Building a metal-responsive promoter with synthetic regulatory elements.

A fusion gene consisting of the promoter region from the mouse metallothionein-I gene joined to the coding region of the herpes simplex virus thymidine kinase gene is efficiently regulated by zinc in a transient assay when transfected into baby hamster kidney cells. Analysis of similar plasmids in which the metallothionein-I promoter region was mutated indicated the presence of multiple metal regulatory elements (MREs) between -176 and -44 base pairs from the cap site. To further investigate the function of MREs, we inserted a synthetic DNA fragment containing the sequence of MRE-a (the element between -55 and -44 base pairs) into the nonresponsive promoter of the thymidine kinase gene in various positions and configurations. Little or no induction by zinc was observed with single insertions of the regulatory sequence, whereas many different constructions having two copies of MRE-a were inducible. The precise position of the two MREs relative to each other or to the thymidine kinase promoter elements had a relatively small effect on the efficiency of induction, but the inducibility could be further increased by the introduction of more MRE-a sequences. MRE-a can function synergistically with the thymidine kinase distal promoter elements, but in the presence of the TATA box alone it functions as a positive, zinc-dependent promoter element.     

Correcting errors in synthetic DNA through consensus shuffling

Although efficient methods exist to assemble synthetic oligonucleotides into genes and genomes, these suffer from the presence of 1–3 random errors/kb of DNA. Here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding protein (MutS). PCR assembly of the remaining fragments yields a new population of full-length sequences enriched for the consensus sequence of the input population. We show that two iterations of consensus shuffling improved a population of synthetic green fluorescent protein (GFPuv) clones from ~60 to >90% fluorescent, and decreased errors 3.5- to 4.3-fold to final values of ~1 error per 3500 bp. In addition, two iterations of consensus shuffling corrected a population of GFPuv clones where all members were non-functional, to a population where 82% of clones were fluorescent. Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences.     

Interaction of trans and cis regulatory elements in the INO1 promoter of Saccharomyces cerevisiae.

Electrophoretic mobility shift assays (EMSA) were used to define the regions of the INO1 promoter that interact with factors present in extracts prepared from the yeast, Saccharomyces cerevisae. These experiments identified three different types of protein:DNA complexes that assemble with the INO1 promoter. Formation of one type of complex depended on functional alleles of previously described regulatory genes, INO2 and INO4, that encode positive regulatory factors. Formation of the INO2/INO4-dependent complexes was increased when extracts prepared from cells grown under derepressing conditions (i.e. absence of inositol and choline). A second type of complex was dependent on the OPI1 gene, that encodes a negative regulator. Computer-aided sequence analysis of the promoters of several genes encoding phospholipid biosynthetic enzymes revealed a highly conserved nine basepair element (5'-ATGTGAAAT-3'), designated 'nonamer' motif, that is similar to the octamer motif identified in the promoters of mammalian immunoglobulin genes. The nonamer motif was shown to form a specific complex with a factor, designated nonamer binding factor (NBF). Extracts prepared from Schizosaccharomyces pombe formed a nonamer-specific complex.     

Ultrasensitive synthetic protein regulatory networks using mixed decoys

Cellular protein interaction networks exhibit sigmoidal input-output relationships with thresholds and steep responses (i.e. ultrasensitivity). Although cooperativity can be a source of ultrasensitivity, we examined whether the presence of "decoy" binding sites that are not coupled to activation could also lead to this effect. To systematically vary key parameters of the system, we designed a synthetic regulatory system consisting of an autoinhibited PDZ domain coupled to an activating SH3 domain binding site. In the absence of a decoy binding site, this system is non-ultrasensitive, as predicted by modeling of this system. Addition of a high-affinity decoy site adds a threshold, but the response is not ultrasensitive. We found that sigmoidal activation profiles can be generated utilizing multiple decoys with mixtures of high and low affinities, where high affinity decoys act to set the threshold and low affinity decoys ensure a sigmoidal response. Placing the synthetic decoy system in a mitotic spindle orientation cell culture system thresholds this physiological activity. Thus, simple combinations of non-activating binding sites can lead to complex regulatory responses in protein interaction networks.     

