Search for a relationship between molecular anomalies of the mutant erythrocyte pyruvate kinase variants and their pathological expression

Summary Anomalies of the mutant pyruvate kinase variants and clinical symptoms have been compared in 22 unrelated patients with congenital red cell pyruvate kinase deficiency. This study suggests that some characteristics of the mutant enzymes could play a role in the intensity of haemolysis, namely residual activity, affinity for the substrate phosphoenolpyruvate and for the allosteric activator fructose 1,6 diphosphate and inhibition by ATP.     

Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells.

Treatment of Ltk^?, mouse L cells deficient in thymidine kinase (tk), with Bam I restriction endonuclease cleaved DNA from herpes simplex virus-1 (HSV-1) produced tk⁺ clones with a frequency of 10^?6/2 μg of HSV-1 DNA. Untreated cells or cells treated with Eco RI restriction endonuclease fragments produced no tk+ clones under the same conditions. The thymidine kinase activities of four independently derived clones were characterized by biochemical and serological techniques. By these criteria, the tk activities were found to be identical to HSV-1 tk and different from host wildtype tk. The tk⁺ phenotype was stable over several hundred cell generations, although the rate of reversion to the tk^? phenotype, as judged by cloning efficiency in the presence of bromodeoxyuridine, was high (1–5 × 10^?3). HSV-1 DNA Bam restriction fragments were separated by gel electrophoresis, and virtually all activity, as assayed by transfection, was found to reside in a 3.4 kb fragment. Transformation efficiency with the isolated fragment is 20 fold higher per gene equivalent than with the unfractionated total Bam digest. These results prove the usefulness of transfection assays as a means for the bioassay and isolation of restriction fragments carrying specific genetic information. Cells expressing HSV-1 tk may also provide a useful model system for the detailed analysis of eucaryotic and viral gene regulation.     

Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor.

Previous studies from our laboratories have demonstrated the feasibility of transferring the thymidine kinase (tk) gene from restriction endonuclease-generated fragments of herpes simplex virus (HSV) DNA to cultured mammalian cells. In this study, high molecular weight DNA from cells containing only one copy of the HSV gene coding for tk was successfully used to transform L+K-cells to the tk+ phenotype. The acquired phenotype was demonstrated to be donor-derived by analysis of the electrophoretic mobility of the tk activity, and the presence of HSV DNA sequences in the recipient cells was demonstrated. In companion experiments, we used high molecular weight DNA derived from tissues and cultured cells of a variety of species to transfer tk activity. The tk+ mouse cells transformed with human DNA were shown to express human type tk activity as determined by isoelectric focusing.     

Cellulose acetate electrophoresis of protein kinases: Detection of the active forms using various substrates

Electrophoresis on cellulose acetate strips was used to analyze protein kinases from normal rat liver. In addition to already well-characterized cAMP-dependent protein kinases type I and II and cAMP-independent casein kinases I and II, this method enabled the detection of several supplementary bands corresponding to kinases which were investigated according to their substrate specificity, activation by cAMP, and inhibition by the specific inhibitor of the catalytic subunit of cAMP-dependent protein kinases or by heparin. Using this rapid, sensitive, and resolutive electrophoretic method, different isozyme patterns could be obtained starting from minute amounts of different types of biological material.     

Chemical and Physical Characterization of Interfacial-Active Lipids from Rhodococcus erythropolis Grown on n-Alkanes

Lipophilic compounds of the culture suspension containing Rhodococcus erythropolis DSM43215 had surfactant properties when the bacteria were cultivated with n-alkanes as the sole carbon source. Thirteen main components from a dichloromethane-methanol extract of the R. erythropolis cultures were isolated and characterized to specify quantitatively their surfactant properties, e.g., minimum surface and interfacial tensions and critical micelle concentrations. The interfacial activity of the organic extract was dominated by α,α-trehalose-6,6′-dicorynomycolates which reduced interfacial tension from 44 to 18 mN/m. Phosphatidylethanolamines which were also present in the organic extract reduced the interfacial tension below 1 mN/m. The trehalose corynomycolates had extremely low critical micelle concentrations in high-salinity solutions, and the interfacial properties were stabile in solutions with a wide range of pH and ionic strength.     

