The two homeodomains of the ZmHox2a gene from maize originated as an internal gene duplication and have evolved different target site specificities.

The maize ZmHox2a gene encodes two homeodomains which originated by a 699 bp duplication within an ancestral precursor. The sequences of the two ZmHox2a homeodomains are highly diverged in the N-terminal arm, while residues in the helical part have mostly been conserved. We show here that both ZmHox2a homeodomains are functional DNA-binding motifs but exhibit different target site specificities. CASTing experiments reveal a TCCT motif recognized by HD1 but a GATC tetranucleotide as the recognition sequence of HD2. Mutation of the central nucleotides in both tetranucleotide core motifs abolishes DNA binding. A domain swap experiment indicates that target site specificity is achieved in a combinatorial manner by the contributions of the diverged N-terminal arms together with the slightly different recognition helices. Computer modelling suggests that K47 and H54 in the recognition helices preferentially contact the bases at the 3'-terminus of the tetranucleotide target sequences.     

DNA targeting and cleavage by an engineered metalloprotein dimer

Nature has illustrated through numerous examples that protein dimerization has structural and functional advantages. We previously reported the design and characterization of an engineered “metallohomeodomain” protein (C2) based on a chimera of the EF-hand Ca-binding motif and the helix–turn–helix motif of homeodomains (Lim and Franklin in Protein Sci. 15:2159–2165, 2004). This small metalloprotein binds the hard metal ions Ca(II) and Ln(III) and interacts with DNA with modest sequence preference and affinity, yet exhibits only residual DNA cleavage activity. Here we have achieved substantial improvement in function by constructing a covalent dimer of this C2 module (F2) to create a larger multidomain protein. As assayed via fluorescence spectroscopy, this F2 protein binds Ca(II) more avidly (25-fold) than C2 on a per-domain basis; in gel shift selection experiments, metallated F2 exhibits a specificity toward 5′-TAATTA-3′ sequences. Finally, Ca₂F2 cleaves plasmid DNA and generates a linear product in a Ca(II)-dependent way, unlike the CaC2 monomer. To the best of our knowledge this activation of Ca(II) in the context of an EF-hand binding motif is unique and represents a significant step forward in the design of artificial metallonucleases by utilizing biologically significant metal ions.     

Structural feature of sorghum chloroplast psbA gene and regulation effects of its 5'-noncoding region*

Comparative analysis reveals remarkahle homology between the sequences of bothpsbA gene nucleotides and the inferred amino acids of sorghum, a C₄ plant, and those of rice, a C₃ plant. The 5′-noncoding region of sorghumpsbA gene contains the conservative promoter elements, “—35” element and “—10” element, like the prokaryote and the promoter element, TATA box, like the eukaryote. As compared with that of the rice, an extra sequence of 7 bp is found in the leader sequence of the mRNA in the former. Using anin vitro system, it has been demonstrated that protein factor exists in sorghum chloroplast protein extract which specifically hinds to the 5′-noncoding region ofpsbA gene. Measurement of the expression of luciferase shows a 2–5 time greater reaction of the expression plasmids pALqs which contain leader region of sorghumpsbA gene than that of the expression plasmids pALqr which contain leader region of ricepsbA gene inE. coli. Project supported by the Chinese National “863” and “973” Projects     

