An evaluation of the phylogenetic position of the dinoflagellateCrypthecodinium cohnii based on 5S rRNA characterization

Summary Partial nucleotide sequences for the 5S and 5.8S rRNAs from the dinoflagellateCrypthecodinium cohnii have been determined, using a rapid chemical sequencing method, for the purpose of studying dinoflagellate phylogeny. The 5S RNA sequence shows the most homology (75%) with the 5S sequences of higher animals and the least homology (< 60%) with prokaryotic sequences. In addition, it lacks certain residues which are highly conserved in prokaryotic molecules but are generally missing in eukaryotes. These findings suggest a distant relationship between dinoflagellates and the prokaryotes. Using two different sequence alignments and several different methods for selecting an optimum phylogenetic tree for a collection of 5S sequences including higher plants and animals, fungi, and bacteria in addition to theC. cohnii sequence, the dinoflagellate lineage was joined to the tree at the point of the plant-animal divergence, well above the branching point of the fungi. This result is of interest because it implies that the well-documented absence in dinoflagellates of histones and the typical nucleosomal subunit structure of eukaryotic chromatin is the result of secondary loss. and not anindication of an extremely primitive state, as was previously suggested. Computer simulations of 5S RNA evolution have been carried out in order to demonstrate that the above-mentioned phylogenetic placement is not likely to be the result of random sequence convergence.We have also constructed a phylogeny for 5.8S RNA sequences in which plants, animals, fungi and the dinoflagellates are again represented. While the order of branching on this tree is the same as in the 5S tree for the organisms represented, because it lacks prokaryotes, the 5.8S tree cannot be considered a strong independent confirmation of the 5S result. Moreover, 5.8S RNA appears to have experienced very different rates of evolution in different lineages indicating that it may not be the best indicator of evolutionary relationships.We have also considered the existing biological data regarding dinoflagellate evolution in relation to our molecular phylogenetic evidence.     

Production of extrachromosomal r-determinant circles from integrated R100.1: Involvement of the E. coli recombination system

Summary The drug resistance plasmid R100.1 can integrate into the E. coli chromosome at several sites on the plasmid. Many of the resulting Hfr strains continuously produce extrachromosomal circular forms of the r-determinant. These r-det plasmids seem incapable of stable autonomous replication. We show that their presence in the cell requires the continuous activity of functional recA and recC genes but does not require the lexA function.The production of r-det circular forms is correlated with an increased copy number of r-det sequences, relative to RTF sequences. This copy number increase is, however, also found in a recA ^- backgound where no circular forms of r-det are found. These results show that a specific replication of r-det sequences, not present in the wild-type R100.1 plasmid, occurs in these R-Hfr strains. They suggest that a rec promoted recombination, posterior to the specific replication event, is needed for the production of circular r-det forms.     

Retention of human skin fibroblast fatty acid modifications during maintenance culture

Summary The fatty acid composition of cultured human skin fibroblasts was modified by adding either oleic or linoleic acid to the growth medium. After the cultures became confluent, they were washed and transferred to different maintenance media in order to determine the stability of the various fatty acyl modifications. Some changes in fatty acid composition occurred under all conditions. When the maintenance medium was supplemented with fatty acid, the cellular neutral lipid and phospholipid fatty acyl composition were altered markedly within 16 to 24 hr. If no supplemental fatty acid was available during the maintenance period, however, the modified fatty acyl compositions were sufficiently retained so that appreciable differences between the cells enriched with oleate and linoleate persisted for at least 48 to 72 hr. This considerable degree of stability occurred when either 10% delipidized fetal bovine serum or 10% fetal bovine serum containing its inherent lipids were present in the maintenance medium. Although the triglyceride content of the fatty acid-modified cells was quite labile, neither the cholesterol nor phospholipid content changed appreciably during culture in any of the maintenance media. Since the fatty acid compositional differences persisted during several days of maintenance under certain conditions, these modified cultures appear to be a useful experimental system for assessing the effect of lipid structure on fairly long-term cellular functions. This work was supported by Arteriosclerosis Specialized Center of Research Grant HL14230 from the National Heart, Lung and Blood Institute, National Institutes of Health.     

Processing and fermentation of Jerusalem artichoke for ethanol production

Summary Processing and fermentation trials on Jerusalem artichoke (Helianthus tuberosus L.) tubers, and on pure inulin media were carried out. Acid and thermal treatments, pure and mixed cultures of yeast, and enzyme preparations were investigated. Best ethanol yields on either substrate were obtained with pH 2 thermal treatments, resulting in 131.6 liters ethanol per metric ton fresh tuber.     

