A gibberellin-regulated gene from wheat with sequence homology to cathepsin B of mammalian cells

A previous report described several cDNAs corresponding to mRNAs which accumulated in wheat aleurone layers treated with gibberellic acid (GA) (Baulcombe and Buffard, 1983). The protein sequence deduced from one of these clones (2529) has extensive similarity to the thiol protease, cathepsin B from mammalian cells. Southern analysis of wheat DNA has shown that the 2529 mRNA is encoded by a small family of genes carried on the group 4 chromosome. The nucleotide sequence of a member of the gene family expressed at a low level in aleurone layers and the use of a primer extension assay to identify a clone of a member of the gene family producing an abundant mRNA are reported. The 2529 mRNA accumulates in the scutellum and the aleurone layer of germinating grains where its expression is regulated by GA. In the scutellum the expression was restricted to the parenchyma, suggesting that the 2529 product may have a role other than for mobilization of the endosperm.     

A novel extracellular carbohydrase produced by Lipomyces tetrasporus

Summary The ascosporogenous yeast Lipomyces tetrasporus produced an unusual extracellular carbohydrase. It was purified to homogeneity using ammonium sulphate precipitation and DEAE Bio-gel A ion-exchange chromatography. While retaining highest activity on low-molecular-weight saccharides such as maltose and nigerose, it displays considerable activity towards polymeric substrates including soluble starch. It is particularly unusual in that it also hydrolyses dextran and has a very high affinity for this substrate. The enzyme has an exo-lytic mode of action with the only hydrolysis product, glucose, being released in the -anomeric form. Optimum activity occurs at pH 4.5 and at 50°C. It is a glycoprotein, and has an M _r value of 150 000 (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) — 183 000 (fast protein liquid chromatography) and a pI of 6.0. Offprint requests to: C. T. Kelly     

Transgenic animals as a tool for studying the effect of the c-myc proto-oncogene on cardiac development

Transgenic animals provide a model system to elucidate the role of specific proteins in development. This model is now being used increasingly in the cardiovascular system to study cardiac growth and differentiation. During cardiac myocyte development a transition occurs from hyperplastic to hypertrophic growth. In the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc functions to regulate myocyte proliferation and/or differentiation, we examined the in vivo effect of increasing c-myc expression during fetal development and of preventing the decrease in c-myc mRNA expression that normally occurs during myocyte development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. Increased c-myc mRNA expression is associated with both atrial and ventricular enlargement in the transgenic mice. This increase in cardiac mass is secondary to myocyte hyperplasia, with the transgenic hearts containing greater than twice as many myocytes as nontransgenic hearts. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth, and suggest a regulatory role for the c-myc protooncogene in cardiac myogenesis.     

Purification of ribulose 1,5-bisphosphate carboxylase/oxygenase and of carboxysomes from Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa (Montagu)

The bacterial symbionts of many marine invertebrates contain ribulose 1,5-bisphosphate (RuBP) carboxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50–90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small subunits, with molecular weights of 53,110 and 11,100 respectively. Particulate RuBP carboxylase activity was associated with polyhedral bodies 50–100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were inferred to be carboxysomes. Native electrophoresis of isolated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl-sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 21% of the total carboxysomal protein. The most abundant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function.Abbreviations FPLC fast performance liquid chromatography - IB isolation buffer - PAGE polyacrylamide gel electrophoresis - RuBP carboxylase - ribulose 1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl-sulphate     

Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.     

Clinical and molecular heterogeneity of phenylalanine hydroxylase deficiencies in France

RFLPs of 68 normal and 74 mutant alleles at the phenylalanine hydroxylase (PAH) locus were determined in 37 French kindreds. A total of 23 haplotypes, including 18 normal and 16 mutant alleles, were observed. Two-thirds of all mutant alleles were confined within only four haplotypes, while the last third was accounted for by 12 haplotypes, including eight haplotypes absent from Caucasian pedigrees reported thus far. Several mutant haplotypes were present in typical phenylketonuria only, others were present in variants only, and some were present in both. In addition, a particular mutant haplotype (haplotype 2) was found to harbor different mutations in our series, resulting in either typical phenylketonuria or in mild hyperphenylalaninemias. The diploid combination of so many mutant haplotypes in PAH-deficient patients and of compound heterozygosity at the PAH locus in southern Europe might account for the broad spectrum of individual phenotypes observed in France.     

Effect of length changes on sensitivity of helical artery strips to adenosine

The relationship between length and the responses of helical strips of rabbit femoral and coronary arteries to vasoactive agents was investigated by conducting concentration--response determinations at the length for maximum active force (Lmax) and a shorter length. The mean effective dose (ED50) values for norepinephrine (NE) were smaller when femoral artery strips were set at Lmax, in comparison to the values at the shorter length. Maximal relaxation of femoral artery strips by adenosine was greater when the strips were set at Lmax. However, adenosine ED50 values were smaller at Lmax only in groups of strips in which responses at Lmax were obtained prior to those at the shorter length. Experiments with coronary artery strips did not demonstrate consistent relationships between strip length and ED50 values for acetylcholine (ACh) or adenosine. The results of experiments with artery strips from normotensive and those from one-kidney, one-clip hypertensive rabbits were similar. Thus, the femoral artery data indicate that helical artery strip preparations may exhibit length-dependent sensitivity to a vasodilator agent as well as vasoconstrictor agents under certain experimental conditions. However, the coronary artery data suggest that length may not affect sensitivity of isolated artery preparations from all vascular beds in the same manner.     

Genetic Controls over Activities of Tyrosinase and Dopachrome Conversion Factor in Murine Melanocytes

We evaluated the three catalytic activities of tyrosinase and one activity of dopachrome conversion factor (DCF) in extracts made from skins of 6-day-old yellow and nonyellow mice. At least one of the catalytic activities of tyrosinase and of DCF correlate with the color of pigment being produced in the hair follicles of the mice. We use these data to evaluate existing hypotheses about the mechanism of the interacting genetic controls over melanogenesis.