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Chatterjee, I, Ibanez-Ventoso C, Vijay P, Singaravelu G, Baldi C, Bair J, Ng S, Smolyanskaya A, Driscoll M, Singson A.  2013.  Dramatic fertility decline in aging C. elegans males is associated with mating execution deficits rather than diminished sperm quality. Exp. Gerontol.. 48:1156–1166. Abstract
Although much is known about female reproductive aging, fairly little is known about the causes of male reproductive senescence. We developed a method that facilitates culture maintenance of Caenorhabditis elegans adult males, which enabled us to measure male fertility as populations age, without profound loss of males from the growth plate. We find that the ability of males to sire progeny declines rapidly in the first half of adult lifespan and we examined potential factors that contribute towards reproductive success, including physical vigor, sperm quality, mating apparatus morphology, and mating ability. Of these, we find little evidence of general physical decline in males or changes in sperm number, morphology, or capacity for activation, at time points when reproductive senescence is markedly evident. Rather, it is the loss of efficient mating ability that correlates most strongly with reproductive senescence. Low insulin signaling can extend male ability to sire progeny later in life, although insulin impact on individual facets of mating behavior is complex. Overall, we suggest that combined modest deficits, predominantly affecting the complex mating behavior rather than sperm quality, sum up to block effective C. elegans male reproduction in middle adult life.
Chatterjee, M, Tabi Z, Galli M, Malcomber S, Buck A, Muszynski M, Gallavotti A.  2014.  The boron efflux transporter ROTTEN EAR is required for maize inflorescence development and fertility. Plant Cell. (26):2962-2977. AbstractWebsite
Although boron has a relatively low natural abundance, it is an essential plant micronutrient. Boron deficiencies cause major crop losses in several areas of the world, affecting reproduction and yield in diverse plant species. Despite the importance of boron in crop productivity, surprisingly little is known about its effects on developing reproductive organs. We isolated a maize (Zea mays) mutant, called rotten ear (rte), that shows distinct defects in vegetative and reproductive development, eventually causing widespread sterility in its inflorescences, the tassel and the ear. Positional cloning revealed that rte encodes a membrane-localized boron efflux transporter, co-orthologous to the Arabidopsis thaliana BOR1 protein. Depending on the availability of boron in the soil, rte plants show a wide range of phenotypic defects that can be fully rescued by supplementing the soil with exogenous boric acid, indicating that rte is crucial for boron transport into aerial tissues. rte is expressed in cells surrounding the xylem in both vegetative and reproductive tissues and is required for meristem activity and organ development.We show that low boron supply to the inflorescences results in widespread defects in cell and cell wall integrity, highlighting the structural importance of boron in the formation of fully fertile reproductive organs.
Chaudhuri, S, Messing J.  1994.  Allele-specific parental imprinting of dzr1, a posttranscriptional regulator of zein accumulation. Proceedings of the National Academy of Sciences of the United States of America. 91:4867-71. AbstractWebsite
Parental imprinting describes the phenomenon of unequivalent gene function based on transmission from the female or male parent. We have discovered parental imprinting of an allele of the dzr1 locus that posttranscriptionally regulates the accumulation of 10-kDa zein in the maize endosperm. The imprinted allele of MO17 inbred origin, dzr1 + MO17, conditions low accumulation of the 10-kDa zein and is dominant when transmitted through the female but recessive when transmitted through the male. Analyzing endosperms with equal parental contributions of dzr1 + MO17 ruled out the possibility that the unequivalent phenotype of dzr1 + MO17 was due to parental dosage imbalance in the triploid endosperm. Second-generation studies show that the dominant or recessive phenotype of dzr1 + MO17 is determined at every generation based on immediate parental origin with no grandparental effect.
