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PubMed2004

Identification of amylase inhibitor deficient mutants in pigeonpea (Cajanus cajan (L.) Millisp.).

We have developed and analyzed several mutant lines (M6 generation) of pigeonpea (Cajanus cajan (L.) Millsp.) for the content of defensive proteins and antinutritional factors. Inhibitors of proteinase and of amylase, lectins, and raffinose family oligosaccharides were analyzed in mature seeds of different pigeonpea accessions (untreated) and compared with mutant lines. Proteinase inhibitor profiles were similar in terms of number and intensities of activity bands but they differ marginally in the activity units in pigeonpea accessions and mutants. Pigeonpea mutants showed significant differences in amylase inhibitor profiles as well as activity units from those of pigeonpea accessions. Interestingly, two mutants (A6-5-1 and A7-3-2) were identified to have absence of amylase inhibitor isoforms. Hemagglutinating activity and raffinose family oligosaccharides content were found to be significantly higher in mutants than in accessions. It is evident from the results that proteinase inhibitors of pigeonpea are stable while amylase inhibitors, lectins, and raffinose family oligosaccharides show altered expression upon mutagen treatments. These mutants will be ideal candidates for further evaluation.

Amylases↗
PubMed1991

A suppressor of a yeast splicing mutation (prp8-1) encodes a putative ATP-dependent RNA helicase.

Five small nuclear RNAs (snRNAs) are required for nuclear pre-messenger RNA splicing: U1, U2, U4, U5 and U6. The yeast U1 and U2 snRNAs base-pair to the 5' splice site and branch-point sequences of introns respectively. The role of the U5 and U4/U6 small nuclear ribonucleoprotein particles (snRNPs) in splicing is not clear, though a catalytic role for the U6 snRNA has been proposed. Less is known about yeast splicing factors, but the availability of genetic techniques in Saccharomyces cerevisiae has led to the identification of mutants deficient in nuclear pre-mRNA splicing (prp2-prp27). Several PRP genes have now been cloned and their protein products characterized. The PRP8 protein is a component of the U5 snRNP and associates with the U4/U6 snRNAs/snRNP to form a multi-snRNP particle believed to be important for spliceosome assembly. We have isolated extragenic suppressors of the prp8-1 mutation of S. cerevisiae and present here the preliminary characterization of one of these suppressors, spp81. The predicted amino-acid sequence of the SPP81 protein shows extensive similarity to a recently identified family of proteins thought to possess ATP-dependent RNA helicase activity. The possible role of this putative helicase in nuclear pre-mRNA splicing is discussed.

Adenosine Triphosphate↗
PubMed2003

Requirement for the PI3K/Akt pathway in MEK1-mediated growth and prevention of apoptosis: identification of an Achilles heel in leukemia.

The Raf/MEK/ERK kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using DeltaMEK1:ER, a conditionally active form of MEK1 which responds to either beta-estradiol or the estrogen receptor antagonist 4 hydroxy-tamoxifen (4HT), we previously documented the ability of this dual specificity protein kinase to abrogate the cytokine-dependency of human (TF-1) and murine (FDC-P1 and FL5.12) hematopoietic cells lines. Here we demonstrate the ability of DeltaMEK1:ER to activate the phosphatidylinositol 3-kinase (PI3K)/Akt/p70 ribosomal S6 kinase (p70(S6K)) pathway and the importance of this pathway in MEK1-mediated prevention of apoptosis. MEK1-responsive cells can be maintained long term in the presence of beta-estradiol, 4HT or IL-3. Removal of hormone led to the rapid cessation of cell proliferation and the induction of apoptosis in a manner similar to cytokine deprivation of the parental cells. Stimulation of DeltaMEK1:ER by 4HT resulted in ERK, PI3K, Akt and p70(S6K) activation. Treatment with PI3K, Akt and p70(S6K) inhibitors prevented MEK-responsive growth. Furthermore, the apoptotic effects of PI3K/Akt/p70(S6K) inhibitors could be enhanced by cotreatment with MEK inhibitors. Use of a PI3K inhibitor and a constitutively active form of Akt, [DeltaAkt(Myr(+))], indicated that activation of PI3K was necessary for MEK1-responsive growth and survival as activation of Akt alone was unable to compensate for the loss of PI3K activity. Cells transduced by MEK or MEK+Akt displayed different sensitivities to signal transduction inhibitors, which targeted these pathways. These results indicate a requirement for the activation of the PI3K pathway during MEK-mediated transformation of certain hematopoietic cells. These experiments provide important clues as to why the identification of mutant signaling pathways may be the Achilles heel of leukemic cell growth. Leukemia treatment targeting multiple signal transduction pathways may be more efficacious than therapy aimed at inhibiting a single pathway.

