Donal A. Hickey

A universal DNA mini-barcode for biodiversity analysis

BackgroundThe goal of DNA barcoding is to develop a species-specific sequence library for all eukaryotes. A 650 bp fragment of the cytochrome c oxidase 1 (CO1) gene has been used successfully for species-level identification in several animal groups. It may be difficult in practice, however, to retrieve a 650 bp fragment from archival specimens, (because of DNA degradation) or from environmental samples (where universal primers are needed).ResultsWe used a bioinformatics analysis using all CO1 barcode sequences from GenBank and calculated the probability of having species-specific barcodes for varied size fragments. This analysis established the potential of much smaller fragments, mini-barcodes, for identifying unknown specimens. We then developed a universal primer set for the amplification of mini-barcodes. We further successfully tested the utility of this primer set on a comprehensive set of taxa from all major eukaryotic groups as well as archival specimens.ConclusionIn this study we address the important issue of minimum amount of sequence information required for identifying species in DNA barcoding. We establish a novel approach based on a much shorter barcode sequence and demonstrate its effectiveness in archival specimens. This approach will significantly broaden the application of DNA barcoding in biodiversity studies.

COMPOSITIONAL BIAS MAY AFFECT BOTH DNA-BASED AND PROTEIN-BASED PHYLOGENETIC RECONSTRUCTIONS

Abstract. It is now well-established that compositional bias in DNA sequences can adversely affect phylogenetic analysis based on those sequences. Phylogenetic analyses based on protein sequences are generally considered to be more reliable than those derived from the corresponding DNA sequences because it is believed that the use of encoded protein sequences circumvents the problems caused by nucleotide compositional biases in the DNA sequences. There exists, however, a correlation between AT/GC bias at the nucleotide level and content of AT- and GC-rich codons and their corresponding amino acids. Consequently, protein sequences can also be affected secondarily by nucleotide compositional bias. Here, we report that DNA bias not only may affect phylogenetic analysis based on DNA sequences, but also drives a protein bias which may affect analyses based on protein sequences. We present a striking example where common phylogenetic tools fail to recover the correct tree from complete animal mitochondrial protein-coding sequences. The data set is very extensive, containing several thousand sites per sequence, and the incorrect phylogenetic trees are statistically very well supported. Additionally, neither the use of the LogDet/paralinear transform nor removal of positions in the protein alignment with AT- or GC-rich codons allowed recovery of the correct tree. Two taxa with a large compositional bias continually group together in these analyses, despite a lack of close biological relatedness. We conclude that even protein-based phylogenetic trees may be misleading, and we advise caution in phylogenetic reconstruction using protein sequences, especially those that are compositionally biased.

A DNA-Based Approach to the Identification of Insect Species Used for Postmorten Interval Estimation

Insect larvae found on a corpse can be used for estimating postmortem intervals. Here, we describe a molecular method for rapid identification of these insects. Specific insect DNA fragments were amplified using the polymerase chain reaction (PCR), followed by direct DNA sequencing of the amplification products. We sequenced 2300 base pairs of mitochondrial DNA from each of three blowfly species: Phormia regina, Phaenicia sericata and Lucilia illustris. All three species are important in forensic entomology. We found 118 nucleotide differences between the L. illustris and P. sericata sequences, 186 between L. illustris and P. regina, and 192 between P. sericata and P. regina. Based on these abundant DNA sequence differences, we can unambiguously identify the immature larval stages of these insects. These DNA sequence differences were also used to predict species-specific, diagnostic restriction sites in the amplified DNA, and these predictions were verified by digestion with nine restriction enzymes. The DNA sequences reported here encode the mitochondrial COI, COII and tRNA-leucine genes.

Nucleotide Composition Bias Affects Amino Acid Content in Proteins Coded by Animal Mitochondria

Abstract. We show that in animal mitochondria homologous genes that differ in guanine plus cytosine (G + C) content code for proteins differing in amino acid content in a manner that relates to the G + C content of the codons. DNA sequences were analyzed using square plots, a new method that combines graphical visualization and statistical analysis of compositional differences in both DNA and protein. Square plots divide codons into four groups based on first and second position A + T (adenine plus thymine) and G + C content and indicate differences in amino acid content when comparing sequences that differ in G + C content. When sequences are compared using these plots, the amino acid content is shown to correlate with the nucleotide bias of the genes. This amino acid effect is shown in all protein-coding genes in the mitochondrial genome, including cox I, cox II, and cyt b, mitochondrial genes which are commonly used for phylogenetic studies. Furthermore, nucleotide content differences are shown to affect the content of all amino acids with A + T- and G + C-rich codons. We speculate that phylogenetic analysis of genes so affected may tend erroneously to indicate relatedness (or lack thereof) based only on amino acid content.

