Molecular Consequences of Ds Insertion Into and Excision From the Helix-Loop-Helix Domain of the Maize R Gene

Abstract The R and B proteins of maize are required to activate the transcription of several genes in the anthocyanin biosynthetic pathway. To determine the structural requirements for R function in vivo, we are exploiting its sensitive mutant phenotype to identify transposon (Ds) insertions that disrupt critical domains. Here we report that the ability of the r-m1 allele to activate transcription of at least three structural genes is reduced to only 2% of wild-type activity because of a 396-bp Ds element in helix 2 of the basic helix-loop-helix (bHLH) motif. Residual activity likely results from the synthesis of a mutant protein that contains seven additional amino acids in helix 2. This protein is encoded by a transcript where most of the Ds sequence has been spliced from pre-mRNA. Two phenotypic classes of stable derivative alleles, very pale and extremely pale, condition <1% of wild-type activity as a result of the presence of two- and three-amino-acid insertions, respectively, at the site of Ds excision. Localization of these mutant proteins to the nucleus indicates a requirement for an intact bHLH domain after nuclear import. The fact that deletion of the entire bHLH domain has only a minor effect on R protein activity while these small insertions virtually abolish activity suggests that deletion of the bHLH domain may bypass a requirement for bHLH-mediated protein-protein interactions in the activation of the structural genes in the anthocyanin biosynthetic pathway.

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Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance

Thrombosis and Haemostasis ◽

10.1160/th16-03-0223 ◽

2016 ◽

Vol 116 (12) ◽

pp. 1022-1031 ◽

Cited By ~ 8

Author(s):

Yuki Takagi ◽

Moe Murata ◽

Toshihiro Kozuka ◽

Yukiko Nakata ◽

Ryo Hasebe ◽

...

Keyword(s):

Surface Plasmon Resonance ◽

Residual Activity ◽

Base Substitution ◽

Enzyme Linked Immunosorbent Assay ◽

Dependent Manner ◽

Missense Mutations ◽

Wild Type ◽

Gene Encoding ◽

Single Base

SummaryAntithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0%, in contrast to values of all variants were maintained at above 80%. The thrombin–AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12%, whereas that of tested variants was 37%–56%. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin–TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin–TM affinity- dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM- mediated anticoagulation.

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Deletion of the Mycobacterium tuberculosis α-Crystallin-Like hspX Gene Causes Increased Bacterial Growth In Vivo

Infection and Immunity ◽

10.1128/iai.74.2.861-868.2006 ◽

2006 ◽

Vol 74 (2) ◽

pp. 861-868 ◽

Cited By ~ 82

Author(s):

Yanmin Hu ◽

Farahnaz Movahedzadeh ◽

Neil G. Stoker ◽

Anthony R. M. Coates

Keyword(s):

Mycobacterium Tuberculosis ◽

Constitutive Expression ◽

Active Role ◽

Structural Genes ◽

Wild Type ◽

Major Antigen ◽

Oxide Stress ◽

Unusual Phenotype

ABSTRACT Hypervirulent mutants of Mycobacterium tuberculosis, whose growth rates are higher in vivo, have now been reported to have mutations in both regulatory and structural genes, but the basis for this unusual phenotype is not understood. One hypervirulence gene, dosR (devR, Rv2031c), activates transcription of approximately 50 genes in this pathogen in response to hypoxia and nitric oxide stress. The most dramatic activation (∼80-fold) is activation of the hspX (acr, Rv2031c) gene, which encodes a 16-kDa α-crystallin-like protein that is a major antigen. In this study we found that a Δacr mutant exhibited increased growth following infection of BALB/c mice in vivo and in both resting and activated macrophages in vitro (as measured by the number of CFU). The increased growth in macrophages was equal to that of a ΔdosR mutant, while introduction of a constitutively expressed hspX gene reduced the ΔdosR virulence to wild-type levels. These results suggest that the increased number of CFU of the ΔdosR mutant was largely due to loss of hspX expression. We also confirmed that constitutive expression of hspX slows growth in vitro, and we propose that hspX plays an active role in slowing the growth of M. tuberculosis in vivo immediately following infection.

