ABSTRACT
High-throughput DNA barcoding has become essential in ecology and evolution, but some technical questions still remain. Increasing the number of PCR cycles above the routine 20-30 cycles is a common practice when working with old-type specimens, which provide little amounts of DNA, or when facing annealing issues with the primers. However, increasing the number of cycles can raise the number of artificial mutations due to polymerase errors. In this work, we sequenced 20 COI libraries in the Illumina MiSeq platform. Libraries were prepared with 40, 45, 50, 55, and 60 PCR cycles from four individuals belonging to four species of four genera of cephalopods. We found no relationship between the number of PCR cycles and the number of mutations despite using a nonproofreading polymerase. Moreover, even when using a high number of PCR cycles, the resulting number of mutations was low enough not to be an issue in the context of high-throughput DNA barcoding (but may still remain an issue in DNA metabarcoding due to chimera formation). We conclude that the common practice of increasing the number of PCR cycles should not negatively impact the outcome of a high-throughput DNA barcoding study in terms of the occurrence of point mutations.
Subject(s)
Cephalopoda/genetics , DNA Barcoding, Taxonomic/methods , High-Throughput Nucleotide Sequencing/methods , Mutation , Polymerase Chain Reaction/methods , Animals , Cephalopoda/classification , Gene LibraryABSTRACT
Several studies on 5S ribosomal DNA (5S rDNA) have been focused on a subset of the following features in mostly one organism: number of copies, pseudogenes, secondary structure, promoter and terminator characteristics, genomic arrangements, types of non-transcribed spacers and evolution. In this work, we systematically analyzed 5S rDNA sequence diversity in available metazoan genomes, and showed organism-specific and evolutionary-conserved features. Putatively functional sequences (12,766) from 97 organisms allowed us to identify general features of this multigene family in animals. Interestingly, we show that each mammal species has a highly conserved (housekeeping) 5S rRNA type and many variable ones. The genomic organization of 5S rDNA is still under debate. Here, we report the occurrence of several paralog 5S rRNA sequences in 58 of the examined species, and a flexible genome organization of 5S rDNA in animals. We found heterogeneous 5S rDNA clusters in several species, supporting the hypothesis of an exchange of 5S rDNA from one locus to another. A rather high degree of variation of upstream, internal and downstream putative regulatory regions appears to characterize metazoan 5S rDNA. We systematically studied the internal promoters and described three different types of termination signals, as well as variable distances between the coding region and the typical termination signal. Finally, we present a statistical method for detection of linkage among noncoding RNA (ncRNA) gene families. This method showed no evolutionary-conserved linkage among 5S rDNAs and any other ncRNA genes within Metazoa, even though we found 5S rDNA to be linked to various ncRNAs in several clades.
Subject(s)
Evolution, Molecular , RNA, Ribosomal, 5S/genetics , Animals , Base Sequence , Consensus Sequence , Gene Dosage , Genetic Linkage , Inverted Repeat Sequences , Molecular Sequence Data , Promoter Regions, Genetic , RNA, Small Nuclear/genetics , Sequence Analysis, DNA , Sequence Homology, Nucleic AcidABSTRACT
The linkage between 5S ribosomal DNA and other multigene families has been detected in many eukaryote lineages, but whether it provides any selective advantage remains unclear. In this work, we report the occurrence of linked units of 5S ribosomal DNA (5S rDNA) and U1 small nuclear DNA (U1 snDNA) in 10 razor shell species (Mollusca: Bivalvia: Pharidae) from four different genera. We obtained several clones containing partial or complete repeats of both multigene families in which both types of genes displayed the same orientation. We provide a comprehensive collection of razor shell 5S rDNA clones, both with linked and nonlinked organisation, and the first bivalve U1 snDNA sequences. We predicted the secondary structures and characterised the upstream and downstream conserved elements, including a region at -25 nucleotides from both 5S rDNA and U1 snDNA transcription start sites. The analysis of 5S rDNA showed that some nontranscribed spacers (NTSs) are more closely related to NTSs from other species (and genera) than to NTSs from the species they were retrieved from, suggesting birth-and-death evolution and ancestral polymorphism. Nucleotide conservation within the functional regions suggests the involvement of purifying selection, unequal crossing-overs and gene conversions. Taking into account this and other studies, we discuss the possible mechanisms by which both multigene families could have become linked in the Pharidae lineage. The reason why 5S rDNA is often found linked to other multigene families seems to be the result of stochastic processes within genomes in which its high copy number is determinant.
Subject(s)
Bivalvia/genetics , RNA, Ribosomal, 5S/genetics , RNA, Small Nuclear/genetics , Animals , Base Composition/genetics , Base Sequence , Bivalvia/classification , Evolution, Molecular , Gene Order , Molecular Sequence Data , Nucleic Acid Conformation , Phylogeny , RNA, Ribosomal, 5S/chemistry , RNA, Small Nuclear/chemistryABSTRACT
Thyroid adenomas containing adipose tissue are rare. Only scattered case reports have been recorded. We describe an additional case of an adenolipoma of the thyroid gland, incidentally found in residual gland subsequent to excision of a papillary carcinoma.
Subject(s)
Adenoma/pathology , Lipoma/pathology , Thyroid Neoplasms/pathology , Female , Humans , Middle AgedABSTRACT
The clinicopathologic features of a giant adrenal myelolipoma (diameter 16 cm, weight 900 g) are presented. The ultrasonographic and the computed tomographic appearance of adrenal myelolipoma are reviewed. Diagnostic fine-needle aspiration biopsy is recommended for avoidance of unnecessary surgical treatment.
Subject(s)
Adrenal Gland Neoplasms , Myelolipoma , Adrenal Gland Neoplasms/diagnosis , Adrenal Gland Neoplasms/epidemiology , Adrenal Glands/pathology , Diagnostic Imaging , Humans , Male , Middle Aged , Myelolipoma/diagnosis , Myelolipoma/epidemiologyABSTRACT
A case of paraneoplastic hypercalcaemic syndrome is reported in a patient with Hodgkin's disease. This was detected eight months before widespread lymphadenopathy became apparent. Lymphocyte depleted Hodgkin's disease was diagnosed. PTH (parathyroid hormone) activity was suppressed and PTHRP (PTH related protein) was less than 5 pmol/l. 1,25(OH)2D3 was in the normal range. Plasma calcium values returned to normal after the administration of indomethacin. Thus the pathogenesis of the hypercalcaemia in this patient could be associated with the synthesis of prostaglandins.
