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A R Place

Publications and source records attributed to A R Place.

36 records · Page 2Linked to original sources
PubMed1985

UGA nonsense mutation in the alcohol dehydrogenase gene of Drosophila melanogaster.

A mutant gene, which we have designated AdhnB, codes for a defective form of the enzyme alcohol dehydrogenase in Drosophila melanogaster. We show that the polypeptide encoded by AdhnB is approximately 2000 Mr smaller than the protein synthesized under the direction of the wild-type alcohol dehydrogenase gene. In contrast, the alcohol dehydrogenase mRNA produced by both genes is the same size. We cloned and sequenced a portion of the protein-coding region of AdhnB and compared it to the same region in the wild-type gene. We found a single base substitution: a change of the TGG tryptophan codon at amino acid 235 to a TGA termination codon. This nonsense mutation accounts for the observed reduction in size of the alcohol dehydrogenase polypeptide. In further studies, we found that the steady-state levels of alcohol dehydrogenase mRNA in flies carrying the AdhnB gene and the wild-type alcohol dehydrogenase gene were indistinguishable. However, the steady-state level of alcohol dehydrogenase polypeptide was reduced to 1% of wild-type levels in flies with the AdhnB gene. Moreover, the rate of alcohol dehydrogenase synthesis in mutant flies was reduced to 50% of that found in wild type. The aberration in AdhnB thus affects both the rate of synthesis and the rate of degradation of the alcohol dehydrogenase peptide. AdhnB is the first reported nonsense mutant in Drosophila.

Alcohol Oxidoreductases↗
PubMed1984

Purification and characterization of the lactate dehydrogenase (LDH-B4) allozymes of Fundulus heteroclitus.

In order to evaluate whether structural differences exist between allelic variants of a B-type lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) in the minnow Fundulus heteroclitus, the allozymes (LDH-Ba4, LDH-Ba/Bb, and LDH-Bb4) were purified to homogeneity by affinity chromatography. Each variant was characterized as to holoenzyme and subunit molecular mass, isoelectric point (pI), thermal and urea stability, and susceptibility to proteolysis. Differences in electrophoretic mobilities were due to a lower pI for LDH-Ba4 (pI = 6.6) than for LDH-Bb4 (pI = 7.2). Stability to inactivation by heat, urea, and proteolysis was in each case: LDH-Bb4 greater than LDH-Ba/Bb greater than LDH-Ba4. Inactivation by trypsin may involve the arginine-rich catalytic loop of lactate dehydrogenase (50). The results suggest that the allozymes differ in their conformational flexibility.

Animals↗
PubMed1984

Kinetic characterization of the lactate dehydrogenase (LDH-B4) allozymes of Fundulus heteroclitus.

In order to evaluate whether functional differences exist between allelic variants (allozymes) of the B-type lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) in the fish Fundulus heteroclitus, the kinetic properties were examined. Differences in product inhibition between allelic isozymes were observed. LDH-Bb4 was found to be inhibited by lower concentrations of both lactate and pyruvate than either LDH-Ba/Bb or LDH-Ba4. While the pH and temperature dependence for maximal catalysis (Vmax) in both the forward and reverse directions were indistinguishable among the allozymes, reaction velocities at low substrate concentrations (Vmax/Km) were significantly different. These differences could be explained by LDH-Bb4 having a greater substrate affinity than LDH-Ba4 at cold temperatures while the reverse was true at elevated temperatures. At a given pH, LDH-Bb4 showed a greater reaction rate at lower temperatures (e.g. 10 degrees C) than LDH-Ba4. The phenomenon was reversed at higher temperatures (e.g. greater than 35 degrees C). When reaction rates were compared at constant relative alkalinity, that is, a constant [OH-]/[H+] ratio, similar findings were obtained. The response for the heterozygous allozyme, LDH-Ba/Bb, was not the arithmetic average of the two homotetrameric allozymes. The biological significance of these allozymic differences is discussed.

Alleles↗
PubMed1983

Formaldehyde mutagenesis in Drosophila. Molecular analysis of ADH-negative mutants.

The Adh gene from 4 formaldehyde-generated ADH-negative mutants of Drosophila melanogaster has been cloned and sequenced. All 4 mutants bear small deletions within the gene, ranging in size from 6 to 34 base pairs. 2 of the deletions lie within a 65-base pair intervening sequence and are accompanied by other aberrations. The other two are within the protein coding region of the gene. Some of these aberrations may be explained by a slipped mispairing mechanism.

