Doris Ursic

Phenotypic suppression and misreading in Saccharomyces cerevisiae

A NUMBER of antibiotics and other inhibitors have been useful in genetic and biochemical analyses of the protein-synthesising machinery of prokaryotic and eukaryotic organisms. Aminoglycoside antibiotics have been shown to be particularly helpful in this respect, especially in identifying ribosomal protein cistrons in bacteria. The aminoglycosides cause extensive misreading of the RNA code words in vitro1 and suppress many nonsense and missense mutations in E. coli2 phenotypically. The misreading observed in cell-free translation is believed to be the basis for the phenotypic suppression, although the exact mechanism is not known. Apart from a report of suppression of a single mutation in yeast by streptomycin3, there have been no demonstrations of phenotypic suppression in eukaryotic organisms. Earlier studies of mistranslation in vitro in eukaryotic systems indicated that this phenomenon is rare. Streptomycin did not cause misreading with cytoplasmic ribosomes of rat liver4, rabbit spleen5, chicken liver6 or yeast7; neomycin had no effect in rabbit reticulocyte extracts4 and only a slight effect in the yeast and chicken liver systems6,7. These results suggested that translation in higher organisms functions with higher fidelity than that in bacteria. Nevertheless, aminoglycoside antibiotics have recently been shown to cause extensive translational misreading in vitro in systems derived from Tetrahymena8, wheat embryo9, and cultured human cells10.

The yeast SEN3 gene encodes a regulatory subunit of the 26S proteasome complex required for ubiquitin-dependent protein degradation in vivo.

The yeast Sen1 protein was discovered by virtue of its role in tRNA splicing in vitro. To help determine the role of Sen1 in vivo, we attempted to overexpress the protein in yeast cells. However, cells with a high-copy SEN1-bearing plasmid, although expressing elevated amounts of SEN1 mRNA, show little increase in the level of the encoded protein, indicating that a posttranscriptional mechanism limits SEN1 expression. This control depends on an amino-terminal element of Sen1. Using a genetic selection for mutants with increased expression of Sen1-derived fusion proteins, we identified mutations in a novel gene, designated SEN3. SEN3 is essential and encodes a 945-residue protein with sequence similarity to a subunit of an activator of the 20S proteasome from bovine erythrocytes, called PA700. Earlier work indicated that the 20S proteasome associates with a multisubunit regulatory factor, resulting in a 26S proteasome complex that degrades substrates of the ubiquitin system. Mutant sen3-1 cells have severe defects in the degradation of such substrates and accumulate ubiquitin-protein conjugates. Most importantly, we show biochemically that Sen3 is a subunit of the 26S proteasome. These data provide evidence for the involvement of the 26S proteasome in the degradation of ubiquitinated proteins in vivo and for a close relationship between PA700 and the regulatory complexes within the 26S proteasome, and they directly demonstrate that Sen3 is a component of the yeast 26S proteasome.

SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

Inactivation of the yeast Sen1 protein affects the localization of nucleolar proteins

A mutation in the Saccharomyces cerevisiae SEN1 gene causes accumulation of end-matured, intron-containing pre-tRNAs. Cells containing the thermosensitive sen1-1 mutation exhibit reduced tRNA splicing endonuclease activity. However, Sen1p is not the catalytic subunit of this enzyme. We have used Sen1p-specific antibodies for cell fractionation studies and immunofluorescent microscopy and determined that Sentp is a low abundance protein of about 239 kDa. It localizes to the nucleus with a granular distribution. We verified that a region in SEN1 containing a putative nuclear localization signal sequence (NLS) is necessary for nuclear targeting. Furthermore, we found that inactivation of Sen1p by temperature shift of a strain carrying sen1-1 leads to mislocalization of two nucleolar proteins, Nopt and Ssb1 Possible mechanisms are discussed for several related nuclear functions of Sen1p, including tRNA splicing and the maintenance of a normal crescent-shaped nucleolus.

A Drosophila melanogaster gene encodes a protein homologous to the mouse t complex polypeptide 1

We have isolated and sequenced a cDNA from Drosophila melanogaster that is homologous to the mouse Tcp-1 gene encoding the t complex polypeptide 1, TCP-1. The Drosophila gene maps by in situ hybridization to bands 94B1-2 of the polytene chromosomes. It shares 66% nucleotide sequence identity with the mouse gene. The predicted Drosophila protein consists of 557 amino acids and shares 72% identity with the mouse polypeptide. The TCP-1 polypeptide appears to be highly conserved in evolution from mammals to simple eukaryotes because the Drosophila gene probe also detects related sequences in DNA from the yeast, Saccharomyces cerevisiae. The presence of TCP-1-related polypeptides in organisms such as Drosophila and yeast should facilitate biochemical and genetic analysis of its function.

