Distribution and characterization of a Sandhoff disease-associated 50-kb deletion in the gene encoding the human β-hexosaminidase β-chain

Summary A 50-kb deletion was demonstrated in the gene encoding for the -subunit of human hexosaminidase (HEXB), using field inversion gel electrophoresis (FIGE) of SfiI-digested chromosomal DNA from patients with Sandhoff disease. We investigated 14 patients from different parts of Europe and found no deletion in 5 patients, 2 patients homozygous for the deletion, and 7 patients with the deletion in one allele. The distribution of the 50-kb deletion was approximately in agreement with the Hardy-Weinberg equilibrium. The deletion was characterized using chromosomal DNA from one of the two homozygous patients. Restriction fragments were hybridized with a 1.6-kb (almost complete) and a 0.4-kb (5) HEXB cDNA clone. It appeared that the deletion started in intron 5, extending in the 5 direction and causing the loss of exon 1–5 and the promoter area of the HEXB gene.     

Alanine dehydrogenase of the β-lactam antibiotic producer Streptomyces clavuligerus

l-Alanine dehydrogenase was found in extracts of the antibiotic producer Streptomyces clavuligerus. The enzyme was induced by ammonia, and the level of induction was dependend on the extracellular concentration. l-Alanine was the only amino acid able to induce alanine dehydrogenase. The enzyme was characterized from a 38-fold purified preparation. Pyruvate (K _m =1.1 mM), ammonia (K _m =20 mM) and NADH (K _m =0.14 mM) were required for the reductive amination, and l-alanine (K _m =9.1 mM) and NAD (K _m =0.5 mM) for the oxidative deaminating reaction. The aminating reaction was inhibited by alanine, serine and NADPH. Alanine inhibited uncompetitively with respect to NADH (K _i =1.6 mM) and noncompetitively with respect to ammonia (K _i =2.0 mM) and pyruvate (K _i =3.0 mM). In the aminating reaction 3-hydroxypyruvate, glyoxylate and 2-oxobutyrate could partially (6–7%) substitute pyruvate. Alanine dehydrogenase from S. clavuligerus differed with respect to its molecular weight (92000) and its kinetic properties from those described for other microorganisms.Abbreviation Alanine-DH l-alanine:NAD oxidoreductase     

Characterization of the oat1 gene of Penicillium chrysogenum encoding an ω-aminotransferase: induction by L-lysine, L-ornithine and L-arginine and repression by ammonium

The Penicillium chrysogenum oat1 gene, which encodes a class III omega-aminotransferase, was cloned and characterized. This enzyme converts lysine into 2-aminoadipic semialdehyde, and plays an important role in the biosynthesis of 2-aminoadipic acid, a precursor of penicillin and other beta-lactam antibiotics. The enzyme is related to ornithine-5-aminotransferases and to the lysine-6-aminotransferases encoded by the lat genes found in bacterial cephamycin gene clusters. Expression of oat1 is induced by lysine, ornithine and arginine, and repressed by ammonium ions. AreA-binding GATA and GATT sequences involved in regulation by ammonium, and an 8-bp direct repeat associated with arginine induction in Emericella (Aspergillus nidulans and Saccharomyces cerevisiae, were found in the oat1 promoter region. Deletion of the oat1 gene resulted in the loss of omega-aminotransferase activity. The null mutants were unable to grow on ornithine or arginine as sole nitrogen sources and showed reduced growth on lysine. Complementation of the null mutant with the oat1 gene restored normal levels of omega-aminotransferase activity and the ability to grow on ornithine, arginine and lysine. The role of the oat1 gene in the biosynthesis of 2-aminoadipic acid is discussed.     

Identification and purification of O -acetyl-l-serine sulphhydrylase in Penicillium chrysogenum

We have demonstrated that Penicillium chrysogenum possesses the l-cysteine biosynthetic enzyme O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-l-serine sulphhydrylase and O-acetyl-l-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-l-serine as substrate for the formation of l-cysteine. The purified␣enzyme did not catalyse the formation of l-homocysteine from O-acetyl-l-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyl-l-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold in relation to the cell-free extract. Two bands, showing exactly the same intensity, were present on a sodium dodecyl sulphate/polyacrylamide gel, and the molecular masses of these were estimated to be 59 kDa and 68 kDa respectively. The K _m value for O-acetyl-l-serine and V _max of O-acetyl-l-serine sulphhydrylase were estimated to be 1.3 mM and 14.9 μmol/mg protein⁻¹ h⁻¹ respectively. The activity of the purified enzyme had a temperature optimum of approximately 45 °C, which is much higher than the actual temperature for penicillin synthesis. Furthermore, O-acetyl-l-serine sulphhydrylase activity was to have a maximum in the range of pH 7.0–7.4. Received: 20 March 1998 / Received revision: 27 July 1998 / Accepted: 12 August 1998     

