The DED1 gene, which encodes a putative RNA helicase, has been implicated in nuclear pre-messenger RNA splicing in the yeast Saccharomyces cerevisiae. It is shown here by genetic and biochemical analysis that translation, rather than splicing, is severely impaired in two newly isolated ded1 conditional mutants. Preliminary evidence suggests that the protein Ded1p may be required for the initiation step of translation, as is the distinct DEAD-box protein, eukaryotic initiation factor 4A (eIF4A). The DED1 gene could be functionally replaced by a mouse homolog, PL10, which suggests that the function of Ded1p in translation is evolutionarily conserved.

The DBP5 gene encodes a putative RNA helicase of unknown function in the yeast Saccharomyces cerevisiae. It is shown here that Dbp5p is an ATP-dependent RNA helicase required for polyadenylated [poly(A) ⍣ ] RNA export. Surprisingly, Dbp5p is present predominantly, if not exclusively, in the cytoplasm, and is highly enriched around the nuclear envelope. This observation raises the possibility that Dbp5p may play a role in unloading or remodeling messenger RNA particles (mRNPs) upon arrival in the cytoplasm and in coupling mRNP export and translation. The functions of Dbp5p are likely to be conserved, since its potential homologues can be found in a variety of eukaryotic cells.

In Saccharomyces cerevisiae, ribosomal biogenesis takes place primarily in the nucleolus, in which a single 35S precursor rRNA (pre-rRNA) is first transcribed and sequentially processed into 25S, 5.8S, and 18S mature rRNAs, leading to the formation of the 40S and 60S ribosomal subunits. Although many components involved in this process have been identified, our understanding of this important cellular process remains limited. Here we report that one of the evolutionarily conserved DEAD-box protein genes in yeast, DBP3, is required for optimal ribosomal biogenesis. DBP3 encodes a putative RNA helicase, Dbp3p, of 523 amino acids in length, which bears a highly charged amino terminus consisting of 10 tandem lysine-lysine-X repeats ([KKX] repeats). Disruption of DBP3 is not lethal but yields a slow-growth phenotype. This genetic depletion of Dbp3p results in a deficiency of 60S ribosomal subunits and a delayed synthesis of the mature 25S rRNA, which is caused by a prominent kinetic delay in pre-rRNA processing at site A 3 and to a lesser extent at sites A 2 and A 0 . These data suggest that Dbp3p may directly or indirectly facilitate RNase MRP cleavage at site A 3 . The direct involvement of Dbp3p in ribosomal biogenesis is supported by the finding that Dbp3p is localized predominantly in the nucleolus. In addition, we show that the [KKX] repeats are dispensable for Dbp3p's function in ribosomal biogenesis but are required for its proper localization. The [KKX] repeats thus represent a novel signaling motif for nuclear localization and/or retention.In eukaryotes, ribosomal biogenesis takes place primarily in a specialized subnuclear compartment termed the nucleolus, where rRNA genes are localized and first transcribed into a single precursor rRNA (pre-rRNA). In Saccharomyces cerevisiae, this pre-rRNA is rapidly associated with a large number of ribosomal and nonribosomal proteins and sequentially processed through a number of intermediates by both exo-and endonucleases into mature 25S, 18S, and 5.8S rRNAs which are found in 60S (25S and 5.8S rRNAs) and 40S (18S rRNA) ribosomal subunits (reviewed in reference 79). During transcription and processing, pre-rRNA is also chemically modified by methylation on base and ribose moieties and by pseudouridylation. Although this highly complicated rRNA-processing pathway has been studied with many organisms, the genetically tractable yeast system has so far provided the most extensive information regarding the various cis-and trans-acting factors crucial to this process.In yeast, rRNA is first transcribed by RNA polymerase I as a 35S pre-rRNA which contains the mature 18S, 5.8S, and 25S rRNAs separated by two internal transcribed spacers, ITS1 and ITS2, and flanked by two external transcribed spacers, 5Ј ETS and 3Ј ETS (Fig. 1) (reviewed in reference 74). Processing of this 35S pre-rRNA occurs initially at sites A 0 , A 1 , and A 2 to yield the 20S pre-rRNA and the 27SA 2 pre-rRNA. Processing of the 20S pre-rRNA takes place in the cytoplasm, where site D is cleaved to form the ...

Kansas City, Kansas and V.A. Hospital Kansas City, Missouri, U S A .Multispecific, cross-reactive sera can be demonstrated by absorption studies to be of two types; a single antibody that is multivalent, and some that are mixtures of monospecific and multispecific antibody populations. The second type can be separated by a differential absorption technique. The characteristics of these sera that can be separated and the techniques are discussed.