The Molecular Phylogeny of a Nematode-Specific Clade of Heterotrimeric G-Protein α-Subunit Genes

In animal olfactory systems, odorant molecules are detected by olfactory receptors (ORs). ORs are part of the G-protein-coupled receptor (GPCR) superfamily. Heterotrimeric guanine nucleotide binding G-proteins (G-proteins) relay signals from GPCRs to intracellular effectors. G-proteins are comprised of three peptides. The G-protein α subunit confers functional specificity to G-proteins. Vertebrate and insect Gα-subunit genes are divided into four subfamilies based on functional and sequence attributes. The nematode Caenorhabditis elegans contains 21 Gα genes, 14 of which are exclusively expressed in sensory neurons. Most individual mammalian cells express multiple distinct GPCR gene products, however, individual mammalian and insect olfactory neurons express only one functional odorant OR. By contrast C. elegans expresses multiple ORs and multiple Gα subunits within each olfactory neuron. Here we show that, in addition to having at least one member of each of the four mammalian Gα gene classes, C. elegans and other nematodes also possess two lineage-specific Gα gene expansions, homologues of which are not found in any other organisms examined. We hypothesize that these novel nematode-specific Gα genes increase the functional complexity of individual chemosensory neurons, enabling them to integrate odor signals from the multiple distinct ORs expressed on their membranes. This neuronal gene expansion most likely occurred in nematodes to enable them to compensate for the small number of chemosensory cells and the limited emphasis on cephalization during nematode evolution. [Reviewing Editor: Dr. John Oakeshott] Damien M. O’Halloran and David A. Fitzpatrick contributed equally to this work.     

Genes for all metals—a bacterial view of the Periodic Table

Bacterial chromosomes have genes for transport proteins for inorganic nutrient cations and oxyanions, such as NH₄ ⁺, K⁺, Mg²⁺, Co²⁺, Fe³⁺, Mn²⁺, Zn²⁺ and other trace cations, PO₄ ^3-, SO₄ ^2- and less abundant oxyanions. Together these account for perhaps a few hundred genes in many bacteria. Bacterial plasmids encode resistance systems for toxic metal and metalloid ions including Ag⁺ AsO₂ ^-, AsO₄ ^3-, Cd²⁺, Co²⁺, CrO₄ ²⁻, Cu²⁺, Hg²⁺, Ni²⁺, Pb²⁺, TeO₃ ²⁻, TI⁺ and Zn²⁺. Most resistance systems function by energy-dependent efflux of toxic ions. A few involve enzymatic (mostly redox) transformations. Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. The Cd²⁺-resistance cation pump of Gram-positive bacteria is membrane P-type ATPase, which has been labeled with ³²P from [γ-³²P]ATP and drives ATP-dependent Cd²⁺ (and Zn²⁺) transport by membrane vesicles. The genes defective in the human hereditary diseases of copper metabolism, Menkes syndrome and Wilson’s disease, encode P-type ATPases that are similar to bacterial cadmium ATPases. The arsenic resistance system transports arsenite [As(III)], alternatively with the ArsB polypeptide functioning as a chemiosmotic efflux transporter or with two polypeptides, ArsB and ArsA, functioning as an ATPase. The third protein of the arsenic resistance system is an enzyme that reduces intracellular arsenate [As(V)] to arsenite [As(III)], the substrate of the efflux system. In Gram-negative cells, a three polypeptide complex functions as a chemiosmotic cation/protein exchanger to efflux Cd²⁺, Zn²⁺ and Co²⁺. This pump consists of an inner membrane (CzcA), an outer membrane (CzcC) and a membrane-spanning (CzcB) protein that function together. Received 08 August 1997/ Accepted in revised form 01 November 1997     

How Informative Is Wright's Estimator of the Number of Genes Affecting a Quantitative Character?

S. Wright suggested an estimator, m, of the number of loci, m, contributing to the difference in a quantitative character between two differentiated populations, which is calculated from the phenotypic means and variances in the two parental populations and their F1 and F2 hybrids. The same method can also be used to estimate m contributing to the genetic variance within a single population, by using divergent selection to create differentiated lines from the base population. In this paper we systematically examine the utility and problems of this technique under the influences of unequal allelic effects and initial allele frequencies, and linkage, which are known to lead m to underestimate m. In addition, we examine the effects of population size and selection intensity during the generations of selection. During selection, the estimator m rapidly approaches its expected value at the selection limit. With reasonable assumptions about unequal allelic effects and initial allele frequencies, the expected value of m without linkage is likely to be on the order of one-third of the number of genes. The estimates suffer most seriously from linkage. The practical maximum expectation of m is just about the number of chromosomes, considerably less than the "recombination index" which has been assumed to be the upper limit. The estimates are also associated with large sampling variances. An estimator of the variance of m derived by R. Lande substantially underestimates the actual variance. Modifications to the method can ameliorate some of the problems. These include using F3 or later generation variances or the genetic variance in the base population, and replicating the experiments and estimation procedure. However, even in the best of circumstances, information from m is very limited and can be misleading.     

