PU.1 as an essential activator for the expression of gp91phox gene in human peripheral neutrophils, monocytes, and B lymphocytes

We have reported a deficiency of a 91-kDa glycoprotein component of the phagocyte NADPH oxidase (gp91phox) in neutrophils, monocytes, and B lymphocytes of a patient with X chromosome-linked chronic granulomatous disease. Sequence analysis of his gp91phox gene revealed a single-base mutation (C → T) at position −53. Electrophoresis mobility-shift assays showed that both PU.1 and hematopoietic-associated factor 1 (HAF-1) bound to the inverted PU.1 consensus sequence centered at position −53 of the gp91phox promoter, and the mutation at position −53 strongly inhibited the binding of both factors. It was also indicated that a mutation at position −50 strongly inhibited PU.1 binding but hardly inhibited HAF-1 binding, and a mutation at position −56 had an opposite binding specificity for these factors. In transient expression assay using HEL cells, which express PU.1 and HAF-1, the mutations at positions −53 and −50 significantly reduced the gp91phox promoter activity; however, the mutation at position −56 did not affect the promoter activity. In transient cotransfection study, PU.1 dramatically activated the gp91phox promoter in Jurkat T cells, which originally contained HAF-1 but not PU.1. In addition, the single-base mutation (C → T) at position −52 that was identified in a patient with chronic granulomatous disease inhibited the binding of PU.1 to the promoter. We therefore conclude that PU.1 is an essential activator for the expression of gp91phox gene in human neutrophils, monocytes, and B lymphocytes.

The MITE family Heartbreaker (Hbr): Molecular markers in maize

Transposable elements are ubiquitous in plant genomes, where they frequently comprise the majority of genomic DNA. The maize genome, which is believed to be structurally representative of large plant genomes, contains single genes or small gene islands interspersed with much longer blocks of retrotransposons. Given this organization, it would be desirable to identify molecular markers preferentially located in genic regions. In this report, the features of a newly described family of miniature inverted repeat transposable elements (MITEs) (called Heartbreaker), including high copy number and polymorphism, stability, and preference for genic regions, have been exploited in the development of a class of molecular markers for maize. To this end, a modification of the AFLP procedure called transposon display was used to generate and display hundreds of genomic fragments anchored in Hbr elements. An average of 52 markers were amplified for each primer combination tested. In all, 213 polymorphic fragments were reliably scored and mapped in 100 recombinant inbred lines derived from a cross between the maize inbreds B73 × Mo17. In this mapping population, Hbr markers are distributed evenly across the 10 maize chromosomes. This procedure should be of general use in the development of markers for other MITE families in maize and in other plant and animal species where MITEs have been identified.

Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein

There is still a lack of information on the specific characteristics of DNA-binding proteins from hyperthermophiles. Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archaeon Sulfolobus islandicus. orf56 has not been characterised yet but low sequence similarily to several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein. The DNA-binding properties of ORF56, expressed in Escherichia coli, have been investigated by EMSA experiments and by fluorescence anisotropy measurements. Recombinant ORF56 binds to double-stranded DNA, specifically to an inverted repeat located within the promoter of orf56. Binding to this site could down-regulate transcription of the orf56 gene and also of the overlapping orf904 gene, encoding the putative initiator protein of plasmid replication. By gel filtration and chemical crosslinking we have shown that ORF56 is a dimeric protein. Stoichiometric fluorescence anisotropy titrations further indicate that ORF56 binds as a tetramer to the inverted repeat of its target binding site. CD spectroscopy points to a significant increase in ordered secondary structure of ORF56 upon binding DNA. ORF56 binds without apparent cooperativity to its target DNA with a dissociation constant in the nanomolar range. Quantitative analysis of binding isotherms performed at various salt concentrations and at different temperatures indicates that approximately seven ions are released upon complex formation and that complex formation is accompanied by a change in heat capacity of –6.2 kJ/mol. Furthermore, recombinant ORF56 proved to be highly thermostable and is able to bind DNA up to 85°C.

