VITAMIN B12 AND THE MACROMOLECULAR COMPOSITION OF EUGLENA : II. Recovery from Unbalanced Growth Induced by Vitamin B12 Deficiency

When vitamin B₁₂ is added to B₁₂-deficient cultures of Euglena gracilis, the cells undergo two relatively synchronous cell divisions within a shorter than usual period of time, apparently as a result of a transitory shortening of the cell cycle. The first cell division pulse, occurring 4.5 h after addition of B₁₂, is preceded by the completion of DNA duplication, but appears to involve no net synthesis of RNA or protein. Before the second round of cell division at about 11 h, a significant amount of DNA synthesis is observed. This time it is accompanied by a minor increase in the RNA and protein content of the culture. The cellular contents of RNA and protein were observed to decrease steadily after the resumption of cell division in B₁₂-depleted cultures receiving the vitamin. Ultimately all three macromolecules returned to their nondeficient, plateau stage levels; by this time, cell division had ceased.     

Micro-complement fixation: a quantitative estimator of protein evolution

The quantitative immunological technique of micro-complement fixation (MC'F) has been routinely used during the past decade to assess evolutionary relationships among living vertebrate species. The large data base that has been generated, along with the excellent correlations between immunologically measured genetic distances and paleontologically derived estimates of divergence times, have formed the basis for the albumin molecular clock. Immunological distance (ID) involves a logarithmic transformation of experimentally measured antibody concentrations. The justification for this transformation has rested entirely on empirical correlations. Consequently, several other transformations have been proposed as giving better fits to particular data sets. We derive, from first principles, the relationship between ID and the amino acid sequence replacements (AAR) between compared albumins. ID is shown to be a linear estimator of AAR. This ID-AAR relationship is based on a proposed process of antibody assortment and exclusion. We present experimental data confirming that such an antibody assortment-exclusion process occurs in MC'F. This process can explain both the high sensitivity and the quantitative phylogenetic nature of the MC'F assay. The assortment-exclusion process also predicts a divergence limit beyond which MC'F data no longer provide robust phylogenetic data.     

Expression of maternal and paternal histone genes during early cleavage stages of the echinoderm hybrid Strongylocentrotus purpuratus × Lytechinus pictus

Interspecific hybrids of the sea urchins Strongylocentrotus purpuratus (♀) and Lytechinus pictus (♂) were used to estimate the contributions of the maternal and paternal genomes to histone mRNA synthesis during early development. Radiolabeled histone mRNAs from the two sea urchin species were identified by hybridization to cloned histone genes from both S. purpuratus and L. pictus and shown to be electrophoretically distinguishable. The synthesis of maternal and paternal histone mRNA in these hybrid embryos is evident as early as the two-cell stage. By at least the 16-cell stage, both maternal and paternal histone mRNAs are associated with polysomes. The relative amounts of the maternal and paternal histone mRNAs synthesized by the zygote appear to be similar.     

Polyoma Virus Infection of Retinoic Acid-Induced Differentiated Teratocarcinoma Cells

The mouse teratocarcinoma stem cell line, F9, becomes permissive for productive polyoma infection upon treatment with retinoic acid. Through the use of M13-polyoma recombinant single-stranded DNA probes, spliced and unspliced early viral RNA were detected after polyoma infection of retinoic acid-treated and untreated F9 cultures.     

Blockage of methanogenesis in marine sediments by the nitrification inhibitor 2-chloro-6-(trichloromethyl) pyridine (Nitrapyrin or N-Serve)

Nitrapyrin or N-Serve [2-chloro-6-(trichloromethyl)pyridine] blocked methanogenesis associated with slurries of marine sediments. Both nitrapyrin and chloroform, an established inhibitor of methanogenic bacteria, were effective at micromolar concentrations. Chemical hydrolysis of nitrapyrin resulted in the release of three molar equivalents of chloride ions and the loss of its ability to inhibit methane production. Thus, the potency of nitrapyrin in blocking methanogenesis seemed to depend upon its trichloromethyl moiety; this conclusion was supported in experiments with other substituted pyridine compounds.     

Restriction endonuclease cleavage map of pKM101: Relationship to parental plasmid R46

Summary A detailed restriction endonuclease cleavage map of the plasmid pKM101 has been constructed. pKM101 plasmids containing individual Tn5 insertions were used to facilitate the ordering of restriction fragments generated by enzymes cleaving pKM101 at multiple sites. By restriction enzyme analysis, pKM101 (35.4 kilobases) appears to have arisen from its clinically-isolated parent by deletion of a single DNA region which codes for three of the four drug resistances carried by R46.     