LEA Gene Introns: is the Intron of Dehydrin Genes a Characteristic of the Serine-Segment?

Dehydrins, which belong to group 2 LEA proteins, are a family of intrinsically unstructured plant proteins that accumulate during the late stages of embryogenesis and in response to abiotic stresses. We have previously reported that the OpsDHN1 gene, encoding an SK₃-type acidic dehydrin protein from Opuntia streptacantha, contains an intron inserted within the sequence encoding the S-motif. Herein, we present an in silico analysis of intron sequences in dehydrin genes from mono- and dicotyledonous plants that reveals a preference for insertion within the nucleotide sequence encoding the S-motif. Sequence comparison of ten Dhn genes from Arabidopsis thaliana and the orthologous genes in Arabidopsis lyrata revealed that introns maintain considerable sequence identity and conserve the insertion pattern. Furthermore, syntenic regions were identified among eight orthologous genes of A. thaliana and A. lyrata, showing that correlated gene arrangements are conserved between these Arabidopsis species. Our study shows that most SKn-type dehydrins contain one intron that is conserved in phase and location; this intron is linked to the nucleotide sequence that encodes the S-motif.     

Expression of Psychrophilic Genes in Mesophilic Hosts: Assessment of the Folding State of a Recombinant α-Amylase

α-Amylase from the antarctic psychrophile Alteromonas haloplanktis is synthesized at 0 ± 2°C by the wild strain. This heat-labile α-amylase folds correctly when overexpressed in Escherichia coli, providing the culture temperature is sufficiently low to avoid irreversible denaturation. In the described expression system, a compromise between enzyme stability and E. coli growth rate is reached at 18°C.Psychrophilic enzymes possess specific properties, such as high activity at low temperatures and weak thermal stability, which promise to allow the use of these enzymes as industrial biocatalysts, as biotechnological tools, or for fundamental research (6, 8, 11). For instance, substantial energy savings can be obtained if heating is not required during large-scale processes which take advantage of the efficient catalytic capacity of cold-adapted enzymes in the range 0 to 20°C. The pronounced heat lability of psychrophilic enzymes also allows their selective inactivation in a complex mixture, as illustrated by an antarctic bacterial alkaline phosphatase which is available for molecular biology research (7). Finally, psychrophilic enzymes represent the lower natural limit of protein stability (3) and are useful tools for studies in the field of protein folding.Large-scale fermentation of psychrophilic microorganisms suffers from two main drawbacks, however: the low production levels of wild strains and the prohibitive cost of growing wild strains at low temperatures. A possible alternative is to overexpress the gene coding for a psychrophilic protein in a mesophilic host for which efficient expression systems have been designed. In this context, two crucial questions remain to be solved: (i) what is the folding state of an enzyme normally synthesized at 0°C when it is expressed by the mesophilic genetic machinery at higher temperatures, and (ii) is there a temperature at which a compromise can be reached between the stability of the psychrophilic enzyme and the mesophilic growth rate? To address these questions, the heat-labile α-amylase from the antarctic psychrophile Alteromonas haloplanktis (2, 4) was expressed in Escherichia coli at various temperatures.

Construction of the expression vector and α-amylase production.

The α-amylase gene (2) was cloned downstream from the lacZ promoter in pUC12 by ligating the SmaI site of the polylinker to the HpaI site located 60 nucleotides upstream from the formylmethionine codon. This construction is devoid of the C-terminal peptide cleaved by the wild strain following α-amylase secretion. The recombinant enzyme was expressed in E. coli RR1 with the constitutive assistance of lacZ (without IPTG [isopropyl-β-d-thiogalactopyranoside] induction) in a medium containing 16 g of bactotryptone, 16 g of yeast extract, 5 g of NaCl, 2.5 g of K₂HPO₄, 0.1 μM CaCl₂, and 100 mg of ampicillin per liter. The effect of the culture temperature on α-amylase production by E. coli is illustrated in Fig. ​Fig.1.1. Within the range of temperatures used, maximal enzyme production was reached below 18°C, whereas higher temperatures induced a gradual decrease of α-amylase activity in cultures. Three independent cultures were pooled for the purification of the recombinant enzymes produced at 18 and 25°C. Open in a separate windowFIG. 1Temperature dependence of α-amylase production by E. coli. Results are expressed as percent mean maximal activity recorded at 18°C.

