<p>In order to use Acinetobacter baylyi ADP1 as a host for molecularly enhanced triacylglyceride (TAG) production, it is important to understand key enzymatic steps involved in TAG biosynthesis and use. Four different thioesterase genes were cloned from Acinetobacter baylyi ADP1 and expressed in E. coli to investigate their contribution to the free fatty acid (FFAs) accumulation. Overexpression of the genes tesA? (a leaderless form of the gene tesA) and tesC resulted in increased accumulation of FFAs when compared with the host strain. The TesA? thioesterase tended to produce shorter chain and unsaturated FFAs, myristic acid and oleic acid, respectively, compared to TesC and other investigated thioesterases. We identified four crucial one-step enzymatic reactions encoded by the genes lip1, lip2, acr1 and fadE, mutations that were predicted to have an effect on the TAG accumulation. Among a series of generated strains, a strain that had the lip1, lip2 and fadE mutations was constructed showing a 2.44-fold increase in the TAG production compare to the wild type. To achieve high yields of microbial biofuel production using Acinetobacter baylyi ADP1, we constructed and expressed two three-gene operons in E. coli K19 and A. baylyi strains having inactivated ?-oxidation pathway to observe operon effects on FFAs and overall lipid productions. E. coli RU35 produced 0.04 ?g of TAG/mg of dry biomass and E. coli RU14 produced 0.03 ?g of TAG/mg of dry biomass. E. coli K19 by itself does not possess any DGAT activity, hence the production of TAGs in the engineered strains is mediated by enzyme WS/DGAT encoded by the two plasmids. In all A. baylyi strains the introduction of the operon decreased TAGs accumulation by 16-63%. The decrease in accumulation of TAGs in the A. baylyi strains is predicted to be due to the action of the TesA? thioesterase, which is apparently very efficient in competing with WS/DGAT for acyl-CoA. This study demonstrates that finding of a thioesterase which has a low affinity to acyl-CoA and a high affinity to acyl-ACP is critical for the successful increase of TAGs in A. baylyi.

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Polycyclic aromatic hydrocarbons (PAHs), complex organic molecules that are the by-products of carbon-based fuel combustion, are carcinogenic upon biotransformation in the body. Cytochrome P450 1A1 (CYP1A1) is part of the enzyme system primarily responsible for the metabolism and biotransformation of PAHs in humans. In HepG2 cells (a human hepatoma cell line), it has been established previously in this laboratory that 3-methylcholanthrene (MC), a PAH, induces CYP1A1 activity 20 to 30 fold over control values. Also in those studies, MC’s ability to induce this activity was significantly reduced by compounds that inhibit the synthesis of complex sphingolipids. However, addition of the synthetic short-chain sphingolipid C2-ceramide after inducer and inhibitor treatment restored CYP1A1 inducibility. This produced the current laboratory hypothesis that complex sphingolipids are involved in the signal transduction pathway used by MC to induce CYP1A1 activity. This research investigated the role that sphingolipids play in the inducibility of CYP1A1 by MC. Initially, the induction of CYP1A1 was examined by MC as a transcriptional event in these cells. Western blot analysis was used to determine whether CYP1A1 protein amounts fluctuate with CYP1A1 activity per the treatments used. Further studies included quantitative real-time reverse transcriptase PCR to quantitate the amount of CYP1A1 mRNA that resulted from each treatment. It was found that the action of the inhibitor of sphingolipid synthesis seems to be dependent on the presence of a heavy metal. It was also found that C2-ceramide, the prototype complex sphingolipid used, causes synergistic induction of CYP1A1 enzyme activity and that this synergism apparently results from actions at the level of the gene. Thus, it can be concluded that complex sphingolipids are involved in the transcriptional regulation of CYP1A1 expression.

