Double Knockout Strain Research Articles

The unfolded protein response represses differentiation through the RPD3-SIN3 histone deacetylase.

In Saccharomyces cerevisiae, splicing of HAC1 mRNA is initiated in response to the accumulation of unfolded proteins in the endoplasmic reticulum by the transmembrane kinase-endoribonuclease Ire1p. Spliced Hac1p (Hac1ip) is a negative regulator of differentiation responses to nitrogen starvation, pseudohyphal growth, and meiosis. Here we show that the RPD3-SIN3 histone deacetylase complex (HDAC), its catalytic activity, recruitment of the HDAC to the promoters of early meiotic genes (EMGs) by Ume6p, and the Ume6p DNA-binding site URS1 in the promoters of EMGs are required for nitrogen-mediated negative regulation of EMGs and meiosis by Hac1ip. Co-immunoprecipitation experiments demonstrated that Hac1ip can interact with the HDAC in vivo. Systematic analysis of double deletion strains revealed that HAC1 is a peripheral component of the HDAC. In summary, nitrogen-induced synthesis of Hac1ip and association of Hac1ip with the HDAC are physiological events in the regulation of EMGs by nutrients. These data also define for the first time a gene class that is under negative control by the UPR, and provide the framework for a novel mechanism through which bZIP proteins repress transcription.

Anticancer drug resistance induced by disruption of the Saccharomyces cerevisiae NPR2 gene: a novel component involved in cisplatin- and doxorubicin-provoked cell kill.

The therapeutic potential of antitumor drugs is seriously limited by the manifestation of cellular drug resistance. We used the budding yeast Saccharomyces cerevisiae as a model system to identify novel mechanisms of resistance to one of the most active anticancer agents, cisplatin. We pinpointed NPR2 (nitrogen permease regulator 2) as a gene whose disruption conferred resistance to cisplatin. In addition, we observed a 4-fold cross-resistance of yeast npr2Delta cells (i.e., cells from which the NPR2 gene had been disrupted) to the anticancer drug doxorubicin, in combination with hypersensitivity to cadmium chloride. Furthermore, npr2Delta cells displayed unaltered cellular cisplatin and doxorubicin accumulation and showed an enhanced rate of spontaneous mutation compared with the isogenic parent. These data indicate that the npr2Delta phenotype overlaps that of the sky1Delta cells that we characterized previously (Mol Pharmacol 61:659-666, 2002). Therefore, we generated yeast npr2Delta sky1Delta double-knockout cells and performed clonogenic survival assays for cisplatin and doxorubicin, which revealed that NPR2 and SKY1 (SR-protein-specific kinase from budding yeast) are epistatic. The double-knockout strain was just as resistant to cisplatin and doxorubicin as the single-knockout strain that was most resistant to either drug. In conclusion, we identified NPR2 as a novel component involved in cell kill provoked by cisplatin and doxorubicin, and our data support the hypothesis that NPR2 and SKY1 may use mutual regulatory routes to mediate the cytotoxicity of these anticancer drugs.

Vaccine and skin testing properties of two avirulent Mycobacterium bovis mutants with and without an additional esat-6 mutation

Setting: Molecular techniques are now available to develop new live tuberculosis vaccines by producing avirulent strains of the Mycobacterium tuberculosis complex with known genes deleted. Objectives: Determine if removal of esat-6 from new live tuberculosis vaccines with known attenuating mutations affects their vaccine efficacy and if it could enable the development of discriminating diagnostic tests. Design: Remove the esat-6 gene by allelic exchange from two illegitimate mutants of Mycobacterium bovis that had previously been shown to have similar vaccine efficacy to BCG in a guinea pig vaccination model. Determine the effect this removal has on virulence, vaccine efficacy and skin test reactivity in guinea pigs. Results: Two double knockout strains of M. bovis were produced and their virulence and vaccine efficacy were compared to their parent strains. Removal of the esat-6 gene had no significant effect on vaccine efficacy. In skin tests, animals inoculated with the double knockout strains reacted to PPD but not ESAT-6, whereas those inoculated with the parent strains had similar skin test reactivity to both PPD and esat-6. Conclusion: Removal of esat-6 from new live tuberculosis vaccine candidates has no significant effect on vaccine properties but does enable the use of skin tests to distinguish between vaccination and infection.

