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Ebright, YW, Chen Y, Ludescher RD, Ebright RH.  1993.  N-(iodoacetyl)-p-phenylenediamine-EDTA: a reagent for high-efficiency incorporation of an EDTA-metal complex at a rationally selected site within a protein.. Bioconjugate chemistry. 4(3):219-25. Abstract
We have developed a highly efficient procedure to incorporate an EDTA:metal complex at a rationally selected site within a full-length protein. Our procedure has two steps: In step one, we use site-directed mutagenesis to introduce a unique solvent-accessible cysteine residue at the site of interest. In step two, we derivatized the resulting protein with N-(iodoacetyl)-p-phenylenediamine-EDTA:metal, a novel haloacetyl derivative of EDTA:metal. We have used this procedure to incorporate each of three EDTA:metal complexes at amino acid 2 of the helix-turn-helix motif of the sequence-specific DNA binding protein Cro: a radioactive and nucleolytic EDTA:metal complex (EDTA:55Fe), a radioactive EDTA:metal complex (EDTA:63Ni), and a fluorescent and heavy-atom EDTA:metal complex (EDTA:Eu). Incorporation of EDTA:metal was highly efficient (> 80% for EDTA:55Fe and EDTA:63Ni; 60% for EDTA:Eu) and highly site-specific (> 99%). We have analyzed DNA affinity cleaving by the Cro derivative having EDTA:55Fe at amino acid 2 of the helix-turn-helix motif. The Cro derivative cleaves DNA at base pairs -4 to 6 of the DNA half site in the protein-DNA complex, indicating that amino acid 2 of the helix-turn-helix motif of Cro is close to base pairs -4 to 6 of the DNA half site in the Cro-DNA complex in solution.(ABSTRACT TRUNCATED AT 250 WORDS)
Ebright, RH, Ebright YW, Gunasekera A.  1989.  Consensus DNA site for the Escherichia coli catabolite gene activator protein (CAP): CAP exhibits a 450-fold higher affinity for the consensus DNA site than for the E. coli lac DNA site.. Nucleic acids research. 17(24):10295-305. Abstract
We have synthesized two 40 base pair DNA fragments; one fragment contains the consensus DNA site for CAP (fragment 'ICAP'); the other fragment contains the E. coli lac promoter DNA site for CAP (fragment 'LCAP'). We have investigated the binding of CAP to the two DNA fragments using the nitrocellulose filter binding assay. Under standard conditions [( NaCl] = 200 mM, pH = 7.3), CAP exhibits a 450-fold higher affinity for ICAP than for LCAP. The salt dependence of the binding equilibrium indicates that CAP makes eight ion pairs with ICAP, but only six ion pairs with LCAP. Approximately half of the difference in binding free energy for interaction of CAP with ICAP vs. LCAP is attributable to this difference in ion-pair formation. The pH dependence of the binding equilibrium indicates that the eight CAP-ICAP ion pairs and the six CAP-LCAP ion pairs do not involve His residues of CAP.
