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.

Dissection of the genetic background of childhood onset progressive myoclonic epilepsies

The progressive myoclonic epilepsies (PMEs) are a clinically and etiologically heterogeneous group of symptomatic epilepsies characterized by myoclonus, tonic-clonic seizures, psychomotor regression and ataxia. Different disorders have been classified as PMEs. Of these, the group of neuronal ceroid lipofuscinoses (NCLs) comprise an entity that has onset in childhood, being the most common cause of neurodegeneration in children. The primary aim of this thesis was to dissect the molecular genetic background of patients with childhood onset PME by studying candidate genes and attempting to identify novel PME-associated genes. Another specific aim was to study the primary protein properties of the most recently identified member of the NCL-causing proteins, MFSD8. To dissect the genetic background of a cohort of Turkish patients with childhood onset PME, a screen of the NCL-associated genes PPT1, TPP1, CLN3, CLN5, CLN6, MFSD8, CLN8 and CTSD was performed. Altogether 49 novel mutations were identified, which together with 56 mutations found by collaborators raised the total number of known NCL mutations to 364. Fourteen of the novel mutations affect the recently identified MFSD8 gene, which had originally been identified in a subset of mainly Turkish patients as the underlying cause of CLN7 disease. To investigate the distribution of MFSD8 defects, a total of 211 patients of different ethnic origins were evaluated for mutations in the gene. Altogether 45 patients from nine different countries were provided with a CLN7 molecular diagnosis, denoting the wide geographical occurrence of MFSD8 defects. The mutations are private with only one having been established by a founder-effect in the Roma population from the former Czechoslovakia. All mutations identified except one are associated with the typical clinical picture of variant late-infantile NCL. To address the trafficking properties of MFSD8, lysosomal targeting of the protein was confirmed in both neuronal and non-neuronal cells. The major determinant for this lysosomal sorting was identified to be an N-terminal dileucine based signal (9-EQEPLL-14), recognized by heterotetrameric AP-1 adaptor proteins, suggesting that MFSD8 takes the direct trafficking pathway en route to the lysosomes. Expression studies revealed the neurons as the primary cell-type and the hippocampus and cerebellar granular cell layer as the predominant regions in which MFSD8 is expressed. To identify novel genes associated with childhood onset PME, a single nucleotide polymorphism (SNP) genomewide scan was performed in three small families and 18 sporadic patients followed by homozygosity mapping to determine the candidate loci. One of the families and a sporadic patient were positive for mutations in PLA2G6, a gene that had previously been shown to cause infantile neuroaxonal dystrophy. Application of next-generation sequencing of candidate regions in the remaining two families led to identification of a homozygous missense mutation in USP19 for the first and TXNDC6 for the second family. Analysis of the 18 sporadic cases mapped the best candidate interval in a 1.5 Mb region on chromosome 7q21. Screening of the positional candidate KCTD7 revealed six mutations in seven unrelated families. All patients with mutations in KCTD7 were reported to have early onset PME, rapid disease progression leading to dementia and no pathologic hallmarks. The identification of KCTD7 mutations in nine patients and the clinical delineation of their phenotype establish KCTD7 as a gene for early onset PME. The findings presented in this thesis denote MFSD8 and KCTD7 as genes commonly associated with childhood onset symptomatic epilepsy. The disease-associated role of TXNDC6 awaits verification through identification of additional mutations in patients with similar phenotypes. Completion of the genetic spectrum underlying childhood onset PMEs and understanding of the gene products functions will comprise important steps towards understanding the underlying pathogenetic mechanisms, and will possibly shed light on the general processes of neurodegeneration and nervous system regulation, facilitating the diagnosis, classification and possibly treatment of the affected cases.

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.

The function and regulation of the U11-48K protein in U12-dependent splicing

The removal of noncoding sequences, or introns, from the eukaryotic messenger RNA precursors is catalyzed by a ribonucleoprotein complex known as the spliceosome. In most eukaryotes, two distinct classes of introns exist, each removed by a specific type of spliceosome. The major, U2-type introns account for over 99 % of all introns, and are almost ubiquitous. The minor, U12-type introns are found in most but not all eukaryotes, and reside in conserved locations in a specific set of genes. Due to their slow excision rates, the U12-type introns are expected to be involved in the regulation of the genes containing them by inhibiting the maturation of the messenger RNAs. However, little information is currently available on how the activity of the U12-dependent spliceosome itself is regulated. The levels of many known splicing factors are regulated through unproductive alternative splicing events, which lead to inclusion of premature STOP codons, targeting the transcripts for destruction by the nonsense-mediated decay pathway. These alternative splice sites are typically found in highly conserved sequence elements, which also contain binding sites for factors regulating the activation of the splice sites. Often, the activation is achieved by binding of products of the gene in question, resulting in negative feedback loops. In this study, I show that U11-48K, a protein factor specific to the minor spliceosome, specifically recognizes the U12-type 5' splice site sequence, and is essential for proper function of the minor spliceosome. Furthermore, the expression of U11-48K is regulated through a feedback mechanism, which functions through conserved sequence elements that activate alternative splicing and nonsense-mediated decay. This mechanism is conserved from plants to animals, highlighting both the importance and early origin of this mechanism in regulating splicing factors. I also show that the feedback regulation of U11-48K is counteracted by a component of the major spliceosome, the U1 small nuclear ribonucleoprotein particle, as well as members of the hnRNP F/H protein family. These results thus suggest that the feedback mechanism is finely tuned by multiple factors to achieve precise control of the activity of the U12-dependent spliceosome.

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.

