A set of primers for amplification of mitochondrial DNA in Picea abies and other conifer species

Thirteen primer pairs generating intraspecific length and/or presence‐absence polymorphism in Picea abies have been obtained from a P. abies mtDNA library, using different methodologies (agarose gel electrophoresis, polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP), single‐strand conformation (SSCP). Poly‐morphism tests were extended successfully to other Picea species (P. omorika, P. engelmanii and P. glauca) and species belonging to other conifer genera (Abies alba, Larix laricina and Pinus pinaster). This set of PCR‐based mitochondrial markers can provide promising tools for studying phylogeography or phylogeny in P. abies and other conifer species in which the mt genome is generally the only one to be transmitted via the female gamete.     

Brief communication: mitochondrial DNA variation suggests extensive gene flow from Polynesian ancestors to indigenous Melanesians in the northwestern Bismarck Archipelago

Archaeological, linguistic, and genetic studies show that Austronesian (AN)-speaking Polynesian ancestors came from Asia/Taiwan to the Bismarck Archipelago in Near Oceania more than 3,600 years ago, and then expanded into Remote Oceania. However, it remains unclear whether they extensively mixed with indigenous Melanesians who had populated the Bismarck Archipelago before their arrival. To examine the extent of admixture between Polynesian ancestors and indigenous Melanesians, mitochondrial DNA (mtDNA) variations in the D-loop region and the cytochrome oxidase and lysine transfer RNA (COII/tRNA(Lys)) intergenic 9-bp deletion were analyzed in the following three Oceanian populations: 1) Balopa Islanders as AN-speaking Melanesians living in the northwestern end of the Bismarck Archipelago, 2) Tongans as AN-speaking Polynesians, and 3) Gidra as non-Austronesian-speaking Melanesians in the southwestern lowlands of Papua New Guinea. Phylogenetic analysis of mtDNA sequences revealed that more than 60% of mtDNA sequences in the Balopa Islanders were very similar to those in Tongans, suggesting an extensive gene flow from Polynesian ancestors to indigenous Melanesians. Furthermore, analysis of pairwise difference distributions for the D-loop sequences with the 9-bp deletion and the Polynesian motif (i.e., T16217C, A16247G, and C16261T) suggested that the expansion of Polynesian ancestors possessing these variations occurred approximately 7,000 years ago.     

Cryptic human growth hormone gene sequences direct gonadotroph-specific expression in transgenic mice

Our laboratory reported previously that chimeric genes encoding either rat somatostatin (SS) or human GH (hGH), but containing the identical mouse metallothionein-I (MT) promoter/enhancer sequences and hGH 3'-flanking sequences, were selectively expressed in the gonadotrophs of transgenic mice. The experiments reported here were designed to identify the DNA sequences responsible for this unexpected cell-specific expression within the anterior pituitary. We produced new transgenic mice expressing fusion genes that tested separately the requirement of the MT or 3'-hGH sequences for gonadotroph expression. A fusion gene that retained the original MT and SS sequences, with a simian virus 40 polyadenylation signal exchanged for the 3'-hGH sequences, no longer directed strong pituitary expression, but was active in the liver. In contrast, a cytomegalovirus promoter/enhancer-SS-hGH fusion gene was expressed at the same high level in the anterior pituitaries of transgenic mice as the originally studied MT-SS-hGH gene. Immunohistochemical analysis indicated that pituitary expression of the cytomegalovirus promoter/enhancer-SS-hGH fusion gene was also restricted to gonadotroph cells in adult mice. These studies indicate that sequences within the 3'-flanking region of the hGH gene can direct expression of chimeric genes to pituitary cells that do not normally produce growth hormone.     

Tissue-specific and expression of porcine growth hormone gene in BAC transgenic mice

One of the primary goals of traditional livestock breeding is to improve growth rate and optimise body size. Growth rate can be significantly increased by integrating a growth hormone (GH) transgene under the control of a ubiquitous promoter, but while such animals do demonstrate increased growth there are also serious deleterious side-effects to the animals health. Here we report the generation and initial characterization of transgenic mice that carried a porcine BAC encoding the porcine GH gene. We show that GH expression is restricted specifically to the pituitary, is associated with elevated IGF-1 levels, and results in growth enhancement. No negative effects to the health of the transgenic animals were detected. This initial characterisation supports the use of BAC pGH transgene in livestock studies.     