Five-year perspective of the large-scale growth of mammalian cells in suspension culture

The Cell Culture Center at the University of Alabama in Birmingham was set up to produce large quantities of cells and their products from suspension cultured cell lines. This system has now been in operation for over five years and has been effective in producing large quantities of mammalian cells of murine and human origin. This article describes the system and some growth parameters which have been of importance for large-scale mammalian cell growth.     

The behaviour of male orchid bees (Apidae,Hymenoptera, Insecta) and the question of leks

The male territories of two species of orchid bees, Eulaema meriana and Euglossa imperialis, are described. These territories consist of a perch, where the males display on the trunk of a tree, and a route flown from and back to the perch. Territories are located in treefalls or other large light gaps in tropical forest. The territories of these two species differ in the height of the perch and amount of light reaching it, size of the perch tree, and period of activity. A definition of lek is given. These male euglossines form facultative leks in large treefalls. The relationships between male territories, female foraging ranges, and collection of aromatic material by males are discussed.     

Scanning electron microscopy of the disruption of tobacco hornworm, Manduca sexta, midgut by Bacillus thuringiensis endotoxin

The ingestion of Bacillus thuringiensis crystal endotoxin by Manduca sexta causes the destruction of both goblet and columnar cells of the midgut. One hour after ingestion, the microvilli show pathological effects. Nearly complete destruction of the goblet and columnar cells has taken place after 4 hr exposure to the toxin.     

Purification and preliminary characterization of two asclepains from the latex of Asclepias syriaca L. (milkweed).

Two groups of asclepains have been isolated from Asclepias syriaca L. (milk-weed) latex and a representative of each has been purified. Asclepains A3 and B5 are homogeneous proteins with molecular weights of 23 000 and 21 000, respectively. Both require a reducing and chelating agent for maximum activity and hydrolyze ester, amide and peptide bonds. The optimum pH for hydrolysis of casein is 7.5 to 8.5 for asclepain A3 and 7.0 to 7.5 for asclepain B5. Both enzymes are autolytic when active and are inhibited by p-chloromercuribenzoate, iodoacetic acid and sodium tetrathionate. Asclepains A3 and B5 each contain one titratable SH group per molecule and no bound carbohydrate. Each of the two enzymes has leucine as the N-terminal amino acid. There are notable differences in their amino acid compositions.     

Characterization of a site-specific restriction endonuclease from Rhodopseudomonas sphaeroides.

A type II restriction endonuclease, RshI, has been partially purified from photoheterotrophically grown Rhodopseudomonas sphaeroides strain 2.4.1. The enzyme preparation, after a single DE-52 column fractionation, is free of 5' exonuclease and phosphatase activities but contains a trace of 3' exonuclease activity. Based upon deoxyribonucleic acid (DNA) sequencing data in the vicinity of the enzyme-promoted cleavage of pBR322 DNA, we have concluded that RshI probably recognizes the palinodromic hexanucleotide sequence 5'-CGATCG-3' and cleaves between the T and C. lambda cI857 DNA contains three RshI sites, two of which lie in the replaceable region. The plasmid pBR322, which carries resistances to ampicillin and tetracycline, contains a single RshI site in the ampicillin resistance determinant. Insertion of DNA into the RshI site of pBR322 results in loss of ampicillin resistance but retention of tetracycline resistance, thereby providing a convenient screening procedure for recombinant plasmids.     

Role of bovine serum albumin in the nutrition of Mycobacterium tuberculosis.

Bovine serum albumin promotes the growth of small inocula of Mycobacterium tuberculosis in media containing unesterified fatty acids. Albumin binds fatty acids present in concentrations toxic for the organisms. In the present study, additional roles of albumin were investigated. When present in a basal medium, fatty acid-free albumin could be utilized by M. tuberculosis as a sole source of carbon. Since albumin could not substitute for the amino acids in basal medium as a nitrogen source, it was concluded that the protein component in albumin was not utilized as a nutrient by the organisms. An ether extract of fatty acid-free albumin supported a small but significant amount of growth. Analysis of the lipids in fatty acid-free albumin by gas chromatography revealed the presence of 686 microgram of fatty acid per g of albumin. Although a small amount of growth occurred when a lipid extract of albumin was present in the medium, growth stimulation was dependent in major part on the presence of undenatured albumin in the medium. Lipids, when bound to albumin, can serve as a nontoxic source of carbon and energy.