Scherrer and Jost’s symposium: the gene concept in 2008

Reconsideration of the term “gene” should take into account (a) the potential clash between hierarchical levels of information discussed in the 1970s by Gregory Bateson, (b) the contrast between conventional and genome phenotypes discussed in the 1980s by Richard Grantham, and (c) the emergence in the 1990s of a new science—Evolutionary Bioinformatics—that views genomes as channels conveying multiple forms of information through the generations. From this perspective, there is conceptual continuity between the functional “gene” of Mendel and today’s GenBank sequences. If the function attributed to a gene can change specifically as the result of a DNA mutation, then the mutated part of DNA can be considered as part of the gene. Conversely, even if appearing to locate within a gene, a mutation that does not change the specific function is not part of the gene, although it may change some other function to which the DNA sequence contributes. This strict definition is impractical, but serves as a guide to more workable, context-dependent, definitions. The gene is either (1) The DNA sequence that is transcribed, (2) The latter plus the immediate 5′ and 3′ sequences that, when mutated, specifically affect the function, (3) The latter two, plus any remote sequences that, when mutated, specifically affect the function. Attempts, such as that of Scherrer and Jost, to redefine Mendel’s “gene,” may be too narrowly focused on regulation to the exclusion of other important themes.     

Characterization of Hoxd1 Protein–DNA-Binding Specificity Using Affinity Chromatography and Random DNA Oligomer Selection

1. Hoxd1 is member of the labial subfamily of Hox genes that has a conserved 60 amino acid homeodomain region. The homeodomain is an important determining factor in the binding of the protein to specific DNA sequence(s). DNA-binding specificity for the Hoxd1 protein has not been determined previously.2. We have employed a rapid affinity chromatography method to determine optimal DNA binding sequences for the 109 amino acid Hoxd1 peptide, comprising the homeodomain and the entire carboxy terminal region of the Hoxd1 protein.3. Labial Hox proteins have intrinsically weak DNA-binding activity that has been attributed to the nonbasic residues at positions 2 and 3 in the N-terminal arm of the homeodomain. The presence of the Hoxd1 carboxy terminal region negated the influence of the nonbasic residues and facilitated Hoxd1 DNA-binding specificity.4. DNA sequences bound to the Hoxd1 peptide-affinity column were separated from a random pool of oligonucleotide sequences by gradient elution and enriched by polymerase chain reaction. Preferred sequences were identified on 5w and 3 of a TAAT core, extending the binding site to T/AT/gTAATTGTA.5. Stability and specificity of optimal DNA-binding sequence for Hoxd1 homeodomain were determined by equilibrium and kinetic studies. Dissociation coefficient constant (K _D) was estimated to be 8.6 × 10^–9 M and the DNA–Hoxd1 homeodomain complex has a half life (t _1/2) of 12.7 min.6. A molecular model of Hoxd1 homeodomain–-DNA interaction based on the X-ray coordinates of Antennapedia homeodomain–DNA complex has revealed novel interactions of key Hoxd1 residues at the protein–DNA interface.     

Interchange of DNA-binding modes in the deformed and ultrabithorax homeodomains: a structural role for the N-terminal arm

The deformed (Dfd) and ultrabithorax (Ubx) homeoproteins regulate developmental gene expression in Drosophila melanogaster by binding to specific DNA sequences within its genome. DNA binding is largely accomplished via a highly conserved helix-turn-helix DNA-binding domain that is known as a homeodomain (HD). Despite nearly identical DNA recognition helices and similar target DNA sequence preferences, the in vivo functions of the two proteins are quite different. We have previously revealed differences between the two HDs in their interactions with DNA. In an effort to define the individual roles of the HD N-terminal arm and recognition helix in sequence-specific binding, we have characterized the structural details of two Dfd/Ubx chimeric HDs in complex with both the Dfd and Ubx-optimal-binding site sequences. We utilized hydroxyl radical cleavage of DNA to assess the positioning of the proteins on the binding sites. The effects of missing nucleosides and purine methylation on HD binding were also analyzed. Our results show that both the Dfd and Ubx HDs have similar DNA-binding modes when in complex with the Ubx-optimal site. There are subtle but reproducible differences in these modes that are completely interchanged when the Dfd N-terminal arm is replaced with the corresponding region of the Ubx HD. In contrast, we showed previously that the Dfd-optimal site sequence elicits a very different binding mode for the Ubx HD, while the Dfd HD maintains a mode similar to that elicited by the Ubx-optimal site. Our current methylation interference studies suggest that this alternate binding mode involves interaction of the Ubx N-terminal arm with the minor groove on the opposite face of DNA relative to the major groove that is occupied by the recognition helix. As judged by hydroxyl radical footprinting and the missing nucleoside experiment, it appears that interaction of the Ubx recognition helix with the DNA major groove is reduced. Replacing the Dfd N-terminal arm with that of Ubx does not elicit a complete interchange of the DNA-binding mode. Although the position of the chimera relative to DNA, as judged by hydroxyl radical footprinting, is similar to that of the Dfd HD, the missing nucleoside and methylation interference patterns resemble those of the Ubx HD. Repositioning of amino acid side-chains without wholesale structural alteration in the polypeptide appears to occur as a function of N-terminal arm identity and DNA-binding site sequence. Complete interchange of binding modes was achieved only by replacement of the Dfd N-terminal arm and the recognition helix plus 13 carboxyl-terminal residues with the corresponding residues of Ubx. The position of the N-terminal arm in the DNA minor groove appears to differ in a manner that depends on the two base-pair differences between the Dfd and Ubx-optimal-binding sites. Thus, N-terminal arm position dictates the binding mode and the interaction of the recognition helix with nucleosides in the major groove.     

