The transition point for protein synthesis in late S-G2 may be delayed by genome bromosubstitution without affecting the time of entry into mitosis

Bromosubstitution for most of the S period in synchronous populations of Allium cepa L. meristematic cells resulted in a delay in the late S-G2 transition point where protein synthesis is needed for later mitotic entrance to occur. This retardation in the position of the transition point was not accompanied by the expected delay in the entrance into mitosis, suggesting that such protein synthesis is a requisite, but not a timer for prophase triggering.     

Identification of Culturable Oligotrophic Bacteria within Naturally Occurring Bacterioplankton Communities of the Ligurian Sea by 16S rRNA Sequencing and Probing

Abstract Typical marine bacteria (i.e., obligately oligotrophic) that were numerically dominant members of naturally occurring marine communities were identified by cloning and sequencing the amplified 16S rRNA genes obtained from dilution cultures of the original samples. The data reported here refer to two different habitats of a marine pelagic environment (28 miles offshore, in the northwestern Mediterranean Sea). The samples were taken from the water column at two representative layers, i.e., the 30-m depth, corresponding to the chlorophyll maximum layer, and the 1800-m depth, representative of a deep, oligotrophic environment. Three major lineages were found in the 16S rDNA clone libraries prepared from the two samples, two of which could be assigned to the Vibrio and the Rhodobacter groups. The third lineage was a distant relative of the genus Flavobacterium, but it was not closely related to any marine isolate. Six oligonucleotide probes, either complementary to the conserved sequence domains or selectively hybridizing to the clone sequences, were designed for use as hybridization group-specific and strain-specific probes. A single-mismatch discrimination between certain probes and nontarget sequences was demonstrated by detecting the probes' specificity at different hybridization and washing conditions. The screening of the clone libraries with the obtained probes revealed that neither the 30-m sample higher dilution nor the 1800-m one were pure cultures. While some representatives of the Vibrio group were found in both the surface and the deep sample, the members of the Flavobacterium and Rhodobacter lineages were detected only in the deep and the euphotic layers, respectively. We suggest an approach for analyzing autochthonous marine bacteria able to grow in unamended seawater. Received: 19 May 1998; Accepted: 29 October 1998     

Potent inhibitory effects of the 5'-triphosphates of (E)-5-(2-bromovinyl)-2'-deoxyuridine and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil on DNA polymerase gamma

(E)-5-(2-Bromovinyl)-2'-deoxyuridine 5'-triphosphate (BrVdUTP) and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil 5'-triphosphate (BrVarafUTP), which are known as specific inhibitors of herpes simplex viral (type 1 and 2) DNA polymerase, were found to be strong inhibitors of DNA polymerase gamma from human KB and murine myeloma cells. In fact BrVdUTP and BrVarafUTP were found to be stronger inhibitors of DNA polymerase gamma than of other DNA polymerases having viral (herpes simplex virus or retrovirus) origin or cellular (eukaryotic alpha and beta, or prokaryotic) origin. The mode of inhibition of DNA polymerase gamma by BrVdUTP and BrVarafUTP was competitive with respect to dTTP, the normal substrate. Whereas BrVdUTP was an efficient substrate for DNA polymerase gamma and other DNA polymerases that were examined, BrVarafUTP failed to serve as a substrate for DNA synthesis. Ki values for BrVdUTP (40 nM) and BrVarafUTP (7 nM) with DNA polymerase gamma, as determined with (rA)n.(dT) as the template.primer, were much smaller than the Km values for dTTP (0.16 microM and 0.71 microM for murine and human DNA polymerase gamma, respectively). Thus, the affinity of BrVdUTP or BrVarafUTP for DNA polymerase gamma was much stronger than that of dTTP.     

Molecular alteration of a muscarinic acetylcholine receptor system during synaptogenesis

Biochemical properties of the muscarinic acetylcholine receptor system of the avian retina were found to change during the period when synapses form in ovo. Comparison of ligand binding to membranes obtained before and after synaptogenesis showed a significant increase in the affinity, but not proportion, of the high affinity agonist-binding state. There was no change in receptor sensitivity to antagonists during this period. Pirenzepine binding, which can discriminate muscarinic receptor subtypes, showed the presence of a single population of low affinity sites (M2) before and after synaptogenesis. The change in agonist binding was not due to the late development of receptor function; tests for receptor-stimulated phosphatidylinositol turnover and for modulation of agonist binding by guanylylimidodiphosphate showed functional coupling to be present several days prior to the onset of synapse formation. However, detergent-solubilization of membranes eliminated differences in agonist binding between receptors from embryos and hatched chicks, suggesting a developmental change in interactions of the receptor with functionally related membrane components. A possible basis for altered interactions was obtained from isoelectric point data showing that the muscarinic receptor population underwent a transition from a predominantly low pI form (4.25) in 13 day embryos to a predominantly high pI form (4.50) in newly hatched chicks. The possibility that biochemical changes in the muscarinic receptor play a role in differentiation of the system by controlling receptor position on the surface of nerve cells is discussed.     

Evolution of the immunodominant domain of the circumsporozoite protein gene from Plasmodium vivax. Implications for vaccines

Recent work directed toward the development of a malarial vaccine has focused on the identification and production of the immunodominant repeating peptide of the circumsporozoite protein of the human malaria parasites as an antigen. An important factor which relates to the usefulness of this antigen in a vaccine is the rate at which the molecule changes in sequence. We have determined the sequence and arrangement of the repeating epitope of the circumsporozoite protein gene from a Plasmodium vivax isolate from La Paz, El Salvador (Sal-I). This is compared with a portion of the previously published sequence of the circumsporozoite protein gene from a P. vivax isolate from Belém, Brazil. The genes appear to be very similar in the repeat region. There are 20 similar repeating units in the El Salvador strain and only 19 units are conserved in the Brazilian strain. Following this there are degenerate repeats in both strains. Even the pattern of silent mutations in the repeat area are similar; however, they are not necessarily in the identical location and appear to have shifted. The data suggest that the repeat region of these genes may be evolving by an accelerated mechanism(s). Such a phenomenon could severely decrease the long-term efficacy of a repeat-based anti-sporozoite vaccine.     

A numerical taxonomic study of Flavobacterium-Cytophaga strains from dairy sources

Phenotypic data on 203 Gram-negative non-fermentative bacteria of the Flavobacterium-Cytophaga group isolated from milk and butter were analyzed by numerical taxonomic techniques. Twenty reference strains including species of Flavobacterium, Cytophaga and strains of Pseudomonas paucimobilis were included in the study. Using the matching coefficient of Sokal & Michener with antibiotic susceptibility data included, 139 isolates were recovered in nine clusters. Six of these clusters were linked at or above the 85% S level while three were linked at or above the 79% S level. The largest cluster, representing 46.3% of the isolates, could be equated with Flavobacterium sp. Group IIb. Other clusters could be equated with Flavobacterium sp. L 16/1 (22.7% of isolates), F. balustinum (10.8% of isolates), F. breve (4.4%), F. multivorum (3.5%) and Cytophaga johnsonae (1.5%). The cluster resembling Flavobacterium sp. L 16/1 and a smaller unclassified cluster, were exceptional in being susceptible to the antibiotics cephalothin and penicillin G.