Chen, S, Gunasekera A, Zhang X, Kunkel TA, Ebright RH, Berman HM.  2001.  Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: alteration of DNA binding specificity through alteration of DNA kinking.. Journal of molecular biology. 314(1):75-82. Abstract
The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' ("primary kink"). CAP recognizes the base-pair immediately 5' to the primary-kink site, T:A(6), through an "indirect-readout" mechanism involving sequence effects on the energetics of primary-kink formation. CAP recognizes the base-pair immediately 3' to the primary-kink site, G:C(7), through a "direct-readout" mechanism involving formation of a hydrogen bond between Glu181 of CAP and G:C(7). Here, we report that substitution of the carboxylate side-chain of Glu181 of CAP by the one-methylene-group-shorter carboxylate side-chain of Asp changes DNA binding specificity at position 6 of the DNA half site, changing specificity for T:A(6) to specificity for C:G(6), and we report a crystallographic analysis defining the structural basis of the change in specificity. The Glu181-->Asp substitution eliminates the primary kink and thus eliminates indirect-readout-based specificity for T:A(6). The Glu181-->Asp substitution does not eliminate hydrogen-bond formation with G:C(7), and thus does not eliminate direct-readout-based specificity for G:C(7).
Chen, Y, Ebright RH.  1993.  Phenyl-azide-mediated photocrosslinking analysis of Cro-DNA interaction.. Journal of molecular biology. 230(2):453-60. Abstract
Using phenyl-azide-mediated photocrosslinking, we show that the alpha carbon of amino acid 2 of the helix-turn-helix motif of bacteriophage lambda Cro is within 12 A of the bottom-strand nucleotides at positions 2 and 3 of the DNA half site in the Cro-DNA complex in solution. This result is in excellent agreement with the crystallographic structure of the Cro-DNA complex. The results of phenyl-azide-mediated photocrosslinking analysis of Cro-DNA interaction, together with the previously reported results of phenyl-azide-mediated photocrosslinking analysis of CAP-DNA interaction, establish that phenyl-azide-mediated photocrosslinking is generalizable and provide information regarding the structural requirements for phenyl-azide-mediated photocrosslinking. Comparison of the results of phenyl-azide-mediated photocrosslinking to the results of EDTA: iron-mediated affinity cleaving indicates that phenyl-azide-mediated photocrosslinking yields superior resolution.
Chen, H, Tang H, Ebright RH.  2003.  Functional interaction between RNA polymerase alpha subunit C-terminal domain and sigma70 in UP-element- and activator-dependent transcription.. Molecular cell. 11(6):1621-33. Abstract
We show that the Escherichia coli RNA polymerase (RNAP) alpha subunit C-terminal domain (alphaCTD) functionally interacts with sigma(70) at a subset of UP-element- and activator-dependent promoters, we define the determinants of alphaCTD and sigma(70) required for the interaction, and we present a structural model for the interaction. The alphaCTD-sigma(70) interaction spans the upstream promoter and core promoter, thereby linking recognition of UP-elements and activators in the upstream promoter with recognition of the -35 element in the core promoter. We propose that the alphaCTD-sigma(70) interaction permits UP-elements and activators not only to "recruit" RNAP through direct interaction with alphaCTD, but also to "remodel" RNAP-core-promoter interaction through indirect, alphaCTD-bridged interactions with sigma(70).
Chen, Y, Ebright YW, Ebright RH.  1994.  Identification of the target of a transcription activator protein by protein-protein photocrosslinking.. Science (New York, N.Y.). 265(5168):90-2. Abstract
Here it is shown, with the use of protein-protein photocrosslinking, that the carboxyl-terminal region of the alpha subunit of RNA polymerase (RNAP) is in direct physical proximity to the activating region of the catabolite gene activator protein (CAP) in the ternary complex of the lac promoter, RNAP, and CAP. These results strongly support the proposal that transcription activation by CAP involves protein-protein contact between the carboxyl-terminal region of the alpha subunit and the activating region of CAP.
