Interaction between the <Emphasis Type="Italic">UCP2</Emphasis>–866G/A,mtDNA 10398G/A and <Emphasis Type="Italic">PGC1α</Emphasis> p.Thr394Thr and p.Gly482Ser polymorphisms in type 2 diabetes susceptibility in North Indian population

In the recent past, we have observed a possible role of 10398A and 16189C mtDNA and PGC1α p.Thr394Thr (rs2970847) and p.Gly482Ser (rs8192673) variant genotypes providing susceptibility/protection against type 2 diabetes mellitus (T2DM) in two North Indian population groups. These initial observations encouraged us to look at the candidate genes in combination with –866G/A (rs659366) polymorphism in uncoupling protein 2 (UCP2) in a single study of a relatively large sample size, constituted of both the cohorts, to unravel an interesting outcome of an additive interaction in-between the studied genes. In a total of 1,686 individuals (762 cases and 924 controls) belonging to Indo-European linguistic group from North India, a comparison of risk genotype combinations of: UCP2–866GG, mtDNA 10398A and PGC1α p.Thr394Thr or p.Gly482Ser against the protective genotypes: UCP2–866XA, mtDNA 10398G and PGC1α p.Thr394Thr (nominal P value = 1.75 × 10⁻¹⁴, Odds ratio, OR = 5.29, 3.40–8.22 at 95% CI) or PGC1α p.Gly482Ser (nominal p value = 4.42 × 10⁻²⁴, OR = 8.59, 5.53–13.35 at 95% CI), showed a highly significant difference and increased ORs. In a complex disease, it is always encouraging to find an additive interaction of multiple small effects of the studied candidate gene variations. An erratum to this article is available at .     

Molecular identification and the features of genetic diversity in interspecific hybrids of Amur sturgeon ( Acipenser schrenckii × A. baerii, A. baerii × A. schrenckii, A. schrenckii × A. ruthenus , and A. ruthenus × A. schrenckii ) based on variability of multilocus RAPD markers

The method of polymerase chain reaction with random primers (RAPD PCR) was used to identify the progeny of the crosses between three sturgeon species, Amur sturgeon (Acipenser schrenckii Brandt, 1869), Siberian sturgeon (A. baerii Brandt, 1869), and sterlet (A. ruthenus Linnaeus, 1758). Using ten primers, genetic variation in 70 yearlings, produced in seven individual crosses: Acipenser schrenckii × A. schrenckii, A. baerii × A. baerii, A. ruthenus × A. ruthenus, A. schrenckii × A. baerii, A. baerii × A. schrenckii, A. schrenckii × A. ruthenus, and A. ruthenus × A. schrenckii was described and evaluated. It was demonstrated that the samples composed of hybrids from individual crosses were more variable than the samples of parental species. On the other hand, pooled samples of hybrids from two cross directions were genetically less variable than the pooled samples of their parents. The three main features of the hybrid RAPD profiles identified included: (1) preservation of marker DNA fragments of both parents in one genome; (2) presence of specific DNA fragments, absent from both parents; and (3) dependence of the frequency of some DNA fragments from the cross direction. Multidimensional scaling clearly distinguishes in the space of three coordinates the individuals of original species and the hybrid progeny with differentiation in the groups of direct and backcross hybrids. Analysis of relationships (UPGMA and NJ) pointed to substantial differentiation between the species, as well as between the species and hybrid progeny. Close genetic relationships between direct and backcross hybrids were demonstrated. Multilocus RAPD markers in association with statistical methods are considered to be the useful tool for discrimination of interspecific hybrids of sturgeon. Possible reasons for the differences in the hybrid RAPD profiles are discussed. Original Russian Text ? K.V. Rozhkovan, G.N. Chelomina, E.I. Rachek, 2008, published in Genetika, 2008, Vol. 44, No. 11, pp. 1453–1460.     

