fragment containing the Streptomyces choA gene, which codes a secreted cholesterol oxidase, was determined. A single open reading frame encodes a mature cholesterol oxidase of 504 amino acids, with a calculated Mr of 54,913. The leader peptides extend over 42 amino acids and have the characteristics of a signal sequence, including basic amino acids near the amino terminus and a hydrophobic core near the signal cleavage site. Analyses of the total amino acid composition and amino acid sequencing of the first 21 amino acids from the N terminus of the purified extracellular enzyme agree with the values deduced from nucleotide sequencing data.