Stochastic simulations of the origins and implications of long-tailed distributions in gene expression

Gene expression noise results in protein number distributions ranging from long-tailed to Gaussian. We show how long-tailed distributions arise from a stochastic model of the constituent chemical reactions and suggest that, in conjunction with cooperative switches, they lead to more sensitive selection of a subpopulation of cells with high protein number than is possible with Gaussian distributions. Single-cell-tracking experiments are presented to validate some of the assumptions of the stochastic simulations. We also examine the effect of DNA looping on the shape of protein distributions. We further show that when switches are incorporated in the regulation of a gene via a feedback loop, the distributions can become bimodal. This might explain the bimodal distribution of certain morphogens during early embryogenesis.

Structural plasticity and enzyme action: Crystal structures of Mycobacterium tuberculosis peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA in order to avoid the toxicity resulting from its accumulation and to free the tRNA available for further rounds in protein synthesis. The structure of the enzyme from Mycobacteritan tuberculosis has been determined in three crystal forms. This structure and the structure of the enzyme frorn Escherichia coli in its crystal differ substantially on account of the binding of the C terminus of the E. coli enzyme to the peptide-binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body, of the molecule and a polypepticle Y stretch involving a loop and a helix. This stretch is in the open conformation when the enzyme is in the free state as in the crystals of M. tuberculosis peptidyl-tRNA hydrolase. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the E. coli crystal mimics the peptide-bound enzyme molecule. The peptide stretch referred to earlier now closes on the bound peptide. Concurrently, a channel connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of two residues. Thus, the crystal structure of M. tuberculosis peptidyl-tRNA hydrolase when compared with the crystal structure of the E. coli enzyme, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule. (c) 2007 Elsevier Ltd. All rights reserved.

Characterization of the human SLC22A18 gene promoter and its regulation by the transcription factor Sp1

SLC22A18, a poly-specific organic cation transporter, is paternally imprinted in humans and mice. It shows loss-of-heterozygosity in childhood and adult tumors, and gain-of-imprinting in hepatocarcinomas and breast cancers. Despite the importance of this gene, its transcriptional regulation has not been studied, and the promoter has not yet been characterized. We therefore set out to identify the potential cis-regulatory elements including the promoter of this gene. The luciferase reporter assay in human cells indicated that a region from -120 by to +78 by is required for the core promoter activity. No consensus TATA or CHAT boxes were found in this region, but two Sp1 binding sites were conserved in human, chimpanzee, mouse and rat. Mutational analysis of the two Sp1 sites suggested their requirement for the promoter activity. Chromatin-immunoprecipitation showed binding of Sp1 to the promoter region in vivo. Overexpression of Sp1 in Drosophila Sp1-null SL2 cells suggested that Sp1 is the transactivator of the promoter. The human core promoter was functional in mouse 3T3 and monkey COS7 cells. We found a CpG island which spanned the core promoter and exon 1. COBRA technique did not reveal promoter methylation in 10 normal oral tissues, 14 oral tumors, and two human cell lines HuH7 and A549. This study provides the first insight into the mechanism that controls expression of this imprinted tumor suppressor gene. A COBRA-based assay has been developed to look for promoter methylation in different cancers. The present data will help to understand the regulation of this gene and its role in tumorigenesis. (C) 2008 Elsevier B.V. All rights reserved.

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa

Purpose: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. Methods: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. Results: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease.

Characterization of the human SLC22A18 gene promoter and its regulation by the transcription factor Sp1

SLC22A18, a poly-specific organic cation transporter, is paternally imprinted in humans and mice. It shows loss-of-heterozygosity in childhood and adult tumors, and gain-of-imprinting in hepatocarcinomas and breast cancers. Despite the importance of this gene, its transcriptional regulation has not been studied, and the promoter has not yet been characterized. We therefore set out to identify the potential cis-regulatory elements including the promoter of this gene. The luciferase reporter assay in human cells indicated that a region from -120 by to +78 by is required for the core promoter activity. No consensus TATA or CHAT boxes were found in this region, but two Sp1 binding sites were conserved in human, chimpanzee, mouse and rat. Mutational analysis of the two Sp1 sites suggested their requirement for the promoter activity. Chromatin-immunoprecipitation showed binding of Sp1 to the promoter region in vivo. Overexpression of Sp1 in Drosophila Sp1-null SL2 cells suggested that Sp1 is the transactivator of the promoter. The human core promoter was functional in mouse 3T3 and monkey COS7 cells. We found a CpG island which spanned the core promoter and exon 1. COBRA technique did not reveal promoter methylation in 10 normal oral tissues, 14 oral tumors, and two human cell lines HuH7 and A549. This study provides the first insight into the mechanism that controls expression of this imprinted tumor suppressor gene. A COBRA-based assay has been developed to look for promoter methylation in different cancers. The present data will help to understand the regulation of this gene and its role in tumorigenesis. (C) 2008 Elsevier B.V. All rights reserved.