Animals↗
PubMed2003

The role of p53 in chemosensitivity and radiosensitivity.

The role of p53 as a central mediator of the DNA damage and other cellular stress responses is well established. The ultimate growth-suppressive function of p53 in part explains its ability to confer chemosensitivity and radiosensitivity upon tumor cells. Recent work in the field has added complexity to our understanding, in terms of identifying novel regulators of p53 stability and function, elucidation of the importance of the p53 family towards p53 function, a growing list of transcriptional targets as well as transcription-independent apoptotic effects and mechanisms, tissue specificity of the p53 response, a molecular understanding of p53-dependent therapeutic sensitization, and efforts towards molecular targeting of the p53 pathway. p53 remains an attractive target for drug development in cancer because its alteration provides a fundamental difference between normal and cancer cells. Strategies are emerging for the identification of mutant p53-specific therapies, therapies targeted at mutant p53-expressing tumors, as well as therapies that target various aspects of the p53 life cycle to enhance chemosensitization. The tools of molecular imaging are beginning to accelerate the pace of discovery and preclinical testing of p53 in animal models. The future holds promise for specific, individualized targeting of mutant or wild-type p53, or its transcriptional targets, in combination therapies with other cancer-specific drugs, to maximize tumor cell killing while protecting normal cells from toxic side effects.

DNA Damage↗
PubMed2002

Alteration of reaction and substrate specificity of a bacterial type III polyketide synthase by site-directed mutagenesis.

RppA, which belongs to the type III polyketide synthase family, catalyses the synthesis of 1,3,6,8-tetrahydroxynaphthalene (THN), which is the key intermediate of melanin biosynthesis in the bacterium Streptomyces griseus. The reaction of THN synthesis catalysed by RppA is unique in the type III polyketide synthase family, in that it selects malonyl-CoA as a starter substrate. The Cys-His-Asn catalytic triad is also present in RppA, as in plant chalcone synthases, as revealed by analyses of active-site mutants having amino acid replacements at Cys(138), His(270) and Asn(303) of RppA. Site-directed mutagenesis of the amino acid residues that are likely to form the active-site cavity revealed that the aromatic ring of Tyr(224) is essential for RppA to select malonyl-CoA as a starter substrate, since substitution of Tyr(224) by amino acids other than Phe and Trp abolished the ability of RppA to accept malonyl-CoA as a starter, whereas the mutant enzymes Y224F and Y224W were capable of synthesizing THN via the malonyl-CoA-primed reaction. Of the site-directed mutants generated, A305I was found to produce only a triketide pyrone from hexanoyl-CoA as starter substrate, although wild-type RppA synthesizes tetraketide and triketide pyrones in the hexanoyl-CoA-primed reaction. The kinetic parameters of Ala(305) mutants and identification of their products showed that the substitution of Ala(305) by bulky amino acid residues restricted the number of elongations of the growing polyketide chain. Both Tyr(224) (important for starter substrate selection) and Ala(305) (important for intermediate elongation) were found to be conserved in three other RppAs from Streptomyces antibioticus and Streptomyces lividans.