Sequence preference in DNA binding: de novo designed helix–turn–helix metallopeptides recognize a family of DNA target sites

The DNA-binding behavior and target sequences of two designed metallopeptides have been investigated with an iterative electrophoresis mobility shift assay followed by PCR amplification, and by circular dichroism spectroscopy. Peptides P3W and P5b were designed based on the structural similarity of the helix–turn–helix motif of homeodomains and the EF-hand motifs of calmodulin, as previously described for P3W. Like P3W, P5b binds both Eu(III) (K _d=12.6±1.9 μM) and Ca(II) (K _d=70±8 μM) with reasonable affinity. Binding selection from a library of randomized 8-mer DNA oligonucleotide sequences identified one target family for CaP5b [5′-pur-T-pur-G-(G/C)-3′], and two target sites for CaP3W [5′-(A/T)-G-G-G-(T/C)-3′ and 5′-A-T-(G/T)-T-G-3′]. Circular dichroism studies indicate that unlike EuP3W, EuP5b is poorly folded in the absence of DNA. In the presence of DNA containing target-binding sites for both peptides, both EuP3W and EuP5b increase in helical content, in the latter case significantly. These results suggest that EuP5b binding to target DNA involves an induced-fit mechanism. These small chimeric metallopeptides have been found to bind selectively to DNA targets, analogous to natural protein–DNA interactions. This corroborates our earlier conclusions (J. Am. Chem. Soc. 125:6656, 2003) that sequence-preferential DNA cleavage by Ce(IV)P3W was due to sequence recognition. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Expression and function of the homeodomain-containing protein Hex in thyroid cells

The homeodomain-containing protein Hex (also named Prh) is expressed in primitive endoderm (during the early phases of development), in some endoderm-derived tissues and in endothelial and hematopoietic precursors. Hex expression is extinguished during terminal differentiation of endothelial and hematopoietic cells as well as in adult lung. Previous investigations have demonstrated that Hex is expressed during early thyroid gland development. No information has been reported on Hex expression in adult thyroid gland or on the function of this protein in follicular thyroid cells. These issues represent the focus of the present study. We demonstrate that Hex mRNA is present in rat and human adult thyroid gland as well as in differentiated follicular thyroid cell lines. In FRTL-5 cells TSH reduces Hex expression. In thyroid cell lines transformed by several oncogenes Hex expression is completely abolished. By using co-transfection assays we demonstrate that Hex is a repressor of the thyroglobulin promoter and that it is able to abolish the activating effects of both TTF-1 and Pax8. These data would suggest that Hex may play an important role in thyroid cell differentiation. Protein–DNA interaction experiments indicate that Hex is able to bind sites of the thyroglobulin promoter containing either the core sequence 5′-TAAT-3′ or 5′-CAAG-3′. The DNA binding specificity of the Hex homeodomain, therefore, is more ‘relaxed’ than that observed in the majority of other homeodomains.     

