James M. Wilhelm

Phenotypic suppression of nonsense mutants in yeast by aminoglycoside antibiotics

STREPTOMYCIN, an aminoglycoside antibiotic, can reverse the mutant phenotypes of many nonsense and missense mutations in Escherichia coli and in bacteriophage T4. This phenomenon has been called phenotypic suppression, since the mutant phenotype returns after removal of the drug1. The most likely explanation for phenotypic suppression is that streptomycin promotes mistranslation in vivo, and that acceptable amino acids are inserted into the growing polypeptide chain at the site of the mutant codon. Consistent with this view is the observation that streptomycin causes E. coli ribosomes to mistranslate RNA in vitro2,3. Streptomycin and neomycin have however been found to have no effect in stimulating ribosomes from eukaryotic cells to mistranslate RNA in vitro4,5. A subclass of the aminoglycoside antibiotics has been shown6,7 to stimulate eukaryotic ribosomes to misread RNA. The highly active molecules are distinguished in that they contain the drug fragment paromamine (or 3′-deoxyparomamine). We have therefore examined the capacity of various aminoglycosides to suppress mutations phenotypically in the eukaryotic yeast, Saccharomyces cerevisiae. The results presented here show that paromomycin, which contains paromamine, is capable of phenotypic suppression of the nonsense mutations in S. cerevisiae.

Mistranslation in a eucaryotic organism

Previous work from our laboratory has demonstrated that a subclass of the aminoglycoside antibiotics, those containing the drug fragment paromamine, stimulates mistranslation in cell-free protein-synthesizing systems derived from eucaryotic cells. We report here experiments which show that the ciliate Tetrahymena thermophila (formerly T. pyriformis, syngen 1) is sensitive to the paromamine-containing aminoglycoside antibiotics. The drugs are active with respect to growth inhibition, inhibition of protein synthesis in the whole organism, inhibition of protein synthesis in vitro and the stimulation of mistranslation in cell-free protein-synthesizing systems. Because of their misreading properties, these drugs may be useful in isolating and propagating strains carrying mutations which can be translationally suppressed (that is, missense and nonsense mutations).

Altered 40 S ribosomal subunits in omnipotent suppressors of yeast.

The five suppressors SUP35, SUP43, SUP44, SUP45 and SUP46, each mapping at a different chromosomal locus in the yeast Saccharomyces cerevisiae, suppress a wide range of mutations, including representatives of all three types of nonsense mutations, UAA, UAG and UGA. We have demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosomes from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin. Furthermore, ribosomal subunit mixing experiments established that the 40 S ribosomal subunit, and this subunit only, is responsible for the higher levels of misreading. Thus, the gene products of SUP35, SUP44, SUP45 and SUP46 are components of the 40 S subunit or are enzymes that modify the subunit. In addition, a protein from the 40 S subunit of the SUP35 suppressor has an altered electrophoretic mobility; this protein is distinct from the altered protein previously uncovered in the 40 S subunit of the SUP46 suppressor. In contrast to the ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46, the ribosomes from the SUP43 suppressor do not significantly misread polyuridylate templates in vitro, suggesting that this locus may not encode a ribosomal component or that the misreading is highly specific.

Mechanisms of action of aminoglycoside antibiotics in eucaryotic protein synthesis.

Tetrahymena thermophila is a eucaryotic organism that is highly susceptible to growth inhibition by aminoglycoside antibiotics. Concentrations of paromomycin, gentamicin G418, and hygromycin B at 22, 10, and 17 microM, respectively, inhibited growth by 50%. A combination of in vitro and in vivo methods was used to determine the mechanisms of action of these aminoglycoside antibiotics on protein synthesis in T. thermophila. Analysis of polysome profiles from paromomycin- and gentamicin G418-treated cells showed clear, progressive depletions of polysomes concomitant with an inhibition of in vivo [14C] lysine incorporation. In vitro, paromomycin and gentamicin G418, which are disubstituted 2-deoxystreptamine-containing molecules, were not very effective inhibitors of either the translocation of peptidyl-tRNA or the elongation of nascent polypeptide chains on polysomes. In contrast, we found that the translocation of phe-tRNA on polyuridylate programmed ribosomes was susceptible to inhibition by paromomycin. We conclude that the primary inhibitory action of paromomycin and gentamicin G418 was at (i) an early stage of elongation after initiation, (ii) the initiation stage of translation, or (iii) a stage of translation before initiation. Hygromycin B, which is a monosubstituted 2-deoxystreptamine-containing aminoglycoside, potently inhibited the elongation of nascent chains during the translation of polysomes. In addition, the in vitro translation of polysomes from two hygromycin B-resistant mutants was resistant to the inhibition of elongation caused by hygromycin B.