Acyl Coenzyme A↗
PubMed2002

Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii.

Two forms of the protozoan parasite Toxoplasma gondii are associated with intermediate hosts such as humans: rapidly growing tachyzoites are responsible for acute illness, whereas slowly dividing encysted bradyzoites can remain latent within the tissues for the life of the host. In order to identify genetic factors associated with parasite differentiation, we have used a strong bradyzoite-specific promoter (identified by promoter trapping) to drive the expression of T. gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) in stable transgenic parasites, providing a stage-specific positive/negative selectable marker. Insertional mutagenesis has been carried out on this parental line, followed by bradyzoite induction in vitro and selection in 6-thioxanthine to identify misregulation mutants. Two different mutants fail to induce the HXGPRT gene efficiently during bradyzoite differentiation. These mutants are also defective in other aspects of differentiation: they replicate well under bradyzoite growth conditions, lysing the host cell monolayer as effectively as tachyzoites. Expression of the major bradyzoite antigen BAG1 is reduced, and staining with Dolichos biflorus lectin shows reduced cyst wall formation. Microarray hybridizations show that these mutants behave more like tachyzoites at a global level, even under bradyzoite differentiation conditions.

Animals↗
PubMed1971

Host-gene control of C-type tumor virus-expression and tumorigenesis: relevance of studies in inbred mice to cancer in man and other species.

C-type RNA viruses are tumorigenic in mice and other species. They are widespread and transmitted in an intimate relationship with host-cell genetic material. Tumorigenesis is no doubt a complex biological phenomenon, and involves both genetic and environmental factors. Among the genetic factors, single-locus determination is probably exceptional; yet, a number of genes are known in inbred mice and other species, including man, that can exert a major influence. Clearly, studies with inbred mice apply outside the murine model and justify generalizations on the etiology of cancer. The types of cancer observed in man are similar to those of inbred strains of mice, and their stochastic and often familial occurrences suggest that genetic factors play important roles in both man and mouse. A number of heritable disorders predipsose to tumorigenesis. Investigations of the relationship between various polymorphic phenotypes and human tumors should be intensified. For instance, a study of high-risk human-tumor groups could aid considerably not only in the recognition of etiological agents but also of underlying host-gene controlled mechanisms. Indeed epidemiological studies have already led to the identification of mutant genes that influence the incidence of human cancer. Such studies may be guided by findings in inbred strains of mice. There is good evidence in inbred mice that mutations in host chromosomes determine whether or not genes coding for RNA oncogene viral "functions" become expressed as cancer. Although an etiological role of C-type RNA virus has not yet been demonstrated in human cancer, studies of the interaction of viral and host-cell genomes in mice and other species, and the visualization of C-type virus in human tumors have provided good reasons for suspecting the presence in man of oncogenic genes (oncogenes) homologous to those of the C-type viral genomes, which are well studied in mice, hamsters, cats, rats, and chickens.

Animals↗
PubMed1974

Amino-acid sequence of dihydrofolate reductase from a methotrexate-resistant mutant of Streptococcus faecium and identification of methionine residues at the inhibitor binding site.

The amino-acid sequence of dihydrofolate reductase (7,8-dihydrofolate:NADP(+) oxidoreductase, EC 1.5.1.4) from S. faecium var Durans strain A is reported, and methionine residues 28 and 50 are shown to be protected by the inhibitor aminopterin from carboxymethylation by iodoacetate which occurs in absence of the inhibitor. Comparison of the sequence with that of the Escherichia coli reductase reveals two domains of considerable homology, one (the N-terminal region) presumably concerned with dihydrofolate and inhibitor binding and the other with dinucleotide binding. No significant sequence homology was found between larger dehydrogenases and the dihydrofolate reductases, which must, therefore, have evolved from a different ancestral protein.

Amino Acid Sequence↗
PubMed1977

Identification of amber and ochre mutants of the human virus Ad2+ND1.