Influence of loop sequence on relative stability of bimolecular triplex DNA

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Directional cloning of native PCR products with preformed sticky ends (Autosticky PCR)

A novel method for the directional cloning of native PCR products was developed. Abasic sites in DNA templates make DNA polymerases stall at the site during synthesis of the complementary strand. Since the 5′ ends of PCR product strands contain built-in amplification primers, abasic sites within the primers result in the formation of 5′ single-stranded overhangs at the ends of the PCR product, enabling its direct ligation to a suitably cleaved cloning vector without any further modification. This “autosticky PCR” (AS-PCR) overcomes the problems caused by end sensitivity of restriction enzymes, or internal restriction sites within the amplified sequences, and enables the generation of essentially any desired 5′ overhang. Received: 11 August 1998 / Accepted: 2 October 1998     

DNA methylation pattern in pig in vivo produced embryos

DNA methylation/demethylation pattern, determined by 5-methylcytosine (5-MeC) immunostaining, was evaluated in porcine “in vivo” produced embryos from zygote up to the blastocyst stage. In one-cell stage embryos, only the maternal pronucleus showed a positive labeling whilst the paternal pronucleus showed almost no labeling. The intensity of labeling is high until the late morula stage. Blastocysts containing less than 100 cells showed the same intensity of labeling in both the inner cell mass (ICM) nuclei and the trophectodermal (TE) cell nuclei. Interestingly, with further cell multiplication, cells of the ICM became more intensively labeled when compared to TE cells. This distinct methylation pattern is even more profound in blastocysts containing about 200–300 cells and is not caused by the difference in the cell volume of ICM and TE cells.An erratum to this article can be found at     

Structural feature of sorghum chloroplastpsbA gene and regulation effects of its 5′-noncoding region

Comparative analysis reveals remarkahle homology between the sequences of bothpsbA gene nucleotides and the inferred amino acids of sorghum, a C₄ plant, and those of rice, a C₃ plant. The 5′-noncoding region of sorghumpsbA gene contains the conservative promoter elements, “—35” element and “—10” element, like the prokaryote and the promoter element, TATA box, like the eukaryote. As compared with that of the rice, an extra sequence of 7 bp is found in the leader sequence of the mRNA in the former. Using anin vitro system, it has been demonstrated that protein factor exists in sorghum chloroplast protein extract which specifically hinds to the 5′-noncoding region ofpsbA gene. Measurement of the expression of luciferase shows a 2–5 time greater reaction of the expression plasmids pALqs which contain leader region of sorghumpsbA gene than that of the expression plasmids pALqr which contain leader region of ricepsbA gene inE. coli.     

Conservation in wheat high-molecular-weight glutenin gene promoter sequences: comparisons among loci and among alleles of the GLU-B1-1 locus

 The high-molecular-weight glutenin (HMW) genes and encoded subunits are known to be critical for wheat quality characteristics and are among the best-studied cereal research subjects. Two lines of experiments were undertaken to further understand the structure and high expression levels of the HMW-glutenin gene promoters. Cross hybridizations of clones of the paralogous x-type and y-type HMW-glutenin genes to a complete set of six genes from a single cultivar showed that each type hybridizes best within that type. The extent of hybridization was relatively restricted to the coding and immediate flanking DNA sequences. Additional DNA sequences were determined for four published members of the HMW-glutenin gene family (encoding subunits Ax2^*, Bx7, Dx5, and Dy10) and showed that the flanking DNA of the examined genes diverge at approximately −1200 bp 5′ to the start codon and 200–400 bp 3′ to the stop codon. These divergence sites may indicate the boundaries of sequences important in gene expression. In addition, promoter sequences were determined for alleles of the Bx gene (Glu-B1-1), a gene reported to show higher levels of expression than other HMW-glutenin genes and with variation among cultivars. The sequences of Bx promoters from three cultivars and one wild tetraploid wheat indicated that all Bx alleles had few differences and contained a duplicated portion of the promoter sequence “cereal-box” previously suspected as a factor in higher levels of expression. Thus, the “cereal-box” duplication preceeded the origin of hexaploid wheat, and provides no evidence to explain the variations in Bx subunit synthesis levels. One active Bx allele contained a 185-bp insertion that evidently resulted from a transposition event. Received: 5 August 1997 / Accepted: 6 November 1997     

