HistoChoice as an alternative to formalin fixation of undecalcified bone specimens.

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visualized more easily in HistoChoice fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.     

Aberrant Splicing and Transcription Termination Caused by P Element Insertion into the Intron of a Drosophila Gene

Insertional mutagenesis screens using the P[lacZ, rosy(+)] (PZ) transposable element have provided thousands of mutant lines for analyzing genes of varied function in the fruitfly, Drosophila melanogaster. As has been observed with other P elements, many of the PZ-induced mutations result from insertion of the P element into the promoter or 5'' untranslated regions of the affected gene. We document here a novel mechanism for mutagenesis by this element. We show that sequences present within the element direct aberrant splicing and termination events that produce a mRNA composed of 5'' sequences from the mutated gene (in this case, pipsqueak) and 3'' sequences from within the P[lacZ, rosy(+)] element. These truncated RNAs could yield proteins with dominant mutant effects.     

Characterization of DNA topoisomerase activity in two strains of Mycoplasma fermentans and in Mycoplasma pirum.

DNA topoisomerases (topos) are essential enzymes that participate in many cellular processes involving DNA. The presence of the DNA-gyrase genes in various mycoplasmas has been reported elsewhere. However, the characterization of DNA topo activity in mycoplasmas has not been previously undertaken. In this study, we characterized the topo activity in extracts of Mycoplasma fermentans K7 and incognitus and in Mycoplasma pirum, as well as in partially purified extract of M. fermentans K7. The topo activity in these microorganisms had the following properties. (i) The relaxation of supercoiled DNA was ATP dependent. (ii) ATP independent relaxation activity was not detected. (iii) Supercoiling of relaxed topoisomers was not observed. (iv) The relaxation activity was inhibited by DNA gyrase and topo IV antagonists (novobiocin and oxolinic acid) and by eukaryotic topo II (m-AMSA [4'-(9-acridylamino)methanesulfon-m-anisidide]) and topo I antagonists (camptothecin). Other eukaryotic topo II antagonists (teniposide and etoposide) did not affect the topo relaxation activity. (v) Two polypeptides of 66 and 180 kDa were found to be associated with the mycoplasma topo activity. These results suggest that the properties of the topo enzyme in these mycoplasma species resemble those of the bacterial topo IV and the eukaryotic and the bacteriophage T4 topo II. The findings that mycoplasma topo is inhibited by both eukaryotic topo II and topo I antagonists and that m-AMSA and camptothecin inhibited the growth of M. fermentans K7 in culture support our conclusion that these mycoplasma species have topo with unique properties.     

Mechanism of activation of the mouse c-mos oncogene by the LTR of an intracisternal A-particle gene.

In the mouse myeloma XRPC-24 the DNA of an intracisternal A-particle (IAP) is inserted within the coding region of c-mos. This insertion splits the c-mos into a 3' rc-mos and a 5' rc-mos separated by approximately 4.7 kb of IAP DNA. The insertion is in a head-to-head orientation and brings the 5' LTR of the IAP in juxtaposition to the 3' rc-mos such that the IAP and the 3' rc-mos are transcribed in opposite directions. The intact c-mos gene is usually dormant, whereas the 3' rc-mos is actively transcribed and is capable of transforming NIH3T3 cells. In an effort to understand the nature of this activation we mapped the 5' ends of the 3' rc-mos mRNA present in XPRC-24. We found two main mRNA start sites, one mapping to the junction of the 3' rc-mos and the 5' LTR, and the other located 10 nucleotides upstream to this junction, within the 5' LTR. This result indicates that the 3' rc-mos in XRPC-24 was activated by insertion of a promoter provided by the LTR of an IAP genome. Furthermore, the 5' LTR appears to possess promoter activities in two directions. This conclusion was confirmed by the fact that this 5' LTR, in both orientations, was able to activate the bacterial gene coding for chloramphenicol acetyltransferase (CAT) in the modular vector pSVOCAT.     

