Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector

Small molecule-regulated transcription has broad utility and would benefit from an easily delivered self-contained regulatory cassette capable of robust, tightly controlled target gene expression. We describe the delivery of a modified dimerizer-regulated gene expression system to cells on a single retrovirus. A transcription factor cassette responsive to the natural product dimerizer rapamycin was optimized for retroviral delivery by fusing a highly potent chimeric activation domain to the rapamycin-binding domain of FKBP-rapamycin-associated protein (FRAP). This improvement led to an increase in both the potency and maximal levels of gene expression induced by rapamycin, or nonimmunosuppressive rapamycin analogs. The modified transcription factor cassette was incorporated along with a target gene into a single rapamycin-responsive retrovirus. Cell pools stably transduced with the single virus system displayed negligible basal expression and gave induction ratios of at least three orders of magnitude in the presence of rapamycin or a nonimmunosuppressive rapamycin analog. Levels of induced gene expression were comparable to those obtained with the constitutive retroviral long terminal repeat and the single virus system performed well in four different mammalian cell lines. Regulation with the dimerizer-responsive retrovirus was tight enough to allow the generation of cell lines displaying inducible expression of the highly toxic diphtheria toxin A chain gene. The ability to deliver the tightly inducible rapamycin system in a single retrovirus should facilitate its use in the study of gene function in a broad range of cell types.

Quantitative assessment of enzyme specificity in vivo: P2 recognition by Kex2 protease defined in a genetic system

The specificity of the yeast proprotein-processing Kex2 protease was examined in vivo by using a sensitive, quantitative assay. A truncated prepro-α-factor gene encoding an α-factor precursor with a single α-factor repeat was constructed with restriction sites for cassette mutagenesis flanking the single Kex2 cleavage site (-SLDKR↓EAEA-). All of the 19 substitutions for the Lys (P2) residue in the cleavage site were made. The wild-type and mutant precursors were expressed in a yeast strain lacking the chromosomal genes encoding Kex2 and prepro-α-factor. Cleavage of the 20 sites by Kex2, expressed at the wild-type level, was assessed by using a quantitative-mating assay with an effective range greater than six orders of magnitude. All substitutions for Lys at P2 decreased mating, from 2-fold for Arg to >106-fold for Trp. Eviction of the Kex2-encoding plasmid indicated that cleavage of mutant sites by other cellular proteases was not a complicating factor. Mating efficiencies of strains expressing the mutant precursors correlated well with the specificity (kcat/KM) of purified Kex2 for comparable model peptide substrates, validating the in vivo approach as a quantitative method. The results support the conclusion that KM, which is heavily influenced by the nature of the P2 residue, is a major determinant of cleavage efficiency in vivo. P2 preference followed the rank order: Lys > Arg > Thr > Pro > Glu > Ile > Ser > Ala > Asn > Val > Cys > AsP > Gln > Gly > His > Met > Leu > Tyr > Phe > Trp.

A resistant genetic background leading to incomplete penetrance of intestinal neoplasia and reduced loss of heterozygosity in ApcMin/+ mice

Previous studies of Min/+ (multiple intestinal neoplasia) mice on a sensitive genetic background, C57BL/6 (B6), showed that adenomas have lost heterozygosity for the germ-line ApcMin mutation in the Apc (adenomatous polyposis coli) gene. We now report that on a strongly resistant genetic background, AKR/J (AKR), Min-induced adenoma multiplicity is reduced by about two orders of magnitude compared with that observed on the B6 background. Somatic treatment with a strong mutagen increases tumor number in AKR Min/+ mice in an age-dependent manner, similar to results previously reported for B6 Min/+ mice. Immunohistochemical analyses indicate that Apc expression is suppressed in all intestinal tumors from both untreated and treated AKR Min/+ mice. However, the mechanism of Apc inactivation in AKR Min/+ mice often differs from that observed for B6 Min/+ mice. Although loss of heterozygosity is observed in some tumors, a significant percentage of tumors showed neither loss of heterozygosity nor Apc truncation mutations. These results extend our understanding of the effects of genetic background on Min-induced tumorigenesis in several ways. First, the AKR strain carries modifiers of Min in addition to Mom1. This combination of AKR modifiers can almost completely suppress spontaneous intestinal tumorigenesis associated with the Min mutation. Second, even on such a highly resistant genetic background, tumor formation continues to involve an absence of Apc function. The means by which Apc function is inactivated is affected by genetic background. Possible scenarios are discussed.

