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Carrieri, D, McNeely K, De Roo AC, Bennette N, Pelczer I, Dismukes CG.  2009.  Identification and quantification of water-soluble metabolites by cryoprobe-assisted nuclear magnetic resonance spectroscopy applied to microbial fermentation. Magnetic Resonance in Chemistry. 47:S138-S146.Website
Zhang, XP, Ebright RH.  1990.  Identification of a contact between arginine-180 of the catabolite gene activator protein (CAP) and base pair 5 of the DNA site in the CAP-DNA complex.. Proceedings of the National Academy of Sciences of the United States of America. 87(12):4717-21. Abstract
We have used site-directed mutagenesis to replace amino acid 1 of the recognition alpha-helix of the catabolite gene activator protein (CAP), Arg-180, with glycine and with alanine. Substitution of Arg-180 of CAP eliminated specificity between G.C, A.T, C.G, and T.A at base pair 5 of the DNA half-site. The effect was position-specific: substitution of Arg-180 did not eliminate specificity between G.C, A.T, C.G, and T.A at base pair 7 of the DNA half-site. We conclude, in agreement with the model for the structure of the CAP-DNA complex [Weber, I. & Steitz, T. (1984) Proc. Natl. Acad. Sci. USA 81, 3973-3977; and Ebright, R., Cossart, P., Gicquel-Sanzey, B. & Beckwith, J. (1984) Proc. Natl. Acad. Sci. USA 81, 7274-7278], that Arg-180 of CAP makes a specificity-determining contact with base pair 5 of the DNA half-site in the CAP-DNA complex. The identification of the contact by Arg-180 in this report, in conjunction with the identification of the contact by Glu-181 in a previous report [Ebright, R., Cossart, P., Gicquel-Sanzey, B. & Beckwith, J. (1984) Nature (London) 311, 232-235], provides information sufficient to define the orientation of the helix-turn-helix motif of CAP with respect to DNA in the CAP-DNA complex.
Nakamura, Y, Haines N, Chen J, Okajima T, Furukawa K, Urano T, Stanley P, Irvine KD, Furukawa K.  2002.  Identification of a Drosophila gene encoding xylosylprotein beta4-galactosyltransferase that is essential for the synthesis of glycosaminoglycans and for morphogenesis. The Journal of biological chemistry. 277:46280-8. AbstractWebsite
In mammals, the xylosylprotein beta4-galactosyltransferase termed beta4GalT7 (XgalT-1, EC ) participates in proteoglycan biosynthesis through the transfer of galactose to the xylose that initiates each glycosaminoglycan chain. A Drosophila cDNA homologous to mammalian beta4-galactosyltransferases was identified using a human beta4GalT7 cDNA as a probe in a BLAST analysis of expressed sequence tags. The Drosophila cDNA encodes a type II membrane protein with 322 amino acids and shows 49% identity to human beta4GalT7. Extracts from L cells transfected with the cDNA exhibited marked galactosyltransferase activity specific for a xylopyranoside acceptor. Moreover, transfection with the cloned cDNA restored glycosaminoglycan synthesis in beta4GalT7-deficient Chinese hamster ovary cells. In transfectant lysates the properties of Drosophila and human beta4GalT7 resembled each other, except that Drosophila beta4GalT7 showed a less restricted specificity and was active at a wider range of temperatures. Drosophila beta4GalT7 is expressed throughout development, with higher expression levels in adults. Reduction of Drosophila beta4GalT7 levels using expressed RNA interference (RNAi) in imaginal discs resulted in an abnormal wing and leg morphology similar to that of flies with defective Hedgehog and Decapentaplegic signaling, which are known to depend on intact proteoglycan biosynthesis. Immunohistochemical analysis of tissues confirmed that both heparan sulfate and chondroitin sulfate biosynthesis were impaired. Our results demonstrate that Drosophila beta4GalT7 has the in vitro and in vivo properties predicted for an ortholog of human beta4GalT7 and is essential for normal animal development through its role in proteoglycan biosynthesis.