Solvent effects on the energy landscapes and folding kinetics of polyalanine

The effect of a solvation on the thermodynamics and kinetics of polyalanine (Ala12) is explored on the basis of its energy landscapes in vacuum and in an aqueous solution. Both energy landscapes are characterized by two basins, one associated with α-helical structures and the other with coil and β-structures of the peptide. In both environments, the basin that corresponds to the α-helical structure is considerably narrower than the basin corresponding to the β-state, reflecting their different contributions to the entropy of the peptide. In vacuum, the α-helical state of Ala12 constitutes the native state, in agreement with common helical propensity scales, whereas in the aqueous medium, the α-helical state is destabilized, and the β-state becomes the native state. Thus solvation has a dramatic effect on the energy landscape of this peptide, resulting in an inverted stability of the two states. Different folding and unfolding time scales for Ala12 in hydrophilic and hydrophobic chemical environments are caused by the higher entropy of the native state in water relative to vacuum. The concept of a helical propensity has to be extended to incorporate environmental solvent effects.

Decrease in Phosphoribulokinase Activity by Antisense RNA in Transgenic Tobacco. Relationship between Photosynthesis, Growth, and Allocation at Different Nitrogen Levels1

To study the direct effects of photosynthesis on allocation of biomass by altering photosynthesis without altering leaf N or nitrate content, phosphoribulokinase (PRK) activity was decreased in transgenic tobacco (Nicotiana tabacum L.) with an inverted tobacco PRK cDNA and plants were grown at different N levels (0.4 and 5 mm NH4NO3). The activation state of PRK increased as the amount of enzyme was decreased genetically at both levels of N. At high N a 94% decrease in PRK activity had only a small effect (20%) on photosynthesis and growth. At low N a 94% decrease in PRK activity had a greater effect on leaf photosynthesis (decreased by up to 50%) and whole-plant photosynthesis (decreased by up to 35%) than at high N. These plants were up to 35% smaller than plants with higher PRK activities because they had less structural dry matter and less starch, which was decreased by 3- to 4-fold, but still accumulated to 24% to 31% of dry weight; young leaves contained more starch than older leaves in older plants. Leaves had a higher ion and water content, and specific leaf area was higher, but allocation between shoot and root was unaltered. In conclusion, low N in addition to a 94% decrease in PRK by antisense reduces the activity of PRK sufficient to diminish photosynthesis, which limits biomass production under conditions normally considered sink limited.

Dynamics of mobile element activity in chalcone synthase loci in the common morning glory (Ipomoea purpurea)

Mobile element dynamics in seven alleles of the chalcone synthase D locus (CHS-D) of the common morning glory (Ipomoea purpurea) are analyzed in the context of synonymous nucleotide sequence distances for CHS-D exons. By using a nucleotide sequence of CHS-D from the sister species Ipomoea nil (Japanese morning glory [Johzuka-Hisatomi, Y., Hoshino, A., Mori, T., Habu, Y. & Iida, S. (1999) Genes Genet. Syst. 74, 141–147], it is also possible to determine the relative frequency of insertion and loss of elements within the CHS-D locus between these two species. At least four different types of transposable elements exist upstream of the coding region, or within the single intron of the CHS-D locus in I. purpurea. There are three distinct families of miniature inverted-repeat transposable elements (MITES), and some recent transpositions of Activator/Dissociation (Ac/Ds)-like elements (Tip100), of some short interspersed repetitive elements (SINEs), and of an insertion sequence (InsIpCHSD) found in the neighborhood of this locus. The data provide no compelling evidence of the transposition of the mites since the separation of I. nil and I. purpurea roughly 8 million years ago. Finally, it is shown that the number and frequency of mobile elements are highly heterogeneous among different duplicate CHS loci, suggesting that the dynamics observed at CHS-D are locus-specific.

Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands

The crystal structures of the ligand-binding domain (LBD) of the vitamin D receptor complexed to 1α,25(OH)2D3 and the 20-epi analogs, MC1288 and KH1060, show that the protein conformation is identical, conferring a general character to the observation first made for retinoic acid receptor (RAR) that, for a given LBD, the agonist conformation is unique, the ligands adapting to the binding pocket. In all complexes, the A- to D-ring moieties of the ligands adopt the same conformation and form identical contacts with the protein. Differences are observed only for the 17β-aliphatic chains that adapt their conformation to anchor the 25-hydroxyl group to His-305 and His-397. The inverted geometry of the C20 methyl group induces different paths of the aliphatic chains. The ligands exhibit a low-energy conformation for MC1288 and a more strained conformation for the two others. KH1060 compensates this energy cost by additional contacts. Based on the present data, the explanation of the superagonist effect is to be found in higher stability and longer half-life of the active complex, thereby excluding different conformations of the ligand binding domain.

Top-down free-energy minimization on protein potential energy landscapes

The hierarchical properties of potential energy landscapes have been used to gain insight into thermodynamic and kinetic properties of protein ensembles. It also may be possible to use them to direct computational searches for thermodynamically stable macroscopic states, i.e., computational protein folding. To this end, we have developed a top-down search procedure in which conformation space is recursively dissected according to the intrinsic hierarchical structure of a landscape's effective-energy barriers. This procedure generates an inverted tree similar to the disconnectivity graphs generated by local minima-clustering methods, but it fundamentally differs in the manner in which the portion of the tree that is to be computationally explored is selected. A key ingredient is a branch-selection algorithm that takes advantage of statistically predictive properties of the landscape to guide searches down the tree branches that are most likely to lead to the physically relevant macroscopic states. Using the computational folding of a β-hairpin-forming peptide as an example, we show that such predictive properties indeed exist and can be used for structure prediction by free-energy global minimization.

Tomato Ve disease resistance genes encode cell surface-like receptors

In tomato, Ve is implicated in race-specific resistance to infection by Verticillium species causing crop disease. Characterization of the Ve locus involved positional cloning and isolation of two closely linked inverted genes. Expression of individual Ve genes in susceptible potato plants conferred resistance to an aggressive race 1 isolate of Verticillium albo-atrum. The deduced primary structure of Ve1 and Ve2 included a hydrophobic N-terminal signal peptide, leucine-rich repeats containing 28 or 35 potential glycosylation sites, a hydrophobic membrane-spanning domain, and a C-terminal domain with the mammalian E/DXXXLφ or YXXφ endocytosis signals (φ is an amino acid with a hydrophobic side chain). A leucine zipper-like sequence occurs in the hydrophobic N-terminal signal peptide of Ve1 and a Pro-Glu-Ser-Thr (PEST)-like sequence resides in the C-terminal domain of Ve2. These structures suggest that the Ve genes encode a class of cell-surface glycoproteins with receptor-mediated endocytosis-like signals and leucine zipper or PEST sequences.

Light regulation of type IV pilus-dependent motility by chemosensor-like elements in Synechocystis PCC6803

To optimize photosynthesis, cyanobacteria move toward or away from a light source by a process known as phototaxis. Phototactic movement of the cyanobacterium Synechocystis PCC6803 is a surface-dependent phenomenon that requires type IV pili, cellular appendages implicated in twitching and social motility in a range of bacteria. To elucidate regulation of cyanobacterial motility, we generated transposon-tagged mutants with aberrant phototaxis; mutants were either nonmotile or exhibited an “inverted motility response” (negative phototaxis) relative to wild-type cells. Several mutants contained transposons in genes similar to those involved in bacterial chemotaxis. Synechocystis PCC6803 has three loci with chemotaxis-like genes, of which two, Tax1 and Tax3, are involved in phototaxis. Transposons interrupting the Tax1 locus yielded mutants that exhibited an inverted motility response, suggesting that this locus is involved in controlling positive phototaxis. However, a strain null for taxAY1 was nonmotile and hyperpiliated. Interestingly, whereas the C-terminal region of the TaxD1 polypeptide is similar to the signaling domain of enteric methyl-accepting chemoreceptor proteins, the N terminus has two domains resembling chromophore-binding domains of phytochrome, a photoreceptor in plants. Hence, TaxD1 may play a role in perceiving the light stimulus. Mutants in the Tax3 locus are nonmotile and do not make type IV pili. These findings establish links between chemotaxis-like regulatory elements and type IV pilus-mediated phototaxis.

