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Ebright, YW, Chen Y, Kim Y, Ebright RH.  1996.  S-[2-(4-azidosalicylamido)ethylthio]-2-thiopyridine: radioiodinatable, cleavable, photoactivatible cross-linking agent.. Bioconjugate chemistry. 7(3):380-4. Abstract
S-[2-(4-Azidosalicylamido)ethylthio]-2-thiopyridine (AET) contains a 2-thiopyridyl moiety, which permits cysteine-specific incorporation into protein through a cleavable disulfide bond, and a 4-azidosalicylamido moiety, which permits radioiodination and photoactivatible cross-linking. In contrast to the related compound S-[2-[N-[4-(4-azidosalicylamido)butyl]carbomoyl]ethylthio]-2 -thiopyridine [APDP; Zecherle, G., Oleinikov, A., and Traut, R. (1992) Biochemistry 31, 9526], AET contains a relatively short linker arm between the 2-thiopyridyl moiety and the 4-azidosalicylamido moiety. In a previous paper, it was shown that AET could be used in site-specific protein-protein photocross-linking to identify nearest-neighbor protein domains within a multiprotein complex [Chen, Y., Ebright, Y., and Ebright, R. (1994) Science 265, 90]. In this paper, the synthesis, radioiodination, and incorporation into protein of AET are described.
Hassan, HM, Degen D, Jang K H, Ebright RH, Fenical W.  2015.  Salinamide F, new depsipeptide antibiotic and inhibitor of bacterial RNA polymerase from a marine-derived Streptomyces sp.. The Journal of antibiotics. 68(3):206-9.
Acton, TB, Mead J, Steiner AM, Vershon AK.  2000.  Scanning Mutagenesis of Mcm1: Residues Required for DNa Binding, DNa Bending, and Transcriptional Activation by a MADS-box Protein. Mol Cell Biol. 20:1-11. Abstract
MCM1 is an essential gene in the yeast Saccharomyces cerevisiae and is a member of the MADS-box family of transcriptional regulatory factors. To understand the nature of the protein-DNA interactions of this class of proteins, we have made a series of alanine substitutions in the DNA-binding domain of Mcm1 and examined the effects of these mutations in vivo and in vitro. Our results indicate which residues of Mcm1 are important for viability, transcriptional activation, and DNA binding and bending. Substitution of residues in Mcm1 which are highly conserved among the MADS-box proteins are lethal to the cell and abolish DNA binding in vitro. These positions have almost identical interactions with DNA in both the serum response factor-DNA and alpha2-Mcm1-DNA crystal structures, suggesting that these residues make up a conserved core of protein-DNA interactions responsible for docking MADS-box proteins to DNA. Substitution of residues which are not as well conserved among members of the MADS-box family play important roles in contributing to the specificity of DNA binding. These results suggest a general model of how MADS-box proteins recognize and bind DNA. We also provide evidence that the N-terminal extension of Mcm1 may have considerable conformational freedom, possibly to allow binding to different DNA sites. Finally, we have identified two mutants at positions which are critical for Mcm1-mediated DNA bending that have a slow-growth phenotype. This finding is consistent with our earlier results, indicating that DNA bending may have a role in Mcm1 function in the cell.
Calviño, M, Bruggmann R, Messing J.  2008.  Screen of Genes Linked to High-Sugar Content in Stems by Comparative Genomics. Rice. 1:166-176. AbstractWebsite
One of the great advantages of the fully sequenced rice genome is to serve as a reference for other cereal genomes in particular for identifying genes linked to unique traits. A trait of great interest is reduced lignocellulose in the stem of related species in favor of fermentable sugars as a source of biofuels. While sugarcane is one of the most efficient biofuel crops, little is known about the underlying gene repertoire involved in it. Here, we take advantage of the natural variation of sweet and grain sorghum to uncover genes that are conserved in rice, sorghum, and sugarcane but differently expressed in sweet versus grain sorghum by using a microarray platform and the syntenous alignment of rice and sorghum genomic regions containing these genes. Indeed, enzymes involved in carbohydrate accumulation and those that reduce lignocellulose can be identified.