The use of mRNA display to select high-affinity protein-binding peptides

We report the use of “mRNA display,” an in vitro selection technique, to identify peptide aptamers to a protein target. mRNA display allows for the preparation of polypeptide libraries with far greater complexity than is possible with phage display. Starting with a library of ≈1013 random peptides, 20 different aptamers to streptavidin were obtained, with dissociation constants as low as 5 nM. These aptamers function without the aid of disulfide bridges or engineered scaffolds, yet possess affinities comparable to those for monoclonal antibody–antigen complexes. The aptamers bind streptavidin with three to four orders of magnitude higher affinity than those isolated previously by phage display from lower complexity libraries of shorter random peptides. Like previously isolated peptides, they contain an HPQ consensus motif. This study shows that, given sufficient length and diversity, high-affinity aptamers can be obtained even from random nonconstrained peptide libraries. By engineering structural constraints into these ultrahigh complexity peptide libraries, it may be possible to produce binding agents with subnanomolar binding constants.

Formation of temporal-feature maps by axonal propagation of synaptic learning

Computational maps are of central importance to a neuronal representation of the outside world. In a map, neighboring neurons respond to similar sensory features. A well studied example is the computational map of interaural time differences (ITDs), which is essential to sound localization in a variety of species and allows resolution of ITDs of the order of 10 μs. Nevertheless, it is unclear how such an orderly representation of temporal features arises. We address this problem by modeling the ontogenetic development of an ITD map in the laminar nucleus of the barn owl. We show how the owl's ITD map can emerge from a combined action of homosynaptic spike-based Hebbian learning and its propagation along the presynaptic axon. In spike-based Hebbian learning, synaptic strengths are modified according to the timing of pre- and postsynaptic action potentials. In unspecific axonal learning, a synapse's modification gives rise to a factor that propagates along the presynaptic axon and affects the properties of synapses at neighboring neurons. Our results indicate that both Hebbian learning and its presynaptic propagation are necessary for map formation in the laminar nucleus, but the latter can be orders of magnitude weaker than the former. We argue that the algorithm is important for the formation of computational maps, when, in particular, time plays a key role.

Substrate-specific inhibition of RecQ helicase

The RecQ helicases constitute a small but highly conserved helicase family. Proteins in this family are of particular interest because they are critical to maintenance of genomic stability in prokaryotes and eukaryotes. Eukaryotic RecQ helicase family members have been shown to unwind not only DNA duplexes but also DNAs with alternative structures, including structures stabilized by G quartets (G4 DNAs). We report that Escherichia coli RecQ can also unwind G4 DNAs, and that unwinding requires ATP and divalent cation. RecQ helicase is comparably active on duplex and G4 DNA substrates, as measured by direct comparison of protein activity and by competition assays. The porphyrin derivative, N-methyl mesoporphyrin IX (NMM), is a highly specific inhibitor of RecQ unwinding activity on G4 DNA but not duplex DNA: the inhibition constant (Ki) for NMM inhibition of G4 DNA unwinding is 1.7 µM, approximately two orders of magnitude below the Ki for inhibition of duplex DNA unwinding (>100 µM). NMM may therefore prove to be a valuable compound for substrate-specific inhibition of other RecQ family helicases in vitro and in vivo.

An evaluation of the performance of cDNA microarrays for detecting changes in global mRNA expression

The cDNA microarray is one technological approach that has the potential to accurately measure changes in global mRNA expression levels. We report an assessment of an optimized cDNA microarray platform to generate accurate, precise and reliable data consistent with the objective of using microarrays as an acquisition platform to populate gene expression databases. The study design consisted of two independent evaluations with 70 arrays from two different manufactured lots and used three human tissue sources as samples: placenta, brain and heart. Overall signal response was linear over three orders of magnitude and the sensitivity for any element was estimated to be 2 pg mRNA. The calculated coefficient of variation for differential expression for all non-differentiated elements was 12–14% across the entire signal range and did not vary with array batch or tissue source. The minimum detectable fold change for differential expression was 1.4. Accuracy, in terms of bias (observed minus expected differential expression ratio), was less than 1 part in 10 000 for all non-differentiated elements. The results presented in this report demonstrate the reproducible performance of the cDNA microarray technology platform and the methods provide a useful framework for evaluating other technologies that monitor changes in global mRNA expression.