Cloning and restriction fragment length polymorphism analysis of a cDNA for swine PIT-1, a gene controlling growth hormone expression*

A partial swine cDNA which encodes the functional domain of PIT-1 was isolated by the polymerse chain reaction (PCR). The swine PIT-1 cDNA clone is 95% identical at the protein level to the rat Pit-1 gene. Thus, Pit-l's known function in control of rat growth hormone and prolactin expression is likely to be conserved in swine. This swine cDNA clone was used to investigate genetic variability at PIT-1 in several American and Chinese breeds. Polymorphic BamIII fragments were found in pure-bred Meishan animals (n= 13), but only monomorphic fragments in five American breeds (n= 36).     

Co‐amplification of cytochrome b and D‐loop mtDNA fragments for the identification of degraded DNA samples

We propose a simple and effective approach to simultaneously co‐amplify both cytochrome b and D‐loop fragments to evaluate DNA preservation and to monitor possible contaminations in the analysis of degraded animal DNA samples. We have applied this approach to over 200 ancient salmon samples and 25 ancient whale DNA samples, clearly demonstrating its multiple benefits for analysis of degraded DNA samples, and the ease in which co‐amplification can be optimized for different taxa. This simple, cost‐efficient and genomic DNA‐saving approach can be used routinely in the analysis of minute and degraded DNA samples in wildlife forensics, food inspection, conservation biology and ancient faunal remains.     

Optimization of TaDREB3 gene expression in transgenic barley using cold‐inducible promoters

Constitutive over‐expression of the TaDREB3 gene in barley improved frost tolerance of transgenic plants at the vegetative stage of plant development, but leads to stunted phenotypes and 3‐ to 6‐week delays in flowering compared to control plants. In this work, two cold‐inducible promoters with contrasting properties, the WRKY71 gene promoter from rice and the Cor39 gene promoter from durum wheat, were applied to optimize expression of TaDREB3. The aim of the work was to increase plant frost tolerance and to decrease or prevent negative developmental phenotypes observed during constitutive expression of TaDREB3. The OsWRKY71 and TdCor39 promoters had low‐to‐moderate basal activity and were activated by cold treatment in leaves, stems and developing spikes of transgenic barley and rice. Expression of the TaDREB3 gene, driven by either of the tested promoters, led to a significant improvement in frost tolerance. The presence of the functional TaDREB3 protein in transgenic plants was confirmed by the detection of strong up‐regulation of cold‐responsive target genes. The OsWRKY71 promoter–driven TaDREB3 provides stronger activation of the same target genes than the TdCor39 promoter. Analysis of the development of transgenic plants in the absence of stress revealed small or no differences in plant characteristics and grain yield compared with wild‐type plants. The WRKY71–TaDREB3 promoter–transgene combination appears to be a promising tool for the enhancement of cold and frost tolerance in crop plants but field evaluation will be needed to confirm that negative development phenotypes have been controlled.     

Evaluation of an antisense RNA transgene for inhibiting growth hormone gene expression in transgenic rats

We compared the levels of growth hormone (GH) mRNA in the pituitary, plasma GH concentration, and altered phenotype in rats heterozygous and homozygous for an antisense RNA transgene targeted to the rat GH gene, with those in nontransgenic rats. We initially investigated whether the transgene promoter, which is connected to four copies of a thyroid hormone response element (TRE) that increases promoter activity, affected in vivo transgene expression in the pituitary of the transgenic rats. Plasma GH concentration correlated negatively with T, injection in surgically thyroidectomized heterozygous transgenic rats. There was a reduction of about ?35–40% in GH mRNA levels in the pituitary of homozygous animals compared with those in non-transgenic rats. Plasma GH concentration was significantly ?25–32 and ?29–41% lower in heterozygous and homozygous transgenic rats, respectively, compared with that in nontransgenic animals. Furthermore, the growth rates in homozygous transgenic rats were reduced by ?72–81 and ?51–70% compared with those of their heterozygous and nontransgenic littermates, respectively. The results of these studies suggested that the biological effect of GH in vivo is modulated dose-dependently by the antisense RNA transgene. The rat GH gene can therefore be targeted by antisense RNA produced from a transgene, as reflected in the protein and RNA levels. © 1995 Wiley-Liss, Inc.     

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole‐mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker‐mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2‐kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5‐kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8‐kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters. Dev. Genet. 25:158–167, 1999. © 1999 Wiley‐Liss, Inc.     