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.
Ebright, RH.  1991.  Identification of amino acid-base pair contacts by genetic methods.. Methods in enzymology. 208:620-40.
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.
Nguyen, TA, Brescic J, Vinyard DJ, Chandrasekar T, Dismukes CG.  2012.  Identification of an oxygenic reaction center psbADC operon in the cyanobacterium Gloeobacter violaceus PCC 7421.. Molecular biology and evolution. 29(1):35-8. Abstract
Gloeobacter violaceus, the earliest diverging oxyphotobacterium (cyanobacterium) on the 16S ribosomal RNA tree, has five copies of the photosystem II psbA gene encoding the D1 reaction center protein subunit. These copies are widely distributed throughout the 4.6 Mbp genome with only one copy colocalizing with other PSII subunits, in marked contrast to all other psbA genes in all publicly available sequenced genomes. A clustering of two other psb genes around psbA3 (glr2322) is unique to Gloeobacter. We provide experimental proof for the transcription of a psbA3DC operon, encoding three of the five reaction center core subunits (D1, D2, and CP43). This is the first example of a transcribed gene cluster containing the D1/D2 or D1/D2/CP43 subunits of PSII in an oxygenic phototroph (prokaryotic or eukaryotic). Implications for the evolution of oxygenic photosynthesis are discussed.
Bae, Y-K, Lyman-Gingerich J, Barr MM, Knobel KM.  2008.  Identification of Genes Involved in the Ciliary Trafficking of C. Elegans PKD-2. Dev Dyn. 237:2021-2029. Abstract
Ciliary membrane proteins are important extracellular sensors, and defects in their localization may have profound developmental and physiological consequences. To determine how sensory receptors localize to cilia, we performed a forward genetic screen and identified 11 mutants with defects in the ciliary localization (cil) of C. elegans PKD-2, a transient receptor potential polycystin (TRPP) channel. Class A cil mutants exhibit defects in PKD-2::GFP somatodendritic localization while Class B cil mutants abnormally accumulate PKD-2::GFP in cilia. Further characterization reveals that some genes mutated in cil mutants act in a tissue-specific manner while others are likely to play more general roles in such processes as intraflagellar transport (IFT). To this end, we identified a Class B mutation that disrupts the function of the cytoplasmic dynein light intermediate chain gene xbx-1. Identification of the remaining mutations will reveal novel molecular pathways required for ciliary receptor localization and provide further insight into mechanisms of ciliary signaling.
Sekelsky, JJ, McKim KS, Messina L, French RL, Hurley WD, Arbel T, Chin GM, Deneen B, Force SJ, Hari KL et al..  1999.  Identification of novel Drosophila meiotic genes recovered in a P- element screen. Genetics. 152:529-42. AbstractWebsite
The segregation of homologous chromosomes from one another is the essence of meiosis. In many organisms, accurate segregation is ensured by the formation of chiasmata resulting from crossing over. Drosophila melanogaster females use this type of recombination-based system, but they also have mechanisms for segregating achiasmate chromosomes with high fidelity. We describe a P-element mutagenesis and screen in a sensitized genetic background to detect mutations that impair meiotic chromosome pairing, recombination, or segregation. Our screen identified two new recombination-deficient mutations: mei-P22, which fully eliminates meiotic recombination, and mei-P26, which decreases meiotic exchange by 70% in a polar fashion. We also recovered an unusual allele of the ncd gene, whose wild-type product is required for proper structure and function of the meiotic spindle. However, the screen yielded primarily mutants specifically defective in the segregation of achiasmate chromosomes. Although most of these are alleles of previously undescribed genes, five were in the known genes alphaTubulin67C, CycE, push, and Trl. The five mutations in known genes produce novel phenotypes for those genes.
