Publications

1994
Pendergrast, PS, Ebright YW, Ebright RH.  1994.  High-specificity DNA cleavage agent: design and application to kilobase and megabase DNA substrates.. Science (New York, N.Y.). 265(5174):959-62. Abstract
Strategies to cleave double-stranded DNA at specific DNA sites longer than those of restriction endonucleases (longer than 8 base pairs) have applications in chromosome mapping, chromosome cloning, and chromosome sequencing--provided that the strategies yield high DNA-cleavage efficiency and high DNA-cleavage specificity. In this report, the DNA-cleaving moiety copper:o-phenanthroline was attached to the sequence-specific DNA binding protein catabolite activator protein (CAP) at an amino acid that, because of a difference in DNA bending, is close to DNA in the specific CAP-DNA complex but is not close to DNA in the nonspecific CAP-DNA complex. The resulting CAP derivative, OP26CAP, cleaved kilobase and megabase DNA substrates at a 22-base pair consensus DNA site with high efficiency and exhibited no detectable nonspecific DNA-cleavage activity.
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.
Das, OP, Messing J.  1994.  Variegated phenotype and developmental methylation changes of a maize allele originating from epimutation. Genetics. 136:1121-41. AbstractWebsite
Two instances of genetic transmission of spontaneous epimutation of the maize P-rr gene were identified. Transmission gave rise to two similar, moderately stable alleles, designated P-pr-1 and P-pr-2, that exhibited Mendelian behavior. Both isolates of P-pr conditioned a variable and variegated phenotype, unlike the uniform pigmentation conditioned by P-rr. Extensive genomic analysis failed to reveal insertions, deletions or restriction site polymorphisms between the new allele and its progenitor. However, methylation of the P gene was increased in P-pr relative to P-rr, and was greatly reduced (though not lost) in a revertant to uniform pigmentation. Variability in pigmentation conditioned by P-pr correlated with variability in transcript levels of the P gene, and both correlated inversely with variability in its methylation. Part of the variability in methylation could be accounted for by a developmental decrease in methylation in all tissues of plants carrying P-pr. We hypothesize that the variegated phenotype results from a general epigenetic pathway which causes a progressive decrease in methylation and increase in expression potential of the P gene as a function of cell divisions in each meristem of the plant. This renders all tissues chimeric for a functional gene; chimerism is visualized as variegation only in pericarp due to the tissue specificity of P gene expression. Therefore, this allele that originates from epimutation may exemplify an epigenetic mechanism for variegation in maize.
1993
Irvine, KD, Botas J, Jha S, Mann RS, Hogness DS.  1993.  Negative autoregulation by Ultrabithorax controls the level and pattern of its expression. Development. 117:387-99. AbstractWebsite
The Drosophila homeotic gene Ultrabithorax (Ubx) encodes transcriptional regulatory proteins (UBX) that specify thoracic and abdominal segmental identities. Ubx autoregulation was examined by manipulating UBX levels, both genetically and with an inducible transgene, and monitoring the effect of these manipulations on the expression of Ubx and Ubx-lacZ reporter genes. Positive autoregulation by Ubx is restricted to the visceral mesoderm, while in other tissues Ubx negatively autoregulates. In some cases, negative autoregulation stabilizes UBX levels, while in others it modulates the spatial and temporal patterns of UBX expression. This modulation of UBX expression may enable Ubx to specify distinct identities in different segments. The upstream control region of Ubx contains multiple autoregulatory elements for both positive and negative autoregulation.