Array-based gene expression, CGH and tissue data defines a 12q24 gain in neuroblastic tumors with prognostic implication

Neuroblastoma has successfully served as a model system for the identification of neuroectoderm-derived oncogenes. However, in spite of various efforts, only a few clinically useful prognostic markers have been found. Here, we present a framework, which integrates DNA, RNA and tissue data to identify and prioritize genetic events that represent clinically relevant new therapeutic targets and prognostic biomarkers for neuroblastoma.

Purification and properties of uridine hydrolase from mung-bean (Phaseolus radiatus) seedlings

The occurrence in plants of an enzyme system catalyzing the cleavage of uridine has been demonstrated. The enzyme from Phaseolus radiatus was purified about 132-fold with 24% recovery by a combination of procedures involving mild acid treatment, ammonium sulphate fractionation, negative adsorption on calcium phosphate gel and DEAE-cellulose chromatography. The enzyme cleaves uridine to uracil and ribose in the absence of phosphate indicating that the mechanism of cleavage was hydrolytic rather than phosphorolytic. The enzyme is specific to uridine and does not act on other purine and pyrimidine compounds. The enzyme shows maximum activity at pH 7.4 and has a temperature optimum of 45 °. It does not require metal ions for activity. Inhibition of the enzyme by p-chloromercuribenzoate as well as N-ethylmaleimide and the reversal of p-chloromercuribenzoate inhibition by sulfhydryl agents indicate the probable involvement of readily oxidizable sulfhydryl groups in enzyme activity.

Part I: Enzyme replacement versus neurotrophic factor viral vector-mediated gene therapy of Parkinson’s disease, Part II: The effects of orally dosed tolcapone and entacapone on dopamine metabolism in the dorsal striatum and nucleus accumbens in rats

Part I: Parkinson’s disease is a slowly progressive neurodegenerative disorder in which particularly the dopaminergic neurons of the substantia nigra pars compacta degenerate and die. Current conventional treatment is based on restraining symptoms but it has no effect on the progression of the disease. Gene therapy research has focused on the possibility of restoring the lost brain function by at least two means: substitution of critical enzymes needed for the synthesis of dopamine and slowing down the progression of the disease by supporting the functions of the remaining nigral dopaminergic neurons by neurotrophic factors. The striatal levels of enzymes such as tyrosine hydroxylase, dopadecarboxylase and GTP-CH1 are decreased as the disease progresses. By replacing one or all of the enzymes, dopamine levels in the striatum may be restored to normal and behavioral impairments caused by the disease may be ameliorated especially in the later stages of the disease. The neurotrophic factors glial cell derived neurotrophic factor (GDNF) and neurturin have shown to protect and restore functions of dopaminergic cell somas and terminals as well as improve behavior in animal lesion models. This therapy may be best suited at the early stages of the disease when there are more dopaminergic neurons for neurotrophic factors to reach. Viral vector-mediated gene transfer provides a tool to deliver proteins with complex structures into specific brain locations and provides long-term protein over-expression. Part II: The aim of our study was to investigate the effects of two orally dosed COMT inhibitors entacapone (10 and 30 mg/kg) and tolcapone (10 and 30 mg/kg) with a subsequent administration of a peripheral dopadecarboxylase inhibitor carbidopa (30 mg/kg) and L- dopa (30 mg/kg) on dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens of freely moving rats using dual-probe in vivo microdialysis. Earlier similarly designed studies have only been conducted in the dorsal striatum. We also confirmed the result of earlier ex vivo studies regarding the effects of intraperitoneally dosed tolcapone (30 mg/kg) and entacapone (30 mg/kg) on striatal and hepatic COMT activity. The results obtained from the dorsal striatum were generally in line with earlier studies, where tolcapone tended to increase dopamine and DOPAC levels and decrease HVA levels. Entacapone tended to keep striatal dopamine and HVA levels elevated longer than in controls and also tended to elevate the levels of DOPAC. Surprisingly in the nucleus accumbens, dopamine levels after either dose of entacapone or tolcapone were not elevated. Accumbal DOPAC levels, especially in the tolcapone 30 mg/kg group, were elevated nearly to the same extent as measured in the dorsal striatum. Entacapone 10 mg/kg elevated accumbal HVA levels more than the dose of 30 mg/kg and the effect was more pronounced in the nucleus accumbens than in the dorsal striatum. This suggests that entacapone 30 mg/kg has minor central effects. Also our ex vivo study results obtained from the dorsal striatum suggest that entacapone 30 mg/kg has minor and transient central effects, even though central HVA levels were not suppressed below those of the control group in either brain area in the microdialysis study. Both entacapone and tolcapone suppressed hepatic COMT activity more than striatal COMT activity. Tolcapone was more effective than entacapone in the dorsal striatum. The differences between dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens may be due to different properties of the two brain areas.