Differential fluorescence labeling of cysteinyl clusters uncovers high tissue levels of thionein

The isolation of thionein (T) from tissues has not been reported heretofore. T contains 20 cysteinyl residues that react with 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide to form fluorescent adducts. In metallothionein (MT) the cysteinyl residues, which are bound to zinc, do not react. However, they do react in the presence of a chelating agent such as EDTA. The resultant difference in chemical reactivity provides a means to measure T in the absence of EDTA, (MT + T) in its presence, and, of course, MT by difference. The 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide derivative of T can be isolated from tissue homogenates by HPLC and quantified fluorimetrically with a detection limit in the femtomolar range and a linear response over 3 orders of magnitude. Analysis of liver, kidney, and brain of rats reveals almost as much T as MT. Moreover, in contrast to earlier views, MT in tissue extracts appears to be less stable than T. The existence of T in tissues under normal physiological conditions has important implications for its function both in zinc metabolism and the redox balance of the cell.

Strong DNA binding by covalently linked dimeric Lac headpiece: Evidence for the crucial role of the hinge helices

The combined structural and biochemical studies on Lac repressor bound to operator DNA have demonstrated the central role of the hinge helices in operator bending and the induction mechanism. We have constructed a covalently linked dimeric Lac-headpiece that binds DNA with four orders of magnitude higher affinity as compared with the monomeric form. This enabled a detailed biochemical and structural study of Lac binding to its cognate wild-type and selected DNA operators. The results indicate a profound contribution of hinge helices to the stability of the protein–DNA complex and highlight their central role in operator recognition. Furthermore, protein–DNA interactions in the minor groove appear to modulate hinge helix stability, thus accounting for affinity differences and protein-induced DNA bending among the various operator sites. Interestingly, the in vitro DNA-binding affinity of the reported dimeric Lac construct can de readily modulated by simple adjustment of redox conditions, thus rendering it a potential artificial gene regulator.

Importance of anchor genomes for any plant genome project

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.

Biological impact on mineral dissolution: Application of the lichen model to understanding mineral weathering in the rhizosphere

Microorganisms modify rates and mechanisms of chemical and physical weathering and clay growth, thus playing fundamental roles in soil and sediment formation. Because processes in soils are inherently complex and difficult to study, we employ a model based on the lichen–mineral system to identify the fundamental interactions. Fixed carbon released by the photosynthetic symbiont stimulates growth of fungi and other microorganisms. These microorganisms directly or indirectly induce mineral disaggregation, hydration, dissolution, and secondary mineral formation. Model polysaccharides were used to investigate direct mediation of mineral surface reactions by extracellular polymers. Polysaccharides can suppress or enhance rates of chemical weathering by up to three orders of magnitude, depending on the pH, mineral surface structure and composition, and organic functional groups. Mg, Mn, Fe, Al, and Si are redistributed into clays that strongly adsorb ions. Microbes contribute to dissolution of insoluble secondary phosphates, possibly via release of organic acids. These reactions significantly impact soil fertility. Below fungi–mineral interfaces, mineral surfaces are exposed to dissolved metabolic byproducts. Through this indirect process, microorganisms can accelerate mineral dissolution, leading to enhanced porosity and permeability and colonization by microbial communities.

Population structure and recent evolution of Plasmodium falciparum

Plasmodium falciparum is the agent of malignant malaria, one of mankind's most severe maladies. The parasite exhibits antigenic polymorphisms that have been postulated to be ancient. We have proposed that the extant world populations of P. falciparum have derived from one single parasite, a cenancestor, within the last 5,000–50,000 years. This inference derives from the virtual or complete absence of synonymous nucleotide polymorphisms at genes not involved in immune or drug responses. Seeking to conciliate this claim with extensive antigenic polymorphism, we first note that allele substitutions or polymorphisms can arise very rapidly, even in a single generation, in large populations subject to strong natural selection. Second, new alleles can arise not only by single-nucleotide mutations, but also by duplication/deletion of short simple-repeat DNA sequences, a process several orders of magnitude faster than single-nucleotide mutation. We analyze three antigenic genes known to be extremely polymorphic: Csp, Msp-1, and Msp-2. We identify regions consisting of tandem or proximally repetitive short DNA sequences, including some previously unnoticed. We conclude that the antigenic polymorphisms are consistent with the recent origin of the world populations of P. falciparum inferred from the analysis of nonantigenic genes.

