Survival of Legionella pneumophila in the cold-water ciliate Tetrahymena vorax.

The processing of phagosomes containing Legionella pneumophila and Escherichia coli were compared in Tetrahymena vorax, a hymenostome ciliated protozoan that prefers lower temperatures. L. pneumophila did not multiply in the ciliate when incubated at 20 to 22 degrees C, but vacuoles containing L. pneumophila were retained in the cells for a substantially longer time than vacuoles with E. coli. Electron micrographs showed no evidence of degradation of L. pneumophila cells through 12 h, while E. coli cells in the process of being digested were observed in vacuoles 75 min after the addition of the bacterium. T. vorax ingested L. pneumophila normally, but by 10 to 15 min, the vacuolar membrane appeared denser than that surrounding nascent or newly formed phagosomes. In older vacuoles, electron-dense particles lined portions of the membrane. Acidification of the phagosomes indicated by the accumulation of neutral red was similar in T. vorax containing L. pneumophila or E. coli. This ciliate could provide a model for the analysis of virulence-associated intracellular events independent of the replication of L. pneumophila.     

Isolation of a yeast mutant deficient in pyruvate carboxylase activity

To improve our understanding of the catalytic mechanism and regulatory properties of pyruvate carboxylase (EC 6.4.1.1), an important biotin-dependent enzyme, we have sought to isolate mutants in Saccharomyces cerevisiae which are defective in pyruvate carboxylase activity. One mutant was isolated which was unable to grow on glucose minimal medium unless supplemented with aspartate. Although the enzyme had only 25% of the wild type pyruvate carboxylase activity, Western analysis and RNase protection analysis demonstrated that the mutant gene was expressed at approximately 70% of the wild type level. On the basis of genetic crosses and complementation tests, we have attributed the defect to mutations in the PYC gene encoding pyruvate carboxylase.     

Prohibitin, an evolutionarily conserved intracellular protein that blocks DNA synthesis in normal fibroblasts and HeLa cells.

Genes that act inside the cell to negatively regulate proliferation are of great interest because of their implications for such processes as development and cancer, but these genes have been difficult to clone. This report details the cloning and analysis of cDNA for prohibitin, a novel mammalian antiproliferative protein. Microinjection of synthetic prohibitin mRNA blocks entry into S phase in both normal fibroblasts and HeLa cells. Microinjection of an antisense oligonucleotide stimulates entry into S phase. By sequence comparison, the prohibitin gene appears to be the mammalian analog of Cc, a Drosophila gene that is vital for normal development.     

Rhizobium meliloti exopolysaccharides: genetic analyses and symbiotic importance.

Genetic experiments have indicated that succinoglycan (EPS I), the acidic Calcofluor-binding exopolysaccharide, of the nitrogen-fixing bacterium Rhizobium meliloti strain Rm1021 is required for nodule invasion and possibly for later events in nodule development on alfalfa and other hosts. Fourteen exo loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. We have identified two loci, exoR and exoS, that are involved in the regulation of EPS I synthesis in the free-living state. Certain exo mutations which completely abolish EPS I production are lethal in an exoR95 or exoS96 background. Histochemical analyses of the expression of exo genes during nodulation using exo::TnphoA fusions have indicated that the exo genes are expressed most strongly in the invasion zone. In addition, we have discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for exopolysaccharides in symbiosis are discussed.     

A study of the application of Kohonen-type neural networks to the Travelling Salesman Problem

It is observed that animals often have to resolve difficult tasks of optimization and that this process can be studied by applying the formal framework of neural networks to a simple problem such as the Travelling Salesman Problem. Existing work is reviewed with particular emphasis on recent studies using self-organizing networks. An algorithm is described in which general principles developed by Kohonen are applied to the Travelling Salesman Problem. Simulation results are given for problem sets of up to 10,000 cities. The routes generated are reported as being slightly longer than those produced by simulating annealing; compute time is lower and scales less than quadratically with problem size. It is suggested that the ability to perform optimization is an emergent computational property not just of the Kohonen model but of any mechanism capable of producing topology-preserving mappings, including mechanisms within the brain.     

Genetic counselor training: a review and considerations for the future.

The first training program for genetic counselors began in 1969. Since then a number of other programs have been developed and more than 650 individuals have graduated from these programs. This article reviews the development and current status of training opportunities for genetic counselors. Twelve programs that currently grant a master's-level degree in genetic counseling are reviewed. Other areas, such as certification and licensure, that reflect genetic counseling training or such issues of professional growth as continuing education and career advances are addressed.     

Separation of large DNA restriction fragments on a size-exclusion column by a nonideal mechanism

Double-strand DNA (dsDNA) restriction fragments were chromatographed on the DuPont Bioseries GF-250 column. Two anomolous chromatographic properties were observed. (1) A triphasic dependence of retention on dsDNA chain length was observed. Small DNA fragments (less than 500 base pairs) displayed typical size exclusion, intermediate size DNA (800-5000 base pairs) eluted in the void volume, and larger DNA fragments were increasingly retained. (2) The void volume for nucleic acids was less than that for large polypeptides. The retention of moderately large DNA fragments increased linearly as the square root of the chain length over the range 5.5 to 50 kilobase pairs (ca. 3-30 X 10(6) Mr). A number of eluant manipulations were carried out in order to examine the mechanism by which the larger DNA fragments were being retained and separated. Evidence was not obtained to support either ion exchange or reverse phase as the retention mechanism. The usefulness of such a column for molecular biological manipulations is illustrated by the rapid isolation of homogeneous viral DNA fragments resected from their cloning vectors with restriction endonucleases.     

All nod genes of Rhizobium meliloti are involved in alfalfa nodulation by exo mutants.

Nodulation of alfalfa by exoB mutants of Rhizobium meliloti occurred without root hair curling or infection thread formation. nod exoB double mutants had the same nodulation deficiency as single nod mutants. Therefore, all the known nod genes are involved in nodule induction by exoB mutants, which apparently occurs via intercellular invasion.