The complete mitochondrial genome of Alligator mississippiensis and the separation between recent archosauria (birds and crocodiles)

The complete mitochondrial genome of the alligator, Alligator mississippiensis, was sequenced. The size of the molecule is 16,642 nucleotides. Previously reported rearrangements of tRNAs in crocodile mitochondrial genomes were confirmed and, relative to mammals, no other deviations of gene order were observed. The analysis of protein-coding genes of the alligator showed an evolutionary rate that is roughly the same as in mammals. Thus, the evolutionary rate in the alligator is faster than that in birds as well as that in cold-blooded vertebrates. This contradicts hypotheses of constant body temperatures or high metabolic rate being correlated with elevated molecular evolutionary rates. It is commonly acknowledged that birds are the closest living relatives to crocodiles. Birds and crocodiles represent the only archosaurian survivors of the mass extinction at the Cretaceous/Tertiary boundary. On the basis of mitochondrial protein- coding genes, the Haemothermia hypothesis, which defines birds and mammals as sister groups and thus challenges the traditional view, could be rejected. Maximum-likelihood branch length data of amino acid sequences suggest that the divergence between the avian and crocodilian lineages took place at approximately equal to 254 MYA.      

Avian embryonic thermoregulation: role of Q10 in interpretation of endothermic reactions

1. Incubated chicken eggs (D14 to D21) were placed separately in transparent acrylic glass chambers which were immersed in a 37.5°C water bath for 3 h. The chambers were then moved for 3 h to another water bath controlled at 34.5°C. Oxygen consumption and temperature of the allantoic fluid (T_af) were measured at 5-min intervals for the whole experiment using an oxygen analyzer and CrNi–Ni-thermocouples, respectively. Heat production (HP) was calculated, using an assumed RQ of 0.72.

2. At 37.5°C the relationship between HP and embryonic age follows a sigmoid curve. Between D18 (HP 7.25 J g⁻¹ h; 2.01 W kg⁻¹) and D19 (HP 7.21 J g⁻¹ h⁻¹; 2.00 W kg⁻¹) this function had a plateau phase with a duration of about 24 h.

3. During the cooling process, HP decreased continuously and the relationship between T_a and HP could be described by an exponential function. From the results, it was possible to calculate the relationships between T_af, as a measure of body core temperature, and Q₁₀; the lower the T_af the higher the Q₁₀.

4. Because the actual measured HP is the result of the negative effect of Q₁₀ on HP and the stimulating influence of the CNS-generated HP, a Q₁₀ of more than 2.0 demonstrates the absence of endothermy. Chicken embryos aged between 14 and 21 d have a Q₁₀ of less than 2.0 at body temperatures (T_af) between 34 and 30°C. It is postulated that in chicken embryos of this age endothermic reactions may occur.

5. The Q₁₀-method is suitable for investigating the prenatal development of endothermic reactions in avian embryos.     

Mikrobiologische Bodenuntersuchungen im Lunzer Gebiet

Ohne ZusammenfassungAusgeführt mit Unterstützung der Notgemeinschaft der Deutschen Wissenschaft.     

Tubulin glycylases are required for primary cilia,control of cell proliferation and tumor development in colon

TTLL3 and TTLL8 are tubulin glycine ligases catalyzing posttranslational glycylation of microtubules. We show here for the first time that these enzymes are required for robust formation of primary cilia. We further discover the existence of primary cilia in colon and demonstrate that TTLL3 is the only glycylase in this organ. As a consequence, colon epithelium shows a reduced number of primary cilia accompanied by an increased rate of cell division in TTLL3-knockout mice. Strikingly, higher proliferation is compensated by faster tissue turnover in normal colon. In a mouse model for tumorigenesis, lack of TTLL3 strongly promotes tumor development. We further demonstrate that decreased levels of TTLL3 expression are linked to the development of human colorectal carcinomas. Thus, we have uncovered a novel role for tubulin glycylation in primary cilia maintenance, which controls cell proliferation of colon epithelial cells and plays an essential role in colon cancer development.     

Complete Genome Sequence and Pathogenicity of Two Swine Parainfluenzavirus 3 Isolates from Pigs in the United States

