Essential Genes in the Hdf6 Region of Chromosome I in Caenorhabditis Elegans

In this paper we describe the analysis of essential genes in the hDf6 region of chromosome I of Caenorhabditis elegans. Nineteen complementation groups have been identified which are required for the growth, survival or fertility of the organism (essential genes). Since ten of these genes were represented by more than one allele, a Poisson calculation predicts a minimum estimate of 25 essential genes in hDf6. The most mutable gene in this region was let-354 with seventeen alleles. An average mutation rate of 5 x 10(-5) mutations/gene/chromosome screened was calculated for an ethyl methanesulfonate dose of 15 mM. Mutations were recovered by screening for lethal mutations using the duplication sDp2 for recovery. Our analysis shows that duplications are very effective for maintenance and mapping of large numbers of lethal mutations. Approximately 600 lethal mutations were mapped in order to identify the 54 that are in the deficiency hDf6. The hDf6 region appears to have a lower proportion of early arresting mutations than other comparably sized regions of the genome.     

The surface coat of bloodstream forms of Trypanosoma musculi from mice

Iodination and immunoprecipitation techniques together with indirect fluorescent antibody tests identified two polypeptides (SP) of molecular weights 88,000–92,000 and 66,000–70,000 in the surface coat of bloodstream forms of the mouse trypanosome, Trypanosoma musculi. As parasites multiply and enter the early plateau phase of infection the 88,000–92,000 SP is present while the 66,000–70,000 SP is only detectable after the mid-plateau phase. Western blotting of parasite extracts showed that the 88,000–92,000 SP was present throughout the course of infection, but it appears to become masked by the 66,000–70,000 SP or possibly immunoglobulin from about 16 days after infection. Based on results when Western blots of parasite extracts were probed with antibodies affinity purified against the 88,000–92,000 SP, the two SP appear to be immunologically related and the smaller may be a cleavage product of the larger. This would explain why affinity purified antibodies to each SP bound to trypanosomes collected 8 days after infection, when only the 88,000–92,000 is detectable in parasite extracts. However, the failure of antibodies affinity purified against the 66,000–70,000 SP to bind to the 88,000–92,000 SP in Western blots suggests that the smaller SP has some epitopes that are immunologically distinct from those of the larger SP.     

An example of Leber hereditary optic neuropathy not involving a mutation in the mitochondrial ND4 gene.

A large Australian family afflicted with Leber's Hereditary Optic Neuropathy (LHON) is analyzed at the nucleotide sequence level in this report. Biochemical assays of platelet mitochondria isolated from members of this family have demonstrated a significant decrease in the specific activity of Complex I (NADH-ubiquinol oxidoreductase) of the electron transport chain. It is shown here, however, that neither this biochemical lesion nor the optic neuropathy are due to the mutation at nucleotide position 11,778 of the mitochondrial ND4 gene first identified by Wallace et al. in several LHON pedigrees. Furthermore, extensive DNA sequencing studies reveal no candidate mutations within the mitochondrial ND3 gene, the ND4L/ND4 genes, or the contiguous tRNA genes. These studies provide the first direct evidence that not all LHON lineages--even those associated with a biochemical defect in mitochondrial respiratory chain Complex I--carry a mutation in the ND4 gene. Members of the Australian LHON family exhibit neurological abnormalities in addition to the well-characterized ophthalmological changes. It is hypothesized that LHON may be a syndrome or set of related diseases in which the clinical abnormalities are a function, at least in part, of the mitochondrial Complex I gene in which the proximate mutation occurs.     

Conjugal Transfer of Megaplasmid 2 between Rhizobium meliloti Strains in Alfalfa Nodules

A DNA fragment containing the RP4 mob function, as well as the gentamicin and spectinomycin resistance genes, was inserted by gene replacement onto the megaplasmid 2 (pM2) of Rhizobium meliloti 0540 (Inf⁻ EPS⁻), resulting in PG101 (Inf⁻ EPS⁻). The self-transfer of pM2 and the mobilization of pM2 by plasmid RP4-4 were investigated during conjugation between PG101 and R. meliloti 2526 (Nod⁻). In filter conjugations, pM2 was readily mobilized by RP4-4. In addition to this, the self-transfer of one megaplasmid (pM) was detected at a frequency of 3 × 10⁻⁷. Bacteria isolated from the nodules of alfalfa and coinoculated with strains PG101 and 2526 showed that pM2 was mobilized at a frequency of approximately 7 × 10⁻⁵. Bacterial cell numbers were too low in the nodules for detection of the self-transfer of pM2 to occur. No pM2 transfer was detected in the inoculum. A comparison of the transfer frequencies for the various conjugation conditions revealed that pM2 transfer occurred as frequently in the nodules as in filter conjugations. These results indicate that the nodule creates conditions for gene transfer that are comparable to optimal laboratory conditions.     

