The glyceraldehyde-3-phosphate dehydrogenase gene family in the nematode, Caenorhabditis elegans: isolation and characterization of one of the genes.

The isolation and genomic sequence of one of possibly four glyceraldehyde-3-phosphate dehydrogenase genes in the nematode, Caenorhabditis elegans is presented. The complete nucleotide sequence of the coding as well as the noncoding flanking regions of this gene has been determined. The deduced amino-acid sequence agrees with the sequence of typical glyceraldehyde-3-phosphate dehydrogenase enzymes and its molecular weight of 36,235 agrees with its size determined previously (Yarbrough, P. and Hecht, R. (1984) J. Biol. Chem. 259, 14711-14720). That this isolated gene encodes a nematode glyceraldehyde-3-phosphate dehydrogenase is additionally confirmed by demonstrating its immunoreactivity to an anti-nematode glyceraldehyde-3-phosphate dehydrogenase antibody after its expression as a fusion protein with dihydrofolate reductase. Codon utilization follows a pattern typical of other expressed nematode genes. The gene is split by two introns that are highly conserved in comparison to other introns observed in C. elegans. The placement of one of these introns is conserved with respect to the chicken glyceraldehyde-3-phosphate dehydrogenase gene. Within the 5' flanking sequence homology to actin and the homology 2 block of the major myosin gene (unc-54) is noted. It is of interest that the 3' flanking region contains a CAAAT box, followed by a TATAAT box, before an open reading frame of a closely linked gene that also contains a small AT-rich intron with the nematode consensus splice junction.

Genomic organization of the glyceraldehyde-3-phosphate dehydrogenase gene family of Caenorhabditis elegans.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDHase) is encoded by four genes designated gpd-1 through gpd-4 in the nematode Caenorhabditis elegans. gpd-1 has been isolated and sequenced, and is shown here to have a nearly identical copy (gpd-4) with respect to coding and regulatory flanking sequence information as well as to the placement of its two introns. Both genes, which are separated by 250,000 to 300,000 base-pairs were assigned to chromosome II by in situ hybridization and physically linked to a DNA polymorphism located near unc-4 on the genetic map. The genes gpd-2 and gpd-3 are also nearly identical with each other but differ from the gpd-1 and gpd-4 pair with respect to the positions of their two introns and a cluster of amino acid changes within the amino-terminal region of the enzyme. Furthermore, one gene from each pair (gpd-4 and gpd-2) exhibits a single amino acid substitution at positions heretofore known to be conserved in all other systems so far examined including the extreme thermophiles. gpd-2 and gpd-3 are organized as a direct tandem repeat separated by only 244 base-pairs. They have been assigned to an 85,200 base-pair contig that maps to the left end of the X chromosome. The absence of gpd-3 from C. elegans var. Bergerac was used as a marker to map the gpd-2,3 gene pair near unc-20. Northern analyses have shown that gpd-1 and gpd-4 are preferentially expressed in embryos, while the expression of gpd-2 and gpd-3 increases during postembryonic development. These analyses indicate that the gpd-1,4 gene pair encodes the minor isoenzyme, GAPDHase-1, present in all cells of the nematode while the other gene pair (gpd-2,3) encodes the major isoenzyme, GAPDHase-2, preferentially expressed in the bodywall muscle. The G + T-rich and T-rich regions essential for vertebrate beta-globin polyadenylation were also observed for gpd-3.

Conservation of gene organization and trans-splicing in the glyceraldehyde-3-phosphate dehydrogenase-encoding genes of Caenorhabditis briggsae.

