Wall-less prokaryotes from fall flowers in central United States and Maryland

Four spiroplasma strains and eleven isolates tentatively identified as acholeplasmas were obtained from fall flowers in Colorado, Nebraska, Illinois, and Maryland. Although the acholeplasma isolates were heterogeneous, all showed antigenic sharing with a group of unnamed organisms (L1 and related strains) isolated in othe studies from flowers in Florida. The W20 and W24 isolates from Nebraska were partially related to the L1 group by DNA-DNA homology and polyacrylamide gel electrophoresis (PAGE) analyses. A Colorado spiroplasma (W13) was identifed as a new strain of group IV complex. Three spiroplasma strains from flowers in Maryland old fields represent a new serovar with closest affinity to subgroup I-4 and to the LB12 and N525 serovars of group I. Widespread occurrence of acholeplasmas on flowers in this study, and on plant surfaces in general, suggests that, like spiroplasmas they probably will be found to reside in arthropods.     

Characterization of isolated Fe-loaded rat hepatocytes prepared by collagenase perfusion

Summary Hepatocytes from livers of rats loaded by Fe-dextran treatment were isolated by an in situ collagenase perfusion technique and evaluated for their biochemical, cytochemical, and morphological characteristics in cell culture. Iron loads 15 times higher than in normal rat liver cells isolated in the same way were retained in the preparations with 40% present as hemosiderin. A simple centrifugation-mathematical approach is described for the calculation of Fe content in the hepatocyte (95%) and reticuloendothelial (5%) fractions in the isolates. The cells were cultured for 22 h without loss of protein synthesis capability or significant changes in cell count, viability, endogenous glutamate-oxaloacetate transaminase (GOT) or Fe and were morphologically similar in most respects to unloaded (normal) hepatocytes similarly cultured. Studies are in progress to assess the utility of these preparations as a model for Fe mobilization from Fe-loaded animals. This work is supported by National Institutes of Health Grants AM 25647-03 (M. Dawson, Principal Investigator) and GM 28158-01 (C. Tyson, Principal Investigator). The technical assistance of Mr. Jack E. Dabbs, Mr. Charles Hart, and Mr. Randy Douglas is acknowledged.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

The baboon endogenous virus genome. II. Provirus sequence variations in baboon cell DNA.

Restriction analysis of the approximately 100 integrated baboon endogenous virus (BaEV) proviruses in baboon cells and tissues has revealed two major sequence variations, both in the gag gene region of the genome. One, a 150 nucleotide pair insert, is present in a small proportion of the proviral DNAs and some baboons, but is present in the majority of the proviral DNAs of other baboons. The second, a Bam HI recognition sequence located 2.25 kb from the proviral 5' end, is missing or modified in approximately one-half of the integrated genomes. We consider the possibility that accumulation of proviruses not containing the 0.15 kb insert is correlated with viral activation and expression since it is this form that is a replication intermediate in freshly infected permissive cells. It is evident from these initial studies that the organization of the multiple BaEV proviruses in baboon DNA has undergone modification during evolution.     

A sequence specific endonuclease from Micrococcus radiodurans

A new sequence specific endonuclease, MraI has been purified from Micrococcus radiodurans. This enzyme cleaves bacteriophage λ DNA at three sites, adenovirus type 2 DNA at more than 12 sites and has a unique site on ΦX174 DNA. It has no sites on SV40, PM2 and pBR322 DNA. The three sites on phage λ DNA are different from those cleaved by SmaI, XmaI and XorII. The sites of cleavage are located at 0.424, 0.447 and 0.834 fractional lengths on the physical map of λ DNA. MraI is shown to be an isoschizomer of SacII and SstII recognizing the palindromic nucleotide sequence ′5-CCGC↓GG-3′. The enzyme shows an absolute requirement of Mg²⁺, but is active in the absence of added 2-mercaptoethanol. The enzyme shows activity at a broad range of temperature and pH with an optimum at 45°C and pH 7.0. MraI represents the first restriction enzyme from a bacterium whose DNA lacks modified methylated bases.     

Morphogenetic effects of light and guanine derivatives on the fruiting myxobacterium Stigmatella aurantiaca.

When low cell densities of the myxobacterium Stigmatella aurantiaca were starved on an inorganic salts and agar medium, cell aggregation and fruiting body formation showed a striking dependency upon the presence of light. This dependency was not manifested when sufficient amounts of guanosine or guanine nucleotides were added to the medium. Light interacted cooperatively with suboptimal concentrations of guanine compounds to promote development. None of the other purine or pyrimidine derivatives, with the exception of adenine, stimulated development. However, aggregates that formed in the presence of adenine did not mature into fruiting bodies and instead disaggregated.     

Effects of specific cations on aggregation and fruiting body morphology in the myxobacterium Stigmatella aurantiaca.

The cation requirements for fruiting body formation in the myxobacterium Stigmatella aurantiaca on agarose were determined. Calcium alone caused the cells to aggregate into interconnecting ridges. Under these conditions, stalk formation was severely depressed but sporangia frequently formed. The combination of magnesium and manganese was necessary for optimal formation of discrete aggregates (rather than ridges) and stalks. Manganese inhibited sporangium development. The inclusion of calcium into the magnesium-manganese medium overcame the inhibition by manganese and stimulated the production of multiple sporangia.     

Perturbation of lipid metabolism in L-M cultured cells by elaidic acid supplementation: formation of fatty alcohols

Supplementation of culture medium with elaidic acid (400 μg/flask) in L-M cells results in the formation of an otherwise undetected lipid component. We have identified this lipid component to be a mixture of free fatty alcohols containing primarily elaidyl alcohol with cetyl, stearoyl, and oleoyl alcohols as minor constituents. Formation of fatty alcohols by fatty acid supplementation seems to be specific with $\text{trans}$ fatty acids (i.e., elaidate, $\text{trans}$ vaccenate, and linolelaidate); addition of stearate and oleate to the L-M cells does not produce fatty alcohols. The fatty alcohols accumulated by the $\text{trans}$ fatty acid supplementation are associated with both the particulate and supernatant fractions of the cells.     

Fluorescent thin-layer peptide mapping for protein identification and comparison in the subnanomole range

Conditions and simple precautions are presented for carrying out highly reproducible and sensitive peptide mapping by thin-layer chromatography and subsequent electrophoresis of subnanomole amounts of tryptic digest on silica gel G or GHL plates. The fluorogenic reagent “fluorescamine” is employed for visualization under long-wavelength ultraviolet illumination. Permanent photorecording of high-contrast images, using readily available filters, is substituted for subjective hand scoring of plates. Contrast reversal is used to produce peptide maps suitable for half-tone reproduction.