Rat liver beta-glucuronidase. cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells.

A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3' untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells.     

Ancestral polymorphism and linkage disequilibrium at the het-6 region in pseudohomothallic Neurospora tetrasperma

In species of Neurospora, non-self recognition is mediated by at least 11 heterokaryon (het) incompatibility loci. Previously, we identified ancient allelic variation at het-c in pseudohomothallic N. tetrasperma, which confirmed outcrossing in this species. Here, we report distinct ancestral alleles at het-6 and un-24, two closely linked genes with het incompatibility function in N. crassa. The pattern of variation at het-6 and un-24 in N. tetrasperma is similar to that observed for N. crassa, where two ancestral allele specificities exist for each locus, Oak Ridge (het-6(OR), un-24(OR)) and Panama (het-6(PA), un-24(PA)). Only het-6(OR)/un-24(OR) and het-6(PA)/un-24(PA) allele combinations have been observed. The absence of recombinant haplotypes (e.g., het-6(OR)/un-24(PA)) appears to derive from an ancestral chromosomal rearrangement that limits recombination. Allelic variation at het-6 and un-24 in N. tetrasperma provides further evidence of outcrossing in this predominantly selfing species and indicates that selection maintains ancient allelic diversity at het loci.     

Culturing the epiblast cells of the pig blastocyst

Summary Pig epiblast cells that had been separated from other early embryonic cells were cultured in vitro. A three-step dissection protocol was used to isolate the epiblast from trophectoderm and primitive endoderm before culturing. Blastocysts collected at 7 to 8 days postestrus were immunodissected to obtain the inner cell mass (ICM) and destroy trophectodermal cells. The ICM was cultured for 2 to 3 days on STO feeder cells. The epiblast was then physically dissected free of associated primitive endoderm. Epiblast-derived cells, grown on STO feeders, produced colonies of small cells resembling mouse embryonic stem cells. This primary cell morphology changed as the colonies grew and evolved into three distinct colony types (endodermlike, neural rosette, or complex). Cell cultures derived from these three colony types spontaneously differentiated into numerous specialized cell types in STO co-culture. These included fibroblasts, endodermlike cells, neuronlike cells, pigmented cells, adipogenic cells, contracting muscle cells, dome-forming epithelium, ciliated epithelium, tubule-forming epithelium, and a round amoeboid cell type resembling a plasmacyte after Wright staining. The neuronlike cells, contracting muscle cells, and tubule-forming epithelium had normal karyotypes and displayed finite or undefined life spans upon long-term STO co-culture. The dome-forming epithelium had an indefinite life span in STO co-culture and also retained a normal karyotype. These results demonstrate the in vitro pluripotency of pig epiblast cells and indicate the epiblast can be a source for deriving various specialized cell cultures or cell lines.     

Quantitation of picomole levels of N-acetyl- and N-glycolylneuraminic acids by a HPLC-adaptation of the thiobarbituric acid assay

A simple HPLC adaptation of the periodate-TBA assay for free N-acetyl- and N-glycolylneuraminic acids greatly extends the sensitivity and increases the specificity of this standard colorimetric assay. The method, employing a C18 reverse-phase column eluted isocratically with a phosphoric acid-MeOH buffer, is linear over a range of 2 pmol to 20 nmol. Analyses can be performed directly on cell lysates and digests without prior purification of released sialic acids from contaminating salts and biological materials. Interference from 2-deoxysugars is completely eliminated as the chromophore from these compounds is completely resolved from that derived from sialic acids. The application of the technique to quantify cell-surface and total cellular TBA-reactive sialic acids on the surfaces of a variety of tumor cells is described. Additionally, the extent of desialylation of erythrocytes necessary to expose the T antigen is determined.     

Transmission of a bacterial consortium in Eisenia fetida egg capsules

The earthworm Eisenia fetida harbours Verminephrobacter eiseniae within their excretory nephridia. This symbiont is transferred from the parent into the egg capsules where the cells are acquired by the developing earthworm in a series of recruitment steps. Previous studies defined V. eiseniae as the most abundant cell type in the egg capsules, leaving approximately 30% of the bacteria unidentified and of unknown origin. The study presented here used terminal restriction fragment length polymorphism analysis together with cloning and sequencing of 16S rRNA genes to define the composition of the bacterial consortium in E. fetida egg capsules from early to late development. Newly formed capsules of E. fetida contained three bacterial types, a novel Microbacteriaceae member, a Flexibacteriaceae member and the previously described V. eiseniae. Fluorescent in situ hybridization (FISH) using specific and general rRNA probes demonstrated that the bacteria are abundant during early development, colonize the embryo and appear in the adult nephridia. As the capsules mature, Herbaspirillum spp. become abundant although they were not detected within the adult worm. These divergent taxa could serve distinct functions in both the adult earthworm and in the egg capsule to influence the competitive ability of earthworms within the soil community.     