Ebright, YW, Chen Y, Pendergrast PS, Ebright RH.  1992.  Incorporation of an EDTA-metal complex at a rationally selected site within a protein: application to EDTA-iron DNA affinity cleaving with catabolite gene activator protein (CAP) and Cro.. Biochemistry. 31(44):10664-70. Abstract
We have developed a simple procedure to incorporate an EDTA-metal complex at a rationally selected site within a full-length protein. Our procedure has two steps: In step 1, we use site-directed mutagenesis to introduce a unique solvent-accessible cysteine residue at the site of interest. In step 2, we derivatize the resulting protein with S-(2-pyridylthio)cysteaminyl-EDTA-metal, a novel aromatic disulfide derivative of EDTA-metal. We have used this procedure to incorporate an EDTA-iron complex at amino acid 2 of the helix-turn-helix motif of each of two helix-turn-helix motif sequence-specific DNA binding proteins, catabolite gene activator protein (CAP) and Cro, and we have analyzed EDTA-iron-mediated DNA affinity cleavage by the resulting protein derivatives. The CAP derivative cleaves DNA at base pair 2 of the DNA half-site in the protein-DNA complex, and the Cro derivative cleaves DNA at base pairs -3 to 5 of the DNA half-site in the protein-DNA complex. We infer that amino acid 2 of the helix-turn-helix motif of CAP is close to base pair 2 of the DNA half-site in the CAP-DNA complex in solution and that amino acid 2 of the helix-turn-helix motif of Cro is close to base pairs -3 to 5 of the DNA half-site in the Cro-DNA complex in solution.(ABSTRACT TRUNCATED AT 250 WORDS)
Ebright, RH.  1986.  Evidence for a contact between glutamine-18 of lac repressor and base pair 7 of lac operator.. Proceedings of the National Academy of Sciences of the United States of America. 83(2):303-7. Abstract
Glutamine-18 of the lac repressor (lacR) has been substituted by glycine, by serine, and by leucine. The specificities of wild-type lacR and of the three substituted lacR variants have been analyzed with respect to base pairs 5, 6, 7, 8, 9, and 10 of the lac operator (lacO). The data indicate that [Gly18]lacR, [Ser18]lacR, and [Leu18]lacR lose the ability to distinguish between the O+ base pair G . C and the Oc base pairs T . A and A . T at position 7 of lacO (KdOc/KdO+ approximately equal to 1). In contrast, the three substituted variants retain the ability to discriminate O+ from Oc at each other position, by factors of 9 to 37. Therefore, I propose that glutamine-18 contacts base pair 7 of lacO. These data suggest that the interaction between the helix-turn-helix motif and DNA may be very similar or identical in lacR and the catabolite gene activator protein.
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.
Ebright, RH, Cossart P, Gicquel-Sanzey B, Beckwith J.  1984.  Mutations that alter the DNA sequence specificity of the catabolite gene activator protein of E. coli.. Nature. 311(5983):232-5. Abstract
Three mutations that alter the DNA sequence specificity of the catabolite gene activator protein (CAP) from AA-TGTGA--T---TCA-ATW to AA-TGTAA--T---TCA-ATW have been isolated. All three mutations affect the same amino acid of CAP, glutamic acid 181. We propose that it is this amino acid of CAP that makes contacts with base pairs 7 and 16 of the symmetrical recognition site.
Ebright, RH.  1998.  RNA polymerase-DNA interaction: structures of intermediate, open, and elongation complexes.. Cold Spring Harbor symposia on quantitative biology. 63:11-20.
Ebright, RH.  1993.  Transcription activation at Class I CAP-dependent promoters.. Molecular microbiology. 8(5):797-802. Abstract
Catabolite gene activator protein (CAP)-dependent promoters can be grouped into three classes, based on the requirement for transcription activation and the position of the DNA site for CAP. Class I CAP-dependent promoters require only CAP for transcription activation and have the DNA site for CAP located upstream of the DNA site for RNA polymerase. Amino acids 156 to 162 of the promoter-proximal subunit of CAP are essential for transcription activation at Class I CAP-dependent promoters, but are not essential for DNA binding, and are not essential for DNA bending. In the structure of the CAP-DNA complex, these amino acids are located in a surface loop and form a cluster on the surface of the CAP-DNA complex. Amino acids 261, 265, and 270 of the alpha subunit of RNA polymerase are essential for response to transcription activation by CAP at Class I CAP-dependent promoters. Several lines of evidence indicate that transcription activation at Class I CAP-dependent promoters requires a direct protein-protein contact between amino acids 156 to 162 of the promoter-proximal subunit of CAP and a molecule of RNA polymerase bound adjacent to CAP on the same face of the DNA helix. It is a strong possibility that this direct protein-protein contact involves amino acids 261 and 265 of the alpha subunit of RNA polymerase.
