Mycoparasitic interaction relieves binding of the Cre1 carbon catabolite repressor protein to promoter sequences of the ech42 (endochitinase-encoding) gene in Trichoderma harzianum

The fungus Trichoderma harzianum is a potent mycoparasite of various plant pathogenic fungi. We have studied the molecular regulation of mycoparasitism in the host/mycoparasite system Botrytis cinerea/T. harzianum. Protein extracts, prepared from various stages of mycoparasitism, were used in electrophoretic mobility-shift assays (EMSAs) with two promoter fragments of the ech-42 (42-kDa endochitinase-encoding) gene of T. harzianum. This gene was chosen as a model because its expression is triggered during mycoparasitic interaction [Carsolio, C., Gutierrez, A., Jimenez, B., van Montagu, M. & Herrera-Estrella, A. (1994) Proc. Natl. Acad. Sci. USA 91, 10903–10907]. All cell-free extracts formed high-molecular weight protein–DNA complexes, but those obtained from mycelia activated for mycoparasitic attack formed a complex with greater mobility. Competition experiments, using oligonucleotides containing functional and nonfunctional consensus sites for binding of the carbon catabolite repressor Cre1, provided evidence that the complex from nonmycoparasitic mycelia involves the binding of Cre1 to both fragments of the ech-42 promoter. The presence of two and three consensus sites for binding of Cre1 in the two ech-42 promoter fragments used is consistent with these findings. In contrast, the formation of the protein–DNA complex from mycoparasitic mycelia is unaffected by the addition of the competing oligonucleotides and hence does not involve Cre1. Addition of equal amounts of protein of cell-free extracts from nonmycoparasitic mycelia converted the mycoparasitic DNA–protein complex into the nonmycoparasitic complex. The addition of the purified Cre1::glutathione S-transferase protein to mycoparasitic cell-free extracts produced the same effect. These findings suggest that ech-42 expression in T. harzianum is regulated by (i) binding of Cre1 to two single sites in the ech-42 promoter, (ii) binding of a “mycoparasitic” protein–protein complex to the ech-42 promoter in vicinity of the Cre1 binding sites, and (iii) functional inactivation of Cre1 upon mycoparasitic interaction to enable the formation of the mycoparasitic protein–DNA complex.

Multiple independent transpositions of mitochondrial DNA control region sequences to the nucleus

Transpositions of mtDNA sequences to the nuclear genome have been documented in a wide variety of individual taxa, but little is known about their taxonomic frequency or patterns of variation. We provide evidence of nuclear sequences homologous to the mtDNA control region in seven species of diving ducks (tribe Aythyini). Phylogenetic analysis places each nuclear sequence as a close relative of the mtDNA haplotypes of the specie(s) in which it occurs, indicating that they derive from six independent transposition events, all occurring within the last ≈1.5 million years. Relative-rate tests and comparison of intraspecific variation in nuclear and mtDNA sequences confirm the expectation of a greatly reduced rate of evolution in the nuclear copies. By representing mtDNA haplotypes from ancestral populations, nuclear insertions may be valuable in some phylogenetic analyses, but they also confound the accurate determination of mtDNA sequences. In particular, our data suggest that the presumably nonfunctional but more slowly evolving nuclear sequences often will not be identifiable by changes incompatible with function and may be preferentially amplified by PCR primers based on mtDNA sequences from related taxa.

A herpes simplex virus 1 recombinant lacking the glycoprotein G coding sequences is defective in entry through apical surfaces of polarized epithelial cells in culture and in vivo

During infection of a new host, the first surfaces encountered by herpes simplex viruses are the apical membranes of epithelial cells of mucosal surfaces. These cells are highly polarized, and the protein composition of their apical and basolateral membranes are very different, so that different viral entry pathways have evolved for each surface. To determine whether the viral glycoprotein G (gG) is specifically required for efficient infection of a particular surface of polarized cells, apical and basal surfaces were infected with wild-type virus or a gG deletion mutant. After infection of polarized cells in culture, the gG− virus was deficient in infection of apical surfaces but was able to infect cells through basal membranes, replicate, and spread into surrounding cells. The gG-dependent step in apical infection was a stage beyond attachment. After in vivo infection of apical surfaces of epithelial cells of nonscarified mouse corneas, infection by glycoprotein C− or gG− virus was considerably reduced as compared with that observed after infection with wild-type virus. In contrast, when corneas were scarified, allowing virus access to other cell surfaces, the gG and glycoprotein C deletion mutants infected eyes as efficiently as wild-type viruses. A secondary mutation allowing infection of apical surfaces by gG− virus arose readily during passage of the virus in nonpolarized cells, indicating that either the gG-dependent step of apical infection can be bypassed or that another viral protein can acquire the same function.