East Asian contributions to Dutch traditional and western commercial chickens inferred from mtDNA analysis

Understanding the complex origin of domesticated populations is of vital importance for understanding, preserving and exploiting breed genetic diversity. Here, we aim to assess Asian contributions to European traditional breeds and western commercial chickens for mitochondrial genetic diversity. To this end, a 365‐bp fragment of the chicken mtDNA D‐loop region of 16 Dutch fancy breeds (113 individuals) was surveyed, comprising almost the entire breed diversity of The Netherlands. We also sequenced the same fragment for 160 commercial birds representing all important commercial types from multiple commercial companies that together represent more than 50% of the worldwide commercial value. We identified 20 different haplotypes. The haplotypes clustered into five clades. The commonest clade (E‐clade) supposedly originates from the Indian subcontinent. In addition, both in commercial chicken and Dutch fancy breeds, many haplotypes were found with a clear East Asian origin. However, the erratic occurrence of many different East Asian mitochondrial clades indicates that there were many independent instances where breeders used imported exotic chickens for enhancing local breeds. Nucleotide diversity and haplotype diversity analyses showed the influence of the introgression of East Asian chicken on genetic diversity. All populations that had haplotypes of multiple origin displayed high inferred diversity, as opposed to most populations that had only a single mitochondrial haplotype signature. Most fancy breeds were found to have a much lower within‐population diversity compared to broilers and layers, although this is not the case for mitochondrial estimates in fancy breeds that have multiple origin haplotypes.     

Comprehensive investigation of nucleotide diverdity in yaks

To understand the maternal genetic diversity of Tianzhu white yak better, we analyzed mtDNA D‐loop sequences of 209 Tianzhu white yaks, which are from the central region of Tianzhu white yak habitat. Accordingly, a total of 45 haplotypes were identified in Tianzhu white yaks in this study, and 18 of them were unique. The nucleotide diversity and haplotype diversity of population studied were 0.024 ± 0.003 and 0.946 ± 0.007 respectively, revealing that Tianzhu white yak possess a relatively high genetic diversity. The phylogenetic analysis, combining D‐loop sequences in this study with 533 previous published D‐loop sequences of 13 yak breeds, indicated that Tianzhu white yaks fell mainly into haplogroup A and that a small portion belonged to haplogroups B, C, D and E. Moreover, six haplotypes of 20 individuals identified in Tianzhu white yak were in the taurine haplogroup, indicating hybridization between Bos taurus and Tianzhu white yaks. In summary, this study supplies a comprehensive maternal genetic pattern for Tianzhu white yak and provides a basic reference for future breeding programs to conserve the purebred Tianzhu white yak.     

Expression of human growth hormone in transgenic rice cell suspension culture

Human growth hormone (hGH), a pituitary-derived polypeptide, evidences a wide range of biological functions, including protein synthesis, cell proliferation, and metabolism. A synthetic hGH gene (shGH) has been synthesized on the basis of plant-optimized codon usage via an overlap PCR strategy and located in a plant expression vector under the control of the rice amylase 3D (Ramy3D) promoter, which is induced by sugar starvation. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Donjin) via particle bombardment transformation methods. The integration of the shGH gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification and shGH expression in transgenic rice suspension cells was confirmed via Northern blot analysis. The shGH protein was detected in the transgenic rice cell suspension culture medium following induction with sugar starvation, using Western blot analysis. The quantity of shGH that accumulated in the transgenic rice cell suspension medium was 57 mg/l. The shGH accumulated in the transgenic rice cell suspension culture medium evidenced a biological activity similar to that of Escherichia coli-derived recombinant hGH. These results indicate that the shGH was generated and accumulated in the transgenic rice cell suspension culture medium, and manifested biological activity.     

Cloning and differential expression of the growth hormone in Pampus argenteus

The growth hormone (GH) is a pluripotent hormone produced by the pituitary in vertebrates. It plays important roles in the growth, development, and metabolism of vertebrates.We cloned GH cDNA sequence of Pampus argenteus (GenBank: KT257176). Multi‐sequence analysis revealed P. argenteus GH cDNA contained four conservative cysteine residues positions (Cys⁶⁹, Cys¹⁷⁷, Cys¹⁹⁴, and Cys²⁰²) and shared more than 51.5% identity with homologues from other reported bony fish GHs, except that of Lepisosteus osseus. We used semi‐quantitative RT‐PCR and quantitative real‐time PCR to detect GH expression in 10 tissues and GH expression levels in the pituitary at six different growth stages, and also detected GH content in serum at different growth stages . qPCR showed that GH mRNA was detected in the liver, muscle, kidney, intestine, pituitary, olfactory bulb, stomach, heart, gill, and ovary. The highest level of P. argenteus GH mRNA was observed in the pituitary (P < 0.01, n = 3). At different growth stages, P. argenteus GH expression first increased, decreased, and increased again. GH gene expression levels and the variations of serum GH levels of P. argenteus were consistent with the growth rate and associated with the sexual maturity. In addition, in situ hybridization was used to locate the GH expression in pituitary. In situ hybridization showed that the GH‐positive cells were round, oval, or irregular and often gathered into groups or presented branches along the nerve fibers.     

