HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain.

Huntington disease (HD) is associated with the expansion of a polyglutamine tract, greater than 35 repeats, in the HD gene product, huntingtin. Here we describe a novel huntingtin interacting protein, HIP1, which co-localizes with huntingtin and shares sequence homology and biochemical characteristics with Sla2p, a protein essential for function of the cytoskeleton in Saccharomyces cerevisiae. The huntingtin-HIP1 interaction is restricted to the brain and is inversely correlated to the polyglutamine length in huntingtin. This provides the first molecular link between huntingtin and the neuronal cytoskeleton and suggests that, in HD, loss of normal huntingtin-HIP1 interaction may contribute to a defect in membrane-cytoskeletal integrity in the brain.

The E1B 19K/Bcl-2-binding protein Nip3 is a dimeric mitochondrial protein that activates apoptosis.

Nip3 (nineteen kD interacting protein-3) is an E1B 19K and Bcl-2 binding protein of unknown function. Nip3 is detected as both a 60- and 30-kD protein in vivo and in vitro and exhibits strong homologous interaction in a yeast two-hybrid system indicating that it can homodimerize. Nip3 is expressed in mitochondria and a mutant (Nip3(163)) lacking the putative transmembrane domain and COOH terminus does not dimerize or localize to mitochondria. Transient transfection of epitope-tagged Nip3 in Rat-1 fibroblasts and MCF-7 breast carcinoma induces apoptosis within 12 h while cells transfected with the Nip3(163) mutant have a normal phenotype, suggesting that mitochondrial localization is necessary for induction of cell death. Nip3 overexpression increases the sensitivity to apoptosis induced by granzyme B and topoisomerase I and II inhibitors. After transfection, both Nip3 and Nip3(163) protein levels decrease steadily over 48 h indicating that the protein is rapidly degraded and this occurs in the absence of cell death. Bcl-2 overexpression initially delays the onset of apoptosis induced by Nip3 but the resistance is completely overcome in longer periods of incubation. Nip3 protein levels are much higher and persist longer in Bcl-2 expressing cells. In conclusion, Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression.

The C. elegans orthologue ceBNIP3 interacts with CED-9 and CED-3 but kills through a BH3- and caspase-independent mechanism.

We have studied ceBNIP3, the orthologue of BNIP3 in C. elegans. Sequence analysis reveals that the different domains of BNIP3 have been conserved throughout evolution. ceBNIP3 contains a C-terminal transmembrane (TM) domain, a conserved domain (CD) of 19 amino acids, a BCL-2 homology-3 (BH3)-like domain and a PEST sequence. ceBNIP3 is expressed primarily as a 25 kDa monomer and a 50 kDa homodimer. After transfection, ceBNIP3 protein is rapidly degraded through a ubiquitin-dependent pathway by the proteasome. Like BNIP3, the TM domain of ceBNIP3 mediates the localization of the protein to mitochondria and is also necessary for homodimerization and cell death in mammalian cells. Neither the putative BH3 domain nor conserved domain is necessary for killing. ceBNIP3 protein interacts with CED-9 and BCL-XL, but unlike other pro-apoptotic BCL-2 family members, the BH3-like domain does not participate in dimerization. The ceBNIP3 TM domain mediates interaction with both CED-9 and BCL-XL. ceBNIP3 interacts with CED-3 but co-expression of CED-3 and ceBNIP3 does not significantly enhance induction of cell death in the presence or absence of CED-4. ceBNIP3 kills mammalian cells by a caspase-independent mechanism. In conclusion, we find that although ceBNIP3 interacts with CED-9 and CED-3 it kills by a BH3- and caspase-independent mechanism.

Specificity of the yeast rev3 delta antimutator and REV3 dependency of the mutator resulting from a defect (rad1 delta) in nucleotide excision repair.

