Publications

1996
1995
Kim, J, Irvine KD, Carroll SB.  1995.  Cell recognition, signal induction, and symmetrical gene activation at the dorsal-ventral boundary of the developing Drosophila wing. Cell. 82:795-802. AbstractWebsite
Appendage formation in insects and vertebrates depends upon signals from both the anterior-posterior and dorsal-ventral (DV) axes. In Drosophila, wing formation is organized symmetrically around the DV boundary of the growing wing imaginal disc and requires interactions between dorsal and ventral cells. Compartmentalization of the wing disc, dorsal cell behavior, and the expression of two dorsally expressed putative signaling molecules, fringe (fng) and Serrate (Ser), are regulated by the apterous selector gene. Here, we demonstrate that fng and Ser have distinct roles in a novel cell recognition and signal induction process. fng serves as a boundary-determining molecule such that Ser is induced wherever cells expressing fng and cells not expressing fng are juxtaposed. Ser in turn triggers the expression of genes involved in wing growth and patterning on both sides of the DV boundary.
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.
Rongo, C, Gavis ER, Lehmann R.  1995.  Localization of oskar RNA regulates oskar translation and requires Oskar protein. Development. 121:2737-46. AbstractWebsite
The site of oskar RNA and protein localization within the oocyte determines where in the embryo primordial germ cells form and where the abdomen develops. Initiation of oskar RNA localization requires the activity of several genes. We show that ovaries mutant for any of these genes lack Oskar protein. Using various transgenic constructs we have determined that sequences required for oskar RNA localization and translational repression map to the oskar 3'UTR, while sequences involved in the correct temporal activation of translation reside outside the oskar 3'UTR. Upon localization of oskar RNA and protein at the posterior pole, Oskar protein is required to maintain localization of oskar RNA throughout oogenesis. Stable anchoring of a transgenic reporter RNA at the posterior pole is disrupted by oskar nonsense mutations. We propose that initially localization of oskar RNA permits translation into Oskar protein and that subsequently Oskar protein regulates its own RNA localization through a positive feedback mechanism.
Tang, H, Severinov K, Goldfarb A, Ebright RH.  1995.  Rapid RNA polymerase genetics: one-day, no-column preparation of reconstituted recombinant Escherichia coli RNA polymerase.. Proceedings of the National Academy of Sciences of the United States of America. 92(11):4902-6. Abstract
We present a simple, rapid procedure for reconstitution of Escherichia coli RNA polymerase holoenzyme (RNAP) from individual recombinant alpha, beta, beta', and sigma 70 subunits. Hexahistidine-tagged recombinant alpha subunit purified by batch-mode metal-ion-affinity chromatography is incubated with crude recombinant beta, beta', and sigma 70 subunits from inclusion bodies, and the resulting reconstituted recombinant RNAP is purified by batch-mode metal-ion-affinity chromatography. RNAP prepared by this procedure is indistinguishable from RNAP prepared by conventional methods with respect to subunit stoichiometry, alpha-DNA interaction, catabolite gene activator protein (CAP)-independent transcription, and CAP-dependent transcription. Experiments with alpha (1-235), an alpha subunit C-terminal deletion mutant, establish that the procedure is suitable for biochemical screening of subunit lethal mutants.
Ebright, RH, Busby S.  1995.  The Escherichia coli RNA polymerase alpha subunit: structure and function.. Current opinion in genetics & development. 5(2):197-203. Abstract
Recent work has established that the Escherichia coli RNA polymerase alpha subunit consists of an amino-terminal domain containing determinants for interaction with the remainder of RNA polymerase, a carboxy-terminal domain containing determinants for interaction with DNA and interaction with transcriptional activator proteins, and a 13-36 amino acid unstructured and/or flexible linker. These findings suggest a simple, integrated model for the mechanism of involvement of alpha in promoter recognition and transcriptional activation.