Ebright, RH, Gunasekera A, Zhang XP, Kunkel TA, Krakow JS.  1990.  Lysine 188 of the catabolite gene activator protein (CAP) plays no role in specificity at base pair 7 of the DNA half site.. Nucleic acids research. 18(6):1457-64. Abstract
Two similar, but not identical, models have been proposed for the amino acid-base pair contacts in the CAP-DNA complex ('model I,' Weber, I. and Steitz, T., Proc. Natl. Acad. Sci. USA, 81, 3973-3977, 1984; 'model II,' Ebright, et al., Proc. Natl. Acad. Sci. USA, 81, 7274-7278, 1984). The most important difference between the two models involves Lys188 of CAP. Model I predicts that Lys188 of CAP makes a specificity determining contact with base pair 7 of the DNA half site. In contrast, model II predicts that Lys188 makes no contact with base pair 7 of the DNA half site. In the present work, we have used site-directed mutagenesis to replace Lys188 of CAP by Asn, an amino acid unable to make the putative contact. We have assessed the specificities of the following proteins, both in vitro and in vivo: wild-type CAP, [Asn188]CAP, [Val181]CAP, and [Val181;Asn188]CAP. The results indicate that Lys188 makes no contribution to specificity at base pair 7 of the DNA half site. We propose, contrary to model I, that Lys188 makes no contact with base pair 7 of the DNA half site.
Edgell, MH, Hardies SC, Loeb DD, Shehee WR, Padgett RW, Burton FH, Comer MB, Casavant NC, Funk FD, Hutchison CA.  1987.  The L1 family in mice. Progress in clinical and biological research. 251:107-29.Website
Emtage, L, Chang H, Tiver R, Rongo C.  2009.  MAGI-1 modulates AMPA receptor synaptic localization and behavioral plasticity in response to prior experience. PLoS One. 4:e4613. AbstractWebsite
It is well established that the efficacy of synaptic connections can be rapidly modified by neural activity, yet how the environment and prior experience modulate such synaptic and behavioral plasticity is only beginning to be understood. Here we show in C. elegans that the broadly conserved scaffolding molecule MAGI-1 is required for the plasticity observed in a glutamatergic circuit. This mechanosensory circuit mediates reversals in locomotion in response to touch stimulation, and the AMPA-type receptor (AMPAR) subunits GLR-1 and GLR-2, which are required for reversal behavior, are localized to ventral cord synapses in this circuit. We find that animals modulate GLR-1 and GLR-2 localization in response to prior mechanosensory stimulation; a specific isoform of MAGI-1 (MAGI-1L) is critical for this modulation. We show that MAGI-1L interacts with AMPARs through the intracellular domain of the GLR-2 subunit, which is required for the modulation of AMPAR synaptic localization by mechanical stimulation. In addition, mutations that prevent the ubiquitination of GLR-1 prevent the decrease in AMPAR localization observed in previously stimulated magi-1 mutants. Finally, we find that previously-stimulated animals later habituate to subsequent mechanostimulation more rapidly compared to animals initially reared without mechanical stimulation; MAGI-1L, GLR-1, and GLR-2 are required for this change in habituation kinetics. Our findings demonstrate that prior experience can cause long-term alterations in both behavioral plasticity and AMPAR localization at synapses in an intact animal, and indicate a new, direct role for MAGI/S-SCAM proteins in modulating AMPAR localization and function in the wake of variable sensory experience.
