Linkage and Association Between CA Repeat Polymorphism of the TNFR2 Gene and Obesity Phenotypes in Two Independent Caucasian Populations

Previously, our group has reported a suggestive linkage evidence of 1p36 with body mass index (BMI) (LOD = 2.09). The tumor necrosis factor receptor 2 (TNFR2) at 1p36 is an excellent positional and functional candidate gene for obesity. In this study, we have investigated the linkage and association between the TNFR2 gene and obesity phenotypes in two large independent samples, using the quantitative transmission disequilibrium tests (QTDT). The first group was made up of 1 836 individuals from 79 multi-generation pedigrees. The second group was a randomly ascertained set of 636 individuals from 157 US Caucasian nuclear families. Obesity phenotypes tested include BMI, fat mass, and percentage fat mass (PFM). A significant result (P = 0.0056) was observed for linkage with BMI in the sample of the multigenerational pedigrees. Our data support the TNFR2 gene as a quantitative trait locus (QTL) underlying BMI variation in the Caucasian populations.     

Transgenic Acacia sinuata from Agrobacterium tumefaciens-mediated transformation of hypocotyls

Transgenic herbicide tolerant Acacia sinuata plants were produced by transformation with the bar gene conferring phosphinothricin resistance. Precultured hypocotyl explants were infected with Agrobacterium tumefaciens strain EHA105 in the presence of 100 μM acetosyringone and shoots regenerated on MS (Murashige and Skoog, 1962, Physiol Plant 15:473–497) medium with 13.3 μM benzylaminopurine, 2.6 μM indole-3-acetic acid, 1 g l⁻¹ activated charcoal, 1.5 mg l⁻¹ phosphinothricin, and 300 mg l⁻¹ cefotaxime. Phosphinothricin at 1.5 mg l⁻¹ was used for the selection. Shoots surviving selection on medium with phosphinothricin expressed GUS. Following Southern hybridization, eight independent shoots regenerated of 500 cocultivated explants were demonstrated to be transgenic, which represented transformation frequency of 1.6%. The transgenics carried one to four copies of the transgene. Transgenic shoots were propagated as microcuttings in MS medium with 6.6 μM 6-benzylaminopurine and 1.5 mg l⁻¹ phosphinothricin. Shoots elongated and rooted in MS medium with gibberellic acid and indole-3-butyric acid, respectively both supplemented with 1.5 mg l⁻¹ phosphinothricin. Micropropagation of transgenic plants by microcuttings proved to be a simple means to bulk up the material. Several transgenic plants were found to be resistant to leaf painting with the herbicide Basta.     

Agrobacterium-mediated transformation of seedling-derived maize callus

Efficient production of seedling-derived Type I callus was demonstrated for several corn genotypes including commercial inbred lines. Seeds were germinated on MS-based medium containing 10 mg l(-1) picloram and 3 mg l(-1) 6-benzylaminopurine, which induced the development of axillary buds in the area of coleoptilar node. Nodal sections of 7-10-day old seedlings were isolated, split longitudinally, and placed on callus induction medium supplemented with 2.2 mg l(-1) picloram and 0.5 mg l(-1) 2,4-dichlorophenoxyacetic acid. For lines L4 and L9 the frequency of embryogenic callus induction was 38-42% based on calli per split nodal section. Frequency of callus induction from split nodal sections of seeds germinated on media without growth regulators was 0-3%. Seedling-derived callus of five genotypes was used for Agrobacterium-mediated transformation. Two constructs containing the green fluorescence protein gene and genes for either neomycin phosphotransferase II or glyphosate selection were used in transformation experiments. Transformation frequency varied from 2 to 11% and about 60% of the T(0) plants had 1-2 copies of transgenes.     

Strategies to obtain stable transgenic plants from non-embryogenic lines: complementation of the nn 1 mutation of the NORK gene in Medicago sativa MN1008

Strategies to introduce genes into non-embryogenic plants for complementation of a mutation are described and tested on tetraploid alfalfa (Medicago sativa). Genes conditioning embryogenic potential, a mutant phenotype, and a gene to complement the mutation can be combined using several different crossing and selection steps. In the successful strategy used here, the M. sativa genotype MnNC-1008(NN) carrying the recessive non-nodulating mutant allele nn ₁ was crossed with the highly embryogenic alfalfa line Regen S and embryogenic hybrid individuals were identified from the F1 progeny. After transformation of these hybrids with the wild-type gene (NORK), an F2 generation segregating for the mutation and transgene were produced. Plants homozygous for the mutant allele and carrying the wild-type NORK transgene could form root nodules after inoculation with Sinorhizobium meliloti demonstrating successful complementation of the nn ₁ mutation.     

Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola

Transgenic plants offer advantages for biomolecule production because plants can be grown on a large scale and the recombinant macromolecules can be easily harvested and extracted. We introduced an Aspergillus phytase gene into canola (Brassica napus) (line 9412 with low erucic acid and low glucosinolates) by Agrobacterium-mediated transformation. Phytase expression in transgenic plant was enhanced with a synthetic phytase gene according to the Brassica codon usage and an endoplasmic reticulum (ER) retention signal KDEL that confers an ER accumulation of the recombinant phytase. Secretion of the phytase to the extracellular fluid was also established by the use of the tobacco PR-S signal peptide. Phytase accumulation in mature seed accounted for 2.6% of the total soluble proteins. The enzyme can be glycosylated in the seeds of transgenic plants and retain a high stability during storage. These results suggest a commercial feasibility of producing a stable recombinant phytase in canola at a high level for animal feed supplement and for reducing phosphorus eutrophication problems.     

Agrobacterium tumefaciens-mediated transformation of Rhipsalidopsis gaertneri

A protocol for Agrobacterium tumefaciens-mediated genetic transformation of Rhipsalidopsis cv. CB5 was developed. Calluses derived from phylloclade explants and sub-cultured onto fresh callus induction medium over a period of 9–12 months were co-cultivated with A. tumefaciens LBA4404. Plasmid constructs carrying the nptII gene, as a selectable marker, and the reporter uidA gene were used. Transformed Rhipsalidopsis calluses with a vigorous growth phenotype were obtained by extended culture on media containing 600 mg l⁻¹ kanamycin. After 9 months of a stringent selection pressure, the removal of kanamycin from the final medium together with the culture of the transformed calluses under nutritional stress led to the formation of several transgenic adventitious shoots. Transformation was confirmed by GUS staining (for uidA gene), ELISA analysis and Southern blot hybridization (for the nptII gene). With this approach, a transformation efficiency of 22.7% was achieved. Overall results described in this study demonstrate that Agrobacterium-mediated transformation is a promising approach for this cactus species.     

Refining the application of direct embryogenesis in sugarcane: effect of the developmental phase of leaf disc explants and the timing of DNA transfer on transformation efficiency

A rapid in vitro protocol using direct somatic embryogenesis and microprojectile bombardment was investigated to establish the developmental phases most suitable for efficient sugarcane transformation. Immature leaf roll disc explants with and without pre-emergent inflorescence tissue were compared. It was shown that for effective transformation to occur, explants should be cultured for several days to allow initiation of embryo development prior to bombardment. Leaf roll discs with pre-emergent inflorescences showed a higher degree of embryogenic competence than non-flowering explants, and transformation efficiency was higher when explants containing floral initials were bombarded. Despite the occurrence of high numbers of phenotypically negative plants, combining the use of inflorescent leaf roll discs with direct embryogenic regeneration has the potential to improve the speed and efficiency of transgenesis in sugarcane.     

Higher intensity of purifying selection on >90% of the human genes revealed by the intrinsic replacement mutation rates

For over 3 decades, the rate of replacement mutations has been assumed to be equal to, and estimated from, the rate of "strictly" neutral sequence divergence in noncoding regions and in silent-codon positions where mutations do not alter the amino acid encoded. This assumption is fundamental to estimating the fraction of harmful protein mutations and to identifying adaptive evolution at individual codons and proteins. We show that the assumption is not justifiable because a much larger fraction of codon positions is involved in hypermutable CpG dinucleotides as compared with the introns, leading to a higher expected replacement mutation rate per site in a vast majority of the genes. Consideration of this difference reveals a higher intensity of purifying natural selection than previously inferred in human genes. We also show that a much smaller number of genes are expected to be evolving with positive selection than that predicted using sequence divergence at intron and silent positions in the human genome. These patterns indicate the need for using new approaches for estimating rates of amino acid-altering mutations in order to find positively selected genes and codons in genomes that contain hypermutable CpG's.