Zhong, H, McCord R, Vershon AK.  1999.  Identification of Target Sites of the Alpha2-Mcm1 Repressor Complex in the Yeast Genome. Genome Res. 9:1040-1047. Abstract
The alpha2 and Mcm1 proteins bind DNA as a heterotetramer to repress transcription of cell-type-specific genes in the yeast Saccharomyces cerevisiae. Based on the DNA sequence requirements for binding by the alpha2-Mcm1 complex, we have searched the yeast genome for all potential alpha2-Mcm1 binding sites. Genes adjacent to the sites were examined for expression in the different cell mating types. These sites were further analyzed by cloning the sequences into a heterologous promoter and assaying for alpha2-Mcm1-dependent repression in vivo and DNA-binding affinity in vitro. Fifty-nine potential binding sites were identified in the search. Thirty-seven sites are located within or downstream of coding region of the gene. None of the sites assayed from this group are functional repressor sites in vivo or bound by the alpha2-Mcm1 complex in vitro. Among the remaining 22 sites, six are in the promoters of known alpha-specific genes and two other sites have an alpha2-Mcm1-dependent role in determining the direction of mating type switching. Among the remaining sequences, we have identified a functional site located in the promoter region of a previously uncharacterized gene, SCYJL170C. This site functions to repress transcription of a heterologous promoter and the alpha2-Mcm1 complex binds to the site in vitro. SCYJL170C is repressed by alpha2-Mcm1 in vivo and therefore using this method we have identified a new a-specific gene, which we call ASG7.
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.
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.
Meibom, KL, Kallipolitis BH, Ebright RH, Valentin-Hansen P.  2000.  Identification of the subunit of cAMP receptor protein (CRP) that functionally interacts with CytR in CRP-CytR-mediated transcriptional repression.. The Journal of biological chemistry. 275(16):11951-6. Abstract
At promoters of the Escherichia coli CytR regulon, the cAMP receptor protein (CRP) interacts with the repressor CytR to form transcriptionally inactive CRP-CytR-promoter or (CRP)(2)-CytR-promoter complexes. Here, using "oriented heterodimer" analysis, we show that only one subunit of the CRP dimer, the subunit proximal to CytR, functionally interacts with CytR in CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes. Our results provide information about the architecture of CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes and rule out the proposal that masking of activating region 2 of CRP is responsible for the transcriptional inactivity of the complexes.
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.
Messing, J, Llaca V.  1998.  Importance of anchor genomes for any plant genome project. Proceedings of the National Academy of Sciences of the United States of America. 95:2017-20. AbstractWebsite
Progress in agricultural and environmental technologies is hampered by a slower rate of gene discovery in plants than animals. The vast pool of genes in plants, however, will be an important resource for insertion of genes, via biotechnological procedures, into an array of plants, generating unique germ plasms not achievable by conventional breeding. It just became clear that genomes of grasses have evolved in a manner analogous to Lego blocks. Large chromosome segments have been reshuffled and stuffer pieces added between genes. Although some genomes have become very large, the genome with the fewest stuffer pieces, the rice genome, is the Rosetta Stone of all the bigger grass genomes. This means that sequencing the rice genome as anchor genome of the grasses will provide instantaneous access to the same genes in the same relative physical position in other grasses (e.g., corn and wheat), without the need to sequence each of these genomes independently. (i) The sequencing of the entire genome of rice as anchor genome for the grasses will accelerate plant gene discovery in many important crops (e.g., corn, wheat, and rice) by several orders of magnitudes and reduce research and development costs for government and industry at a faster pace. (ii) Costs for sequencing entire genomes have come down significantly. Because of its size, rice is only 12% of the human or the corn genome, and technology improvements by the human genome project are completely transferable, translating in another 50% reduction of the costs. (iii) The physical mapping of the rice genome by a group of Japanese researchers provides a jump start for sequencing the genome and forming an international consortium. Otherwise, other countries would do it alone and own proprietary positions.