Chen, S, Vojtechovsky J, Parkinson GN, Ebright RH, Berman HM.  2001.  Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: DNA binding specificity based on energetics of DNA kinking.. Journal of molecular biology. 314(1):63-74. Abstract
The catabolite activator protein (CAP) makes no direct contact with the consensus base-pair T:A at position 6 of the DNA half-site 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' but, nevertheless, exhibits strong specificity for T:A at position 6. Binding of CAP results in formation of a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site. The consensus base-pair T:A at position 6 and the consensus base-pair G:C at position 7 form a T:A/G:C step, which is known to be associated with DNA flexibility. It has been proposed that specificity for T:A at position 6 is a consequence of formation of the DNA kink between positions 6 and 7, and of effects of the T:A(6)/G:C(7) step on the geometry of DNA kinking, or the energetics of DNA kinking. In this work, we determine crystallographic structures of CAP-DNA complexes having the consensus base-pair T:A at position 6 or the non-consensus base-pair C:G at position 6. We show that complexes containing T:A or C:G at position 6 exhibit similar overall DNA bend angles and local geometries of DNA kinking. We infer that indirect readout in this system does not involve differences in the geometry of DNA kinking but, rather, solely differences in the energetics of DNA kinking. We further infer that the main determinant of DNA conformation in this system is protein-DNA interaction, and not DNA sequence.
Cho, E, Feng Y, Rauskolb C, Maitra S, Fehon RG, Irvine KD.  2006.  Delineation of a Fat tumor suppressor pathway. Nature Genetics. 38:1142-50. AbstractWebsite
Recent studies in Drosophila melanogaster of the protocadherins Dachsous and Fat suggest that they act as ligand and receptor, respectively, for an intercellular signaling pathway that influences tissue polarity, growth and gene expression, but the basis for signaling downstream of Fat has remained unclear. Here, we characterize functional relationships among D. melanogaster tumor suppressors and identify the kinases Discs overgrown and Warts as components of a Fat signaling pathway. fat, discs overgrown and warts regulate a common set of downstream genes in multiple tissues. Genetic experiments position the action of discs overgrown upstream of the Fat pathway component dachs, whereas warts acts downstream of dachs. Warts protein coprecipitates with Dachs, and Warts protein levels are influenced by fat, dachs and discs overgrown in vivo, consistent with its placement as a downstream component of the pathway. The tumor suppressors Merlin, expanded, hippo, salvador and mob as tumor suppressor also share multiple Fat pathway phenotypes but regulate Warts activity independently. Our results functionally link what had been four disparate groups of D. melanogaster tumor suppressors, establish a basic framework for Fat signaling from receptor to transcription factor and implicate Warts as an integrator of multiple growth control signals.
Cho, E, Irvine KD.  2004.  Action of fat, four-jointed, dachsous and dachs in distal-to-proximal wing signaling. Development. 131:4489-500. AbstractWebsite
In the Drosophila wing, distal cells signal to proximal cells to induce the expression of Wingless, but the basis for this distal-to-proximal signaling is unknown. Here, we show that three genes that act together during the establishment of tissue polarity, fat, four-jointed and dachsous, also influence the expression of Wingless in the proximal wing. fat is required cell autonomously by proximal wing cells to repress Wingless expression, and misexpression of Wingless contributes to proximal wing overgrowth in fat mutant discs. Four-jointed and Dachsous can influence Wingless expression and Fat localization non-autonomously, consistent with the suggestion that they influence signaling to Fat-expressing cells. We also identify dachs as a gene that is genetically required downstream of fat, both for its effects on imaginal disc growth and for the expression of Wingless in the proximal wing. Our observations provide important support for the emerging view that Four-jointed, Dachsous and Fat function in an intercellular signaling pathway, identify a normal role for these proteins in signaling interactions that regulate growth and patterning of the proximal wing, and identify Dachs as a candidate downstream effector of a Fat signaling pathway.
Clark, RM, Linton E, Messing J, Doebley JF.  2004.  Pattern of diversity in the genomic region near the maize domestication gene tb1. Proceedings of the National Academy of Sciences of the United States of America. 101:700-7. AbstractWebsite
Domesticated maize and its wild ancestor (teosinte) differ strikingly in morphology and afford an opportunity to examine the connection between strong selection and diversity in a major crop species. The tb1 gene largely controls the increase in apical dominance in maize relative to teosinte, and a region of the tb1 locus 5' to the transcript sequence was a target of selection during maize domestication. To better characterize the impact of selection at a major "domestication" locus, we have sequenced the upstream tb1 genomic region and systematically sampled nucleotide diversity for sites located as far as 163 kb upstream to tb1. Our analyses define a selective sweep of approximately 60-90 kb 5' to the tb1 transcribed sequence. The selected region harbors a mixture of unique sequences and large repetitive elements, but it contains no predicted genes. Diversity at the nearest 5' gene to tb1 is typical of that for neutral maize loci, indicating that selection at tb1 has had a minimal impact on the surrounding chromosomal region. Our data also show low intergenic linkage disequilibrium in the region and suggest that selection has had a minor role in shaping the pattern of linkage disequilibrium that is observed. Finally, our data raise the possibility that maize-like tb1 haplotypes are present in extant teosinte populations, and our findings also suggest a model of tb1 gene regulation that differs from traditional views of how plant gene expression is controlled.
