Induction of β-N-acetylhexosaminidase in Aspergillus oryzae

Summary Extracellular -N-acetylhexosaminidase in basic specific activity 1.5 U/mg protein was induced 15 – 35 times (up to 50 U/mg protein) by mixture of chitooligomers (crude chitin hydrolysate), 10 – 20 times (20 – 30 U/mg protein) by N-acetylglucosamine, and 10 times (14 U/mg protein) by chitosan in Aspergillus oryzae. Addition of NaCl (15 – 23 g/l) to the cultivation medium enhanced the induction in 10 – 20 %.     

The Higher Structure of Chromatin in the LCR of the β-Globin Locus Changes during Development

The β-globin locus control region (LCR) is able to enhance the expression of all globin genes throughout the course of development. However, the chromatin structure of the LCR at the different developmental stages is not well defined. We report DNase I and micrococcal nuclease hypersensitivity, chromatin immunoprecipitation analyses for histones H2A, H2B, H3, and H4, and 3C (chromatin conformation capture) assays of the normal and mutant β-globin loci, which demonstrate that nucleosomes at the DNase I hypersensitive sites of the LCR could be either depleted or retained depending on the stages of development. Furthermore, MNase sensitivity and 3C assays suggest that the LCR chromatin is more open in embryonic erythroblasts than in definitive erythroblasts at the primary- and secondary-structure levels; however, the LCR chromatin is packaged more tightly in embryonic erythroblasts than in definitive erythroblasts at the tertiary chromatin level. Our study provides the first evidence that the occupancy of nucleosomes at a DNase I hypersensitive site is a developmental stage-related event and that embryonic and adult cells possess distinct chromatin structures of the LCR.     

Restriction enzyme analysis of the β-globin gene in DNA from β°-thalassaemic subjects from Ferrara

The map of restriction sites including and surrounding the δ- and β-globin genes has been established for three Ferrara β°-thalassaemic subjects. The fragments obtained using nine restriction enzymes do not show any differences from normal DNA. Among others, restriction enzymes giving short fragments at the 5′ and 3′ ends of the β-globin structural gene have been employed. The results obtained for the thalassaemic DNA are identical to those for control DNA, thus excluding the presence of extensive deletions in or adjacent to the coding regions of the β-globin gene in Ferrara β°-thalassaemia.     

Genetic Heterogeneity of the β-Globin Gene in Various Geographic Populations of Yunnan in Southwestern China

Objectives

The aim of this study was to investigate the geographic distribution of β-globin gene mutations in different ethnic groups in Yunnan province.

Methods

From 2004 to 2014, 1,441 subjects with hemoglobin disorders, identified by PCR-reverse dot blot and DNA sequencing, were studied according to ethnicity and geographic origin. Haplotypes were examined among 41 unrelated thalassemia chromosomes.

Results

Eighteen β-thalassemia mutations and seven hemoglobin variants were identified for 1,616 alleles in 22 different ethnic groups from all 16 prefecture-level divisions of Yunnan. The prevalence of β-thalassemia was heterogeneous and regionally specific. CD 41-42 (-TCTT) was the most prevalent mutation in the populations of northeastern Yunnan. CD 17 (A>T) was the most common mutation in the populations of southeastern Yunnan, especially for the Zhuang minority, whereas Hb E (CD 26, G>A) was the most prevalent mutation in populations of southwestern Yunnan, especially for the Dai minority. Among the seven types of haplotypes identified, CD 17 (A>T) was mainly linked to haplotype VII (+ - - - - - +) and IVS-II-654 (C>T) was only linked to haplotype I (+ - - - - + +).

Conclusion

Our data underline the heterogeneity of β-globin gene mutations in Yunnan. This distribution of β-globin mutations in the geographic regions and ethnic populations provided a detailed ethnic basis and evolutionary view of humans in southern China, which will be beneficial for genetic counseling and prevention strategies.     