Der Bienenfresser(Merops apiaster L.) in ?sterreich

Ohne Zusammenfassung     

DnaK-dependent ribosome biogenesis in Escherichia coli? : competition for dominance between the alleles dnaK756 and dnaK ?+

The Escherichia coli chaperone DnaK is vital for many cellular functions, including ribosome biogenesis at high temperature. Thus, the dnaK756-ts (λ ^R ) mutant, at the non-permissive temperature, is inhibited at a late stage of ribosome assembly, yielding 21S, 32S and 45S precursor particles. This defect, unlike the λ resistance and thermosensitivity phenotypes, is not complemented by lysogenisation with a transducing phage λ dnaK ⁺ bearing the wild-type dnaK gene. However this dominant phenotype becomes recessive when dnaK ⁺ is expressed from a medium-copy-number plasmid. On the other hand, an excess of DnaK causes an unexpected dominant-lethal effect of the dnaK756 allele near non-permissive temperatures. This interplay between the dnaK ⁺ and dnaK756 alleles supports the idea of that DnaK oligomers form in the cell. Received: 28 April 1998 / Accepted: 24 July 1998     

Retrofitting BACs with G418 resistance, luciferase, and oriP and EBNA-1 – new vectors for in vitro and in vivo delivery

Background  

Bacterial artificial chromosomes (BACs) have been used extensively for sequencing the human and mouse genomes and are thus readily available for most genes. The large size of BACs means that they can generally carry intact genes with all the long range controlling elements that drive full levels of tissue-specific expression. For gene expression studies and gene therapy applications it is useful to be able to retrofit the BACs with selectable genes such as G418 resistance, reporter genes such as luciferase, and oriP/EBNA-1 from Epstein Barr virus which allows long term episomal maintenance in mammalian cells.     

Somatic embryogenesis and plant regeneration in diploid Allium fistulosum × A. cepa F1 hybrid onions

Procedures were developed for disinfestation of non-dormant basal plate tissue excised from field grown basal plate tissue of diploid Allium fistulosum × A. cepa F₁ hybrid onions. Contamination levels varied with the season and vegetative development of plant material. Callus initiated from basal plate tissue and immature inflorescences of the F₁ hybrids was maintained on a BDS-based medium containing 0.75 mg/l picloram and 2.0 mg/l BA. When this medium was supplemented with vitamins and glycine, and with proline at 2.5 gm/1, somatic embryos began to form. Their development continued on a BDS-based shoot promotion medium containing 0.03 mg/l picloram and 0.32 mg/l 2iP supplemented with vitamins, glycine and proline. Genotypes differed significantly in the numbers of structures regenerated. Plantlets from somatic embryos were rooted into BDS or half-strength BDS medium without growth substances and were successfully transferred to sterilized potting mix in plastic commercial corsage boxes.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - 2iP N6-(2-isopentenyl)adenine - NAA 1-naphthylacetic acid - BDS Gamborg's B5 medium modified by Dunstan and Short (1977a)     

Synthesis and transporter binding properties of (R)-2β,3β- and (R)-2α,3α-diaryltropanes

(R)-2-Aryl-2-tropinone (9) was synthesized from (R)-2-carbomethoxy-3-tropinone (5) and was used as the key intermediate for the synthesis of (R)-2β,3β- and (R)-2α,3α-diaryltropanes. Inhibition of radioligand binding studies at the dopamine, serotonin, and norepinephrine transporters showed that the (R)-3β-(4-methylphenyl)-2β-phenyltropane (3b, RTI-422) possessed an IC₅₀ value of 1.96 nM at the dopamine transporter and was highly selective for this transporter relative to the serotonin and norepinephrine transporters.     

