Involvement of heme in the transcriptional activation of CYPIIB1/B2 gene by phenobarbitone in rat liver—Studies with succinylacetone

Earlier studies in this laboratory had implicated heme to function as a positive modulator of phenobarbitonemediated activation of CYPIIB1/B2 gene transcription in rat liver. However, recent reports have indicated that succinylacetone, a specific inhibitor of δ-aminolevulinate dehydrase, does not affect this process. The present studies indicate that succinylacetone does inhibit the phenobarbitone-mediated increase in CYPIIB1/B2 mRNAs and their transcription in rat liver at early time points (45 min to 3 h), but the inhibition is not pronounced at later time points (16 h). Succinylacetone is a weaker inhibitor of heme biosynthesis than CoCl2, 3-amino-1,2,4-triazole, or thioacetamide used earlier in this laboratory. Succinylacetone induces δ-aminolevulinate synthase, whereas the other compounds depress the levels of the enzyme. There is a good correlation between the amount of freshly synthesized nuclear heme pool and the activation of CYPIIB1/B2 transcription by phenobarbitone. A model implicating a nuclear heme pool regulating the transcription of δ-aminolevulinate synthase, CYPIIB1/ B2, and heme oxygenase genes is proposed.

Studies on Causes of Multiple Sclerosis : From Genes to Transcriptome

Multiple sclerosis (MS) is the most common cause of neurological disability in young adults, affecting more than two million people worldwide. It manifests as a chronic inflammation in the central nervous system (CNS) and causes demyelination and neurodegeneration. Depending on the location of the demyelinated plaques and axonal loss, a variety of symptoms can be observed including deficits in vision, coordination, balance and movement. With a typical age of onset at 20-40 years, the social and economic impacts of MS on lives of the patients and their families are considerable. Unfortunately the current treatments are relatively inefficient and the development of more effective treatments has been impeded by our limited understanding of the causes and pathogenesis of MS. Risk of MS is higher in biological relatives of MS patients than in the general population. Twin and adoption studies have shown that familial clustering of MS is explained by shared genetic factors rather than by shared familial environment. While the involvement of the human leukocyte antigen (HLA) genes was first discovered four decades ago, additional genetic risk factors have only recently been identified through genome-wide association studies (GWAS). Current evidence suggests that MS is a highly polygenic disease with perhaps hundreds of common variants with relatively modest effects contributing to susceptibility. Despite extensive research, the majority of these risk factors still remain to be identified. In this thesis the aim was to identify novel genes and pathways involved in MS. Using genome-wide microarray technology, gene expression levels in peripheral blood mononuclear cells (PBMC) from 12 MS patients and 15 controls were profiled and more than 600 genes with altered expression in MS were identified. Three of five selected findings, DEFA1A3, LILRA4 and TNFRSF25, were successfully replicated in an independent sample. Increased expression of DEFA1A3 in MS is a particularly interesting observation, because its elevated levels have previously been reported also in several other autoimmune diseases. A systematic review of seven microarray studies was then performed leading to identification of 229 genes, in which either decreased or increased expression in MS had been reported in at least two studies. In general there was relatively little overlap across the experiments: 11 of the 229 genes had been reported in three studies and only HSPA1A in four studies. Nevertheless, these 229 genes were associated with several immunological pathways including interleukin pathways related to type 2 and type 17 helper T cells and regulatory T cells. However, whether these pathways are involved in causing MS or related to secondary processes activated after disease onset remains to be investigated. The 229 genes were also compared with loci identified in published MS GWASs. Single nucleotide polymorphisms (SNP) in 17 of the 229 loci had been reported to be associated with MS with P-value less than 0.0001 including variants in CXCR4 and SAPS2, which were the only loci where evidence for correlation between the associated variant and gene expression was found. The CXCR4 variant was further tested for association with MS in a large case-control sample and the previously reported suggestive association was replicated (P-value is 0.0004). Finally, common genetic variants in candidate genes, which had been selected on the basis of showing association with other autoimmune diseases (MYO9B) or showing differential expression in MS in our study (DEFA1A3, LILRA4 and TNFRSF25), were tested for association with MS, but no evidence of association was found. In conclusion, through a systematic review of genome-wide expression studies in MS we have identified several promising candidate genes and pathways for future studies. In addition, we have replicated a previously suggested association of a SNP variant upstream of CXCR4 with MS. Keywords: autoimmune disease, common variant, CXCR4, DEFA1A3, HSPA1A,gene expression, genetic association, GWAS, MS, multiple sclerosis, systematic review

Interfamilial transfer of amber suppressor gene for the isolation of amber mutants of mycobacteriophage I3

A suppressor-containing strain of Mycobacterium smegmatis SN2 was isolated by transferring an amber suppressor carried on the plasmid of Pseudomonas pseudoalcaligenes ERA through transformation. Amber mutants of mycobacteriophage I3 were isolated.