Induction of the Synthesis of Endo-1,4-β-xylanase and β-Galactosidase in Original and Recombinant Strains of the Fungus Penicillium canescens

The induction of synthesis of the secreted enzymes endo-1,4--xylanase (EC 3.2.1.8) and -galactosidase (EC 3.2.1.23) in original and recombinant Penicillium canescens strains has been studied. In all producer strains, the synthesis of these enzymes was induced by arabinose and its metabolite arabitol. The two enzymes differed in the concentration of arabinose required for induction: the synthesis of -galactosidase was most pronounced at 1 mM, whereas maximum synthesis of endo-1,4--xylanase was observed at 5–10 mM. An increase in the number of endo-1,4--xylanase copies in the high-copy-number strain of the fungus suppressed the synthesis of -galactosidase; the synthesis of endo-1,4--xylanase in the high-copy-number recombinant producing -galactosidase was affected to a lesser extent. The amount of enzymes synthesized did not depend on the saccharide used as the sole source of carbon for growing the mycelium prior to its transfer to the inducer-containing medium.     

Molecular Analysis of the β-Globin Gene Cluster in the Niokholo Mandenka Population Reveals a Recent Origin of the βS Senegal Mutation

A large and ethnically well-defined Mandenka sample from eastern Senegal was analyzed for the polymorphism of the beta-globin gene cluster on chromosome 11. Five RFLP sites of the 5' region were investigated in 193 individuals revealing the presence of 10 different haplotypes. The frequency of the sickle-cell anemia causing mutation (beta(S)) in the Mandenka estimated from this sample is 11.7%. This mutation was found strictly associated with the single Senegal haplotype. Approximately 600 bp of the upstream region of the beta-globin gene were sequenced for a subset of 94 chromosomes, showing the presence of four transversions, five transitions, and a composite microsatellite polymorphism. The sequence of 22 beta(S) chromosomes was also identical to the previously defined Senegal haplotype, suggesting that this mutation is very recent. Monte Carlo simulations (allowing for a specific balancing selection model, a logistic growth of the population, and variable initial frequencies of the Senegal haplotype) were used to estimate the age of the beta(S) mutation. Resulting maximum-likelihood estimates are 45-70 generations (1,350-2,100 years) for very different demographic scenarios. Smallest confidence intervals (25-690 generations) are obtained under the hypothesis that the Mandenka population is large (N(e) >5,000) and stationary or that it has undergone a rapid demographic expansion to a current size of >5,000 reproducing individuals, which is quite likely in view of the great diversity found on beta(A) chromosomes.     

Synthesis of β-galactosidase inEscherichia coli in the presence of phenylalanine analogues

The effect of phenylalanine analogues (p-F-phenylalanine, phenylserine and furylalanine) is described on the synthesis of inducible β-galactosidase inEscherichia coli ML-30 and phenylalanine requiring mutant ML-48. The incorporation of these analogues into the enzyme molecule results in the formation of a protein sensitive to a different extent to heat, urea and trypsin. The influence of the analogues on the ability to concentrate inducer inside the cells is also described. The different effect of the analogues on the synthesis and stability of the enzyme is discussed.     

Gene Conversion and Functional Divergence in the β-Globin Gene Family

Different models of gene family evolution have been proposed to explain the mechanism whereby gene copies created by gene duplications are maintained and diverge in function. Ohta proposed a model which predicts a burst of nonsynonymous substitutions following gene duplication and the preservation of duplicates through positive selection. An alternative model, the duplication–degeneration–complementation (DDC) model, does not explicitly require the action of positive Darwinian selection for the maintenance of duplicated gene copies, although purifying selection is assumed to continue to act on both copies. A potential outcome of the DDC model is heterogeneity in purifying selection among the gene copies, due to partitioning of subfunctions which complement each other. By using the d_N/d_S () rate ratio to measure selection pressure, we can distinguish between these two very different evolutionary scenarios. In this study we investigated these scenarios in the -globin family of genes, a textbook example of evolution by gene duplication. We assembled a comprehensive dataset of 72 vertebrate -globin sequences. The estimated phylogeny suggested multiple gene duplication and gene conversion events. By using different programs to detect recombination, we confirmed several cases of gene conversion and detected two new cases. We tested evolutionary scenarios derived from Ohtas model and the DDC model by examining selective pressures along lineages in a phylogeny of -globin genes in eutherian mammals. We did not find significant evidence for an increase in the ratio following major duplication events in this family. However, one exception to this pattern was the duplication of -globin in simian primates, after which a few sites were identified to be under positive selection. Overall, our results suggest that following gene duplications, paralogous copies of -globin genes evolved under a nonepisodic process of functional divergence.[Reviewing Editor: Martin Kreitman]     

