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Purpose: Development of an ad hoc protocol for the preimplantion genetic diagnosis of propionic acidemia in a couple carrying the mutations c.737G>T (G246V) and c.1218del14ins12 (ins/del) in the PCCB gene. Propionic acidemia is an autosomal recessive metabolic disorder where the body is unable to process certain parts of proteins and lipids. Symptoms manifest few days after birth and sometimes progress to more serious medical problems, including heart abnormalities, coma and death., Methods: Four short tandem repeat markers closely linked to the PCCB gene were tested, in order to support the direct mutation detection diagnosis. Multiplex fluorescent heminested polymerase chain reaction followed by fragment analysis and minisequencing was used., Results: Fourteen single blastomeres from nine embryos were tested and two carrier embryos were transferred, resulting in the birth of two healthy boys., Conclusions: Preimplantation genetic diagnosis represents a valid reproductive option for couples affected of propionic acidemia, in order to avoid transmission to offspring.

Hereditary angiooedema is an autosomal dominant disease caused by mutations in the SERPING1 gene. It is characterized by oedemas in different parts of the body, being particularly dangerous when swelling involves the upper airway. Preimplantation genetic diagnosis (PGD) was performed in a couple where the woman carries a deletion of 2.9Kb that includes exon 4 of the SERPING1 gene. Four polymorphic short tandem repeat markers were tested in order to establish the disease-bearing haplotype and three of them were fully informative. Amplification efficiency at the preclinical work up ranged from 71% to 100% for each locus and allele drop out rates were between 0% and 20% for the polymorphic markers. The couple underwent PGD using fluorescent multiplex heminested polymerase chain reaction. Six embryos were biopsied and five of them were diagnosed as healthy. Two embryos were transferred and a singleton pregnancy was achieved, resulting in the birth of a healthy boy., (Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Hypokalaemic periodic paralysis is a rare dominant inherited disease where a person suffers sudden falls of circulating potassium concentrations, producing muscle weakness and sometimes severe paralysis. Attacks can occur as frequently as several times a day or once in a year. The age of onset is usually adolescence but symptoms can appear as early as 10 years of age. Muscle weakness can compromise vital functions such as breathing or swallowing and heart arrhythmias are also frequent during attacks. Preimplantation genetic diagnosis, an early form of prenatal diagnosis for couples at risk of transmitting inherited diseases, was used to prevent the transmission of this disease. Six polymorphic short tandem repeat or microsatellite markers (STR) closely linked to the CACNA1S gene were tested. Three fully informative markers were chosen to establish the disease-bearing haplotype in the family and to determine the genetic status of five embryos by multiplex fluorescent heminested PCR. Four of the five embryos tested were diagnosed as non-affected and one as affected. Two embryos were transferred resulting in a singleton pregnancy and the birth of a healthy girl., (2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Purpose: Description of the confluence of different molecular techniques to detect three different mutations in one cell. The man carries a 20 base pair insertion in exon 12 of the POR gene (c.1551_1552ins20), and the woman carries a point mutation in exon 8 of the POR gene (c.859G>C) plus a triplet repeat expansion in the HTT gene., Methods: Huntington Disease (HD) had to be diagnosed using short tandem repeat (STR) markers linked to the HTT gene. The mutation c.1551_1552ins20 was analyzed by fragment size and c.859G>C was minisequenced. Furthermore, STR markers linked to the POR gene were included to support the diagnosis of P450 oxidoreductase (POR) deficiency., Results: Nine embryos were diagnosed in total: three as POR deficiency affected, two as HD affected, one as POR deficiency and HD affected, and two as carriers of the paternal POR deficiency mutation and healthy for HD. These two last embryos were transferred but no pregnancy was achieved., Conclusions: A successful procedure combining direct and indirect methods for the detection of three different mutations in a single cell has been achieved for the first time.