Production of fertile somatic hybrids of Gossypium hirsutum?+?G. bickii and G. hirsutum?+?G. stockii via protoplast fusion

Fertile somatic hybrids were obtained via symmetric electrofusion of protoplasts from two combinations of tetraploid cotton (G. hirsutum cv. Coker 201, AD genome) and diploid wild cottons G. bickii (G genome) and G. stockii (E genome), respectively. Observation by morphological, flow cytometric analysis, chromosome counting and RAPD analysis of the tested hybrids of Coker 201 + G. bickii and Coker 201 + G. stockii confirmed the regenerated plants as hybrid status. Cytological investigation of the metaphase root-tip cells revealed there were 78 chromosomes in the hybrids. Flow cytometric analysis showed the tested plants had a relative DNA contents close to the total DNA contents of the two parents. RAPD analysis revealed the hybrids contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the hybrids was intermediate between the two fusion partners. The hybrid plants were successfully transferred to the soil, and they bloomed and set bolls. It is sure that the new hexaploids developed by cell fusion would contribute to cotton breeding through backcrossing with the elite genotypes of G. hirsutum.     

A ThCAP gene from Tamarix hispida confers cold tolerance in transgenic Populus (P. davidiana?×?P. bolleana)

The ThCAP gene, which encodes a cold acclimation protein, was isolated from a Tamarix hispida NaCl-stress root cDNA library; its expression patterns were then assayed by qRT-PCR in different T. hispida tissues treated with low temperature (4°C), salt (400 mM NaCl), drought (20% PEG6000) and exogenous abscisic acid (100 μM). Induction of ThCAP gene was not only responsive to different stress conditions but was also organ specific. When transgenic Populus (P. davidiana × P. bolleana) plants were generated, expressing ThCAP under regulation of the cauliflower mosaic virus CaMV 35S promoter, they had a greater resistance to low temperature than non-transgenic seedlings, suggesting that ThCAP might play an important role in cold tolerance.     

Beobachtungen an einer Nonnensteinschm?tzer-Population(Oenanthe p. pleschanka [Lepechin])

Ohne Zusammenfassung     

Phylogeny and expression profiling of CAD and CAD-like genes in hybrid Populus (P. deltoides × P. nigra): evidence from herbivore damage for subfunctionalization and functional divergence

Background  

Cinnamyl Alcohol Dehydrogenase (CAD) proteins function in lignin biosynthesis and play a critical role in wood development and plant defense against stresses. Previous phylogenetic studies did not include genes from seedless plants and did not reflect the deep evolutionary history of this gene family. We reanalyzed the phylogeny of CAD and CAD-like genes using a representative dataset including lycophyte and bryophyte sequences. Many CAD/CAD-like genes do not seem to be associated with wood development under normal growth conditions. To gain insight into the functional evolution of CAD/CAD-like genes, we analyzed their expression in Populus plant tissues in response to feeding damage by gypsy moth larvae (Lymantria dispar L.). Expression of CAD/CAD-like genes in Populus tissues (xylem, leaves, and barks) was analyzed in herbivore-treated and non-treated plants by real time quantitative RT-PCR.     

Comparative study (AFLP and morphology) of three species of Prosopis of the Section Algarobia: P. juliflora, P. pallida, and P. limensis. Evidence for resolution of the “P. pallida–P. juliflora complex”

The problems of delimitation of species of Prosopis originate from the few morphological discontinuities which exist among some of them; some, however, originated as a result of wide distribution of germplasm without proper knowledge of the species, in particular, much material catalogued as P. juliflora, but being of other species, was distributed for reforestation projects worldwide. This work tests the morphological results obtained for P. pallida and P. limensis of the Peruvian–Ecuadorian coast and for P. juliflora of the Caribbean Basin of Colombia and Venezuela utilizing a study of AFLPs and a study of the morphology of plantlets developed in a conventional garden study. The phenogram obtained for the AFLPs demonstrates each of the three species to be a well differentiated cluster and the molecular variance between them is significantly greater than the variance within each species. Study of the plantlets also indicates statistically significant differences for four morphological characters between P. juliflora and the other two species (P. pallida and P. limensis). These results, in addition to the morphological differentiation evident between adult plants of P. pallida and P. limensis and the clear separation of these two species from P. juliflora, corroborate the genetic identity of the three taxa analyzed.     