Hypoxia-inducible factor 1alpha is essential for cell cycle arrest during hypoxia.

A classical cellular response to hypoxia is a cessation of growth. Hypoxia-induced growth arrest differs in different cell types but is likely an essential aspect of the response to wounding and injury. An important component of the hypoxic response is the activation of the hypoxia-inducible factor 1 (HIF-1) transcription factor. Although this transcription factor is essential for adaptation to low oxygen levels, the mechanisms through which it influences cell cycle arrest, including the degree to which it cooperates with the tumor suppressor protein p53, remain poorly understood. To determine broadly relevant aspects of HIF-1 function in primary cell growth arrest, we examined two different primary differentiated cell types which contained a deletable allele of the oxygen-sensitive component of HIF-1, the HIF-1alpha gene product. The two cell types were murine embryonic fibroblasts and splenic B lymphocytes; to determine how the function of HIF-1alpha influenced p53, we also created double-knockout (HIF-1alpha null, p53 null) strains and cells. In both cell types, loss of HIF-1alpha abolished hypoxia-induced growth arrest and did this in a p53-independent fashion. Surprisingly, in all cases, cells lacking both p53 and HIF-1alpha genes have completely lost the ability to alter the cell cycle in response to hypoxia. In addition, we have found that the loss of HIF-1alpha causes an increased progression into S phase during hypoxia, rather than a growth arrest. We show that hypoxia causes a HIF-1alpha-dependent increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27; we also find that hypophosphorylation of retinoblastoma protein in hypoxia is HIF-1alpha dependent. These data demonstrate that the transcription factor HIF-1 is a major regulator of cell cycle arrest in primary cells during hypoxia.

Identification of the tRNA-Dihydrouridine Synthase Family

5,6-Dihydrouridine (D) is a modified base found abundantly in the D-loops of tRNA from Archaea, Bacteria, and Eukarya. D is thought to be formed post-transcriptionally by the reduction of uridines in tRNA transcripts. Despite its abundance, no enzymes that catalyze D-formation have been identified. Using comparative genomics and computational methods we have identified members of the cluster of orthologous genes, COG0042, as putative dihydrouridine synthase encoding genes. Escherichia coli contains three COG0042 family members (yjbN, yhdG, and yohI). Strains were created where one, two, or all three of the COG0042 genes were deleted. Purified tRNA samples were investigated from the three single and the three double knockout strains, as well as from the triple deletion strain. The results showed that the COG0042 gene family is responsible for tRNA-dihydrouridine synthase activity in E. coli. They also suggest that the COG0042-encoded family members act site-specifically on the tRNA D-loop and contain non-redundant catalytic functions in vivo.

Identification and characterization of CIA/ASF1 as an interactor of bromodomains associated with TFIID.

General transcription initiation factor IID (TFIID) plays a central and critical role in transcription initiation from both naked and chromatin templates. Although interaction between several DNA-binding proteins and TFIID were identified and well characterized, functional linkage between TFIID and chromatin factors has remained to be elucidated. Here we show the identification and characterization of human CIA/hASF1 (identified previously as a histone chaperone) as an interactor of two tandem bromodomain modules of human (h)TAF(II)250/CCG1, the largest subunit of TFIID. Although yeast (y)TAF(II)145, a homologue of hTAF(II)250/CCG1 in Saccharomyces cerevisiae, lacks bromodomains, glutathione S-transferase pull-down and immunoprecipitation assays revealed that Asf1p (antisilencing function 1), the counterpart of CIA in S. cerevisiae, interacts with Bdf1p (bromodomain factor 1), which is reported to serve as the missing bromodomain in yTAF(II)145. Furthermore, yeast strain lacking the BDF1 gene shows the Spt phenotype that is shown also by the ASF1 gene disruptant, and a double-knockout strain of both genes shows synthetic lethality, indicating that ASF1 genetically interacts with bromodomains associated with yTFIID. We also found that Asf1p coprecipitates with yTFIID subunits from yeast whole-cell extract, and overexpression of yTFIID subunits suppress the Spt phenotype caused by gene disruption of the ASF1. This study describes the functional linkage between TFIID and a histone chaperone.

