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At least 595 records · Page 33Linked to original sources
PubMed1997

Identification and characterization of ssb and uup mutants with increased frequency of precise excision of transposon Tn10 derivatives: nucleotide sequence of uup in Escherichia coli.

A Lac+ papillation assay was used to identify mutants (tex) of Escherichia coli that exhibit an increased frequency of precise excision of a lacZ::Tn10dKan insertion. Three tex strains had suffered mutations in the gene (ssb) encoding the essential single-stranded DNA-binding protein SSB, which resulted in the following alterations in the 177-residue protein: G4D; L10F, P24S; and V102M. The phenotypes of these ssb mutants indicated that they were largely unaffected in other functions mediated by SSB, such as DNA replication, recombination, and repair. Strains with multicopy ssb+ exhibited a decreased frequency of Tn10dKan precise excision. Three other tex mutants had insertion mutations in the locus designated uup at 21.75 min on the linkage map. The nucleotide sequence of uup was determined, and the gene was inferred to encode a 625-amino-acid hydrophilic protein that belongs to the superfamily of ABC-domain proteins (with two pairs of the Walker A and B motifs), which are postulated to be involved in coupling ATP hydrolysis with other biological processes. The uup gene product shares extensive homology with the deduced sequences of two proteins of Haemophilus influenzae. The uup gene is also situated immediately upstream of (and is transcribed in the same direction as) the paraquat-inducible SoxRS-regulated pqi-5 gene, two reported promoters for which are situated within the uup coding sequence.

ATP-Binding Cassette Transporters↗
PubMed2000

Identification and characterization of constitutively active Smad2 mutants: evaluation of formation of Smad complex and subcellular distribution.

Smads mediate activin, transforming growth factor beta (TGFbeta), and bone morphogenetic protein signaling from receptors to nuclei. According to the current model, activated activin/TGFbeta receptors phosphorylate the carboxyl-terminal serines of Smad2 and Smad3 (SSMS-COOH); phosphorylated Smad2/3 oligomerizes with Smad4, translocates to the nucleus, and modulates transcription of defined genes. To test key features of this model in detail, we explored the construction of constitutively active Smad2 mutants. To mimic phosphorylated Smad2, we made two Smad2 mutants with acidic amino acid substitutions of carboxyl-terminal serines: Smad2-2E (Ser465, 467Glu) and Smad2-3E (Ser464, 465, 467Glu). The mutants enhanced basal transcriptional activity in a mink lung epithelial cell line, L17. In a Smad4-deficient cell line, SW480.7, Smad2-2E did not affect basal signaling; however, cotransfection with full-length Smad4, but not transfection of Smad4 alone, resulted in enhanced basal transcriptional activity, suggesting that the constitutively active Smad2 mutant also requires Smad4 for function. In vitro protein interaction analysis revealed that Smad2-2E bound more tightly to Smad4 than did wild-type Smad2; dissociation constants were 270 +/- 66 nM for wild-type Smad2:Smad4 complexes and 79 +/- 18 nM for Smad2-2E:Smad4 complexes. Determination of the subcellular localization of Smad2 revealed that a greater percentage of Smad2-2E was localized in the nucleus than wild-type Smad2. These results suggest that Smad2 phosphorylation results in both tighter binding to Smad4 and increased nuclear concentration; those changes may be responsible for transcriptional activation by Smad2.

Animals↗
PubMed2004

[Generation and identification of rice T-DNA insertional mutant lines].

By using the matured embryos of Japonica rice variety Zhonghua No. 11 as explants, rice transformation was performed by Agrobacterium-mediated co-cultivation method, resulting in 1489 independent transgenic rice plants that carry a T-DNA insertion. Genomic DNA gel-blot and PCR analyses showed that 69.8% of the total lines contain the inserted T-DNA. The flanking sequence of T-DNA in transgenic rice plants was analyzed using Tail-PCR. In addition, we have evaluated 1066 T1 transgenic lines on heading days, plant height and panicles per hill, and found different types of mutants from a number of lines.

DNA, Bacterial↗
PubMed2006

[Identification of the rice (Oryza sativa L.) mutant msp1-4 and expression analysis of its UDT1 and GAMYB genes].