The yeast REV3 gene has been predicted to encode a DNA polymerase specializing in translesion synthesis. This polymerase likely participates in spontaneous mutagenesis, as rev3 mutants have an antimutator phenotype. Translesion synthesis also may be necessary for the mutator caused by a RAD1 (nucleotide excision repair) deletion (rad1 delta). To further examine the role of REV3 in spontaneous mutagenesis, we characterized SUP4-o mutations that arose spontaneously in strains having combinations of normal or mutant REV3 and RAD1 alleles. The largest fraction of the rev3 delta-dependent mutation rate decrease was observed for single base-pair substitutions and deletions, although the rates of all mutational classes detected in the RAD1 background were reduced by at least 30%. Interestingly, inactivation of REV3 was associated with a doubling of the number of sites at which the retrotransposon Ty inserted. rev3 delta also greatly diminished the magnitude of the rad1 delta mutator, but not to the rev3 delta antimutator level, implicating REV3-dependent and independent processes in the rad1 delta mutator effect. However, the specificity of the rev3 delta antimutator suggested that the same REV3-dependent processes gave rise to the majority of spontaneous mutations in the RAD1 and rad1 delta strains.

Specificities of the Saccharomyces cerevisiae rad6, rad18, and rad52 mutators exhibit different degrees of dependence on the REV3 gene product, a putative nonessential DNA polymerase.

The Saccharomyces cerevisiae rad6, rad18, and rad52 mutants exhibit DNA repair deficiencies and distinct mutator phenotypes. DNA replication past unrepaired spontaneous damage might contribute to the specificities of these mutators. Because REV3 is thought to encode a DNA polymerase that specializes in translesion synthesis, we determined the REV3 dependence of the rad mutator specificities. Spontaneous mutagenesis at a plasmid-borne SUP4-o locus was examined in isogenic strains having combinations of normal or mutant REV3 and RAD6, RAD18, or RAD52 alleles. For the rad6 and rad18 mutators, the mutation rate increase relied largely, but not exclusively, on REV3 whereas the rad52 mutator was entirely REV3 dependent. The influence of REV3 on the specificity of the rad6 mutator differed markedly depending on the mutational class examined. However, the requirement of rev3 for the production of G.C-->T.A transversions by the rad18 mutator, which induces only these substitutions, was similar to that for rad6-mediated G.C-->T.A transversion. This supports a role for the Rad6-Rad18 protein complex in the control of spontaneous mutagenesis. The available data imply that the putative Rev3 polymerase can process a variety of spontaneous DNA lesions that normally are substrates for error-free repair.

Elimination of the yeast RAD6 ubiquitin conjugase enhances base-pair transitions and G.C----T.A transversions as well as transposition of the Ty element: implications for the control of spontaneous mutation.

The RAD6 gene of the yeast Saccharomyces cerevisiae encodes an enzyme that conjugates ubiquitin to other proteins. Defects in RAD6 confer a mutator phenotype due, in part, to an increased rate of transposition of the yeast Ty element. To further delineate the role of protein ubiquitination in the control of spontaneous mutagenesis in yeast, we have characterized 202 mutations that arose spontaneously in the SUP4-o gene carried on a centromere vector in a RAD6 deletion strain. The resulting mutational spectrum was compared to that for 354 spontaneous SUP4-o mutations isolated in the isogenic wild-type parent. This comparison revealed that the rad6 mutator enhanced the rate of single base-pair substitution, as well as Ty insertion, but did not affect the rates of the other mutational classes detected. Relative to the wild-type parent, Ty inserted at considerably more SUP4-o positions in the rad6 strain with a significantly smaller fraction detected at a transposition hotspot. These findings suggest that, in addition to the rate of transposition, protein ubiquitination might influence the target site specificity of Ty insertion. The increase in the substitution rate accounted for approximately 90% of the rad6 mutator effect but only the two transitions and the G. C----T.A transversion were enhanced. Analysis of the distribution of these events within SUP4-o suggested that the site specificity of the substitutions was influenced by DNA sequence context. Transformation of heteroduplex plasmid DNAs into the two strains demonstrated that the rad6 mutator did not reduce the efficiency of correcting mismatches that could give rise to the transitions or transversion nor did it bias restoration of the mismatches to the incorrect base-pairs. These results are discussed in relation to possible mechanisms that might link ubiquitination of proteins to spontaneous mutation rates.