The genes encoding body-wall-specific glyceraldehyde-3-phosphate dehydrogenase from Caenorhabditis briggsae were sequenced and compared to the homologous genes from Caenorhabditis elegans. The direct tandem organization of these genes, gpd-2 and gpd-3, and the size and location of the two introns in each gene are the same in C. elegans and C. briggsae. Primer-extension studies demonstrated that the two genes in C. briggsae are trans-splice differentially with the same splice leader (SL) RNAs as are observed in C. elegans. The gdp-2 gene is trans-spliced with SL1 while gdp-3 is trans-spliced with SL2. Significant sequence conservation was observed within the promoter regions of each species and may indicate those regions responsible for body-wall-muscle-specific gene expression and/or differential trans-splicing. Comparisons of the sequences suggest that the tandem repeat of the genes has been subjected to concerted evolution and that C. briggsae and C. elegans diverged much earlier than would be anticipated based on morphological similarities alone. Finally, an open reading frame found several hundred nucleotides upstream from gpd-2, in both species, appears to be homologous to the ATP synthase subunit, ATPase inhibitor protein, from bovine mitochondria.

A squash technique demonstrating embryonic nuclear cleavage of the nematode Caenorhabditis elegans.

A simple squash technique was developed which permits the observation of individual nuclei during embryogenesis of Caenorhabditis elegans. The technique consists of placing several two-cell stage embryos on a subbed slide in a droplet of M-9 salt buffer and incubating them in a sealed humidity chamber at 16.4 degrees C for increasing time intervals. The embryos are then squashed, fixed, and stained with Hoechst 33258. Rate of cleavage at 25.0 degrees C is 1.8 times faster than that at 16.4 degrees C. This yields superimposable growth curves upon correction for temperature. An initial lag in the rate of nuclear cleavage is followed by a burst of cell proliferation, which continues and then slows before 550-580 cells are produced at 4 to 5 hr at 25 degrees C. The squash size increases with cell number and reaches a maximum at about the 400-cell stage when early morphogenesis begins. The second half of embryogenesis is characterized by histogenesis in which the cells are held more tightly together, individual nuclei become less distinct, and the squash size decreases to a minimum as a small worm is formed.

Simple adaptations to extend the range of flow cytometry five orders of magnitude for the DNA analysis of uni-and multicellular systems.

Procedures and instrumentation are described to extend the capability of a cytometry system to record samples that exhibit a wide range of fluorescence such as multicellular systems. The method employs a log amplifier in combination with a set of neutral density filters that reduces the incident light reaching the photomultiplier tube. With any given filter, signals within an intensity range of 200-fold can be measured; different filters can be used to obtain an extended overall range. Polystyrene fluorescent microspheres and a variety of mithramycin stained biological samples ranging from yeast cells to Paramecium were processed by the system. The relative DNA content of individual multicellular embryos was determined for a heterogeneous population of embryonic stages isolated from the nematode, Caenorhabditis elegans. As part of the evaluation of the procedure, the practical upper limit of range extension was determined. The most intense fluorescent signal was produced when untreated pecan pollen stained with ethidium bromide fluoresced with a factor (8.4 +/- 1.3) X 10(4) more than ethidium bromide stained E. coli cells.

Two isoenzymes of glyceraldehyde-3-phosphate dehydrogenase in Caenorhabditis elegans. Isolation, properties, and immunochemical characterization.

Two glyceraldehyde-3-phosphate dehydrogenases have been separated and purified from the nematode Caenorhabditis elegans. As defined by starch gel electrophoresis, the faster-migrating isoenzyme, glyceraldehyde-3-phosphate dehydrogenase-2, increases its activity during postembryonic development. In contrast, the slower-migrating isoenzyme, glyceraldehyde-3-phosphate dehydrogenase-1, is enriched in isolated embryos. Both isoenzymes were initially purified by ammonium sulfate fractionation, gel filtration, and NAD+-agarose affinity chromatography. The separation of both isoenzymes as well as their purification to homogeneity was obtained by preparative chromatofocusing. The subunit molecular weight of each isoenzyme is 38,500 +/- 500. A tetrameric native molecular weight of 157,000 +/- 2000 was determined for glyceraldehyde-3-phosphate dehydrogenase-2. Monospecific rabbit polyclonal antibodies were initially raised against the major isoenzyme and subsequently used to characterize both isoenzymes. Staphylococcus aureas V8 protease digests of each isoenzyme were separated electrophoretically and stained immunochemically, providing evidence that the two isoenzymes differed in their amino acid sequences. Developmental immunocytochemical studies suggest that the embryonic-enriched isoenzyme, glyceraldehyde-3-phosphate dehydrogenase-1, is present in all cells. The second isoenzyme, exhibiting the major activity during postembryonic larval development, may define a body-wall-muscle specific activity which is located within the actin-containing I and A zones of the nematode's sarcomeres.