Induction of xylanase in Aspergillus tamarii by methyl β-d-xyloside

Aspergillus tamarii produced extracellular xylanase and intracellular β-xylosidase inductively in washed glucose-grown mycelia incubated with xylan and methyl β-d-xyloside, a synthetic glycoside. Methyl β-d-xyloside was a more effective inducer than xylan at the same concentration for both enzymes. Glucose and cycloheximide were found to inhibit xylanase production by methyl β-d-xyloside. Methyl β-d-xyloside was hydrolyzed to xylose by mycelial extract in vitro. Received: 23 May 1996 / Received revision: 5 September 1996 / Accepted: 13 October 1996     

Mutations Affecting Synthesis of β-Galactosidase Activity in the Yeast KLUYVEROMYCES LACTIS

Fifty-one mutants of Kluyveromyces lactis that cannot grow on lactose (Lac^-) were isolated and characterized. All of the mutations are in nuclear genes, are recessive in their wild-type allele and define seven complementation groups, which we designate lac3 through lac9. Strains bearing mutations in lac3, lac5, lac7, lac8 and lac9 are also unable to grow on galactose (Gal^-). Since the Gal^- and Lac^- phenotype co-segregate, they are probably due to a single mutation. Strains bearing mutations in any of the seven complementation groups grow normally on glucose. However, strains bearing mutations in lac3, lac5 and lac6 do not grow on glucose if lactose is also present in the medium. Likewise, strains bearing mutations in lac3 and lac5 do not grow on glucose in the presence of galactose. Complementation groups lac4 and lac5 are loosely linked and map within a cluster of auxotrophic mutations on a chromosome that we designate chromosome 2. The remaining five groups are unlinked. Thus, there is no evidence for clustering of Lac genes into an operon-like regulatory unit.——To further characterize the nature of the Lac^- phenotype, the basal and inducible level of β-galactosidase activity were measured. All mutants had nearly normal basal enzyme levels, except those in lac4, which had barely detectable levels. Inducible enzyme levels varied from barely detectable levels in mutants bearing lac4 mutations up to four-fold inducible levels in strains bearing mutations in other complementation groups. In all cases, however, induction levels were below the 30-fold level obtained in wild-type cells. Three strains bearing lac5 mutations contain increased enzyme activity in the absence of inducer, indicating constitutive synthesis of β-galactosidase. In summary, these data indicate that several genes are necessary for synthesis of β-galactosidase activity.     

Synthesis of β-Tubanol

Synthesis of β-tubanol (VIa) was achieved by the bromination of dihydro-β-tubanol acetate (VIIb) followed by dehydrobromination and the subsequent hydrolysis. 2,2-Dimethyl-5-hydroxy-chromanone as well as its derivatives (IV) and the corresponding chromanols (V) and chromans (VII) were also prepared.     

Induction of β-oxidation enzymes and microbody proliferation in Aspergillus nidulans

Aspergillus nidulans is able to grow on oleic acid as sole carbon source. Characterization of the oleate-induced β-oxidation pathway showed the presence of the two enzyme activities involved in the first step of this catabolic system: acyl-CoA oxidase and acyl-CoA dehydrogenase. After isopicnic centrifugation in a linear sucrose gradient, microbodies (peroxisomes) housing the β-oxidation enzymes, isocitrate lyase and catalase were clearly resolved from the mitochondrial fraction, which contained fumarase. Growth on oleic acid was associated with the development of many microbodies that were scattered throughout the cytoplasm of the cells. These microbodies (peroxisomes) were round to elongated, made up 6% of the cytoplasmic volume, and were characterized by the presence of catalase. The β-oxidation pathway was also induced in acetate-grown cells, although at lower levels; these cells lacked acyl-CoA oxidase activity. Nevertheless, growth on acetate did not cause a massive proliferation of microbodies in A. nidulans. Received: 8 March 1996 / Accepted: 5 August 1996     