Specific molecular recognition of mixed nucleic acid sequences: An aromatic dication that binds in the DNA minor groove as a dimer

Phenylamidine cationic groups linked by a furan ring (furamidine) and related compounds bind as monomers to AT sequences of DNA. An unsymmetric derivative (DB293) with one of the phenyl rings of furamidine replaced with a benzimidazole has been found by quantitative footprinting analyses to bind to GC-containing sites on DNA more strongly than to pure AT sequences. NMR structural analysis and surface plasmon resonance binding results clearly demonstrate that DB293 binds in the minor groove at specific GC-containing sequences of DNA in a highly cooperative manner as a stacked dimer. Neither the symmetric bisphenyl nor bisbenzimidazole analogs of DB293 bind significantly to the GC containing sequences. DB293 provides a paradigm for design of compounds for specific recognition of mixed DNA sequences and extends the boundaries for small molecule-DNA recognition.

Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene

The Arabidopsis thaliana NPR1 has been shown to be a key regulator of gene expression during the onset of a plant disease-resistance response known as systemic acquired resistance. The npr1 mutant plants fail to respond to systemic acquired resistance-inducing signals such as salicylic acid (SA), or express SA-induced pathogenesis-related (PR) genes. Using NPR1 as bait in a yeast two-hybrid screen, we identified a subclass of transcription factors in the basic leucine zipper protein family (AHBP-1b and TGA6) and showed that they interact specifically in yeast and in vitro with NPR1. Point mutations that abolish the NPR1 function in A. thaliana also impair the interactions between NPR1 and the transcription factors in the yeast two-hybrid assay. Furthermore, a gel mobility shift assay showed that the purified transcription factor protein, AHBP-1b, binds specifically to an SA-responsive promoter element of the A. thaliana PR-1 gene. These data suggest that NPR1 may regulate PR-1 gene expression by interacting with a subclass of basic leucine zipper protein transcription factors.

Transcription coupled repair of 8-oxoguanine in murine cells: The Ogg1 protein is required for repair in nontranscribed sequences but not in transcribed sequences

To assess the role of the Ogg1 DNA glycosylase in the transcription-coupled repair (TCR) of the mutagenic lesion, 7,8-dihydro-8oxoguanine (8-OxoG), we have investigated the removal of this lesion in wild-type and ogg1−/− null mouse embryo fibroblast (MEF) cell lines. We used nonreplicating plasmids containing a single 8-OxoG·C base pair in a different assay that allowed us to study the removal of 8-OxoG located in a transcribed sequence (TS) or in a nontranscribed sequence (NTS). The results show that the removal of 8-OxoG in a wild-type MEF cell line is faster in the TS than in the NTS, indicating TCR of 8-OxoG in murine cells. In the homozygous ogg1−/− MEF cell line, 8-OxoG was not removed from the NTS whereas there was still efficient 8-OxoG repair in the TS. Expression of the mouse Ogg1 protein in the homozygous ogg1−/− cell line restored the ability to remove 8-OxoG in the NTS. Therefore, we have demonstrated that Ogg1 is essential for the repair of 8-OxoG in the NTS but is not required in the TS. These results indicate the existence of an Ogg1-independent pathway for the TCR of 8-OxoG in vivo.

Native protein sequences are close to optimal for their structures

How large is the volume of sequence space that is compatible with a given protein structure? Starting from random sequences, low free energy sequences were generated for 108 protein backbone structures by using a Monte Carlo optimization procedure and a free energy function based primarily on Lennard–Jones packing interactions and the Lazaridis–Karplus implicit solvation model. Remarkably, in the designed sequences 51% of the core residues and 27% of all residues were identical to the amino acids in the corresponding positions in the native sequences. The lowest free energy sequences obtained for ensembles of native-like backbone structures were also similar to the native sequence. Furthermore, both the individual residue frequencies and the covariances between pairs of positions observed in the very large SH3 domain family were recapitulated in core sequences designed for SH3 domain structures. Taken together, these results suggest that the volume of sequence space optimal for a protein structure is surprisingly restricted to a region around the native sequence.

Independent and combined analyses of sequences from all three genomic compartments converge on the root of flowering plant phylogeny

Plant phylogenetic estimates are most likely to be reliable when congruent evidence is obtained independently from the mitochondrial, plastid, and nuclear genomes with all methods of analysis. Here, results are presented from separate and combined genomic analyses of new and previously published data, including six and nine genes (8,911 bp and 12,010 bp, respectively) for different subsets of taxa that suggest Amborella + Nymphaeales (water lilies) are the first-branching angiosperm lineage. Before and after tree-independent noise reduction, most individual genomic compartments and methods of analysis estimated the Amborella + Nymphaeales basal topology with high support. Previous phylogenetic estimates placing Amborella alone as the first extant angiosperm branch may have been misled because of a series of specific problems with paralogy, suboptimal outgroups, long-branch taxa, and method dependence. Ancestral character state reconstructions differ between the two topologies and affect inferences about the features of early angiosperms.