Electrophoresis of proteins and nucleic acids on acrylamide-agarose gels lacking covalent crosslinking

The preparation of acrylamide-agarose gels lacking covalent crosslinking with methylenebisacrylamide is described. These hybrid gels melt at 85 degrees C and, consequently, allow quantitative analysis of tritium-labeled protein after electrophoresis. Recovery of tritium-labeled ribonucleic acids extracted from hybrid gels is 20 to 25% greater than from standard acrylamide-methylenebisacrylamide gels. Standard curves of electrophoretic mobilities as a function of molecular weights of dissociated proteins and ribonucleic acids are compared for acrylamide-agarose gels and acrylamide-methylenebisacrylamide gels.     

No activation of new initiation points for deoxyribonucleic acid replication in BALB/c 3T3 cells transformed by Kirsten sarcoma virus.

BALB/c 3T3 cells were transformed by Kirsten sarcoma virus, and five clones were isolated in soft agar. Average replicon sizes of the transformed cell lines were estimated by the method of fiber-autoradiography (J. A. Huberman and A. D. Riggs, J. Mol. Biol.32:327-341, 1968) and found to be the same size as the nontransformed 3T3 cells, analyzed in parallel. The results indicate that, unlike simian virus 40 and Epstein-Barr virus, Kirsten sarcoma virus does not activate new initiation points for cellular deoxyribonucleic acid replication in murine sarcoma virus-transformed BALB/c 3T3 cells.     

Cellular and extracellular siderophores of Aspergillus nidulans and Penicillium chrysogenum.

Aspergillus nidulans and Penicillium chrysogenum produce specific cellular siderophores in addition to the well-known siderophores of the culture medium. Since this was found previously in Neurospora crassa, it is probably generally true for filamentous ascomycetes. The cellular siderophore of A. nidulans is ferricrocin; that of P. chrysogenum is ferrichrome. A. nidulans also contains triacetylfusigen, a siderophore without apparent biological activity. Conidia of both species lose siderophores at high salt concentrations and become siderophore dependent. This has also been found in N. crassa, where lowering of the water activity has been shown to be the causal factor. We used an assay procedure based on this dependency to reexamine the extracellular siderophores of these species. During rapid mycelial growth, both A. nidulans and P. chrysogenum produced two highly active, unidentified siderophores which were later replaced by a less active or inactive product--coprogen in the case of P. chrysogenum and triacetylfusigen in the case of A. nidulans. N. crassa secreted coprogen only. Fungal siderophore metabolism is varied and complex.     

Continuous open flow-through system as a model for oil degradation in the arctic ocean

A continuous flow-through system incubated in situ was used to model oil biodegradation in Arctic coastal waters. High numbers of oil-degrading microorganisms were found in the Arctic coastal waters examined in this study. The microbial community underlying oil slicks increased and showed a population shift to a greater percentage of hydrocarbon-utilizing microorganisms. Microbial populations and oil biodegradation were increased by the addition of nitrogen and phosphorus. Both abiotic and biodegradative losses were lower than expected, perhaps due to the unusually harsh, ice-dominated Arctic summer, during which these tests were conducted. Chromatographic and spectrometric analyses showed that residual oils contained similar percentages of individual components and classes of hydrocarbons, regardless of the amount of degradation, indicating that most components of the oil were being degraded at similar rates.     

3H-Spiroperidol binding and dopamine-stimulated adenylate cyclase: evidence for multiple classes of receptors in primate brain regions.

Studies of displacement by agonist and antagonist drugs of ³H-spiroperidol binding in brain regions of $\text{Cebus}$ and rhesus monkeys revealed one type of receptor in caudate nucleus and a second type of receptor in both frontal and anterior limbic cortex. Compared with caudate, the cortical regions were more sensitive to clozapine and loxapine, equally sensitive to fluphenazine and relatively less sensitive to haloperidol. Also, the cortical regions were insensitive to molindone. Parallel studies using the dopamine-stimulated adenylate cyclase have demonstrated three types of receptors, one in caudate, a second in frontal cortex, and a third in anterior limbic cortex. In each region studied, relative sensitivities to drug using these two methods differed, suggesting that in each of these regions only a relatively small portion of ³H-spiroperidol receptors are coupled to adenylate cyclase.