Transgenic Populations Research Articles

Differential equations models for interacting wild and transgenic mosquito populations

We formulate and study continuous-time models, based on systems of ordinary differential equations, for interacting wild and transgenic mosquito populations. We assume that the mosquito mating rate is either constant, proportional to total mosquito population size, or has a Holling-II-type functional form. The focus is on the model with the Holling-II-type functional mating rate that incorporates Allee effects, in order to account for mating difficulty when the size of the total mosquito populations is small. We investigate the existence and stability of both boundary and positive equilibria. We show that the Holling-II-type model is the more realistic and, by means of numerical simulations, that it exhibits richer dynamics.

Transformation by T-DNA integration causes highly sterile phenotype independent of transgenes in Arabidopsis thaliana

Agrobacterium tumefaciens-mediated gene transformation caused highly sterile phenotype in T1 transgenic populations of Arabidopsis thaliana. The phenomenon occurred independent of the genes and construct types used for transformation. The occurring frequency is less than 10% and the phenotype is inheritable. Intensive examination elucidated that the sterility is due to indehiscence or delayed dehiscence of the anthers during the flowering stage, resulting from the reduced or disordered endothecial secondary wall thickening of the anthers in the sterile flowers. Exogenous jasmonic acid application cannot rescue the sterile phenotype. Additionally, by using the Escherichia coli uidA gene encoding the beta-glucuronidase as a reporter gene, we indicated that the Cauliflower mosaic virus 35S promoter was not constitutively active as expected previously in the reproductive organs of Arabidopsis. These results contribute significantly to the plant community by suggesting that more careful examination and statistical analysis are needed while studying gain-of-function phenotypes of genes, especially for genes that might be involved in reproductive development.

Forcing neural progenitor cells to cycle is insufficient to alter cell-fate decision and timing of neuronal differentiation in the spinal cord

BackgroundDuring the development of the nervous system, neural progenitor cells can either stay in the pool of proliferating undifferentiated cells or exit the cell cycle and differentiate. Two main factors will determine the fate of a neural progenitor cell: its position within the neuroepithelium and the time at which the cell initiates differentiation. In this paper we investigated the importance of the timing of cell cycle exit on cell-fate decision by forcing neural progenitors to cycle and studying the consequences on specification and differentiation programs.ResultsAs a model, we chose the spinal progenitors of motor neurons (pMNs), which switch cell-fate from motor neurons to oligodendrocytes with time. To keep pMNs in the cell cycle, we forced the expression of G1-phase regulators, the D-type cyclins. We observed that keeping neural progenitor cells cycling is not sufficient to retain them in the progenitor domain (ventricular zone); transgenic cells instead migrate to the differentiating field (mantle zone) regardless of cell cycle exit. Cycling cells located in the mantle zone do not retain markers of neural progenitor cells such as Sox2 or Olig2 but upregulate transcription factors involved in motor neuron specification, including MNR2 and Islet1/2. These cycling cells also progress through neuronal differentiation to axonal extension. We also observed mitotic cells displaying all the features of differentiating motor neurons, including axonal projection via the ventral root. However, the rapid decrease observed in the proliferation rate of the transgenic motor neuron population suggests that they undergo only a limited number of divisions. Finally, quantification of the incidence of the phenotype in young and more mature neuroepithelium has allowed us to propose that once the transcriptional program assigning neural progenitor cells to a subtype of neurons is set up, transgenic cells progress in their program of differentiation regardless of cell cycle exit.ConclusionOur findings indicate that maintaining neural progenitor cells in proliferation is insufficient to prevent differentiation or alter cell-fate choice. Furthermore, our results indicate that the programs of neuronal specification and differentiation are controlled independently of cell cycle exit.