The branched-chain amino acid transaminase gene family in Arabidopsis encodes plastid and mitochondrial proteins.

Branched-chain amino acid transaminases (BCATs) play a crucial role in the metabolism of leucine, isoleucine, and valine. They catalyze the last step of the synthesis and/or the initial step of the degradation of this class of amino acids. In Arabidopsis, seven putative BCAT genes are identified by their similarity to their counterparts from other organisms. We have now cloned the respective cDNA sequences of six of these genes. The deduced amino acid sequences show between 47.5% and 84.1% identity to each other and about 30% to the homologous enzymes from yeast (Saccharomyces cerevisiae) and mammals. In addition, many amino acids in crucial positions as determined by crystallographic analyses of BCATs from Escherichia coli and human (Homo sapiens) are conserved in the AtBCATs. Complementation of a yeast Deltabat1/Deltabat2 double knockout strain revealed that five AtBCATs can function as BCATs in vivo. Transient expression of BCAT:green fluorescent protein fusion proteins in tobacco (Nicotiana tabacum) protoplasts shows that three isoenzymes are imported into chloroplasts (AtBCAT-2, -3, and -5), whereas a single enzyme is directed into mitochondria (AtBCAT-1).

Individual somatic H1 subtypes are dispensable for mouse development even in mice lacking the H1(0) replacement subtype.

H1 linker histones are involved in facilitating the folding of chromatin into a 30-nm fiber. Mice contain eight H1 subtypes that differ in amino acid sequence and expression during development. Previous work showed that mice lacking H1(0), the most divergent subtype, develop normally. Examination of chromatin in H1(0-/-) mice showed that other H1s, especially H1c, H1d, and H1e, compensate for the loss of H1(0) to maintain a normal H1-to-nucleosome stoichiometry, even in tissues that normally contain abundant amounts of H1(0) (A. M. Sirotkin et al., Proc. Natl. Acad. Sci. USA 92:6434-6438, 1995). To further investigate the in vivo role of individual mammalian H1s in development, we generated mice lacking H1c, H1d, or H1e by homologous recombination in mouse embryonic stem cells. Mice lacking any one of these H1 subtypes grew and reproduced normally and did not exhibit any obvious phenotype. To determine whether one of these H1s, in particular, was responsible for the compensation present in H1(0-/-) mice, each of the three H1 knockout mouse lines was bred with H1(0) knockout mice to generate H1c/H1(0), H1d/H1(0), or H1e/H1(0) double-knockout mice. Each of these doubly H1-deficient mice also was fertile and exhibited no anatomic or histological abnormalities. Chromatin from the three double-knockout strains showed no significant change in the ratio of total H1 to nucleosomes. These results suggest that any individual H1 subtype is dispensable for mouse development and that loss of even two subtypes is tolerated if a normal H1-to-nucleosome stoichiometry is maintained. Multiple compound H1 knockouts will probably be needed to disrupt the compensation within this multigene family.

Yeast Lacking Superoxide Dismutase(s) Show Elevated Levels of “Free Iron” as Measured by Whole Cell Electron Paramagnetic Resonance

A current hypothesis explaining the toxicity of superoxide anion in vivo is that it oxidizes exposed [4Fe-4S] clusters in certain vulnerable enzymes causing release of iron and enzyme inactivation. The resulting increased levels of "free iron" catalyze deleterious oxidative reactions in the cell. In this study, we used low temperature Fe(III) electron paramagnetic resonance (EPR) spectroscopy to monitor iron status in whole cells of the unicellular eukaryote, Saccharomyces cerevisiae. The experimental protocol involved treatment of the cells with desferrioxamine, a cell-permeant, Fe(III)-specific chelator, to promote oxidation of all of the "free iron" to the Fe(III) state wherein it is EPR-detectable. Using this method, a small amount of EPR-detectable iron was detected in the wild-type strain, whereas significantly elevated levels were found in strains lacking CuZn-superoxide dismutase (CuZn-SOD) (sod1 delta), Mn-SOD (sod2 delta), or both SODs, throughout their growth but particularly in stationary phase. The accumulation was suppressed by expression of wild-type human CuZn-SOD (in the sod1 delta mutant), by pmr1, a genetic suppressor of the sod delta mutant phenotype (in the sod1 delta sod2 delta double knockout strain), and by anaerobic growth. In wild-type cells, an increase in the EPR-detectable iron pool could be induced by treatment with paraquat, a redox-cycling drug that generates superoxide. Cells that were not pretreated with desferrioxamine had Fe(III) EPR signals that were equally as strong as those from treated cells, indicating that "free iron" accumulated in the ferric form in our strains in vivo. Our results indicate a relationship between superoxide stress and iron handling and support the above hypothesis for superoxide-related oxidative damage.

