Journal Article
Ge, Z, Bergonci T, Zhao Y, Zou Y, Du S, Liu M-C, Luo X, Ruan H, García-Valencia LE, Zhong S et al..  2017.  <em>Arabidopsis</em> pollen tube integrity and sperm release are regulated by RALF-mediated signaling. Science. 358(6370):1596. AbstractWebsite
In plants, sperm cells travel through the pollen tube as it grows toward the ovule. Successful fertilization depends on the pollen tube rupturing to release the sperm cells (see the Perspective by Stegmann and Zipfel). Ge et al. and Mecchia et al. elucidated the intercellular cross-talk that maintains pollen tube integrity during growth but destroys it at just the right moment. The signaling peptides RALF4 and RALF19, derived from the pollen tube, maintain its integrity as it grows. Once in reach of the ovule, a related signaling peptide, RALF34, which derives from female tissues, takes over and causes rupture of the pollen tube.Science, this issue p. 1596, p. 1600; see also p. 1544In flowering plants, fertilization requires complex cell-to-cell communication events between the pollen tube and the female reproductive tissues, which are controlled by extracellular signaling molecules interacting with receptors at the pollen tube surface. We found that two such receptors in Arabidopsis, BUPS1 and BUPS2, and their peptide ligands, RALF4 and RALF19, are pollen tube–expressed and are required to maintain pollen tube integrity. BUPS1 and BUPS2 interact with receptors ANXUR1 and ANXUR2 via their ectodomains, and both sets of receptors bind RALF4 and RALF19. These receptor-ligand interactions are in competition with the female-derived ligand RALF34, which induces pollen tube bursting at nanomolar concentrations. We propose that RALF34 replaces RALF4 and RALF19 at the interface of pollen tube–female gametophyte contact, thereby deregulating BUPS-ANXUR signaling and in turn leading to pollen tube rupture and sperm release.
Bolot, S, Abrouk M, Masood-Quraishi U, Stein N, Messing J, Feuillet C, Salse J.  2009.  The 'inner circle' of the cereal genomes. Curr Opin Plant Biol. 12:119-25. AbstractWebsite
Early marker-based macrocolinearity studies between the grass genomes led to arranging their chromosomes into concentric 'crop circles' of synteny blocks that initially consisted of 30 rice-independent linkage groups representing the ancestral cereal genome structure. Recently, increased marker density and genome sequencing of several cereal genomes allowed the characterization of intragenomic duplications and their integration with intergenomic colinearity data to identify paleo-duplications and propose a model for the evolution of the grass genomes from a common ancestor. On the basis of these data an 'inner circle' comprising five ancestral chromosomes was defined providing a new reference for the grass chromosomes and new insights into their ancestral relationships and origin, as well as an efficient tool to design cross-genome markers for genetic studies.
Jiao, X, Doamekpor S, Bird JG, Nickels BE, Tong L, Hart RP, Kiledjian M.  2017.  5′-end Nicotinamide Adenine Dinucleotide cap in human cells promotes RNA decay through DXO-mediated deNADding.. Cell. 168(6):1015-1027.
McKim, KS, Jang JK, Sekelsky JJ, Laurencon A, Hawley RS.  2000.  mei-41 is required for precocious anaphase in Drosophila females. Chromosoma. 109:44-49.
Hari, KL, Santerre A, Sekelsky JJ, McKim KS, Boyd JB, Hawley RS.  1995.  The mei-41 gene of D. melanogaster is a structural and function homolog of the human ataxia telangiectasia gene. Cell. 82:815-821.