Transgenic wheat plants expressing an oat arginine decarboxylase cDNA exhibit increases in polyamine content in vegetative tissue and seeds

We generated and characterized a transgenic wheat (Triticum aestivum L.) population expressing constitutively an oat arginine decarboxylase (ADC) cDNA. Transgenic wheat plants expressing the transgene had a significant increase in ADC activity with no concomitant changes in ODC activity. We measured 2-fold increases in putrescine, spermidine and spermine content in leaves of transgenic plants compared to wild-type. These plants also exhibited significant increases in the three polyamines in seeds (7-fold putrescine, 2.5-fold spermidine and 1.8-fold spermine). This significant polyamine accumulation was heritable in subsequent generations. In order to characterize molecularly this population we cloned a partial TaADC cDNA. This sequence was used as a probe to ascertain the presence of two distinct ADC transcripts in wheat. Further DNA analyses confirmed the presence of at least two distinct ADC genes in the wheat genome. Our results demonstrate that polyamine levels can be altered in transgenic wheat plants resulting in phenotypically normal and fertile plants. This transgenic wheat population will serve as useful germplasm to embark on further in depth studies to ascertain the role of polyamines in abiotic stress responses.

Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum)

A TaqMan quantitative real-time PCR detection system was developed to examine transgene copy number in cotton. GhUBC1, a gene validated to be present as a single copy per haploid Gossypium hirsutum genome, was used as the endogenous reference to estimate copy number of GFP and selection marker NPTII in 28 T0 plants. This system was found to be more accurate than genomic Southern blot hybridization and could effectively tell homozygotes from heterozygotes in a T1 transgenic cotton population. Therefore it is suitable for efficient and cost effective early screening of transgenic seedlings and identifying transgene homozygotes in segregation populations.

The overexpression of asparagine synthetase A from E. coli affects the nitrogen status in leaves of lettuce (Lactuca sativa L.) and enhances vegetative growth

The asparagine synthetase A (EC 6.3.1.1) of E. coli (AS-A) mainly uses ammonia to produce asparagine, a key nitrogen transporter in plants. The AS-A encoding gene (asnA) was expressed constitutively in lettuce cultivar ‘Cortina’ under the control of pMAC, a chimerical promoter, to induce phenotypical alterations of plant growth and quality as a consequence of nitrogen status changes. Nine fertile transgenic lines harbouring independent T-DNA insertions were recovered. Primary transformants shared new visible traits such as a higher leaf number and wider leaf surface than the wild-type. The progeny of three primary transformants stably maintained these phenotypes, to which the synthesis of both asnA transcript and protein were associated. In pMAC:asnA plants, seed germination, formation and development of leaves, bolting and flowering occurred earlier than non-transformed plants. Twenty-eight days after sowing (das), transgenic plants showed a ca. 1.3 increase of leaf area and dry weight as compared to the wild-type. Moreover, the contents of asparagine, aspartic acid and glutamine, but not that of glutamic acid, of pMAC:asnA young plants (21 das) were greater than the wild-type. The level of total soluble protein was higher in transgenic than in non-transformed leaves borne on plants at 35, 50 and 75 das. A decrease of nitrate was also measured in pMAC:asnA leaves with respect to non-transformed ‘Cortina’, in transgenic populations at 60 das. In pMAC:asnA genotypes, the altered content of nitrogen transport amino acids, the tolerance to increasing doses of ammonium and phosphinothricin indirectly proved the AS-A enzymatic activity in lettuce.

EFFECT OF THE INTERNAL AND EXTERNAL FACTORS ON YIELD AND QUALITATIVE-QUANTITATIVE CHARACTERISTICS OF CHAMOMILE ESSENTIAL OIL