A rice male-sterile mutant msp1-4 (MULTIPLE SPOROCYTE) with japonica cultivar '9522' background, was obtained in M(3) population treated with (60)Co gamma-ray. Results of genetic analysis indicated that the male-sterile phenotype was controlled by a single recessive locus. To map this locus, an F(2) population was constructed from the cross between the msp1-4 (japonica) and 'LongTeFu B' (indica). This locus was mapped between the two InDel markers, WY-4 and WY-8, with physical distance of 247 kb. A deletion with 10 base pairs between 758 bp and 767 bp in MSP1 open reading frame was confirmed by sequence analysis, which led to pre-termination of MSP1 translation. Phenotype analysis of msp1-4 indicated that it was similar to the msp1 mutant. To insight the expression change of rice anther developmental genes in this mutant, semi-quantitative RT-PCR analysis was carried out. The results showed that the expression level of rice UDT1 and GAMYB were reduced in msp1-4, implying that UDT1 and GAMYB are possibly the downstream genes of MSP1 gene in rice pollen development.

Flowers↗
PubMed2004

Mu dI1(Ap lac) mutagenesis of Yersinia pestis plasmid pFra and identification of temperature-regulated loci associated with virulence.

The F1 capsule of Yersinia pestis, encoded by the 100 kb plasmid pFra, is often assumed to be essential for full virulence of Y. pestis. However, virulent strains of Y. pestis that are F1- and either pFra+ or pFra- have been reported. To assess the role of pFra-encoded factors in virulence, mutants in pFra with insertions of the defective transposing bacteriophage Mu dl(Ap lac) were obtained, by using the wild type (wt) and the pLcr-cured derivative of strain C092. Mutants that exhibited temperature regulation of lactose fermentation and retarded electrophoretic mobility of pFra were selected. A total of 15 insertion mutants were isolated in the wt strain (12 of which had a single insertion in the genome, in pFra); and 24 mutants in the isogenic pLcr- derivative. Four of the pLcr+ mutants, and none of the pLcr- mutants, were F1-. All F1- mutants were decreased in virulence for mice compared to the wt parent; and five of the F1+ mutants also were significantly attenuated in mice. Fusion end-joints of insert DNA were cloned into Escherichia coli by using pMLB524, a vector for rescuing operon fusions of lacZ. Recombinants were obtained which contained pFra inserts ranging from < 2kb to approximately 36 kb, and the insertions occurred at several sites on pFra. All of the four F1- mutants tested mapped within the F1 capsule operon (caf1). The remaining five attenuated mutants sequenced were F1+ and mapped outside of but near the operon. Sequencing and complete analysis of the pFra insertions mutants could facilitate identification of new potential virulence factors.

Animals↗
PubMed2004

Skeletal dysplasia and male infertility locus on mouse chromosome 9.

In mice and humans, growth insufficiency and male infertility are common disorders that are genetically and phenotypically complex. We describe a spontaneously arising mouse mutant, chagun, that is affected by both dwarfism and male infertility. Dwarfism disproportionately affects long bones and is characterized by a defect in the proliferative zone of chondrocytes in the growth plate. Gonads of mutant males are small, with apparent germ cell loss and no evidence of mature sperm. The locus responsible for chagun is recessive and maps to distal chromosome 9, in a region homologous to human chromosome 3. This location is consistent with chagun defining a novel locus. Identification of the mutant gene will uncover the basis for another type of skeletal dysplasia and male infertility.

Animals↗
PubMed1999

Immunophenotype of mutant ras p21 and early response to radiotherapy in cancer of the uterine cervix.

Previous studies have postulated that ras gene mutations may influence cellular response to radiotherapy. However, clinical studies have often been limited by the cumbersome methodology associated with DNA analysis. The availability of ELISA method has eventually made clinical evaluation of ras gene mutation feasible. In this study ras mutation by in vitro identification of four mutant forms of p21 ras in cervical tumor tissue extracts was analyzed. Mutant ras proteins were evaluated by an Enzyme Linked Immunosorbent Assay (ELISA). Expression of ras p21 mutations was studied in 101 patients, and a correlation between pre-treatment experimental analyses and the clinical status of the patient after radiotherapy (up to 16 months follow up) was established. There was no correlation between the presence of Val 12 p21 and tumor response to radiotherapy. Yet, presence of the other three mutant proteins had significant relationship to treatment outcome. Detection of Arg 12 mutation was more common in patients who either had residual disease or developed recurrences (28%) as compared to those remaining disease-free (1.5%). The presence of the Arg 12 mutation therefore correlated to poor prognosis (r = 0.445, p = 0.0000). Similarly, the Asp 12 mutation was also more common in patients with residual/recurrent disease (25%) as compared to patients remaining disease-free (3%). Asp 12 mutation also showed a correlation to treatment outcome (r = 0.337, p = 0.00057). Asp 13 mutation was more frequent in patients with residual or recurrent disease (28%) as compared to those remaining disease-free (4.6%). On the basis of laboratory evidence ras genes appear to be involved as modulators of tumor response to radiation therapy. This understanding of the involvement of specific genes in radioresistance will result in the improvement of potential therapies that can be targeted at specific genes, through approaches such as selective inhibition by anti-sense oligonucleotides.

