DNA-RNA hybridization.

Interest in nucleic acid hybridization stems mainly from its great power as a tool in biological research. It is used in several quite distinct ways. Because of the high degree of specificity that they show, hybridization techniques can be used to measure the amount of one specific sequence within a very heterogeneous mixture of sequences. Measurements of 1/10(6)-10(7) have been recorded. In extension of this, various properties of a specific sequence can often be studied. Secondly, because the kinetics of nucleic acid hybridization are quite well understood, it can be used to characterize both a pure sequence and a very complex mixture of sequences, like the genome of a vertebrate. Thirdly, again because of its specificity, it can be used to measure homologies between different populations of nucleic acids. Lastly, in conjunction with other techniques, it can be used as a basis for the fractionation of nucleic acid populations and the purification of specific sequences. Specific examples of these applications are given, with special reference to the organization of the genome in higher eukaryotes.     

Purification of the receptor for platelet-derived growth factor from porcine uterus

The receptor for platelet-derived growth factor has been purified to homogeneity on a large scale from porcine uterus. The purification procedure utilizes solubilization of uterus membranes by Triton X-100, followed by sequential chromatographies on wheat germ agglutinin-Sepharose, fast protein liquid chromatography Mono-Q, and anti-phosphotyrosine-Sepharose. About 160 micrograms of homogeneous and functionally active 170-kDa receptor could be purified from 5 kg of uterus tissue. The pure receptor responded to platelet-derived growth factor stimulation by autophosphorylation, indicating that the receptor has a kinase domain as an integral part of the molecule. A rabbit antiserum was produced against the pure receptor, which specifically recognizes the intact 170-kDa receptor.     

Trait-based analyses for the detection of linkage between marker loci and quantitative trait loci in crosses between inbred lines

Summary Methods are presented for determining linkage between a marker locus and a nearby locus affecting a quantitative trait (quantitative trait locus=QTL), based on changes in the marker allele frequencies in selection lines derived from the F-2 of a cross between inbred lines, or in the high and low phenotypic classes of an F-2 or BC population. The power of such trait-based (TB) analyses was evaluated and compared with that of methods for determining linkage based on the mean quantitative trait value of marker genotypes in F-2 or BC populations [marker-based (MB) analyses]. TB analyses can be utilized for marker-QTL linkage determination in situations where the MB analysis is not applicable, including analysis of polygenic resistance traits where only a part of the population survives exposure to the Stressor and analysis of marker-allele frequency changes in selection lines. TB analyses may be a useful alternative to MB analyses when interest is centered on a single quantitative trait only and costs of scoring for markers are high compared with costs of raising and obtaining quantitative trait information on F-2 or BC individuals. In this case, a TB analysis will enable equivalent power to be obtained with fewer individuals scored for the marker, but more individuals scored for the quantitative trait. MB analyses remain the method of choice when more than one quantitative trait is to be analyzed in a given population.Contribution from the ARO, Bet Dagan, Israel. No. 1698-E, 1986 series     

G-418, an elongation inhibitor of 80 S ribosomes

The mode of action of the aminoglycoside G-418 was studied in wheat-germ, cell-free translation systems programmed with rat-liver polyadenylated RNA. Incorporation of amino acids into protein was effectively inhibited by G-418 in the microM concentration range. The inhibition pattern obtained was not uniform. The synthesis of polypeptides with higher molecular weights was more inhibited than that of smaller polypeptides. An identical inhibition pattern within a similar range of concentrations was obtained with cycloheximide, a known elongation inhibitor. Translation activity was abolished when the wheat-germ 80 S ribosomes were removed and could be partially reconstructed upon addition of the ribosomes. Incubation with G-418 prior to isolation yielded ribosomes defective in their reconstruction ability. The inhibition pattern was not uniform and exhibited again the same relationship between the size of a polypeptide and the extent of inhibition of its synthesis. Therefore, we suggest that in wheat-germ, cell-free translation systems G-418 affects the 80 S ribosomes and inhibits the elongation cycle.     

Enrichment and Isolation of Naphthalenesulfonic Acid-Utilizing Pseudomonads

Naphthalenesulfonate-degrading bacteria were obtained by continuous enrichment from a naphthalene-degrading population from sewage. In addition to naphthalene, Pseudomonas sp. A3 can utilize 2-naphthalenesulfonate (2NS) and Pseudomonas sp. C22 can utilize both 1-naphthalenesulfonate (1NS) and 2NS as sole carbon sources. In a mixture of 1NS and 2NS, the former substrate is utilized by strain C22 only after complete consumption of 2NS. During exponential growth, approximately 10% of the organic carbon of naphthalenesulfonates is temporarily excreted. These unidentified metabolites can readily be used by other bacteria, which, by supplying strain C22 with vitamins, allow optimal growth in stable mixed cultures. The degradative capability of Pseudomonas sp. A3 for 2NS was irreversibly lost under nonselective growth conditions and could be transferred from the wild type to a distinguishable cured strain of the wild type.     

Referate

Ohne Zusammenfassung     

Allgemeines,Genetik, Cytologie,Physiologie

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Elevated amounts of methotrexate-binding protein,different from normal dihydrofolate reductase,in a petunia MTXR-cell line

Summary A petunia cell line, 1ECB, was previously isolated by the stepwise selection procedure, for resistance to methotrexate (MTX), an antimetabolite for the enzyme dihydrofolate reductase (DHFR). Using ammonium sulfate precipitates of cell lysates of cell line 1ECB and its parental cell line (WT), it was found that the mutant has an increase of 400 fold in ³H-MTX binding capacity and a decrease in the affinity for MTX binding, at two orders of magnitude, in comparison with the WT. In addition, the DHFR specific activity in the mutant increased only moderately (5- to 10-fold), this activity is extremely sensitive to MTX inhibition as compared to the WT. It is evident that the MTX resistance of line 1ECB results mainly from overproduction of an MTX-binding protein which differs from the WT DHFR by four biochemical criteria. This protein may serve as a trap for the excess amounts of MTX to which the cells are exposed.     

A soluble form of the interleukin 4 receptor in biological fluids

Murine biological fluids and murine cell culture supernatants were analyzed for the presence of soluble murine interleukin 4 receptor (sIL4R) with the use of two monoclonal antibodies directed against the receptor. Mouse urine, serum, ascitic fluid, and cell culture supernatants contained varying levels of immunoreactive protein. All of the immunoreactive protein possessed interleukin 4 (IL 4) binding activity. Following partial purification of ascitic fluid a protein was isolated that binds IL 4 with high affinity. This data is consistent with the fact that murine biological fluids contain a soluble version of the murine IL 4 receptor that arises via secretion of the soluble receptor and/or via shedding of the extracellular portion of the full-length receptor from the cell surface.