Rapid genomic identification of transgenic integration sites for mouse insertional mutants.
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Animals↗
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Identification and characterization of Trichoplusia ni furin truncated mutant.
We report a new prohormone convertase gene of insect origin, Trichoplusia ni furin, which was cloned from BTI-Tn-5B-4 (Tn5) insect cells. We constructed a truncated mutant that lacked Cys-rich repeated segments. Using baculovirus expression system and standard enzymatic assay, we obtained recombinant Tn furin and evaluated aspects of its function.
Animals↗
Identification and characterization of collaborating oncogenes in compound mutant mice.
We have used proviral tagging in tumor-prone transgenic mice to identify collaborating oncogenes and genes contributing to tumor progression. This has yielded a series of oncogenes that could be assigned to different complementation groups in transformation: the myc, Pim, Bmi1, and Frat1 complementation groups. Frat1 is involved in tumor progression and appears to function in the Wnt signaling pathway. Overexpression of Fratl confers a growth advantage to transplanted tumor cells in vivo and to cells grown in vitro at high density. Frat1 might exert its activity by impairing the kinase activity of Gsk3beta, which is involved in the degradation of beta-catenin. Pim genes appear to act in tumor initiation and show strong synergism with myc in lymphomagenesis. Overexpression of Pim1 can also overcome some of the proliferative defects caused by defective interleukin signaling supporting a role of Pim1 in cell proliferation. We have applied proviral tagging in compound mutant Emu-myc/Pim1-/-/Pim2-/- mice to identify genes that can complement for the loss of Pim1 and Pim2 and, therefore, are able to synergize with c-myc in lymphomagenesis. A number of new as well as known genes have been found by this "complementation tagging." The latter included c-kit, Tp12, and cyclin D2, suggesting that Pim kinases might act upstream of or parallel to these known proto-oncogenes.
Animals↗
Ligand-independent oligomerization of natriuretic peptide receptors. Identification of heteromeric receptors and a dominant negative mutant.
Activation of many single-transmembrane receptors requires ligand-induced receptor oligomerization. We have examined the oligomerization of the atrial natriuretic peptide receptor, NPR-A, using epitope-tagged receptor in a co-immunoprecipitation assay. Unlike other single-transmembrane receptors, NPR-A oligomerized in a ligand-independent fashion. Extracellular receptor sequences were both necessary and sufficient for oligomer formation. NPR-A was also able to oligomerize with the related natriuretic peptide receptor, NPR-B. A truncated NPR-A lacking most of the cytoplasmic domain blocked activation of the full-length receptor, presumably through formation of an inactive heteromer. These results indicate that oligomerization of this single-transmembrane receptor is important for the transduction of a conformational change across the plasma membrane but are not consistent with models in which natriuretic peptide receptor oligomerization serves merely to bring intracellular domains together.
Amino Acid Sequence↗
Identification and properties of the proline664-leucine mutant LDL receptor in South Africans of Indian origin.
The incidence of familial hypercholesterolemia (FH) is high among South African Indians. The proline664-leucine low density lipoprotein (LDL)-receptor mutation was detected in four apparently unrelated Indian FH families in South Africa. This mutation was originally described in an FH subject (MM) of Indian (Gujerat province) origin (Soutar et al. 1989. Proc. Natl. Acad. Sci. 86: 4166-4170). All four South African families trace their origin to the vicinity of Surat in the Gujerat province of India. Haplotype analyses revealed that both LDL receptor genes in one of the homozygous patients are the same as those in the subject MM. The phenotype of the mutant protein was analyzed in skin fibroblasts of homozygous patients. [35S]methionine pulse-chase experiments revealed that the receptor precursors were slowly processed to mature receptors. Mature mutant receptors were degraded at faster than normal rates. This mutation, which is in the epidermal growth factor (EGF)-precursor-like domain of the LDL receptor, was previously reported to yield binding-defective receptors. Here we report that the affinity of the mutant LDL receptor for both LDL and beta-very low density lipoprotein (beta-VLDL) was normal and that the steady-state level of mutant receptors was about 20% of normal. Thus, the disease FH in these subjects is presumably due to the low steady-state level of receptor molecules that are functionally normal but exhibit accelerated turnover.
Adolescent↗
Protein synthesis in yeast. Identification of an altered elongation factor in thermolabile mutants of the yeast Saccharomyces cerevisiae.
