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- Novel large deletions in the human alpha-globin gene cluster: Clarifying the HS-40 long-range regulatory role in the native chromosome environmentPublication . Coelho, Andreia; Isabel, Picanço; Filomena, Seuanes; Seixas, Maria Teresa; Paula, FaustinoGlobin genes, which encode the protein subunits of hemoglobin (Hb), are organized in two different gene clusters and present a coordinated and differential pattern of expression during development. Concerning the human α-globin gene cluster (located at chromosome region 16p13.3), four upstream highly conserved elements known as multispecies conserved sequences (MCS-R1-4) or DNase I hypersensitive sites (HSs) are implicated in the long-range regulation of downstream gene expression. However, only the absence of the MCS-R2 site (HS-40) has proven to drastically downregulate the expression of those genes, and consequently, it has been regarded as the major and crucial distal regulatory element. In this study, Multiplex Ligation-dependent Probe Amplification was used to screen for deletions in the telomeric region of the short arm of chromosome 16, in an attempt to explain the α-thalassemia or the HbH disease present in a group of Portuguese patients. We report four novel and five uncommon deletions that remove the α-globin distal regulatory elements and/or the complete α-globin gene cluster. Interestingly, one of them occurred de novo and removes all HSs except HS-10, while other eliminates only the HS-40 site, the latter being replaced by the insertion of a 39 nucleotide orphan sequence. Our results demonstrate that HS-10 alone does not significantly enhance the α-globin gene expression. The absence of HS-40 in homozygosity, found in a patient with Hb H disease, strongly downregulates the expression of α-globin genes but it is not associated with a complete absence of α-globin chain production. The study of naturally occurring deletions in this region is of great interest to understand the role of each upstream regulatory element in the native human erythroid environment.
- A novel molecular method for identification of Oenococcus oeni and its specific detection in winePublication . Marques, Ana P.; Zé-Zé, Líbia; San-Romão, Maria Vitória; Tenreiro, RogérioOenococcus oeni is a species of lactic acid bacteria with economic interest in winemaking. Using both in silico and in vitro analyses, a molecular method was developed that allows the identification of O. oeni isolates and its detection from wine samples. The method is based on the amplification of 16S rRNA gene with universal primers followed by restriction with the endonuclease FseI, generating two fragments of 326 and 1233 bp. Among wine bacteria, the FseI recognition sequence is only found in the 16S rRNA gene of O. oeni, ensuring the specificity of the method. The use of Whatman FTA cards for DNA extraction and purification is an efficient and interesting alternative to current methods, as samples can be easily collected at wineries by a non-specialized technician, stored at room temperature and sent in a mail envelope to the analytical laboratory for processing. The proposed method, with a detection limit between 102 and 103 cfu/mL and a full turnaround time of ca. 8 h, ensures the rapid and reliable detection of O. oeni in wine samples during winemaking surveillance and wine quality control.