Although the U3 small nucleolar RNA (snoRNA), a member of the box C/D class of snoRNAs, was identified with the spliceosomal small nuclear RNAs (snRNAs) over 30 years ago, its function and its associated protein components have remained more elusive. The U3 snoRNA is ubiquitous in eukaryotes and is required for nucleolar processing of pre-18S ribosomal RNA in all organisms where it has been tested. Biochemical and genetic analyses suggest that U3 pre-rRNA base-pairing interactions mediate endonucleolytic pre-rRNA cleavages. Here we have purified a large ribonucleoprotein (RNP) complex from Saccharomyces cerevisiae that contains the U3 snoRNA and 28 proteins. Seventeen new proteins (Utp1 17) and Rrp5 were present, as were ten known components. The Utp proteins are nucleolar and specifically associated with the U3 snoRNA. Depletion of the Utp proteins impedes production of the 18S rRNA, indicating that they are part of the active pre-rRNA processing complex. On the basis of its large size (80S; calculated relative molecular mass of at least 2,200,000) and function, this complex may correspond to the terminal knobs present at the 5' ends of nascent pre-rRNAs. We have termed this large RNP the small subunit (SSU) processome.

8-Nitro-2′-deoxyguanosine (8-nitrodG) is a relatively unstable, mutagenic lesion of DNA that is increasingly believed to be associated with tissue inflammation. Due to the lability of the glycosidic bond, 8-nitrodG cannot be incorporated into oligodeoxynucleotides (ODNs) by chemical DNA synthesis and thus very little is known about its physicochemical properties and base-pairing preferences. Here we describe the synthesis of 8-nitro-2′-O-methylguanosine, a ribonucleoside analogue of this lesion, which is sufficiently stable to be incorporated into ODNs. Physicochemical studies demonstrated that 8-nitro-2′-O-methylguanosine adopts a syn conformation about the glycosidic bond; thermal melting studies and molecular modelling suggest a relatively stable syn-8-nitroG·anti-G base pair. Interestingly, when this lesion analogue was placed in a primer-template system, extension of the primer by either avian myeloblastosis virus reverse transcriptase (AMV-RT) or human DNA polymerase β (pol β), was significantly impaired, but where incorporation opposite 8-nitroguanine did occur, pol β showed a 2:1 preference to insert dA over dC, while AMV-RT incorporated predominantly dC. The fact that no 8-nitroG·G base pairing is seen in the primer extension products suggests that the polymerases may discriminate against this pairing system on the basis of its poor geometric match to a Watson–Crick pair.

The levels of staphylococcal enterotoxin B (SEB) produced by various naturally occurring toxinogenic strains of Staphylococcus aureus are highly variable. The SEB gene (seb) from a high-producer strain, S6, has previously been cloned and characterized. Cloning and nucleotide sequence analysis of the upstream region of the seb gene from DU4916 and COL (medium-and low-level toxin-producer strains, respectively) showed that their sequence was identical to that of the seb gene from strain S6. Strains carrying the cloned seb gene from DU4916 and COL produced similar levels of SEB protein and mRNA to those produced by strains carrying the cloned seb gene from strain S6. An RNA encoded by the 8-lysin gene (hid) has been shown to regulate the genes for a number of extracellular proteins, including SEB. Northern (RNA) blot analysis showed that variable levels of hld RNA were present in various SEB-producer strains, with the order being S6 > DU4916 > COL. Our results suggest that differences in host factor(s), including the hld RNA, are responsible for the production of different amounts of SEB by many naturally occurring strains.The genes encoding several staphylococcal enterotoxins have been cloned and characterized (1-3, 6, 12, 17). Staphylococcal enterotoxin B (SEB) consists of a single polypeptide of 239 amino acids and has a molecular weight of 28,336 (9, 12). The naturally occurring SEB-producer (Seb+) strains S6, DU4916, and COL excrete approximately 375, 50, and 12 pLg of SEB per ml, respectively, into the culture media (20, 21). The SEB gene (seb) has been previously cloned from the high Seb+ strain S6 (17). The promoter sequence of the seb gene has been identified by nuclease Si mapping and by comparison with the promoter consensus sequences of Escherichia coli (8). An upstream element located between nucleotides 58 and 93 upstream of the transcription start site has been shown to be required for the transcription of the seb gene (12, 13). Southern hybridization experiments have shown that all the Seb+ strains tested carry a single copy of the seb gene (11). Northern (RNA) hybridization analysis of SEB mRNA from a number of naturally occurring Seb+ strains has shown that the seb gene is regulated at the level of transcription or mRNA stability (8). A polycistronic locus, termed the accessory gene regulator (agr), coordinately regulates the synthesis of a number of extracellular proteins in Staphylococcus aureus, including SEB (14,16,18). Although the synthesis of several exoproteins is decreased in agr mutants, the production of some exoproteins such as protein A and coagulase is increased in these strains (10,15,17). The activator of the agr system has been shown to be the b-lysin transcript (10). In this paper we demonstrate that host factors in the parental strains, including hld RNA, rather than differences in upstream sequences are responsible for the differential expression of the seb gene in various strains.Cloning of the seb gene from S. aureus DU4916 and COL. The seb gene from strains DU4916 and COL w...

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