Hursh, DA, Padgett RW, Gelbart WM.  1993.  Cross regulation of decapentaplegic and Ultrabithorax transcription in the embryonic visceral mesoderm of Drosophila. Development (Cambridge, England). 117:1211-22. AbstractWebsite
The Drosophila decapentaplegic gene (dpp) encodes a TGF-beta family member involved in signal transduction during embryonic midgut formation. The shortvein (shv) class of cis-regulatory dpp mutants disrupt expression in parasegments 4 and 7 (ps4 and ps7) of the embryonic visceral mesoderm (VM) surrounding the gut and cause abnormalities in gut morphogenesis. We demonstrate that cis-regulatory elements directing expression in ps4 and ps7 are separable and identify DNA fragments that generate ps4 and ps7 expression patterns using reporter gene constructs. dpp reporter gene expression in both ps4 and ps7 is autoregulated as it requires endogenous dpp+ activity. Reporter gene ps7 expression requires the wild-type action of Ultra-bithorax (Ubx), and abdominal-A. Furthermore, the expression of certain Ubx reporter genes is coincident with dpp in the VM. Both the mis-expression of Ubx reporter genes in the developing gastric caecae at ps4 and its normal expression in ps7 are dependent upon endogenous dpp+ activity. We conclude that dpp both responds to and regulates Ubx in ps7 of the visceral mesoderm and that Ubx autoregulation within this tissue may be indirect as it requires more components than have previously been thought.
Padgett, RW, Wozney JM, Gelbart WM.  1993.  Human BMP sequences can confer normal dorsal-ventral patterning in the Drosophila embryo. Proceedings of the National Academy of Sciences of the United States of America. 90:2905-9. AbstractWebsite
The type beta transforming growth factor family is composed of a series of processed, secreted growth factors, several of which have been implicated in important regulatory roles in cell determination, inductive interactions, and tissue differentiation. Among these factors, the sequence of the DPP protein from Drosophila is most similar to two of the vertebrate bone morphogenetic proteins, BMP2 and BMP4. Here we report that the human BMP4 ligand sequences can function in lieu of DPP in Drosophila embryos. We introduced the ligand region from human BMP4 into a genomic fragment of the dpp gene in place of the Drosophila ligand sequences and recovered transgenic flies by P-element transformation. We find that this chimeric dpp-BMP4 transgene can completely rescue the embryonic dorsal-ventral patterning defect of null dpp mutant genotypes. We infer that the chimeric DPP-BMP4 protein can be processed properly and, by analogy with the action of other family members, can activate the endogenous DPP receptor to carry out the events necessary for dorsal-ventral patterning. Our evidence suggests that the DPP-BMP4 signal transduction pathway has been functionally conserved for at least 600 million years.
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.  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.
Chen, Y, Ebright RH.  1993.  Phenyl-azide-mediated photocrosslinking analysis of Cro-DNA interaction.. Journal of molecular biology. 230(2):453-60. Abstract
Using phenyl-azide-mediated photocrosslinking, we show that the alpha carbon of amino acid 2 of the helix-turn-helix motif of bacteriophage lambda Cro is within 12 A of the bottom-strand nucleotides at positions 2 and 3 of the DNA half site in the Cro-DNA complex in solution. This result is in excellent agreement with the crystallographic structure of the Cro-DNA complex. The results of phenyl-azide-mediated photocrosslinking analysis of Cro-DNA interaction, together with the previously reported results of phenyl-azide-mediated photocrosslinking analysis of CAP-DNA interaction, establish that phenyl-azide-mediated photocrosslinking is generalizable and provide information regarding the structural requirements for phenyl-azide-mediated photocrosslinking. Comparison of the results of phenyl-azide-mediated photocrosslinking to the results of EDTA: iron-mediated affinity cleaving indicates that phenyl-azide-mediated photocrosslinking yields superior resolution.
Zhou, Y, Zhang X, Ebright RH.  1993.  Identification of the activating region of catabolite gene activator protein (CAP): isolation and characterization of mutants of CAP specifically defective in transcription activation.. Proceedings of the National Academy of Sciences of the United States of America. 90(13):6081-5. Abstract
We have isolated 21 mutants of catabolite gene activator protein (CAP) defective in transcription activation at the lac promoter but not defective in DNA binding. The amino acid substitutions in the mutants map to a single region of CAP: amino acids 156-162. As assessed in vitro, the substituted CAP variants are nearly completely unable to activate transcription at the lac promoter but bind to DNA with the same affinity and bend DNA to the same extent as wild-type CAP. Our results establish that amino acids 156-162 are critical for transcription activation at the lac promoter but not for DNA binding and DNA bending. In the structure of CAP, amino acids 156-162 are part of a surface loop. We propose that this surface loop makes a direct protein-protein contact with RNA polymerase at the lac promoter.