Amino Acid Substitution↗
PubMed2006

Mutagenesis as an unbiased approach to identify novel contraceptive targets.

To accommodate diverse personal needs in family planning, diverse contraceptive approaches are desirable. This goal requires identification of new contraceptive targets. Phenotype-driven mutagenesis is an unbiased approach to identify novel genes and functions in reproductive processes. The ReproGenomics Program at The Jackson Laboratory is a United States National Institutes of Health resource for production, identification and distribution of mutant mouse models of infertility that can be used for identification of potential targets for contraception. The strategy of this program is whole genome, random ENU mutagenesis, coupled with a phenotype screen for breeding failure as the only phenotype. A three-generation breeding scheme selects recessive mutations affecting reproductive functions. G3 males and females that fail to reproduce by natural mating to wild-type animals undergo secondary phenotype screens to assess gonad and accessory organ histology, hormone production, gamete production and gamete function in fertilization. The genetic transmission of the infertility trait in each family is confirmed and each mutation is genetically mapped to a defined chromosome region, facilitating identification of candidate genes from sequence and expression databases. Genes essential for fertility in both males and females and acting both meiotically and post-meiotically have been identified by this strategy. Phenotypes include male infertility with normal sperm count, but failure in fertilization of oocytes. Phenotype descriptions of each mutation are posted on the program website, . These unique reproductive mutant mouse resources will lead to new discoveries in andrology (and gynecology) research, as well as reproductive medicine. Dissection of gene function in known and newly discovered reproductive pathways will expand our focus to reveal novel targets for contraception.

Alkylating Agents↗
PubMed1998

A novel fluorescence-based genetic strategy identifies mutants of Saccharomyces cerevisiae defective for nuclear pore complex assembly.

Nuclear pore complexes (NPCs) are large proteinaceous portals for exchanging macromolecules between the nucleus and the cytoplasm. Revealing how this transport apparatus is assembled will be critical for understanding the nuclear transport mechanism. To address this issue and to identify factors that regulate NPC formation and dynamics, a novel fluorescence-based strategy was used. This approach is based on the functional tagging of NPC proteins with the green fluorescent protein (GFP), and the hypothesis that NPC assembly mutants will have distinct GFP-NPC signals as compared with wild-type (wt) cells. By fluorescence-activated cell sorting for cells with low GFP signal from a population of mutagenized cells expressing GFP-Nup49p, three complementation groups were identified: two correspond to mutant nup120 and gle2 alleles that result in clusters of NPCs. Interestingly, a third group was a novel temperature-sensitive allele of nup57. The lowered GFP-Nup49p incorporation in the nup57-E17 cells resulted in a decreased fluorescence level, which was due in part to a sharply diminished interaction between the carboxy-terminal truncated nup57pE17 and wt Nup49p. Interestingly, the nup57-E17 mutant also affected the incorporation of a specific subset of other nucleoporins into the NPC. Decreased levels of NPC-associated Nsp1p and Nup116p were observed. In contrast, the localizations of Nic96p, Nup82p, Nup159p, Nup145p, and Pom152p were not markedly diminished. Coincidentally, nuclear import capacity was inhibited. Taken together, the identification of such mutants with specific perturbations of NPC structure validates this fluorescence-based strategy as a powerful approach for providing insight into the mechanism of NPC biogenesis.

Alleles↗
PubMed2003

The molecular analysis of leaf senescence--a genomics approach.

Senescence in green plants is a complex and highly regulated process that occurs as part of plant development or can be prematurely induced by stress. In the last decade, the main focus of research has been on the identification of senescence mutants, as well as on genes that show enhanced expression during senescence. Analysis of these is beginning to expand our understanding of the processes by which senescence functions. Recent rapid advances in genomics resources, especially for the model plant species Arabidopsis, are providing scientists with a dazzling array of tools for the identification and functional analysis of the genes and pathways involved in senescence. In this review, we present the current understanding of the mechanisms by which plants control senescence and the processes that are involved.