Defective in Cuticular Ridges (DCR) of Arabidopsis thaliana, a Gene Associated with Surface Cutin Formation, Encodes a Soluble Diacylglycerol Acyltransferase

A key step in the triacylglycerol (TAG) biosynthetic pathway is the final acylation of diacylglycerol (DAG) by DAG acyltransferase. In silico analysis has revealed that the DCR (defective in cuticular ridges) (At5g23940) gene has a typical HX4D acyltransferase motif at the N-terminal end and a lipid binding motif VX(2)GF at the middle of the sequence. To understand the biochemical function, the gene was overexpressed in Escherichia coli, and the purified recombinant protein was found to acylate DAG specifically in an acyl-CoA-dependent manner. Overexpression of At5g23940 in a Saccharomyces cerevisiae quadruple mutant deficient in DAG acyltransferases resulted in TAG accumulation. At5g23940 rescued the growth of this quadruple mutant in the oleate-containing medium, whereas empty vector control did not. Lipid particles were localized in the cytosol of At5g23940-transformed quadruple mutant cells, as observed by oil red O staining. There was an incorporation of 16-hydroxyhexadecanoic acid into TAG in At5g23940-transformed cells of quadruple mutant. Here we report a soluble acyl-CoA-dependent DAG acyltransferase from Arabidopsis thaliana. Taken together, these data suggest that a broad specific DAG acyltransferase may be involved in the cutin as well as in the TAG biosynthesis by supplying hydroxy fatty acid.

RAD51C: a novel cancer susceptibility gene is linked to Fanconi anemia and breast cancer

Germline mutations in many of the genes that are involved in homologous recombination (HR)-mediated DNA double-strand break repair (DSBR) are associated with various human genetic disorders and cancer. RAD51 and RAD51 paralogs are important for HR and in the maintenance of genome stability. Despite the identification of five RAD51 paralogs over a decade ago, the molecular mechanism(s) by which RAD51 paralogs regulate HR and genome maintenance remains obscure. In addition to the known roles of RAD51C in early and late stages of HR, it also contributes to activation of the checkpoint kinase CHK2. One recent study identifies biallelic mutation in RAD51C leading to Fanconi anemia-like disorder. Whereas a second study reports monoallelic mutation in RAD51C associated with increased risk of breast and ovarian cancer. These reports show RAD51C is a cancer susceptibility gene. In this review, we focus on describing the functions of RAD51C in HR, DNA damage signaling and as a tumor suppressor with an emphasis on the new roles of RAD51C unveiled by these reports.

Development of tissue culture and virus-induced gene silencing for Calendula officinalis

Calendula officinalis is grown widely as an ornamental plant across Europe. It belongs to the large. Asteraceae family. In this study, the aim was to explore the possibilities to use Calendula officinalis as a new model organism for flower development and secondary mechanism studies in Asteraceae. Tissue culture of Calendula officinalis was established using nine different cultivars. Murashige & Skoog (MS) medium with four different combinations of plant growth regulators were tested. Of all these combinations, the medium containing 1mg/l BAP, 0.1 mg/l IAA, and 1mg/l Zeatin achieved highest frequency of adventitious shoot regeneration from hypocotyl and cotyledon explants. Virus-induced gene silencing is a recent developed genetic tool for charactering the gene functions in plants, and extends the range of host plants that are not accessible for Agrobacterium transformation. Here, tobacco rattle virus (TRV)-based VIGS technique was tested in calendula (cv. Single Orange). We used TRV carrying Gerbera hybrid phytoene desaturase (PDS) gene fragment to induce PDS silencing in calendula. Vacuum infiltration and syringe infiltration methods both resulted in photo-bleaching phenotypes in leaves, bracts and petals. Loss-of-function phenotypes occurred on calendula 13 days post-infiltration. In conclusion, the data indicates that calendula explants can be regenerated through tissue culture which is a prerequisite for development of stable transformation methods. However, further optimization is still needed to improve the frequency. In addition, VIGS was applied to silence PDS marker gene expression indicating that this method has potential for gene functional studies in future.