Thermophilic prokaryotes have characteristic patterns of codon usage, amino acid composition and nucleotide content

A number of recent studies have shown that thermophilic prokaryotes have distinguishable patterns of both synonymous codon usage and amino acid composition, indicating the action of natural selection related to thermophily. On the other hand, several other studies of whole genomes have illustrated that nucleotide bias can have dramatic effects on synonymous codon usage and also on the amino acid composition of the encoded proteins. This raises the possibility that the thermophile-specific patterns observed at both the codon and protein levels are merely reflections of a single underlying effect at the level of nucleotide composition. Moreover, such an effect at the nucleotide level might be due entirely to mutational bias. In this study, we have compared the genomes of thermophiles and mesophiles at three levels: nucleotide content, codon usage and amino acid composition. Our results indicate that the genomes of thermophiles are distinguishable from mesophiles at all three levels and that the codon and amino acid frequency differences cannot be explained simply by the patterns of nucleotide composition. At the nucleotide level, we see a consistent tendency for the frequency of adenine to increase at all codon positions within the thermophiles. Thermophiles are also distinguished by their pattern of synonymous codon usage for several amino acids, particularly arginine and isoleucine. At the protein level, the most dramatic effect is a two-fold decrease in the frequency of glutamine residues among thermophiles. These results indicate that adaptation to growth at high temperature requires a coordinated set of evolutionary changes affecting (i) mRNA thermostability, (ii) stability of codon-anticodon interactions and (iii) increased thermostability of the protein products. We conclude that elevated growth temperature imposes selective constraints at all three molecular levels: nucleotide content, codon usage and amino acid composition. In addition to these multiple selective effects, however, the genomes of both thermophiles and mesophiles are often subject to superimposed large changes in composition due to mutational bias.

Genomic and proteomic adaptations to growth at high temperature.

Most positively selected mutations cause changes in metabolism, resulting in a better-adapted phenotype. But as well as acting on the information content of genes, natural selection may also act directly on nucleic acid and protein molecules. We review the evidence for direct temperature-dependent natural selection acting on genomes, transcriptomes and proteomes.

GENOMIC ORGANIZATION AND EXPRESSION OF A TRYPSIN GENE FROM THE SPRUCE BUDWORM, CHORISTONEURA FUMIFERANA

A 7 kb (kilobase) genomic fragment containing a trypsin gene from the spruce budworm Choristoneura fumiferana was isolated and sequenced. The coding sequence is interrupted by two introns; these occur at the same positions as the first two introns of mammalian trypsin genes. The three exons encode 256 amino acids. The deduced protein sequence displays all of the structural features that characterize trypsin enzymes in other eukaryotic organisms. Genomic Southern hybridization showed that there is only one copy of the trypsin gene in the Choristoneura genome. This gene is expressed in the insect midgut, where the pH is extremely high. The complete lack of Lysine residues may be an adaptation to the high pH conditions. Extensive sequencing of the flanking regions did not reveal the presence of any linked trypsin-encoding genes. Instead, several short repetitive sequences and a sequence homologous to a Drosophila reverse transcriptase gene was identified in this genomic region.

Thermal adaptation of the small subunit ribosomal RNA gene : A comparative study

We carried out a comprehensive survey of small subunit ribosomal RNA sequences from archaeal, bacterial, and eukaryotic lineages in order to understand the general patterns of thermal adaptation in the rRNA genes. Within each lineage, we compared sequences from mesophilic, moderately thermophilic, and hyperthermophilic species. We carried out a more detailed study of the archaea, because of the wide range of growth temperatures within this group. Our results confirmed that there is a clear correlation between the GC content of the paired stem regions of the 16S rRNA genes and the optimal growth temperature, and we show that this correlation cannot be explained simply by phylogenetic relatedness among the thermophilic archaeal species. In addition, we found a significant, positive relationship between rRNA stem length and growth temperature. These correlations are found in both bacterial and archaeal rRNA genes. Finally, we compared rRNA sequences from warm-blooded and cold-blooded vertebrates. We found that, while rRNA sequences from the warm-blooded vertebrates have a higher overall GC content than those from the cold-blooded vertebrates, this difference is not concentrated in the paired regions of the molecule, suggesting that thermal adaptation is not the cause of the nucleotide differences between the vertebrate lineages.

Isolation and characterization of a full-length trypsin-encoding cDNA clone from the Lepidopteran insect, Choristoneura fumiferana.

The protease-encoding genes of Lepidopteran insects are of interest because they are adapted to functioning at very high pH optima, in the range of pH 10-12. Here, we report the isolation and sequence characterization of a trypsin-encoding cDNA clone from the spruce budworm, Choristoneura fumiferana.

Higher level phylogeny of mosquitoes (Diptera: Culicidae): mtDNA data support a derived placement for Toxorhynchites

We assess the potential of complete coding sequences of the mitochondrial cytochrome oxidase genes (COI and COII) and the intervening tRNA-Leucine gene for use in mosquito higher-level systematics, and apply this data to an outstanding question: the phylogenetic affinities of Toxorhynchites. Traditionally placed in its own subfamily and regarded as sister group to Culicinae, recent morphological data instead have suggested that this distinctive genus belongs well within the Culicinae. Published molecular systematic studies seemingly conflict with this new morphological data or are ambiguous. The mitochondrial gene data that we present show good potential for elucidating suprageneric relationships in Culicidae, and strongly support the placement of Toxorhynchites well within the Culicinae. Reexamination of published data sets suggests that there is no substantive conflict among data sets on this issue.