New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites.

We describe the production of new alleles of the LEU2, URA3 and TRP1 genes of Saccharomyces cerevisiae by in vitro mutagenesis. Each new allele, which lacks restriction enzyme recognition sequences found in the pUC19 multicloning site, was used to construct a unique series of yeast-Escherichia coli shuttle vectors derived from the plasmid pUC19. For each gene a 2 mu vector (YEplac), an ARS1 CEN4 vector (YCplac) and an integrative vector (YIplac) was constructed. The features of these vectors include (i) small size; (ii) unique recognition site for each restriction enzyme found in the pUC19 multicloning site; (iii) screening for plasmids containing inserts by color assay; (iv) high plasmid yield; (v) efficient transformation of S. cerevisiae. These vectors should allow greater flexibility with regard to DNA restriction fragment manipulation and subcloning.

Cloning and nucleotide sequence analysis of the Saccharomyces cerevisiae RAD4 gene required for excision repair of UV-damaged DNA.

The RAD4 gene of Saccharomyces cerevisiae is required for the incision step of excision repair. We have cloned the RAD4 gene and determined its nucleotide sequence. RAD4 encodes a somewhat basic protein of 754 amino acids (aa) with an Mr of 87,173. RAD4 contains several groups of 4-7 consecutive basic aa residues that could be involved in DNA binding and it also contains an alpha-helix-turn-alpha-helix motif for DNA binding. Like several other DNA repair proteins of S. cerevisiae, the C terminus of RAD4 protein is highly acidic.

Genetic transformation of yeast.

Genetic transformation was first described by Griffith in 1928 and has since been demonstrated in a variety of organisms, including many species of fungi. This review focuses on the history and technology of the transformation of Saccharomyces cerevisiae. The application of protocols developed for S. cerevisiae to other important yeast species is discussed. The protocols for transformation by spheroplasting, LiAc/ssDNA/PEG, and electroporation are compared, and possible mechanisms for transformation are discussed.

Production of digoxigenin-labelled RNA probes and the detection of cytokine mRNA in rat spleen and brain by in situ hybridization.

Non-radioactive in situ hybridization is a sensitive method for determining the site of production for secretory molecules such as cytokines. We report here on the central and peripheral induction of proinflammatory cytokines by endotoxin, and outline procedures for the generation and application of rat-specific digoxigenin (Dig)-labelled RNA probes for the localization of mRNA by in situ hybridization. Rats were injected either intravenously (i.v.) or intracerebroventricularly (i.c.v.) with vehicle or lipopolysaccharide (LPS) and sacrificed at various time intervals post-injection. Rats were then perfused with 4% paraformaldehyde and the spleens and brains were removed and cryoprotected in 30% sucrose. Dig-labelled, rat-specific, antisense and sense RNA probes were generated by in vitro transcription from PCR-derived templates. Positive staining with all the antisense probes was cytoplasmic, whereas the sense probes showed no staining. Numerous tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) mRNA positive cells were observed in the marginal zone and in the red pulp of the spleen after iv LPS injections, whereas sections from saline-treated animals showed minimal cytokine mRNA expression. Cells positive for TNF-alpha and IL-1beta mRNA were detectable in the brain after i.c.v. injections of LPS, but not after icv injection of vehicle. An antisense probe for c-fos was utilized in these studies as a positive control for our procedure due to its anatomically specific expression in the rat brain after LPS. In conclusion we have demonstrated that in situ hybridization with Dig-labelled RNA probes is an efficient, sensitive and reliable tool to localize cytokine mRNA production in rat tissue.