α-Amylase purification.

The gram-negative A. haloplanktis was cultivated at 4°C, and α-amylase was purified from the culture supernatants by ion-exchange chromatography on DEAE-agarose followed by gel filtration on Sephadex G-100 and Ultrogel AcA54 as previously described (2, 4). The recombinant α-amylases were purified by the protocol developed for the wild-type enzyme except that concentration by ammonium sulfate precipitation at 70% saturation was required before the first chromatographic step. Recombinant enzyme production at 18 and 25°C ranged between 60 and 100 mg/liter of culture, which corresponds to a 10-fold improvement over production by the wild strain.

Characterization of the recombinant α-amylases.

N- and C-terminal amino acid sequences (determined on an Applied Biosystems Procise analyzer and by carboxypeptidase Y digestion, respectively) of α-amylase produced at 18 and 25°C indicated that the signal peptide is correctly cleaved in E. coli and that no additional posttranslational cleavage occurred. The isoelectric point (5.5) and the molecular mass (49,340 Da as determined from the sequence and 49,342 ± 8 Da as determined from electrospray mass spectroscopy measurements) were identical to the values recorded for the wild-type enzyme. Dynamic light scattering (DynaPro-801; DLS Instruments) also showed that the purified recombinant enzymes are homogeneous, without any evidence of aggregated forms.

Comparison of the wild-type and recombinant α-amylases.

Several properties of the wild-type enzyme produced at 4°C and the recombinant α-amylase expressed in E. coli at 18°C were compared (Table ​(Table1).1).

TABLE 1

Kinetic parameters, dissociation constants, and free thiol groups for the wild-type and recombinant α-amylases

Over expression of wild type or a catalytically dead mutant of Sirtuin 6 does not influence NFκB responses

SIRT6 is involved in inflammation, aging and metabolism potentially by modulating the functions of both NFκB and HIF1α. Since it is possible to make small molecule activators and inhibitors of Sirtuins we wished to establish biochemical and cellular assays both to assist in drug discovery efforts and to validate whether SIRT6 represents a valid drug target for these indications. We confirmed in cellular assays that SIRT6 can deacetylate acetylated-histone H3 lysine 9 (H3K9Ac), however this deacetylase activity is unusually low in biochemical assays. In an effort to develop alternative assay formats we observed that SIRT6 overexpression had no influence on TNFα induced nuclear translocation of NFκB, nor did it have an effect on nuclear mobility of RelA/p65. In an effort to identify a gene expression profile that could be used to identify a SIRT6 readout we conducted genome-wide expression studies. We observed that overexpression of SIRT6 had little influence on NFκB-dependent genes, but overexpression of the catalytically inactive mutant affected gene expression in developmental pathways.     

Phylogenetic and Functional Analysis of Gut Microbiota of a Fungus-Growing Higher Termite: Bacteroidetes from Higher Termites Are a Rich Source of β-Glucosidase Genes

Fungus-growing termites, their symbiotic fungi, and microbiota inhibiting their intestinal tract comprise a highly efficient cellulose-hydrolyzing system; however, little is known about the role of gut microbiota in this system. Twelve fosmid clones with β-glucosidase activity were previously obtained by functionally screening a metagenomic library of a fungus-growing termite, Macrotermes annandalei. Ten contigs containing putative β-glucosidase genes (bgl1–10) were assembled by sequencing data of these fosmid clones. All these contigs were binned to Bacteroidetes, and all these β-glucosidase genes were phylogenetically closed to those from Bacteroides or Dysgonomonas. Six out of 10 β-glucosidase genes had predicted signal peptides, indicating a transmembrane capability of these enzymes to mediate cellulose hydrolysis within the gut of the termites. To confirm the activities of these β-glucosidase genes, three genes (bgl5, bgl7, and bgl9) were successfully expressed and purified. The optimal temperature and pH of these enzymes largely resembled the environment of the host’s gut. The gut microbiota composition of the fungus-growing termite was also determined by 454 pyrosequencing, showing that Bacteroidetes was the most dominant phylum. The diversity and the enzyme properties of β-glucosidases revealed in this study suggested that Bacteroidetes as the major member in fungus-growing termites contributed to cello-oligomer degradation in cellulose-hydrolyzing process and represented a rich source for β-glucosidase genes.     