McKim, KS, Hayashi-Hagihara A.  1998.  mei-W68 in Drosophila melanogaster encodes a Spo11 homolog: evidence that the mechanism for initiating meiotic recombination is conserved. Genes & Dev.. 12:2932-42. AbstractWebsite
Meiotic recombination requires the action of several gene products in both Saccharomyces cerevisiae and Drosophila melanogaster. Genetic studies in D. melanogaster have shown that the mei-W68 gene is required for all meiotic gene conversion and crossing-over. We cloned mei-W68 using a new genetic mapping method in which P elements are used to promote crossing-over at their insertion sites. This resulted in the high-resolution mapping of mei-W68 to a 18-kb region that contains a homolog of the S. cerevisiae spo11 gene. Molecular analysis of several mutants confirmed that mei-W68 encodes an spo11 homolog. Spo11 and MEI- W68 are members of a family of proteins similar to a novel type II topoisomerase. On the basis of this and other lines of evidence, Spo11 has been proposed to be the enzymatic activity that creates the double- strand breaks needed to initiate meiotic recombination. This raises the possibility that recombination in Drosophila is also initiated by double-strand breaks. Although these homologous genes are required absolutely for recombination in both species, their roles differ in other respects. In contrast to spo11, mei-W68 is not required for synaptonemal complex formation and does have a mitotic role.
Giunta, KL, Jang JK, Manheim EM, Subramanian G, McKim KS.  2002.  subito encodes a kinesin-like protein required for meiotic spindle pole formation in Drosophila melanogaster. Genetics. 160:1489-1501.
Revyakin, A, Liu C, Ebright RH, Strick TR.  2006.  Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching.. Science (New York, N.Y.). 314(5802):1139-43. Abstract
Using single-molecule DNA nanomanipulation, we show that abortive initiation involves DNA "scrunching"--in which RNA polymerase (RNAP) remains stationary and unwinds and pulls downstream DNA into itself--and that scrunching requires RNA synthesis and depends on RNA length. We show further that promoter escape involves scrunching, and that scrunching occurs in most or all instances of promoter escape. Our results support the existence of an obligatory stressed intermediate, with approximately one turn of additional DNA unwinding, in escape and are consistent with the proposal that stress in this intermediate provides the driving force to break RNAP-promoter and RNAP-initiation-factor interactions in escape.
Lee, N K, Kapanidis AN, Wang Y, Michalet X, Mukhopadhyay J, Ebright RH, Weiss S.  2005.  Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation.. Biophysical journal. 88(4):2939-53. Abstract
Fluorescence resonance energy transfer (FRET) between a donor (D) and an acceptor (A) at the single-molecule level currently provides qualitative information about distance, and quantitative information about kinetics of distance changes. Here, we used the sorting ability of confocal microscopy equipped with alternating-laser excitation (ALEX) to measure accurate FRET efficiencies and distances from single molecules, using corrections that account for cross-talk terms that contaminate the FRET-induced signal, and for differences in the detection efficiency and quantum yield of the probes. ALEX yields accurate FRET independent of instrumental factors, such as excitation intensity or detector alignment. Using DNA fragments, we showed that ALEX-based distances agree well with predictions from a cylindrical model of DNA; ALEX-based distances fit better to theory than distances obtained at the ensemble level. Distance measurements within transcription complexes agreed well with ensemble-FRET measurements, and with structural models based on ensemble-FRET and x-ray crystallography. ALEX can benefit structural analysis of biomolecules, especially when such molecules are inaccessible to conventional structural methods due to heterogeneity or transient nature.
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.
Severinov, K, Nair S.  2012.  The action of microcin C and mechanisms of bacterial resistance to it. Future Microbiol. 7:281-289.