The form and structure of chamomile is affected by genetic factors and environmental conditions. From carbohydrates as primary metabolites which are formed in photosynthesis, secondary metabolites useful for plant immunology and communication are synthesized. Nowadays, chamomile is a most favored and much used medicinal plant throughout the world. Phytotherapeutically useful are inflorescences whereby substantial part of their curative effects is determined by the essential oil content and composition. In regard to qualitative-quantitative characteristics of essential oil with a wide range of biodiversity, several chamomile chemotypes are characterized. Besides the major influence of genetic factors, the environment has an important effect on essential oil accumulation and composition. The environmental control is modified by chamomile plant ontogeny, and many other factors outside the scope of this review, such as light intensity, day length, temperature, nutrition, irrigation, plant growth regulators, tissue cultures and their transgenic transformation, intraspecific interactions, population dynamics, parasites, diseases, pest control, interspecific competition and harvest management are also known to affect yield of chamomile essential oil and its composition. INTRODUCTION Nowadays, chamomile is a most favored and much used medicinal plant throughout the world. Phytotherapeutically useful are primarily inflorescences (flower heads or anthodia) and chamomile drug (Chamomillae Flos). A substantial part of drug effects are determined by the essential oil content and its composition. The form, structure and morphological characteristics of chamomile plants, their essential oil accumulation and quality are affected by genetic background and environmental conditions. The first time the influence of the defined factors were determined by Putievsky in 1994 on herb yield, essential oil and its composition. New recognition of these in regard to chamomile is as follows: • Genetics (origin, breeding and ploidisation, variation and biodiversity), • Morphogenesis (flowering, harvest), • Abiotic Factors (light intensity, day length, temperature, nutrition, irrigation, plant growth regulators, tissue cultures and their genetically transformation), • Biotic Factors (intraspecific interactions, population dynamics, parasites, diseases, pest control, interspecific competition and harvest management).

Transplant conditions determine the contribution of homeostatically expanded donor CD8 memory cells to host lymphoid reconstitution following syngeneic HCT

To investigate the ability of donor CD8 memory (CD8 TM) cells to expand in recipients following syngeneic hematopoietic stem cell transplants (HCT). The influence of clinically important transplant parameters--conditioning level, delayed infusion and dose--on the homeostatic expansion and overall contribution of donor CD8 TM to host CD8 reconstitution was determined. Lymphopenia-induced CD8 TM homeostatic expansion was examined in a syngeneic murine HCT model. Antigen specific CD8 TM included both T-cell receptor transgenic and nontransgenic populations. An ex vivo technique using antigen, interleukin (IL)-2, and IL-15 was used to generate homogenous transgenic CD8 TM (i.e., central memory) and was adapted to enrich the heterogeneous nontransgenic CD8 population specific for a nonameric epitope. Both transgenic and naturally occurring CD8 memory populations, derived in vivo or generated ex vivo, underwent a similar kinetic pattern of homeostatic expansion following transplantation into ablatively conditioned syngeneic recipients. Transplant parameters, i.e., lower conditioning, delayed infusion, and lower donor CD8 cell numbers shortened the period of expansion and lowered the steady-state numbers. The pattern of CD8 TM expansion was dependent on conditioning levels, time of infusion, and dose. Transplantation of varying donor CD8 TM numbers demonstrated there was a maximal donor cell contribution to host CD8 reconstitution. The application of multiple well-defined memory CD8 populations supports the notion that these findings are characteristic of memory CD8 cells in general.

Transgenic Potato Lines Expressing cry1Ba1 or cry1Ca5 Genes are Resistant to Potato Tuber Moth

Modified cry1Ca5 and cry1Ba1 genes, under the transcriptional control of a CaMV 35S promoter, were individually transferred into potato (Solanum tuberosum L.) cv. Iwa using Agrobacterium-mediated transformation. Thirty-six and thirty-eight independently derived cry1Ca5- and cry1Ba1-transgenic plants were regenerated respectively. Multiplex-PCR confirmed the presence of the nptII selectable marker gene and the specific cry gene in all regenerated lines. In greenhouse experiments, approximately 90% of each transgenic population produced phenotypically normal plants. More than 90% of the cry1Ca5-transgenic lines gave 100% larval mortality of potato tuber moth (PTM), Phthorimaea operculella (Zeller), on excised greenhouse-grown leaf and tuber bioassays. In contrast, only 40–50% of the cry1Ba1-transgenic lines gave 50 to 100% of larval mortality of PTM using the same bioassays, although all lines except one significantly inhibited larval growth. Southern blot analysis for eight selected cry1Ca5-transgenic lines revealed that they contained 1 to 6 copies of the cry gene. The amount of Cry1C protein, quantified by ELISA, ranged from 0.26 to 8.42 μg per g of leaf tissue and from 0.23 to 1.02 μg per g of tuber tissue. No relationship was apparent between cry1Ca5 gene copy number and amount of Cry protein expressed in leaves and tubers. Southern blot analysis for seven selected cry1Ba1-transgenic lines revealed that they contained 2 to 8 copies of the cry1Ba1 gene. Both the cry1Ca5 and cry1Ba1 genes offer additional sources of resistance that can be deployed with other effective cry genes to control tuber moth in potatoes.