Salmonella enterica Serovar Typhimurium Lacking hfq Gene Confers Protective Immunity against Murine Typhoid

Salmonella enterica is an important enteric pathogen and its various serovars are involved in causing both systemic and intestinal diseases in humans and domestic animals. The emergence of multidrug-resistant strains of Salmonella leading to increased morbidity and mortality has further complicated its management. Live attenuated vaccines have been proven superior over killed or subunit vaccines due to their ability to induce protective immunity. Of the various strategies used for the generation of live attenuated vaccine strains, focus has gradually shifted towards manipulation of virulence regulator genes. Hfq is a RNA chaperon which mediates the binding of small RNAs to the mRNA and assists in post-transcriptional gene regulation in bacteria. In this study, we evaluated the efficacy of the Salmonella Typhimurium Dhfq strain as a candidate for live oral vaccine in murine model of typhoid fever. Salmonella hfq deletion mutant is highly attenuated in cell culture and animal model implying a significant role of Hfq in bacterial virulence. Oral immunization with the Salmonella hfq deletion mutant efficiently protects mice against subsequent oral challenge with virulent strain of Salmonella Typhimurium. Moreover, protection was induced upon both multiple as well as single dose of immunizations. The vaccine strain appears to be safe for use in pregnant mice and the protection is mediated by the increase in the number of CD4(+) T lymphocytes upon vaccination. The levels of serum IgG and secretory-IgA in intestinal washes specific to lipopolysaccharide and outer membrane protein were significantly increased upon vaccination. Furthermore, hfq deletion mutant showed enhanced antigen presentation by dendritic cells compared to the wild type strain. Taken together, the studies in murine immunization model suggest that the Salmonella hfq deletion mutant can be a novel live oral vaccine candidate.

Post-transcriptional Repair of a Split Heat Shock Protein 90 Gene by mRNA trans-Splicing

Heat shock protein 90 participates in diverse biological processes ranging from protein folding, cell cycle, signal transduction and development to evolution in all eukaryotes. It is also critically involved in regulating growth of protozoa such as Dictyostelium discoideum, Leishmania donovani, Plasmodium falciparum, Trypanosoma cruzi, and Trypanosoma evansi. Selective inhibition of Hsp90 has also been explored as an intervention strategy against important human diseases such as cancer, malaria, or trypanosomiasis. Giardia lamblia, a simple protozoan parasite of humans and animals, is an important cause of diarrheal disease with significant morbidity and some mortality in tropical countries. Here we show that the G. lamblia cytosolic hsp90 ( glhsp90) is split in two similar sized fragments located 777 kb apart on the same scaffold. Intrigued by this unique arrangement, which appears to be specific for the Giardiinae, we have investigated the biosynthesis of GlHsp90. We used genome sequencing to confirm the split nature of the giardial hsp90. However, a specific antibody raised against the peptide detected a product with a mass of about 80 kDa, suggesting a post-transcriptional rescue of the genomic defect. We show evidence for the joining of the two independent Hsp90 transcripts in-trans to one long mature mRNA presumably by RNA splicing. The splicing junction carries hallmarks of classical cis-spliced introns, suggesting that the regular cis-splicing machinery may be sufficient for repair of the open reading frame. A complementary 26-nt sequence in the ``intron'' regions adjacent to the splice sites may assist in positioning the two pre-mRNAs for processing. This is the first example of post-transcriptional rescue of a split gene by trans-splicing.

Dendritic Poly(ether imine) Based Gene Delivery Vector

The nonviral vector based gene delivery approach is attractive due to advantages associated with molecular-level modifications suitable for optimization of vector properties. In a new class of nonviral gene delivery systems, we herein report the potential of poly(ether Mine) (PETIM) dendrimers to mediate an effective gene delivery function. PETIM dendrimer, constituted with tertiary amine branch points, n-propyl ether linkers and primary amines at their peripheries, exhibits significantly reduced toxicities, over a broad concentration range. The dendrimer complexes pDNA effectively, protects DNA from endosomal damages, and delivers to the cell nucleus. Gene transfection studies, utilizing a reporter plasmid pEGFP-C1 and upon complexation with dendrimer, showed a robust expression of the encoded protein. The study shows that PETIM dendrimers are hitherto unknown novel gene delivery vectors, combining features of poly(ethylene imine)-based polymers and dendrimers, yet are relatively nontoxic and structurally precise.

Cardiac remodeling in Drosophila arises from changes in actin gene expression and from a contribution of lymph gland-like cells to the heart musculature

Understanding the basis of normal heart remodeling can provide insight into the plasticity of the cardiac state, and into the potential for treating diseased tissue. In Drosophila, the adult heart arises during metamorphosis from a series of events, that include the remodeling of an existing cardiac tube, the elaboration of new inflow tracts, and the addition of a layer of longitudinal muscle fibers. We have identified genes active in all these three processes, and studied their expression in order to characterize in greater detail normal cardiac remodeling. Using a Transglutaminase-lacZ transgenic line, that is expressed in the inflow tracts of the larval and adult heart, we confirm the existence of five inflow tracts in the adult structure. In addition, expression of the Actin87E actin gene is initiated in the remodeling cardiac tube, but not in the longitudinal fibers, and we have identified an Act87E promoter fragment that recapitulates this switch in expression. We also establish that the longitudinal fibers are multinucleated, characterizing these cells as specialized skeletal muscles. Furthermore, we have defined the origin of the longitudinal fibers, as a subset of lymph gland cells associated with the larval dorsal vessel. These studies underline the myriad contributors to the formation of the adult Drosophila heart, and provide new molecular insights into the development of this complex organ. (C) 2011 Elsevier Ireland Ltd. All rights reserved.