Codelia, VA, Irvine KD.  2012.  Hippo Signaling Goes Long Range. Cell. 150:669-670.Website
Codelia, V, Sun G, Irvine KD.  2014.  Regulation of YAP by Mechanical Strain through Jnk and Hippo Signaling. Current Biology. 24:2012-2017.Website
Colavita, A, Krishna S, Zheng H, Padgett RW, Culotti JG.  1998.  Pioneer axon guidance by UNC-129, a C. elegans TGF-β. Science (New York, NY). 281:706-9. AbstractWebsite
The unc-129 gene, like the unc-6 netrin gene, is required to guide pioneer motoraxons along the dorsoventral axis of Caenorhabditis elegans. unc-129 encodes a member of the transforming growth factor-beta (TGF-beta) superfamily of secreted signaling molecules and is expressed in dorsal, but not ventral, rows of body wall muscles. Ectopic expression of UNC-129 from ventral body wall muscle disrupts growth cone and cell migrations that normally occur along the dorsoventral axis. Thus, UNC-129 mediates expression of dorsoventral polarity information required for axon guidance and guided cell migrations in C. elegans.
Colombie, N, Cullen CF, Brittle AL, Jang JK, Earnshaw WC, Carmena M, McKim K, Ohkura H.  2008.  Dual roles of Incenp crucial to the assembly of the acentrosomal metaphase spindle in female meiosis. Development. 135:3239-46.Website
Correia, T, Papayannopoulos V, Panin V, Woronoff P, Jiang J, Vogt TF, Irvine KD.  2003.  Molecular genetic analysis of the glycosyltransferase Fringe in Drosophila. Proceedings of the National Academy of Sciences of the United States of America. 100:6404-9. AbstractWebsite
Fringe proteins are beta1,3-N-acetylglucosaminyltransferases that modulate signaling through Notch receptors by modifying O-linked fucose on epidermal growth factor domains. Fringe is highly conserved, and comparison among 18 different Fringe proteins from 11 different species identifies a core set of 84 amino acids that are identical among all Fringes. Fringe is only distantly related to other glycosyltransferases, but analysis of the predicted Drosophila proteome identifies a set of four sequence motifs shared among Fringe and other putative beta1,3-glycosyltransferases. To gain functional insight into these conserved sequences, we genetically and molecularly characterized 14 point mutations in Drosophila fringe. Most nonsense mutations act as recessive antimorphs, raising the possibility that Fringe may function as a dimer. Missense mutations identify two distinct motifs that are conserved among beta1,3-glycosyltransferases, and that can be modeled onto key motifs in the crystallographic structures of bovine beta1,4-galactosyltransferase 1 and human glucuronyltransferase I. Other missense mutations map to amino acids that are conserved among Fringe proteins, but not among other glycosyltransferases, and thus may identify structural motifs that are required for unique aspects of Fringe activity.
Cowperthwaite, M, Park W, Xu Z, Yan X, Maurais SC, Dooner HK.  2002.  Use of the transposon Ac as a gene-searching engine in the maize genome. Plant Cell. 14:713–726. AbstractWebsite
1300 independent Ac transposants. The majority of transposed Ac elements are linked to either the bz or the wx donor loci on chromosome 9. A few of the insertions produce obvious visible phenotypes, but most of them do not, suggesting that these populations will be more useful for reverse genetics than for forward transposon mutagenesis. An inverse polymerase chain reaction method was adapted for the isolation of DNA adjacent to the transposed Ac elements (tac sites). Most Ac insertions were into unique DNA. By sequencing tac sites and comparing the sequences to existing databases, insertions were identified in a number of putative maize genes. The expression of most of these genes was confirmed by RNA gel blot analysis. We report here the isolation and characterization of the first 46 tac sites from the two insertion libraries.