Interactions of Sen1, Nrd1, and Nab3 with Multiple Phosphorylated Forms of the Rpb1 C-Terminal Domain in Saccharomyces cerevisiae

ABSTRACT The Saccharomyces cerevisiae SEN1 gene codes for a nuclear, ATP-dependent helicase which is embedded in a complex network of protein-protein interactions. Pleiotropic phenotypes of mutations in SEN1 suggest that Sen1 functions in many nuclear processes, including transcription termination, DNA repair, and RNA processing. Sen1, along with termination factors Nrd1 and Nab3, is required for the termination of noncoding RNA transcripts, but Sen1 is associated during transcription with coding and noncoding genes. Sen1 and Nrd1 both interact directly with Nab3, as well as with the C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II. It has been proposed that Sen1, Nab3, and Nrd1 form a complex that associates with Rpb1 through an interaction between Nrd1 and the Ser5-phosphorylated (Ser5-P) CTD. To further study the relationship between the termination factors and Rpb1, we used two-hybrid analysis and immunoprecipitation to characterize sen1-R302W, a mutation that impairs an interaction between Sen1 and the Ser2-phosphorylated CTD. Chromatin immunoprecipitation indicates that the impairment of the interaction between Sen1 and Ser2-P causes the reduced occupancy of mutant Sen1 across the entire length of noncoding genes. For protein-coding genes, mutant Sen1 occupancy is reduced early and late in transcription but is similar to that of the wild type across most of the coding region. The combined data suggest a handoff model in which proteins differentially transfer from the Ser5- to the Ser2-phosphorylated CTD to promote the termination of noncoding transcripts or other cotranscriptional events for protein-coding genes.

Sen1p Performs Two Genetically Separable Functions in Transcription and Processing of U5 Small Nuclear RNA in Saccharomyces cerevisiae

The Saccharomyces cerevisiae SEN1 gene codes for a nuclear-localized superfamily I helicase. SEN1 is an ortholog of human SETX (senataxin), which has been implicated in the neurological disorders ataxia-ocular apraxia type 2 and juvenile amyotrophic lateral sclerosis. Pleiotropic phenotypes conferred by sen1 mutations suggest that Sen1p affects multiple steps in gene expression. Sen1p is embedded in a protein–protein interaction network involving direct binding to multiple partners. To test whether the interactions occur independently or in a dependent sequence, we examined interactions with the RNA polymerase II subunit Rpb1p, which is required for transcription, and Rnt1p, which is required for 3′-end maturation of many noncoding RNAs. Mutations were identified that impair one of the two interactions without impairing the other interaction. The effects of the mutants on the synthesis of U5 small nuclear RNA were analyzed. Two defects were observed, one in transcription termination and one in 3′-end maturation. Impairment of the Sen1p–Rpb1p interaction resulted in a termination defect. Impairment of the Sen1p–Rnt1p interaction resulted in a processing defect. The results suggest that the Sen1p–Rpb1p and Sen1p–Rnt1p interactions occur independently of each other and serve genetically separable purposes in targeting Sen1p to function in two temporally overlapping steps in gene expression.

Agrobacterium tumefaciens T-DNA integrates into multiple sites of the sunflower crown gall genome

SummaryWe analyzed the integration of a tumor inducing (Ti) plasmid into an octopine producing crown gall tumor of sunflower, line PSCG 15955. A continuous Ti plasmid segment (T-DNA) of about 19.5 kilo base pairs (kbp) is transferred and integrated into a small number of sites of the plant DNA.The number of T-DNA integration sites in our tumor line was estimated by two different methods. First, cloned fragments of the T-DNA were hybridized to tumor DNA and the hybridization patterns were observed. The number of T-DNA integration sites in line PSCG 15955 was found to be approximately eight.Second, a library was constructed from total DNA of tumor line PSCG 15955 by molecular cloning using the bacteriophage lambda vector Charon 4A. Recombinant phages having sequence homologies to the Ti plasmid were selected. The lower limit on the number of integration sites was three because we obtained three different right hand side genomic clones of plant/T-DNA hybrids. The initial screening of the library also revealed two left hand border clones. Hybridization of these five distinct recombinant clones to uninfected sunflower DNA shows that the cloned T-DNA segments are covalently bonded to plant DNA. The left and right hand plant boundary sequences are homologous to either unique and or repeated plant DNA segments.

Detecting phosphorylation-dependent interactions with the C-terminal domain of RNA polymerase II subunit Rpb1p using a yeast two-hybrid assay.

Rpb1p, the largest subunit of S. cerevisiae RNA polymerase II, contains a repetitive structure called the C-terminal domain (CTD). The CTD serves as a scaffold for the regulated association and dissociation of more than a hundred proteins involved in RNA synthesis. Phosphorylation of two serine residues (Ser2 and Ser5) in the repeating units of the CTD change dynamically during the pre-initiation, initiation, elongation, and termination of transcription to control the binding and release of transcriptional components. A modification of the well established yeast two-hybrid assay for protein-protein interactions is described that detects interactions between phosphorylated forms of the CTD and proteins whose interactions with the CTD depend on phosphorylation. The efficacy of the approach was established by first showing that two-hybrid fusions containing the CTD are phosphorylated at Ser2 and Ser5 residues. Interactions between the CTD and three known CTD-binding proteins were analyzed. The results suggest that the modified two-hybrid system accurately assays CTD-binding and provides a new and convenient assay for CTD-binding proteins.

A new antibiotic with known resistance factors, G418, inhibits plant cells

Abstract Growth rates of two lines of tobacco ( Nicotiana tabacum ) cell suspension cultures were measured in the presence or absence of G418, a new 2-deoxystreptamine antibiotic related to Gentamycin. Cell growth rates of N . tabacum cv. Burley were inhibited at drug concentrations as low as 1.65 × 10−7 M. At 4 × 10−7 M, the doubling time was increased from 1.5 days (control) to 2.3 days (treatment). The drug was lethal to cells at 4 × 10−6 M, and inhibition was irreversible. Cells of N . tabacum cv. Wisconsin 38 also were inhibited by the drug, although at slightly higher concentrations (ca. 2–5 fold). In view of our findings, G418 and its associated resistance factors could be of great value in plant genetic engineering.