Uncovering spatial topology represented by rat hippocampal population neuronal codes

Hippocampal population codes play an important role in representation of spatial environment and spatial navigation. Uncovering the internal representation of hippocampal population codes will help understand neural mechanisms of the hippocampus. For instance, uncovering the patterns represented by rat hippocampus (CA1) pyramidal cells during periods of either navigation or sleep has been an active research topic over the past decades. However, previous approaches to analyze or decode firing patterns of population neurons all assume the knowledge of the place fields, which are estimated from training data a priori. The question still remains unclear how can we extract information from population neuronal responses either without a priori knowledge or in the presence of finite sampling constraint. Finding the answer to this question would leverage our ability to examine the population neuronal codes under different experimental conditions. Using rat hippocampus as a model system, we attempt to uncover the hidden "spatial topology" represented by the hippocampal population codes. We develop a hidden Markov model (HMM) and a variational Bayesian (VB) inference algorithm to achieve this computational goal, and we apply the analysis to extensive simulation and experimental data. Our empirical results show promising direction for discovering structural patterns of ensemble spike activity during periods of active navigation. This study would also provide useful insights for future exploratory data analysis of population neuronal codes during periods of sleep.     

The synthetic integron: an in vivo genetic shuffling device

As the field of synthetic biology expands, strategies and tools for the rapid construction of new biochemical pathways will become increasingly valuable. Purely rational design of complex biological pathways is inherently limited by the current state of our knowledge. Selection of optimal arrangements of genetic elements from randomized libraries may well be a useful approach for successful engineering. Here, we propose the construction and optimization of metabolic pathways using the inherent gene shuffling activity of a natural bacterial site-specific recombination system, the integron. As a proof of principle, we constructed and optimized a functional tryptophan biosynthetic operon in Escherichia coli. The trpA-E genes along with ‘regulatory’ elements were delivered as individual recombination cassettes in a synthetic integron platform. Integrase-mediated recombination generated thousands of genetic combinations overnight. We were able to isolate a large number of arrangements displaying varying fitness and tryptophan production capacities. Several assemblages required as many as six recombination events and produced as much as 11-fold more tryptophan than the natural gene order in the same context.     

Detection of mRNAs containing regulatory peptide coding sequences using synthetic oligodeoxynucleotides

To understand the regulation of the production of peptide hormones, it is vital to elucidate their biosynthetic pathways. We chose to study a major regulatory peptide, vasoactive intestinal peptide (VIP), a peptide possessing both neurotransmitter and neurohormone actions. To identify the specific peptide mRNA we are using, as hybridization probes, radiolabeled synthetic oligodcoxynucleotides with sequence complementary to the predicted peptide mRNA sequence. Employing this approach, we identified and partially purified a ～ 1600-base long mRNA containing VIP related sequences which can be translated in vitro into VIP-immunoreactive polypeptides. Such mRNA was detected in normal VIP producing tissue (rat brain), as well as in a tumor producing VIP (human buccal tumor). This mRNA differs in size from a known VIP-mRNA identified in human neuro-blastoma cells, suggesting the possibility of different VIP-mRNAs in different cell types.     

Uncovering gene regulatory networks during mouse fetal germ cell development

The commitment of germ cells to either oogenesis or spermatogenesis occurs during fetal gonad development: germ cells enter meiosis or mitotic arrest, depending on whether they reside within an ovary or a testis, respectively. Despite the critical importance of this step for sexual reproduction, gene networks underlying germ cell development have remained only partially understood. Taking advantage of the W(v) mouse model, in which gonads lack germ cells, we conducted a microarray study to identify genes expressed in fetal germ cells. In addition to distinguishing genes expressed by germ cells from those expressed by somatic cells within the developing gonads, we were able to highlight specific groups of genes expressed only in female or male germ cells. Our results provide an important resource for deciphering the molecular pathways driving proper germ cell development and sex determination and will improve our understanding of the etiology of human germ cell tumors that arise from dysregulation of germ cell differentiation.