Sequence variation at the major histocompatibility complex locus DQ beta in beluga whales (Delphinapterus leucas) [published erratum appears in Mol Biol Evol 1995 Nov;12(6):1174]

Genetic variation at the Major Histocompatibility Complex locus DQ beta was analyzed in 233 beluga whales (Delphinapterus leucas) from seven populations: St. Lawrence Estuary, eastern Beaufort Sea, eastern Chukchi Sea, western Hudson Bay, eastern Hudson Bay, southeastern Baffin Island, and High Arctic and in 12 narwhals (Monodon monoceros) sympatric with the High Arctic beluga population. Variation was assessed by amplification of the exon coding for the peptide binding region via the polymerase chain reaction, followed by either cloning and DNA sequencing or single-stranded conformation polymorphism analysis. Five alleles were found across the beluga populations and one in the narwhal. Pairwise comparisons of these alleles showed a 5:1 ratio of nonsynonymous to synonymous substitutions per site leading to eight amino acid differences, five of which were nonconservative substitutions, centered around positions previously shown to be important for peptide binding. Although the amount of allelic variation is low when compared with terrestrial mammals, the nature of the substitutions in the peptide binding sites indicates an important role for the DQ beta locus in the cellular immune response of beluga whales. Comparisons of allele frequencies among populations show the High Arctic population to be different (P < or = .005) from the other beluga populations surveyed. In these other populations an allele, Dele-DQ beta*0101-2, was found in 98% of the animals, while in the High Arctic it was found in only 52% of the animals. Two other alleles were found at high frequencies in the High Arctic population, one being very similar to the single allele found in narwhal.      

Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects

We report the complete sequence of a paralogous copy of elongation factor-1 alpha (EF-1 alpha) in the honeybee, Apis mellifera (Hymenoptera: Apidae). This copy differs from a previously described copy in the positions of five introns and in 25% of the nucleotide sites in the coding regions. The existence of two paralogous copies of EF-1 alpha in Drosophila and Apis suggests that two copies of EF-1 alpha may be widespread in the holometabolous insect orders. To distinguish between a single, ancient gene duplication and parallel, independent fly and bee gene duplications, we performed a phylogenetic analysis of hexapod EF-1 alpha sequences. Unweighted parsimony analysis of nucleotide sequences suggests an ancient gene duplication event, whereas weighted parsimony analysis of nucleotides and unweighted parsimony analysis of amino acids suggests the contrary: that EF-1 alpha underwent parallel gene duplications in the Diptera and the Hymenoptera. The hypothesis of parallel gene duplication is supported both by congruence among nucleotide and amino acid data sets and by topology-dependent permutation tail probability (T-PTP) tests. The resulting tree topologies are also congruent with current views on the relationships among the holometabolous orders included in this study (Diptera, Hymenoptera, and Lepidoptera). More sequences, from diverse orders of holometabolous insects, will be needed to more accurately assess the historical patterns of gene duplication in EF-1 alpha.      

Identification of genes involved in siderophore transport in Streptomyces coelicolor A3(2)

The potential iron siderophore transporter genes have been determined from the genome sequence of Streptomyces coelicolor A3(2). One of these gene clusters, cdtABC, was disrupted and characterized to determine its role in the uptake of the siderophores produced by S. coelicolor. Resistance to the siderophore-like antibiotics, salmycin and albomycin, was tested in the parent and cdtABC mutant, showing that the parent, but not the mutant, was sensitive to salmycin, while both were resistant to albomycin. Ferrioxamine competition assays against salmycin suggest that the uptake of salmycin is via a ferrioxamine transport system. However, Fe-55 ferrioxamine B uptake experiments did not reveal any difference between the parent and mutant. This suggests that CdtABC specifically transports salmycin, while ferrioxamine uptake maybe substituted by another transport system.     