Cruz, JW, Sharp JD, Hoffer ED, Maehigashi T, Vvedenskaya IO, Konkimalla A, Husson RN, Nickels BE, Dunham C, Woychik NA.  2015.  Growth-regulating Mycobacterium tuberculosis VapC-mt4 toxin is an isoacceptor-specific tRNase. Nat Commun. 6:7480.
Cruz-Alvarez, M, Kirihara JA, Messing J.  1991.  Post-transcriptional regulation of methionine content in maize kernels. Molecular & general genetics : MGG. 225:331-9. AbstractWebsite
Message levels for a methionine-rich 10 kDa zein were determined in three inbred lines of maize and their reciprocal crosses at various stages during endosperm development. Inbred line BSSS-53, which overexpresses the 10 kDa protein in mature kernels, was shown to have higher mRNA levels in developing endosperm, as compared to inbred lines W23 and W64A. Differences in mRNA levels could not be explained by differences in transcription rate of the 10 kDa zein gene, indicating differential post-transcriptional regulation of this storage protein in the different inbred lines analyzed. Among progeny segregating for the BSSS-53 allele of the 10 kDa zein structural gene Zps10/(22), mRNA levels are independent of Zps10/(22) segregation, indicating that post-transcriptional regulation of mRNA levels takes place via a trans-acting mechanism. In the same progeny, mRNA levels are also independent of allelic segregation of the regulatory locus Zpr10/(22). Thus, the trans-acting factor encoded by Zpr10/(22) determines accumulation of 10 kDa zein at a translational or post-translational step. Multiple trans-acting factors are therefore involved in post-transcriptional regulation of the methionine-rich 10 kDa zein.
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Das, P, Maduzia LL, Padgett RW.  1999.  Genetic approaches to TGFβ signaling pathways. Cytokine & growth factor reviews. 10:179-86.Website
Das, A, Shah SJ, Fan B, Paik D, DiSanto DJ, Hinman A M, Cesario JM, Battaglia RA, Demos N, McKim KS.  2016.  Spindle Assembly and Chromosome Segregation Requires Central Spindle Proteins in Drosophila Oocytes.. Genetics. 202(1):61-75. Abstract
Oocytes segregate chromosomes in the absence of centrosomes. In this situation, the chromosomes direct spindle assembly. It is still unclear in this system which factors are required for homologous chromosome bi-orientation and spindle assembly. The Drosophila kinesin-6 protein Subito, although nonessential for mitotic spindle assembly, is required to organize a bipolar meiotic spindle and chromosome bi-orientation in oocytes. Along with the chromosomal passenger complex (CPC), Subito is an important part of the metaphase I central spindle. In this study we have conducted genetic screens to identify genes that interact with subito or the CPC component Incenp. In addition, the meiotic mutant phenotype for some of the genes identified in these screens were characterized. We show, in part through the use of a heat-shock-inducible system, that the Centralspindlin component RacGAP50C and downstream regulators of cytokinesis Rho1, Sticky, and RhoGEF2 are required for homologous chromosome bi-orientation in metaphase I oocytes. This suggests a novel function for proteins normally involved in mitotic cell division in the regulation of microtubule-chromosome interactions. We also show that the kinetochore protein, Polo kinase, is required for maintaining chromosome alignment and spindle organization in metaphase I oocytes. In combination our results support a model where the meiotic central spindle and associated proteins are essential for acentrosomal chromosome segregation.
