In the framework of a gene flow assessment, we investigated the natural hybridization rate between
(AADD genome) and
(AA genome). The latter species, a diploid progenitor of
, is spontaneously ...present in South Africa. Reciprocal crosses were performed without emasculation between
and
Neither examination of the morphological characteristics nor flow cytometry analysis of the 335 plants resulting from the
×
cross showed any hybrid features. Of the 148 plants produced from the
×
cross, three showed a hybrid phenotype, and their hybrid status was confirmed by SSR markers. Analysis of DNA content by flow cytometry and morphological traits clearly showed that two of these plants were triploid (AAD). The third plant had a flow cytometry DNA content slightly higher than
In addition, its morphological characteristics (plant architecture, presence and size of petal spots, leaf shape) led us to conclude that this plant was AAAD thus resulting from fertilization with an unreduced AA gamete of the female
parent. Fluorescent
Hybridization (FISH) and meiotic behavior confirmed this hypothesis. To the best of our knowledge, this is the first description of such gametes in
, and it opens new avenues in breeding programs. Furthermore, this plant material could provide a useful tool for studying the expression of genes duplicated in the A and D cotton genome.
A method using PCR amplification and primer extension with fluorescent oligonucleotides was developed to analyze T-cell repertoires. The sizes of the hypervariable CDR3-like regions of the murine ...T-cell antigen receptor β chains were measured for all possible Vβ-Jβcombinations. This analysis shows that β chains are distributed into at least 2000 groups, a value that provides a lower limit to their complexity. The CDR3 sizes appear to be dependent on the Jβand especially the Vβsegment used and correlates with amino acid sequence motifs in the corresponding CDR1 region. This feature of T-cell receptors is discussed.
A PCR-based method that determines VDJ junction size patterns in 24 human TCR V beta subfamilies was used to analyze T cells infiltrating sequential malignant melanoma biopsies for the presence of ...clonal expansions. Infiltrating T cell populations were found to present clonal expansions over a more or less complex polyclonal background. Two clones from a single patient were sequenced and detected in three different tumor sites (skin biopsies), whereas only one of them was also present in peripheral blood. Biopsies from this patient did not show major repertoire changes during in vivo IL-2 treatment. In contrast, in biopsies from a second patient, the expression of all the detected V beta subfamilies was increased and a larger number of clones expanded, probably as a result of therapy. A similar evolution was found among infiltrating T cells cultured in vitro from a third patient for several weeks in the presence of IL-2, where the largely polyclonal repertoire of fresh T cells (from invaded lymph nodes) was dramatically reduced to mainly clonal expansions in all V beta subfamilies detected. The high resolution method used here enables a rapid, comprehensive, qualitative, and semiquantitative description of the T cell repertoire of heterogeneous cell populations. Its use in conjunction with a functional analysis of clones detected within these populations should provide a better understanding of the evolution of the T cell repertoire among tumor-infiltrating lymphocytes during the progression of the disease and as a response to immunotherapy.
Patients with mitral valve disease can develop pulmonary artery hypertension that persists after mitral valve replacement. In 1987, nitric oxide (NO) was reported to be an important factor accounting ...for the biologic activity of endothelium-derived relaxing factor. Inhaled NO was subsequently reported to be a selective pulmonary vasodilator in animals and patients. Therefore we investigated the vasodilating effect of inhaled NO in patients with mild pulmonary artery hypertension after mitral valve replacement. Six patients who underwent mitral valve replacement for mitral stenosis presented with a mean pulmonary artery pressure greater than 25 mmHg within 24 h after surgery. During mechanical ventilation at FIO2 0.5, NO (36.8-38.4 ppm) was breathed for 10 min. Hemodynamic data were recorded before NO, after 10 min of NO inhalation, and 30 min after the end of NO inhalation. Statistically significant (P < 0.05) hemodynamic response to inhaled NO included a transient decrease in systolic (-10%), diastolic (-8%), and mean (-10%) pulmonary artery pressures; a decrease in pulmonary vascular resistance (-22%); an increase in mixed venous hemoglobin O2 saturation (+6%); and a decrease in arteriovenous O2 content difference (-7%). During NO inhalation, there was no change in systemic arterial or pulmonary wedge pressures. Methemoglobin levels remained < 1%. Inhalation of this concentration of NO for 10 min causes transient pulmonary artery vasodilation and hemodynamic improvement in patients with mild chronic pulmonary artery hypertension after mitral valve replacement.
