A Statistical Approach to Identify Ancient Template DNA

One of the key problems in the study of ancient DNA is that of authenticating sequences obtained from PCR amplifications of highly degraded samples. Contamination of ancient samples and postmortem damage to endogenous DNA templates are the major obstacles facing researchers in this task. In particular, the authentication of sequences obtained from ancient human remains is thought by many to be rather challenging. We propose a novel approach, based on the c statistic, that can be employed to help identify the sequence motif of an endogenous template, based on a sample of sequences that reflect the nucleotide composition of individual template molecules obtained from ancient tissues (such as cloned products from a PCR amplification). The c statistic exploits as information the most common form of postmortem damage observed among clone sequences in ancient DNA studies, namely, lesion-induced substitutions caused by cytosine deamination events. Analyses of simulated sets of templates with miscoding lesions and real sets of clone sequences from the literature indicate that the c-based approach is highly effective in identifying endogenous sequence motifs, even when they are not present among the sampled clones. The proposed approach is likely to be of general use to researchers working with DNA from ancient tissues, particularly from human remains, where authentication of results has been most challenging. [Reviewing Editor: Dr. Magnus Nordborg]     

Duplicated sequence motif in the long terminal repeat of maedi-visna virus extends cell tropism and is associated with neurovirulence

Maedi-visna virus (MVV) is a lentivirus of sheep causing chronic inflammatory disease of the lungs (maedi) and the nervous system (visna). We have previously shown that a duplicated sequence in the long terminal repeat (LTR) of MVV is a determinant of cell tropism. Here, we demonstrate that deletion of a CAAAT sequence from either one of the repeats resulted in poor virus growth in sheep choroid plexus cells. A duplication in the LTR encompassing the CAAAT sequence was found in four neurological field cases that were sequenced, but no duplication was present in the LTRs from seven maedi cases; one maedi isolate was mixed. These results indicate that the duplication in the LTR is associated with neurovirulence.     

Effects of the Hekla 4 tephra on vegetation in Northwest Iceland

Vegetation plays a key role in preventing the remobilisation of tephra and aeolian activity following tephra fall. Recent volcanic eruptions in Iceland have highlighted the consequences of tephra fall for ecosystems and human health. Improved understanding of the mechanisms behind ecosystem recovery following tephra fall is particularly important for Iceland. Today?~42% of the country is classified as desert and unvegetated and sparsely vegetated areas are unable to trap tephra fall and prevent subsequent wind erosion. This paper presents palaeoenvironmental reconstructions before and after the Hekla 4 tephra from two lakes in Northwest Iceland, from within a woodland in the lowland, and in open woodland under stress at the highland margin. The c. 4,200 cal bp. Hekla 4 tephra is one of the most extensive Icelandic Holocene tephra layers and the eruption produced an estimated?~9 km³ of tephra. The palaeoecological reconstructions provide an insight into the responses of two relatively stable ecosystems to thick tephra deposits during a period of cooling climate. The understory vegetation in the lowland woodland was buried by the tephra, however Betula pubescens trees were not severely affected and the woodland recovered relatively quickly. In contrast, open woodland at the highland margin that was already at its ecological limit, shifted to dwarf shrub heath, a more resilient vegetation community in response to the tephra fall and cooling climate.     

Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species.

The gene encoding a subtilisin-like serine proteinase in the psychrotrophic Vibrio sp. PA44 has been successfully cloned, sequenced and expressed in Escherichia coli. The gene is 1593 basepairs and encodes a precursor protein of 530 amino acid residues with a calculated molecular mass of 55.7 kDa. The enzyme is isolated, however, as an active 40.6-kDa proteinase, without a 139 amino acid residue N-terminal prosequence. Under mild conditions the enzyme undergoes a further autocatalytic cleavage to give a 29.7-kDa proteinase that retains full enzymatic activity. The deduced amino acid sequence of the enzyme has high homology to proteinases of the proteinase K family of subtilisin-like proteinases. With respect to the enzyme characteristics compared in this study the properties of the wild-type and recombinant proteinases are the same. Sequence analysis revealed that especially with respect to the thermophilic homologues, aqualysin I from Thermus aquaticus and a proteinase from Thermus strain Rt41A, the cold-adapted Vibrio-proteinase has a higher content of polar/uncharged amino acids, as well as aspartate residues. The thermophilic enzymes had a higher content of arginines, and relatively higher number of hydrophobic amino acids and a higher aliphatic index. These factors may contribute to the adaptation of these proteinases to different temperature conditions.     

ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism

Macroautophagy/autophagy is an essential catabolic process that targets a wide variety of cellular components including proteins, organelles, and pathogens. ATG7, a protein involved in the autophagy process, plays a crucial role in maintaining cellular homeostasis and can contribute to the development of diseases such as cancer. ATG7 initiates autophagy by facilitating the lipidation of the ATG8 proteins in the growing autophagosome membrane. The noncanonical isoform ATG7(2) is unable to perform ATG8 lipidation; however, its cellular regulation and function are unknown. Here, we uncovered a distinct regulation and function of ATG7(2) in contrast with ATG7(1), the canonical isoform. First, affinity-purification mass spectrometry analysis revealed that ATG7(2) establishes direct protein–protein interactions (PPIs) with metabolic proteins, whereas ATG7(1) primarily interacts with autophagy machinery proteins. Furthermore, we identified that ATG7(2) mediates a decrease in metabolic activity, highlighting a novel splice-dependent function of this important autophagy protein. Then, we found a divergent expression pattern of ATG7(1) and ATG7(2) across human tissues. Conclusively, our work uncovers the divergent patterns of expression, protein interactions, and function of ATG7(2) in contrast to ATG7(1). These findings suggest a molecular switch between main catabolic processes through isoform-dependent expression of a key autophagy gene.     

Natural phosphatidylcholine is actively translocated across the plasma membrane to the surface of mammalian cells

The cell surface of eukaryotic cells is enriched in choline phospholipids, whereas the aminophospholipids are concentrated at the cytosolic side of the plasma membrane by the activity of one or more P-type ATPases. Lipid translocation has been investigated mostly by using short chain lipid analogs because assays for endogenous lipids are inherently complicated. In the present paper, we optimized two independent assays for the translocation of natural phosphatidylcholine (PC) to the cell surface based on the hydrolysis of outer leaflet phosphoglycerolipids by exogenous phospholipase A2 and the exchange of outer leaflet PC by a transfer protein. We report that PC reached the cell surface in the absence of vesicular traffic by a pathway that involved translocation across the plasma membrane. In erythrocytes, PC that was labeled at the inside of the plasma membrane was translocated to the cell surface with a half-time of 30 min. This translocation was probably mediated by an ATPase, because it required ATP and was vanadate-sensitive. The inhibition of PC translocation by glibenclamide, an inhibitor of various ATP binding cassette transporters, and its reduction in erythrocytes from both Abcb1a/1b and Abcb4 knockout mice, suggest the involvement of ATP binding cassette transporters in natural PC cell surface translocation. The relative importance of the outward translocation of PC as compared with the well characterized fast inward translocation of phosphatidylserine for the overall asymmetric phospholipid organization in plasma membranes remains to be established.     

Simultaneous mutations in CA and Vif of Maedi-Visna virus cause attenuated replication in macrophages and reduced infectivity in vivo

Maedi-visna virus (MVV) is a lentivirus of sheep sharing several key features with the primate lentiviruses. The virus causes slowly progressive diseases, mainly in the lungs and the central nervous system of sheep. Here, we investigate the molecular basis for the differential growth phenotypes of two MVV isolates. One of the isolates, KV1772, replicates well in a number of cell lines and is highly pathogenic in sheep. The second isolate, KS1, no longer grows on macrophages or causes disease. The two virus isolates differ by 129 nucleotide substitutions and two deletions of 3 and 15 nucleotides in the env gene. To determine the molecular nature of the lesions responsible for the restrictive growth phenotype, chimeric viruses were constructed and used to map the phenotype. An L120R mutation in the CA domain, together with a P205S mutation in Vif (but neither alone), could fully convert KV1772 to the restrictive growth phenotype. These results suggest a functional interaction between CA and Vif in MVV replication, a property that may relate to the innate antiretroviral defense mechanisms in sheep.     

Screening for restriction endonucleases in aerobic,thermophilic eubacteria

Summary A total of 216 Icelandic aerobic, heterotrophic, thermophiles belonging to three different genera were screened for type II restriction endonucleases. The frequency of positive strains was 44% for both Thermus and Bacillus but 63% for Rhodothermus. Approximately half of the enzymes from each group were characterised and a total of 14 different restriction enzymes were found. In all cases they were isoschizomers of known enzymes. Thermus contained 9 different types, Bacillus 6 and Rhodothermus had 3. This is the first time that isoschizomers of BspEI, BglI, EagI and EcoRV are found in Thermus and BstBI and EcoRV are found in Rhodothermus.