The trishanku gene and terminal morphogenesis in Dictyostelium discoideum

P>Multicellular development in the social amoeba Dictyostelium discoideum is triggered by starvation. It involves a series of morphogenetic movements, among them being the rising of the spore mass to the tip of the stalk. The process requires precise coordination between two distinct cell types-presumptive (pre-) spore cells and presumptive (pre-) stalk cells. Trishanku (triA) is a gene expressed in prespore cells that is required for normal morphogenesis. The triA- mutant shows pleiotropic effects that include an inability of the spore mass to go all the way to the top. We have examined the cellular behavior required for the normal ascent of the spore mass. Grafting and mixing experiments carried out with tissue fragments and cells show that the upper cup, a tissue that derives from prestalk cells and anterior-like cells (ALCs), does not develop properly in a triA- background. A mutant upper cup is unable to lift the spore mass to the top of the fruiting body, likely due to defective intercellular adhesion. If wild-type upper cup function is provided by prestalk and ALCs, trishanku spores ascend all the way. Conversely, Ax2 spores fail to do so in chimeras in which the upper cup is largely made up of mutant cells. Besides proving that under these conditions the wild-type phenotype of the upper cup is necessary and sufficient for terminal morphogenesis in D. discoideum, this study provides novel insights into developmental and evolutionary aspects of morphogenesis in general. Genes that are active exclusively in one cell type can elicit behavior in a second cell type that enhances the reproductive fitness of the first cell type, thereby showing that morphogenesis is a cooperative process.

Evolution of sequence specificity in a restriction endonuclease by a point mutation

Restriction endonucleases (REases) protect bacteria from invading foreign DNAs and are endowed with exquisite sequence specificity. REases have originated from the ancestral proteins and evolved new sequence specificities by genetic recombination, gene duplication, replication slippage, and transpositional events. They are also speculated to have evolved from nonspecific endonucleases, attaining a high degree of sequence specificity through point mutations. We describe here an example of generation of exquisitely site-specific REase from a highly-promiscuous one by a single point mutation.

Evolution of domain combinations in protein kinases and its implications for functional diversity

Protein kinases phosphorylating Ser/Thr/Tyr residues in several cellular proteins exert tight control over their biological functions. They constitute the largest protein family in most eukaryotic species. Protein kinases classified based on sequence similarity in their catalytic domains, cluster into subfamilies, which share gross functional properties. Many protein kinases are associated or tethered covalently to domains that serve as adapter or regulatory modules,naiding substrate recruitment, specificity, and also serve as scaffolds. Hence the modular organisation of the protein kinases serves as guidelines to their functional and molecular properties. Analysis of genomic repertoires of protein kinases in eukaryotes have revealed wide spectrum of domain organisation across various subfamilies of kinases. Occurrence of organism-specific novel domain combinations suggests functional diversity achieved by protein kinases in order to regulate variety of biological processes. In addition, domain architecture of protein kinases revealed existence of hybrid protein kinase subfamilies and their emerging roles in the signaling of eukaryotic organisms. In this review we discuss the repertoire of non-kinase domains tethered to multi-domain kinases in the metazoans. Similarities and differences in the domain architectures of protein kinases in these organisms indicate conserved and unique features that are critical to functional specialization. (C) 2009 Elsevier Ltd. All rights reserved.

Confirmation of linkage and refinement of the RP28 locus for autosomal recessive retinitis pigmentosa on chromosome 2p14-p15 in an Indian family

PURPOSE: To report the linkage analysis of retinitis pigmentosa (RP) in an Indian family. METHODS: Individuals were examined for symptoms of retinitis pigmentosa and their blood samples were withdrawn for genetic analysis. The disorder was tested for linkage to known 14 adRP and 22 arRP loci using microsatellite markers. RESULTS: Seventeen individuals including seven affecteds participated in the study. All affected individuals had typical RP. The age of onset of the disease ranged from 8-18 years. The disorder in this family segregated either as an autosomal recessive trait with pseudodominance or an autosomal dominant trait. Linkage to an autosomal recessive locus RP28 on chromosome 2p14-p15 was positive with a maximum two-point lod score of 3.96 at theta=0 for D2S380. All affected individuals were homozygous for alleles at D2S2320, D2S2397, D2S380, and D2S136. Recombination events placed the minimum critical region (MCR) for the RP28 gene in a 1.06 cM region between D2S2225 and D2S296. CONCLUSIONS : The present data confirmed linkage of arRP to the RP28 locus in a second Indian family. The RP28 locus was previously mapped to a 16 cM region between D2S1337 and D2S286 in a single Indian family. Haplotype analysis in this family has further narrowed the MCR for the RP28 locus to a 1.06 cM region between D2S2225 and D2S296. Of 15 genes reported in the MCR, 14 genes (KIAA0903, OTX1, MDH1, UGP2, VPS54, PELI1, HSPC159, FLJ20080, TRIP-Br2, SLC1A4, KIAA0582, RAB1A, ACTR2, and SPRED2) are either expressed in the eye or retina. Further study needs to be done to test which of these genes is mutated in patients with RP linked to the RP28 locus.