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Regulation of scute function by extramacrochaete in vitro and in vivo

Development ◽

10.1242/dev.120.12.3595 ◽

1994 ◽

Vol 120 (12) ◽

pp. 3595-3603 ◽

Cited By ~ 2

Author(s):

C.V. Cabrera ◽

M.C. Alonso ◽

H. Huikeshoven

Keyword(s):

Expression Patterns ◽

Transcription Activation ◽

Loss Of Function ◽

Wild Type ◽

Helix Loop Helix ◽

In The Wild ◽

Yeast Assay ◽

Number Of Cells

The pattern of adult sensilla in Drosophila is established by the dosage-sensitive interaction of two antagonistic groups of genes. Sensilla development is promoted by members of the achaete-scute complex and the daughterless gene whereas it is suppressed by whereas extramacrochaete (emc) and hairy. All these genes encode helix-loop-helix proteins. The products of the achaete-scute complex and daughterless interact to form heterodimers able to activate transcription. In this report, we show that (1) extra-macrochaete forms heterodimers with the achaete, scute, lethal of scute and daughterless products; (2) extramacrochaete inhibits DNA-binding of Achaete, Scute and Lethal of Scute/Daughterless heterodimers and Daughterless homodimers and (3) extramacrochaete inhibits transcription activation by heterodimers in a yeast assay system. In addition, we have studied the expression patterns of scute in wild-type and extramacrochaete mutant imaginal discs. Expression of scute RNA during imaginal development occurs in groups of cells, but high levels of protein accumulate in the nuclei of only a subset of the RNA-expressing cells. The pattern is dynamic and results in a small number of protein-containing cells that correspond to sensillum precursors. extramacrochaete loss-of-function alleles develop extra sensilla and correspondingly display a larger number of cells with scute protein. These cells appear to arise from those that in the wild type already express scute RNA; hence, extramacrochaete is a repressor of scute function whose action may take place post-transcriptionally.

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MiR858b Inhibits Proanthocyanidin Accumulation by the Repression of DkMYB19 and DkMYB20 in Persimmon

Frontiers in Plant Science ◽

10.3389/fpls.2020.576378 ◽

2020 ◽

Vol 11 ◽

Author(s):

Sichao Yang ◽

Meng Zhang ◽

Liqing Xu ◽

Zhengrong Luo ◽

Qinglin Zhang

Keyword(s):

Expression Patterns ◽

Transient Transformation ◽

Structural Genes ◽

Wild Type ◽

Rna Ligase ◽

Key Factor ◽

R2r3 Myb ◽

Posttranscriptional Level

Persimmon proanthocyanidin (PA) biosynthesis is controlled by structural genes and regulated by transcription factors (TFs). MicroRNAs are a key factor involved in regulating gene expression at the posttranscriptional level whose functions in persimmon PA biosynthesis are poorly understood. Here, we identified a microRNA, miR858b, that putatively targets two R2R3-MYB TFs, DkMYB19 and DkMYB20. DkMYB19, DkMYB20, and miR858b showed divergent expression patterns during fruit development, and the interaction between miR858b and DkMYB19 or DkMYB20 was experimentally validated by 5′ RNA ligase-mediated RACE, LUC enzyme activity analysis, and GFP signal detection. The DkMYB19 localized to the nucleus as well as the cytoplasm and DkMYB20 localized to the nucleus. The overexpression of miR858b led to the downregulation of DkMYB19 and DkMYB20, which reduced the content of PA, whereas a reduction in miR858b activity upregulated DkMYB19 and DkMYB20, resulting in a high content of PA in leaves transiently expressing a small tandem target mimic construct for blocking miR858 (STTM858b) in vivo. The transient transformation of miR858b in fruit discs in vitro also reduced the content of PA, while the content of PA increased under the transient transformation of fruit discs with STTM858b, DkMYB19, or DkMYB20. A similar phenomenon was observed upon the overexpression of miR858b in wild-type (WT) Arabidopsis and DkMYB19 or DkMYB20 in persimmon leaf calli. These findings suggested that miR858b repressed the expression of DkMYB19 and DkMYB20, which contributed to the PA accumulation in persimmon.

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Melanocyte development in vivo and in neural crest cell cultures: crucial dependence on the Mitf basic-helix-loop-helix-zipper transcription factor

Development ◽

10.1242/dev.124.12.2377 ◽

1997 ◽

Vol 124 (12) ◽

pp. 2377-2386 ◽

Cited By ~ 10

Author(s):

K. Opdecamp ◽

A. Nakayama ◽

M.T. Nguyen ◽

C.A. Hodgkinson ◽

W.J. Pavan ◽

...