Although human adenoviruses grow poorly in monkey cells, this defect can be overcome either by coinfection of cells with simian virus 40 (SV40) or by insertion of the relevant portion of the SV40 genome into the adenovirus genome to form an adenovirus-SV40 hybrid virus. The nondefective adenovirus-2-SV40 hybrid virus, Ad2+ND1, contains an insertion of 17% of the SV40 genome, which codes for at least part of a 30,000 dalton protein. A set of Ad2+ND1 host-range mutants that have lost the ability to grow in monkey cells and behave as point mutants fail to synthesize the 30,000 dalton ND1 protein. Translation in vitro of SV40-specific mRNA from mutant-infected cells produces unique short polypeptides instead of the 30,000 dalton protein. Here we show that this set of host-range mutants includes both ochre and amber nonsense mutations. When the SV40-specific mRNA from the host-range mutants is translated in vitro to produce the polypeptide fragments, yeast suppressor tRNA can partially restore synthesis of wild-type-size 30,000 dalton protein. By this assay, one mutant is ochre and two are amber.

Adenoviridae↗
PubMed1989

Assay of vacuolar pH in yeast and identification of acidification-defective mutants.

As part of a genetic analysis of the biogenesis and function of the vacuole (lysosome) in the yeast Saccharomyces cerevisiae, assays of vacuolar pH were developed and used to identify mutants defective in vacuolar acidification. Vacuoles were labeled with 6-carboxyfluorescein with the membrane-permeant precursor 6-carboxyfluorescein diacetate. Dual-excitation flow cytometry was used to calibrate the pH-dependence of 6-carboxyfluorescein fluorescence in vivo. Vacuoles in wild-type yeast were mildly acidic, pH 6.2, in cells grown under several different conditions. Cultures labeled with 6-carboxyfluorescein were screened by fluorescence-ratio microscopy to detect mutants that had defects related to vacuolar acidification. A recessive nuclear mutation, vph1-1, caused an abnormally high vacuolar pH of 6.9, as assayed by flow cytometry, and eliminated vacuolar uptake of the weak base quinacrine. Acidification in a pep12::LEU2 mutant appeared defective by fluorescence-ratio microscopy and quinacrine-uptake assays, but the vacuolar pH in the pep12::LEU2 mutant was nearly normal (pH 6.3) in flow cytometric assays.

Hydrogen-Ion Concentration↗
PubMed1997

The surface region of the bifunctional vaccinia RNA modifying protein VP39 that interfaces with Poly(A) polymerase is remote from the RNA binding cleft used for its mRNA 5' cap methylation function.

VP39 is a single-domain, bifunctional viral protein, which acts at both ends of nascent mRNA. At the 5' end, it acts as a cap-specific 2'-O-methyltransferase. At the 3' end, it acts as a poly(A) polymerase processivity factor, requiring its direct association with poly(A) polymerase. Although crystallographic and biochemical data show the catalytic center and associated binding sites for VP39's methyltransferase function to be juxtaposed around a superficial cleft on the protein surface, surface regions required for VP39's mRNA 3' end modifying functions are not known. Here, we identify a surface region that interfaces directly with poly(A) polymerase, taking three independent approaches: (i) development of a direct in vitro dimerization assay, which is applied to numerous VP39 point mutants; (ii) identification of sites within VP39 that become protected from protease cleavage upon dimerization and further mutagenesis based upon these data; (iii) site-specific photo-cross-linking of VP39 to VP55. We find that the dimerization interface lies on a surface region remote from the methyltransferase cleft and contains a 3-5-residue "hot-spot," which is very sensitive to amino acid substitutions. Various other sites within VP39 consistently became hypersensitive to protease cleavage upon interaction with VP55, indicating the occurrence of extensive conformational changes.

Amino Acid Sequence↗
PubMed2001

Identification of a dominant negative mutant of Sprouty that potentiates fibroblast growth factor- but not epidermal growth factor-induced ERK activation.