The evolution of genomic imprinting

We explore three possible pathways for the evolution of genomic imprinting. (1) Imprinting may be advantageous in itself when imprinted and unimprinted alleles of a locus confer different phenotypes. If a segment of DNA is imprinted in the gametes of one sex but not in those of the other, it might lead to effects correlated with sexual dimorphism. More fundamentally, in certain organisms, sex determination might have evolved because of imprinting. When imprinting leads to chromosome elimination or inactivation and occurs in some embryos but not in others, two classes of embryos, differing in the number of functional gene copies, would result. A model for sex determination based on inequality in the actual or effective copy-number of particular noncoding, regulatory sequences of DNA has been proposed (Chandra, Proc. natn. Acad. Sci. U.S.A. 82. 1165–1169 and 6947–6949, 1985). Maternal control of offspring sex is another possible consequence of imprinting; this would indicate a potential role for imprinting in sex ratio evolution. (2) Genes responsible for imprinting may have pleiotropic effects and they may have been selected for reasons other than their imprinting ability. Lack of evidence precludes further consideration of this possibility. (3) Imprinting could have co-evolved with other traits. For instance, gamete-specific imprinting could lead to a lowered fitness of androgenetic or gynogenetic diploids relative to the fitness of ‘normal’ diploids. This in turn would reinforce the evolution of anisogamy. The reversibility of imprinting raises the possibility of occasional incomplete or improper erasure. If the site of imprinting is the egg – as appears to be the case with the human X (Chandra and Brown, Nature 253. 165–168, 1975) – either improper imprinting or improper erasure could lead to unusual patterns of inheritance (as in the fragile-X syndrome) or fitness effects skipping generations.

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.

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.

Studies on molecular evolution and structural features of double-headed inhibitors of ?-amylase and trypsin in plants

Plant seeds usually have high concentrations of proteinase and amylase inhibitors. These inhibitors exhibit a wide range of specificity, stability and oligomeric structure. In this communication, we report analysis of sequences that show statistically significant similarity to the double-headed alpha-amylase/trypsin inhibitor of ragi (Eleusine coracana). Our aim is to understand their evolutionary and structural features. The 14 sequences of this family that are available in the SWISSPROT database form three evolutionarily distinct branches. The branches relate to enzyme specificities and also probably to the oligomeric state of the proteins and not to the botanical class of the plant from which the enzymes are derived. This suggests that the enzyme specificities of the inhibitors evolved before the divergence of commercially cultivated cereals. The inhibitor sequences have three regions that display periodicity in hydrophobicity. It is likely that this feature reflects extended secondary structure in these segments. One of the most variable regions of the polypeptide corresponds to a loop, which is most probably exposed in the native structure of the inhibitors and is responsible for the inhibitory property.

Variational principles in evolution

For a one-locus selection model, Svirezhev introduced an integral variational principle by defining a Lagrangian which remained stationary on the trajectory followed by the population undergoing selection. It is shown here (i) that this principle can be extended to multiple loci in some simple cases and (ii) that the Lagrangian is defined by a straightforward generalization of the one-locus case, but (iii) that in two-locus or more general models there is no straightforward extension of this principle if linkage and epistasis are present. The population trajectories can be constructed as trajectories of steepest ascent in a Riemannian metric space. A general method is formulated to find the metric tensor and the surface-in the metric space on which the trajectories, which characterize the variations in the gene structure of the population, lie. The local optimality principle holds good in such a space. In the special case when all possible linkage disequilibria are zero, the phase point of the n-locus genetic system moves on the surface of the product space of n higher dimensional unit spheres in a certain Riemannian metric space of gene frequencies so that the rate of change of mean fitness is maximum along the trajectory. In the two-locus case the corresponding surface is a hyper-torus.

