Identification of a core promoter and a novel isoform of the human TSCI gene transcript and structural comparison with mouse homolog

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with loci on chromosome 9q34.12 (TSC1) and chromosome 16p13.3 (TSC2). Genes for both loci have been isolated and characterized. The promoters of both genes have not been characterized so far and little is known about the regulation of these genes. This study reports the characterization of the human TSC1 promoter region for the first time. We have identified a novel alternative isoform in the 5' untranslated region (UTR) of the TSC1 gene transcript involving exon 1. Alternative isoforms in the 5' UTR of the mouse Tsc1 gene transcript involving exon I and exon 2 have also been identified. We have identified three upstream open reading frames (uORFs) in the 5' UTR of the TSC1/Tsc1 gene. A comparative study of the 5' UTR of TSC1/Tsc1 gene has revealed that there is a high degree of similarity not only in the sequence but also in the splicing pattern of both human and mouse TSC1 genes. We have used PCR methodology to isolate approximately 1.6 kb genomic DNA 5' to the TSC1 cDNA. This sequence has directed a high level of expression of luciferase activity in both HeLa and HepG2 cells. Successive 5' and 3' deletion analysis has suggested that a -587 bp region, from position +77 to -510 from the transcription start site (TSS), contains the promoter activity. Interestingly, this region contains no consensus TATA box or CAAT box. However, a 521-bp fragment surrounding the TSS exhibits the characteristics of a CpG island which overlaps with the promoter region. The identification of the TSC1 promoter region will help in designing a suitable strategy to identify mutations in this region in patients who do not show any mutations in the coding regions. It will also help to study the regulation of the TSC1 gene and its role in tumorigenesis. (C) 2003 Elsevier B.V. All rights reserved.

NETGEM: Network Embedded Temporal GEnerative Model for gene expression data

Background: Temporal analysis of gene expression data has been limited to identifying genes whose expression varies with time and/or correlation between genes that have similar temporal profiles. Often, the methods do not consider the underlying network constraints that connect the genes. It is becoming increasingly evident that interactions change substantially with time. Thus far, there is no systematic method to relate the temporal changes in gene expression to the dynamics of interactions between them. Information on interaction dynamics would open up possibilities for discovering new mechanisms of regulation by providing valuable insight into identifying time-sensitive interactions as well as permit studies on the effect of a genetic perturbation. Results: We present NETGEM, a tractable model rooted in Markov dynamics, for analyzing the dynamics of the interactions between proteins based on the dynamics of the expression changes of the genes that encode them. The model treats the interaction strengths as random variables which are modulated by suitable priors. This approach is necessitated by the extremely small sample size of the datasets, relative to the number of interactions. The model is amenable to a linear time algorithm for efficient inference. Using temporal gene expression data, NETGEM was successful in identifying (i) temporal interactions and determining their strength, (ii) functional categories of the actively interacting partners and (iii) dynamics of interactions in perturbed networks. Conclusions: NETGEM represents an optimal trade-off between model complexity and data requirement. It was able to deduce actively interacting genes and functional categories from temporal gene expression data. It permits inference by incorporating the information available in perturbed networks. Given that the inputs to NETGEM are only the network and the temporal variation of the nodes, this algorithm promises to have widespread applications, beyond biological systems. The source code for NETGEM is available from https://github.com/vjethava/NETGEM

Heterologous expression of P5CS gene in chickpea enhances salt tolerance without affecting yield

Vigna Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) cDNA was transferred to chickpea (Cicer arietinum L.) cultivar Annigeri via Agrobacterium tumefaciens mediated transformation. Following selection on hygromycin and regeneration, 60 hygromycin-resistant plants were recovered. Southern blot analysis of five fertile independent lines of T0 and T1 generation revealed single and multiple insertions of the transgene. RT-PCR and Western blot analysis of T0 and T1 progeny demonstrated that the P5CS gene is expressed and produced functional protein in chickpea. T1 transgenic lines accumulated higher amount of proline under 250 mM NaCl compared to untransformed controls. Higher accumulation of Na(+) was noticed in the older leaves but negligible accumulation in seeds of T1 transgenic lines as compared to the controls. Chlorophyll stability and electrolyte leakage indicated that proline overproduction helps in alleviating salt stress in transgenic chickpea plants. The T1 transgenics lines were grown to maturity and set normal viable seeds under continuous salinity stress (250 mM) without any reduction in plant yield in terms of seed mass.