Xu, A, Haines N, Dlugosz M, Rana NA, Takeuchi H, Haltiwanger RS, Irvine KD.  2007.  In vitro reconstitution of the modulation of Drosophila Notch-ligand binding by Fringe. The Journal of biological chemistry. 282:35153-62. AbstractWebsite
Notch signaling plays critical roles in animal development and physiology. The activation of Notch receptors by their ligands is modulated by Fringe-dependent glycosylation. Fringe catalyzes the addition of N-acetylglucosamine in a beta1,3 linkage onto O-fucose on epidermal growth factor-like domains. This modification of Notch by Fringe influences the binding of Notch ligands to Notch receptors. However, prior studies have relied on in vivo glycosylation, leaving unresolved the question of whether addition of N-acetylglucosamine is sufficient to modulate Notch-ligand interactions on its own, or whether instead it serves as a precursor to subsequent post-translational modifications. Here, we describe the results of in vitro assays using purified components of the Drosophila Notch signaling pathway. In vitro glycosylation and ligand binding studies establish that the addition of N-acetylglucosamine onto O-fucose in vitro is sufficient both to enhance Notch binding to the Delta ligand and to inhibit Notch binding to the Serrate ligand. Further elongation by galactose does not detectably influence Notch-ligand binding in vitro. Consistent with these observations, carbohydrate compositional analysis and mass spectrometry on Notch isolated from cells identified only N-acetylglucosamine added onto Notch in the presence of Fringe. These observations argue against models in which Fringe-dependent glycosylation modulates Notch signaling by acting as a precursor to subsequent modifications and instead establish the simple addition of N-acetylglucosamine as a basis for the effects of Fringe on Drosophila Notch-ligand binding.
Oh, H, Irvine KD.  2009.  In vivo analysis of Yorkie phosphorylation sites. Oncogene. 28:1916-27. AbstractWebsite
The co-activator Yorkie (Yki) mediates transcriptional regulation effected by the Drosophila Fat-Warts (Wts)-Hippo (Hpo) pathways. Yki is inhibited by Wts-mediated phosphorylation, and a Wts phosphorylation site at Ser168 has been identified. Here we identify two additional Wts phosphorylation sites on Yki, and examine the respective contribution of all three sites to Yki nuclear localization and activity. Our results show that although Ser168 is the most critical site, all three phosphorylation sites influence Yki localization and activity in vivo, and can be sites of regulation by Wts. Thus, investigations of the role of Yki and its mammalian homolog Yes-associated protein (YAP) in development and oncogenesis should include evaluations of additional sites. The WW domains of Yki are not required for its phosphorylation, but instead are positively required for its activity. We also identify two potential sites of phosphorylation by an unknown kinase, which could influence phosphorylation of Ser168 by Wts, suggesting that there are additional mechanisms for regulating Yki/YAP activity.
Carrieri, D, Ananyev GM, Brown T, Dismukes GC.  2007.  In vivo bicarbonate requirement for water oxidation by Photosystem II in the hypercarbonate-requiring cyanobacterium Arthrospira maxima. J Inorg Biochem. 101:1865-74. AbstractWebsite
While the presence of inorganic carbon in the form of (bi)carbonate has been known to be important for activity of Photosystem II (PSII), the vast majority of studies on this "bicarbonate effect" have been limited to in vitro studies of isolated thylakoid membranes and PSII complexes. Here we report an in vivo requirement for bicarbonate that is both reversible and selective for this anion for efficient water oxidation activity in the hypercarbonate-requiring cyanobacterium Arthrospira (Spirulina) maxima, originally isolated from highly alkaline soda lakes. Using a non-invasive internal probe of PSII charge separation (variable fluorescence), primary electron acceptor (Q(A)(-)/Q(A)) reoxidation rate, and flash-induced oxygen yield, we report the largest reversible bicarbonate effect on PSII activity ever observed, which is due to the requirement for bicarbonate at the water-oxidizing complex. Temporal separation of this donor side bicarbonate requirement from a smaller effect of bicarbonate on the Q(A)(-) reoxidation rate was observed. We expect the atypical way in which Arthrospira manages intracellular pH, sodium, and inorganic carbon concentrations relative to other cyanobacteria is responsible for this strong in vivo bicarbonate requirement.
