Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network

Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.     

Isolation and characterization of Patnopecten mid-gut gland endo-beta-xylosidase active on peptidochondroitin sulfate

An endo-beta-xylosidase acting on the linkage region of peptidochondroitin sulfate was isolated from the mid-gut gland of the mollusc Patnopecten and purified about 375-fold, using a combination of ammonium sulfate fractionation, gel filtration on Sephacryl S-200, and DEAE-Sephacel chromatography. The pH optimum and the isoelectric point of this enzyme were 4.0 and 7.0, respectively. The molecular weight, estimated by gel filtration through Sephacryl S-200, was 78,000. The purified enzyme was completely free from protease, exoglycosidases, sulfatase, and phosphatase. This enzyme hydrolyzed the xylosyl serine linkage of the linkage region of various glycosaminoglycans, that is chondroitin sulfate, dermatan sulfate and heparan sulfate, all possessing a very small peptide segment, but not proteoglycans. It was concluded that this endo-beta-xylosidase was involved in the catabolism of proteoglycans.     

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA.     

Analysis of a monoclonal rat antibody directed to the alpha-chain variable region (V alpha 3) of the mouse T cell antigen receptor

Three rat mAb, RR3-15, RR3-16, and RR3-18, were established by fusing spleen cells from a rat immunized with the male Ag-specific cytolytic T cell clone, OH6, to mouse myeloma cells. The mAb was identified by their capacity to focus the cytolytic activity of the OH6 CTL clone on nonspecific target cells via FcR-FcR interaction. That all three mAb recognized the OH6 TCR was confirmed by immunoprecipitation studies in which each antibody precipitated a 90 kDa disulfide-linked heterodimer characteristic of the TCR. Surface immunofluorescence staining of a panel of T cell lines and splenic T cell populations showed that RR3-16 reacted not only to the OH6 T cell clone but also to a minor fraction of normal T cells. This reactivity was found to be due to the expression of a gene in the V alpha 3 family. However, RR3-16 did not react with all T cell lines and clones known to express genes from the V alpha 3 family. cDNA sequences of three independent RR3-16+ T cell hybridomas analyzed by polymerase chain reaction were identical to the previously published V alpha 3 sequence of the CTL clone C9. Thus, the mAb RR3-16 is specific for a single member of the TCR V alpha 3 gene family. Analysis of the expression of RR3-16+ TCR in CD4+ and CD8+ subsets of peripheral T cells demonstrated preferential expression on CD8+ T cells, suggesting regulated expression of this particular TCR V alpha gene.     

Demonstration of an endo-beta-galactosidase and an endo-beta-xylosidase that degrade the proteoglycan linkage region

Purified proteochondroitin sulfate was incubated at pH 4.0 with a rabbit liver lysosomal enzyme fraction. The formed degradation products were isolated by gel filtration followed by reduction with NaB3H4. After acid hydrolysis of the reduced digest, the hydrolysate was passed through Dowex 50W-X8 and 1-X2 columns. The separated neutral sugars were then subjected to paper chromatography, which demonstrated that galactose and xylose had been at the reducing termini of chondroitin sulfate after incubation with the liver lysosomal preparation. These results suggest the presence in the liver lysosomal preparation of an endo-beta-galactosidase and an endo-beta-xylosidase which act at the linkage region of proteochondroitin sulfate.     

A human polyadenylation factor is a G protein beta-subunit homologue.

Cleavage stimulation factor (CstF) is one of the multiple factors required for polyadenylation of mammalian pre-mRNAs in vitro. We have shown previously that this factor is composed of three distinct subunits of 77, 64, and 50 kDa, and that the 64-kDa subunit can be UV-cross-linked to RNA in a polyadenylation signal (AAUAAA)-dependent manner. By molecular cloning, the 64-kDa subunit was shown to contain a ribonucleoprotein-type RNA binding domain and a novel repeat structure. To study the functions of the other subunits, we have now isolated cDNAs encoding the 50-kDa subunit of human CstF. This subunit shares extensive homology with mammalian G protein beta-subunits and has a characteristic repeat structure (transducin repeat), in which an approximately 44-amino acid-long sequence is repeated seven times. To our knowledge, the 50-kDa subunit is the first example of a functional beta-subunit-like protein in vertebrates. Possible roles of the transducin repeat, both in CstF function specifically and in other beta-subunit homologues more generally, are discussed.