Lentiviral vector-mediated genetic modification of human neural progenitor cells for ex vivo gene therapy

Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.

Isolation and characterization of the cytoplasmic male sterility-associated orf456 gene of chili pepper (Capsicum annuum L.)

Cytoplasmic male sterility (CMS) in plants is known to be associated with novel open reading frames (ORFs) that result from recombination events in the mitochondrial genome. In this study Southern and Northern blot analyses using several mitochondrial DNA probes were conducted to detect the presence of differing band patterns between male fertile and CMS lines of chili pepper (Capsicum annuum L.). In the CMS pepper, a novel ORF, termed orf456, was found at the 3'-end of the coxll gene. Western blot analysis revealed the expression of an approximately 17-kDa product in the CMS line, and the intensity of expression of this protein was severely reduced in the restorer pepper line. To investigate the functional role of the ORF456 protein in plant mitochondria, we carried out two independent experiments to transform Arabidopsis with a mitochondrion-targeted orf456 gene construct by Agrobacterium-mediated transformation. About 45 % of the T1 transgenic population showed the male-sterile phenotype and no seed set. Pollen grains from semi-sterile T1 plants were observed to have defects on the exine layer and vacuolated pollen phenotypes. It is concluded that this newly discovered orf456 may represent a strong candidate gene--from among the many CMS-associated mitochondrial genes--for determining the male-sterile phenotype of CMS in chili pepper.

Transgenic Overexpression of RasGRP1 in Mouse Epidermis Results in Spontaneous Tumors of the Skin

RasGRP1 is a guanine nucleotide exchange factor for Ras and a receptor of the second messenger diacylglycerol and its ultrapotent analogues, the phorbol esters. We have recently shown expression of RasGRP1 in the epidermal keratinocytes where it can mediate Ras activation in response to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, a well-known mouse skin tumor promoter. To explore the participation of RasGRP1 in skin carcinogenesis, we targeted the overexpression of RasGRP1 to basal epidermal keratinocytes using the keratin 5 promoter. These transgenic mice were viable and indistinguishable from their littermates, with normal differentiation and skin architecture. However, a percentage of the adult transgenic population developed spontaneous skin tumors, mainly squamous cell papillomas. The transgene was detected in the tumors as well as in primary keratinocytes isolated from transgenic mice. The transgenic keratinocytes also displayed elevated levels of active, GTP-loaded Ras compared with the levels observed in keratinocytes derived from wild-type littermates. We noticed a correlation between tumor incidence and wounding, which suggests that RasGRP1 overexpression may confer sensitivity to promotional stimuli, like wound repair mechanisms. Interestingly, we also found elevated levels of granulocyte colony-stimulating factor in conditioned media derived from transgenic keratinocytes subjected to in vitro wounding. Taken together, these data are the first to provide evidence of a novel role for RasGRP1 in skin carcinogenesis and suggest that RasGRP1 may participate in tumorigenesis through modulation of Ras and autocrine pathways.

The Rapid Ex Vivo Generation of an Immunodominant Antigen-Specific Memory CD8 Population and Its Subsequent Homeostatic Expansion in Ablatively-Conditioned HCT Recipients.