Direct detection of Salmonella without pre-enrichment in milk, ice-cream and fruit juice by PCR against hilA gene

A novel PCR based assay was devised to specifically detect contamination of any Salmonella serovar in milk, fruit juice and ice-cream without pre-enrichment. This method utilizes primers against hilA gene which is conserved in all Salmonella serovars and absent from the close relatives of Salmonella. An optimized protocol, in terms time and money, is provided for the reduction of PCR contaminants from milk, ice-cream and juice through the use of routine laboratory chemicals. The simplicity, efficiency (time taken 3-4 h) and sensitivity (to about 5-10 CFU/ml) of this technique confers a unique advantage over other previously used time consuming detection techniques. This technique does not involve pre-enrichment of the samples or extensive sample processing, which was a pre-requisite in most of the other reported studies. Hence, this assay can be ideal for adoption, after further fine tuning, by food quality control for timely detection of Salmonella contamination as well as other food-borne pathogens (with species specific primers) in food especially milk, ice-cream and fruit juice. (C) 2011 Elsevier Ltd. All rights reserved.

Why Is Less Cationic Lipid Required To Prepare Lipoplexes from Plasmid DNA than Linear DNA in Gene Therapy?

The most important objective of the present study was to explain why cationic lipid (CL)-mediated delivery of plasmid DNA (pDNA) is better than that of linear DNA in gene therapy, a question that, until now, has remained unanswered. Herein for the first time we experimentally show that for different types of CLs, pDNA, in contrast to linear DNA, is compacted with a large amount of its counterions, yielding a lower effective negative charge. This feature has been confirmed through a number of physicochemical and biochemical investigations. This is significant for both in vitro and in vivo transfection studies. For an effective DNA transfection, the lower the amount of the CL, the lower is the cytotoxicity. The study also points out that it is absolutely necessary to consider both effective charge ratios between CL and pDNA and effective pDNA charges, which can be determined from physicochemical experiments.

Loading of single-walled carbon nanotubes in cationic cholesterol suspensions significantly improves gene transfection efficiency in serum

We present here a series of cholesterol based cationic lipid suspensions that solubilize single-walled carbon nanotubes (SWCNT) efficiently in water. Each cationic lipid formulation was characterized in terms of their energy minimized molecular structures, bilayer widths of the aggregates based on X-ray diffraction. Then these aggregates were investigated pertaining to their DNA binding and release efficiency, effect of CNT inclusion on the stability of cationic cholesterol lipid-DNA complexes, Zeta potential values and changes in the chiro-optical property of DNA, effect on Raman spectral shift and changes in morphology by SEM and AFM. Each cationic lipid formulation was optimized for the amount of SWCNT solubilized in water, lipid-DNA ratio, amount of the plasmid DNA that can be transfected and the effect on the cellular toxicity. The resulting SWCNT-lipid formulations were then used for in vitro transfection of pEGFP-C3 in A549 (human alveolar basal epithelial) cells and HeLa (human cervical cancer) cells. Advantageously, the CNT-loaded formulations confer an excellent transfection efficiency even in high percentages of blood serum and showed significantly better gene transfer efficiency compared to one of the potent, well-known commercial transfection reagent, Lipofectamine2000.

Mechanism of Fragility at BCL2 Gene Minor Breakpoint Cluster Region during t(14;18) Chromosomal Translocation

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Breaks in chromosome 18 are localized at the 3'-UTR of BCL2 gene or downstream and are mainly clustered in either the major breakpoint region or the minor breakpoint cluster region (mcr). The recombination activating gene (RAG) complex induces breaks at IgH locus of chromosome 14, whereas the mechanism of fragility at BCL2 mcr remains unclear. Here, for the first time, we show that RAGs can nick mcr; however, the mechanism is unique. Three independent nicks of equal efficiency are generated, when both Mg2+ and Mn2+ are present, unlike a single nick during V(D)J recombination. Further, we demonstrate that RAG binding and nicking at the mcr are independent of nonamer, whereas a CCACCTCT motif plays a critical role in its fragility, as shown by sequential mutagenesis. More importantly, we recapitulate the BCL2 mcr translocation and find that mcr can undergo synapsis with a standard recombination signal sequence within the cells, in a RAG-dependent manner. Further, mutation to the CCACCTCT motif abolishes recombination within the cells, indicating its vital role. Hence, our data suggest a novel, physiologically relevant, nonamer-independent mechanism of RAG nicking at mcr, which may be important for generation of chromosomal translocations in humans.