Cell-free extracts from the wild type yeast strain (A364A) and from a group of noncomplementing mutants that are conditionally defective in translation were preincubated at a restrictive temperature prior to incubation at a permissive temperature for protein synthesis. Results of these experiments showed that upon exposure to the restrictive temperature (39 degrees C), all five of the noncomplementing mutants lost ability to incorporate amino acid into protein. The wild type parent strain retained better than 80% of the activity under identical conditions of heat treatment. Mutant extracts could be revived to incorporate amino acid by the addition of the purified yeast elongation factor 3. Factors 1 and 2 had no effect. The heat-treated extract from one mutant did not supplement the activity of the other mutant. Although all five of the mutants were inactivated by preincubation at 39 degrees C, each showed a variable rate and extent of thermolability. Heat-treated mutant extracts were fully active in polyphenylalanine synthesis with liver ribosomes but not with the yeast ribosomes. Since liver ribosomes do not require factor 3, this assay then confirms that factor 3 is the thermolabile component in this group of noncomplementing mutants.
Fungal Proteins↗
Direct identification of the amino acid changes in two mutant lac repressors.
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Amino Acid Sequence↗
Identification of the amino acid substitutions in two mutant forms of the recA protein from Escherichia coli: recA441 and recA629.
We have identified the amino acid substitutions in two mutant forms of the recA protein from Escherichia coli. The recA441 mutant, which shows constitutive expression of the recA-mediated SOS response at 42 degrees C, contains two amino acid substitutions, glutamic acid to lysine at residue 38 and isoleucine to valine at residue 298. The recA629 mutant is an unusual pseudorevertant of recA441 that is no longer capable of spontaneous expression of SOS functions at 42 degrees C. Purified recA629 protein is cold-labile for several of the wild-type enzymatic activities and is shown here to contain three amino acid substitutions, the two found in the recA441 protein at residues 38 and 298, as well as an aspartic acid-to-glycine change at residue 32. The mutation at residue 32 was verified by restriction digestion of the 5' region of the recA629 structural gene.
Alleles↗
[Identification of a polymyxin preparation obtained from a mutant Bacillus polymyxa strain].
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Bacillus↗
Segregation of mutant ovalbumins and ovalbumin-globin fusion proteins in Xenopus oocytes. Identification of an ovalbumin signal sequence.
The intramolecular signals for chicken ovalbumin secretion were examined by producing mutant proteins in Xenopus oocytes. An ovalbumin complementary DNA clone was manipulated in vitro, and constructs containing altered protein-coding sequences and either the simian virus 40 (SV40) early promoter or Herpes simplex thymidine kinase promoter, were microinjected into Xenopus laevis oocytes. The removal of the eight extreme N-terminal amino acids of ovalbumin had no effect on the segregation of ovalbumin with oocyte membranes nor on its secretion. A protein lacking amino acids 2 to 21 was sequestered in the endoplasmic reticulum but remained strongly associated with the oocyte membranes rather than being secreted. Removal of amino acids 231 to 279, a region previously reported to have membrane-insertion function, resulted in a protein that also entered the endoplasmic reticulum but was not secreted. Hybrid proteins containing at their N terminus amino acids 9 to 41 or 22 to 41 of ovalbumin fused to the complete chimpanzee alpha-globin polypeptide were also sequestered by oocyte membranes. We conclude that the ovalbumin "signal" sequence is internally located within amino acids 22 to 41, and we speculate that amino acids 9 to 21 could be important for the completion of ovalbumin translocation through membranes.
Amino Acid Sequence↗
Selection of T-DNA-tagged male and female gametophytic mutants by segregation distortion in Arabidopsis.
As a strategy for the identification of T-DNA-tagged gametophytic mutants, we have used T-DNA insertional mutagenesis based on screening for distorted segregation ratios by antibiotic selection. Screening of approximately 1000 transgenic Arabidopsis families led to the isolation of eight lines showing reproducible segregation ratios of approximately 1:1, suggesting that these lines are putative gametophytic mutants caused by T-DNA insertion at a single locus. Genetic analysis of T-DNA transmission through reciprocal backcrosses with wild type showed severe reductions in genetic transmission of the T-DNA through the male and/or female gametes. Direct evidence for mutant phenotypes in these lines was investigated by DAPI staining of mature pollen grains and by the analysis of seed set and embryo sac morphology in cleared ovules. One line, termed limpet pollen, showed a novel pollen phenotype in that the generative cell failed to migrate inward after pollen mitosis I, such that the generative or sperm cells remained against the pollen wall. Two other lines, andarta and tistrya, were defective in female transmission and showed an early arrest of embryo sac development with the viable megaspore not initiating the nuclear division cycles. These data demonstrate the efficacy of a segregation ratio distortion strategy for the identification of T-DNA-tagged gametophytic mutants in Arabidopsis.