Studies of DNA bound RNA molecules isolated from nucleoids of Escherichia coli.

Methods are developed for studying RNA molecules bound directly to DNA in bacterial nucleoids. It is found that among the 1000-3000 nascent RNA chains that normally are attached to the DNA via their associated RNA polymerase molecules, 74 +/- 14 chains per nucleoid can be bound differently. These chains unlike the other nascent RNAs remained bound to the DNA after the chromosome was deproteinized and sheared. Sensitive assays using radioactive labels detected no RNA polymerase involved in the RNA-DNA linkage. The linkage was stable at low temperatures, but the RNA separated from the DNA at high temperature. The bound RNA molecules were heterodisperse (weight average length 1200 bases). Pulse-chase experiments and studies of the fate of these RNA molecules in rifampicin treated cells demonstrated that they are nascent RNAs, degraded or released from the DNA in vivo with kinetics similar to that of the total nascent RNA. Hybridization analyses showed that the chains are composed at least in part of nascent rRNA and known mRNA molecules. Some, but not more than 5% of the bound chains, contained sequences of about 300 nucleotides in length, bound to the DNA in an RNase resistant form.

Muscle differentiation in normal and cleavage-arrested mutant embryos of Caenorhabditis elegans.

The differentiation of body-wall muscle cells was studied in the nematode Caenorhabditis elegans. Specific antibodies to myosin and paramyosin, major protein constituents of differentiated muscle, react with mesodermal cells in wild-type embryos towards the end of the first half of embryogenesis. Immunoreactive cells (2-16) first appear in embryos with 400-450 of the 550 cells present at hatching. Such embryos have developed at 25.5 degrees C for 4-4 1/2 hr beyond the two-cell stage. As development proceeds, a maximum of 81 immunoreactive cells forms four columns running anterior-posterior. Each column is composed of two lines of tightly opposed round cells, which then elongate into spindle-shaped cells. Mutant embryos in which cleavage arrests prematurely also generate cells that produce myosin and paramyosin. The initiation of muscle differentiation appears to be independent of the number of cell or nuclear divisions within a lineage or of the proliferation of other cells. These results suggest that the biosynthesis of muscle-specific proteins by nematode embryonic muscle cells is regulated by mechanisms intrinsic to these cells.

Determinants of enzyme thermostability observed in the molecular structure of Thermus aquaticus D-glyceraldehyde-3-phosphate dehydrogenase at 25 Angstroms Resolution.

The crystal structure of holo D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the extreme thermophile Thermus aquaticus has been solved at 2.5 Angstroms resolution. To study the determinants of thermostability, we compare our structure to four other GAPDHs. Salt links, hydrogen bonds, buried surface area, packing density, surface to volume ratio, and stabilization of alpha-helices and beta-turns are analyzed. We find a strong correlation between thermostability and the number of hydrogen bonds between charged side chains and neutral partners. These charged-neutral hydrogen bonds provide electrostatic stabilization without the heavy desolvation penalty of salt links. The stability of thermophilic GAPDHs is also correlated with the number of intrasubunit salt links and total hydrogen bonds. Charged residues, therefore, play a dual role in stabilization by participating not only in salt links but also in hydrogen bonds with a neutral partner. Hydrophobic effects allow for discrimination between thermophiles and psychrophiles, but not within the GAPDH thermophiles. There is, however, an association between thermostability and decreasing enzyme surface to volume ratio. Finally, we describe several interactions present in both our GAPDH and a hyperthermophilic GAPDH that are absent in the less thermostable GAPDHs. These include a four-residue salt link network, a hydrogen bond near the active site, an intersubunit salt link, and several buried Ile residues.