Various mitogenic stimuli such as epidermal growth factor (EGF), fibroblast growth factor (FGF), and phorbol 12,13-dibutyrate (PDBu) activate the Ras-Raf-MEK-ERK pathway, but the regulatory mechanism of this pathway remains to be investigated. Here we found that in 293 cells, mammalian Sprouty2 and Sprouty4 were rapidly induced by EGF, FGF, and PDBu in an ERK pathway-dependent manner. Forced expression of Sprouty2 and Sprouty4 inhibited FGF-induced ERK activation but did not affect EGF- or PDBu-induced ERK activation. To examine whether endogenous Sproutys were also selective inhibitors, we generated a dominant negative form of Sprouty2 (Y55A) and Sprouty4 (Y53A) in which conserved tyrosine residues were mutated. These mutants reverted the suppressive effect of both Sprouty2 and Sprouty4 but not that of RasGAP or SPRED (Sprouty-related EVH1 domain-containing protein), another Sprouty-related Ras suppressor. Expression of dominant negative Sprouty2 and Sprouty4 enhanced and prolonged FGF- but not EGF-induced ERK activation in 293 cells. In PC12 cells, endogenous Sprouty4 was also induced by FGF. Overexpression of wild-type Sprouty4 blocked FGF-induced differentiation, whereas Y53A-Sprouty4 enhanced it. These observations suggest that endogenous Sprouty2 and Sprouty4 are physiological negative feedback regulators of growth factor-mediated ERK pathway and that there are Sprouty-sensitive and -insensitive ERK activation pathways. Finding a dominant negative form of Sproutys will facilitate the study of the molecular mechanism and physiological function of Sproutys.

Animals↗
PubMed2001

The propeptide of the transforming growth factor-beta superfamily member, macrophage inhibitory cytokine-1 (MIC-1), is a multifunctional domain that can facilitate protein folding and secretion.

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-beta (TGF-beta) superfamily. While it is synthesized in a pre-pro form, it is unique among superfamily members because it does not require its propeptide for correct folding or secretion of the mature peptide. To investigate factors that enable these propeptide independent events to occur, we constructed MIC-1/TGF-beta1 chimeras, both with and without a propeptide. All chimeras without a propeptide secreted less efficiently compared with the corresponding constructs with propeptide. Folding and secretion were most affected after replacement of the predicted major alpha-helix in the mature protein, residues 56-68. Exchanging the human propeptide in this chimera with either the murine MIC-1 or TGF-beta1 propeptide resulted in secretion of the unprocessed, monomeric chimera, suggesting a specific interaction between the human MIC-1 propeptide and mature peptide. Propeptide deletion mutants enabled identification of a region between residues 56 and 78, which is important for the interaction between the propeptide and the mature peptide. Cotransfection experiments demonstrated that the propeptide must be in cis with the mature peptide for this phenomenon to occur. These results suggest a model for TGF-beta superfamily protein folding.

Activins↗
PubMed2003

Phenotype-based identification of mouse chromosome instability mutants.

There is increasing evidence that defects in DNA double-strand-break (DSB) repair can cause chromosome instability, which may result in cancer. To identify novel DSB repair genes in mice, we performed a phenotype-driven mutagenesis screen for chromosome instability mutants using a flow cytometric peripheral blood micronucleus assay. Micronucleus levels were used as a quantitative indicator of chromosome damage in vivo. Among offspring derived from males mutagenized with the germline mutagen N-ethyl-N-nitrosourea (ENU), we identified a recessive mutation conferring elevated levels of spontaneous and radiation- or mitomycin C-induced micronuclei. This mutation, named chaos1 (chromosome aberration occurring spontaneously 1), was genetically mapped to a 1.3-Mb interval on chromosome 16 containing Polq, encoding DNA polymerase theta. We identified a nonconservative mutation in the ENU-derived allele, making it a strong candidate for chaos1. POLQ is homologous to Drosophila MUS308, which is essential for normal DNA interstrand crosslink repair and is unique in that it contains both a helicase and a DNA polymerase domain. While cancer susceptibility of chaos1 mutant mice is still under investigation, these data provide a practical paradigm for using a forward genetic approach to discover new potential cancer susceptibility genes using the surrogate biomarker of chromosome instability as a screen.