Yagi, M, Wolf KV, Baesjou PJ, Bernasek SL, Dismukes CG.  2001.  Selective Photoproduction of O2 from the Mn4O4 Cubane Core: A Structural and Functional Model for the Photosynthetic Water-Oxidizing Complex. Angewandte Chemie. 113:3009-3012.Website
Yu, Y, Dubey M, Bernasek SL, Dismukes CG.  2007.  Self-Assembled Monolayer of Organic Iodine on a Au Surface for Attachment of Redox-Active Metal Clusters. Langmuir. 23:8257-8263. AbstractWebsite
Bae, Y-K, Barr MM.  2008.  Sensory Roles of Neuronal Cilia: Cilia Development, Morphogenesis, and Function in C. Elegans. Front Biosci. 13:5959-5974. Abstract
In the free-living nematode Caenorhabditis elegans, cilia are found on the dendritic endings of sensory neurons. C. elegans cilia are classified as 'primary' or 'sensory' according to the '9+0' axonemal ultrastructure (nine doublet outer microtubules with no central microtubule pair) and lack of motility, characteristics of '9+2' cilia. The C. elegans ciliated nervous system allows the animal to perceive environmental stimuli and make appropriate developmental, physiological, and behavioral decisions. In vertebrates, the biological significance of primary cilia had been largely neglected. Recent findings have placed primary/sensory cilia in the center of cellular signaling and developmental processes. Studies using genetic model organisms such as C. elegans identified the link between ciliary dysfunction and human ciliopathies. Future studies in the worm will address important basic questions regarding ciliary development, morphogenesis, specialization, and signaling functions.
Geraghty, DE, Messing J, Rubenstein I.  1982.  Sequence analysis and comparison of cDNAs of the zein multigene family. The EMBO journal. 1:1329-35. AbstractWebsite
The nucleotide sequence of two zein cDNAs in hybrid plasmids A20 and B49 have been determined. The insert in A20 is 921 bp long including a 5' non-coding region of 60 nucleotides, preceded by what is believed to be an artifactual sequence of 41 nucleotides, and a 3' non-coding region of 87 nucleotides. The B49 insert is 467 bp long and includes approximately one-half the protein coding sequence as well as a 3' non-coding region of 97 nucleotides. These sequences have been compared with the previously published sequence of another zein clone, A30 . A20 and A30 , both encoding 19 000 mol. wt. zeins , have approximately 85% homology at the nucleotide level. The B49 sequence, corresponding to a 22 000 mol. wt. zein, has approximately 65% homology to either A20 or A30 . All three zeins share common features including nearly identical amino acid compositions. In addition, the tandem repeats of 20 amino acids first seen in A30 are also present in A20 and B49 .
Messing, J, Bharti AK, Karlowski WM, Gundlach H, Kim HR, Yu Y, Wei F, Fuks G, Soderlund CA, Mayer KF et al..  2004.  Sequence composition and genome organization of maize. Proceedings of the National Academy of Sciences of the United States of America. 101:14349-54. AbstractWebsite
Zea mays L. ssp. mays, or corn, one of the most important crops and a model for plant genetics, has a genome approximately 80% the size of the human genome. To gain global insight into the organization of its genome, we have sequenced the ends of large insert clones, yielding a cumulative length of one-eighth of the genome with a DNA sequence read every 6.2 kb, thereby describing a large percentage of the genes and transposable elements of maize in an unbiased approach. Based on the accumulative 307 Mb of sequence, repeat sequences occupy 58% and genic regions occupy 7.5%. A conservative estimate predicts approximately 59,000 genes, which is higher than in any other organism sequenced so far. Because the sequences are derived from bacterial artificial chromosome clones, which are ordered in overlapping bins, tagged genes are also ordered along continuous chromosomal segments. Based on this positional information, roughly one-third of the genes appear to consist of tandemly arrayed gene families. Although the ancestor of maize arose by tetraploidization, fewer than half of the genes appear to be present in two orthologous copies, indicating that the maize genome has undergone significant gene loss since the duplication event.