Immediate hypersensitivity to Parthenium hysterophorus. I. Association of HLA antigens and Parthenium rhinitis

Lymphocytes collected from rhinitis subjects with strong positive skin reactions to the pollen allergens of Parthenium hysterophorus (American feverfew) having moderate to high titres of Parthenium-specific serum IgE were analysed for association of HLA-antigens covering 13 specificities of HLA-A, 17 specificities of HLA-B and eight specificities of HLA-DR loci by the NIH two-stage microlymphocytotoxicity assay. Comparison of the phenotypic frequencies of HLA-A and B antigens between Parthenium rhinitis subjects (n= 22) and control subjects (n= 137) did not suggest any significant association when tested for these antigen specificities. A significant correlation in the association of HLA-DR3 antigen with a relative risk of 11·33, however, was observed in Parthenium rhinitis subjects (n= 30) when compared to controls (n= 50).

Drosophila “enhancer-trap” transposants: Gene expression in chemosensory and motor pathways and identification of mutants affected in smell and taste ability

We have isolated about a thousandDrosophila P-element transposants that allow thein situ detection of genomic enhancer elements by a histochemical assay for β-galactosidase activity. We summarize the β-galactosidase staining patterns of over 200 such transposants in the adult. Our aim was to identify genes that are likely to be involved in the chemosensory and motor pathways ofDrosophila. Based on β-galactosidase expression patterns in the tissues of our interest, we have chosen some strains for further analysis. Behavioral tests on a subset of the transposants have, in addition, identified several strains defective in their chemosensory responses.

Developmental regulation of cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) gene expression in rat liver

The expression of cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) genes has been studied as a function of development in rat liver. The levels of cytochrome P-450 (b+e) mRNAs and their transcription rates are too low for detection in the 19-day old fetal liver before or after phenobarbitone treatment. However, glutathione transferase (Ya+Yc) mRNAs can be detected in the fetal liver as well as their induction after phenobarbitone treatment can be demonstrated. These mRNAs contents as well as their inducibility with phenobarbitone are lower in maternal liver than that of adult nonpregnant female rat liver. Steroid hormone administration to immature rats blocks substantially the phenobarbitone mediated induction of the two mRNA families as well as their transcription. It is suggested that steroid hormones constitute one of the factors responsible for the repression of the cytochrome P-450 (b+e) and glutathione transferase (Ya+Yc) genes in fetal liver.

Uncoupling Protein 2 and 3 Gene Polymorphisms and Their Association with Type 2 Diabetes in Asian Indians

Background: This study examined the association of -866G/A, Ala55Val, 45bpI/D, and -55C/T polymorphisms at the uncoupling protein (UCP) 3-2 loci with type 2 diabetes in Asian Indians. Methods: A case-control study was performed among 1,406 unrelated subjects (487 with type 2 diabetes and 919 normal glucose-tolerant NGT]), chosen from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in Southern India. The polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism and direct sequencing. Haplotype frequencies were estimated using an expectation-maximization algorithm. Linkage disequilibrium was estimated from the estimates of haplotypic frequencies. Results: The genotype (P = 0.00006) and the allele (P = 0.00007) frequencies of Ala55Val of the UCP2 gene showed a significant protective effect against the development of type 2 diabetes. The odds ratios (adjusted for age, sex, and body mass index) for diabetes for individuals carrying Ala/Val was 0.72, and that for individuals carrying Val/Val was 0.37. Homeostasis insulin resistance model assessment and 2-h plasma glucose were significantly lower among Val-allele carriers compared to the Ala/Ala genotype within the NGT group. The genotype (P = 0.02) and the allele (P = 0.002) frequencies of -55C/T of the UCP3 gene showed a significant protective effect against the development of diabetes. The odds ratio for diabetes for individuals carrying CT was 0.79, and that for individuals carrying TT was 0.61. The haplotype analyses further confirmed the association of Ala55Val with diabetes, where the haplotypes carrying the Ala allele were significantly higher in the cases compared to controls. Conclusions: Ala55Val and -55C/T polymorphisms at the UCP3-2 loci are associated with a significantly reduced risk of developing type 2 diabetes in Asian Indians.