There is currently significant interest in the transplant field to develop adoptive-transfer strategies utilizing T cell populations to provide immediate immune function as well as long-term immune reconstitution following hematopoietic cell transplantation (HCT). Presumably, these pre-selected T cell populations could then be further expanded in the transplant recipient as a consequence of lymphopenia-induced proliferation. However, clinical application of adoptive transfer strategies has been limited by practical (time, expense) and technical (isolation and expansion of antigen-specific T cell populations) difficulties, hence more efficient approaches need to be identified. Recent reports have demonstrated the feasibility for the rapid ex vivo generation of transgenic memory CD8 populations. We investigated the potential of applying this ex vivo approach to generate and expand an immunodominant antigen-specific memory population from primary CD8 T cells. CD8 cells recognizing the mouse H60 epitope were selected as the antigen-specific CD8 population. The H60 glycoprotein is the ligand for NKG2D and the LTFNYRNL peptide is an immunodominant minor transplantation antigen. H60 is expressed by BALB.B (H2b) hematopoietic cells and recognized by C57BL/6 (B6) CD8 cells within the context of the H2Kb molecule. CD8 T cells from normal B6 spleens were positively selected using Miltenyi beads. The purified CD8 cells (97%) were then cultured with bone marrow-derived B6 DC, rmIL-2, and H60 peptide (1μM) for 3 days. Cells were harvested and re-cultured with rmIL-15 for 2–4 days. The resultant CD8 population was enriched 10 fold for tetramer-stained H60+ CD8 T cells (average: 3.0% of CD8 T cells). The H60+ CD8 cells displayed a memory phenotype as characterized by CD44+, Ly6C+, CD62Lintermed, and CD25lo expression. We hypothesized these H60+ CD8 T cells could be further expanded in transplant recipients by lymphopenia-induced proliferation. To determine the expansion and persistence of H60+ TM post-HCT, H60+-enriched CD8 cells were co-transplanted with T cell-depleted B6 bone marrow into 9.0Gy-conditioned syngeneic recipients. The phenotype and number of H60+ cells in recipient spleens and bone marrow were assessed beginning 3 days post-HCT. Notably, the H60+ CD8 cells maintained their memory phenotype and persisted at least 2 months post-transplant. The ex vivo-generated H60+ TM underwent a relative expansion of 1.5–2 fold as assessed in recipient spleens, similar to the post-transplant expansion of H60+ CD8 TM derived in vivo from B6 mice primed to BALB.B cells. Moreover, this post-HCT expansion was also similar to that by an ex vivo-generated, transgenic CD8 TM population. Both (ex vivo and in vivo generated) H60+ TM populations also exhibited expansion (1.5–2 fold) in the bone marrow. In total, an immunodominant antigen-specific CD8 TM population was selectively generated and enriched ex vivo and found to undergo expansion following transplant into ablatively conditioned HCT recipients. The similarities in expansion and persistence between ex vivo generated H60 and in vivo primed H60 populations suggest this approach may have useful applications towards the development of successful adoptive transfer strategies.

The cyanogenic glucoside composition of Zygaena filipendulae (Lepidoptera: Zygaenidae) as effected by feeding on wild-type and transgenic lotus populations with variable cyanogenic glucoside profiles

Zygaena larvae sequester the cyanogenic glucosides linamarin and lotaustralin from their food plants (Fabaceae) as well as carry out de novo biosynthesis of these compounds. In this study, Zygaena filipendulae were reared on wild-type Lotus corniculatus and wild-type and transgenic L. japonicus plants with differing content and ratios of the cyanogenic glucosides linamarin and lotaustralin and of the cyanoalkenyl glucosides rhodiocyanoside A and D. LC-MS analyses, free choice feeding experiments and developmental studies were used to examine the effect of varying content and ratios of these secondary metabolites on the feeding preferences, growth and development of Z. filipendulae. Larvae reared on cyanogenic L. corniculatus developed faster compared to larvae reared on L. japonicus although free choice feeding trials demonstrated that the latter plant source was the preferred food plant. Larvae reared on acyanogenic L. corniculatus showed decelerated development. Analysis of different life stages and tissues demonstrate that Z. filipendulae strive to maintain certain threshold content and ratios of cyanogenic glucosides regardless of the composition of the food plants. Despite this, the ratios of cyanogenic glucosides in Z. filipendulae remain partly affected by the ratio of the food plant due to the high proportion of sequestering that takes place.