The aim of this study was to analyze proliferative verrucous leukoplakia (PVL) and oral squamous cell carcinoma (OSCC) for the possible presence of Epstein-Barr virus (EBV). We studied three groups: Sub-Group 1 was composed of 10 patients with PVL, (6 of whom had developed OSCC); Sub-Group 2 comprised 5 patients with OSCC but no preceding PVL; and Sub-Group 3 were 5 controls with clinically normal oral mucosa. Oral biopsies from all cases were examined for Epstein-Barr virus (EBV) by nested PCR. EBV was detected in 60% of Sub-Group 1 patients (PVL ) and in 40% of Sub-Group 2 (OSCC), but in 0% of Sub-Group 3 (controls).

Purpose: To analyze proliferative verrucous leukoplakia (PVL) for the presence of human papillomavirus (HPV) in different stages of the disease., Materials and Methods: We studied 13 patients with PVL. In 10 patients (76.9%), a lesional biopsy was taken and frozen at -40 degrees C. Four patients were instructed to mouth rinse with sterile sera. The biopsy and rinse samples were analyzed for HPV by PCR., Results: We did not detect HPV infection in the PVL tissue or in the oral rinse of any of the 13 patients in any stage of the disease analyzed, neither in oral squamous cell carcinoma nor in the simple hyperkeratosis., Conclusion: There was no association between PVL and HPV infection in our patients.

The yeast Saccharomyces cerevisiae has been widely used for the implementation of DNA chip technologies. For this reason and due to the extensive use of this organism for basic and applied studies, yeast DNA chips are being used by many laboratories for expression or genomic analyses. While membrane arrays (macroarrays) offer several advantages, for many laboratories they are not affordable. Here we report that a cluster of four Spanish molecular-biology yeast laboratories, with relatively small budgets, have developed a complete set of probes for the genome of S. cerevisiae. These have been used to produce a new type of macroarray on a nylon surface. The macroarrays have been evaluated and protocols for their use have been optimized.

The yeast Saccharomyces cerevisiae is one of the most popular model organisms. It was the first eukaryote whose genome was sequenced. Since then many functional analysis projects have tried to find the function of many genes and to understand its metabolism in a holistic way. Apart from basic science this microorganism is of great interest in several biotechnology processes, such as winemaking. Only global studies of the cell as a whole can help us to understand many of the technical problems facing winemaking. DNA chip technology is one of the most promising tools for the analysis of cell physiology. Yeast has been the model organism for the development of this technique. Many of the studies can be applied to improve our knowledge of wine strains. Nevertheless wine strains are quite different in some aspects from the laboratory reference strains so a particular study of wine strains and especially during the winemaking process is needed. During the past two years some groups have started this study and the first results have been published. We review here the current state of the knowledge of wine yeast and the capacity of DNA chip technology for its improvement.

The olive fly, Bactrocera oleae, is the key pest on olives in the Mediterranean area. The pest can destroy, in some cases, up to 70% of the olive production. Its control relies mainly on chemical treatments, sometimes applied by aircraft over vast areas, with their subsequent ecological and toxicological side effects. Bacillus thuringiensis is a spore-forming soil bacterium which produces a protein crystal toxic to some insects, including the orders of Lepidoptera, Diptera, and Coleoptera and other invertebrates. The aim of this study was to search for isolates toxic to B. oleae. Several hundred B. thuringiensis isolates were obtained from olive groves and olive presses in different areas of Greece, Sardinia (Italy), and Spain and from cooperating scientists throughout the world. Some isolates were found toxic only to adults or larvae and some to both stages of the olive fly. In addition, the most toxic isolates were assayed on Opius concolor Szepl. (Hym. Braconidae), the most important parasitoid of the olive fruit fly. Only 3 isolates out of 14 gave significant mortality against this parasitoid. Several of the most toxic crystalliferous isolates may contain novel toxins since they gave no PCR products when probed with primers specified for 39 known toxin genes., (Copyright 1999 Academic Press.)