Keyword(s):

Neural Crest ◽

Cell Cultures ◽

Neural Crest Cell ◽

Tyrosine Kinase Receptor ◽

Wild Type ◽

Helix Loop Helix ◽

Migration Pathway ◽

Endothelin 3 ◽

Crest Cell

The more than 20 different Mitf mutations in the mouse are all associated with deficiencies in neural crest-derived melanocytes that range from minor functional disturbances with some alleles to complete absence of mature melanocytes with others. In the trunk region of wild-type embryos, Mitf-expressing cells that coexpressed the melanoblast marker Dct and the tyrosine kinase receptor Kit were found in the dorsolateral neural crest migration pathway. In contrast, in embryos homozygous for an Mitf allele encoding a non-functional Mitf protein, Mitf-expressing cells were extremely rare, no Dct expression was ever found, and the number of Kit-expressing cells was much reduced. Wild-type neural crest cell cultures rapidly gave rise to cells that expressed Mitf and coexpressed Kit and Dct. With time in culture, Kit expression was increased, and pigmented, dendritic cells developed. Addition of the Kit ligand Mgf or endothelin 3 or a combination of these factors all rapidly increased the number of Dct-positive cells. Cultures from Mitf mutant embryos initially displayed Mitf-positive cells similar in numbers and Kit-expression as did wild-type cultures. However, Kit expression did not increase with time in culture and the mutant cells never responded to Mgf or endothelin 3, did not express Dct, and never showed pigment. In fact, even Mitf expression was rapidly lost. The results suggest that Mitf first plays a role in promoting the transition of precursor cells to melanoblasts and subsequently, by influencing Kit expression, melanoblast survival.

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Cloning of hexokinase structural genes from Saccharomyces cerevisiae mutants with regulatory mutations responsible for glucose repression.

Molecular and Cellular Biology ◽

10.1128/mcb.5.11.3035 ◽

1985 ◽

Vol 5 (11) ◽

pp. 3035-3040 ◽

Cited By ~ 26

Author(s):

K D Entian ◽

F Hilberg ◽

H Opitz ◽

D Mecke

Keyword(s):

Structural Gene ◽

Glucose Repression ◽

Regulatory System ◽

Structural Genes ◽

Wild Type ◽

Tetrad Analysis ◽

Catalytic Center ◽

Regulatory Mutations ◽

Pii Proteins

The regulatory hexokinase PII mutants isolated previously (K.-D. Entian and K.-U. Fröhlich, J. Bacteriol. 158:29-35, 1984) were characterized further. These mutants were defective in glucose repression. The mutation was thought to be in the hexokinase PII structural gene, but it did not affect the catalytic activity of the enzyme. Hence, a regulatory domain for glucose repression was postulated. For further understanding of this regulatory system, the mutationally altered hexokinase PII proteins were isolated from five mutants obtained independently and characterized by their catalytic constants and bisubstrate kinetics. None of these characteristics differed from those of the wild type, so the catalytic center of the mutant enzymes remained unchanged. The only noticeable difference observed was that the in vivo modified form of hexokinase PII, PIIM, which has been described recently (K.-D. Entian and E. Kopetzki, Eur. J. Biochem. 146:657-662, 1985), was absent from one of these mutants. It is possible that the PIIM modification is directly connected with the triggering of glucose repression. To establish with certainty that the mutation is located in the hexokinase PII structural gene, the genes of these mutants were isolated after transforming a hexokinaseless mutant strain and selecting for concomitant complementation of the nuclear function. Unlike hexokinase PII wild-type transformants, glucose repression was not restored in the hexokinase PII mutant transformants. In addition mating experiments with these transformants followed by tetrad analysis of sporulated diploids gave clear evidence of allelism to the hexokinase PII structural gene.

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Selective Removal of Aberrant Extender Units by a Type II Thioesterase for Efficient FR-008/Candicidin Biosynthesis in Streptomyces sp. Strain FR-008

Applied and Environmental Microbiology ◽

10.1128/aem.01012-08 ◽

2008 ◽

Vol 74 (23) ◽

pp. 7235-7242 ◽

Cited By ~ 24

Author(s):

Yongjun Zhou ◽

Qingqing Meng ◽

Delin You ◽

Jialiang Li ◽

Shi Chen ◽

...

Keyword(s):

Biosynthetic Pathway ◽

Biochemical Analysis ◽

Wild Type Strain ◽

Type I ◽

Type Ii ◽

Wild Type ◽

Acyl Carrier Proteins ◽

Peptide Synthetase ◽

In The Wild

ABSTRACT Gene fscTE, encoding a putative type II thioesterase (TEII), was associated with the FR-008/candicidin gene cluster. Deletion of fscTE reduced approximately 90% of the FR-008/candicidin production, while the production level was well restored when fscTE was added back to the mutant in trans. FscTE was unable to compensate for the release of the maturely elongated polyketide as site-directed inactivation of the type I thioesterase (TEI) totally abolished FR-008/candicidin production. Direct biochemical analysis of FscTE in parallel with its homologue TylO from the tylosin biosynthetic pathway demonstrated their remarkable preferences for acyl-thioesters (i.e., propionyl-S-N-acetylcysteamine [SNAC] over methylmalonyl-SNAC and acetyl-SNAC over malonyl-SNAC) and thus concluded that TEII could maintain effective polyketide biosynthesis by selectively removing the nonelongatable residues bound to acyl carrier proteins. Overexpression of FscTE under the strong constitutive ermE*p promoter in the wild-type strain did not suppress FR-008/candicidin formation, which confirmed its substrate specificity in vivo. Furthermore, successful complementation of the fscTE mutant was obtained with fscTE and tylO, whereas no complementation was detected with nonribosomal peptide synthetase (NRPS) TEII tycF and srfAD, reflecting substrate specificities of TEIIs distinctive from those of either polyketide synthases or NRPSs.