Analysis by isopycnic centrifugation of isolated nucleoids of Escherichia coli.

The isolated, formaldehyde-fixed nucleoid of E. coli has been analyzed by isopycnic centrifugation in CsCl density gradients. The membrane-free nucleoid bands at a density of 1.69 +/- 0.02 g/cm3. The membrane-associated nucleoid bands at a density of 1.46 +/- 0.02 g/cm3. Both species sediment to equilibrium as nearly monodisperse bands in CsCl, suggesting that the nucleoid components of DNA, RNA and protein are present in relatively constant ratios. These ratios are constant regardless of the position of the nucleoids in the heterogeneous sedimentation profile of a preparative sucrose gradient. The fixed nucleoids remain condensed during isopycnic centrifugation and there is no detectable loss of RNA from the nucleoid.

Conditional absence of mitosis-specific antigens in a temperature-sensitive embryonic-arrest mutant of Caenorhabditis elegans.

A monoclonal antibody, specific to phosphoproteins in mitotic HeLa cells was found to crossreact with a similar set of proteins in embryos of the nematode, Caenorhabditis elegans. In C. elegans, as in mammalian cells, the highly conserved antigenic epitope is associated with a family of high molecular weight polypeptides. The antigenic reactivity of these multiple proteins also depends on their phosphorylation, since antibody binding is reduced after alkaline phosphatase treatment. The antigens are detected at the centrosomes, and in the nuclear region and surrounding cytoplasm of mitotic cells. The significance of these antigens is emphasized by their absence at restrictive temperature in embryos of the temperature-sensitive embryonic-arrest mutant, emb-29V. Furthermore, temperature shift-down experiments suggest that the emb-29 mutation defines a cell division cycle function that affects an essential activity required for progression into M phase.

A novel set of uncoordinated mutants in Caenorhabditis elegans uncovered by cold-sensitive mutations.

A set of uncoordinated (Unc) cold-sensitive (cs) mutants was isolated at a stringent condition of 11 degrees C. About half of the 13 independently isolated cs-Unc mutants were alleles of three X-linked Unc mutants that exhibited the "kinker" phenotype. The remaining four isolates identified new mutants that exhibited "kinker," "coiler," or severe paralytic phenotypes. The temperature-sensitive period (TSP) for each gene was determined. As a homozygous or heterozygous dominant, unc-125 exhibited a TSP throughout all stages of development. Its severe paralysis was immediately observed upon a shift down to 11 degrees C and reversed upon a shift up to 23 degrees C. The reversible thermolability of the unc-125 gene product indicated that it may function in a multicomponent process involved in neuro-excitation.

Thyroid status and muscarinic receptor density and affinity in rat intestinal smooth muscle.

Hypothyroid and hyperthyroid states are associated with constipation and diarrhea, respectively. To determine the possible involvement of the cholinergic system in these disorders we studied the effect of thyroid status (thyroidectomy or thyroxin injection for 10-20 weeks) on intestinal smooth muscle muscarinic receptors. Receptor density and affinity were determined in the ileum and colon with [3H]-quinuclidinyl benzilate (QNB), and compared with the responsiveness of isolated muscle strips to cumulative doses of bethanechol. Neither the affinity nor the density of muscarinic receptors was significantly altered by chronic hypo- or hyperthyroidism: KD approximately 1nM; Bmax approximately 200 fmole/mg protein. Similarly, there was no change in functional response of the ileum or colon to cholinergic stimulation. The results suggest that chronic hypothyroidism or hyperthyroidism do not alter muscarinic receptors in rat ileal or colonic muscle.

