Initiation of replication in the Chinese hamster dihydrofolate reductase domain

Two-dimensional (2-D) gel analysis of replication intermediates in the Chinese hamster dihydrofolate reductase domain has suggested that nascent chains can initiate at any of a large number of sites scattered throughout a ～50 kb “initiation locus” (although the level of initiation detected at any given site within this region was relatively low). This result contrasts markedly with data from anin vitro strand switching assay suggesting that >80% of initiations occur within a single 500 bp fragment lying within the initiation locus. In an effort to reconcile these two disparate views of the initiation reaction, we have questioned the validity of our 2-D gel data in several ways. We show here that: 1) the number of replication bubbles detected in the DHFR locus in the early S period is markedly increased when the cells are released from a synchronizing agent that inhibits initiationper se, rather than from aphidicolin, which is a chain elongation inhibitor; 2) initiation in the DHFR domain occurs only during the first 90 min of the S period, as would be expected of an early-firing origin; 3) a pulse of³H-thymidine moves through the structures observed on 2-D gels with the kinetics expected ofbonafide replication intermediates; and 4) preparations of replication intermediates that are subsequently analyzed on 2-D gels appear, by electron microscopy, to represent the typical theta structures and single-forked molecules expected of bidirectional origins of replication; no unusual structures (e.g., microbubbles) were seen.     

Lampyridae (Coleoptera): A plethora of mollicute associations

Beetles (Coleoptera) harbor many species ofAcholeplasma andSpiroplasma (division Tenericutes, class Mollicutes). Mollicutes were isolated from guts and/or hemocoels of firefly beetles (Lampyridae) from the United States (Maryland and West Virginia), Ecuador, and Tobago. Firefly beetles were frequent hosts for the group XIV spiroplasma, isolated from Ellychnia corrusca, and the group XIX spiroplasma, isolated fromPhoturis spp. The most unusual feature of the firefly-mollicute association is the carriage of four Mycoplasma species. Recent phylogenetic studies indicate that these species are members of a clade that includes a vertebrate pathogen,Mycoplasma mycoides. The high rate of occurrence ofMycoplasma species (which are, otherwise, infrequent in insects) in lampyrid beetles suggests that the association is significant. The unusual light-producing physiology of lampyrids (which is dependent on large pools of energy) and the production of large amounts of cardenolides from cholesterol (a critical growth factor for many mollicutes) may favor colonization by mollicutes. Offprint requests to: K. J. Hackett.     

Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon

To facilitate efficient allelic exchange of genetic information into a wild-type strain background, we improved upon and merged approaches using a temperature-sensitive plasmid and a counter-selectable marker in the chromosome. We first constructed intermediate strains of Escherichia coli K12 in which we replaced wild-type chromosomal sequences, at either the fimB-A or lacZ-A loci, with a newly constituted DNA cassette. The cassette consists of the sacB gene from Bacillus subtilis and the neomycin (kanamycin) resistance gene of Tn5, but, unlike another similar cassette, it lacks IS1 sequences. We found that sucrose sensitivity was highly dependent on incubation temperature and sodium chloride concentration. The DNA to be exchanged into the chromosome was first cloned into derivatives of plasmid pMAK705, a temperature-sensitive pSC101 replicon. The exchanges were carried out in two steps, first selecting for plasmid integration by standard techniques. In the second step, we grew the plasmid integrates under non-selective conditions at 42 degrees C, and then in the presence of sucrose at 30 degrees C, allowing positive selection for both plasmid excision and curing. Despite marked locus-specific strain differences in sucrose sensitivity and in the growth retardation due to the integrated plasmids, the protocol permitted highly efficient exchange of cloned DNA into either the fim or lac chromosomal loci. This procedure should allow the exchange of any DNA segment, in addition to the original or mutant allelic DNA, into any non-essential parts of the E. coli chromosome.     

Virus removal during groundwater recharge: effects of infiltration rate on adsorption of poliovirus to soil.

Studies were conducted to determine the influence of infiltration rate on poliovirus removal during groundwater recharge with tertiary-treated wastewater effluents. Experiments were conducted at a uniquely designed, field-situated test recharge basin facility through which some 62,000 m3 of sewage had been previously applied. Recharge at high infiltration rates (75 to 100 cm/h) resulted in the movement of considerable numbers of seeded poliovirus to the groundwater. Moderately reduced infiltration rates (6 cm/h) affected significantly improved virus removal. Very low infiltration rates (0.5 to 1.0 cm/h), achieved by partial clogging of the test basin, yielded the greatest virus removal efficiencies.     

Physical Factors That Affect In Vitro Autographa californica Nuclear Polyhedrosis Virus Infection

Of the physical parameters tested for in vitro baculovirus infection, multiplicity of infection was most important in governing percent cell infection. Most plaques formed within the first 5 min of incubation. Efficiency of infection, however, was low, and the virus titer did not diminish during prolonged incubation. Efficiency of infection improved markedly when cells or virus were preincubated with selected polyanions and polycations. Precise regulation of the pH, osmotic pressure, and ionic composition of the cell culture medium also promoted maximum in vivo infection.     