Significance of Stereochemistry of 3′-Terminal Phosphorothioate-modified Primer in DNA Polymerase-mediated Chain Extension

Influence of stereochemistry of the 3′-terminal phosphorothioate (PS)-modified primers was studied in a single base extension (SBE) assay to evaluate any improvements in specificity. SBE reactions were catalyzed by members of the high fidelity Pfu family of DNA polymerases with (exo+) or without (exo−) 3′ → 5′ exonucleolytic activity. The diastereomerically pure PS-labeled primers used in these studies were obtained either by the stereospecific chemical synthesis invented in our laboratory or by the more conventional ion-exchange chromatographic method for separation of a mixture of diastereomers (R_P and S_P). When the SBE reaction was performed in the presence of mispaired 2′-deoxyribonucleoside triphosphates (dNTPs), the “racemic” 3′-phosphorothioate primer mixture resulted in a lower level of 3′ → 5′ exonuclease-mediated cleavage products in comparison to the SBE reactions carried out with the corresponding unmodified primer. When the diastereomerically pure R_P 3′-phosphorothioate primer was examined, the results were largely the same as for the racemic 3′-phosphorothioate primer mixture. In contrast, a 3′-PS primer of S_P configuration displayed significantly improved performance in the SBE reaction. This included the lack of 3′ → 5′ proofreading products, less mispriming, and improved yield of incorporation of the correct nucleotide.     

Hemidesmosome Formation Is Initiated by the β4 Integrin Subunit,Requires Complex Formation of β4 and HD1/Plectin,and Involves a Direct Interaction between β4 and the Bullous Pemphigoid Antigen 180

Hemidesmosomes (HDs) are stable anchoring structures that mediate the link between the intermediate filament cytoskeleton and the cell substratum. We investigated the contribution of various segments of the β4 integrin cytoplasmic domain in the formation of HDs in transient transfection studies using immortalized keratinocytes derived from an epidermolysis bullosa patient deficient in β4 expression. We found that the expression of wild-type β4 restored the ability of the β4-deficient cells to form HDs and that distinct domains in the NH₂- and COOH-terminal regions of the β4 cytoplasmic domain are required for the localization of HD1/plectin and the bullous pemphigoid antigens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the β4 cytoplasmic domain was dispensable for HD formation, although it may be involved in the efficient localization of BP180. Using the yeast two-hybrid system, we could demonstrate a direct interaction between β4 and BP180 which involves sequences within the COOH-terminal part of the CS and the third fibronectin type III (FNIII) repeat. Immunoprecipitation studies using COS-7 cells transfected with cDNAs for α6 and β4 and a mutant BP180 which lacks the collagenous extracellular domain confirmed the interaction of β4 with BP180. Nevertheless, β4 mutants which contained the BP180-binding region, but lacked sequences required for the localization of HD1/plectin, failed to localize BP180 in HDs. Additional yeast two- hybrid assays indicated that the 85 COOH-terminal residues of β4 can interact with the first NH₂-terminal pair of FNIII repeats and the CS, suggesting that the cytoplasmic domain of β4 is folded back upon itself. Unfolding of the cytoplasmic domain may be part of a mechanism by which the interaction of β4 with other hemidesmosomal components, e.g., BP180, is regulated.