Homeostatic Proliferation as an Isolated Variable Reverses CD8+ T Cell Anergy and Promotes Tumor Rejection

Although recent work has suggested that lymphopenia-induced homeostatic proliferation may improve T cell-mediated tumor rejection, there is little direct evidence isolating homeostatic proliferation as an experimental variable, and the mechanism by which improved antitumor immunity occurs via homeostatic proliferation is poorly understood. An adoptive transfer model was developed in which tumor-specific 2C/RAG2(-/-) TCR transgenic CD8+ T cells were introduced either into the lymphopenic environment of RAG2(-/-) mice or into P14/RAG2(-/-) mice containing an irrelevant CD8+ TCR transgenic population. RAG2(-/-), but not P14/RAG2(-/-) recipients supported homeostatic proliferation of transferred T cells as well as tumor rejection. Despite absence of tumor rejection in P14/RAG2(-/-) recipients, 2C cells did become activated, as reflected by CFSE dilution and CD44 up-regulation. However, these cells showed poor IFN-gamma and IL-2 production upon restimulation, consistent with T cell anergy and similar to the hyporesponsiveness induced by administration of soluble peptide Ag. To determine whether homeostatic proliferation could uncouple T cell anergy, anergic 2C cells were transferred into RAG(-/-) recipients, which resulted in vigorous homeostatic proliferation, recovery of IL-2 production, and acquisition of the ability to reject tumors. Taken together, our data suggest that a major mechanism by which homeostatic proliferation supports tumor rejection is by maintaining and/or re-establishing T cell responsiveness.

Transgenic oilseed rape along transportation routes and port of Vancouver in western Canada

The occurrence of transgenic herbicide-resistant oilseed rape (Brassica napus) in ruderal (non-crop disturbed) areas has not been investigated previously in Canada. The primary objective of this study was to document their occurrence in two main ruderal areas (along railways and roads) in the province of Saskatchewan, where half of all oilseed rape is grown, and at the port of Vancouver, British Columbia on the west coast of Canada, where most oilseed rape destined for export is transported by rail. During the 2005 growing season, leaf samples of oilseed rape plants were collected at randomly-selected sites along railways and roads across Saskatchewan ecoregions and at Vancouver; infestation area, density, and plant height of oilseed rape were measured at each site. The presence of the glyphosate and glufosinate resistance traits was determined using test strips. The infestation area of oilseed rape, averaged across 155 sampled sites in the Saskatchewan survey, was markedly smaller in populations along railways than roads; in contrast, infestation area averaged across 54 sites in the Vancouver survey was greater for populations along railways than roads. In both surveys, mean plant density was greater for populations found along railways than roads. Two-thirds of oilseed rape plants sampled across Saskatchewan ecoregions and at Vancouver were transgenic, although the relative proportion of plants with the glyphosate or glufosinate resistance trait varied between surveys. Frequency of occurrence of transgenic plants in ruderal areas was similar to the proportion of the oilseed rape area planted with transgenic cultivars in the recent preceding years. A single transgenic B. rapa x B. napus hybrid was found along a road in Vancouver, confirming the relatively high probability of hybridization between these two Brassica species. With current control measures, transgenic oilseed rape populations may persist and spread in these ruderal areas.

Cellular and Molecular Mechanisms of Synovial Joint and Articular Cartilage Formation

Synovial joints and articular cartilage play crucial roles in the skeletal function, but relatively little is actually known about their embryonic development. Here we first focused on the interzone, a thin mesenchymal cell layer forming at future joint sites that is widely thought to be critical for joint and articular cartilage development. To determine interzone cell origin and fate, we microinjected the vital fluorescent dye DiI at several peri-joint sites in chick limbs and monitored the behavior and fate of labeled cells over time. Peri-joint mesenchymal cells located immediately adjacent to incipient joints migrated, became part of the interzone, and were eventually found in epiphyseal articular layer and joint capsule. Interzone cells isolated and reared in vitro expressed typical phenotypic markers, including GDF-5, Wnt-14, and CD-44, and differentiated into chondrocytes over time. To determine the molecular mechanisms of articular chondrocyte formation, we carried out additional studies on the ets transcription factor family member ERG and its alternatively spliced variant C-1-1 that we previously found to be expressed in developing avian articular chondrocytes. We cloned the human counterpart of avian C-1-1 (ERGp55Delta81) and conditionally expressed it in transgenic mice under cartilage-specific Col2 gene promotor-enhancer control. The entire transgenic mouse limb chondrocyte population exhibited an immature articular-like phenotype and a virtual lack of growth plate formation and chondrocyte maturation compared to wild-type littermate. Together, our studies reveal that peri-joint mesenchymal cells take part in interzone and articular layer formation, interzone cells can differentiate into chondrocytes, and acquisition of a permanent articular chondrocyte phenotype is aided and perhaps dictated by ets transcription factor ERG.