Loeb, DD, Padgett RW, Hardies SC, Shehee WR, Comer MB, Edgell MH, Hutchison CA.  1986.  The sequence of a large L1Md element reveals a tandemly repeated 5' end and several features found in retrotransposons. Mol Cell Biol. 6:168-82. AbstractWebsite
The complete nucleotide sequence of a 6,851-base pair (bp) member of the L1Md repetitive family from a selected random isolate of the BALB/c mouse genome is reported here. Five kilobases of the element contains two overlapping reading frames of 1,137 and 3,900 bp. The entire 3,900-bp frame and the 3' 600 bp of the 1,137-bp frame, when compared with a composite consensus primate L1 sequence, show a ratio of replacement to silent site differences characteristic of protein coding sequences. This more closely defines the protein coding capacity of this repetitive family, which was previously shown to possess a large open reading frame of undetermined extent. The relative organization of the 1,137- and 3,900-bp reading frames, which overlap by 14 bp, bears resemblance to protein-coding, mobile genetic elements. Homology can be found between the amino acid sequence of the 3,900-bp frame and selected domains of several reverse transcriptases. The 5' ends of the two L1Md elements described in this report have multiple copies, 4 2/3 copies and 1 2/3 copy, of a 208-bp direct tandem repeat. The sequence of this 208-bp element differs from the sequence of a previously defined 5' end for an L1Md element, indicating that there are at least two different 5' end motifs for L1Md.
Song, R, Llaca V, Linton E, Messing J.  2001.  Sequence, regulation, and evolution of the maize 22-kD alpha zein gene family. Genome research. 11:1817-25. AbstractWebsite
We have isolated and sequenced all 23 members of the 22-kD alpha zein (z1C) gene family of maize. This is one of the largest plant gene families that has been sequenced from a single genetic background and includes the largest contiguous genomic DNA from maize with 346,292 bp to date. Twenty-two of the z1C members are found in a roughly tandem array on chromosome 4S forming a dense gene cluster 168,489-bp long. The twenty-third copy of the gene family is also located on chromosome 4S at a site approximately 20 cM closer to the centromere and appears to be the wild-type allele of the floury-2 (fl2) mutation. On the basis of an analysis of maize cDNA databases, only seven of these genes appear to be expressed including the fl2 allele. The expressed genes in the cluster are interspersed with nonexpressed genes. Interestingly, some of the expressed genes differ in their transcriptional regulation. Gene amplification appears to be in blocks of genes explaining the rapid and compact expansion of the cluster during the evolution of maize.
Fingerman, I, Nagaraj V, Norris D, Vershon AK.  2003.  Sfp1 Plays a key role in Yeast Ribosome Biogenesis. Eukaryot Cell. 2:1061-1068. Abstract
Sfp1, an unusual zinc finger protein, was previously identified as a gene that, when overexpressed, imparted a nuclear localization defect. sfp1 cells have a reduced size and a slow growth phenotype. In this study we show that SFP1 plays a role in ribosome biogenesis. An sfp1 strain is hypersensitive to drugs that inhibit translational machinery. sfp1 strains also have defects in global translation as well as defects in rRNA processing and 60S ribosomal subunit export. Microarray analysis has previously shown that ectopically expressed SFP1 induces the transcription of a large subset of genes involved in ribosome biogenesis. Many of these induced genes contain conserved promoter elements (RRPE and PAC). Our results show that activation of transcription from a reporter construct containing two RRPE sites flanking a single PAC element is SFP1 dependent. However, we have been unable to detect direct binding of the protein to these elements. This suggests that regulation of genes containing RRPEs is dependent upon Sfp1 but that Sfp1 may not directly bind to these conserved promoter elements; rather, activation may occur through an indirect mechanism.