Characterization of bullous pemphigoid antigen: A unique basement membrane protein of stratified squamous epithelia

Bullous pemphigoid (BP) antigen is a normal basement membrane zone antigen of epidermis and other stratified squamous epithelia. It is defined immunologically by antibodies in the sera of patients with the subepidermal blistering disease BP. In this study we sought to demonstrate that epidermal cells synthesize this antigen, to determine the immunological specificity of BP antibodies and to characterize this antigen. Cultured human epidermal cells (HEC) and a spontaneously transformed mouse epidermal cell line (Pam) both demonstrated BP antigen by indirect immunofluorescence. To characterize the antigen, these cells were radiolabeled with ³⁵S-methionine or ¹⁴C-amino acids and extracts were immunoprecipitated using nine different BP sera. Immunoprecipitated proteins were identified using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and fluorography. All nine BP sera precipitated a protein with disulfide-linked chains of apparent molecular weight approximately 220 kd. Eight normal human sera and six pemphigus vulgaris sera, as well as antibodies directed against fibronectin and laminin, did not precipitate this protein. Furthermore, it was not precipitated by BP sera from radiolabeled extracts of fibroblasts. The protein was soluble in Tris-HCI buffered saline but was not secreted into the culture medium. These studies demonstrate that BP antigen is synthesized by epidermal cells in culture, different patients with BP have antibodies against the same protein, and BP antigen can be identified on SDS-PAGE as a high molecular weight protein consisting of disulfide-linked chains of approximate molecular weight 220 kd.     

Synaptic structure in the visceral ganglion of the lamellibranch mollusc,Spisula solida

Summary An examination using the electron microscope was carried out on the visceral ganglion of the marine bivalve mollusc Spisula solida. A range of fixation, block staining and section staining technique was used to study the structure of chemical synapses. Phosphotungstic acid employed as a block stain specifically stained pre- and post-synaptic structures associated with the membrane at synapses as well as one class of granular vesicle. The specialised contacts were however shown to be rare and in many parts completely absent. Many axons, containing several types of vesicle, were shown to be varicose and it is proposed that they may function in a similar way to the unspecialised varicose terminals of vertebrate autonomic neurons. The role of membrane specialisations in intercellular adhesion is discussed. This study concludes that many synapses may be morphologically unidentified using present criteria.     

Bacteriophage T4 Head Maturation: Release of Progeny DNA from the Host Cell Membrane

We have presented a new approach to studying bacteriophage T4 head maturation. Using a modified M-band technique, we have shown that progeny deoxyribonucleic acid (DNA) was synthesized on the host cell membrane throughout infection. This DNA was released from the membrane later in infection as the result of formation of the phage head; detachment of the DNA required the action of gene products 20, 21, 22, 23, 24, 31, 16, 17 and 49, known to be necessary for normal head formation. Gene products 2, 4, 50, 64, 65, 13 and 14, also involved in head morphogenesis were not required to detach progeny DNA from the membrane; the presence of the phage tail and tail fibers also was not required. DNA was released in the form of immature heads and initially was sensitive to deoxyribonuclease (DNase). Conversion to DNase resistance followed rapidly. The amount of phage precursors present at the time of DNA synthesis determined the time of onset and detachment rate of DNA from the M band as well as the kinetics by which the detached DNA become DNase resistant.     

Transport and metabolism of galactose in rat kidney cortex

1. Analysis of transport of d-galactose was complicated by metabolism of the compound but appeared to have two components: a substrate-saturable component and a diffusion component. At low substrate concentration (<1mm) active transport was observed. Accumulation of galactose was largely independent of Na(+) concentration. The apparent K(m) for this component was 0.2mm. At substrate concentrations above 1mm the active transport system appeared saturated and further increases in substrate concentration resulted in a linear increase in the rate of galactose accumulation, but no concentration gradient was formed. 2. d-[1-(14)C]Galactose (2mm) was metabolized to (14)CO(2) by rat kidney-cortex slices incubated at 37 degrees C, at the rate of 68nmol/h per 100mg of tissue. 3. Intracellular components from such incubations were separated into a neutral fraction, the only major labelled component being galactose, and a phosphorylated fraction. 4. Phosphorylated metabolites found in galactose-incubated slices increased with increasing substrate concentration and achieved a limiting value of 0.42mm after 60min of incubation. 5. Galactose uptake was inhibited by anaerobiosis, dinitrophenol and phlorrhizin. 6. Methyl alpha-d-glucoside and d-glucose partially inhibited galactose uptake only at ratios of 100:1. 7. The presence of pyruvate did not decrease galactose metabolism although it did decrease production of (14)CO(2) from [1-(14)C]galactose. Gluconeogenesis occurred in the presence of pyruvate and (14)C from galactose was found in glucose. 8. Rat kidney-cortex slices metabolized 2mm-[1-(14)C]galactonate to (14)CO(2) at a rate of 20nmol/h per 100mg of tissue.