Optimizing the stability of single-chain proteins by linker length and composition mutagenesis

Linker length and composition were varied in libraries of single-chain Arc repressor, resulting in proteins with effective concentrations ranging over six orders of magnitude (10 μM–10 M). Linkers of 11 residues or more were required for biological activity. Equilibrium stability varied substantially with linker length, reaching a maximum for glycine-rich linkers containing 19 residues. The effects of linker length on equilibrium stability arise from significant and sometimes opposing changes in folding and unfolding kinetics. By fixing the linker length at 19 residues and varying the ratio of Ala/Gly or Ser/Gly in a 16-residue-randomized region, the effects of linker flexibility were examined. In these libraries, composition rather than sequence appears to determine stability. Maximum stability in the Ala/Gly library was observed for a protein containing 11 alanines and five glycines in the randomized region of the linker. In the Ser/Gly library, the most stable protein had seven serines and nine glycines in this region. Analysis of folding and unfolding rates suggests that alanine acts largely by accelerating folding, whereas serine acts predominantly to slow unfolding. These results demonstrate an important role for linker design in determining the stability and folding kinetics of single-chain proteins and suggest strategies for optimizing these parameters.

On the origin of and phylogenetic relationships among living amphibians

The phylogenetic relationships among the three orders of modern amphibians (Caudata, Gymnophiona, and Anura) have been estimated based on both morphological and molecular evidence. Most morphological and paleontological studies of living and fossil amphibians support the hypothesis that salamanders and frogs are sister lineages (the Batrachia hypothesis) and that caecilians are more distantly related. Previous interpretations of molecular data based on nuclear and mitochondrial rRNA sequences suggested that salamanders and caecilians are sister groups to the exclusion of frogs. In an attempt to resolve this apparent conflict, the complete mitochondrial genomes of a salamander (Mertensiella luschani) and a caecilian (Typhlonectes natans) were determined (16,656 and 17,005 bp, respectively) and compared with previously published sequences from a frog (Xenopus laevis) and several other groups of vertebrates. Phylogenetic analyses of the mitochondrial data supported with high bootstrap values the monophyly of living amphibians with respect to other living groups of tetrapods, and a sister group relationship of salamanders and frogs. The lack of phylogenetically informative sites in the previous rRNA data sets (because of its shorter size and higher among-site rate variation) likely explains the discrepancy between our results and those based on previous molecular data. Strong support of the Batrachia hypothesis from both molecule- and morphology-based studies provides a robust phylogenetic framework that will be helpful to comparative studies among the three living orders of amphibians and will permit better understanding of the considerably divergent vertebral, brain, and digit developmental patterns found in frogs and salamanders.

Overexpression of a Homeobox Gene, LeT6, Reveals Indeterminate Features in the Tomato Compound Leaf1

The cultivated tomato (Lycopersicon esculentum) has a unipinnate compound leaf. In the developing leaf primordium, major leaflet initiation is basipetal, and lobe formation and early vascular differentiation are acropetal. We show that engineered alterations in the expression of a tomato homeobox gene, LeT6, can cause dramatic changes in leaf morphology. The morphological states are variable and unstable and the phenotypes produced indicate that the tomato leaf has an inherent level of indeterminacy. This is manifested by the production of multiple orders of compounding in the leaf, by numerous shoot, inflorescence, and floral meristems on leaves, and by the conversion of rachis-petiolule junctions into “axillary” positions where floral buds can arise. Overexpression of a heterologous homeobox transgene, kn1, does not produce such phenotypic variability. This indicates that LeT6 may differ from the heterologous kn1 gene in the effects manifested on overexpression, and that 35S-LeT6 plants may be subject to alterations in expression of both the introduced and endogenous LeT6 genes. The expression patterns of LeT6 argue in favor of a fundamental role for LeT6 in morphogenesis of leaves in tomato and also suggest that variability in homeobox gene expression may account for some of the diversity in leaf form seen in nature.