Genes for the ‘H’ subunit of human ferritin are present on a number of human chromosomes

Summary DNa has been extracted from hamster-human and mouse-human hybrid cell lines, restricted with EcoRI, and hybridised to a probe for the H subunit of human ferritin, pDBR2. Sequences highly homologous to this probe have been found on at least eight human chromosomes: 1, 2, 3, 6p216cen, 11, 14, 20, and Xq23–25Xqter. Only the gene on chromosome 11 appears to be expressed in these hybrids Southern blotting of DNA from somatic cell hybrids containing different subsets of human chromosomes. The study shows that H subunit sequences are found on at least nine different chromosomes.     

Expression of Cell Cycle-Related Genes During Neuronal Apoptosis: Is there a Distinct Pattern?

An emerging hypothesis considers the process of neuronal apoptosis as a consequence of unscheduled and unsynchronized induction of cell cycle mediators. Induction of several cell cycle genes precedes neuronal apoptosis and may be involved in determination of cell fate. We have now characterized changes in expression of cell cycle genes during apoptosis induced by oxidative stress in chick post-mitotic sympathetic neurons. Induction of cyclin B occurred prior to the commitment of neurons to both dopamine- and peroxide-triggered apoptosis. Both the neuronal death and the rise in cyclin B were inhibited by antioxidant treatment, suggesting a functional role for cyclin B induction during neuronal apoptosis. Induction of the cyclin dependent kinase CDK5 protein coincided with the time point when neurons were irreversibly committed to die. Expression of other cell cycle mediators such as cyclin D1 and the cyclin dependent kinases CDC2 and CDK2 was undetected and not induced by exposure to oxidative stress. Comparative analysis of the profile of cell cycle mediators induced during neuronal apoptosis of different neuronal cell populations revealed no distinct pattern of events. There are no cell cycle stage-specific mediators that are ultimately stimulated during neuronal apoptosis, suggesting that multiple pathways of re-activating the dormant cell-cycle, converge to determine entry into apoptosis. Nevertheless, the existence of some cell cycle mediators, that were not reported so far to be induced in post mitotic neurons during oxidative stress, substantiate them as part of the strong differentiating forces.     

The “Missing Heritability” of Psychiatric Disorders: Elusive Genes or Non-Existent Genes?

The psychiatric genetics field is currently undergoing a crisis due to the decades-long failure to uncover the genes believed to cause the major psychiatric disorders. Since 2009, leading researchers have explained these negative results on the basis of the “missing heritability” argument, which holds that more effective research methods must be developed to uncover presumed missing genes. According to the author, problems with the missing heritability argument include genetic determinist beliefs, a reliance on twin research, the use of heritability estimates, and the failure to seriously consider the possibility that presumed genes do not exist. The author concludes that decades of negative results support a finding that genes for the major psychiatric disorders do not appear to exist, and that research attention should be directed away from attempts to uncover “missing heritability” and toward environmental factors and a reassessment of previous genetic interpretations of psychiatric family, twin, and adoption studies.     

Analysis of DNA–Protein Contacts in a Complex between Methyltransferase SsoII and a Promoter Region of the SsoII Restriction–Modification Genes

Heterocyclic bases and phosphate groups involved in the DNA–methyltransferase SsoII (M·SsoII) interaction were identified in the regulatory DNA region localized within the promoter region of the SsoII restriction–modification genes by footprinting with the use of formic acid, hydrazine, dimethyl sulfate, and N-ethyl-N-nitrosourea as modifying agents. It has been established that the enzyme interacts with three guanines, one adenine, two thymines, and three phosphate groups of each strand of the DNA duplex. These heterocyclic bases and phosphate groups are disposed symmetrically within the 15-mer inverted repeat of the regulatory DNA region. It has been demonstrated by footprinting with dimethyl sulfate that the C7 atoms of guanines interacting with the enzyme are exposed to the DNA major groove. Two theoretical models were built describing the contacts in a complex between M·SsoII and the regulatory DNA region.     

Demethylation of MicroRNA-124a Genes Attenuated Proliferation of Rheumatoid Arthritis Derived Fibroblast-Like Synoviocytes and Synthesis of Tumor Necrosis Factor-α