Differential transcription of multiple copies of a silk worm gene encoding tRNA

Ten different tRNAGly1 genes from the silk worm, Bombyx mori, have been cloned and characterized. These genes were transcribed in vitro in homologous nuclear extracts from the posterior silk gland (PSG) or nuclear extracts derived from the middle silk gland or ovarian tissues. Although the transcription levels were much higher in the PSG nuclear extracts, the transcriptional efficiency of the individual genes followed a similar pattern in all the extracts. Based on the levels of in vitro transcription, the ten tRNAGly1 genes could be divided into three groups, viz., those which were transcribed at very high levels (e.g., clone pR8), high to medium levels (e.g., pBmil, pBmpl, pBmhl, pBmtl) and low to barely detectable levels (e.g., pBmsl, pBmjl and pBmkl). The coding sequences of all these tRNA genes being identical, the differential transcription suggested that the flanking sequences modulate their transcriptional efficiency. The presence of positive and negative regulatory elements in the 5' flanking regions of these genes was confirmed by transcription competition experiments. A positive element was present in the immediate upstream A + T-rich sequences in all the genes, but no consensus sequences correlating to the transcriptional status could be generated. The presence of negative elements on the other hand was indicated only in some of the genes and therefore may have a role in the differential transcription of these tRNAGly genes in vivo.

Specificity in the regulation of eukaryotic gene transcription

The regulation of eukaryotic gene transcription poses major challenges in terms of the innumerable protein factors required to ensure tissue or cell-type specificity. While this specificity is sought to be explained by the interaction of cis-acting DNA elements and thetrans-acting protein factor(s), considerable amount of degeneracy has been observed in this interaction. Immunoglobulin heavy chain gene expression in B cells and liver-specific gene expression are discussed as examples of this complexity in this article. Heterodimerization and post-translational modification of transcription factors and the organization of composite promoter elements are strategies by which diverse sets of genes can be regulated in a specific manner using a finite number of protein factors

An ensemble of B-DNA dinucleotide geometries lead to characteristic nucleosomal DNA structure and provide plasticity required for gene expression

Background: A nucleosome is the fundamental repeating unit of the eukaryotic chromosome. It has been shown that the positioning of a majority of nucleosomes is primarily controlled by factors other than the intrinsic preference of the DNA sequence. One of the key questions in this context is the role, if any, that can be played by the variability of nucleosomal DNA structure. Results: In this study, we have addressed this question by analysing the variability at the dinucleotide and trinucleotide as well as longer length scales in a dataset of nucleosome X-ray crystal structures. We observe that the nucleosome structure displays remarkable local level structural versatility within the B-DNA family. The nucleosomal DNA also incorporates a large number of kinks. Conclusions: Based on our results, we propose that the local and global level versatility of B-DNA structure may be a significant factor modulating the formation of nucleosomes in the vicinity of high-plasticity genes, and in varying the probability of binding by regulatory proteins. Hence, these factors should be incorporated in the prediction algorithms and there may not be a unique `template' for predicting putative nucleosome sequences. In addition, the multimodal distribution of dinucleotide parameters for some steps and the presence of a large number of kinks in the nucleosomal DNA structure indicate that the linear elastic model, used by several algorithms to predict the energetic cost of nucleosome formation, may lead to incorrect results.

The effect of progesterone replacement on gene expression in the corpus luteum during induced regression and late luteal phase in the bonnet monkey (Macaca radiata)

Background: In higher primates, although LH/CG play a critical role in the control of corpus luteum (CL) function, the direct effects of progesterone (P4) in the maintenance of CL structure and function are unclear. Several experiments were conducted in the bonnet monkey to examine direct effects of P4 on gene expression changes in the CL, during induced luteolysis and the late luteal phase of natural cycles. Methods: To identify differentially expressed genes encoding PR, PR binding factors, cofactors and PR downstream signaling target genes, the genome-wide analysis data generated in CL of monkeys after LH/P-4 depletion and LH replacement were mined and validated by real-time RT-PCR analysis. Initially, expression of these P4 related genes were determined in CL during different stages of luteal phase. The recently reported model system of induced luteolysis, yet capable of responsive to tropic support, afforded an ideal situation to examine direct effects of P4 on structure and function of CL. For this purpose, P4 was infused via ALZET pumps into monkeys 24 h after LH/P4 depletion to maintain mid luteal phase circulating P4 concentration (P4 replacement). In another experiment, exogenous P4 was supplemented during late luteal phase to mimic early pregnancy. Results: Based on the published microarray data, 45 genes were identified to be commonly regulated by LH and P4. From these 19 genes belonging to PR signaling were selected to determine their expression in LH/P-4 depletion and P4 replacement experiments. These 19 genes when analyzed revealed 8 genes to be directly responsive to P4, whereas the other genes to be regulated by both LH and P4. Progesterone supplementation for 24 h during the late luteal phase also showed changes in expression of 17 out of 19 genes examined. Conclusion: These results taken together suggest that P4 regulates, directly or indirectly, expression of a number of genes involved in the CL structure and function.