Rabbit heavy chain haplotypes — Allotypic determinants expressed byVH-CH recombinants

This report summarizes our current understanding of the heavy chain haplotypes found in our laboratories' rabbits. Independently derived data from several laboratories have been synthesized into a consistent picture of the linked inheritance of allotypic markers found on the different heavy chain classes and subclasses of rabbit immunoglobulins in pedigreed rabbits, including the families of three apparentVH-CH recombinants. In one recombinant, the entire group ofCH markers (C, C, and C) recombined with the set ofVH. Although in the other two recombinants all CH markers may also have recombined as a group, in one of these only IgG and IgACH genes were informative; in the other recombinant, only the IgG allotypes were informative. Some allotypic determinants found on IgM molecules (conformational) appear only when a specific variable region allotype (VHa) is combined with a specific constant region allotype (C). New combinations ofVHa and C allotypes were generated in two of the genetic recombinants and led to new conformational determinants. The gains and losses observed lend support to the hypothesis that the determinants result from conformations generated by the combination of allotype-specificVH and C protein sequences. Conceivably, DNA events that joinVH to diversity (D)- and joining (J)-coding sequences or mRNA processing events that splice J to C could be involved in generating the sequences that form allotype-specific determinants.     

Effects of butylated hydroxyanisole on the aryl hydrocarbon hydroxylase of rats and mice

The effects of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on the aryl hydrocarbon hydroxylase (AHH) activities in the liver, lung and skin of rats and mice have been studied to examine the possible mechanisms of the anticarcinogenic actions of these compounds. Both compounds inhibit the hydroxylase activities of hepatic microsomes and nuclei, with BHA a more potent inhibitor than BHT. The AHH of lung microsomes is inhibited to a lesser extent by BHA and BHT than that of the liver. The AHH activities of both liver and lung microsomes become less susceptible to the inhibition after pretreatment of the animals with 3-methylcholanthrene (MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but phenobarbital (PB) pretreatment does not produce such an effect. In skin homogenates, however, the AHH activities of control rats and mice are not inhibited by BHA and BHT. The only skin sample which is inhibited by BHA and BHT is that from TCDD-pretreated mice. It has been established that the extent of inhibition with different samples is related to the concentration of BHA in the incubation but not to the amounts or specific activities of microsomes used. Double reciprocal plots suggest that BHA exerts a mixed inhibition on the hydroxylase of liver microsomes with a K_i of 7.7 μM. Analysis of the metabolites of benzo[a]pyrene (BP) shows that BHA inhibits the formation of various metabolites uniformly without changing the regio-selectivity of the enzyme system. The mechanism of inhibition has also been studied with a reconstituted AHH system consisting of cytochrome P-450 (P-450), reductase and phospholipid. The system with P-450 isolated from PB-induced microsomes is inhibited to a much greater extent than that with MC-induced P-450. The results indicate that the inhibitory action of BHA is dependent on the species of the animal, tissue types and treatment with inducers.     

Specificity of Insertion by the Translocatable Tetracycline-Resistance Element Tn10

Genetic analysis of 131 independent transpositions of the tetracycline-resistance element Tn10 from a single site in phage P22 into the histidine operon of Salmonella typhimurium reveals that Tn10 insertions are not randomly distributed along this chromosomal target. The insertions occur in 22 different "clusters"; insertions within each cluster are very tightly linked in recombination tests. Tn10 insertions are not evenly distributed among the identified clusters. The existence of these clusters suggests that this chromosomal target contains particular genetic signals that guide Tn10 to particular preferred positions for insertion. Insertions within each cluster occur in both orientations with roughly equal frequency.--The relationship among different insertions within each cluster has been examined. The resolution of genetic mapping places an upper limit of about 50 basepairs on the distance between different insertions within a cluster. Different insertions within a cluster usually have the same reversion frequency; however, heterogeneity in reversion frequency has been detected in at least two clusters. For most clusters, the available data are consistent with the simple possibility that all insertions within a cluster are at identical positions; however, the data do not exclude other possibilities.     

Mutagenic effect on L5178Y mouse lymphoma cells by growth in ethylene oxide-sterilized polycarbonate flasks

Summary The mutation frequency of L5178Y mouse lymphoma cells to resistance to 5′-bromo-2′-deoxyuridine increased 6-to 14-fold after growth in ethylene oxide-sterilized polycarbonate culture flasks compared to growth in glass flasks. No comparable increase was observed when L5178Y cells were growth in identical polycarbonate culture flasks sterilized by autoclaving.     

Transdetermination of Drosophila imaginal discs cultured in vitro

Imaginal discs of Drosophila melanogaster undergo transdetermination when cultured in vivo in the abdominal cavity of adult female hosts. We report here that leg discs cultured in vitro, in a recently developed system, also undergo transdetermination. Whether cultured in vivo or in vitro, leg discs produce a similar range of specific transdetermined structures. Moreover, in comparison to discs cultured in vivo, the discs cultured in vitro exhibit a similar correlation between the amount of growth and the total frequency of transdetermination.     

Protoplast induction inMicrasterias andCosmarium

Summary Protoplasts of the desmidsMicrasterias angulosa, M. denticulata, M. thomasiana andCosmarium turpinii were obtained by incubating cells in Waris' liquid medium + 0.3 M mannitol + 2% Cellulysin for 1–3 hours. One osmotically fragile protoplast was formed at the isthmus from the joint contents of both semicells. The resultant protoplasts were bright green and remained so for more than 5 days in the osmotically protective medium. The protoplast yield was better than 80%. The empty cell walls were not digested by the Cellulysin or by autolytic enzymes.     