One pot synthesis of GDP‐mannose by a multi‐enzyme cascade for enzymatic assembly of lipid‐linked oligosaccharides

Glycosylation of proteins is a key function of the biosynthetic‐secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell‐cell adhesion, blood‐group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein‐based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose‐1‐phosphate‐guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1‐domain polyphosphate kinase 2 (1D‐Ppk2) expressed in E. coli for the cell‐free production and regeneration of GDP‐mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP‐mannose is produced at various conditions, that is pH 7–8, temperature 25–35°C and co‐factor concentrations of 5–20 mM MgCl₂. The maximum reaction rate of GDP‐mannose achieved was 2.7 μM/min at 30°C and 10 mM MgCl₂ producing 566 nmol GDP‐mannose after a reaction time of 240 min. With respect to the initial GDP concentration (0.8 mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane‐deleted Alg1 (ALG1ΔTM), the first mannosyltransferase in the ER‐associated lipid‐linked oligosaccharide (LLO) assembly. Thereby, in a one‐pot reaction, phytanyl‐PP‐(GlcNAc)₂‐Man₁ was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl‐PP‐(GlcNAc)₂‐Man₁ can serve as a substrate for the synthesis of LLO for the cell‐free in vitro glycosylation of proteins. A high‐performance anion exchange chromatography method with UV and conductivity detection (HPAEC‐UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established kinetic model enabled the optimization of the GDP‐mannose regenerating cascade and can further be used to study coupling of the GDP‐mannose cascade with glycosyltransferases. Overall, the study envisages a first step towards the development of a platform for the cell‐free production of LLOs as precursors for in vitro glycoengineering of proteins.     

Mortality in over 350,000 Insured Swedish Dogs from 1995–2000: II. Breed-Specific Age and Survival Patterns and Relative Risk for Causes of Death

This study continues analysis from a companion paper on over 350,000 insured Swedish dogs up to 10 years of age contributing to more than one million dog-years at risk during 1995–2000. The age patterns for total and diagnostic mortality and for general causes of death (trauma, tumour, locomotor, heart and neurological) are presented for numerous breeds. Survival estimates at five, eight and 10 years of age are calculated. Survival to 10 years of age was 75% or more in Labrador and golden retrievers, miniature and toy poodles and miniature dachshunds and lowest in Irish wolfhounds (91% dead by 10 years). Multivariable analysis was used to estimate the relative risk for general and more specific causes of death between breeds accounting for gender and age effects, including two-way interactions. Older females had tumour as a designated cause of death more often than males in most breeds, but not in the Bernese mountain dog. Information presented in this and the companion paper inform our understanding of the population level burden of disease, and support decision-making at the population and individual level about health promotion efforts and treatment and prognosis of disease events.     

AsnI: a novel class II restriction endonuclease from Arthrobacter sp., strain N-CM, recognizing 5'-AT/TAAT-3'

A new class II restriction endonuclease, AsnI, with a novel sequence specificity was isolated from the Gram-positive eubacterium Arthrobacter species, strain N-CM. AsnI recognizes the unambiguously defined palindromic hexanucleotide (Formula: see text) consisting of A- and T-residues. The novel enzyme in the presence of Mg2+ cleaves specifically both strands as indicated by the arrows. The staggered cuts generate 5'-protruding ends with single-stranded 5'-TA-3' dinucleotide extensions. The novel enzyme may be a useful tool for cloning experiments by complementation of the few enzymes such as PstI and PvuI cutting only once in the Ampr-gene of plasmids pBR322 and pBR328.     

Live imaging of <Emphasis Type="Italic">Drosophila</Emphasis>gonad formation reveals roles for Six4 in regulating germline and somatic cell migration

Background  

Movement of cells, either as amoeboid individuals or in organised groups, is a key feature of organ formation. Both modes of migration occur during Drosophila embryonic gonad development, which therefore provides a paradigm for understanding the contribution of these processes to organ morphogenesis. Gonads of Drosophila are formed from three distinct cell types: primordial germ cells (PGCs), somatic gonadal precursors (SGPs), and in males, male-specific somatic gonadal precursors (msSGPs). These originate in distinct locations and migrate to associate in two intermingled clusters which then compact to form the spherical primitive gonads. PGC movements are well studied, but much less is known of the migratory events and other interactions undergone by their somatic partners. These appear to move in organised groups like, for example, lateral line cells in zebra fish or Drosophila ovarian border cells.