Characterization of messenger RNA for aldolase B in adult and fetal human liver

High molecular weight cellular RNA was isolated from adult and fetal human liver tissue by a procedure of ethanol precipitation in concentrated guanidine-HCl solutions. About 5 mg of RNA were obtained from one gram of liver. RNA was fractionated by sucrose gradient ultracentrifugation. Aldolase B neosynthesized in a reticulocyte lysate cell-free system under the direction of total or fractionated RNA was purified by immunoaffinity microchromatography. Messenger RNA specifying synthesis of aldolase B exhibited a sedimentation coefficient of 16 S both in adult and fetal liver. This enzyme represented 1.3 % of the total neosynthesized proteins in adult liver, 0.1 % in the liver of a 6-month-old fetus and less than 0.01 % in the liver of a 4.5 month-old fetus.     

Molecules,fossils, and the origin of tetrapods

Summary Since the discovery of the coelacanth, Latimeria chalumnae, more than 50 years ago, paleontologists and comparative morphologists have debated whether coelacanths or lungfishes, two groups of lobe-finned fishes, are the closest living relatives of land vertebrates (Tetrapoda). Previously, Meyer and Wilson (1990) determined partial DNA sequences from two conservative mitochondrial genes and found support for a close relationship of lungfishes to tetrapods. We present additional DNA sequences from the 12S rRNA mitochondria gene for three species of the two lineages of lungfishes that were not represented in the first study: Protopterus annectens and Protopterus aethiopicus from Africa and Neoceratodus forsteri (kindly provided by B. Hedges and L. Maxson) from Australia. This extended data set tends to group the two lepidosirenid lungfish lineages (Lepidosiren and Protopterus) with Neoceratodus as their sister group. All lungfishes seem to be more closely related to tetrapods than the coelacanth is. This result appears to rule out the possibility that the coelacanth lineage gave rise to land vertebrates. The common ancestor of lungfishes and tetrapods might have possessed multiple morphological traits that are shared by lungfishes and tetrapods [Meyer and Wilson (1990) listed 14 such traits]. Those traits that seem to link Latimeria and tetrapods are arguably due to convergent evolution or reversals and not to common descent. In this way, the molecular tree facilitates an evolutionary interpretation of the morphological differences among the living forms. We recommended that the extinct groups of lobe-finned fishes be placed onto the molecular tree that has lungfishes and not the coelacanth more closely related to tetrapods. The placement of fossils would help to further interpret the sequence of morphological events and innovations associated with the origin of tetrapods but appears to be problematic because the quality of fossils is not always high enough, and differences among paleontologists in the interpretation of the fossils have stood in the way of a consensus opinion for the branching order among lobefinned fishes. Marshall and Schultze (1992) criticized the morphological analysis presented by Meyer and Wilson (1990) and suggest that 13 of the 14 morphological traits that support the sister group relationship of lungfishes and tetrapods are not shared derived characters. Here we present further alternative viewpoints to the ones of Marshall and Schultze (1992) from the paleontological literature. We argue that all available information (paleontological, neontological, and molecular data) and rigorous cladistic methodology should be used when relating fossils and extant taxa in a phylogenetic framework. Offprint requests to: Axel Meyer     

The switching gene swi6 affects recombination and gene expression in the mating-type region of Schizosaccharomyces pombe

Summary The products of 11 switching (swi) genes are required for efficient mating-type (MT) switching in homothallic (h ⁹⁰) strains of Schizosaccharomyces pombe. The MT region of h ⁹⁰ comprises three cassette genes: the expression site mat1: 1 and two silent loci, mat2: 2 and mat3: 3. Besides reducing MT switching, the swi6 mutation leads to deletions in the MT region caused by intrachromosomal cross-overs between two paired cassettes. These deletions only arise if DNA double-strand breaks are present at mat1: 1, which initiate MT switching. Furthermore, swi6 allows meiotic recombination in the K region, a region of 16 kb between mat2: 2 and mat3: 3; in wild-type strains no recombination occurs in K. swi6 also allows the simultaneous expression of two different cassettes in the same haploid cell. Thus swi6 may have an influence on the general chromatin structure in the MT region.