DNA Fingerprinting and Analysis of Population Structure in the Chestnut Blight Fungus, Cryphonectria Parasitica

We analyzed DNA fingerprints in the chestnut blight fungus, Cryphonectria parasitica, for stability, inheritance, linkage and variability in a natural population. DNA fingerprints resulting from hybridization with a dispersed moderately repetitive DNA sequence of C. parasitica in plasmid pMS5.1 hybridized to 6-17 restriction fragments per individual isolate. In a laboratory cross and from progeny from a single perithecium collected from a field population, the presence/absence of 11 fragments in the laboratory cross and 12 fragments in the field progeny set segregated in 1:1 ratios. Two fragments in each progeny set cosegregated; no other linkage was detected among the segregating fragments. Mutations, identified by missing bands, were detected for only one fragment in which 4 of 43 progeny lacked a band present in both parents; no novel fragments were detected in any progeny. All other fragments appeared to be stably inherited. Hybridization patterns did not change during vegetative growth or sporulation. However, fingerprint patterns of single conidial isolates of strains EP155 and EP67 were found to be heterogenous due to mutations that occurred during culturing in the laboratory since these strains were first isolated in 1976-1977. In a population sample of 39 C. parasitica isolates, we found 33 different fingerprint patterns with pMS5.1. Most isolates differed from all other isolates by the presence or absence of several fragments. Six fingerprint patterns each occurred twice. Isolates with identical fingerprints occurred in cankers on the same chestnut stems three times; isolates within the other three pairs were isolated from cankers more than 5 m apart. The null hypothesis of random mating in this population could not be rejected if the six putative clones were removed from the analysis. Thus, a rough estimate of the clonal fraction of this population is 6 in 39 isolates (15.4%).     

Interaction of acyl coenzyme A substrates and analogues with pig kidney medium-chain acyl-coA dehydrogenase

Several alkylthio coenzyme A (CoA) derivatives (from ethyl- to hexadecyl-SCoA) have been synthesized to probe the substrate binding site in the flavoprotein medium-chain acyl-CoA dehydrogenase from pig kidney. All bind to apparently equivalent sites with a stoichiometry of four per tetramer. A plot of log Kd vs: hydrocarbon chain length is linear from 2 to 16 carbons with a free energy of binding of 390 cal/methylene group. These data suggest an acyl-binding site of moderate hydrophobicity and imply that the observed substrate specificity of the medium-chain dehydrogenase is not achieved simply by the length of the hydrocarbon binding pocket. Extrapolation of the graph to zero chain length predicts a Kd of 1 mM for the CoA moiety. The difference between this value and the experimentally determined value of 206 microM may be attributed to a contribution from the ionization of the sulfhydryl group in CoASH. The interaction of several eight-carbon intermediates of beta-oxidation (trans-2- and trans-3-octenoyl-CoA and L-3-hydroxy- and 3-ketooctanoyl-CoA) with the dehydrogenase has also been studied. All but the L-3-OH derivative bind tightly to the enzyme (with Kd values in the 50-90 nM range) and are very effective inhibitors of the dehydrogenation of octanoyl-CoA. The trans-3-enoyl analogue produces an immediate, intense, long-wavelength band (lambda max = 820 nm), which probably represents a charge-transfer interaction between the delocalized alpha-carbanion donor and oxidized flavin as the acceptor. The L-3-OH analogue is a reductant of the flavin, yielding 3-ketooctanoyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Health evaluation of western arctic King Eiders (Somateria spectabilis)

The western arctic population of King Eiders (Somateria spectabilis) has declined by >50% in recent years. A health assessment was conducted for adult King Eiders breeding on the north slope of Alaska, USA, to evaluate body condition (n=90, 2002-2006) and baseline biochemical and hematologic values (n=20-30, 2005-2006). Body condition for males and females was excellent. Total protein, calcium, alkaline phosphatase, amylase, and globulin were significantly higher in females than in males, likely because of differences in reproductive physiology. These baseline health data can be used to promote conservation of King Eiders and other closely related species of concern.     

Fatty acid esterification in the yolk sac membrane of the avian embryo

The transfer of lipid from the yolk to the avian embryo is mediated by the yolk sac membrane (YSM). Some, but not all, of the published morphological evidence supports the view that the lipid undergoes a cycle of hydrolysis and re-esterification during translocation across the YSM. The present study aims to test this view by investigating the capacity of the YSM to perform esterification of free fatty acids to form acyl-lipids. YSM pieces (area vasculosa), obtained from the chicken embryo at day 10 of development, were incubated in vitro in medium containing [¹⁴C]-palmitic acid. Radioactivity was rapidly incorporated into the tissue lipid indicating a high capacity for esterification. The incorporation was linear with time during the 1-h incubation. Approximately 84% of the incorporated label was recovered in triacylglycerol, 12% was incorporated into phospholipid and less than 1% was detected in cholesteryl ester. [¹⁴C]-palmitic acid was incorporated primarily at the sn-1/3 positions in the triacylglycerol molecule and at the sn-1 position of phospholipid. The incorporation of label into tissue pieces obtained from the non-vascularized peripheral region of the YSM (area vitellina) was much more limited than that observed for the area vasculosa. The results support the hypothesis that yolk lipid is hydrolyzed and re-esterified during transfer across the YSM.Abbreviations YSM yolk sac membrane - VLDL very-low density lipoprotein Communicated by G. Heldmaier