Alcohol Dehydrogenase↗
PubMed1983

The use of enzyme kinetics to predict differences in cellular metabolism, developmental rate, and swimming performance between LDH-B genotypes of the fish, fundulus heteroclitus.

In summary, the LDH-B4 allelic isozymes of F heteroclitus are functionally nonequivalent. The kinetic differences observed can be explained by a greater catalytic efficiency by LDH-B4b at colder temperatures than LDH-Ba4. The situation appears to be reversed at higher temperatures. The LDH-BaBb allelic isozymes is not exactly intermediate to the two homotetramers. These in vitro functional differences can be interpreted as being useful to individuals with specific LDH-B phenotypes. Moreover, predictions concerning their significance at the metabolic and/or whole organism level can be made and tested experimentally. We have found both developmental and physiological differences between LDH-B phenotypes that appear to depend on a significant variation in ATP metabolism between the phenotypes. Although we have not elucidated the mechanism by which ATP metabolism is affected, we have shown that the pattern of ATP differences between phenotypes, corresponds precisely with the pattern of kinetic differences between the LDH-B allelic isozymes. The resolution of the evolutionary significance of these phenomena and their underlying molecular mechanism will shed light on the controversy between exponents of the selectionist and neutralist schools of thought.

Adenosine Triphosphate↗
PubMed1982

Deletions at intervening sequence splice sites in the alcohol dehydrogenase gene of Drosophila.

Two formaldehyde-induced, homozygous viable ADH-negative mutants, Adhfn4 and Adhfn6, possess no material that cross-reacts with antibody directed against ADH, no mature mRNA of wild-type size, and greatly reduced amounts of RNA that hybridizes with an Adh probe. We have cloned the genomic DNA sequences from these mutants in bacteriophage lambda Charon 4 and subcloned the Adh region into plasmid vector pBR327. Restriction analyses revealed one small deletion in each of these mutants and DNA sequencing showed that the splice junctions of the 65-base pair (bp) intervening sequence (IVS) were altered. Both cloned mutant Adh genes, as well as the wild-type gene, are capable of promoting correct specific transcription initiation in HeLa cell nuclear extracts in vitro. We conclude that Adhfn4 and Adhfn6 are defective in RNA processing. Our results provide evidence for the importance of the splice junction sequences in normal ADH RNA processing and stabilization in Drosophila. We also speculate that splicing of ADH RNA proceeds in a nonrandom manner: mutations in one of the intervening sequences appear to cause accumulation of a large ADH RNA containing at least one other IVS.

Alcohol Dehydrogenase↗
PubMed1981

Alcohol dehydrogenase gene of Drosophila melanogaster: relationship of intervening sequences to functional domains in the protein.

The gene that codes for Drosophila alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase EC 1.1.1.1) was identified in a bacteriophage lambda library of genomic Drosophila DNA by using ADH cDNA cloned DNA as a probe. The DNA sequence of the protein encoding region was shown to be in agreement with the amino acid sequence of the ADH. Two intervening DNA sequences (introns) were identified within the protein encoding region: one was 65 nucleotides and located between the codons for amino acid residues 32 and 33, and one was 70 nucleotides and located between the codons for amino acid residues 167 and 168. Both contained the 5' G-T and 3' A-G dinucleotides characteristic of intron boundaries of eukaryotic genes. On the basis of secondary structure predictions, the first 140 amino acid residues of Drosophila ADH are in an alternating beta-sheet/alpha-helix arrangement which is characteristic of the coenzyme binding domain of dehydrogenases. The smaller of the two introns interrupts the domain predicted to bind the adenine portion of the coenzyme.

Alcohol Dehydrogenase↗
PubMed1979

Genetic variation and relative catalytic efficiencies: lactate dehydrogenase B allozymes of Fundulus heteroclitus.

In order to evaluate whether functional differences exist between allelic variants of a B type lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) in the teleost fish Fundulus heteroclitus (Linnaeus), the kinetic properties of pyruvate reduction were examined. While the pH dependence and the temperature dependence for maximal catalysis were indistinguishable among the allozymes, reaction velocities at low pyruvate concentrations were significantly different. At pH values below 8.00, the LDH-BbBb allozyme showed a greater reaction rate at lower temperatures (e.g., 10 degrees C) than LDH-BaBa. The phenomenon was reversed at higher temperatures (e.g., greater than 25 degrees C) for pH values between 6.50 and 7.00. The rates for the heterozygous phenotype, LDH-BaBb, were not the arithmetic average of the two homotetrameric allozymes. When reaction rates were compared at constant relative alkalinity, that is, a constant [OH-]/[H+] ratio, the findings were similar. The differences in the temperature dependence and the pH dependence for pyruvate reduction found between the LDH-B allozymes may reflect a selective adaptation and help explain the geographical variation in the Ldh-B gene frequencies of F. heteroclitus.