ObjectiveTo examine the impact of 5-Aza-2ʹ-deoxycytidine (5-AzadC) on methylation status of miR-124a genes in rheumatoid arthritis (RA) associated fibroblast-like synoviocytes (FLS) and its effect on RA-FLS proliferation and TNF-α expression.ResultsAfter 5-AzadC treatment, the expression of miR-124a was significantly increased compared with the control group (1.545 ± 0.189 vs 0.836 ± 0.166, p = 0.001). On the other hand, 5-AzadC significantly reduced IL-1β-mediated cell proliferation by nearly 2.5 fold (p = 0.006). Also, the level of TNF-α secreted from the cells treated with IL-1β plus 5-AzadC was considerably less than that from the cells treated with IL-1β alone (324.99 ± 22.73 ng/L vs 387.91 ± 58.51 ng/L, p = 0.022). After transfection with miR-124a inhibitor in RA-FLS treated with IL-1β plus 5-AzadC, the cell proliferation was increased by 18.2% and the TNF-α expression was increased by 19.0% (p = 0.001 and 0.011, respectively).ConclusionMethylation of miR-124a genes contributed to IL-1β-mediated RA-FLS proliferation and TNF-α expression.     

Are Noncoding Sequences of Rickettsia prowazekii Remnants of ``Neutralized' Genes?

It has been hypothesized that a large fraction of 24% noncoding DNA in R. prowazekii consists of degraded genes. This hypothesis has been based on the relatively high G+C content of noncoding DNA. However, a comparison with other genomes also having a low overall G+C content shows that this argument would also apply to other bacteria. To test this hypothesis, we study the coding potential in sets of genes, pseudogenes, and intergenic regions. We find that the correlation function and the χ²-measure are clearly indicative of the coding function of genes and pseudogenes. However, both coding potentials make almost no indication of a preexisting reading frame in the remaining 23% of noncoding DNA. We simulate the degradation of genes due to single-nucleotide substitutions and insertions/deletions and quantify the number of mutations required to remove indications of the reading frame. We discuss a reduced selection pressure as another possible origin of this comparatively large fraction of noncoding sequences. Received: 27 December 1999 / Accepted: 5 July 2000     

Molecular Cloning of α-Amylase Genes from DROSOPHILA MELANOGASTER. I. Clone Isolation by Use of a Mouse Probe

A cloned alpha-amylase cDNA sequence from the mouse is homologous to a small set of DNA sequences from Drosophila melanogaster under appropriate conditions of hybridization. A number of recombinant lambda phage that carry homologous Drosophila genomic DNA sequences were isolated using the mouse clone as a hybridization probe. Putative amylase clones hybridized in situ to one or the other of two distinct sites in polytene chromosome 2R and were assigned to one of two classes, A and B. Clone lambda Dm32, representing class A, hybridizes within chromosome section 53CD. Clone lambda Dm65 of class B hybridizes within section 54A1-B1. Clone lambda Dm65 is homologous to a 1450- to 1500-nucleotide RNA species, which is sufficiently long to code for alpha-amylase. No RNA homologous to lambda Dm32 was detected. We suggest that the class B clone, lambda Dm65, contains the functional Amy structural gene(s) and that class A clones contain an amylase pseudogene.     

Sirtuin 3, a New Target of PGC-1α, Plays an Important Role in the Suppression of ROS and Mitochondrial Biogenesis

Background

Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin with a reported association with the human life span. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1α induces several key reactive oxygen species (ROS)-detoxifying enzymes, but the molecular mechanism underlying this is not well understood.

Results

Here we show that PGC-1α strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. Knockdown of PGC-1α led to decreased Sirt3 gene expression. PGC-1α activated the mouse SIRT3 promoter, which was mediated by an estrogen-related receptor (ERR) binding element (ERRE) (−407/−399) mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that ERRα bound to the identified ERRE and PGC-1α co-localized with ERRα in the mSirt3 promoter. Knockdown of ERRα reduced the induction of Sirt3 by PGC-1α in C₂C₁₂ myotubes. Furthermore, Sirt3 was essential for PGC-1α-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1α in C₂C₁₂ myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1α on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1α on mitochondrial biogenesis in C₂C₁₂ myotubes.

Conclusion

Our results indicate that Sirt3 functions as a downstream target gene of PGC-1α and mediates the PGC-1α effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy metabolism and ROS generation. The elucidation of the molecular mechanisms of SIRT3 regulation and its physiological functions may provide a novel target for treating ROS-related disease.     

去乙酰化酶Sirtuin研究进展

依赖于NAD 的去乙酰化酶Sirtuin对细胞的存活、衰老、凋亡等生理活动的调节起到十分重要的作用。Sirtuin系统中的ySir2和SIRT1就目前来说是研究得较为透彻的两个成员。ySir2参与了酵母的交配型基因沉默、端粒的沉默、rDNA重复序列的沉默以及细胞寿命等生理功能。人类SIRT1在细胞存活与代谢等过程中也起到调节作用。本文对Sirtuin的结构、作用机制、底物特异性、影响因子及其功能作了综述。