Misreading of the ribosomal suppressor SUP46 due to an altered 40 S subunit in yeast

Abstract The dominant suppressor, SUP46 , in the yeast Saccharomyces cerevisiae acts on a wide range of different types of mutations. The incorporation of phenylalanine and the misincorporation of leucine in a cell-free system programmed with poly(U) indicated that the ribosomes from a SUP46 strain produce abnormally high rates of translation errors. Furthermore, the cell-free translation system was used to demonstrate that the SUP46 defect resides in the 40 S ribosomal subunit. The growth of SUP46 strains was shown to be unusually sensitive to paromomycin, an aminoglycoside antibiotic that is known to induce translation errors. In addition, paromomycin stimulated mistranslation with SUP46 ribosomes to a greater extent than with normal ribosomes. These results indicate that SUP46 suppression is caused by increased translation errors as a result of the mutationally altered 40 S ribosomal subunit. Paromomycin appears to produce translation errors in SUP46 strains at rates that are too high for cellular growth.

Ultrastructural characterization of human immunodeficiency virus type 1 gag-containing particles assembled in a recombinant adenovirus vector system

The human immunodeficiency virus type 1 (HIV-1) Gag protein was expressed in A549 cells infected with recombinant adenovirus types 4 and 7, each carrying the HIV-1 gag and pro genes. The Gag protein was assembled into enveloped virus-like particles that budded from plasma and vacuolar membranes. The particles, isolated by precipitation and isopycnic density centrifugation, contained both processed and unprocessed Gag-associated proteins.

Immunogenicity of recombinant influenza virus haemagglutinin carrying peptides from the envelope protein of human immunodeficiency virus type 1

Haemagglutinin (HA), the major surface glycoprotein of influenza virus, is a potent immunogen against which viral neutralizing antibodies are directed. Studies of the three-dimensional structure of HA have identified major antigenic sites on the molecule. We have exploited HA as a carrier for small antigenic regions (epitopes) of the HIV-1 envelope (env) glycoprotein. Using recombinant DNA techniques, the epitopes were inserted in-frame into a known antigenic site of HA to produce HA-epitope chimeras. Guinea-pigs and mice immunized with these chimeras in combination with adjuvant generated significant immune responses against the carrier HA and also produced epitope-specific antibodies that recognized the native whole HIV-1 env. One of the chimeras which contained a V3-loop sequence of HIV-1 env elicited neutralizing antibodies against the homologous strain of HIV-1. The antibodies against HA and the inserted epitopes remained at high levels for up to 72 weeks. Remarkably, these responses were generated with low doses of immunogens containing only nanogram quantities of the inserted epitopes. These results suggest the utility of HA as a carrier to allow selective antibody induction against foreign epitopes, and offer a new approach for vaccine development as well as for the production of monospecific antibodies useful in diagnostics and research.

Heteroaryl and Cycloalkyl Sulfonamide Hydroxamic Acid Inhibitors of Matrix Metalloproteinases

Heteroaryl and cycloalkyl sulfonamide-hydroxamic acid MMP inhibitors were investigated. Of these, the pyridyl analogue 2 is the most potent and selective inhibitor of MMP-9 and MMP-13 in vitro.

Non-hinge-binding pyrazolo[1,5-a]pyrimidines as potent B-Raf kinase inhibitors.

As part of our research effort to discover B-Raf kinase inhibitors, we prepared a series of C-3 substituted N-(3-(pyrazolo[1,5-a]pyrimidin-7-yl)phenyl)-3-(trifluoromethyl)benzamides. X-ray crystallography studies revealed that one of the more potent inhibitors (10n) bound to B-Raf kinase without forming a hinge-binding hydrogen bond. With basic amine residues appended to C-3 aryl residues, cellular activity and solubility were enhanced over previously described compounds of this class.

Variation of phenotypic suppression due to the ψ+and ψ− extrachromosomal determinants in yeast

Paromomycin, an aminoglycoside antibiotic, can phenotypically suppress nonsense mutations in the yeast Saccharomyces cerevisiae (Palmer et al., 1979). We report here that the extrachromosomal determinant, ψ+, enhances this phenotypic suppression of all three nonsense mutations. UAG. UAA and UGA, by three-to sevenfold.