Oh, H, Irvine KD.  2008.  In vivo regulation of Yorkie phosphorylation and localization. Development (Cambridge, England). 135:1081-8. AbstractWebsite
Yorkie (Yki), a transcription factor of the Fat and Hippo signaling pathways, is negatively regulated by the Warts kinase. Here, we use Phos-tag gels to characterize Warts-dependent phosphorylation of Yki in vivo, and show that Warts promotes phosphorylation of Yki at multiple sites. We also show that Warts inhibits Yki nuclear localization in vivo, and can promote binding of Yki to 14-3-3 proteins in cultured cells. In vivo assessment of the influence of individual upstream regulators of Warts reveals that some mutants (e.g. fat) have only partial effects on Yki phosphorylation, and weak effects on Yki localization, whereas other genotypes (e.g. ex fat double mutants) have stronger effects on both Yki phosphorylation and localization. We also identify serine 168 as a critical site through which negative regulation of Yki by Warts-mediated phosphorylation occurs, but find that this site is not sufficient to explain effects of Hippo signaling on Yki in vivo. These results identify modulation of subcellular localization as a mechanism of Yki regulation, and establish that this regulation occurs in vivo through multiple sites of Warts-dependent phosphorylation on Yki.
Qian, X, Kumaraswamy GK, Zhang S, Gates C, Ananyev GM, Bryant DA, Dismukes GC.  2015.  Inactivation of nitrate reductase alters metabolic branching of carbohydrate fermentation in the cyanobacterium Synechococcus sp. strain PCC 7002.. Biotechnol Bioeng. 113(5):979-988. Abstract
To produce cellular energy, cyanobacteria reduce nitrate as the preferred pathway over proton reduction (H2 evolution) by catabolizing glycogen under dark anaerobic conditions. This competition lowers H2 production by consuming a large fraction of the reducing equivalents (NADPH and NADH). To eliminate this competition, we constructed a knockout mutant of nitrate reductase, encoded by narB, in Synechococcus sp. PCC 7002. As expected, ΔnarB was able to take up intracellular nitrate but was unable to reduce it to nitrite or ammonia, and was unable to grow photoautotrophically on nitrate. During photoautotrophic growth on urea, ΔnarB significantly redirects biomass accumulation into glycogen at the expense of protein accumulation. During subsequent dark fermentation, metabolite concentrations-both the adenylate cellular energy charge (∼ATP) and the redox poise (NAD(P)H/NAD(P))-were independent of nitrate availability in ΔnarB, in contrast to the wild type (WT) control. The ΔnarB strain diverted more reducing equivalents from glycogen catabolism into reduced products, mainly H2 and d-lactate, by 6-fold (2.8% yield) and 2-fold (82.3% yield), respectively, than WT. Continuous removal of H2 from the fermentation medium (milking) further boosted net H2 production by 7-fold in ΔnarB, at the expense of less excreted lactate, resulting in a 49-fold combined increase in the net H2 evolution rate during 2 days of fermentation compared to the WT. The absence of nitrate reductase eliminated the inductive effect of nitrate addition on rerouting carbohydrate catabolism from glycolysis to the oxidative pentose phosphate (OPP) pathway, indicating that intracellular redox poise and not nitrate itself acts as the control switch for carbon flux branching between pathways.