Estrem, ST, Ross W, Gaal T, Chen ZW, Niu W, Ebright RH, Gourse RL.  1999.  Bacterial promoter architecture: subsite structure of UP elements and interactions with the carboxy-terminal domain of the RNA polymerase alpha subunit.. Genes & development. 13(16):2134-47. Abstract
We demonstrate here that the previously described bacterial promoter upstream element (UP element) consists of two distinct subsites, each of which, by itself, can bind the RNA polymerase holoenzyme alpha subunit carboxy-terminal domain (RNAP alphaCTD) and stimulate transcription. Using binding-site-selection experiments, we identify the consensus sequence for each subsite. The selected proximal subsites (positions -46 to -38; consensus 5'-AAAAAARNR-3') stimulate transcription up to 170-fold, and the selected distal subsites (positions -57 to -47; consensus 5'-AWWWWWTTTTT-3') stimulate transcription up to 16-fold. RNAP has subunit composition alpha(2)betabeta'sigma and thus contains two copies of alphaCTD. Experiments with RNAP derivatives containing only one copy of alphaCTD indicate, in contrast to a previous report, that the two alphaCTDs function interchangeably with respect to UP element recognition. Furthermore, function of the consensus proximal subsite requires only one copy of alphaCTD, whereas function of the consensus distal subsite requires both copies of alphaCTD. We propose that each subsite constitutes a binding site for a copy of alphaCTD, and that binding of an alphaCTD to the proximal subsite region (through specific interactions with a consensus proximal subsite or through nonspecific interactions with a nonconsensus proximal subsite) is a prerequisite for binding of the other alphaCTD to the distal subsite.
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Farace, MG, Hill A, Tripodi M, Padgett RW, Raschella G, Gambari R, Fantoni A, Hutchison CA, Edgell MH.  1984.  Molecular cloning and sequence analysis of a cDNA coding for the mouse alpha-like embryonic globin chain x. Gene. 31:241-5. AbstractWebsite
Cytoplasmic poly(A)+mRNA from 12-day mouse-yolk-sac erythroid cells has been used to prepare a cDNA library in the plasmid pBR322. One clone containing sequences coding for the alpha-like embryonic globin chain x, pHE52, has been identified by hybrid selection and in vitro translation of the complementary mRNA. The nucleotide sequence of pHE52 confirms that it codes for an embryonic alpha-like globin chain. The insert sequence is 316 nucleotides long, contains the codons corresponding to amino acid residues 43-141, and extends into the 3' untranslated region. An analysis of the nucleotide sequence of pHE52 and the other known alpha globins suggests that the adult-embryonic divergence began approx. 400 million years ago reflecting a difference in the evolutionary history of the alpha- and beta-globin gene complexes.
Feklistov, A, Mekler V, Jiang Q, Westblade LF, Irschik H, Jansen R, Mustaev A, Darst SA, Ebright RH.  2008.  Rifamycins do not function by allosteric modulation of binding of Mg2+ to the RNA polymerase active center.. Proceedings of the National Academy of Sciences of the United States of America. 105(39):14820-5. Abstract
Rifamycin antibacterial agents inhibit bacterial RNA polymerase (RNAP) by binding to a site adjacent to the RNAP active center and preventing synthesis of RNA products >2-3 nt in length. Recently, Artsimovitch et al. [(2005) Cell 122:351-363] proposed that rifamycins function by allosteric modulation of binding of Mg(2+) to the RNAP active center and presented three lines of biochemical evidence consistent with this proposal. Here, we show that rifamycins do not affect the affinity of binding of Mg(2+) to the RNAP active center, and we reassess the three lines of biochemical evidence, obtaining results not supportive of the proposal. We conclude that rifamycins do not function by allosteric modulation of binding of Mg(2+) to the RNAP active center.
Feng, Y, Irvine KD.  2007.  Fat and expanded act in parallel to regulate growth through warts. Proceedings of the National Academy of Sciences of the United States of America. 104:20362-7. AbstractWebsite
The conserved Drosophila tumor suppressors Fat and Expanded have both recently been implicated in regulating the activity of the Warts tumor suppressor. However, there has been disagreement as to the nature of the links among Fat, Expanded, and Warts and the significance of these links to growth control. We report here that mutations in either expanded or fat can be rescued to viability simply by overexpressing Warts, indicating that their essential function is their influence on Warts rather than reported effects on endocytosis or other pathways. These rescue experiments also separate the transcriptional from the planar cell polarity branches of Fat signaling and reveal that Expanded does not directly affect polarity. We also investigate the relationship between expanded and fat and show, contrary to prior reports, that they have additive effects on imaginal disk growth and development. Although mutation of fat can cause partial loss of Expanded protein from the membrane, mutation of fat promotes growth even when Expanded is overexpressed and accumulates at its normal subapical location. These observations argue against recent proposals that Fat acts simply as a receptor for the Hippo signaling pathway and instead support the proposal that Fat and Expanded can act in parallel to regulate Warts through distinct mechanisms.