Journal Article↗
PubMed2003

Mutation of Celsr1 disrupts planar polarity of inner ear hair cells and causes severe neural tube defects in the mouse.

We identified two novel mouse mutants with abnormal head-shaking behavior and neural tube defects during the course of independent ENU mutagenesis experiments. The heterozygous and homozygous mutants exhibit defects in the orientation of sensory hair cells in the organ of Corti, indicating a defect in planar cell polarity. The homozygous mutants exhibit severe neural tube defects as a result of failure to initiate neural tube closure. We show that these mutants, spin cycle and crash, carry independent missense mutations within the coding region of Celsr1, encoding a large protocadherin molecule [1]. Celsr1 is one of three mammalian homologs of Drosophila flamingo/starry night, which is essential for the planar cell polarity pathway in Drosophila together with frizzled, dishevelled, prickle, strabismus/van gogh, and rhoA. The identification of mouse mutants of Celsr1 provides the first evidence for the function of the Celsr family in planar cell polarity in mammals and further supports the involvement of a planar cell polarity pathway in vertebrate neurulation.

Animals↗
PubMed2001

Genetic selection in Saccharomyces of mutant mammalian adenylyl cyclases with elevated basal activities.

We show that co-expression of rat Galphas together with type I, II, IV, or VI mammalian adenylyl cyclase (AC) can suppress the growth defect of cyr1 strains of Saccharomyces cerevisiae, which lack a functional endogenous AC. Complemention of cvr1 is not observed in the absence of Galphas, indicating that the mammalian ACs retain their normal regulatory behavior in yeast. Selection for Galphas-independent growth of (cyr1 strains expressing type IV AC yielded several ACIV mutants with enhanced basal activity, each of which had a single amino acid substitution in the conserved C1a or C2a region of the protein. Expression of two of the mutant ACs in HEK293 cells resulted in increased levels of cAMP and elevated adenylyl cyclase activity. Further selection for reverting mutations in one of these constitutively active AC mutants yielded three independent intragenic suppressor mutations. The distribution of the activating and suppressor mutations throughout both C1a and C2a is consistent with a model in which the enhanced basal activity results from an increase in the affinity between C1a and C2a. These results demonstrate the utility of Saccharomyces as a tool for the identification of informative mutant forms of mammalian ACs.

Adenylyl Cyclases↗
PubMed1988

Characterization of Staphylococcus epidermidis mutants sensitive to ultraviolet radiation.

Five UV-sensitive mutants obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment of the Staphylococcus epidermidis W5 strain were characterized phenotypically by assaying their UV- and MNNG-sensitivities, lysogenic inducibility, host-cell reactivation and Weigle reactivation capacities. The results were compared with those of well-characterized Escherichia coli strains, permitting the identification of: 2 mutants that behave as Uvr- Umu-; 1 mutant that appears analogous to Uvr-; 1 mutant that resembles LexA- and 1 mutant that exhibits a RecA- phenotype. The study of these mutants can contribute to the understanding of the repair mechanisms in S. epidermidis.

DNA Damage↗
PubMed1999

The sax1 mutation defines a new locus involved in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana.

In this issue we described a dwarf mutant in Arabidopsis thaliana, sax1, which is affected in brassinosteroid biosynthesis. This primary defect is responsible for alterations in hormone sensitivity of sax1 plants characterized by the hypersensitivity of root elongation to abscisic acid and auxin and the insensitivity of hypocotyl growth to gibberellins and ethylene (Ephritikhine et al., 1999; Plant J. 18, 303-314). In this paper, we report the further characterization of the sax1 mutant aimed at identification of the mutated step in the brassinosteroid biosynthesis pathway. Rescue experiments with various intermediates of the pathway showed that the sax1 mutation alters a very early step catalyzing the oxidation and isomerization of 3 beta-hydroxyl, delta 5,6 precursors to 3-oxo, delta 4,5 steroids. The mapping of the mutation, the physiological properties of the mutant and the rescue experiments indicate that sax1 defines a new locus in the brassinosteroid biosynthesis pathway. The SAX1 protein is involved in brassinosteroid-dependent growth of seedlings in both light and dark conditions.

Arabidopsis↗