Ethanol-hypersensitive and ethanol-dependent cdc? mutants in Schizosaccharomyces pombe

Ethanol-hypersensitive strains (ets mutants), unable to grow on media containing 6% ethanol, were isolated from a sample of mutagenized Schizosaccharomyces pombe wild-type cells. Genetic analysis of these ets strains demonstrated that the ets phenotype is associated with mutations in a large set of genes, including cell division cycle (cdc) genes, largely non-overlapping with the set represented by the temperature conditional method; accordingly, we isolated some ets non-ts cdc ^– mutants, which may identify novel essential genes required for regulation of the S. pombe cell cycle. Conversely, seven well characterized ts cdc ^– mutants were tested for their ethanol sensitivity; among them, cdc1–7 and cdc13–117 exhibited a tight ets phenotype. Ethanol sensitivity was also tested in strains bearing different alleles of the cdc2 gene, and we found that some of them were ets, but others were non-ets; thus, ethanol hypersensitivity is an allele-specific phenotype. Based on the single base changes found in each particular allele of the cdc2 gene, it is shown that a single amino acid substitution in the p34^cdc2 gene product can produce this ets phenotype, and that ethanol hypersensitivity is probably due to the influence of this alcohol on the secondary and/or tertiary structure of the target protein. Ethanol-dependent (etd) mutants were also identified as mutants that can only be propagated on ethanol-containing media. This novel type of conditional phenotype also covers many unrelated genes. One of these etd mutants, etd1-1, was further characterized because of the lethal cdc ^– phenotype of the mutant cells under restrictive conditions (absence of ethanol). The isolation of extragenic suppressors of etd1-1, and the complementation cloning of a DNA fragment encompassing the etd1 ⁺ wild-type gene (or an extragenic multicopy suppressor) demonstrate that current genetic techniques may be applied to mutants isolated by using ethanol as a selective agent.     

Sequence heterogeneity and differential expression of the α-Amy2 gene family in wheat

Summary Within plasmid pUB110 we have identified a 1.2 kb segment necessary and sufficient for driving autonomous replication in Rec⁺ cells at a wild-type copy number. This region can be divided into three functionally discrete segments: a 24 base pair (bp) region that acts as an origin, a 949 bp determinant of an essential replication protein, repU, and a 358 bp incompatibility region, incA, overlapping with the repU gene. The synthesis of the IncA determinant/s proceeds in the direction opposite to that of RepU. The positively (RepU) and negatively (IncA) trans-acting products seem to be involved in the control of plasmid replication. The RepU product has an M_r of 39 kDa, could be overproduced in Escherichia coli, and binds to the pUB110 origin region. Outside the minimal replicon a cis-acting, orientation dependent, 516 bp determinant is required (i) to compete with a coexisting incompatible plasmid and (ii) for segregational stability.     

The mutants compacta ?hnlich, Nitida and Grandiflora define developmental compartments and a compensation mechanism in floral development in Antirrhinum majus

In order to improve our understanding of floral size control we characterised three mutants of Antirrhinum majus with different macroscopic floral phenotypes. The recessive mutant compacta ?hnlich has smaller flowers affected mainly in petal lobe expansion, the dominant mutant Grandiflora has overall larger organs, whilst the semidominant mutation Nitida exhibits smaller flowers in a dose-dependent manner. We developed a cell map in order to establish the cellular phenotypes of the mutants. Changes in organ size were both organ- and region-specific. Nitida and compacta ?hnlich affected cell expansion in proximal and distal petal regions, respectively, suggesting differential regulation between petal lobe regions. Although petal size was smaller in compacta ?hnlich than in wild type, conical cells were significantly bigger, suggesting a compensation mechanism involved in petal development. Grandiflora had larger cells in petals and increased cell division in stamens and styles, suggesting a relationship between genes controlling organ size and organ identity. The level of ploidy in petals of Grandiflora and coan was found to be equivalent to wild type petals and leaves, ruling out an excess of growth via endoreduplication. We discuss our results in terms of current models about control of lateral organ size.     