Evidence that free polysomes are not the precursors of membrane-bound polysomes in rat liver

We tested, in rat liver, the postulate that free polysomes were precursors of membrane-bound polysomes. Three methods were used to isolate free and membrane-bound ribosomes from either post-nuclear or post-mitochondrial supernatants of rat liver. Isolation and quantitation of 28 S and 18 S rRNA allowed determination of the 40 S and 60 S subunit composition of free and membrane-bound ribosomal populations, while pulse labeling of 28 S and 18 S rRNA with [6-¹⁴C]orotic acid and inorganic [³²P]phosphate allowed assessment of relative rates of subunit renewal. Throughout the extra-nuclear compartment, 40 S and 60 S subunits were present in essentially equal numbers, but, free ribosomes contained a stoichiometric excess of 40 S subunits, while membrane-bound ribosomes contained a complementary excess of 60 S subunits. Experiments with labeled precursors showed that throughout the extra-nuclear compartment, 40 S and 60 S subunits accumulated isotopes at essentially equal rates, however, free ribosomes accumulated isotopes faster than membrane-bound ribosomes. Among free ribosomes or polysomes, 40 S subunits accumulated isotopes faster than 60 S subunits, but, this relationship was not seen among membrane-bound ribosomes. Here, 40 S subunits accumulated isotope more slowly than 60 S subunits. This distribution of labeled precursors does not support the postulate that free polysomes are precursors of membrane-bound polysomes, but, these data suggest that membrane-bound polysomes could be precursors of free polysomes.     

Actomyosin interactions with insulin-storage granules in vitro.

Interactions between actomyosin and insulin storage granules isolated from rat islets of Langerhans have been examined in a simple system in vitro, which allows comparison of the sedimentation of the granules in the presence of absence of actomyosin in various conditions. Actomyosin altered granule-sedimentation rates in a manner consistent with the binding of the granules of actomyosin filaments. This interaction was enhanced by addition of ATP (1.5 mM) but unaltered by addition of CaCl2, by calmodulin or by calmodulin in the presence of 10 microM-CaCl2. Addition of EGTA (0.1 mM), cyclic AMP (10 microM) of cytochalasin B (10 microgram/ml) were also without effects in these conditions. Pre-incubation of granules with phospholipase c did not affect granule-actomyosin interaction. Ultrastructural studies showed close contacts between the membranes of the granules and actomyosin filaments. The results indicate the possibility that actomyosin might provide the motile force for granule translocation during the insulin secretory process.     

The Sabinas syndrome

We have defined a new autosomal recessive disorder in patients stemming from a small community in northern Mexico. Diagnosable at birth, its major symptoms include brittle hair, mental retardation, and nail dysplasia. Structural hair abnormalities are seen by both light and electron microscopy. Hair cystine content is reduced while the copper/zinc ratio in hair is increased.     

Comparison of the control and pathways for degradation of the acetylcholine receptor and average protein in cultured muscle cells

In the studies reported here, we investigated whether the degradation of the acetylcholine receptor (AChR) in cultured muscle cells involves similar mechanisms as and is controlled in a manner similar to, the catabolism of the bulk of cell protein. We compared these processes after labeling cell protein with radioactive leucine or phenylalanine for 24 hours, or labeling the acetylcholine receptor with (¹²⁵I)-bungarotoxin. The apparent average half-life of cell protein was 38 ± 2 hours and that of the receptor-toxin complex was 25 ± 1 hours. Incubation in media lacking serum and embryo extract accelerated the degradation of both average protein and the receptor-toxin complex. Insulin reduced the rate of catabolism of both average protein and the receptor-toxin complex toward levels seen in the presence of serum. However, although these two degradative processes seem to be controlled similarly, they probably involve different mechanisms. The protease inhibitors leupeptin and chymostatin, which slowed overall proteolysis in nongrowing muscles and hepatocytes, reduced the degradation of the ACh receptor by 2–11-fold, but had no, or only slight, effects on the catabolism of average protein, even when overall proteolysis was accelerated by omitting serum and embryo extract. Chloroquine, an inhibitor of lysosomal function, also reduced the degradation of AChR, by about 10-fold, but decreased overall protein breakdown by only 20–30%. Incubation of myotubes at lower temperatures reduced both degradative processes, but affected the breakdown of the receptor to a greater extent. Thus the rate-limiting steps in these processes have different activation energies. Incubation with 2-deoxyglucose, an inhibitor of glycolysis, decreased the breakdown of average protein but not that of the receptor-toxin complex. However, the two degradative processes were sensitive to azide, an inhibitor of oxidative phosphorylation. Although the lysosome is the primary site for AChR degradation and perhaps for degradation of other surface proteins, the breakdown of most proteins in myotubes seems to involve a distinct proteolytic system requiring metabolic energy.