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Interaction between opposing modes of phospho-regulation of the proneural proteins Ascl1 and Ngn2

Wellcome Open Research ◽

10.12688/wellcomeopenres.14848.1 ◽

2018 ◽

Vol 3 ◽

pp. 129

Author(s):

Laura J.A. Hardwick ◽

Anna Philpott

Keyword(s):

Inhibitory Effect ◽

Protein Activity ◽

Diverse Range ◽

Temporal Precision ◽

Temporal Regulation ◽

Activating Mutations ◽

Bhlh Domain ◽

Helix Loop Helix ◽

Progenitor Cell Proliferation

From the relatively simple nervous system of Drosophila to the elaborate mammalian cortex, neurogenesis requires exceptional spatial and temporal precision to co-ordinate progenitor cell proliferation and subsequent differentiation to a diverse range of neurons and glia. A limited number of transiently expressed proneural basic-helix-loop-helix (bHLH) transcription factors, for example achaete-scute-complex (as-c) and atonal (ato) in Drosophila and the vertebrate homologues Ascl1 and Neurogenin2 (Ngn2), are able to orchestrate the onset of neuronal determination, context-dependent subtype selection and even influence later aspects of neuronal migration and maturation. Within the last decade, two models have emerged to explain how the temporal activity of proneural determination factors is regulated by phosphorylation at distinct sites. One model describes how cell-cycle associated phosphorylation on multiple sites in the N and C termini of vertebrate proneural proteins limits neuronal differentiation in cycling progenitor cells. A second model describes phosphorylation on a single site in the bHLH domain of Drosophila atonal that acts as a binary switch, where phosphorylation terminates proneural activity. Here we combine activating mutations of phosphorylation sites in the N- and C- termini with an inhibitory phospho-mimetic mutation in the bHLH domain of Ascl1 and Ngn2 proteins, and test their functions in vivo using Xenopus embryos to determine which mode of phospho-regulation dominates. Enhancing activity by preventing N- and C terminal phosphorylation cannot overcome the inhibitory effect of mimicking phosphorylation of the bHLH domain. Thus we have established a hierarchy between these two modes of proneural protein control and suggest a model of temporal regulation for proneural protein activity.

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Binding site for p120/δ-catenin is not required for Drosophila E-cadherin function in vivo

The Journal of Cell Biology ◽

10.1083/jcb.200207160 ◽

2003 ◽

Vol 160 (3) ◽

pp. 313-319 ◽

Cited By ~ 61

Author(s):

Anne Pacquelet ◽

Li Lin ◽

Pernille Rørth

Keyword(s):

Cell Sorting ◽

Residual Activity ◽

Follicle Cell ◽

Wild Type ◽

Border Cell ◽

Epithelial Cadherin ◽

Classical Cadherins ◽

Dependent Processes

Homophilic cell adhesion mediated by classical cadherins is important for many developmental processes. Proteins that interact with the cytoplasmic domain of cadherin, in particular the catenins, are thought to regulate the strength and possibly the dynamics of adhesion. β-catenin links cadherin to the actin cytoskeleton via α-catenin. The role of p120/δ-catenin proteins in regulating cadherin function is less clear. Both β-catenin and p120/δ-catenin are conserved in Drosophila. Here, we address the importance of cadherin–catenin interactions in vivo, using mutant variants of Drosophila epithelial cadherin (DE-cadherin) that are selectively defective in p120ctn (DE-cadherin-AAA) or β-catenin–armadillo (DE-cadherin-Δβ) interactions. We have analyzed the ability of these proteins to substitute for endogenous DE-cadherin activity in multiple cadherin-dependent processes during Drosophila development and oogenesis; epithelial integrity, follicle cell sorting, oocyte positioning, as well as the dynamic adhesion required for border cell migration. As expected, DE-cadherin-Δβ did not substitute for DE-cadherin in these processes, although it retained some residual activity. Surprisingly, DE-cadherin-AAA was able to substitute for the wild-type protein in all contexts with no detectable perturbations. Thus, interaction with p120/δ-catenin does not appear to be required for DE-cadherin function in vivo.

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