The Mex67p-mediated nuclear mRNA export pathway is conserved from yeast to human.

Human TAP is an orthologue of the yeast mRNA export factor Mex67p. In mammalian cells, TAP has a preferential intranuclear localization, but can also be detected at the nuclear pores and shuttles between the nucleus and the cytoplasm. TAP directly associates with mRNA in vivo, as it can be UV-crosslinked to poly(A)+ RNA in HeLa cells. Both the FG-repeat domain of nucleoporin CAN/Nup214 and a novel human 15 kDa protein (p15) with homology to NTF2 (a nuclear transport factor which associates with RanGDP), directly bind to TAP. When green fluorescent protein (GFP)-tagged TAP and p15 are expressed in yeast, they localize to the nuclear pores. Strikingly, co-expression of human TAP and p15 restores growth of the otherwise lethal mex67::HIS3/mtr2::HIS3 double knockout strain. Thus, the human TAP-p15 complex can functionally replace the Mex67p-Mtr2p complex in yeast and thus performs a conserved role in nuclear mRNA export.

Evidence that the RdeA protein is a component of a multistep phosphorelay modulating rate of development in Dictyostelium.

We have isolated an insertional mutant of Dictyostelium discoideum that aggregated rapidly and formed spores and stalk cells within 14 h of development instead of the normal 24 h. We have shown by parasexual genetics that the insertion is in the rdeA locus and have cloned the gene. It encodes a predicted 28 kDa protein (RdeA) that is enriched in charged residues and is very hydrophilic. Constructs with the DNA for the c-Myc epitope or for the green fluorescent protein indicate that RdeA is not compartmentalized. RdeA displays homology around a histidine residue at amino acid 65 with members of the H2 module family of phosphotransferases that participate in multistep phosphoryl relays. Replacement of this histidine rendered the protein inactive. The mutant is complemented by transformation with the Ypd1 gene of Saccharomyces cerevisiae, itself an H2 module protein. We propose that RdeA is part of a multistep phosphorelay system that modulates the rate of development.

13C nuclear magnetic resonance analysis of glucose and citrate end products in an ldhL-ldhD double-knockout strain of Lactobacillus plantarum.

We have examined the metabolic consequences of knocking out the two ldh genes in Lactobacillus plantarum using 13C nuclear magnetic resonance. Unlike its wild-type isogenic progenitor, which produced lactate as the major metabolite under all conditions tested, ldh null strain TF103 mainly produced acetoin. A variety of secondary end products were also found, including organic acids (acetate, succinate, pyruvate, and lactate), ethanol, 2,3-butanediol, and mannitol.

Structural Roles of the Spore Coat Proteins in Dictyostelium discoideum

The integrity of spores formed by mutant strains of Dictyostelium discoideum lacking the major spore coat proteins, SP96, SP70, or SP60, was compared to that of wildtype strains. Single, double, and triple knock-out strains developed normally and produced spores which were indistinguishable from wild-type spores by light or electron microscopy. However, the mutant strains were susceptable to staining with the lectin, ricinA, which recognizes a galactose-rich polysaccharide that is normally hidden by overlying spore coat proteins. The intensity of staining with fluorescently labeled ricinA increased as the spore coat proteins were incrementally lost. While these results indicate that the major outer spore coat proteins are not essential for the construction of a multi-layered spore coat in Dictyostelium, they show that the spores are more porous which might make them at risk to predators before germination.