Das, OP, Messing J.  1994.  Variegated phenotype and developmental methylation changes of a maize allele originating from epimutation. Genetics. 136:1121-41. AbstractWebsite
Two instances of genetic transmission of spontaneous epimutation of the maize P-rr gene were identified. Transmission gave rise to two similar, moderately stable alleles, designated P-pr-1 and P-pr-2, that exhibited Mendelian behavior. Both isolates of P-pr conditioned a variable and variegated phenotype, unlike the uniform pigmentation conditioned by P-rr. Extensive genomic analysis failed to reveal insertions, deletions or restriction site polymorphisms between the new allele and its progenitor. However, methylation of the P gene was increased in P-pr relative to P-rr, and was greatly reduced (though not lost) in a revertant to uniform pigmentation. Variability in pigmentation conditioned by P-pr correlated with variability in transcript levels of the P gene, and both correlated inversely with variability in its methylation. Part of the variability in methylation could be accounted for by a developmental decrease in methylation in all tissues of plants carrying P-pr. We hypothesize that the variegated phenotype results from a general epigenetic pathway which causes a progressive decrease in methylation and increase in expression potential of the P gene as a function of cell divisions in each meristem of the plant. This renders all tissues chimeric for a functional gene; chimerism is visualized as variegation only in pericarp due to the tissue specificity of P gene expression. Therefore, this allele that originates from epimutation may exemplify an epigenetic mechanism for variegation in maize.
Das, P, Maduzia LL, Wang H, Finelli AL, Cho SH, Smith MM, Padgett RW.  1998.  The Drosophila gene Medea demonstrates the requirement for different classes of Smads in dpp signaling. Development (Cambridge, England). 125:1519-28. AbstractWebsite
Signals from transforming growth factor-beta (TGF-beta) ligands are transmitted within the cell by members of the Smad family, which can be grouped into three classes based on sequence similarities. Our previous identification of both class I and II Smads functioning in a single pathway in C. elegans, raised the issue of whether the requirement for Smads derived from different classes is a general feature of TGF-beta signaling. We report here the identification of a new Drosophila class II Smad, Medea, a close homolog of the human tumor-suppressor gene DPC4. Embryos from germline clones of both Medea and Mad (a class I Smad) are ventralized, as are embryos null for the TGF-beta-like ligand decapentaplegic (dpp). Loss of Medea also blocks dpp signaling during later development, suggesting that Medea, like Mad, is universally required for dpp signaling. Furthermore, we show that the necessity for these two closely related, non-redundant Smads, is due to their different signaling properties - upon activation of the Dpp pathway, Mad is required to actively translocate Medea into the nucleus. These results provide a paradigm for, and distinguish between, the requirement for class I and II Smads in Dpp/BMP signaling.
Dasgupta, J, Tyryshkin AM, Kozlov YN, Klimov VV, Dismukes CG.  2006.  Carbonate Complexation of Mn2+ in the Aqueous Phase:  Redox Behavior and Ligand Binding Modes by Electrochemistry and EPR Spectroscopy. The Journal of Physical Chemistry B. 110:5099-5111. AbstractWebsite
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Dasgupta, J, Ananyev GM, Dismukes GC.  2008.  Photoassembly of the water-oxidizing complex in photosystem II. Coordination Chemistry Reviews. 252:347-360. AbstractWebsite
The light-driven steps in the biogenesis and repair of the inorganic core comprising the O-2-evolving center of oxygenic photosynthesis (photosystem II water-oxidation complex, PSII-WOC) are reviewed. These steps, known collectively as photoactivation, involve the photoassembly of the free inorganic cofactors to the cofactor-depleted PSII-(apo-WOC) driven by light and produce the active O-2-evolving core comprised of Mn4CaOxCly. We focus on the functional role of the inorganic components as seen through the competition with non-native cofactors ("inorganic mutants") on water oxidation activity, the rate of the photoassembly reaction, and on structural insights gained from EPR spectroscopy of trapped intermediates formed in the initial steps of the assembly reaction. A chemical mechanism for the initial steps in photoactivation is given that is based on these data. Photoactivation experiments offer the powerful insights gained from replacement of the native cofactors, which together with the recent X-ray structural data for the resting holoenzyme provide a deeper understanding of the chemistry of water oxidation. We also review some new directions in research that photoactivation studies have inspired that look at the evolutionary history of this remarkable catalyst. (c) 2007 Elsevier B.V. All rights reserved.