Messing, J.  2009.  Shotgun DNA sequencing bearing fruits: probing the dynamics of genome size. Intern J Nat & Eng Sci. 3:1-6.
Nickels, BE, Mukhopadhyay J, Garrity SJ, Ebright RH, Hochschild A.  2004.  The sigma 70 subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoter.. Nature structural & molecular biology. 11(6):544-50. Abstract
The sigma(70) subunit of RNA polymerase plays an essential role in transcription initiation. In addition, sigma(70) has a critical regulatory role during transcription elongation at the bacteriophage lambda late promoter, lambda P(R'). At this promoter, sigma(70) mediates a pause in early elongation through contact with a DNA sequence element in the initially transcribed region that resembles a promoter -10 element. Here we provide evidence that sigma(70) also mediates a pause in early elongation at the lac promoter (plac). Like that at lambda P(R'), the pause at plac is facilitated by a sequence element in the initially transcribed region that resembles a promoter -10 element. Using biophysical analysis, we demonstrate that the pause-inducing sequence element at plac stabilizes the interaction between sigma(70) and the remainder of the transcription elongation complex. Bioinformatic analysis suggests that promoter-proximal sigma(70)-dependent pauses may play a role in the regulation of many bacterial promoters.
Pan, G, Feng Y, Ambegaonkar AA, Sun G, Huff M, Rauskolb C, Irvine KD.  2013.  Signal transduction by the Fat cytoplasmic domain.. Development. AbstractWebsite
The large atypical cadherin Fat is a receptor for both Hippo and planar cell polarity (PCP) pathways. Here we investigate the molecular basis for signal transduction downstream of Fat by creating targeted alterations within a genomic construct that contains the entire fat locus, and by monitoring and manipulating the membrane localization of the Fat pathway component Dachs. We establish that the human Fat homolog FAT4 lacks the ability to transduce Hippo signaling in Drosophila, but can transduce Drosophila PCP signaling. Targeted deletion of conserved motifs identifies a four amino acid C-terminal motif that is essential for aspects of Fat-mediated PCP, and other internal motifs that contribute to Fat-Hippo signaling. Fat-Hippo signaling requires the Drosophila Casein kinase 1_ encoded by discs overgrown (Dco), and we characterize candidate Dco phosphorylation sites in the Fat intracellular domain (ICD), the mutation of which impairs Fat-Hippo signaling. Through characterization of Dachs localization and directed membrane targeting of Dachs, we show that localization of Dachs influences both the Hippo and PCP pathways. Our results identify a conservation of Fat-PCP signaling mechanisms, establish distinct functions for different regions of the Fat ICD, support the correlation of Fat ICD phosphorylation with Fat-Hippo signaling, and confirm the importance of Dachs membrane localization to downstream signaling pathways.
Sheehan, B, Klarsfeld A, Ebright R, Cossart P.  1996.  A single substitution in the putative helix-turn-helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence.. Molecular microbiology. 20(4):785-97. Abstract
PrfA, the regulator of virulence-gene expression in the pathogenic bacterium Listeria monocytogenes, displays sequence similarity to members of the CAP-FNR family of transcriptional regulators. To test the functional significance of this similarity, we constructed and analysed substitutions of two amino acids of PrfA predicted to contact DNA, i.e. Ser-184 and Ser-183. Substitution of Ser-184 by Ala reduced DNA binding and virulence-gene activation, and attenuated the virulence in a mouse model of infection. In contrast, substitution of Ser-183 by Ala had the opposite effect in these functional assays. A 17bp DNA sequence, which includes a putative PrfA site, was shown to be sufficient for target-site recognition by PrfA and PrfA-S183A. Our results strongly support the hypothesis that PrfA is a structural and functional homologue of CAP. In addition, they establish a clear correlation between DNA binding by PrfA, virulence-gene activation, and virulence.