Feng, Y, Zhang Y, Ebright RH.  2016.  Structural basis of transcription activation.. Science (New York, N.Y.). 352(6291):1330-3. AbstractWebsite
Class II transcription activators function by binding to a DNA site overlapping a core promoter and stimulating isomerization of an initial RNA polymerase (RNAP)-promoter closed complex into a catalytically competent RNAP-promoter open complex. Here, we report a 4.4 angstrom crystal structure of an intact bacterial class II transcription activation complex. The structure comprises Thermus thermophilus transcription activator protein TTHB099 (TAP) [homolog of Escherichia coli catabolite activator protein (CAP)], T. thermophilus RNAP σ(A) holoenzyme, a class II TAP-dependent promoter, and a ribotetranucleotide primer. The structure reveals the interactions between RNAP holoenzyme and DNA responsible for transcription initiation and reveals the interactions between TAP and RNAP holoenzyme responsible for transcription activation. The structure indicates that TAP stimulates isomerization through simple, adhesive, stabilizing protein-protein interactions with RNAP holoenzyme.
Feng, Y, Degen D, Wang X, Gigliotti M, Liu S, Zhang Y, Das D, Michalchuk T, Ebright YW, Talaue M et al..  2015.  Structural basis of transcription inhibition by CBR hydroxamidines and CBR pyrazoles. Structure. 23:1470-1481. AbstractWebsite
CBR hydroxamidines are small-molecule inhibitors of bacterial RNA polymerase (RNAP) discovered through high-throughput screening of synthetic-compound libraries. CBR pyrazoles are structurally related RNAP inhibitors discovered through scaffold hopping from CBR hydroxamidines. CBR hydroxamidines and pyrazoles selectively inhibit Gram-negative bacterial RNAP and exhibit selective antibacterial activity against Gram-negative bacteria. Here, we report crystal structures of the prototype CBR hydroxamidine, CBR703, and a CBR pyrazole in complex with E. coli RNAP holoenzyme. In addition, we define the full resistance determinant for CBR703, show that the binding site and resistance determinant for CBR703 do not overlap the binding sites and resistance determinants of other characterized RNAP inhibitors, show that CBR703 exhibits no or minimal cross-resistance with other characterized RNAP inhibitors, and show that co-administration of CBR703 with other RNAP inhibitors results in additive antibacterial activities. The results set the stage for structure-based optimization of CBR inhibitors as antibacterial drugs.
Feng, Y, Irvine KD.  2009.  Processing and phosphorylation of the Fat receptor. Proceedings of the National Academy of Sciences of the United States of America. AbstractWebsite
The Drosophila tumor suppressors fat and discs overgrown (dco) function within an intercellular signaling pathway that controls growth and polarity. fat encodes a transmembrane receptor, but post-translational regulation of Fat has not been described. We show here that Fat is subject to a constitutive proteolytic processing, such that most or all cell surface Fat comprises a heterodimer of stably associated N- and C-terminal fragments. The cytoplasmic domain of Fat is phosphorylated, and this phosphorylation is promoted by the Fat ligand Dachsous. dco encodes a kinase that influences Fat signaling, and Dco is able to promote the phosphorylation of the Fat intracellular domain in cultured cells and in vivo. Evaluation of dco mutants indicates that they affect Fat's influence on growth and gene expression but not its influence on planar cell polarity. Our observations identify processing and phosphorylation as post-translational modifications of Fat, correlate the phosphorylation of Fat with its activation by Dachsous in the Fat-Warts pathway, and enhance our understanding of the requirement for Dco in Fat signaling.