The study of gypsy elements in Drosophila subobscura (gypsyDs) indicated that they are transcriptionally active and mobile. From the comparative analysis of a complete gypsyDs element with the canonical gypsy sequence from D. melanogaster (gypsyDm) it can be deduced that while the whole structure is maintained, the gypsyDs ORF3 encodes a non-functional Env protein. The PCR amplification and sequencing of the ORF3 from different laboratory strains and H271 clones show that all gypsyDs sequences studied have frame-shifting mutations in this region. These results support that gypsyDs elements lack functional Env proteins and consequently they lack infective ability. In this way, it can be proposed that gypsyDs elements are degenerate forms of insect retroviruses. Heterogeneous results have been obtained in the study of the presence of gypsyDm sequences in different D. subobscura strains indicating that these sequences are unstable in this species.

We have determined the nucleotide sequence of a 7.5 kb full-size gypsy element from Drosophila subobscura strain H-271. Comparative analyses were carried out on the sequence and molecular structure of gypsy elements of D.subobscura (gypsyDs), D.melanogaster (gypsyDm) and D.virilis (gypsyDv). The three elements show a structure that maintains a common mechanism of expression. ORF1 and ORF2 show typical motifs of gag and pol genes respectively in the three gypsy elements and could encode functional proteins necessary for intracellular expansion. In the three ORF1 proteins an arginine-rich region was found which could constitute a RNA binding motif. The main differences among the gypsy elements are found in ORF3 (env-like gene); gypsyDm encodes functional env proteins, whereas gypsyDs and gypsyDv ORF3s lack some motifs essential for functionality of this protein. On the basis of these results, while gypsyDm is the first insect retrovirus described, gypsyDs and gypsyDv could constitute degenerate forms of these retroviruses. In this context, we have found some evidence that gypsyDm could have recently infected some D.subobscura strains. Comparative analyses of divergence and phylogenetic relationships of gypsy elements indicate that the gypsy elements belonging to species of different subgenera (gypsyDs and gypsyDv) are closer than gypsy elements of species belonging to the same subgenus (gypsyDs and gypsyDm). These data are congruent with horizontal transfer of gypsy elements among different Drosophila spp.

Characterization of sequences homologous to the Drosophila melanogaster gypsy transposable element was carried out in Drosophila subobscura (gypsyDS). They were found to be widely distributed among natural populations of this species. From Southern blot and in situ analyses, these sequences appear to be mobile in this species. GypsyDS sequences are located in both euchromatic and heterochromatic regions. A complete gypsyDS sequence was isolated from a D. subobscura genomic library, and a 1.3-kb fragment which aligns with the ORF2 of the D. melanogaster gypsy element was sequenced. Comparisons of this sequence in three species (D. subobscura, D. melanogaster, and D. virilis) indicate that there is greater similarity between the D. subobscura-D. virilis sequences than between D. subobscura and D. melanogaster. Molecular divergence of gypsy sequences between D. virilis and D. subobscura is estimated at 16 MY, whereas the most likely divergence time of these two species is more than 60 MY. These data strongly suggest that gypsy sequences have been horizontally transferred between these species.

Eight Drosophila species of the obscura subgroup were screened for sequences homologous to the gypsy retrotransposon of D. melanogaster. Molecular characterization of gypsy sequences was first approached through digesting genomic DNAs from these obscura species with appropriate restriction enzymes and subjecting them to Southern blot analysis. The results of this analysis indicate that gypsy-homologous sequences are well conserved among species of the obscura subgroup. With the exception of D. guanche, all other species bear a 7 kb Xho I fragment that represents the complete element in D. melanogaster. Lower molecular weight fragments that could be deleted elements, are shared by different species. Both types of element probably existed before the divergence of this subgroup. Two different species clusters could be established on the basis of hybridization patterns, one represented by D. subobscura and its relative species D. guanche and D. madeirensis, and the other, in which D. obscura, D. tristis and D. subsilvestris are included.