Arabidopsis↗
Identification of two mannoproteins released from cell walls of a Saccharomyces cerevisiae mnn1 mnn9 double mutant by reducing agents.
In this report, we present the identification of the main polypeptides that are extracted from purified cell walls of a Saccharomyces cerevisiae mnn1 mnn9 strain by reducing agents. Treatment of the purified cell walls of this strain with beta-mercaptoethanol releases several mannoproteins, of which three, with apparent sizes of 120, 45, and 40 kDa, are the most abundant. Analysis of the amino-terminal sequences revealed that the 120-kDa mannoprotein is Bar1p, the protease involved in the so-called barrier activity in yeast cells, and that the 45- and 40-kDa mannoproteins are the Kex2-unprocessed and Kex2-processed forms of the gene product of open reading frame (ORF) YJL158c, an ORF that belongs to the PIR (protein with internal repeats) family of genes, composed thus far of PIR1, PIR2/HSP150, and PIR3. Accordingly we have named this gene PIR4, and Pir4 denotes the 40-kDa Kex2-processed form of the mannoprotein. We have characterized Pir4 and have shown the feasibility of using it as a fusion partner for the targeting of recombinant proteins to the cell wall.
Amino Acid Sequence↗
Cloning and sequence analysis of the hemB gene of Staphylococcus aureus.
The hemB gene is a member of the family of genes encoding enzymes of the porphyrin biosynthetic pathway and codes for the enzyme porphobilinogen synthase, which is responsible for the conversion of delta-aminolevulinic acid to porphobilinogen. To clone the hemB gene of Staphylococcus aureus we used Tn917-mediated transposon mutagenesis. Tn917 confers resistance to erythromycin and is carried by plasmid pTV1ts, which has thermosensitive replication. Hem mutants were selected by growth in the presence of kanamycin and erythromycin at 43 degrees C. Preliminary identification of the hem mutants was based on their dwarf colony growth, which could be restored to normal by hemin. DNA extracted from one of the hem mutants was digested with several restriction endonucleases and hybridized to a probe representing the XbaI-AvaI end of Tn917. A BglII-EcoRI fragment of 4.5 kb gave a positive signal and was cloned into pUC18. Transformants were identified by colony hybridization with the Tn917 probe. The positive clones were sequenced, starting from the transposon end. The results allowed us to identify an open reading frame whose nucleotide sequence presented a homology of 63% to the sequence of the hemB gene of Bacillus subtilis and of 55% to the sequence of the hemB gene of Escherichia coli K12. No other nucleotide sequences, except those belonging to known hemB genes, presented significant homologies to our sequence. The cloning of the hemB gene of S. aureus was confirmed by the ability of the gene to complement a hemB mutant of E. coli K12. To our knowledge, this is the first report of the cloning of a hem gene in S. aureus.
Amino Acid Sequence↗
A rapid method to map mutations in Drosophila.
BACKGROUND: Genetic screens in Drosophila have provided a wealth of information about a variety of cellular and developmental processes. It is now possible to screen for mutant phenotypes in virtually any cell at any stage of development by performing clonal screens using the flp/FRT system. The rate-limiting step in the analysis of these mutants is often the identification of the mutated gene, however, because traditional mapping strategies rely mainly on genetic and cytological markers that are not easily linked to the molecular map. RESULTS: Here we describe the development of a single-nucleotide polymorphism (SNP) map for chromosome arm 3R. The map contains 73 polymorphisms between the standard FRT chromosome, and a mapping chromosome that carries several visible markers (rucuca), at an average density of one SNP per 370 kilobases (kb). Using this collection, we show that mutants can be mapped to a 400 kb interval in a single meiotic mapping cross, with only a few hundred SNP detection reactions. Discovery of further SNPs in the region of interest allows the mutation to be mapped with the same recombinants to a region of about 50 kb. CONCLUSION: The combined use of standard visible markers and molecular polymorphisms in a single mapping strategy greatly reduces both the time and cost of mapping mutations, because it requires at least four times fewer SNP detection reactions than a standard approach. The use of this map, or others developed along the same lines, will greatly facilitate the identification of the molecular lesions in mutants from clonal screens.
Animals↗
A complex containing two transcription factors regulates peroxisome proliferation and the coordinate induction of beta-oxidation enzymes in Saccharomyces cerevisiae.