Safrole, eugenol and methyleugenol induce intrachromosomal recombination in yeast.

Deletion of an integrated plasmid, a specific type of intrachromosomal recombination, was evaluated for inducibility with the phenylpropenes safrole, eugenol and methyleugenol in the yeast Saccharomyces cerevisiae. These phenylpropenes are found in food products, spices, pharmaceuticals and clove cigarettes. Safrole and eugenol are known carcinogens in animals and methyleugenol is a suspected carcinogen. These phenylpropenes are not detectable by the Ames assay and most other short-term tests used currently in predictive carcinogenesis. Like safrole, which has been shown to be nonmutagenic with the Ames assay, eugenol and methyleugenol were found to be nonmutagenic with the Ames assay. In contrast, with the yeast assays which screen for intra- and inter-chromosomal recombination in logarithmic phase cultures, all 3 compounds gave a positive dose-related response. These results demonstrate further that the yeast system can be modified easily to detect various genetic endpoints and that it deserves serious consideration as a test system for predictive carcinogenesis.

An analysis of dopa decarboxylase expression during embryogenesis in Drosophila melanogaster.

Dopa decarboxylase (DDC) activity appears near the end of embryogenesis in Drosophila. High titers of 20-OH-ecdysone on the other hand are found at midembryogenesis. Several explanations for this lag were investigated, since this hormone has been shown to induce a rapid increase in DDC activity at pupariation (G. P. Kraminsky, W. C. Clark, M. A. Estelle, R. D. Gietz, B. A. Sage, J. D. O'Connor, and R. B. Hodgetts, 1980, Proc. Natl. Acad. Sci. USA 77, 4175-4179). Using immunological and genetical criteria, it was shown that the same structural gene encodes DDC in embryos, mature larvae, and young adults. This rules out the existence of a distinct embryonic DDC gene unresponsive to 20-OH-ecdysone. Second, no evidence was found to support the hypothesis that a delay in the translation of DDC transcripts, produced in response to the elevated titer of 20-OH-ecdysone at midembryogenesis, caused the lag. Northern analysis of the RNA molecules homologous to cloned genomic sequences revealed that DDC transcripts were present at two different times during embryogenesis. A transcript was found in both ovaries and 0- to 2-hr embryos. However, this species disappeared by 4 hr and DDC transcript levels remained low until late in embryogenesis, when a significant increase occurred. This increase was presumably responsible for the appearance of the enzyme at this time. The northern blotting revealed nine DDC transcript species were present during embryogenesis and hybridization to intron-specific probes indicated that five of these contained at least part of one (or both) of the two introns. Three putative mature mRNA species were identified by their small size, relative abundance, apparent lack of intron sequence, and their presence on polysomes. The two mature species found during the late stages were postulated to differ in the length of their poly(A)+ tails. The third mature species was found only in ovaries and very young embryos and may well be of maternal origin. Data are examined in light of the possibility that this species is derived from a precursor initiated at a novel promotor.

High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier.

A method, using LiAc to yield competent cells, is described that increased the efficiency of genetic transformation of intact cells of Saccharomyces cerevisiae to more than 1 X 10(5) transformants per microgram of vector DNA and to 1.5% transformants per viable cell. The use of single stranded, or heat denaturated double stranded, nucleic acids as carrier resulted in about a 100 fold higher frequency of transformation with plasmids containing the 2 microns origin of replication. Single stranded DNA seems to be responsible for the effect since M13 single stranded DNA, as well as RNA, was effective. Boiled carrier DNA did not yield any increased transformation efficiency using spheroplast formation to induce DNA uptake, indicating a difference in the mechanism of transformation with the two methods.

Applications of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier.