Evolution, Homology Conservation, and Identification of Unique Sequence Signatures in GH19 Family Chitinases

The discovery of GH (Glycoside Hydrolase) 19 chitinases in Streptomyces sp. raises the possibility of the presence of these proteins in other bacterial species, since they were initially thought to be confined to higher plants. The present study mainly concentrates on the phylogenetic distribution and homology conservation in GH19 family chitinases. Extensive database searches are performed to identify the presence of GH19 family chitinases in the three major super kingdoms of life. Multiple sequence alignment of all the identified GH19 chitinase family members resulted in the identification of globally conserved residues. We further identified conserved sequence motifs across the major sub groups within the family. Estimation of evolutionary distance between the various bacterial and plant chitinases are carried out to better understand the pattern of evolution. Our study also supports the horizontal gene transfer theory, which states that GH19 chitinase genes are transferred from higher plants to bacteria. Further, the present study sheds light on the phylogenetic distribution and identifies unique sequence signatures that define GH19 chitinase family of proteins. The identified motifs could be used as markers to delineate uncharacterized GH19 family chitinases. The estimation of evolutionary distance between chitinase identified in plants and bacteria shows that the flowering plants are more related to chitinase in actinobacteria than that of identified in purple bacteria. We propose a model to elucidate the natural history of GH19 family chitinases.

Genetic variation in the LDL receptor-related protein 5 (LRP5) gene : Association with bone health and metabolic parameters

Bone mass accrual and maintenance are regulated by a complex interplay between genetic and environmental factors. Recent studies have revealed an important role for the low-density lipoprotein receptor-related protein 5 (LRP5) in this process. The aim of this thesis study was to identify novel variants in the LRP5 gene and to further elucidate the association of LRP5 and its variants with various bone health related clinical characteristics. The results of our studies show that loss-of-function mutations in LRP5 cause severe osteoporosis not only in homozygous subjects but also in the carriers of these mutations, who have significantly reduced bone mineral density (BMD) and increased susceptibility to fractures. In addition, we demonstrated for the first time that a common polymorphic LRP5 variant (p.A1330V) was associated with reduced peak bone mass, an important determinant of BMD and osteoporosis in later life. The results from these two studies are concordant with results seen in other studies on LRP5 mutations and in association studies linking genetic variation in LRP5 with BMD and osteoporosis. Several rare LRP5 variants were identified in children with recurrent fractures. Sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses revealed no disease-causing mutations or whole-exon deletions. Our findings from clinical assessments and family-based genotype-phenotype studies suggested that the rare LRP5 variants identified are not the definite cause of fractures in these children. Clinical assessments of our study subjects with LPR5 mutations revealed an unexpectedly high prevalence of impaired glucose tolerance and dyslipidaemia. Moreover, in subsequent studies we discovered that common polymorphic LRP5 variants are associated with unfavorable metabolic characteristics. Changes in lipid profile were already apparent in pre-pubertal children. These results, together with the findings from other studies, suggest an important role for LRP5 also in glucose and lipid metabolism. Our results underscore the important role of LRP5 not only in bone mass accrual and maintenance of skeletal health but also in glucose and lipid metabolism. The role of LRP5 in bone metabolism has long been studied, but further studies with larger study cohorts are still needed to evaluate the specific role of LRP5 variants as metabolic risk factors.

Cytosines, but Not Purines, Determine Recombination Activating Gene (RAG)-induced Breaks on Heteroduplex DNA Structures

The sequence specificity of the recombination activating gene (RAG) complex during V(D)J recombination has been well studied. RAGs can also act as structure-specific nuclease; however, little is known about the mechanism of its action. Here, we show that in addition to DNA structure, sequence dictates the pattern and efficiency of RAG cleavage on altered DNA structures. Cytosine nucleotides are preferentially nicked by RAGs when present at single-stranded regions of heteroduplex DNA. Although unpaired thymine nucleotides are also nicked, the efficiency is many fold weaker. Induction of single- or double-strand breaks by RAGs depends on the position of cytosines and whether it is present on one or both of the strands. Interestingly, RAGs are unable to induce breaks when adenine or guanine nucleotides are present at single-strand regions. The nucleotide present immediately next to the bubble sequence could also affect RAG cleavage. Hence, we propose “C(d)C(S)C(S)” (d, double-stranded; s, single-stranded) as a consensus sequence for RAG-induced breaks at single-/double-strand DNA transitions. Such a consensus sequence motif is useful for explaining RAG cleavage on other types of DNA structures described in the literature. Therefore, the mechanism of RAG cleavage described here could explain facets of chromosomal rearrangements specific to lymphoid tissues leading to genomic instability.