<p> DNA is the physical medium for information storage inside of cells. Sub-regions of the DNA composed of linear stretches of nucleic acid sequences (DNA sequences), known as genes are the basic unit of storage in the human genome. Genes contain a myriad different types of information, of which a major class is protein coding genes. As the name suggests these genes hold the information required to make proteins, which then go on to perform cellular function and consequently dictate cellular activity and identity. Genes themselves are further grouped through the physical lineage of being contained on the same macromolecule known as chromosomes. The entire complement of chromosomes makes up the genome of the organism. In the case of humans, which are diploid, the genome is made up of 22 sets of autosomes and one set of sex chromosomes, a total of 46 discrete chromosomes. During interphase of the cell cycle all 46 of these chromosomes are found inside the nucleus in partially condensed, largely discrete regions known as chromosome territories. </p><p> The positioning of chromosome territories and the genes within them is non-random and has previously been demonstrated to be a function of gene expression. In order for a gene to be expressed, its sequence must be accessible to the RNA polymerases (RNAPs) that transcribe it, not tightly compacted around histones in an inaccessible form known as heterochromatin. As different types of cells need different types of proteins to function, and thus different genes to be expressed, the regions of the genome that are found in heterochromatin are cell type specific. The location of gene locus inside the nucleus relative to other sub-nuclear features such as RNAPII foci, known as transcription factories, also dictate expression. This relationship results in the interphase genome forming a cell type specific topology which is the result of the expressed genes. In spite of the persistent observation of cell type specific nuclear topologies, the factors that guide the formation and the function of observed topologies remains unclear. </p><p> Here, we test the relationship between linear and three-dimensional (3D) organization of gene regulation during myogenesis. Our analysis indicates that a subset of human chromosomes is significantly enriched for topologically associated domains (TADs) that contain muscle-specific genes. These lineage-enriched TADs demonstrate an expression dependent pattern of nuclear organization that also affects the positioning of non-enriched TADs. Therefore, lineage-enriched TADs affect cell-specific genome organization during myogenesis. The allelic spatial proximity of one of these domains, which encodes <i>myogenin, </i> reduces transcriptional variability. Moreover, this cell-specific nuclear topology is dependent on cell division. We propose that the linear and spatial organization of gene locus is functionally inter-dependent and that mitosis is critical in establishing this behavior during cellular differentiation. </p><p> We then extend this analysis into murine lymphocyte development. Specifically, we look at naturally occurring suppressive T-regulatory cells (T_regs) that dampen the strength/severity of the immune response and have been demonstrated to be clinically relevant in the emergence and pathogenesis of auto-immune disorders. T-regulatory cells are an interesting model for studying lineage specific nuclear topologies because during an active immune response they are a mixed population of natural T_regs and induced T_regs that phenocopy each other but have different developmental histories. There is also clinical interest in finding a reliable means to make a distinction between these two cell types for the potential treatment of auto-immune disease. By studying features of nuclear organization for two candidate genes, <i>FoxP3</i> and <i>Helios,</i> and the chromosomes they reside on, X and one respectively, we were able to distinguish these two populations of cells on the basis of nuclear topologies. We propose this distinction is the result of differing nuclear topologies in the progenitor population in conjunction with only a sub-set of regions, such as LE-TADs, showing lineage specific localizations. This would lead to remnants of progenitor topologies in terminal lineages. We also make the interesting observation of unexpectedly high rates of coalescence between the active and inactive X chromosomes in cells that specifically express the X-linked gene <i> FoxP3.</i> This finding may lead to transvection based silencing of FoxP3 and the increase in autoimmunity observed in females. </p><p> Collectively, this body of work furthers the understanding of how lineage specific nuclear topologies emerge as a function of gene expression and greatly expands the current understanding of how these topologies exert influence over the expression of genes.</p>

The nucleus accumbens, with its numerous connections, including the ventral tegmental area, substantia nigra, dorsal and medial raphe, ventral and dorsal hippocampus, and gigantocellular reticular nucleus, has been interpreted as serving as a nodal point of informational transfer between these and other brain structures. These neural systems are involved in regulation or modulation of behavioral, emotional, and endocrine states of an individual. Electrical stimulation given to the neural systems stated above produced different evoked potentials in nucleus accumbens. The ventral tegmental area evoked antidromic and orthodromic responses, while the dorsal and medial raphe produced non-fatiguing responses. From an area between the ventral tegmental area and substantia nigra, two stimultaneously evoked potentials in the accumbens were recorded. The dorsal hippocampus produced evoked responses of variable latencies which fatigued in the course of train stimulation, and ventral hippocampus produced antidromic unit responses in accumbens. The gigantocellular reticular nucleus produced two responses, one indicating recruitment while the others one fatigued. The different potentials evoked in the accumbens illustrate the presence of a variety of connections to it with different physiological characteristics. This supports the contention that this nucleus can function as a nodal point of informational transfer between several systems of the brain.

Retroviruses express their repertoire of products from a single primary transcript. However, it appears that the default condition for posttranscriptional processing in a normal cell is to completely splice any intron-containing transcripts. Consequently, the dilemma for the retroviruses is how to export the full length, unspliced and partially spliced transcript which both code for structural proteins, as well as serves as the genome to be packaged into mature virus particles. Many retroviruses (HIV-1, HTLV-l, EIAV, visna) exploit posttranscriptional mechanisms, by which their intron-containing mRNAs circumvent nuclear retention, and are exported to the cytoplasm. For example, HIV-1 RNAs contain at least one functional intron that must enter the cytoplasm to act as templates for the synthesis of proteins. HIV-l regulates expression of its genome through the interaction of a virally encoded trans-acting factor, Rev, with a cis-acting Rev responsive element (RRE). Rev binds unspliced and singly spliced nuclear transcripts containing the RE and shuttles them into the cytoplasm. Other retroviruses (MPMV, SRV) lack Rev-like trans-acting viral proteins. Their transcripts contain a cis-acting element, termed the constitutive transport element (CTE), that allows transport of intron-containing mRNAs. CTE-like elements have also been identified in DNA viruses (Hepatitis B, HSV~1). Retroviral CTEs have been shown to be able to substitute for the Rev/RRE system to allow efficient regulatory control of HIV expression. Although a number of studies have examined what cellular cofactors are involved in Rev/RRE and CTE-mediated transport, it is not clear if common cellular cofactor(s) exist. In these studies we have used a transfection/competition assay to investigate whether Rev/RRE-, SRVCTE~, and MPMVCTE-containing transcripts utilize similar cofactors for nucleocytoplasmic transport. Coexpression of gagMPMVCTE and pCwtRev at various concentrations in the same cell demonstrated that Rev inhibited CTE- mediated transport from the nucleus. Using the same assay we further demonstrated that GTE-mediated export was not inhibited by cotransfection with a pCΔRev(-) mutant clone, a Rev point mutant clone, or luciferase, a non-specific marker protein. Our data suggest that this inhibition is specific for Rev, and for a specific region of the Rev protein. We propose that Rev and the CTEs interact with a common cellular cofactor(s), or that Rev directly interacts with the CTEs.

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No description available.