Merkel, TJ, Dahl JL, Ebright RH, Kadner RJ.  1995.  Transcription activation at the Escherichia coli uhpT promoter by the catabolite gene activator protein.. Journal of bacteriology. 177(7):1712-8. Abstract
Transport and utilization of sugar phosphates in Escherichia coli depend on the transport protein encoded by the uhpT gene. Transmembrane induction of uhpT expression by external glucose 6-phosphate is positively regulated by the promoter-specific activator protein UhpA and the global regulator catabolite gene activator protein (CAP). Activation by UhpA requires a promoter element centered at -64 bp, relative to the start of transcription, and activation by CAP requires a DNA site centered at position -103.5. This DNA site binds the cyclic AMP-CAP complex in vitro, and its deletion from the promoter reduces transcription activity to 7 to 9% of the wild-type level. Ten uhpT promoter derivatives with altered spacing between the DNA site for CAP and the remainder of the promoter were constructed. Their transcription activities indicated that the action of CAP at this promoter is dependent on proper helical phasing of promoter elements, with CAP binding on the same face of the helix as RNA polymerase does. Five CAP mutants defective in transcription activation at class I and class II CAP-dependent promoters but not defective in DNA binding or DNA bending (positive control mutants) were tested for the ability to activate transcription. These CAPpc mutants exhibited little or no defect in transcription activation at uhpT, indicating that CAP action at uhpTp involves a different mechanism than that which is used for its action at other classes of CAP-dependent promoters.
Lund, G, Messing J, Viotti A.  1995.  Endosperm-specific demethylation and activation of specific alleles of alpha-tubulin genes of Zea mays L. Molecular & general genetics : MGG. 246:716-22. AbstractWebsite
We have investigated the methylation status of the alpha-tubulin genes, and the degree of accumulation of their mRNAs in endosperm, embryo and seedling tissues of Zea mays L. We have found that many of the alpha-tubulin genes are differentially demethylated in the endosperm relative to the embryo and seedling. However, only for tub alpha 2 and tub alpha 4 could a correlation between DNA demethylation and increased RNA accumulation be detected. By analyzing the inbred lines W64A and A69Y and their reciprocal crosses, we have also identified in the endosperm two alpha-tubulin genes, tub alpha 3 and tub alpha 4, that are differentially demethylated if transmitted by the maternal germline, but that remain hypermethylated when transmitted by the paternal germline.
Lund, G, Prem Das O, Messing J.  1995.  Tissue-specific DNase I-sensitive sites of the maize P gene and their changes upon epimutation. The Plant Journal. 7:797-807.Website
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, Hawley RS.  1995.  Chromosomal control of meiotic cell division. Science. 270:1595-1601.
Sekelsky, JJ, McKim KS, Chin GM, Hawley RS.  1995.  The Drosophila meiotic recombination gene mei-9 encodes a homologue of the yeast excision repair protein Rad1. Genetics. 141:619-627.
1994
Irvine, KD, Wieschaus E.  1994.  fringe, a Boundary-specific signaling molecule, mediates interactions between dorsal and ventral cells during Drosophila wing development. Cell. 79:595-606. AbstractWebsite
Wing formation in Drosophila requires interactions between dorsal and ventral cells. We describe a new gene, fringe, which is expressed in dorsal cells and encodes for a novel protein that is predicted to be secreted. Wing margin formation and distal wing outgrowth can be induced by the juxtaposition of cells with and without fringe expression, whether at the normal wing margin, at the boundaries of fringe mutant clones in the dorsal wing, or at sites of fringe misexpression in the ventral wing. By contrast, both loss of fringe expression and uniform fringe expression cause wing loss. These observations suggest that fringe encodes a boundary-specific cell-signaling molecule that is responsible for dorsal-ventral cell interactions during wing development.
Xie, T, Finelli AL, Padgett RW.  1994.  The Drosophila saxophone gene: a serine-threonine kinase receptor of the TGF-β superfamily. Science (New York, NY). 263:1756-9. AbstractWebsite
The Drosophila decapentaplegic (dpp) gene encodes a transforming growth factor-beta (TGF-beta)-like protein that plays a key role in several aspects of development. Transduction of the DPP signal was investigated by cloning of serine-threonine kinase transmembrane receptors from Drosophila because this type of receptor is specific for the TGF-beta-like ligands. Here evidence is provided demonstrating that the Drosophila saxophone (sax) gene, a previously identified female sterile locus, encodes a TGF-beta-like type I receptor. Embryos from sax mothers and dpp embryos exhibit similar mutant phenotypes during early gastrulation, and these two loci exhibit genetic interactions, which suggest that they are utilized in the same pathway. These data suggest that sax encodes a receptor for dpp.