Acanthocephalus?amini n. sp. (Acanthocephala: Echinorhynchidae) from the freshwater fish Cichlasoma?urophthalmus (Günther) (Cichlidae) in Mexico

Acanthocephalus amini n. sp. (Palaeacanthocephala: Echinorhynchidae) is described from the intestine of Cichlasoma urophthalmus (Günther) (Pisces: Cichlidae) collected in the Río Champotón, a river in Campeche State, Mexico. It is the fourth species of Acanthocephalus Koelreuther, 1771 described from North American freshwater fishes, although two other species are known from South America. The new species is distinguished from other members of Acanthocephalus by features of its trunk, which is small, clavate, slightly expanded medially and bluntly pointed posteriorly. It is further distinguished by having a cylindrical proboscis armed with 13–14 longitudinal rows of 11–12 stout hooks; the apical and medial proboscis hooks are almost uniform in size and shape, decreasing in size towards the base; the posteriormost hooks are smaller, straighter and more slender than the anterior and middle hooks; and the lateral rows of hooks are more widely spaced, forming a conspicuous longitudinal area devoid of hooks. Furthermore, the lemnisci are saccate and shorter than the proboscis receptacle; and the neck is very short with a thick collar of trunk tegument, which encircles the base of proboscis. In males, the testes are in the middle third of trunk, diagonal, spherical and small relative to the body size, and there are six clavate cement glands. In females, the uterus forms a conspicuous, elongate, cylindrical egg reservoir. The new species is most similar to A. alabamensis Amin & Williams, 1983, but can be distinguished by its swollen, clavate trunk, the largest proboscis hooks being present apically and medially, smaller testes, a shorter male reproductive system relative to body size and females with a prominent uterus. They have different hosts and geographical distribution. The new species can be differentiated from Brasacanthus sphoeroides Thatcher, 2001, a similar species in a monotypic echinorhynchid genus, because the latter is larger, has smaller proboscis hooks and its lemnisci are longer than the receptacle.     

Cleavage-resistant fusion proteins of the M2 muscarinic receptor and Gαi1. Homotropic and heterotropic effects in the binding of ligands

Background

G protein-coupled receptors fused to a Gα-subunit are functionally similar to their unfused counterparts. They offer an intriguing view into the nature of the receptor–G protein complex, but their usefulness depends upon the stability of the fusion.

Methods

Fusion proteins of the M₂ muscarinic receptor and the α-subunit of G_i1 were expressed in CHO and Sf9 cells, extracted in digitonin–cholate, and examined for their binding properties and their electrophoretic mobility on western blots.

Results

Receptor fused to native α_i1 underwent proteolysis near the point of fusion to release a fragment with the mobility of α_i1. The cleavage was prevented by truncation of the α-subunit at position 18. Binding of the agonist oxotremorine-M to the stable fusion protein from Sf9 cells was biphasic, and guanylylimidodiphosphate promoted an apparent interconversion of sites from higher to lower affinity. With receptor from CHO cells, the apparent capacity for N-[³H]methylscopolamine was 60% of that for [³H]quinuclidinylbenzilate; binding at saturating concentrations of the latter was inhibited in a noncompetitive manner at low concentrations of unlabeled N-methylscopolamine.

Conclusions

A stable fusion protein of the M₂ receptor and truncated α_i1 resembles the native receptor–G protein complex with respect to the guanyl nucleotide-sensitive binding of agonists and the noncompetitive binding of antagonists.

General significance

Release of the α-subunit is likely to occur with other such fusion proteins, rendering the data ambiguous or misleading. The properties of a chemically stable fusion protein support the notion that signaling proceeds via a stable multimeric complex of receptor and G protein.