Induction,ultrastructure, isolation,and tissue culture of chlorophyll mutants in carrot

Summary Seeds ofDaucus carota “Danvers” were treated with ethyl methanesulfonate (EMS) for 6-hr periods at concentrations of 0.01, 0.03, 0.1, 0.3, 1.0, and 3.0% to induce plastid mutants. In these treatments, there was a gradual decrease in percent germination from control up to 1.0% EMS and no germinations at 3.0%. The number of chlorophyll mutants increased with dose of mutagen. One mutant plant was isolated from the 0.1% treatment and it had leaf sections of green, white layered on green and pure white; and white and green striped petioles. Histogenic analysis of this mutant showed it to be a GGW chimera, the “all-white” sectors being GWW, arising from displacement of L-II by L-III. Electron micrographs of the white sections showed plastids that had dilated thylakoids typical of PS-I mutants. So called “mixed cells” of normal and mutant plastids were found, suggesting a plastome mutation. Leaf and petiole sections have been successfully cultured through the development of callus, and both green and white plants have been regenerated. Regenerated white plantlets were insensitive to 10 mM methyl viologen (paraquat), whereas green tissues were killed by the herbicide. Support for some of this work was provided by the Miami University Faculty Research Committee and the Comprehensive Employment and Training Act (CETA). This paper will be included as part of the dissertation work of P. D. Miller.     

Ribosomal RNA sequence conservation and gene number in the larval brine shrimp

The haploid genome size of Artemia is determined to be about 0.9·10¹², as evidenced both by Feulgen microspectrophotometry of individual diploid class nuclei, which are but one of five polyploid classes present within the larvae, and by analysis of the reassociation kinetics of the isolated single copy DNA component. Polysomes isolated from 24-h incubation stage larvae contain an average of 10 ribosomes per messenger RNA molecule. Their rRNAs are found to have sedimentation coefficients of 18 S and 26 S, corresponding to molecular weights of 0.70·10⁶ and 1.40·10⁶, respectively, as determined by polyacrylamide electrophoresis and also by sucrose density centrifugation. Denaturation in glyoxal followed by agarose gel electrophoresis shows that unlike deuterostome rRNAs, Artemia 26 S rRNA contains a cryptic nick about midway in the molecule, which is not found in the 18 S molecule. Isolated rRNAs were labelled in vitro with ¹²⁵I and hybridized with filter-immobilized DNA to saturation, which occurred at 0.051% for Xenopus, and at 0.074% for Artemia. From these results, it is calculated that in the haploid Artemia genome there are about 320 copies of the (18 S + 26 S) ribosomal RNA genes. Reciprocal heterologous hybridizations between these two species show that they share about 30% homology between their rDNA coding sequences.     

Immunocytochemical localization of GABAergic neurones at the electron microscopical level

Summary Antibodies prepared to purified brain glutamic acid decarboxylase (GAD), the synthesizing enzyme for the neurotrasmitter, -aminobutyric, acid (GABA), have been utilized with an unlabelled antibody method to localize GABAergic neurones in both light and electron microscopic preparations. A modification of Sternberger's peroxidase-antiperoxidase (PAP) complex is used to localize the site of anti-GAD binding, and the PAP complex is visualized with diaminobenzidine and H₂O₂. The reaction product is visible in both the light and electron microscopes. The ability to localize and identify labelled profiles in the electron microscope provides more functional information than light microscopical preparations. For example, the GAD-positive reaction product occurs mostly in association with synaptic vesicles within axon terminats, and this localization indicates the importance of GAD for the packaging and storage of GABA. The somata and dendrites of neurones giving rise to these terminals are visualized in colchicine-injected material. The GABAergic neurones form axo-somatic, axo-dendritic, axo-axonal and dendro-dendritic synapses in various regions of the rat central nervous system. Pretreatments of animals with anterograde degeneration have shown the significance of some of the GABAergic terminals that form axo-axonal synapses in the spinal cord.An many brain regions, such as the cerebral cortex, hippocampus and olfactory bulb, virtually all of the GABAergic synapses are derived from local circuit neurones. In other regions such as the cerebellum and neostriatum, the GABAergic terminals are derived from both local circuit neurones and the local axon collaterals of projection neurones that have their somata within these regions. A third type of configuration of GABAergic terminals occurs in the globus pallidus and substantia nigra where these terminals are derived from distant brain regions, axon collaterals of projection neurones and from local circuit neurones. Together, these results indicate the complex organization of the GABAergic system of the brain that has been vividly revealed with electron in croscopical immunocytochemistry.     

Conservation of repeated DNA base sequences in theCrustacea: A molecular approach to decapod phylogeny

Summary Analysis of data obtained from molecular hybridization of³H-labeled repetitious DNA has been utilized to reconstruct the broad outlines of phylogenetic relationships among decapod Crustacea. This molecular reconstruction agrees reasonably well with the paleontological record, and with other schemes obtained by comparative morphological and serological approaches. Preliminary evidence is in line with the hypothesis that continuous addition of new repeated sequence families to the genome over long periods of time may in part account for the correlation observed between percent repetitious DNA hybridized and divergence time. It is tentatively concluded that a core of DNA base sequence homology has been highly conserved throughout the evolution of theCrustacea. Demonstration of inter-species sequence homology has important implications to models which relegate a genetic regulatory function to repeated DNAs.