Higher topoisomerase 2 alpha gene transcript levels predict better prognosis in GBM patients receiving temozolomide chemotherapy: identification of temozolomide as a TOP2A inhibitor

The search for molecular markers which predict response to chemotherapy is an important aspect of current neuro-oncology research. MGMT promoter methylation is the only proved marker of glioblastoma. The purpose of this study was to assess the effect of topoisomerase expression on glioblastoma survival and study the mechanisms involved. The transcript levels of all isoforms of the topoisomerase family in all grades of diffuse astrocytoma were assessed. A prospective study of patients with glioblastoma treated by a uniform treatment procedure was performed with the objective of correlating outcome with gene expression. The ability of TOP2A enzyme to relax the super coiled plasmid DNA in the presence of temozolomide was evaluated to assess its effect on TOP2A. The temozolomide cyctotoxicity of TOP2A-silenced U251 cells was assessed. The transcript levels of TOP2A, TOP2B, and TOP3A are upregulated significantly in GBM in comparison with lower grades of astrocytoma and normal brain samples. mRNA levels of TOP2A correlated significantly with survival of the patients. Higher TOP2A transcript levels in GBM patients predicted better prognosis (P = 0.043; HR = 0.889). Interestingly, we noted that temozolomide inhibited TOP2A activity in in-vitro enzyme assays. We also noted that siRNA knock down of TOP2A rendered a glioma cell line resistant to temozolomide chemotherapy. We demonstrated for the first time that temozolomide is also a TOP2A inhibitor and established that TOP2A transcript levels determine the chemosensitivity of glioblastoma to temozolomide therapy. Very high levels of TOP2A are a good prognostic indicator in GBM patients receiving temozolomide chemotherapy.

Genetic analysis of an Indian family with members affected with Waardenburg syndrome and Duchenne muscular dystrophy

Purpose: Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of the eye, skin, and hair. It is caused by mutations in one of the following genes: PAX3 (paired box 3), MITF (microphthalmia-associated transcription factor), EDNRB (endothelin receptor type B), EDN3 (endothelin 3), SNAI2 (snail homolog 2, Drosophila) and SOX10 (SRY-box containing gene 10). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene. The purpose of this study was to identify the genetic causes of WS and DMD in an Indian family with two patients: one affected with WS and DMD, and another one affected with only WS. Methods: Blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to the eight known WS loci, microsatellite markers were selected from the candidate regions and used to genotype the family. Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affected individuals to detect mutations. A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependent probe amplification method using exon-specific probes. Results: Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. Haplotype analysis suggested linkage of the family to the WS4B (EDN3) locus. DNA sequencing identified a novel missense mutation p.T98M in EDN3. A deletion mutation was identified in DMD. Conclusions: This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indian family. The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum of EDN3.

The chbG Gene of the Chitobiose (chb) Operon of Escherichia coli Encodes a Chitooligosaccharide Deacetylase

The chb operon of Escherichia coli is involved in the utilization of the beta-glucosides chitobiose and cellobiose. The function of chbG (ydjC), the sixth open reading frame of the operon that codes for an evolutionarily conserved protein is unknown. We show that chbG encodes a monodeacetylase that is essential for growth on the acetylated chitooligosaccharides chitobiose and chitotriose but is dispensable for growth on cellobiose and chitosan dimer, the deacetylated form of chitobiose. The predicted active site of the enzyme was validated by demonstrating loss of function upon substitution of its putative metal-binding residues that are conserved across the YdjC family of proteins. We show that activation of the chb promoter by the regulatory protein ChbR is dependent on ChbG, suggesting that deacetylation of chitobiose-6-P and chitotriose-6-P is necessary for their recognition by ChbR as inducers. Strains carrying mutations in chbR conferring the ability to grow on both cellobiose and chitobiose are independent of chbG function for induction, suggesting that gain of function mutations in ChbR allow it to recognize the acetylated form of the oligosaccharides. ChbR-independent expression of the permease and phospho-beta-glucosidase from a heterologous promoter did not support growth on both chitobiose and chitotriose in the absence of chbG, suggesting an additional role of chbG in the hydrolysis of chitooligosaccharides. The homologs of chbG in metazoans have been implicated in development and inflammatory diseases of the intestine, indicating that understanding the function of E. coli chbG has a broader significance.

How Does the Spacer Length of Cationic Gemini Lipids Influence the Lipoplex Formation with Plasmid DNA? Physicochemical and Biochemical Characterizations and their Relevance in Gene Therapy