The highly efficient yeast lithium acetate transformation protocol of Schiestl and Gietz (1989) was tested for its applicability to some of the most important needs of current yeast molecular biology. The method allows efficient cloning of genes by direct transformation of gene libraries into yeast. When a random gene pool ligation reaction was transformed into yeast, the LEU2, HIS3, URA3, TRP1 and ARG4 genes were found among the primary transformants at a frequency of approximately 0.1%. The RAD4 gene, which is toxic to Escherichia coli, was also identified among the primary transformants of a ligation library at a frequency of 0.18%. Non-selective transformation using this transformation protocol was shown to increase the frequency of gene disruption three-fold. Co-transformation showed that 30-40% of the transformation-competent cells take up more than one DNA molecule which can be used to enrich for integration and deletion events 30- to 60-fold. Co-transformation was used in the construction of simultaneous double gene disruptions as well as disrupting both copies of one gene in a diploid which occurred at 2-5% the frequency of the single event.

Analysis of the transcription unit adjacent to the 3'-end of the dopa decarboxylase gene in Drosophila melanogaster.

Using strand specific RNA probes, we have ascertained that the transcription unit immediately downstream of the dopa decarboxylase (DDC) gene in Drosophila, is oriented in the opposite direction to DDC. The 3'-termini of these two transcription units lie within 550 bp of one another, and the mature transcripts actually may overlap. We show here that the inclusion of part of the 3'-flanking transcript on a genomic DNA clone originally assumed to include just DDC, accounts for most of the hybridization seen in our previous Northern analysis of very early embryonic RNA. Thus, while DDC-specific transcripts are detectable in the early embryonic stages, they are much less prevalent than we have previously reported.

Interactions between the subunits of casein kinase II.

Casein kinase II (CKII) is a protein serine/threonine kinase known to control the activity of a variety of regulatory nuclear proteins. This enzyme has a tetrameric structure composed of two catalytic (alpha and/or alpha ') subunits and two beta subunits. We have examined the subunit composition of tetrameric complexes of purified bovine CKII by immunoprecipitation using alpha, alpha ', or beta subunit-specific antibodies. These experiments indicate that the enzyme can exist as homotetramers (i.e., alpha 2 beta 2 or alpha 2' beta 2) as well as heterotetramers (i.e. alpha alpha ' beta 2). To further examine subunit interactions between the alpha, alpha ', or beta subunits of CKII, we have utilized the yeast two-hybrid system (Fields, S. and Song, O. (1989) Nature 340: 245-246). For these studies, each subunit of human CKII was expressed in yeast as a fusion with the DNA binding domain or with the transcriptional activation domain of the yeast GAL4 transcriptional activator. These studies demonstrate that the alpha or alpha ' subunits of CKII can interact with the beta subunits of CKII, but not with other alpha or alpha ' subunits. By comparison, the beta subunits of CKII can interact with alpha, alpha ', or beta subunits. These results indicate that the CKII holoenzyme forms because of the ability of beta subunits to dimerize, bringing two heterodimers (alpha beta or alpha ' beta) into a tetrameric complex.

Overlapping transcription units in the dopa decarboxylase region of Drosophila.

The many examples of overlap in the genes of various viruses and bacteria illustrate that the parsimonious utilization of the coding capacity of DNA is relatively common amongst prokaryotes. The recent discovery of a pupal cuticle gene within an intron of the completely unrelated Gart locus in Drosophila shows that overlapping transcription units also exist in higher organisms. However, the prevalence of such phenomena in unknown. We report here a quite different situation of overlap between the 3' termini of a pair of convergent transcription units in another region of the Drosophila genome. This 88-base-pair (bp) genomic region encodes the 3' terminus of the messenger RNA for the enzyme dopa decarboxylase (Ddc) and, in opposite orientation, the 3' terminus of the adjacent gene whose function is unknown. An analysis of the temporal and spatial distribution of the two transcripts within the organism shows that high levels of both transcripts are never concordant. However, within the testes, where the 3' transcript is maximally expressed, low levels of Ddc transcript were detected. This result raises the possibility that a hybrid molecule involving the two transcripts forms in vivo or that transcription interference occurs, with concomitant regulatory implications.