Animals↗
PubMed1978

Genetic bases for protein polymorphism in Fundulus heteroclitus (L.).I. Lactate dehydrogenase (Ldh-B), malate dehydrogenase (Mdh-A), glucosephosphate isomerase (Gpi-B), and phosphoglucomutase (Pgm-A).

Electrophoretic analysis has shown populations of F. heteroclitus to possess variants at four enzyme-coding loci: Ldh-B, Mdh-A, Gpi-B, and Pgm-A. Based on the phenotypic distribution in the F1 generation, each variant segregates as an autosomally inherited codominant allele. A pairwise comparison of the expected phenotypic classes among these loci showed no evidence of strong linkage; however, weak linkage could not be ruled out. Despite the considerable genetic divergence of populations from the geographical extremes of this species, offspring resulting from crosses between individuals from these localities show viabilities similar to those found for crosses of local populations.

Animals↗
PubMed1978

Biochemical genetics of Fundulus heterolitus (L.). I. Temporal and spatial variation in gene frequencies of Ldh-B, Mdh-A, Gpi-B, and Pgm-A.

Natural populations of Fundulus heteroclitus show extensive spatial variation in gene frequencies at four unlinked polymorphic loci. Large clinal changes in gene frequencies were found for Ldh-B, Mdh-A, and Gpi-B, whereas the spatial variation for the Pgm-B locus was small. Since the geographical area over which these clines are found is characterized by a steep thermal gradient, the clines in gene frequency are correlated with a directional change in mean water temperature. Maximum gene diversity of these four loci was correlated with annual fluctuations in water temperature. Temporal stability of the allelic frequencies was established for a 2--4 year period.

Animals↗
PubMedDate not supplied

Neurotensin modulates the composition of pancreatic exocrine secretions in chickens.

The effects of neurotensin on pancreatic exocrine secretion were examined in fasted, conscious White Leghorn hens. A cannula was surgically implanted in the central duct serving the ventral lobe of the pancreas in order to collect pure pancreatic juice. Following recovery, neurotensin was infused intravenously at 3.6 or 10.8 pmol/kg*min. The volume and pH of the pancreatic secretions were recorded and total pancreatic protein concentration, amylase, lipase, trypsin, and chymotrypsin activity were measured every 30 min for 2 hr and compared to secretions following the infusion of 0.9% saline. Our results demonstrated that neurotensin did not affect the pH nor the pancreatic juice protein concentration, but did increase secretion rate following neurotensin infusion at 3.6 pmol/kg*min. Amylase activity was significantly depressed during neurotensin infusions, while lipase (both pancreatic and carboxylester lipase) activity was significantly elevated. The ratio of amylase to lipase activity was especially depressed by neurotensin infusion at 10.8 pmol/kg*min. Insufficient secretory activity prevented a balanced statistical analysis of chymotrypsin activity, but from a pooled analysis, neurotensin had no effect on protease activity in the pancreatic juice. These results support our current research indicating that neurotensin may be a hormonal regulator of postprandial lipid digestion in chickens.

Amylases↗
PubMedDate not supplied

An experimental test of dietary enzyme modulation in pine warblers Dendroica pinus.

Modulation of gut function is important in an ecological and evolutionary context because it likely determines what food items an animal can and cannot eat. We examined how diet affects activity of digestive enzymes in an omnivorous bird, the pine warbler (Dendroica pinus). Pine warblers were fed insect-based, fruit-based, and seed-based diets for approximately 54 d. We then measured activity of amylase, maltase, sucrase, aminopeptidase-N, trypsin, chymotrypsin, carboxypeptidase A, carboxypeptidase B, pancreatic lipase, and carboxyl ester lipase. We predicted that carbohydrase activities would be highest in birds fed the diet highest in carbohydrates (fruit based), protease activities would be highest in those fed the diet highest in protein (insect based), and lipase activities would be highest in those fed the diets highest in lipid (insect based and seed based). Also, we predicted that pine warblers would exhibit greater dietary modulation of enzyme activity than reported for a less omnivorous congener, the yellow-rumped warbler (Dendroica coronata). All predictions were upheld, supporting the hypothesis that pine warblers modulate the activity of digestive enzymes in proportion to demand from substrates in the diet.

Adaptation, Physiological↗