Tan, Q, Linask KL, Ebright RH, Woychik NA.  2000.  Activation mutants in yeast RNA polymerase II subunit RPB3 provide evidence for a structurally conserved surface required for activation in eukaryotes and bacteria.. Genes & development. 14(3):339-48. Abstract
We have identified a mutant in RPB3, the third-largest subunit of yeast RNA polymerase II, that is defective in activator-dependent transcription, but not defective in activator-independent, basal transcription. The mutant contains two amino-acid substitutions, C92R and A159G, that are both required for pronounced defects in activator-dependent transcription. Synthetic enhancement of phenotypes of C92R and A159G, and of several other pairs of substitutions, is consistent with a functional relationship between residues 92-95 and 159-161. Homology modeling of RPB3 on the basis of the crystallographic structure of alphaNTD indicates that residues 92-95 and 159-162 are likely to be adjacent within the structure of RPB3. In addition, homology modeling indicates that the location of residues 159-162 within RPB3 corresponds to the location of an activation target within alphaNTD (the target of activating region 2 of catabolite activator protein, an activation target involved in a protein-protein interaction that facilitates isomerization of the RNA polymerase promoter closed complex to the RNA polymerase promoter open complex). The apparent finding of a conserved surface required for activation in eukaryotes and bacteria raises the possibility of conserved mechanisms of activation in eukaryotes and bacteria.
Charych, EI, Akum BF, Goldberg JS, Jornsten RJ, Rongo C, Zheng JQ, Firestein BL.  2006.  Activity-independent regulation of dendrite patterning by postsynaptic density protein PSD-95. J Neurosci. 26:10164-76. AbstractWebsite
Dendritic morphology determines many aspects of neuronal function, including action potential propagation and information processing. However, the question remains as to how distinct neuronal dendrite branching patterns are established. Here, we report that postsynaptic density-95 (PSD-95), a protein involved in dendritic spine maturation and clustering of synaptic signaling proteins, plays a novel role in regulating dendrite outgrowth and branching, independent of its synaptic functions. In immature neurons, overexpression of PSD-95 decreases the proportion of primary dendrites that undergo additional branching, resulting in a marked reduction of secondary dendrite number. Conversely, knocking down PSD-95 protein in immature neurons increases secondary dendrite number. The effect of PSD-95 is activity-independent and is antagonized by cypin, a nonsynaptic protein that regulates PSD-95 localization. Binding of cypin to PSD-95 correlates with formation of stable dendrite branches. Finally, overexpression of PSD-95 in COS-7 cells disrupts microtubule organization, indicating that PSD-95 may modulate microtubules to regulate dendritic branching. Whereas many factors have been identified which regulate dendrite number, our findings provide direct evidence that proteins primarily involved in synaptic functions can also play developmental roles in shaping how a neuron patterns its dendrite branches.
Walker, SS, Degen D, Nickbarg E, Carr D, Soriano A, Mandal M B, Painter RE, Sheth PR, Xiao L, Sher X et al..  2017.  Affinity selection-mass spectrometry identifies a novel antibacterial RNA polymerase inhibitor.. ACS Chemical Biology. 12:1346-1352. Abstract
The growing prevalence of drug-resistant Gram-negative bacteria is a significant global threat to human health. Rifampicin, an RNA polymerase-targeting agent, is an important part of the antibacterial armamentarium; however the emergence of resistance requires that it be used against only certain infections and usually in combination with another antibiotic. While rifampicin has significant clinical limitations, it does show that bacterial RNA polymerase can be an effective target for antibacterial intervention. To find new RNA polymerase inhibitors we initiated a screen of a collection of antibacterial bioactive molecules using affinity selection-mass spectrometry and purified Escherichia coli core RNA polymerase (subunits α, β, β', ω). Affinity selection screening identified a novel small molecule, MRL-436, that binds selectively to and inhibits RNA polymerase in vitro and inhibits RNA synthesis in the cell. Selection for resistance followed by whole genome sequencing identified a missense mutation in rpoC (β' subunit) and, separately, frameshift mutations in rpoZ (ω subunit) suggesting that MRL-436 targets RNA polymerase in the cell. In addition, cells lacking the rpoZ gene or purified RNA polymerase containing either a specific substitution in β' or lacking ω are selectively resistant to MRL-436. Molecular modeling indicates that the location of the substitution mutation in β' is closely juxtaposed with ω in a region of the complex thought to be important for transcription regulation during cellular response to amino acid starvation.
