A genomic bacterial artificial chromosome (BAC) library of the A genome of wheat has been constructed. Triticum monococcum accession DV92 was selected for this purpose because it is a cultivated diploid wheat and one of the parental lines used in the construction of a saturated genetic map. Leaves from this accession were used to isolate high-molecular-weight DNA from nuclei. This DNA was partially digested with restriction enzyme Hind III, subjected to double size selection, electroeluted and cloned into the pINDIGO451 BAC vector. The library consists of 276 480 clones with an average insert size of 115 kb. Excluding the 1.33% of empty clones and 0.14% of clones with chloroplast DNA, the coverage of this library is 5.6 genome equivalents. With this genome coverage the probability of having any DNA sequence represented in this library is higher than 99.6%. Clones were sorted in 720 384-well plates and blotted onto 15 high-density filters. High-density filters were screened with several single or low-copy clones and five positive BAC clones were selected for further analysis. Since most of the T. monococcum BAC ends included repetitive sequences, a modification was introduced into the classical end-isolation procedure to select low copy sequences for chromosome walking.Key words: bacterial artificial chromosome, BAC library, Triticum monococcum, wheat.Résumé : Une banque génomique du génome A du blé a été produite dans des chromosomes bactériens artificiels (BAC). L'accession DV92 du Triticum monococcum a été choisie pour ces fins car il s'agit d'une lignée diploïde qui est cultivée et d'un des parents employés dans la construction de cartes génétiques saturées. De l'ADN de grand poids moléculaire a été extrait de noyaux des feuilles de ce génotype. Cet ADN a été partiellement digéré avec l'enzyme de restriction HindIII, assujetti à une double sélection en fonction de la taille, électroélué et cloné dans le vecteur BAC pINDIGO451. La banque comprend 276,480 clones dont les inserts ont une taille moyenne de 115 kb. En excluant 1,33 % de clones vides et 0,14 % de clones contenant de l'ADN chloroplastique, cette banque offre une couverture génomique équivalent à 5,6 génomes. Avec une telle couverture, la probabilité qu'une séquence y soit représentée excède 99,6 %. Les clones ont été disposés dans 720 boîtes à 384 puits et placés sur 15 membranes à haute densité. Ces membranes ont été criblées avec des clones à copie unique ou à faible nombre de copies et cinq clones BAC ont été choisis pour des analyses détaillées. Comme la plupart des extrémités de BAC du T. monococcum comprennent de l'ADN répété, une modification a été apportée à la méthode classique d'isolement des séquences terminales afin de sélectionner des séquences à faible nombre de copies pour les fins de marche chromosomique.
We report on three sibs who have autosomal recessive Clericuzio-type poikiloderma neutropenia (PN) syndrome. Recently, this consanguineous family was reported and shown to be informative in identifying the C16orf57 gene as the causative gene for this syndrome. Here we present the clinical data in detail. PN is a distinct and recognizable entity belonging to the group of poikiloderma syndromes among which Rothmund-Thomson is perhaps the best described and understood. PN is characterized by cutaneous poikiloderma, hyperkeratotic nails, generalized hyperkeratosis on palms and soles, neutropenia, short stature, and recurrent pulmonary infections. In order to delineate the phenotype of this rare genodermatosis, the clinical presentation together with the molecular investigations in our patients are reported and compared to those from the literature.
Endosperm texture, i.e. the hardness or softness of the grain, is an important quality criterion in cereals because it determines many grain end-use properties. Grain softness is the dominant trait and is mainly controlled by the Ha locus on the short arm of chromosome 5D in hexaploid bread wheat. Genes for puroindoline a (Pina-D1), puroindoline b (Pinb-D1), and grain softness related protein (Gsp-D1) have been shown to be linked to the Ha locus in dierent mapping populations and have been associated with the expression of grain softness. The study of the linkage relationships among these genes has been limited by the low level of polymorphism in the D genome of hexaploid Triticum aestivum. In the present study, a highly polymorphic Triticum monococcum mapping population was used to analyze linkage relationships among these three genes. Gsp-A m 1 and Pina-A m 1 were found to be completely linked and lie 0.14 cM distal to Pinb-A m 1 in the distal region of the short arm of chromosome 5A m . The tight genetic linkage among these three genes was paralleled by their physical proximity within a single 105-kb clone isolated from a T. monococcum bacterial arti®cial chromosome (BAC) library. A restriction map of this BAC clone showed that Pina-A m
OBJECTIVE -To investigate -cell function and the long-term health of four case subjects presenting with chromosome 6 -associated transient neonatal diabetes (TND).RESEARCH DESIGN AND METHODS -Two unrelated case subjects presenting with paternal uniparental isodisomy of chromosome 6 (UPD6) and two siblings with a paternally inherited duplication of 6q24 were studied. Three case subjects presented with neonatal diabetes that recurred at 4 -17 years, while diabetes was incidentally discovered in the other case subject at 14 years of age. -Cell function was investigated after diabetes relapse by means of an oral glucose tolerance test (OGTT), an intravenous glucose tolerance test (IVGTT), and glucagon tests. The quantitative insulin sensitivity check index (QUICKI) was calculated from fasting blood samples as an estimate of insulin sensitivity.RESULTS --Cell function was investigated at diabetes relapse in two case subjects: the insulin response to both an OGTT and IVGTT was low, whereas the basal levels of C-peptide were normal. No evidence of insulin resistance was found. Residual -cell function was further explored by a glucagon test in all subjects at the age of 16 -28 years and was found to be normal. Final height was within the normal percentiles, whereas one case, who had been poorly controlled since puberty, presented with diabetes-related microvascular complications.CONCLUSIONS -In patients with chromosome 6 -associated TND, the -cell is preserved and able to secrete insulin through the stimulatory G protein pathway while exhibiting a specific defect of insulin secretion after glucose stimulation. This form of diabetes can be managed with insulin or diet, although new therapeutic agents (glucagon-like synthetic analogs) may prove useful in the future. Lack of treatment leads to long-lasting hyperglycemia without the risk of ketoacidosis but associated with microangiopathy in adult life. Diabetes Care 27:2405-2408, 2004T ransient neonatal diabetes (TND) is a rare (1/400,000 live births) but well-recognized disorder manifesting in the early neonatal period with hyperglycemia, dehydration, and minimal ketosis. Most patients are full-term, but growth-retarded, infants. Apparent remission usually occurs by 3 months, and relapse of diabetes has been reported in ϳ50 -60% of cases in late childhood or early adult life (1).Recent progress in molecular analysis has indicated (2-5) that TND is a disease entity, distinct from permanent neonatal diabetes. Three interrelated genetic mechanisms have been ascribed to TND (6 -8): paternal uniparental isodisomy of chromosome 6 (UPD6), paternal duplication of 6q24, and a methylation defect at a CpG island overlapping exon 1 of ZAC (zinc finger protein associated with apoptosis and cell cycle arrest)/HYMAI (imprinted in hydatidiform mole) (9). These imprinted genes may be implicated in the pathogenetic mechanism of diabetes, owing to their hypothetical role in insulin secretion. An inadequate insulin secretory response to glucose stimulation was found in islet cells from ...
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