Expression of the POX1 gene, which encodes peroxisomal acyl coenzyme A oxidase in the yeast Saccharomyces cerevisiae, is tightly regulated and can be induced by fatty acids such as oleate. Previously we have shown that this regulation is brought about by interactions between trans-acting factor(s) and an upstream activating sequence (UAS1) in the POX1 promoter. We recently identified and isolated a transcription factor, Oaf1p, that binds to the UAS1 of POX1 and mediates its induction. A screening strategy has been developed and used to identify eight S. cerevisiae mutants, from three complementation groups, that are defective in the oleate induction of POX1. Characterization of one such mutant led to the identification of Oaf2p, a protein that is 39% identical to Oaf1p. Oaf1p and Oaf2p form a protein complex that is required for the activation of POX1 and FOX3 and for proliferation of peroxisomes. We propose a model in which these two transcription factors heterodimerize and mediate this activation process. The mutants that we have isolated, and further identification of the corresponding defective genes, provide us with an opportunity to characterize the mechanisms involved in the coordinate regulation of peroxisomal beta-oxidation enzymes.
Acetyl-CoA C-Acetyltransferase↗
Improved identification of SUMO attachment sites using C-terminal SUMO mutants and tailored protease digestion strategies.
A growing number of biological processes have been found to be regulated by the covalent attachment of the ubiquitin-like protein SUMO to key cellular targets. A critical step in the process of analyzing the role of SUMO in regulating the activity of these proteins is the identification of the lysine residues that are targeted by this modification. Unfortunately, current methods aimed at mapping these attachment-sites are laborious and often ineffective. We report here the development of a platform that combines the use of different C-terminal SUMO mutants with different protease digestion strategies to enable the rapid and efficient identification of SUMO attachment sites. We successfully apply this approach to several model SUMO substrates as well as to a mixture of SUMO conjugates purified from Saccharomyces cerevisiae. Although we specifically employ this strategy for the identification of SUMO attachment sites in yeast, this general approach can easily be adapted to map the sites of conjugation for other ubiquitin-like proteins from a wide range of organisms.
Amino Acid Sequence↗
A conserved carboxy-terminal subdomain is important for ligand interpretation and transactivation by nuclear receptors.
Nuclear receptors share a highly conserved region located at the very carboxy-terminal part of the ligand-binding domain. Site-directed mutagenesis of conserved hydrophobic residues in this region was reported to create mouse and human glucocorticoid receptors (GRs) and estrogen receptors that cannot transactivate but apparently maintain all the other functions. We constructed analogous mutations in the rat GR to compare the mechanism of deficiency to our recently generated trans-dominant-negative mutant. We found that in the rat GR these carboxy-terminal mutations do not generate trans-dominant-negative receptors. We show that these GR mutants fail to bind dexamethasone properly, and hence receptor transformation and subsequent functions are abolished. Furthermore, we report the identification of a GR mutant that is strongly responsive to the antagonist RU486 but is silent with the agonist dexamethasone. We demonstrate that the reversal of the responsiveness is restricted to GR, since analogous mutations in related receptors do not generate similar phenotypes. Contrary to the case of the progesterone receptor, we show that carboxyl-end truncated GR mutants are not activated by RU486. We conclude that sequence conservation of this subdomain does not necessarily imply functional conservation. Chimeric constructs with GAL4 revealed the importance of protein-protein interactions to exert ligand discrimination, which is mediated by the carboxy-terminal subdomain.
Alanine↗
Detailed biosynthetic pathway to decaprenoxanthin diglucoside in Corynebacterium glutamicum and identification of novel intermediates.
Carotenogenic mutants of Corynebacterium glutamicum were analyzed for their carotenoid content. Mutant MV10 accumulated the same carotenoids as the wild-type, decaprenoxanthin, decaprenoxanthin monoglucoside, and (2R,6R,2'R,6'R)-decaprenoxanthin di-(beta-D)-glucoside, but in three-fold higher amounts. In addition, decaprenoxanthin diglucoside fatty acid esters and the intermediates nonaprene, 2-(3-methyl-2-butenyl)-epsilon,psi-carotene, and sarcinene, 2,2'-bis(3-methyl-2-butenyl)-epsilon,epsilon-carotene were identified as minor carotenoids. The pink mutants MV40 and MV60 synthesized only lycopene. From another pink mutant, MV70, novel C(50)-carotenoids were isolated. By NMR and mass spectroscopy, nonaflavuxanthin, 2-(4-hydroxy-3-methyl-2-butenyl)-1,16-didehydro-1,2-dihydro-psi,psi-carotene, and flavuxanthin, 2,2'-bis(4-hydroxy-3-methyl-2-butenyl)-1,16,1',16'-tetradehydro-1,2,1',2'-tetrahydro-psi,psi-carotene, were identified. The identification of these intermediates revealed the detailed pathway for the formation of decaprenoxanthin derivatives in Corynebacterium glutamicum.
Carotenoids↗