Sun, G, Irvine KD.  2013.  Ajuba Family Proteins Link JNK to Hippo Signaling.. Science signaling. 6:ra81. AbstractWebsite
Wounding, apoptosis, or infection can trigger a proliferative response in neighboring cells to replace damaged tissue. Studies in Drosophila have implicated c-Jun amino-terminal kinase (JNK)-dependent activation of Yorkie (Yki) as essential to regeneration-associated growth, as well as growth associated with neoplastic tumors. Yki is a transcriptional coactivator that is inhibited by Hippo signaling, a conserved pathway that regulates growth. We identified a conserved mechanism by which JNK regulated Hippo signaling. Genetic studies in Drosophila identified Jub (also known as Ajuba LIM protein) as required for JNK-mediated activation of Yki and showed that Jub contributed to wing regeneration after wounding and to tumor growth. Biochemical studies revealed that JNK promoted the phosphorylation of Ajuba family proteins in both Drosophila and mammalian cells. Binding studies in mammalian cells indicated that JNK increased binding between the Ajuba family proteins LIMD1 or WTIP and LATS1, a kinase within the Hippo pathway that inhibits the Yki homolog YAP. Moreover, JNK promoted binding of LIMD1 and LATS1 through direct phosphorylation of LIMD1. These results identify Ajuba family proteins as a conserved link between JNK and Hippo signaling, and imply that JNK increases Yki and YAP activity by promoting the binding of Ajuba family proteins to Warts and LATS.
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.
Carr, EA, Mead J, Vershon AK.  2004.  Alpha1-induced DNa Bending is Required for Transcriptional Activation by the Mcm1-alpha1 Complex. Nucleic Acids Res. 32:2298-2305. Abstract
The yeast Mcm1 protein is a founding member of the MADS-box family of transcription factors that is involved in the regulation of diverse sets of genes through interactions with distinct cofactor proteins. Mcm1 interacts with the Matalpha1 protein to activate the expression of the alpha-cell type-specific genes. To understand the requirement of the cofactor alpha1 for Mcm1-alpha1-dependent transcriptional activation we analyzed the recruitment of Mcm1 to the promoters of alpha-specific genes in vivo and found that Mcm1 is able to bind to the promoters of alpha-specific genes in the absence of alpha1. This suggests the function of alpha1 is more complex than simply recruiting Mcm1. Several MADS-box transcription factors, including Mcm1, induce DNA bending and there is evidence the proper bend may be required for transcriptional activation. We analyzed Mcm1-dependent bending of a Mcm1-alpha1 binding site in the presence and absence of alpha1 and found that Mcm1 alone shows a reduced DNA-bend at this site compared with other Mcm1 binding sites. However, the addition of alpha1 markedly increases the DNA-bend and we present evidence this bend is required for full transcriptional activation. These results support a model in which proper DNA-bending by the Mcm1-alpha1 complex is required for transcriptional activation.
Szeto, L, Fafalios MK, Zhong H, Vershon AK, Broach JR.  1997.  Alpha2p Controls Donor Preference During Mating type Interconversion in Yeast by Inactivating a Recombinational Enhancer of Chromosome IIi. Genes Dev. 11:1899-1911. Abstract
Homothallic strains of Saccharomyces cerevisiae can change mating type as often as every generation by replacing the allele at the MAT locus with a copy of mating type information present at one of two storage loci, HML and HMR, located on either end of chromosome III. Selection of the appropriate donor locus is dictated by a mating type-specific repressor protein, alpha2p: Cells containing alpha2p select HMR, whereas those lacking alpha2p select HML. As a repressor protein, alpha2p binds to DNA cooperatively with the transcriptional activator Mcm1p. Here we show that two alpha2p/Mcm1p-binding sites, DPS1 and DPS2, control donor selection. DPS1 and DPS2 are located approximately 30 kb from the left arm of chromosome III, well removed from HML, HMR, and MAT. Precise deletion of only DPS1 and DPS2 results in random selection of donor loci and in a cells without affecting selection in alpha cells. Reciprocally, deletion of only the alpha2p binding segments in each of these two sites results in selection of the wrong donor loci in alpha cells without affecting preference in a cells. These results suggest that Mcm1p, bound to these two sites in the absence of alpha2p, activates HML as donor. Binding of alpha2p blocks the ability of Mcm1p bound to DPS1 and DPS2 to activate HML, resulting in default selection of HMR as donor. DPS1 and DPS2 also regulate expression of several noncoding RNAs, although deletion of at least one of these RNA loci does not affect donor preference. This suggests that transcriptional activation, rather than transcription of a specific product, is the initiating event in activating the left arm of chromosome III for donor selection.
