We have developed techniques for the detailed analysis of cis-acting sequences in the pre-rRNA of Saccharomyces cerevisiae and used these to study the processing of internal transcribed spacer 1 (ITS1) leading to the synthesis of 5.8S rRNA. As is the case for many eukaryotes, the 5' end of yeast 5.8S rRNA is heterogeneous; we designate the major, short form 5.8S(S), and the minor form (which is seven or eight nucleotides longer) 5.8S(L). These RNAs do not have a precursor/product relationship, but result from the use of alternative processing pathways. In the major pathway, a previously unidentified processing site in ITS1, designated A3, is cleaved. A 10 nucleotide deletion at site A3 strongly inhibits processing of A3 and the synthesis of 5.8S(S); processing is predominantly transferred to the alternative 5.8S(L) pathway. Site A3 lies 76 nucleotides 5' to the end of 5.8S(S), and acts as an entry site for 5'-3' exonuclease digestion which generates the 5' end of 5.8S(S). This pathway is inhibited in strains mutant for XRNlp and RATlp. Both of these proteins have been reported to have 5'-3' exonuclease activity in vitro. Formation of 5.8S(L) is increased by mutations at A3 in cis or in RATlp and XRNlp in trans, and is kinetically faster than 5.8S(S) synthesis.

SUMMARY Many plants produce low-molecular-weight compounds which inhibit the growth of phytopathogenic fungi in vitro. These compounds may be preformed inhibitors that are present constitutively in healthy plants (also known as phytoanticipins), or they may be synthesized in response to pathogen attack (phytoalexins). Successful pathogens must be able to circumvent or overcome these antifungal defenses, and this review focuses on the significance of fungal resistance to plant antibiotics as a mechanism of pathogenesis. There is increasing evidence that resistance of fungal pathogens to plant antibiotics can be important for pathogenicity, at least for some fungus-plant interactions. This evidence has emerged largely from studies of fungal degradative enzymes and also from experiments in which plants with altered levels of antifungal secondary metabolites were generated. Whereas the emphasis to date has been on degradative mechanisms of resistance of phytopathogenic fungi to antifungal secondary metabolites, in the future we are likely to see a rapid expansion in our knowledge of alternative mechanisms of resistance. These may include membrane efflux systems of the kind associated with multidrug resistance and innate resistance due to insensitivity of the target site. The manipulation of plant biosynthetic pathways to give altered antibiotic profiles will also be valuable in telling us more about the significance of antifungal secondary metabolites for plant defense and clearly has great potential for enhancing disease resistance for commercial purposes.

Subnuclear fractionation and coprecipitation by antibodies against the nucleolar protein NOP1 demonstrate that the essential Saccharomyces cerevisiae RNA snR30 is localized to the nucleolus. By using aminomethyl trimethyl-psoralen, snR30 can be cross-linked in vivo to 35S pre-rRNA. To determine whether snR30 has a role in rRNA processing, a conditional allele was constructed by replacing the authentic SNR30 promoter with the GALIO promoter. Repression of snR30 synthesis results in a rapid depletion of snR30 and a progressive increase in cell doubling time. rRNA processing is disrupted during the depletion of snR30; mature 18S rRNA and its 20S precursor underaccumulate, and an aberrant 23S pre-rRNA intermediate can be detected. Initial results indicate that this 23S pre-rRNA is the same as the species detected on depletion of the small nucleolar RNA-associated proteins NOPI and GAR1 and in an snrlO mutant strain. It was found that the 3' end of 23S pre-rRNA is located in the 3' region of ITS1 between cleavage sites A2 and Bi and not, as previously suggested, at the Bi site. snR30 is the fourth small nucleolar RNA shown to play a role in rRNA processing.In eukaryotes, the nucleolus is the center of ribosome biogenesis; imported ribosomal proteins associate with rRNAs, and the mature ribosomal subunits are then exported to the cytoplasm. The 18S, 5.8S, and 25S rRNAs are transcribed as a single transcript, the 35S pre-rRNA, which is processed via a number of intermediates (pre-rRNAs) to the mature rRNAs. It is thought that many of the ribosomal proteins assemble with the pre-rRNA while it is being processed.There is a class of small ribonucleoprotein particles localized to the nucleolus (small nucleolar ribonucleoproteins [snoRNPs]). To date, 10 small nucleolar RNAs (snoRNAs) have been cloned from Saccharomyces cerevisiae (reviewed in reference 31). Seven of these snoRNAs are nonessential, and strains carrying null alleles grow normally. snRlO is not essential, but strains carrying a null allele have a coldsensitive phenotype, and two snoRNAs, U3 and U14 (previously called snR128), are essential for growth.U3 is the most studied of the snoRNAs. Two recent studies have examined its role in rRNA processing in S. cerevisiae. Hughes and Ares (11) constructed a conditional allele of U3 by placing it under the control of a regulated GAL promoter. It was found that depletion of U3 inhibited production of mature 18S and its 20S precursor but 25S and 5.8S continued to be synthesized normally. The processing event, AO, in the 5' external transcribed spacer (ETS) was also abolished by U3 depletion. Beltrame and Tollervey (2) used psoralen cross-linking techniques to map the interaction between U3 and the pre-rRNA. U3 could be crosslinked in vivo to two sites in the ETS of the 35S pre-rRNA. These sites were subsequently analyzed by deletion mutagenesis; deletion of the upstream cross-linking site (+470) gave a phenotype similar to that of U3 depletion, with the loss of synthesis of 18S rRNA, but deletion of the downstream (+655...

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