Heyduk, T, Lee JC, Ebright YW, Blatter EE, Zhou Y, Ebright RH.  1993.  CAP interacts with RNA polymerase in solution in the absence of promoter DNA.. Nature. 364(6437):548-9. Abstract
Protein-protein interactions between transcription activator proteins and RNA polymerase or basal transcription factors have been suggested to be important for transcription activation. Interactions between catabolite gene activator protein (CAP) and RNA polymerase have been proposed based on face-of-helix-dependent transcription activation by CAP and based on face-of-helix-dependent cooperative binding of CAP and RNA polymerase to promoter DNA. Mutants of CAP specifically defective in transcription activation have been isolated (mutants defective in transcription activation, but not defective in DNA binding and DNA bending). All such mutants contain amino-acid substitutions within a surface loop consisting of amino acids 152 to 166 of CAP. Here we use the thermodynamically rigorous technique of fluorescence polarization to show that CAP interacts with RNA polymerase in solution in the absence of promoter DNA (KD,app = 2.8 x 10(-7) M), whereas [Ala158]CAP, a mutant of CAP specifically defective in transcription activation, does not.
Zhou, Y, Busby S, Ebright RH.  1993.  Identification of the functional subunit of a dimeric transcription activator protein by use of oriented heterodimers.. Cell. 73(2):375-9. Abstract
We have constructed heterodimers consisting of two subunits: one CAP subunit that has a nonfunctional activating region but wild-type DNA binding specificity, and one CAP subunit that has a functional activating region but non-wild-type DNA binding specificity. We have oriented the heterodimers on lac promoter DNA by use of promoter derivatives that have DNA sites for CAP consisting of one wild-type half site and one non-wild-type half site, and we have analyzed the abilities of the oriented heterodimers to activate transcription. Our results indicate that transcription. Our results indicate that transcription activation requires the activating region of only one subunit of CAP: the promoter-proximal subunit. The oriented heterodimers method of this report should be generalizable to other dimeric transcription activator proteins.
Schickler, H.  1993.  Repression of the high-methionine zein gene in the maize inbred line Mo17. Plant Journal. 3:221-229..Website
McKim, KS, Jang JK, Theurkauf WE, Hawley RS.  1993.  Mechanical basis of meiotic metaphase arrest. Nature. 362:364-366.
Hawley, RS, McKim KS, Arbel T.  1993.  Meiotic segregation in Drosophila melanogasterfemales: molecules, mechanisms and myths. Ann. Rev. Genet.. 27:281-317.
Hawley, RS, Irick HA, Zitron AE, Haddox DA, Lohe AR, New C, Whitley MD, Arbel T, Jang JK, McKim KS et al..  1993.  There are two mechanisms of achiasmate segregation in Drosophila females, one of which requires heterochromatic homology. Developmental Genetics. 13:440-467.
McKim, KS, Peters K, Rose AM.  1993.  Two types of sites required for meiotic chromosome pairing in Caenorhabditis elegans. Genetics. 134:749-768.