Xu, Y, Guerra TL, Li Z, Ludwig M, Dismukes CG, Bryant DA.  2013.  Altered carbohydrate metabolism in glycogen synthase mutants of Synechococcus sp. strain PCC 7002: Cell factories for soluble sugars.. Metabolic engineering. 16:56-67. Abstract
Glycogen and compatible solutes are the major polymeric and soluble carbohydrates in cyanobacteria and function as energy reserves and osmoprotectants, respectively. Glycogen synthase null mutants (glgA-I glgA-II) were constructed in the cyanobacterium Synechococcus sp. strain PCC 7002. Under standard conditions the double mutant produced no glycogen and more soluble sugars. When grown under hypersaline conditions, the glgA-I glgA-II mutant accumulated 1.8-fold more soluble sugars (sucrose and glucosylglycer-(ol/ate)) than WT, and these cells spontaneously excreted soluble sugars into the medium at high levels without the need for additional transporters. An average of 27% more soluble sugars was released from the glgA-I glgA-II mutant than WT by hypo-osmotic shock. Extracellular vesicles budding from the outer membrane were observed by transmission electron microscopy in glgA-I glgA-II cells grown under hypersaline conditions. The glgA-I glgA-II mutant serves as a starting point for developing cell factories for photosynthetic production and excretion of sugars.
Mathias, JR, Zhong H, Jin Y, Vershon AK.  2001.  Altering the DNA-binding Specificity of the Yeast Matalpha 2 Homeodomain Protein. J Biol Chem. 276:32696-32703. Abstract
Homeodomain proteins are a highly conserved class of DNA-binding proteins that are found in virtually every eukaryotic organism. The conserved mechanism that these proteins use to bind DNA suggests that there may be at least a partial DNA recognition code for this class of proteins. To test this idea, we have investigated the sequence-specific requirements for DNA binding and repression by the yeast alpha2 homeodomain protein in association with its cofactors, Mcm1 and Mata1. We have determined the contribution for each residue in the alpha2 homeodomain that contacts the DNA in the co-crystal structures of the protein. We have also engineered mutants in the alpha2 homeodomain to alter the DNA-binding specificity of the protein. Although we were unable to change the specificity of alpha2 by making substitutions at residues 47, 54, and 55, we were able to alter the DNA-binding specificity by making substitutions at residue 50 in the homeodomain. Since other homeodomain proteins show similar changes in specificity with substitutions at residue 50, this suggests that there is at least a partial DNA recognition code at this position.
