Abstract: BACKGROUND: Hemoglobinopathies are the most common inherited diseases worldwide. Various methods for genotyping of hemoglobin, beta (HBB) gene mutations have been reported, but there is need for a high sample-throughput, cost-effective method for simultaneous screening of several mutations. We report a method that combines the high detectability and dynamic range of chemiluminescence with the high allele-discrimination ability of probe extension reactions for simultaneous genotyping of 15 HBB mutations in a high sample-throughput, dry-reagent format. METHODS: We genotyped the HBB mutations IVSI-110G>A, CD39C>T, IVSI-1G>A, IVSI-6T>C, IVSII-745C>G, IVSII-1G>A, FSC6GAG>G-G, -101C>T, FSC5CCT>C-, IVSI-5G>A, FSC8AAG>-G, -87C>G, IVSII-848C>A, term+6C>G, and HbS (cd6GAG>GTG). The method used comprises the following: (a) duplex PCR that produces fragments encompassing all 15 mutations, (b) probe extension reactions in the presence of fluorescein-modified dCTP, using unpurified amplicons, and (c) microtiter well-based assay of extension products with a peroxidase-antifluorescein conjugate and a chemiluminogenic substrate. We used lyophilized dry reagents to simplify the procedure and assigned the genotype by the signal ratio of the normal-to-mutant-specific probe. RESULTS: We standardized the method by analyzing 60 samples with known genotypes and then validated by blindly genotyping 115 samples with 45 genotypes. The results were fully concordant with sequencing. The reproducibility (including PCR, probe extension reaction, and chemiluminometric assay) was studied for 20 days, and the CVs were 11%-19%. CONCLUSIONS: This method is accurate, reproducible, and cost-effective in terms of equipment and reagents. The application of the method is simple, rapid, and robust. The microtiter well format allows genotyping of a large number of samples in parallel for several mutations.
Abstract: The phylogenetic relationships among the Drosophila melanogaster group species were analyzed using approximately 1700 nucleotide-long sequences of the mitochondrial DNA. Phylogenetic analysis was performed using this region consisting of a part of the cytochrome b (cytb) coding gene, the entire coding sequences of tRNA-Leu, tRNA-Ser and the first subunit of NADH dehydrogenase (NADH1), and a part of the 16S-rRNA gene. The study of these sequences showed that this region of mtDNA is very invariable, as regards with the type of the genes that it contains, as well as the order that they are located on it. The resulting phylogenetic trees reveal a topology that separates the species into three main ancestral lines, leading to the following subgroups: (a) ananassae subgroup, (b) montium subgroup, and (c) melanogaster and Oriental subgroups. The inferred topology complements and generally agrees with previously proposed classifications based on morphological and molecular data.
Abstract: Kappa-casein plays an essential role in the formation, stabilisation and aggregation of milk micelles. Control of kappa-casein expression reflects this essential role, although an understanding of the mechanisms involved lags behind that of the other milk protein genes. We determined the 5'-flanking sequences for the murine, rabbit and human kappa-casein genes and compared them to the published ruminant sequences. The most conserved region was not the proximal promoter region but an approximately 400 bp long region centred 800 bp upstream of the TATA box. This region contained two highly conserved MGF/STAT5 sites with common spacing relative to each other. In this region, six conserved short stretches of similarity were also found which did not correspond to known transcription factor consensus sites. On the contrary to ruminant and human 5' regulatory sequences, the rabbit and murine 5'-flanking regions did not harbour any kind of repetitive elements. We generated a phylogenetic tree of the six species based on multiple alignment of the kappa-casein sequences. This study identified conserved candidate transcriptional regulatory elements within the kappa-casein gene promoter.
Abstract: The phylogenetic relationships among the Drosophila melanogaster subgroup species were analyzed using approximately 1550-nucleotide-long sequences of the Cu,Zn SOD gene. Phylogenetic analysis was performed using separately the whole region and the intron sequences of the gene. The resulting phylogenetic trees reveal virtually the same topology, separating the species into distinct clusters. The inferred topology generally agrees with previously proposed classifications based on morphological and molecular data. The amino acid sequences of the Cu,Zn SOD of the D. melanogaster subgroup species reveal a high-conservation pattern. Only 3.9% of the total amino acid sites are variable, and none affects the major structural elements. Comparison of the Drosophila Cu,Zn SOD amino acid sequences with the Cu,Zn SOD of Bos taurus and Xenopus laevis (whose three-dimensional structure has been elucidated) reveals conservation of all the protein's functionally important amino acids and no substitutions that dramatically change the charge or the polarity of the amino acids.
Abstract: A collection of 64 fig (Ficus carica L.) accessions was characterized through the use of RAPD markers, and results were evaluated in conjunction with morphological and agronomical characters, in order to determine the genetic relatedness of genotypes with diverse geographic origin. The results indicate that fig cultivars have a rather narrow genetic base. Nevertheless, RAPD markers could detect enough polymorphism to differentiate even closely related genotypes (i.e., clones of the same cultivar) and a unique fingerprint for each of the genotypes studied was obtained. No wasteful duplications were found in the collection. Cluster analysis allowed the identification of groups in accordance with geographic origin, phenotypic data and pedigree. Taking into account the limited information concerning fig cultivar development, the results of this study, which provide information on the genetic relationships of genetically distinct material, dramatically increase the fundamental and practical value of the collection and represent an invaluable tool for fig germplasm management.