The zinc content of yeast alcohol dehydrogenase

Analyses for zinc in high specific activity preparations of yeast alcohol dehydrogenase (YADH) indicate a metal content of 1.8–1.9 moles of zinc per mole of enzyme subunit. This zinc content is observed for YADH prepared from Bakers yeast by recrystallization from Am₂SO₄ containing 1 mM EDTA, followed by chromatography on DE-52 and Sephadex-G-200. YADH obtained from Boehringer-Mannheim is characterized by a variable specific activity: preparations with Sp. Ac. = 380–400 U/mg contain 1.8–1.9 moles of zinc per mole of subunit. Dialysis of YADH against EDTA (pH 8.5, 25°, under N₂) reduces the specific activity and zinc content in an approximately linear fashion down to a Sp. Ac. = 150 U/mg, consistent with the preferential loss of a single, weakly bound zinc per subunit which is essential for catalytic activity. Dialysis of YADH against 1 mM ZnCl₂ (pH 6.5–8.5, 25°, under N₂) does not lead to an increase in the zinc content of the enzyme, indicating that under these conditions zinc does not bind adventitiously to YADH. Dialysis against 50 mM CoSO₄ (pH 5.5, 25°, under N2, 60–90 hr) leads to an exchange of ≈ 40% of the enzyme-bound zinc by cobalt. Our preparations of YADH are consistently characterized by a zinc content of ≈ 2 per subunit and we are unable to reduce the zinc content of YADH by dialysis against EDTA without a concomitant loss in enzyme activity, in contrast to reports of one zinc per subunit [Veillon, C. and Sytkowski, A.J., BBRC $\text{67}$: 1499 (1975); Vallee, B.L. and Hoch, F.L., Proc. Nat. Acad. Sci. USA $\text{41}$: 327 (1955)]. The findings reported here, together with the observed structural similarities between YADH and horse liver alcohol dehydrogenase [Jornvall, H., Woenckhaus, C. and Johnscher, G., Eur. J. Biochem. $\text{53}$: 71 (1975)], suggest a role for zinc at both a structural and catalytic site in YADH.     

Process‐based functions for seed retention on animals: a test of improved descriptions of dispersal using multiple data sets

Studies of external seed transport on animals usually assume that the probability of detachment is constant, so that seed retention should show a simple exponential relationship with time. This assumption has not been tested explicitly, and may lead to inaccurate representation of long distance seed dispersal by animals. We test the assumption by comparing the fit to empirical data of simple, two‐parameter functions. Fifty‐two data sets were obtained from five published studies, describing seed retention of 32 plant species on sheep, cattle, deer, goats and mice. Model selection suggested a simple exponential function was adequate for data sets in which seed retention was followed for short periods ( <48 h). The data gathered over longer periods (49–219 days) were best described by the power exponential function, a form of the stretched exponential which allows a changing dropping rate. In these cases the power exponential showed that seed dropping rate decreased with time, suggesting that seeds vary in attachment, with some seeds becoming deeply buried or wound up in the animal's coat. Comparison of fitted parameters across all the data sets also confirmed that seeds with adhesive structures have lower dropping rates than those without. We conclude that the seed dropping rate often changes with time during external transport on animals and that the power exponential is an effective function to describe this change. We advise that, to analyse seed dropping rates adequately, retention should be measured over reasonable time periods – until most seeds are dropped – and both the simple and power exponential functions should be fitted to the resulting data. To increase its utility, we provide functions describing the seed dropping rate and the dispersal kernel resulting from the power exponential relationship.     

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VII. Chemical synthesis by phosphorylation of 6-thioguanosine 5'-monophosphate, 5'-diphosphate and 5'-triphosphate, and their purification and identification by reversed-phase/ion-pair high-performance liquid chromatography and by various enzymatic assays

A fast and reliable two-step method has been established for the chemical synthesis of 6-thioguanosine 5'-monophosphate, 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate starting from the ribonucleoside. In the first step, 6-thioguanosine dissolved in triethyl phosphate, at high yield reacts with phosphorus oxide trichloride to 6-thioguanosine 5'-monophosphate which is purified by anion-exchange chromatography on DEAE-Sephadex using a step gradient of hydrochloric acid. In the second step, 6-thioguanosine 5'-monophosphate dissolved in water, reacts with phosphoric acid in the presence of pyridine/dicyclohexyl carbodiimide and is converted to 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate which are separated from each other and from the 6-thioguanosine 5'-monophosphate by anion-exchange chromatography on DEAE-Sephadex using a gradient of ammonium bicarbonate. Material from each step of the preparation procedure is separated by reversed-phase HPLC chromatography and analyzed for its free ribonucleoside content, 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and small amounts of unidentified phosphorylated compounds. The purity of the final preparations and the identity of each 6-thioguanosine 5'-phosphate are proven by highly specific enzymatic peak-shifting/HPLC analyses using alkaline phosphatase, 5'-nucleotidase, pyruvate kinase, nucleoside diphosphate kinase and combined hexokinase/glucose 6-phosphate dehydrogenase.