Association of methylenetetrahydrofolate reductase gene polymorphisms & colorectal cancer in India

Background & objectives: Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism and involved in DNA synthesis, DNA repair and DNA methylation. The two common functional polymorphisms of MTHFR, 677C -> T and 1298 A -> C have shown to impact several diseases including cancer. This case-control study was undertaken to analyse the association of the MTHFR gene polymorphisms 677 C -> T and 1298 A -> C and risk of colorectal cancer (CRC).Methods: One hundred patients with a confirmed histopathologic diagnosis of CRC and 86 age and gender matched controls with no history of cancer were taken for this study. DNA was isolated from peripheral blood samples and the genotypes were determined by PCR-RFLP. The risk association was estimated by compounding odds ratio (OR) with 95 per cent confidence interval (CI). Results: Genotype frequency of MTHFR 677 CC, CT and TT were 76.7, 22.1 and 1.16 per cent in controls, and 74,25 and 1.0 per cent among patients. The 'T' allele frequency was 12.21 and 13.5 per cent in controls and patients respectively. The genotype frequency of MTHFR 1298 AA, AC, and CC were 25.6, 58.1 and 16.3 per cent for controls and 22, 70 and 8 per cent for patents respectively. The 'C' allele frequency for 1298 A -> C was 43.0 and 45.3 per cent respectively for controls and patients. The OR for 677 CT was 1.18 (95% CI 0.59-2.32, P = 0.642), OR for 1298 AC was 1.68 (95% CI 0.92-3.08, P = 0.092) and OR for 1298 CC was 0.45(95% CI 0.18-1.12, P = 0.081). The OR for the combined heterozygous state (677 CT and 1298 AC) was 1.18(95% CI 0.52-2.64, P =0.697).Interpretation & conclusion: The frequency of the MTHFR 677 TT genotype is rare as compared to 1298 CC genotype in the population studied. There was no association between 677 C -> T and 1298 A -> C polymorphisms and risk of CRC either individually or in combination. The homozygous state for 1298 A -> C polymorphism appears to slightly lower risk of CRC. This needs to be confirmed with a larger sample size.

The transient response of a two-terminal solion to a step-current input

The transition time associated with the time-variation of the voltage across a two-terminal diaphragm-less solion in response to a step-current stimulus has been studied experimentally. A theoretical analysis has also been made by solving the diffusion problem under the appropriate initial and boundary conditions. The behaviour of the theoretically predicted transition times is in agreement with the observed behaviour. The systems under study have been shown to be different from those used hitherto in thin-layer chronopotentiometry.

The transient response of a two-terminal solion to a stepcrrent input

The transition time associated with the time-variation of the voltage across a two-terminal diaphragm-less solion in response to a step-current stimulus has been studied experimentally. A theoretical analysis has also been made by solving the diffusion problem under the appropriate initial and boundary conditions. The behaviour of the theoretically predicted transition times is in agreement with the observed behaviour. The systems under study have been shown to be different from those used hitherto in thin-layer chronopotentiometry.

Drosophila indirect flight muscle specific Act88F actin mutants as a model system for studying congenital myopathies of the human ACTA1 skeletal muscle actin gene

Most human ACTA1 skeletal actin gene mutations cause dominant, congenital myopathies often with severely reduced muscle function and neonatal mortality. High sequence conservation of actin means many mutated ACTA1 residues are identical to those in the Drosophila Act88F, an indirect flight muscle specific sarcomeric actin. Four known Act88F mutations occur at the same actin residues mutated in ten ACTA1 nemaline mutations, A138D/P, R256H/L, G268C/D/R/S and R372C/S. These Act88F mutants were examined for similar muscle phenotypes. Mutant homozygotes show phenotypes ranging from a lack of myofibrils to almost normal sarcomeres at eclosion. Aberrant Z-disc-like structures and serial Z-disc arrays, ‘zebra bodies’, are observed in homozygotes and heterozygotes of all four Act88F mutants. These electron-dense structures show homologies to human nemaline bodies/rods, but are much smaller than those typically found in the human myopathy. We conclude that the Drosophila indirect flight muscles provide a good model system for studying ACTA1 mutations.