Preliminary crystallographic analysis of glyceraldehyde 3-phosphate dehydrogenase from the extreme thermophile Thermus aquaticus.

Crystals have been obtained of glyceraldehyde 3-phosphate dehydrogenase from the extreme thermophile, Thermus aquaticus. This enzyme is stable and active at 363 K, thus its three-dimensional structure should add insight into the structural basis of protein thermostability. Large high-quality crystals were grown using isopropanol and polyethylene glycol at pH 8.4. They crystallize in the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 144.77 (6), b = 148.77 (5), c = 149.50 (7) A, and diffract to beyond 2.8 A. The volume of the unit cell and the packing observed in other GAPDH structures suggest that there are two tetramers per asymmetric unit. With 300 kDa/asymmetric unit expected in this form, its solution represents a challenging molecular replacement problem. A low-resolution data set has been recorded and used to carry out self-rotation, cross-rotation and Patterson-correlation refinement calculations. We found that the Q molecular axes of both tetramers are approximately coincident with the crystallographic a axis, and the non-crystallographic symmetry relating the two tetramers is approximately a rotation of 90 degrees about the a axis.

Molecular relationships between closely related strains and species of nematodes.

Electrophoretic comparisons have been made for 24 enzymes in the Bergerac and Bristol strains of Caenorhabditis elegans and the related species, Caenorhabditis briggsae. No variation was detected between the two strains of C. elegans. In contrast, the two species, C. elegans and C. briggsae exhibited electrophoretic differences in 22 of 24 enzymes. A consensus 5S rRNA sequence was determined for C. elegans and found to be identical to that from C. briggsae. By analogy with other species with relatively well established fossil records it can be inferred that the time of divergence between the two nematode species is probably in the tens of millions of years. The limited anatomical evolution during a time period in which proteins undergo extensive changes supports the hypothesis that anatomical evolution is not dependent on overall protein changes.

Sedation in children: adequacy of two-hour fasting.

OBJECTIVES: (1) To investigate the relationship between the duration of time that children fasted before a procedure and their gastric volume and pH at the time of the procedure. (2) To compare the variables of gastric pH and volume with historical standards. METHODS: We performed 285 gastroscopies for children aged 0.1 to 18.6 years (mean, 7.5 +/- 5.3) between October 1991 and January 1995. Duration of fasting was 0.5 to 24 hours (mean, 6.7 +/- 5.3) after ingestion of clear liquids. Immediately after intravenously administered sedation, the gastric contents were removed endoscopically with suction and direct visualization to ensure complete evacuation. The volume and pH of the gastric contents were measured and analyzed in comparison with the duration of fasting. The values obtained were also compared with historical standards thought to minimize the risk of aspiration pneumonia: gastric volume 0.4 ml or less per kilogram of body weight and pH of 2.5 or greater. RESULTS: There was no significant correlation between duration of fasting and either gastric volume divided by body weight (mean, 0.68 +/- 1.31 ml/kg; range, 0 to 15.23 ml/kg) or pH (mean, 2.03 +/- 1.40; range, 1 to 8). There was less no significant difference in the percentage of children with gastric volume of 0.4 ml/kg or less or with pH of 2.5 or greater between the groups with the following fasting times: 30 minutes to 3 hours, more than 3 hours to 8 hours, and more than 8 hours. CONCLUSIONS: On the basis of the data in this study and a review of the literature, we concluded that (1) fasting longer than 2 hours after ingesting clear liquids does not significantly change gastric volume or pH, (2) there is no advantage in requiring children to fast for longer than 2 hours after clear liquid ingestion before sedation or anesthesia for any procedure, and (3) fewer than half of pediatric patients actually achieve the "desirable" values of a gastric volume of 0.4 ml/kg or less and a pH value of 2.5 pH units or more, regardless of fast duration, even though these values are presented in the literature as a goal to minimize the risk of aspiration pneumonia.