Lipoplexes formed by the pEGFP-C3 plasmid DNA (pDNA) and lipid mixtures containing cationic gemini surfactant of the 1,2-bis(hexadecyl dimethyl ammonium) Acmes family referred to as C16CnC16, where n = 2 3, 5, or 12, and the zwitterionic helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) have been studied from a wide variety of physical, chemical, and biological standpoints. The study has been carried out using several experimental methods, such as zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), cryo-TEM, gene transfection, cell viability/cytotoxicity, and confocal fluorescence microscopy. As reported recently in a communication (J. Am. Chem. Soc. 2011, 133, 18014), the detailed physicochemical and biological studies confirm that, in the presence of the studied series lipid mixtures, plasmid DNA is compacted with a large number of its associated Na+ counterions. This in turn yields a much lower effective negative charge, q(pDNA)(-), a value that has been experimentally obtained for each mixed lipid mixture. Consequently, the cationic lipid (CL) complexes prepared with pDNA and CL/DOPE mixtures to be used in gene transfection require significantly less amount of CL than the one estimated assuming a value of q(DNA)(-) = -2. This drives to a considerably lower cytotoxicity of the gene vector. Depending on the CL molar composition, alpha, of the lipid mixture, and the effective charge ratio of the lipoplex, rho(eff), the reported SAXS data indicate the presence of two or three structures in the same lipoplex, one in the DOPE-rich region, other in the CL-rich region, and another one present at any CL composition. Cryo-TEM and SAXS studies with C16CnC16/DOPE-pDNA lipoplexes indicate that pDNA is localized between the mixed lipid bilayers of lamellar structures within a monolayer of similar to 2 nm. This is consistent with a highly compacted supercoiled pDNA conformation compared with that of linear DNA. Transfection studies were carried out with HEK293T, HeLa, CHO, U343, and H460 cells. The alpha and rho(eff) values for each lipid mixture were optimized on HEK293T cells for transfection, and using these values, the remaining cells were also transfected in absence (-FBS-FBS) and presence (-FBS+FBS) of serum. The transfection efficiency was higher with the CLs of shorter gemini spacers (n = 2 or 3). Each formulation expressed GFP on pDNA transfection and confocal fluorescence microscopy corroborated the results. C16C2C16/DOPE mixtures were the most efficient toward transfection among all the lipid mixtures and, in presence of serum, even better than the Lipofectamine2000, a commercial transfecting agent Each lipid combination was safe and did not show any significant levels of toxicity. Probably, the presence of two coexisting lamellar structures in lipoplexes synergizes the transfection efficiency of the lipid mixtures which are plentiful in the lipoplexes formed by CLs with short spacer (n = 2, 3) than those with the long spacer (n = 5, 12).

Gene expression in osteoblast cells treated with submicron to nanometer hydroxyapatite-mullite eluate particles

The present research focused on determining the effect of hydroxyapatite-20 wt% mullite (H20M) particle eluates on apoptosis and differentiation of human fetal osteoblast (hFOB) cells. The H20M particles (257 +/- 37 nm) were prepared, starting with the production of a nanocomposite using a unique route of spark plasma sintering, followed by a repeated grinding-cryo treatment and elution process. Tetrazolium based cytotoxicity assay results showed a time-and dose-dependent effect of H20M particle eluates on hFOB cytotoxicity. In particular, the results revealed statistically reduced cell viability after hFOB were exposed to the above 10% H20M (257 +/- 37 nm) eluates for 48 h. The apoptotic cell death triggered by H20M treatment was proven by the analysis of molecular markers of apoptosis, that is, the Bcl-2 family of genes. hFOB expression of Bcl-xL and Bcl-xS significantly increased 25.6- and 25.2-fold for 50% of H20M concentrations, respectively. The ratio of Bcl-xL/Bax (4.01) decreased 2-fold for hFOB exposed to 100% of H20M eluates than that for 10% H20M eluate (7.94) treated hFOB cells. On the other hand, the Bcl-xS/Bax ratio for the 10% H20M eluate was 4.15-fold, whereas for 100% H20M eluates, it was 11.55-fold. Specifically, the anti-apoptotic effect of the H20M particle eluates was corroborated by the up-regulation of bone cell differentiation marker genes such as, collagen type I, cbfa, and osteocalcin. In summary, the present work clearly demonstrated that H20M submicron to nanometer composite particle eluates have a minimal effect on hFOB apoptosis and can even up-regulate the expression of bone cell markers at the molecular level.

Mutation analysis of the SLC4A11 gene in Indian families with congenital hereditary endothelial dystrophy 2 and a review of the literature

Purpose: Congenital hereditary endothelial dystrophy 2 (CHED2) is an autosomal recessive disorder caused by mutations in the solute carrier family 4, sodium borate transporter, member 11 (SLC4A11) gene. The purpose of this study was to identify the genetic cause of CHED2 in six Indian families and catalog all known mutations in the SLC4A11 gene. Methods: Peripheral blood samples were collected from individuals of the families with CHED2 and used in genomic DNA isolation. PCR primers were used to amplify the entire coding region including intron-exon junctions of SLC4A11. Amplicons were subsequently sequenced to identify the mutations. Results: DNA sequence analysis of the six families identified four novel (viz., p.Thr262Ile, p.Gly417Arg, p.Cys611Arg, and p.His724Asp) mutations and one known p.Arg869His homozygous mutation in the SLC4A11 gene. The mutation p.Gly417Arg was identified in two families. Conclusions: This study increases the mutation spectrum of the SLC4A11 gene. A review of the literature showed that the total number of mutations in the SLC4A11 gene described to date is 78. Most of the mutations are missense, followed by insertions-deletions. The present study will be helpful in genetic diagnosis of the families reported here.