Finelli, AL, Bossie CA, Xie T, Padgett RW.  1994.  Mutational analysis of the Drosophila tolloid gene, a human BMP-1 homolog. Development (Cambridge, England). 120:861-70. AbstractWebsite
Seven zygotically active genes have been identified in Drosophila that determine the fate of dorsal cells in the developing embryo. decapentaplegic (dpp), a member of the transforming growth factor-beta (TGF-beta) family, appears to play the central role in dorsal ectoderm formation, as mutations in this gene confer the most severe mutant phenotype of this group of genes. dpp's activity is modulated by tolloid, which also has a role in the determination of dorsal cell fate. tolloid encodes a protein that contains a metalloprotease domain and regulatory domains consisting of two EGF motifs and five C1r/s repeats. We have generated several mutant tolloid alleles and have examined their interaction with a graded set of dpp point alleles. Some tolloid alleles act as dominant enhancers of dpp in a trans heterozygote, and are therefore antimorphic alleles. However, a tolloid deficiency shows no such genetic interaction. To characterize the nature of the tolloid mutations, we have sequenced eighteen tolloid alleles. We find that five of the seven alleles that act as dominant enhancers of dpp are missense mutations in the protease domain. We also find that most tolloid alleles that do not interact with dpp are missense mutations in the C-terminal EGF and C1r/s repeats, or encode truncated proteins that delete these repeats. Based on these data, we propose a model in which the tolloid protein functions by forming a complex containing DPP via protein-interacting EGF and C1r/s domains, and that the protease activity of TOLLOID is necessary, either directly or indirectly, for the activation of the DPP complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Finelli, AL, Xie T, Bossie CA, Blackman RK, Padgett RW.  1995.  The tolkin gene is a tolloid/BMP-1 homologue that is essential for Drosophila development. Genetics. 141:271-81. AbstractWebsite
The Drosophila decapentaplegic (dpp) gene, a member of the transforming growth factor beta superfamily of growth factors, is critical for specification of the embryonic dorsal-ventral axis, for proper formation of the midgut, and for formation of Drosophila adult structures. The Drosophila tolloid gene has been shown to genetically interact with dpp. The genetic interactions between tolloid and dpp suggests a model in which the tolloid protein participates in a complex containing the DPP ligand, its protease serving to activate DPP, either directly or indirectly. We report here the identification and cloning of another Drosophila member of the tolloid/bone morphogenic protein (BMP) 1 family, tolkin, which is located 700 bp 5' to tolloid. Its overall structure is like tolloid, with an N-terminal metalloprotease domain, five complement subcomponents C1r/C1s, Uegf, and Bmp1 (CUB) repeats and two epidermal growth factor (EGF) repeats. Its expression pattern overlaps that of tolloid and dpp in early embryos and diverges in later stages. In larval tissues, both tolloid and tolkin are expressed uniformly in the imaginal disks. In the brain, both tolloid and tolkin are expressed in the outer proliferation center, whereas tolkin has another stripe of expression near the outer proliferation center. Analysis of lethal mutations in tolkin indicate it is vital during larval and pupal stages. Analysis of its mutant phenotypes and expression patterns suggests that its functions may be mostly independent of tolloid and dpp.
Fingerman, IM, Sutphen K, Montano SP, Georgiadis MM, Vershon AK.  2004.  Characterization of Critical Interactions Between Ndt80 and MSe DNa Defining a Novel Family of Ig-fold Transcription Factors. Nucleic Acids Res. 32:2947-2956. Abstract
The Ndt80 protein of the yeast Saccharomyces cerevisiae is the founding member of a new sub-family of proteins in the Ig-fold superfamily of transcription factors. The crystal structure of Ndt80 bound to DNA shows that it makes contacts through several loops on one side of the protein that connect beta-strands which form the beta-sandwich fold common to proteins in this superfamily. However, the DNA-binding domain of Ndt80 is considerably larger than many other members of the Ig-fold superfamily and it appears to make a larger number of contacts with the DNA than these proteins. To determine the contribution of each of these contacts and to examine if the mechanism of Ndt80 DNA binding was similar to other members of the Ig-fold superfamily, amino acid substitutions were introduced at each residue that contacts the DNA and assayed for their effect on Ndt80 activity. Many of the mutations caused significant decreases in DNA-binding affinity and transcriptional activation. Several of these are in residues that are not found in other sub-families of Ig-fold proteins. These additional contacts are likely responsible for Ndt80's ability to bind DNA as a monomer while most other members require additional domains or cofactors to recognize their sites.