Llaca, V, Messing J.  1998.  Amplicons of maize zein genes are conserved within genic but expanded and constricted in intergenic regions. The Plant journal : for cell and molecular biology. 15:211-20. AbstractWebsite
The 78,101 base pair long sequence of a cluster of 22-kDa alpha zein genes in the maize inbred BSSS53 was determined. Each zein gene is contained within a repeat unit that varies in length. If such a repeat, or amplicon, is aligned along the entire sequence, a 10.5-fold sequence amplification is delineated. Because of insertions and deletions in intergenic regions, many of the zein genes are spaced over different distances. Only three out of 10 zein-related sequences have an intact open reading frame, indicating an unusual large number of genes unable to contribute to the accumulation of normal-size 22-kDa zein proteins. It is proposed that the seven remaining zein-related sequences be considered gene reserves because of their potential to be restored by gene conversion. Intergenic insertions in the cluster range from 1098 to 14,896 base pairs. Although they are composed of transposable element sequences, they also contain additional open reading frames, two of them showing homology to rice cDNA sequences. The average amplicon is 4423 base pairs long, with the sequence surrounding each zein gene more than 90% conserved. Coincidently, the size of the amplicon is equivalent to the average gene density (one gene within 4640 bp) in the Arabidopsis thaliana genome, one of the smallest in plants. Successive steps of amplification and insertion of DNA might explain to a certain degree how genome size variation has been generated in plants.
Xu, JH, Messing J.  2009.  Amplification of prolamin storage protein genes in different subfamilies of the Poaceae. Theor Appl Genet. AbstractWebsite
Prolamins are seed storage proteins in cereals and represent an important source of essential amino acids for feed and food. Genes encoding these proteins resulted from dispersed and tandem amplification. While previous studies have concentrated on protein sequences from different grass species, we now can add a new perspective to their relationships by asking how their genes are shared by ancestry and copied in different lineages of the same family of species. These differences are derived from alignment of chromosomal regions, where collinearity is used to identify prolamin genes in syntenic positions, also called orthologous gene copies. New or paralogous gene copies are inserted in tandem or new locations of the same genome. More importantly, one can detect the loss of older genes. We analyzed chromosomal intervals containing prolamin genes from rice, sorghum, wheat, barley, and Brachypodium, representing different subfamilies of the Poaceae. The Poaceae commonly known as the grasses includes three major subfamilies, the Ehrhartoideae (rice), Pooideae (wheat, barley, and Brachypodium), and Panicoideae (millets, maize, sorghum, and switchgrass). Based on chromosomal position and sequence divergence, it becomes possible to infer the order of gene amplification events. Furthermore, the loss of older genes in different subfamilies seems to permit a faster pace of divergence of paralogous genes. Change in protein structure affects their physical properties, subcellular location, and amino acid composition. On the other hand, regulatory sequence elements and corresponding transcriptional activators of new gene copies are more conserved than coding sequences, consistent with the tissue-specific expression of these genes.
Ebright, RH, Le Grice SF, Miller JP, Krakow JS.  1985.  Analogs of cyclic AMP that elicit the biochemically defined conformational change in catabolite gene activator protein (CAP) but do not stimulate binding to DNA.. Journal of molecular biology. 182(1):91-107. Abstract
We have measured the effects on catabolite gene activator protein (CAP) of 22 synthetic analogs of cAMP. Each analog was assayed to test three parameters: (1) binding to CAP; (2) induction of the conformational change in CAP; and (3) activation of transcription. Thus we have identified seven cAMP analogs that bind to CAP as well or better than does cAMP, cause the assayed conformational change in CAP, yet exhibit no ability to activate transcription. We designate these analogs class D. The conformational change elicited in CAP by the class D analogs was further investigated by: (1) sensitivity to the proteolytic enzymes chymotrypsin, Staphylococcus aureus V8 protease, subtilisin and trypsin; (2) formation of inter-subunit covalent crosslinks by 5,5'-dithiobis(2-nitrobenzoic acid); and (3) degree of labeling of cysteine by [3H]N-ethylmaleimide. These experiments failed to detect a conformational difference between the CAP-class D and CAP-cAMP complexes. Filter binding and nuclease protection experiments indicate that the class D analogs do not efficiently support the binding of CAP to DNA. From these results, we suggest that there exists a hitherto undetected event dependent on cAMP, and required for CAP to bind to DNA. We suggest that this event involves a change that takes place in proximity to the N6 atom of cAMP. Three possible interpretations are discussed.