Revyakin, A, Allemand JF, Croquette V, Ebright RH, Strick TR.  2003.  Single-molecule DNA nanomanipulation: detection of promoter-unwinding events by RNA polymerase.. Methods in enzymology. 370:577-98.
Revyakin, A, Ebright RH, Strick TR.  2005.  Single-molecule DNA nanomanipulation: improved resolution through use of shorter DNA fragments.. Nature methods. 2(2):127-38.
Chakraborty, A, Mazumder A, Lin M, Hasemeyer A, Xu Q, Wang D, Ebright YW, Ebright RH.  2015.  Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation.. Methods in molecular biology (Clifton, N.J.). 1276:101-31. Abstract
A three-step procedure comprising (1) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (2) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (3) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a cross-linking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP.
Naryshkin, N, Kim Y, Dong Q, Ebright RH.  2001.  Site-specific protein-DNA photocrosslinking. Analysis of bacterial transcription initiation complexes.. Methods in molecular biology (Clifton, N.J.). 148:337-61.
Savage-Dunn, C, Tokarz R, Wang H, Cohen S, Giannikas C, Padgett RW.  2000.  SMA-3 smad has specific and critical functions in DBL-1/SMA-6 TGFβ-related signaling. Dev Biol. 223:70-6. AbstractWebsite
A TGFbeta signal transduction cascade controls body size and male tail morphogenesis in the nematode Caenorhabditis elegans. We have analyzed the function of the sma-3 Smad gene, one of three Smad genes that function in this pathway. Null mutations in sma-3 are at least as severe as null mutations in the ligand and type I receptor genes, dbl-1 and sma-6, indicating that the other Smads do not function in the absence of SMA-3. Furthermore, null mutations in sma-3 do not cause defects in egg laying or in regulation of the developmentally arrested dauer larva stage, indicating no overlapping function with another C. elegans TGFbeta signaling pathway. The sma-3 gene is widely expressed at all developmental stages in hermaphrodites and males. The molecular lesions associated with eight sma-3 alleles of varying severity have been determined. The missense mutations cluster in two previously identified regions important for Smad function.
Padgett, RW, Cho SH, Evangelista C.  1998.  Smads are the central component in transforming growth factor-β signaling. Pharmacology & therapeutics. 78:47-52. AbstractWebsite
Until recently, little was known about how transforming growth factor (TGF)-beta signals are transduced to the nucleus. With the discovery of the Smad proteins initially in Drosophila and C. elegans, the unraveling of the pathway has begun. Nine different vertebrate members also have been reported, indicating that Smads are a conserved component of the TGF-beta pathway. Currently, there are three functional classes of Smads. Class I Smads are phosphorylated by TGF-beta receptors and move to the nucleus. The Class II Smads function with Class I Smads, while Class III Smads antagonize the function of Class I Smads. New evidence shows that Smads bind specific DNA sequences and induce transcription of downstream target genes, thus placing the Smads at the center of the TGF-beta signaling pathway.
Gumienny, TL, Padgett RW.  2003.  A small issue addressed. BioEssays : news and reviews in molecular, cellular and developmental biology. 25:305-8. AbstractWebsite
Cell size is an important determinant of body size. While the genetic mechanisms of cell size regulation have been well studied in yeast, this process has only recently been addressed in multicellular organisms. One recent report by Wang et al. (2002) shows that in the nematode C. elegans, the TGFbeta-like pathway acts in the hypodermis to regulate cell size and consequently body size.1 This finding is an exciting step in discovering the molecular mechanisms that control cell and body size.
Brimblecombe, R, Koo A, Dismukes CG, Swiegers GF, Spiccia L.  2010.  Solar Driven Water Oxidation by a Bioinspired Manganese Molecular Catalyst. Journal of the American Chemical Society. 132:2892-2894. AbstractWebsite