1992
Blatter, EE, Ebright YW, Ebright RH.  1992.  Identification of an amino acid-base contact in the GCN4-DNA complex by bromouracil-mediated photocrosslinking.. Nature. 359(6396):650-2. Abstract
The bZIP DNA-binding proteins are characterized by a 50-amino-acid DNA binding and dimerization motif, consisting of a highly basic DNA-binding region ('b') followed by a leucine zipper dimerization region ('ZIP'). The best characterized bZIP DNA-binding protein is GCN4, a yeast transcriptional activator. GCN4 binds to a 9-base-pair two-fold-symmetric DNA site, 5'-A-4T-3G-2A-1C0T+1C+2A+3T+4-3' (refs 7-10). A detailed model known as the 'induced helical fork' model has been proposed for the structure of the GCN4-DNA complex. Using a site-specific bromouracil-mediated photocrosslinking method, we show here that the alanine at position 238 of GCN4 contacts, or is close to, the thymine 5-methyl of A.T at position +3 of the DNA site in the GCN4-DNA complex. Our results strongly support the induced helical fork model. Our site-specific bromouracil-mediated photocrosslinking method requires no prior information regarding the structure of the protein or the structure of the protein-DNA complex and should be generalizable to DNA-binding proteins that interact with the DNA major groove.
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)
Pendergrast, PS, Chen Y, Ebright YW, Ebright RH.  1992.  Determination of the orientation of a DNA binding motif in a protein-DNA complex by photocrosslinking.. Proceedings of the National Academy of Sciences of the United States of America. 89(21):10287-91. Abstract
We have developed a straightforward biochemical method to determine the orientation of the DNA binding motif of a sequence-specific DNA binding protein relative to the DNA site in the protein-DNA complex. The method involves incorporation of a photoactivatable crosslinking agent at a single site within the DNA binding motif of the sequence-specific DNA binding protein, formation of the derivatized protein-DNA complex, UV-irradiation of the derivatized protein-DNA complex, and determination of the nucleotide(s) at which crosslinking occurs. We have applied the method to catabolite gene activator protein (CAP). We have constructed and analyzed two derivatives of CAP: one having a phenyl azide photoactivatable crosslinking agent at amino acid 2 of the helix-turn-helix motif of CAP, and one having a phenyl azide photoactivatable crosslinking agent at amino acid 10 of the helix-turn-helix motif of CAP. The results indicate that amino acid 2 of the helix-turn-helix motif is close to the top-strand nucleotides of base pairs 3 and 4 of the DNA half site in the CAP-DNA complex, and that amino acid 10 of the helix-turn-helix motif is close to the bottom-strand nucleotide of base pair 10 of the DNA half site in the CAP-DNA complex. The results define unambiguously the orientation of the helix-turn-helix motif relative to the DNA half site in the CAP-DNA complex. Comparison of the results to the crystallographic structure of the CAP-DNA complex [Schultz, S., Shields, S. & Steitz, T. (1991) Science 253, 1001-1007] indicates that the method provides accurate, high-resolution proximity and orientation information.
Ebright, R, Dong Q, Messing J.  1992.  Corrected nucleotide sequence of M13mp18 gene III.. Gene. 114(1):81-3. Abstract
There are seven differences between the actual nucleotide (nt) sequence of bacteriophage M13mp18 gene III and the previously reported nt sequence (which had been compiled based on the nt sequence of wild-type bacteriophage M13 gene III).
Gunasekera, A, Ebright YW, Ebright RH.  1992.  DNA sequence determinants for binding of the Escherichia coli catabolite gene activator protein.. The Journal of biological chemistry. 267(21):14713-20. Abstract
The consensus DNA site for binding of the Escherichia coli catabolite gene activator protein (CAP) is 22 base pairs in length and is 2-fold symmetric: 5'-AAATGTGATCTAGATCACATTT-3'. Positions 4 to 8 of each half of the consensus DNA half-site are the most strongly conserved. In this report, we analyze the effects of substitution of DNA base pairs at positions 4 to 8, the effects of substitution of thymine by uracil and by 5-methylcytosine at positions 4, 6, and 8, and the effect of dam methylation of the 5'-GATC-3' sequence at positions 7 to 10. All DNA sites having substitutions of DNA base pairs at positions 4 to 8 exhibit lower affinities for CAP than does the consensus DNA site, consistent with the proposal that the consensus DNA site is the ideal DNA site for CAP. Specificity for T:A at position 4 appears to be determined solely by the thymine 5-methyl group. Specificity for T:A at position 6 and specificity for A:T at position 8 appear to be determined in part, but not solely, by the thymine 5-methyl group. dam methylation has little effect on CAP.DNA complex formation. The thermodynamically defined consensus DNA site spans 28 base pairs. All, or nearly all, DNA determinants required for maximal affinity for CAP and for maximal thermodynamically defined CAP.DNA ion pair formation are contained within a 28-base pair DNA fragment that has the 22-base pair consensus DNA site at its center. The quantitative data in this report provide base-line thermodynamic data required for detailed investigations of amino acid-base pair and amino acid-phosphate contacts in this protein-DNA complex.
