Mutant

About: Mutant is a research topic. Over the lifetime, 74520 publications have been published within this topic receiving 3477079 citations.

pTAC2, -6, and -12 Are Components of the Transcriptionally Active Plastid Chromosome That Are Required for Plastid Gene Expression

Abstract: Transcription in plastids is mediated by a plastid-encoded multimeric (PEP) and a nuclear-encoded single-subunit RNA polymerase (NEP) and a still unknown number of nuclear-encoded factors. By combining gel filtration and affinity chromatography purification steps, we isolated transcriptionally active chromosomes from Arabidopsis thaliana and mustard (Sinapis alba) chloroplasts and identified 35 components by electrospray ionization ion trap tandem mass spectrometry. Eighteen components, called plastid transcriptionally active chromosome proteins (pTACs), have not yet been described. T-DNA insertions in three corresponding genes, ptac2, -6, and -12, are lethal without exogenous carbon sources. Expression patterns of the plastid-encoded genes in the corresponding knockout lines resemble those of Δrpo mutants. For instance, expression of plastid genes with PEP promoters is downregulated, while expression of genes with NEP promoters is either not affected or upregulated in the mutants. All three components might also be involved in posttranscriptional processes, such as RNA processing and/or mRNA stability. Thus, pTAC2, -6, and -12 are clearly involved in plastid gene expression.

Evidence for a system of general protein glycosylation in Campylobacter jejuni.

Abstract: A genetic locus from Campylobacter jejuni 81-176 (O:23, 36) has been characterized that appears to be involved in glycosylation of multiple proteins, including flagellin. The lipopolysaccharide (LPS) core of Escherichia coli DH5α containing some of these genes is modified such that it becomes immunoreactive with O:23 and O:36 antisera and loses reactivity with the lectin wheat germ agglutinin (WGA). Site-specific mutation of one of these genes in the E. coli host causes loss of O:23 and O:36 antibody reactivity and restores reactivity with WGA. However, site-specific mutation of each of the seven genes in 81-176 failed to show any detectable changes in LPS. Multiple proteins from various cellular fractions of each mutant showed altered reactivity by Western blot analyses using O:23 and O:36 antisera. The changes in protein antigenicity could be restored in one of the mutants by the presence of the corresponding wild-type allele in trans on a shuttle vector. Flagellin, which is known to be a glycoprotein, was one of the proteins that showed altered reactivity with O:23 and O:36 antiserum in the mutants. Chemical deglycosylation of protein fractions from the 81-176 wild type suggests that the other proteins with altered antigenicity in the mutants are also glycosylated.

Functional Synergy between the Transcription Factor Sp1 and the Estrogen Receptor

Abstract: A GC-rich oligonucleotide containing an estrogen responsive element (ERE) half-site from the heat shock protein 27 (Hsp 27) gene promoter (-105 to -84) [ie. GGGCGGG(N)10GGTCA; Sp1(N)10ERE] forms a complex with the Sp1 and estrogen receptor (ER) proteins. Moreover, promoter-reporter constructs containing this sequence (-108 to -84 or -108 to +23) are also estrogen-responsive. Mutation of the ERE half-site in the Hsp 27-derived oligonucleotides did not result in loss of estrogen responsiveness in transient transfection studies, suggesting that estrogen inducibility was mediated through the Sp1-DNA motif. Gel mobility shift assays using 32P-labeled wild type and ERE mutant Sp1(N)10ERE and consensus Sp1 oligonucleotides showed that Sp1 protein formed a DNA-protein complex with all three nucleotides, and the intensities of retarded bands were enhanced by coincubation with wild type ER and 11C-ER, which does not contain the DNA-binding domain. ER mutants in which N-terminal (19C-ER) and C-terminal (15C-ER) regions were deleted did not enhance Sp1-DNA binding or hormone-induced transactivation of GC-rich promoter-reporter constructs in ER-negative MDA-MB-231 cells, whereas both wild type and 11C-ER restored inducibility. Immunoprecipitation studies also confirmed that the Sp1 and ER proteins physically interact. The interaction of the Sp1 and ER proteins and the resulting enhanced Sp1-DNA binding is observed in the presence or absence of estrogen (hormone-independent), whereas transactivation of promoter-reporter constructs is estrogen-dependent. Thus, the results illustrate a new estrogen-dependent transactivation pathway that involves ER-protein interactions and is ERE-independent.

Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

Abstract: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenic luxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in the luxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, the luxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

Functional importance of conserved domains in the flowering-time gene CONSTANS demonstrated by analysis of mutant alleles and transgenic plants

Abstract: CONSTANS promotes flowering of Arabidopsis in response to long-day conditions. We show that CONSTANS is a member of an Arabidopsis gene family that comprises 16 other members. The CO-Like proteins encoded by these genes contain two segments of homology: a zinc finger containing region near their amino terminus and a CCT (CO, CO-Like, TOC1) domain near their carboxy terminus. Analysis of seven classical co mutant alleles demonstrated that the mutations all occur within either the zinc finger region or the CCT domain, confirming that the two regions of homology are important for CO function. The zinc fingers are most similar to those of B-boxes, which act as protein-protein interaction domains in several transcription factors described in animals. Segments of CO protein containing the CCT domain localize GFP to the nucleus, but one mutation that affects the CCT domain delays flowering without affecting the nuclear localization function, suggesting that this domain has additional functions. All eight co alleles, including one recovered by pollen irradiation in which DNA encoding both B-boxes is deleted, are shown to be semidominant. This dominance appears to be largely due to a reduction in CO dosage in the heterozygous plants. However, some alleles may also actively delay flowering, because overexpression from the CaMV 35S promoter of the co-3 allele, that has a mutation in the second B-box, delayed flowering of wild-type plants. The significance of these observations for the role of CO in the control of flowering time is discussed.