Animals↗
PubMed2003

Identification of a topoisomerase I mutant, scsA1, as an extragenic suppressor of a mutation in scaA(NBS1), the apparent homolog of human nibrin in Aspergillus nidulans.

The Mre11-Rad50-Nbs1 protein complex has emerged as a central player in the human cellular DNA damage response, and recent observations suggest that these proteins are at least partially responsible for the linking of DNA damage detection to DNA repair and cell cycle checkpoint functions. Mutations in scaA(NBS1), which encodes the apparent homolog of human nibrin in Aspergillus nidulans, inhibit growth in the presence of the antitopoisomerase I drug camptothecin. This article describes the selection and characterization of extragenic suppressors of the scaA1 mutation, with the aim of identifying other proteins that interfere with the pathway or complex in which the ScaA would normally be involved. Fifteen extragenic suppressors of the scaA1 mutation were isolated. The topoisomerase I gene can complement one of these suppressors. Synergistic interaction between the scaA(NBS1) and scsA(TOP1) genes in the presence of DNA-damaging agents was observed. Overexpression of topoisomerase I in the scaA1 mutant causes increased sensitivity to DNA-damaging agents. The scsA(TOP1) and the scaA(NBS1) gene products could functionally interact in pathways that either monitor or repair DNA double-strand breaks.

Aspergillus nidulans↗
PubMed2006

The control of flowering in time and space.

The transition to flowering is one of the most important developmental decisions made by plants. Classical studies have highlighted the importance of photoperiod in controlling flowering time. More recently, the identification of mutants specifically affected in the photoperiod pathway in the model system Arabidopsis thaliana has enabled the flowering time pathways to be placed in a molecular context. This review highlights recent advances in understanding how photoperiod signals (perceived in the leaves) act at the apex of the plant where the floral stimulus is perceived. The photoperiod pathway acts predominantly through the gene CONSTANS to activate the small signalling molecule FT. While FT transcription is induced in the leaves, it is essential that FT protein is present at the apex of the plant. FT at the apex interacts with the transcription factor FD to induce flowering.

Arabidopsis↗
PubMed1986

Chromogenic identification of oligonucleotide-directed mutants.

We describe a simple plaque color assay for identifying oligonucleotide-directed mutations in cloned DNA fragments. The basis of the method is to: fuse the sequence of interest in-frame to the E.coli lacZ gene to produce a blue plaque phage, mutate the site of interest to a stop codon to generate a white plaque phage, and revert the stop codon and surrounding nucleotides to give a blue plaque phage containing one or more desired amino acid changes. The advantages of this cyclic method are that it produces truncated as well as amino acid substituted protein molecules, it can be repeated to introduce additional mutations, and it eliminates the need for labor intensive screening. Essentially any piece of DNA can be mutated using this method if the fragment has one open reading frame. If there is an open reading frame between the site and the lacZ gene, ATG codons can be inserted at the target site. We have used this method to produce termination and amino acid substitution mutants in the yeast CUP1 gene.

Base Sequence↗
PubMed1994

A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase.

A rapid method for efficiently generating site-directed mutations on a clean sequence background is described. This modification of the megaprimer PCR mutagenesis approach can be performed in one tube in less than 4.5 hours, and does not require purification of intermediate products. High fidelity of DNA sequence replication is obtained by employing Pfu DNA polymerase and limiting the total number of amplification cycles to 30. The mutagenesis efficiency of the procedure is high enough to allow rapid, direct identification of mutants by restriction digest or sequencing techniques.

DNA-Directed DNA Polymerase↗