Use of the SSLP-based method for detection of rare apomictic events in a sexual AtSERK1 transgenic Arabidopsis population

Here we present a screening method to evaluate the potential of genes to transfer aspects of apomixis into sexual crop plants. Based on the assumption that an apomictic progeny is an exact genetic replica of the mother plant we employed a set of single sequence length polymorphism (SSLP) markers to identify individuals displaying heterozygosity fixation in segregating sexual populations as an indication of rare apomictic events. Here we present the results of such a study using the Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 (AtSERK1) gene expressed under the control of the AtLTP1 promoter in sexual Arabidopsis plants. In one of the three tested F2 transgenic populations expressing the AtLTP1::AtSERK1 construct we observed two plants with heterozygosity maintenance for the full set of SSLP markers indicating a possible clonal inheritance. However, as their offspring revealed a close to binomial segregation for a number of heterozygous loci, it was concluded that these two putative apomictic plants either lost their clonal ability in the next generation or resulted from incidental recombination events displaying the genotype of the parent.

Monitoring the escape of transgenic oilseed rape around Japanese ports and roadsides

An investigation was carried out to monitor the escape and spread of oilseed rape (Brassica napus) transgenic plants and the introgression of transgenes to its closely related feral species in Japan. We screened a total of about 7500 feral B. napus, 300 B. rapa, and 5800 B. juncea seedlings from maternal plants in 143 locations at several ports, roadsides, and riverbanks. The presence of glufosinate-resistance or glyphosate-resistance transgenes in these seedlings was confirmed by means of herbicide treatments and also immunochemical and DNA analyses. B. napus plants with herbicide-resistant transgenic seeds were found at five of six major ports and along two of four sampled roadsides in the Kanto District. Transgenic oilseed rape plants have not been commercially cultivated in Japan, suggesting that the transgenes would probably have come from imported transgenic seeds that were spilled during transportation to oilseed processing facilities. No transgenes were detected in seeds collected from B. napus plants growing along riverbanks in the Kanto District or in seeds from closely related species (B. rapa and B. juncea). To our knowledge, this is the first published example of feral, transgenic populations occurring in a nation where the transgenic crop has not been cultivated commercially.

Genetic analysis and high-resolution mapping of a premature senescence genePse(t)in rice (Oryza sativaL.)

A rice mutant, designated pse(t) (premature senescence, tentatively), was isolated from a T-DNA-inserted transgenic population. Senescence advanced more markedly in pse(t) than in wild-type ('Zhonghua 11', japonica) plants. Genetic analysis of pse(t) revealed that the premature senescence mutation was controlled by a single recessive nuclear gene, but that it was not induced by T-DNA insertion. In an effort to understand the genetic and molecular basis underlying premature senescence in rice, a map-based cloning strategy was used to localize Pse(t). High-resolution mapping of the Pse(t) locus was carried out using simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) markers. An F2 population, comprising 1691 pse(t) individuals derived from a cross of the pse(t) mutant with 'Longtepu' (indica), was constructed. Several new polymorphism markers were developed in this study. Genetic linkage analysis showed that the Pse(t) gene was located on the long arm of chromosome 7. It was found that the Pse(t) gene cosegregated with 3 markers and was flanked by markers SS22 and PP21. Thus, the Pse(t) gene is located within a genetic distance of 0.15 cM, corresponding to a physical distance of 220 kb. These findings provide the basic information that can be used for the final isolation of this gene in the rice premature-senescence pathway.