Regulation of expression of a sheep metallothionein 1a-sheep growth hormone fusion gene in transgenic mice.

Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.     

Molecular analysis of gene conversion in spermatids from transgenic mice

Investigations into the mechanisms and properties of gene conversion in mammals are greatly restricted by the inability to recover all the products of a meiosis. Additionally, the study of this process has been hampered by the lack of visible markers to detect gene conversion, especially when the events are rare. In previous work, we developed a transgenic system for detection and quantitation of gene conversion events in the germline of mice (Murti, J.R., Bumbulis, M., Schimenti, J.C., 1992. High frequency germline gene conversion in transgenic mice. Mol. Cell. Biol. 12, 2545–2552) that could be exploited as an assay for recombinogenic chemicals (Murti, J.R, Schimenti, K.J., Schimenti, J.C., 1994. A recombination-based transgenic mouse system for genotoxicity testing. Mutat. Res. 307, 583–595). A specific intrachromosomal gene conversion event between two complementarily defective lacZ genes resulted in the production of β-galactosidase in spermatids, enabling a measurement of conversion frequency. Here, we report that the anticancer drug, cisplatin, increased gene conversion in meiotic stage cells in these transgenic mice. Furthermore, a method was developed for direct molecular analysis of transgene conversion events in single or pooled lacZ-positive spermatids. The ability to identify gametes that have undergone a rare gene conversion event, followed by molecular amplification of the recombinant gene, should make it possible to investigate the mechanisms of genetic recombination in mammals in greater detail than previously possible.     

Tissue-specific expression in transgenic maize of four endosperm promoters from maize and rice

The tissue-specific, developmental, and genetic control of four endosperm-active genes was studied via expression of GUS reporter genes in transgenic maize plants. The transgenes included promoters from the maize granule-bound starch synthase (Waxy) gene (zmGBS), a maize 27 kDa zein gene (zmZ27), a rice small subunit ADP-glucose pyrophosphorylase gene (osAGP) and the rice glutelin 1 gene (osGT1). Most plants had a transgene expression profile similar to that of the endogenous gene: expression in the pollen and endosperm for the zmGBS transgene, and endosperm only for the others. Histological analysis indicated expression initiated at the periphery of the endosperm for zmGBS, zmZ27 and osGT1, while osAGP transgene activity tended to start in the lower portion of the seed. Transgene expression at the RNA level was proportional to GUS activity, and did not influence endogenous gene expression. Genetic analysis showed that there was a positive dosage response with most lines. Activity of the zmGBS transgene was threefold higher in a low starch (shrunken2) genetic background. This effect was not seen with zmZ27 or osGT1 transgenes. The expression of the transgenes is discussed relative to the known behaviour of the endogenous genes, and the developmental programme of the maize endosperm     

Tissue-specific expression and dietary regulation of a chimeric phosphoenolpyruvate carboxykinase/bovine growth hormone gene in transgenic mice

A series of transgenic mice was produced by microinjection of a segment of DNA, containing 460 base pairs of the phosphoenolpyruvate (P-enolpyruvate) carboxykinase promoter-regulatory region ligated to the bovine growth hormone structural gene, into the male pronucleus of fertilized mouse eggs. Founder animals which contained the gene were selected for further analysis and for breeding. The concentration of bovine growth hormone in the serum of animals which were shown to contain the gene ranged from a low of 5 ng/ml serum to approximately 2300 ng/ml serum. Mice with high levels of bovine growth hormone had growth rates double that of their litter mates which did not contain the transgene. The transgene was expressed only in the liver and kidney of the animals studied, and the level of specific mRNA for bovine growth hormone in these tissues could be regulated by diet in a manner similar to the endogenous P-enolpyruvate carboxykinase gene. Feeding the animals a diet high in carbohydrate for 1 week caused a 90% decrease in the concentration of bovine growth hormone in the blood, suggesting that the expression of the chimeric P-enolpyruvate carboxykinase/bovine growth hormone gene is sensitive to insulin. When the same animals were then refed a diet high in protein, but devoid of carbohydrate, the concentration of bovine growth hormone in their blood was induced 30-fold within a week. The administration of dibutyryl cyclic AMP to the transgenic mice caused a 2-fold induction in the level of bovine growth hormone in the serum within 90 min. Thus the region between -460/+73 in the P-enolpyruvate carboxykinase promoter-regulatory region contains sequences which can direct the tissue-specific expression, as well as hormonal and dietary responsiveness, of a linked structural gene.     