McKim, KS, Rose AM.  1994.  Spontaneous duplication loss and breakage in Caenorhabditis elegans. Genome. 37:595-606.Website
Irvine, KD, Wieschaus E.  1994.  Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes. Development (Cambridge, England). 120:827-41. AbstractWebsite
After the onset of gastrulation, the Drosophila germband undergoes a morphological change in which its length along the anterior-posterior axis increases over two-and-a-half fold while its width along the dorsal-ventral axis simultaneously narrows. The behavior of individual cells during germband extension was investigated by epi-illumination and time-lapse video microscopy of living embryos. Cells intercalate between their dorsal and ventral neighbors during extension, increasing the number of cells along the anterior-posterior axis while decreasing the number of cells along the dorsal-ventral axis. Mutations that reduce segmental subdivision of the embryo along the anterior-posterior axis decrease both germband extension and its associated cell intercalation. In contrast, cell intercalation and germband extension are still detected in embryos that lack dorsal-ventral polarity. Characterization of germband extension and cell intercalation in mutant embryos with altered segmentation gene expression indicates that these processes are regionally autonomous and are dependent upon the establishment of striped expression patterns for certain pair-rule genes. Based on these observations, we propose a model for germband extension in which cell intercalation results from the establishment of adhesive differences between stripes of cells by pair-rule genes.
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)
Blatter, EE, Ross W, Tang H, Gourse RL, Ebright RH.  1994.  Domain organization of RNA polymerase alpha subunit: C-terminal 85 amino acids constitute a domain capable of dimerization and DNA binding.. Cell. 78(5):889-96. Abstract
Using limited proteolysis, we show that the Escherichia coli RNA polymerase alpha subunit consists of an N-terminal domain comprised of amino acids 8-241, a C-terminal domain comprised of amino acids 249-329, and an unstructured and/or flexible interdomain linker. We have carried out a detailed structural and functional analysis of an 85 amino acid proteolytic fragment corresponding to the C-terminal domain (alpha CTD-2). Our results establish that alpha CTD-2 has a defined secondary structure (approximately 40% alpha helix, approximately 0% beta sheet). Our results further establish that alpha CTD-2 is a dimer and that alpha CTD-2 exhibits sequence-specific DNA binding activity. Our results suggest a model for the mechanism of involvement of alpha in transcription activation by promoter upstream elements and upstream-binding activator proteins.
Zhou, Y, Pendergrast PS, Bell A, Williams R, Busby S, Ebright RH.  1994.  The functional subunit of a dimeric transcription activator protein depends on promoter architecture.. The EMBO journal. 13(19):4549-57. Abstract
In Class I CAP-dependent promoters, the DNA site for CAP is located upstream of the DNA site for RNA polymerase. In Class II CAP-dependent promoters, the DNA site for CAP overlaps the DNA site for RNA polymerase, replacing the -35 site. We have used an 'oriented heterodimers' approach to identify the functional subunit of CAP at two Class I promoters having different distances between the DNA sites for CAP and RNA polymerase [CC(-61.5) and CC(-72.5)] and at one Class II promoter [CC(-41.5)]. Our results indicate that transcription activation at Class I promoters, irrespective of the distance between the DNA sites for CAP and RNA polymerase, requires the activating region of the promoter-proximal subunit of CAP. In striking contrast, our results indicate that transcription activation at Class II promoters requires the activating region of the promoter-distal subunit of CAP.
Niu, W, Zhou Y, Dong Q, Ebright YW, Ebright RH.  1994.  Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP). I. Saturation and alanine-scanning mutagenesis.. Journal of molecular biology. 243(4):595-602. Abstract
It has been proposed that the surface loop consisting of amino acid residues 152 to 166 of the catabolite gene activator protein (CAP) of Escherichia coli makes direct protein-protein contact with RNA polymerase at the lac promoter. In this work, we have used targeted saturation mutagenesis of codons 152 to 166 of the gene encoding CAP, followed by a screen, to isolate more than 200 independent mutants of CAP defective in transcription activation but not defective in DNA binding. All isolated single-substitution mutants map to just eight amino acid residues; 156, 157, 158, 159, 160, 162, 163 and 164. We propose that these residues define the full extent of the epitope on CAP for the proposed CAP-RNA polymerase interaction. In addition, we have constructed alanine substitutions at each position from residue 152 to 166 of CAP, and we have analyzed the effects on transcription activation at the lac promoter and on DNA binding. Alanine substitution of Thr158 results in an approximately eightfold specific defect in transcription activation. In contrast, alanine substitution of no other residue tested results in a more than twofold specific defect in transcription activation. We conclude that, for Thr158, side-chain atoms beyond C beta are essential for transcription activation at the lac promoter, and we propose that Thr158 OH7 gamma makes direct contact with RNA polymerase in the ternary complex of lac promoter, CAP and RNA polymerase. We conclude further that for no residue other than Thr158 are side-chain atoms beyond C beta essential for transcription activation at the lac promoter.