Dong, Q, Ebright RH.  1992.  DNA binding specificity and sequence of Xanthomonas campestris catabolite gene activator protein-like protein.. Journal of bacteriology. 174(16):5457-61. Abstract
The Xanthomonas campestris catabolite gene activator protein-like protein (CLP) can substitute for the Escherichia coli catabolite gene activator protein (CAP) in transcription activation at the lac promoter (V. de Crecy-Lagard, P. Glaser, P. Lejeune, O. Sismeiro, C. Barber, M. Daniels, and A. Danchin, J. Bacteriol. 172:5877-5883, 1990). We show that CLP has the same DNA binding specificity as CAP at positions 5, 6, and 7 of the DNA half site. In addition, we show that the amino acids at positions 1 and 2 of the recognition helix of CLP are identical to the amino acids at positions 1 and 2 of the recognition helix of CAP:i.e., Arg at position 1 and Glu at position 2.
Zhang, X, Zhou Y, Ebright YW, Ebright RH.  1992.  Catabolite gene activator protein (CAP) is not an "acidic activating region" transcription activator protein. Negatively charged amino acids of CAP that are solvent-accessible in the CAP-DNA complex play no role in transcription activation at lac.. The Journal of biological chemistry. 267(12):8136-9. Abstract
It has been suggested that the catabolite gene activator protein (CAP) uses an "acidic activating region" transcription activation mechanism and that Glu171 of CAP is the critical amino acid of the "acidic activating region" of CAP (Irwin, N., and Ptashne, M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8315-8319). In this paper, we show, contrary to the previously published report, that substitution of Glu171 of CAP fails to result in a specific defect in transcription activation at the lac promoter. Furthermore, in this paper, we show that substitution of each other negatively charged amino acid of CAP that is solvent-accessible in the CAP-DNA complex fails to result in a specific defect in transcription activation at the lac promoter. We conclude that CAP does not use an acidic activating region transcription activation mechanism in transcription activation at the lac promoter.
Ueda, T, Waverczak W, Ward K, Sher N, Ketudat M, Schmidt RJ, Messing J.  1992.  Mutations of the 22- and 27-kD zein promoters affect transactivation by the Opaque-2 protein. The Plant cell. 4:701-9. AbstractWebsite
By utilizing a homologous transient expression system, we have demonstrated that the Opaque-2 (O2) gene product O2 confers positive trans-regulation on a 22-kD zein promoter. This trans-acting function of the O2 protein is mediated by its sequence-specific binding to a cis element (the O2 target site) present in the 22-kD zein promoter. A multimer of a 32-bp promoter fragment containing this O2 target site confers transactivation by O2. A single nucleotide substitution in the O2 target sequence not only abolishes O2 binding in vitro, but also its response to transactivation by O2 in vivo. We have also demonstrated that an amino acid domain including the contiguous basic region and the heptameric leucine repeat is essential for the trans-acting function of the O2 protein. Similar but not identical O2 target sequence motifs can be found in the promoters of zein genes of different molecular weight classes. Conversion of such a motif in the 27-kD zein promoter to an exact O2 target sequence by site-directed mutagenesis was sufficient to increase the binding affinity of the O2 protein in vitro and to confer transactivation by O2 in vivo.