The use of the dideoxy chain termination procedure to sequence linear double-stranded PCR products is hampered by the fast renaturation of the template. We have systematically varied three ...parameters: 1. the annealing temperature (T), to which the sample is transferred after denaturation by boiling, 2. the time course (L) of the labeling reaction, which is carried out at room temperature, 3. the primer:template ratio (R) in the reaction mixture. We have determined an optimal set of conditions, which consists in: 1. a temperature T of -70 degree C, 2. a time course L between 15 and 45 seconds, 3. a ratio R around 20. Under these conditions, several PCR products of different lengths were sequenced on both strands by several investigators.
The lineage relationships of central-memory T cells (T(CM)) cells and effector-memory T cells (T(EM)), as well as their homeostasis and recall capacities, are still controversial. We investigated ...these issues in a murine model using two complementary approaches: T cell receptor repertoire analysis and adoptive transfer experiments of purified H-Y-specific T(CM) and T(EM) populations. Repertoire studies showed that approximately two thirds of T(CM) and T(EM) clones derived from a common naive precursor, whereas the other third was distinct. Both approaches highlighted that T(CM) and T(EM) had drastically distinct behaviors in vivo, both in the absence of antigen or upon restimulation. T(CM) clones were stable in the absence of restimulation and mounted a potent and sustained recall response upon secondary challenge, giving rise to both T(CM) and T(EM), although only a fraction of T(CM) generated T(EM). In contrast, T(EM) persisted for only a short time in the absence of antigen and, although a fraction of them were able to express CD62L, they were unable to mount a proliferative response upon secondary challenge in this model.
The diversity of the T cell repertoire of mature T splenocytes is generated, in the thymus, by pairing of alpha and beta variable domains of the alpha beta TCR and by the rearrangements of various ...gene segments encoding these domains. In the periphery, it results from competition between various T cell subpopulations including recent thymic migrants and long-lived T cells. Quantitative data on the actual size of the T cell repertoire are lacking. Using PCR methods and extensive sequencing, we have measured for the first time the size of the TCR-alpha beta repertoire of naive mouse T splenocytes. There are 5-8 x 105 different nucleotide sequences of BV chains in the whole spleen of young adult mice. We have also determined the size of the BV repertoire in a subpopulation of AV2+ T splenocytes, which allows us to provide a minimum estimate of the alpha beta repertoire. We find that the mouse spleen harbors about 2 x 106 clones of about 10 cells each. This figure, although orders of magnitude smaller than the maximum theoretical diversity (estimated up to 1015), is still large enough to maintain a high functional diversity.
Memory T cells are divided into central and effector subsets with distinct functions and homing capabilities. We analyzed the composition and dynamics of the CD8
+ T cell repertoire of these subsets ...within the peripheral blood of four healthy individuals. Both subsets had largely distinct and autonomous TCRβ repertoires. Their composition remained stable over a 9 month period, during which no cell passage between these subsets was detected despite important size variation of several clones. In one donor, four out of six TCRβ clonotypes specific for the influenza A virus were detected in the central subset only, while the two others were shared. Altogether, these observations suggest that most effector memory T cells may not have derived from the central memory subset.
We have previously described the identification of Artemis, a factor involved in the nonhomologous end joining (NHEJ) phase of V(D)J recombination of T and B cell receptor genes. Null mutations of ...the
Artemis
gene result in a complete absence of T and B lymphocytes that is associated with increased cell radiosensitivity, causing the radiosensitive T
–
B
–
SCID (RS-SCID) condition. We presently report the occurrence of hypomorphic mutations of the
Artemis
gene in four patients from two kindreds. Partially preserved in vivo activity of Artemis is associated with the presence of polyclonal T and B lymphocyte populations, albeit in reduced numbers, along with chromosomal instability and development of EBV-associated lymphoma in two of four patients. This syndrome emphasizes the role of Artemis in the NHEJ pathway of DNA repair and suggests that other, yet ill-defined, conditions associating immunodeficiency and lymphoma could be caused by mutations in genes encoding NHEJ factors.
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