Rolling-circle transposons in eukaryotes

All eukaryotic DNA transposons reported so far belong to a single category of elements transposed by the so-called “cut-and-paste” mechanism. Here, we report a previously unknown category of eukaryotic DNA transposons, Helitron, which transpose by rolling-circle replication. Autonomous Helitrons encode a 5′-to-3′ DNA helicase and nuclease/ligase similar to those encoded by known rolling-circle replicons. Helitron-like transposons have conservative 5′-TC and CTRR-3′ termini and do not have terminal inverted repeats. They contain 16- to 20-bp hairpins separated by 10–12 nucleotides from the 3′-end and transpose precisely between the 5′-A and T-3′, with no modifications of the AT target sites. Together with their multiple diverged nonautonomous descendants, Helitrons constitute ≈2% of both the Arabidopsis thaliana and Caenorhabditis elegans genomes and also colonize the Oriza sativa genome. Sequence conservation suggests that Helitrons continue to be transposed.

The highly recombinogenic bz locus lies in an unusually gene-rich region of the maize genome

The bronze (bz) locus exhibits the highest rate of recombination of any gene in higher plants. To investigate the possible basis of this high rate of recombination, we have analyzed the physical organization of the region around the bz locus. Two adjacent bacterial artificial chromosome clones, comprising a 240-kb contig centered around the Bz-McC allele, were isolated, and 60 kb of contiguous DNA spanning the two bacterial artificial chromosome clones was sequenced. We find that the bz locus lies in an unusually gene-rich region of the maize genome. Ten genes, at least eight of which are shown to be transcribed, are contained in a 32-kb stretch of DNA that is uninterrupted by retrotransposons. We have isolated nearly full length cDNAs corresponding to the five proximal genes in the cluster. The average intertranscript distance between them is just 1 kb, revealing a surprisingly compact packaging of adjacent genes in this part of the genome. At least 11 small insertions, including several previously described miniature inverted repeat transposable elements, were detected in the introns and 3′ untranslated regions of genes and between genes. The gene-rich region is flanked at the proximal and distal ends by retrotransposon blocks. Thus, the maize genome appears to have scattered regions of high gene density similar to those found in other plants. The unusually high rate of intragenic recombination seen in bz may be related to the very high gene density of the region.

Transcriptional regulation of the Rhodococcus rhodochrous J1 nitA gene encoding a nitrilase.

The 1.4-kb downstream region from a nitrilase gene (nitA) of an actinomycete Rhodococcus rhodochrous J1, which is industrially in use, was found to be required for the isovaleronitrile-dependent induction of nitrilase synthesis in experiments using a Rhodococcus-Escherichia coli shuttle vector pK4 in a Rhodococcus strain. Sequence analysis of the 1.4-kb region revealed the existence of an open reading frame (nitR) of 957 bp, which would encode a protein with a molecular mass of 35,100. Deletion of the central and 3'-terminal portion of nitR resulted in the complete loss of nitrilase activity, demonstrating that nitR codes for a transcriptional positive regulator in nitA expression. The deduced amino acid sequence of nitR showed similarity to a positive regulator family including XylS from Pseudomonas putida and AraC from E. coli. By Northern blot analysis, the 1.4-kb transcripts for nitA were detected in R. rhodochrous J1 cells cultured in the presence of isovaleronitrile, but not those cultured in the absence of isovaleronitrile. The transcriptional start site for nitA was mapped to a C residue located 26 bp upstream of its translational start site. Deletion analysis to define the nitA promoter region suggested the possible participation of an inverted repeat sequence, centered on base pair -52, in induction of nitA transcription.