The incompatible interaction between Phytophthora parasitica var. nicotianae race 0 and tobacco is suppressed in transgenic plants expressing antisense lipoxygenase sequences

Nicotiana tabacum 46-8 cultivar displays an incompatible interaction with race 0 of Phytophthora parasitica var. nicotianae (Ppn), a fungal pathogen of most tobacco cultivars. At the plant level, incompatibility is characterized by the induction of lipoxygenase (LOX, EC = 1.13.11.12) activity and localized hypersensitive cell death before defense gene activation. To evaluate the involvement of LOX in the onset of plant defense, tobacco 46-8 plants were genetically engineered using full-length or partial-length antisense (AS) tobacco LOX cDNA constructs. AS expression strongly reduced elicitor- and pathogen-induced LOX activity. Eight independent AS-LOX lines were selected and assayed for their response to Ppn. After root or stem inoculation with race 0, all AS-LOX lines but one displayed a compatible phenotype whereas control transformed plants, not containing the AS-LOX cassette, showed the typical incompatible reaction. The presence of the fungus in transgenic lines was demonstrated by PCR amplification of a Ppn-specific genomic sequence. A linear relationship was found between the extent of LOX suppression and the size of the lesion caused by the fungus. The AS-LOX plants also showed enhanced susceptibility toward the compatible fungus Rhizoctonia solani. The results demonstrate the strong involvement of LOX in the establishment of incompatibility in plant–microorganism interactions, consistent with its role in the defense of host plants.

Mode matches and their locations in the hydrophobic free energy sequences of peptide ligands and their receptor eigenfunctions

Patterns in sequences of amino acid hydrophobic free energies predict secondary structures in proteins. In protein folding, matches in hydrophobic free energy statistical wavelengths appear to contribute to selective aggregation of secondary structures in “hydrophobic zippers.” In a similar setting, the use of Fourier analysis to characterize the dominant statistical wavelengths of peptide ligands’ and receptor proteins’ hydrophobic modes to predict such matches has been limited by the aliasing and end effects of short peptide lengths, as well as the broad-band, mode multiplicity of many of their frequency (power) spectra. In addition, the sequence locations of the matching modes are lost in this transformation. We make new use of three techniques to address these difficulties: (i) eigenfunction construction from the linear decomposition of the lagged covariance matrices of the ligands and receptors as hydrophobic free energy sequences; (ii) maximum entropy, complex poles power spectra, which select the dominant modes of the hydrophobic free energy sequences or their eigenfunctions; and (iii) discrete, best bases, trigonometric wavelet transformations, which confirm the dominant spectral frequencies of the eigenfunctions and locate them as (absolute valued) moduli in the peptide or receptor sequence. The leading eigenfunction of the covariance matrix of a transmembrane receptor sequence locates the same transmembrane segments seen in n-block-averaged hydropathy plots while leaving the remaining hydrophobic modes unsmoothed and available for further analyses as secondary eigenfunctions. In these receptor eigenfunctions, we find a set of statistical wavelength matches between peptide ligands and their G-protein and tyrosine kinase coupled receptors, ranging across examples from 13.10 amino acids in acid fibroblast growth factor to 2.18 residues in corticotropin releasing factor. We find that the wavelet-located receptor modes in the extracellular loops are compatible with studies of receptor chimeric exchanges and point mutations. A nonbinding corticotropin-releasing factor receptor mutant is shown to have lost the signatory mode common to the normal receptor and its ligand. Hydrophobic free energy eigenfunctions and their transformations offer new quantitative physical homologies in database searches for peptide-receptor matches.

Backtracking leukemia to birth: Identification of clonotypic gene fusion sequences in neonatal blood spots

Epidemiological evidence has suggested that some pediatric leukemias may be initiated in utero and, for some pairs of identical twins with concordant leukemia, this possibility has been strongly endorsed by molecular studies of clonality. Direct evidence for a prenatal origin can only be derived by prospective or retrospective detection of leukemia-specific molecular abnormalities in fetal or newborn samples. We report a PCR-based method that has been developed to scrutinize neonatal blood spots (Guthrie cards) for the presence of numerically infrequent leukemic cells at birth in individuals who subsequently developed leukemia. We demonstrate that unique or clonotypic MLL-AF4 genomic fusion sequences are present and detectable in neonatal blood spots from individuals who were diagnosed with acute lymphoblastic leukemia at ages 5 months to 2 years and, therefore, have arisen during fetal hematopoiesis in utero. This result provides unequivocal evidence for a prenatal initiation of acute leukemia in young patients. The method should be applicable to other fusion genes in children with common subtypes of leukemia and will be of value in attempts to unravel the natural history and etiology of this major subtype of pediatric cancer.