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.
Firestein, BL, Rongo C.  2001.  DLG-1 is a MAGUK similar to SAP97 and is required for adherens junction formation. Mol Biol Cell. 12:3465-75. AbstractWebsite
Cellular junctions are critical for intercellular communication and for the assembly of cells into tissues. Cell junctions often consist of tight junctions, which form a permeability barrier and prevent the diffusion of lipids and proteins between cell compartments, and adherens junctions, which control the adhesion of cells and link cortical actin filaments to attachment sites on the plasma membrane. Proper tight junction formation and cell polarity require the function of membrane-associated guanylate kinases (MAGUKs) that contain the PDZ protein-protein interaction domain. In contrast, less is known about how adherens junctions are assembled. Here we describe how the PDZ-containing protein DLG-1 is required for the proper formation and function of adherens junctions in Caenorhabditis elegans. DLG-1 is a MAGUK protein that is most similar in sequence to mammalian SAP97, which is found at both synapses of the CNS, as well as at cell junctions of epithelia. DLG-1 is localized to adherens junctions, and DLG-1 localization is mediated by an amino-terminal domain shared with SAP97 but not found in other MAGUK family members. DLG-1 recruits other proteins and signaling molecules to adherens junctions, while embryos that lack DLG-1 fail to recruit the proteins AJM-1 and CPI-1 to adherens junctions. DLG-1 is required for the proper organization of the actin cytoskeleton and for the morphological elongation of embryos. In contrast to other proteins that have been observed to affect adherens junction assembly and function, DLG-1 is not required to maintain cell polarity. Our results suggest a new function for MAGUK proteins distinct from their role in cell polarity.
Fu, H, Dooner HK.  2002.  Intraspecific violation of genetic colinearity and its implications in maize. Proc. Natl. Acad. Sci. U.S.A.. 99:9573–9578. Abstract
Although allelic sequences can vary extensively, it is generally assumed that each gene in one individual will have an allelic counterpart in another individual of the same species. We report here that this assumption does not hold true in maize. We have sequenced over 100 kb from the bz genomic region of two different maize lines and have found dramatic differences between them. First, the retrotransposon clusters, which comprise most of the repetitive DNA in maize, differ markedly in make-up and location relative to the genes in the bz region. Second, and more importantly, the genes themselves differ between the two lines, demonstrating that genetic microcolinearity can be violated within the same species. Our finding has bearing on the underlying genetic basis of hybrid vigor in maize, and possibly other organisms, and on the measurement of genetic distances.
Fu, H, Zheng Z, Dooner HK.  2002.  Recombination rates between adjacent genic and retrotransposon regions in maize vary by 2 orders of magnitude. Proc. Natl. Acad. Sci. U.S.A.. 99:1082–1087. Abstract
Genetic map length and gene number in eukaryotes vary considerably less than genome size, giving rise to the hypothesis that recombination is restricted to genes. The complex genome of maize contains a large fraction of repetitive DNA, composed principally of retrotransposons arranged in clusters. Here, we assess directly the contribution of retrotransposon clusters and genes to genetic length. We first measured recombination across adjacent homozygous genetic intervals on either side of the bronze (bz) locus. We then isolated and characterized two bacterial artificial chromosome clones containing those intervals. Recombination was almost 2 orders of magnitude higher in the distal side, which is gene-dense and lacks retrotransposons, than in the proximal side, which is gene-poor and contains a large cluster of methylated retrotransposons. We conclude that the repetitive retrotransposon DNA in maize, which constitutes the bulk of the genome, most likely contributes little if any to genetic length.