Inhibition of prostate cancer growth by vitamin D: Regulation of target gene expression

Prostate cancer (PCa) cells express vitamin D receptors (VDR) and 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits the growth of epithelial cells derived from normal, benign prostate hyperplasia, and PCa as well as established PCa cell lines. The growth inhibitory effects of 1,25(OH)(2)D(3) in cell cultures are modulated tissue by the presence and activities of the enzymes 25-hydroxyvitamin D(3) 24-hydroxylase which initiates the inactivation of 1,25(OH)(2)D(3) and 25-hydroxyvitamin D(3) 1alpha-hydroxylase which catalyses its synthesis. In LNCaP human PCa cells 1,25(OH)(2)D(3) exerts antiproliferative activity predominantly by cell cycle arrest through the induction of IGF binding protein-3 (IGFBP-3) expression which in turn increases the levels of the cell cycle inhibitor p21 leading to growth arrest. cDNA microarray analyses of primary prostatic epithelial and PCa cells reveal that 1,25(OH)(2)D(3) regulates many target genes expanding the possible mechanisms of its anticancer activity and raising new potential therapeutic targets. Some of these target genes are involved in growth regulation, protection from oxidative stress, and cell-cell and cell-matrix interactions. A small clinical trial has shown that 1,25(OH)(2)D(3) can slow the rate of prostate specific antigen (PSA) rise in PCa patients demonstrating proof of concept that 1,25(OH)(2)D(3) exhibits therapeutic activity in men with PCa. Further investigation of the role of calcitriol and its analogs for the therapy or chemoprevention of PCa is currently being pursued.     

Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice

Using stable transgenic rice plants, the promoters of 15 genes expressed in rice seed were analysed for their spatial and temporal expression pattern and their potential to promote the expression of recombinant proteins in seeds. The 15 genes included 10 seed storage protein genes and five genes for enzymes involved in carbohydrate and nitrogen metabolism. The promoters for the glutelins and the 13 kDa and 16 kDa prolamins directed endosperm-specific expression, especially in the outer portion (peripheral region) of the endosperm, whilst the embryo globulin and 18 kDa oleosin promoters directed expression in the embryo and aleurone layer. Fusion of the GUS gene to the 26 kDa globulin promoter resulted in expression in the inner starchy endosperm tissue. It should be noted that the 10 kDa prolamin gene was the only one tested that required both the 5' and 3' flanking regions for intrinsic endosperm-specific expression. The promoters from the pyruvate orthophosphate dikinase (PPDK) and ADP-glucose pyrophosphorylase (AGPase) small subunit genes were active not only in the seed, but also in the phloem of vegetative tissues. Within the seed, the expression from these two promoters differed in that the PPDK gene was only expressed in the endosperm, whereas the AGPase small subunit gene was expressed throughout the seed. The GUS reporter gene fused to the alanine aminotransferase (AlaAT) promoter was expressed in the inner portion of the starchy endosperm, whilst the starch branching enzyme (SBE1) and the glutamate synthase (GOGAT) genes were mainly expressed in the scutellum (between the endosperm and embryo). When promoter activities were examined during seed maturation, the glutelin GluB-4, 26 kDa globulin and 10 kDa and 16 kDa prolamin promoters exhibited much higher activities than the others. The seed promoters analysed here exhibited a wide variety of activities and expression patterns, thus providing many choices suitable for various applications in plant biotechnology.     

Stable expression of human growth hormone over 50 generations in transgenic insect larvae

Developments in insect transgenesis using transposons combined with available mass rearing technology for insects such as the Medfly, Ceratitis capitata, provide opportunity for the production of protein for industrial, agricultural and healthcare purposes on a very large scale. In this study, we report the germ-line transformation and expression of a cDNA encoding human growth hormone (hGH) in transgenic Drosophila using the Minos transposon. Production and secretion of a bioactive hGH into the haemolymph of transgenic larvae was demonstrated by immunoblot analysis, ELISA and a proliferation bioassay. Stable expression of hGH was observed over 50 generations. The results indicate that mass reared transgenic diptera with a rapid period of larval growth could provide cost effective production systems for the manufacture of therapeutic and other high value proteins.