Arrayed transposase-binding sequences on the ends of transposon Tn5090/Tn402

The transposon Tn5090/Tn402 encodes a 559 amino acid transposase, TniA, with a DDE motif. Gel mobility shifting and cleavage protection analysis with DNase I and hydroxyl radical probes revealed that TniA binds to multiple repeat sequences on either terminus of Tn5090/Tn402. Four of these TniA-binding 19mers occurred on the left-hand (t) end and two on the right-hand (i) end. Hydroxyl radical cleavage protection demonstrated the presence of 3–6 bp contact sequences on one face of the DNA helix. The binding pattern and organisation of repeats suggested parallels between Tn5090/Tn402 and Mu, which controls its transpositional activity in the assembly step of a higher order transpososome complex. The complex terminal structure and genes of transposase and nucleotide-binding proteins in tandem are hallmarks of the handful of Mu-like elements that are known to date.

Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs

Escherichia coli mRNA translation is facilitated by sequences upstream and downstream of the initiation codon, called Shine–Dalgarno (SD) and downstream box (DB) sequences, respectively. In E.coli enhancing the complementarity between the DB sequences and the 16S rRNA penultimate stem resulted in increased protein accumulation without a significant affect on mRNA stability. The objective of this study was to test whether enhancing the complementarity of plastid mRNAs downstream of the AUG (downstream sequence or DS) with the 16S rRNA penultimate stem (anti-DS or ADS region) enhances protein accumulation. The test system was the tobacco plastid rRNA operon promoter fused with the E.coli phage T7 gene 10 (T7g10) 5′-untranslated region (5′-UTR) and DB region. Translation efficiency was tested by measuring neomycin phosphotransferase (NPTII) accumulation in tobacco chloroplasts. We report here that the phage T7g10 5′-UTR and DB region promotes accumulation of NPTII up to ∼16% of total soluble leaf protein (TSP). Enhanced mRNA stability and an improved NPTII yield (∼23% of TSP) was obtained from a construct in which the T7g10 5′-UTR was linked with the NPTII coding region via a NheI site. However, replacing the T7g10 DB region with the plastid DS sequence reduced NPTII and mRNA levels to 0.16 and 28%, respectively. Reduced NPTII accumulation is in part due to accelerated mRNA turnover.

Identification of sample-specific sequences in mammalian cDNA and genomic DNA by the novel ligation-mediated subtraction (Limes)

The representational difference analysis (RDA) and other subtraction techniques are used to enrich sample-specific sequences by elimination of ubiquitous sequences existing in both the sample of interest (tester) and the subtraction partner (driver). While applying the RDA to genomic DNA of cutaneous lymphoma cells in order to identify tumor relevant alterations, we predominantly isolated repetitive sequences and artificial repeat-mediated fusion products of otherwise independent PCR fragments (PCR hybrids). Since these products severely interfered with the isolation of tester-specific fragments, we developed a considerably more robust and efficient approach, termed ligation-mediated subtraction (Limes). In first applications of Limes, genomic sequences and/or transcripts of genes involved in the regulation of transcription, such as transforming growth factor β stimulated clone 22 related gene (TSC-22R), cell death and cytokine production (caspase-1) or antigen presentation (HLA class II sequences), were found to be completely absent in a cutaneous lymphoma line. On the assumption that mutations in tumor-relevant genes can affect their transcription pattern, a protocol was developed and successfully applied that allows the identification of such sequences. Due to these results, Limes may substitute/supplement other subtraction/comparison techniques such as RDA or DNA microarray techniques in a variety of different research fields.

Cis-acting requirements in flanking DNA for the programmed elimination of mse2.9: a common mechanism for deletion of internal eliminated sequences from the developing macronucleus of Tetrahymena thermophila

During macronuclear development in the ciliated protozoan Tetrahymena thermophila, extensive DNA deletions occur, eliminating thousands of internal eliminated sequences (IESs). Using an rDNA-based transformation assay we have analyzed the role during DNA deletion of DNA flanking mse2.9, an IES within the second intron of a gene encoding an as yet incompletely characterized protein. We establish that a cis-acting sequence for mse2.9 deletion acts at a distance to specify deletion boundaries. A complex sequence element necessary for efficient and accurate mse2.9 deletion is located in the region 47–81 bp from the right side of mse2.9. The ability of a variety of IES flanking sequences to rescue a processing deficient mse2.9 construct indicates that some cis-acting signal is shared among different IESs. In addition, the short intronic sequence that flanks mse2.9 is able to direct efficient and accurate processing. Despite no obvious sequence similarity between mse2.9 and other IESs, we suggest that a common mechanism is used to delete different families of IESs in Tetrahymena.