DNA alignment and Bayesian trees of partial sequences of COI, COIII and the haemocyanin functional unit f-g of 28 octopod species

Background: Octopods have successfully colonised the world's oceans from the tropics to the poles. Yet, successful persistence in these habitats has required adaptations of their advanced physiological apparatus to compensate impaired oxygen supply. Their oxygen transporter haemocyanin plays a major role in cold tolerance and accordingly has undergone functional modifications to sustain oxygen release at sub-zero temperatures. However, it remains unknown how molecular properties evolved to explain the observed functional adaptations. We thus aimed to assess whether natural selection affected molecular and structural properties of haemocyanin that explains temperature adaptation in octopods. Results: Analysis of 239 partial sequences of the haemocyanin functional units (FU) f and g of 28 octopod species of polar, temperate, subtropical and tropical origin revealed natural selection was acting primarily on charge properties of surface residues. Polar octopods contained haemocyanins with higher net surface charge due to decreased glutamic acid content and higher numbers of basic amino acids. Within the analysed partial sequences, positive selection was present at site 2545, positioned between the active copper binding centre and the FU g surface. At this site, methionine was the dominant amino acid in polar octopods and leucine was dominant in tropical octopods. Sites directly involved in oxygen binding or quaternary interactions were highly conserved within the analysed sequence. Conclusions: This study has provided the first insight into molecular and structural mechanisms that have enabled octopods to sustain oxygen supply from polar to tropical conditions. Our findings imply modulation of oxygen binding via charge-charge interaction at the protein surface, which stabilize quaternary interactions among functional units to reduce detrimental effects of high pH on venous oxygen release. Of the observed partial haemocyanin sequence, residue 2545 formed a close link between the FU g surface and the active centre, suggesting a role as allosteric binding site. The prevalence of methionine at this site in polar octopods, implies regulation of oxygen affinity via increased sensitivity to allosteric metal binding. High sequence conservation of sites directly involved in oxygen binding indicates that functional modifications of octopod haemocyanin rather occur via more subtle mechanisms, as observed in this study.

Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3′ end target sequence

Unique, small sequences (sequence tag sites) have been identified at the 3′ ends of most human genes that serve as landmarks in genome mapping. We investigated whether a single copy gene could be isolated directly from total human DNA by transformation-associated recombination (TAR) cloning in yeast using a short, 3′ unique target. A TAR cloning vector was constructed that, when linearized, contained a small amount (381 bp) of 3′ hypoxanthine phosphoribosyltransferase (HPRT) sequence at one end and an 189-bp Alu repeat at the other end. Transformation with this vector along with human DNA led to selective isolations of the entire HPRT gene as yeast artificial chromosomes (YACs) that extended from the 3′ end sequence to various Alu positions as much as 600 kb upstream. These YACs were retrofitted with a NeoR and a bacterial artificial chromosome (BAC) sequence to transfer the YACs to bacteria and subsequently the BACs to mouse cells by using a Neo selection. Most of the HPRT isolates were functional, demonstrating that TAR cloning retains the functional integrity of the isolated material. Thus, this modified version of TAR cloning, which we refer to as radial TAR cloning, can be used to isolate large segments of the human genome accurately and directly with only a small amount of sequence information.

Detection of 400-year-old Yersinia pestis DNA in human dental pulp: An approach to the diagnosis of ancient septicemia

Ancient septicemic plague epidemics were reported to have killed millions of people for 2 millenniums. However, confident diagnosis of ancient septicemia solely on the basis of historical clinical observations is not possible. The lack of suitable infected material has prevented direct demonstration of ancient septicemia; thus, the history of most infections such as plague remains hypothetical. The durability of dental pulp, together with its natural sterility, makes it a suitable material on which to base such research. We hypothesized that it would be a lasting refuge for Yersinia pestis, the plague agent. DNA extracts were made from the dental pulp of 12 unerupted teeth extracted from skeletons excavated from 16th and 18th century French graves of persons thought to have died of plague (“plague teeth”) and from 7 ancient negative control teeth. PCRs incorporating ancient DNA extracts and primers specific for the human β-globin gene demonstrated the absence of inhibitors in these preparations. The incorporation of primers specific for Y. pestis rpoB (the RNA polymerase β-subunit-encoding gene) and the recognized virulence-associated pla (the plasminogen activator-encoding gene) repeatedly yielded products that had a nucleotide sequence indistinguishable from that of modern day isolates of the bacterium. The specific pla sequence was obtained from 6 of 12 plague skeleton teeth but 0 of 7 negative controls (P < 0.034, Fisher exact test). A nucleic acid-based confirmation of ancient plague was achieved for historically identified victims, and we have confirmed the presence of the disease at the end of 16th century in France. Dental pulp is an attractive target in the quest to determine the etiology of septicemic illnesses detected in ancient corpses. Molecular techniques could be applied to this material to resolve historical outbreaks.

Translocation and specific cleavage of bacteriophage T7 DNA in vivo by EcoKI

Infection of Escherichia coli containing the type I restriction enzyme EcoKI by bacteriophage T7 0.3 mutants leads to restriction during the late stages of genome entry and during DNA replication. Patterns of cleavage in vivo suggest that some cutting occurs near the midpoint of two recognition sites, consistent with the idea that EcoKI translocates DNA bidirectionally through itself and cuts when two enzyme molecules collide. Rapid ejection of a 0.3+ T7 genome from a bacteriophage λ particle results in degradation of the infecting DNA by EcoKI, showing that the normal T7 DNA translocation process delays restriction. A unique recognition site inserted at the genomic left end allows EcoKI to function as a molecular motor and to translocate the remaining 39 kilobases of T7 DNA into the cell.

Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation

Changes in DNA methylation during tobacco pollen development have been studied by confocal fluorescence microscopy using a monoclonal anti-5-methylcytosine (anti-m5C) antibody and a polyclonal anti-histone H1 (anti-histone) antibody as an internal standard. The specificity of the anti-m5C antibody was demonstrated by a titration series against both single-stranded DNA and double-stranded DNA substrates in either the methylated or unmethylated forms. The antibody was found to show similar kinetics against both double- and single-stranded DNA, and the fluorescence was proportional to the amount of DNA used. No signal was observed with unmethylated substrates. The extent of methylation of the two pollen nuclei remained approximately constant after the mitotic division that gave rise to the vegetative and generative nuclei. However, during the subsequent development of the pollen, the staining of the generative nucleus decreased until it reached a normalized value of \documentclass[12pt]{minimal} \usepackage{wasysym} \usepackage{amsmath} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}\frac{1}{5}\end{equation*}\end{document} of that of the vegetative nucleus. The use of a confocal microscope makes these data independent of possible focusing artefacts. The anti-histone antibody was used as a control to show that, while the antibody staining directed against 5-methylcytosine changed dramatically during pollen maturation, the histone signal did not. We observed the existence of structural dimorphism amongst tobacco pollen grains, the majority having three pollen apertures and the rest with four. However, the methylation changes observed occurred to the same extent in both subclasses.