Robins, H, Li Y, Padgett RW.  2005.  Incorporating structure to predict microRNA targets. Proceedings of the National Academy of Sciences of the United States of America. 102:4006-9. AbstractWebsite
MicroRNAs (miRNAs) are a recently discovered set of regulatory genes that constitute up to an estimated 1% of the total number of genes in animal genomes, including Caenorhabditis elegans, Drosophila, mouse, and humans [Lagos-Quintana, M., Rauhut, R., Lendeckel, W. & Tuschl, T. (2001) Science 294, 853-858; Lai, E. C., Tomancak, P., Williams, R. W. & Rubin, G.M. (2003) Genome Biol. 4, R42; Lau, N. C., Lim, L. P., Weinstein, E. G. & Bartel, D. P. (2001) Science 294, 858-862; Lee, R. C. & Ambros, V. (2001) Science 294, 862-8644; and Lee, R. C., Feinbaum, R. L. & Ambros, V. (1993) Cell 115, 787-798]. In animals, miRNAs regulate genes by attenuating protein translation through imperfect base pair binding to 3' UTR sequences of target genes. A major challenge in understanding the regulatory role of miRNAs is to accurately predict regulated targets. We have developed an algorithm for predicting targets that does not rely on evolutionary conservation. As one of the features of this algorithm, we incorporate the folded structure of mRNA. By using Drosophila miRNAs as a test case, we have validated our predictions in 10 of 15 genes tested. One of these validated genes is mad as a target for bantam. Furthermore, our computational and experimental data suggest that miRNAs have fewer targets than previously reported.
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)
Work, VH, Radakovits R, Jinkerson RE, Meuser JE, Elliott LG, Vinyard DJ, Laurens LML, Dismukes CG, Posewitz MC.  2010.  Increased Lipid Accumulation in the Chlamydomonas reinhardtii sta7-10 Starchless Isoamylase Mutant and Increased Carbohydrate Synthesis in Complemented Strains. Eukaryotic Cell. 9:1251-1261. AbstractWebsite
The accumulation of bioenergy carriers was assessed in two starchless mutants of Chlamydomonas reinhardtii (the sta6 [ADP-glucose pyrophosphorylase] and sta7-10 [isoamylase] mutants), a control strain (CC124), and two complemented strains of the sta7-10 mutant. The results indicate that the genetic blockage of starch synthesis in the sta6 and sta7-10 mutants increases the accumulation of lipids on a cellular basis during nitrogen deprivation relative to that in the CC124 control as determined by conversion to fatty acid methyl esters. However, this increased level of lipid accumulation is energetically insufficient to completely offset the loss of cellular starch that is synthesized by CC124 during nitrogen deprivation. We therefore investigated acetate utilization and O2 evolution to obtain further insights into the physiological adjustments utilized by the two starchless mutants in the absence of starch synthesis. The results demonstrate that both starchless mutants metabolize less acetate and have more severely attenuated levels of photosynthetic O2 evolution than CC124, indicating that a decrease in overall anabolic processes is a significant physiological response in the starchless mutants during nitrogen deprivation. Interestingly, two independent sta7-10:STA7 complemented strains exhibited significantly greater quantities of cellular starch and lipid than CC124 during acclimation to nitrogen deprivation. Moreover, the complemented strains synthesized significant quantities of starch even when cultured in nutrient-replete medium.
Lai, J, Messing J.  2002.  Increasing maize seed methionine by mRNA stability. The Plant journal : for cell and molecular biology. 30:395-402. AbstractWebsite
The amino acid methionine is a common protein building block that is also important in other cellular processes. Plants, unlike animals, synthesize methionine de novo and are thus a dietary source of this nutrient. A new approach for using maize as a source of nutrient methionine is described. Maize seeds, a major component of animal feeds, have variable levels of protein-bound methionine. This variability is a result of post-transcriptional regulation of the Dzs10 gene, which encodes a seed-specific high-methionine storage protein. Here we eliminate methionine variability by identifying and replacing the cis-acting site for Dzs10 regulation using transgenic seeds. Interestingly, two different mechanisms affect mRNA accumulation, one dependent on and the other independent of the untranslated regions (UTRs) of Dzs10 RNA. Accumulation of chimeric Dzs10 mRNA was not reduced in hybrid crosses and was uncoupled from genomic imprinting by Dzr1, a regulator of Dzs10. Uniform high levels of Dzs10 protein were maintained over five backcross generations of the transgene. The increased level of methionine in these transgenic seeds allowed the formulation of a useful animal feed ration without the addition of synthetic methionine.