Zhou, Y, Merkel TJ, Ebright RH.  1994.  Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP). II. Role at Class I and class II CAP-dependent promoters.. Journal of molecular biology. 243(4):603-10. Abstract
CAP-dependent promoters can be divided into classes based on the position of the DNA site for CAP. In class I CAP-dependent promoters, the DNA site for CAP is located upstream of the DNA site for polymerase; the DNA site for CAP can be located at various distances from the transcription start point, provided that the DNS site for CAP and the DNA site for RNA polymerase are on the same face of the DNA helix. In class II CAP-dependent promoters, the DNA site for CAP overlaps the DNA site for RNA polymerase, replacing the -35 determinants for binding of RNA polymerase. In previous work, we have shown that a surface loop consisting of amino acid residues 152 to 166 of CAP is essential for transcription activation at the best-characterized class I CAP-dependent promoter, the lac promoter, and we proposed that this surface loop makes direct protein-protein contact with RNA polymerase in the ternary complex of lac promoter, CAP, and RNA polymerase. Here, we show that the surface loop consisting of amino acid residues 152 to 166 is essential for transcription activation at other class I CAP-dependent promoters and at a class II CAP-dependent promoter. We show further that the effects of alanine substitutions of residues 152 to 166 are qualitatively identical at the lac promoter and other class I CAP-dependent promoters, but are different at a class II CAP-dependent promoter. We propose that the surface loop consisting of residues 152 to 166 makes identical molecular interactions in transcription activation at all class I CAP-dependent promoters, irrespective of distance between the DNA site for CAP and the transcription start point, but makes a different set of molecular interactions in transcription activation at class II CAP-dependent promoters.
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.
Shang, Z, Ebright YW, Iler N, Pendergrast PS, Echelard Y, McMahon AP, Ebright RH, Abate C.  1994.  DNA affinity cleaving analysis of homeodomain-DNA interaction: identification of homeodomain consensus sites in genomic DNA.. Proceedings of the National Academy of Sciences of the United States of America. 91(1):118-22. Abstract
We have incorporated the DNA-cleaving moiety o-phenanthroline-copper at amino acid 10 of the Msx-1 homeodomain, and we have analyzed site-specific DNA cleavage by the resulting Msx-1 derivative. We show that amino acid 10 of the Msx-1 homeodomain is close to the 5' end of the consensus DNA site 5'-(C/G)TAATTG-3' in the Msx-1-DNA complex. Our results indicate that the orientation of the Msx-1 homeodomain relative to DNA is analogous to the orientation of the engrailed and Antennapedia homeodomains. We show further that DNA affinity cleaving permits identification of consensus DNA sites for Msx-1 in kilobase DNA substrates. The specificity of the approach enabled us to identify an Msx-1 consensus DNA site within the transcriptional control region of the developmental regulatory gene Wnt-1. We propose that incorporation of o-phenanthroline-copper at amino acid 10 of a homeodomain may provide a generalizable strategy to determine the orientation of a homeodomain relative to DNA and to identify homeodomain consensus DNA sites in genomic DNA.
Dong, Q, Blatter EE, Ebright YW, Bister K, Ebright RH.  1994.  Identification of amino acid-base contacts in the Myc-DNA complex by site-specific bromouracil mediated photocrosslinking.. The EMBO journal. 13(1):200-4. Abstract
Myc binds to a 6 bp 2-fold symmetric DNA site: 5'-C-3A-2C-1G+1T+2G+3-3'. Using site-specific 5-bromouracil mediated photocrosslinking, we show that His336 of Myc contacts, or is close to, the thymine 5-methyl group at 2-fold symmetry-related positions -2 and +2 of the DNA site in the Myc-DNA complex. Our results strongly suggest that homologous amino acids of Myc and Max make equivalent contacts in the respective protein-DNA complexes.