Two novel paramyxoviruses, 81-19252 (Texas81) and 92-7783 (ISU92), isolated from the brains of pigs in the United States in the 1980s and 1990s, were characterized. The complete genome of Texas81 virus was 15,456 nucleotides (nt) in length, that of ISU92 was 15,480 nt, and both genomes consisted of six nonoverlapping genes, predicted to encode nine proteins, with conserved and complementary 3′ leader and 5′ trailer regions and conserved gene starts, gene stops, and trinucleotide intergenic sequences similar to those in paramyxoviruses. The corresponding genes from these two viruses were similar in length, except for the F genes, of which the ISU92 form had an additional 24-nt U-rich 3′ untranslated region. The P genes of swine viruses were predicted to produce V and D mRNAs by RNA editing (one to four G insertions in Texas81 and one to nine G insertions in ISU92) or C mRNA by alternative translation initiation. Sequence-specific features related to virus replication and host-specific amino acid signatures indicated that these viruses originated from bovine parainfluenzavirus 3 (bPIV3). Phylogenetic analysis of individual genes suggested that these viruses are novel members of the genus Respirovirus of the Paramyxovirinae subfamily and may be grouped into two subgenotypes of genotype A of bPIV3. Our comprehensive studies revealed that these swine PIV3 are variants of bPIV3 and were possibly transferred from cattle to pigs but failed to establish an active enzootic state. These two viruses were mildly pathogenic to conventionally reared pigs, and results from a limited enzyme-linked immunosorbent assay-based serosurvey of swine farms in Minnesota and Iowa in 2007 and 2008 were negative.Outbreaks of infections with many novel paramyxoviruses causing catastrophic illnesses have been reported all over the world in the last few decades. A large number of diverse host species have been involved, including avian, porcine, canine, bovine, equine, ovine, human, reptilian, and aquatic species (22, 29, 40, 50, 51). Cases of cross-species transmission and pathogen jumping to humans were also reported (10, 20), demonstrating the value of characterizing new animal pathogens, even if their pathogenic potential is currently unknown. Prior to the 1990s, only La Piedad Michoacán paramyxovirus had been well studied as a neurotropic paramyxovirus isolated from pigs. Many paramyxovirus porcine pathogens have been reported since the 1950s in numerous countries, including Japan (55), Canada (18), and Israel (32), as well as the United States (25, 32). There was also a case of concurrent infection with a porcine reproductive and respiratory syndrome virus and a paramyxovirus which was subsequently named SER virus (70) in Germany in the 1990s (28). Four bat-associated paramyxoviruses were reported to cause disease in animals and humans in 1994 (72). Hendra virus and Nipah virus, which caused severe respiratory disease and death in horses and their trainer and severe febrile encephalitis and death in pigs and farmers, respectively, have been classified as members of the genus Henipavirus in the subfamily Paramyxovirinae (7, 9, 20, 30, 48). Some recently isolated viruses, such as Menangle virus (55), Tupaia paramyxovirus (69), Tioman virus (11), Mossman virus (47), J-virus (31, 33), Beilong virus (42), Mapuera virus (34), Tursiops truncatus parainfluenzavirus 1 (PIV1), isolated from bottlenose dolphins (50), and Atlantic salmon paramyxovirus (51), remain unclassified below the subfamily level. All members of the subfamily Paramyxovirinae have six genes in the following order: 3′-N-P-M-F-A-L-5′, where N, P, M, F, A, and L indicate the genes for the nucleocapsid protein, the phosphoprotein, and the matrix, fusion, attachment, and large polymerase proteins, respectively (40).Recently, we reported the antigenic and molecular characterization of glycoprotein genes from two novel swine PIV3 (sPIV3) isolates from the brains of pigs that experienced respiratory and central nervous system disease (57). These two sPIV3 strains were antigenically and genetically very closely related to bovine PIV3 (bPIV3) in the genus Respirovirus (57). However, the pathogenicity of these sPIV3 strains in conventionally reared pigs and the complete genome sequences of these isolates are presently unknown.In bovines, bPIV3 infection results in asymptomatic to severe respiratory disease, but no neurological disease has been reported (16). Limited sequence polymorphism among the bPIV3 strains was detected previously (12, 66). Recently, after an analysis of Australian isolates of bPIV3, two distinct genotypes of bPIV3, A and B, were proposed (29). In this study, we have performed a complete genome sequence analysis of these swine isolates and determined their pathogenicity in conventionally reared pigs. Our analysis indicated that there are two distinct genetic groupings discernible within genotype A, represented by bPIV3 shipping fever strain (bPIV3-SF)-like and bPIV3 strain 910N (bPIV3-910N)-like viruses, with one swine isolate in each of these groups. Several amino acid residues that may reflect the minor population variations in the new host due to cross-species infection were identified. But both swine viruses induced a very mild respiratory illness without any neurological signs in young piglets, suggesting that coinfection with other infectious agents or the presence of other environmental factors may be required to precipitate clinical disease.     

Cooperative adsorption of ezrin on PIP2-containing membranes

By means of the quartz crystal microbalance (QCM) and scanning force microscopy (SFM), the adsorption of ezrin, a member of the ezrin/radixin/moesin protein family, on l-alpha-phosphatidylinositol-4,5-bisphosphate (PIP(2)) containing solid-supported membranes was investigated. An increase in the PIP(2) content in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes resulted in an increased amount of bound ezrin strongly supporting the crucial role of PIP(2) for ezrin recruitment to membranes. No ezrin adsorption to membranes composed of pure POPC was detected. To characterize the binding process in more detail, the kinetics and reversibility of ezrin adsorption were investigated by the QCM technique, showing that the protein remains partly bound after rinsing with pure buffer, which we suspected to be a result of lateral interactions between the proteins. SFM images revealed the formation of two-dimensional ezrin clusters on PIP(2)-doped POPC membranes. Time-elapsed SFM images show that the growth of protein domains occurs from a few nucleation sites. The QCM data in conjunction with the results obtained by SFM led us to propose that the binding process of ezrin occurs in a positive cooperative manner. When lateral interactions of the proteins on the membrane were taken into account, we were able to simulate the kinetics obtained from time-resolved QCM readouts by employing a model developed by Minton. On the basis of the kinetic analysis, we were also able to reconstruct the adsorption isotherm.     

Bacterial extracellular DNA forming a defined network-like structure

It is generally assumed that nucleic acids are localized inside of living cells and that their primary function is the storage of information. In contrast, extracellular DNA is mainly considered as a remnant of lysed cells. Here, we report the formation of extracellular bacterial DNA as a spatial structure. An aquatic bacterium, strain F8, was isolated, which produced a stable filamentous network of extracellular DNA. Different staining and enzymatic techniques confirmed that it was DNA. We were able to amplify the 16S rRNA gene from the extracellular DNA. Restriction endonuclease cleavage and randomly amplified polymorphic DNA analysis of extracellular and genomic DNAs revealed major similarities, but also some differences in both sequences. Our data demonstrate a new function and relevance for extracellular DNA.