Minor Secondary-Structure Variation in the 5"-Untranslated Region of the β-Globin mRNA Changes the Concentration Requirements for eIF2

Nucleotide sequence changes increasing the number of paired bases without producing stable secondary structure in the 5"-untranslated region (5"-UTR) of the -globin mRNA had a slight effect on its translation in rabbit reticulocyte lysate at low mRNA concentration and dramatically decreased translation efficiency at a high concentration. The removal of paired regions restored translation. Addition of purified eIF2 to the lysate resulted in equal translation efficiencies of templates differing in structure of 5"-UTR. A similar effect was observed for p50, a major mRNP protein. Other mRNA-binding initiation factors, eIF4F and eIF4B, had no effect on the dependence of translation efficiency on mRNA concentration. Analysis of the assembly of the 48S initiation complex from its purified components showed that less eIF2 is required for translation initiation on the -globin mRNA than on its derivative containing minor secondary structure elements in 5"-UTR. According to a model proposed, eIF2 not only delivers Met-tRNA, but it also stabilizes the interaction of the 40S ribosome subunit with 5"-UTR, which is of particular importance for translation initiation on templates with structured 5"-UTR.     

Induction of β galactosidase in the yeast Kluyveromyces lactis

Summary The inductive effect of lactose, -methyl-thio-D-galactopyranoside, (TMG) and glucose on galactosidase synthesis in Kluyveromyces lactis has been studied. Whereas TMG gave a five fold stimulation of the rate of -galactosidase synthesis, lactose only gave a small stimulation. Glucose caused represssion at levels above 10^-3M but stimulated -galactosidase synthesis when added at lower concentrations.     

Separation of α- and β-Globin Messenger RNAs

THE 10S RNA fraction of reticulocytes from various species contains the haemoglobin messenger RNA^1–4. When this 10S RNA fraction is added to a cell-free system derived from reticulocytes or Krebs II ascites cells, it directs the synthesis of α and β chains of haemoglobin^5–8. The α and β messenger RNA molecules contained in this fraction, however, have not yet been separated and identified. When reticulocyte. RNA of mouse is subjected to electrophoresis on 6% polyacrylamide gels, the 10S fraction contains two major bands and three minor bands⁹, suggesting that the major lOS RNA bands contain the messenger RNAs for the α- and β-globin chains.     

Synthesis and assembly of soybean β-conglycinin in vitro

The construction of SP6-derived expression plasmids that encode normal and modified -conglycinin subunits is described. With the exception of an additional methionine at their NH₂-terminal ends and the lack of glycans, the normal subunits synthesized at the direction of these plasmids coresponded to mature and subunits isolated from soybean seeds. The subunits assembled into trimers in vitro that were equivalent in size to those formed in vivo. This result shows that the glycans are not required either for protein folding or oligomer assembly. Subunits produced from other plasmids, which had modifications in a highly conserved hydrophobic region in the COOH-terminal end of the subunits, either did not assemble or assembled at an extremely low rate compared to unmodified subunits. Structural changes at the more hydrophilic NH₂-terminal end had mixed effects. Several subunits modified in this region assembled into trimers at rates that were either equal or greater than those for normal subunits. Others assembled less completely than the normal subunits. Our results indicate that the in vitro synthesis and assembly assay will be useful in evaluating structure-function relationships in modified -conglycinin subunits. The results also show that structural changes at the NH₂-terminal end of the subunits are tolerated to a greater extent than modifications in the hydrophobic conserved region in the COOH-terminal half of the subunits, and this information will be useful in efforts to improve soybean quality.