Homologous recombination in the antibiotic producer Penicillium chrysogenum: strain ΔPcku70 shows up-regulation of genes from the HOG pathway

In Penicillium chrysogenum, the industrial producer of the β-lactam antibiotic penicillin, generating gene replacements for functional analyses is very inefficient. Here, we constructed a recipient strain that allows efficient disruption of any target gene via homologous recombination. Following isolation of the Pcku70 (syn. hdfA) gene encoding a conserved eukaryotic DNA-binding protein involved in non-homologous end joining (NHEJ), a Pcku70 knockout strain was constructed using a novel nourseothricin-resistance cassette as selectable marker. In detailed physiological tests, strain ΔPcku70 showed no significant reduction in vegetative growth due to increased sensitivity to different mutagenic substances. Importantly, deletion of the Pcku70 gene had no effect on penicillin biosynthesis. However, strain ΔPcku70 exhibits higher sensitivity to osmotic stress than the parent strain. This correlated well with comparative data from microarray analyses: Genes related to the stress response are significantly up-regulated in the Pcku70 deletion mutant. To demonstrate the applicability of strain ΔPcku70, three genes related to β-lactam antibiotic biosynthesis were efficiently disrupted, indicating that this strain shows a low frequency of NHEJ, thus promoting efficient homologous recombination. Furthermore, we discuss strategies to reactivate Pcku70 in strains successfully used for gene disruptions.     

Evolution and expression analysis of the β-glucosidase (GLU) encoding gene subfamily in maize

The maize ??-glucosidase (ZmGLU1) hydrolyzes cytokinin-conjugates for releasing active cytokinins and thus plays important roles in cytokinin regulatory processes. ZmGLU1 belongs to glycosyl hydrolases 1 (GH1) gene family with a large number of members, and the gene function of other homologs remains to be investigated. In this study, 47 Arabidopsis, 34 rice, 31 brachypodium, 28 sorghum and 26 maize GH1 protein sequences were collected and subsequently used to construct a phylogenetic tree by Neighbor-Joining method. ZmGLU1 together with its 7 paralogs and 4 sorghum homologs were assigned into a distinct group (named GLU subfamily) with far evolutionary distance to other GH1 members. None of the Arabidopsis, rice and brachypodium gene falling into this group indicated a recent evolutionary emergence of GLU subfamily in some Poaceae plants after the divergence of Poaceae species. Phylogeny and comparative genome analysis revealed that GLU subfamily members of maize and sorghum evolved from a common ancestor, and expanded independently in each species by several duplications after maize-sorghum split. Ka/Ks analysis showed that purifying selection played important roles in maintenance of similar functions among the maize GLU paralogs. In addition, the similar protein properties and cytokinin-dependent gene expressions further suggested the similar functions of ZmGLUs in cytokinin activation. However, the organ-dependent expression of ZmGLUs exhibited diverse patterns, which might contribute to their diverse roles in cytokinin homeostasis. Taken together, this work put new insights into the evolution and expression of ZmGLU genes, and provided the foundation for future functional investigations.     

Molecular cloning and characterization of the β-catenin gene from fine-wool sheep

β-Catenin is an evolutionarily conserved molecule that functions as a crucial effector in both cell-to-cell adhesion and Wnt signaling. To gain a better understanding of its role in the development of hair follicles, we cloned the cDNA sequence of the β-catenin gene from the skin of Aohan fine-wool sheep and performed a variety of bioinformatics analyses. We obtained the full-length sequence, which was 4573-bp long and contained a 2346-bp open reading frame encoding a protein of 781 amino acids. The protein had a predicted molecular weight of 85.4 kDa and a theoretical isoelectric point of 5.57. Domain architecture analysis of the β-catenin protein revealed an armadillo repeat region, which is a common feature of β-catenin in other species. The ovine β-catenin gene shares 97.91%, 94.25%, 94.59%, 83.89%, and 89.39% sequence identity with its homologs in Bos taurus, Homo sapiens, Sus scrofa, Gallus gallus, and Mus musculus, respectively, while the amino acid sequence is more than 99% identical with each of these species. The expression of β-catenin mRNA was detected in the heart, liver, spleen, lung, kidney, skin, muscle, and adipose tissue. Expression levels were maximal in the lung and minimal in the muscle, and the difference in expression in these tissues was significant (P < 0.01). Western blot analysis revealed the presence of the β-catenin protein in all tissues examined; expression was lowest in the skin and adipose tissues.     

Isolation and characterization of the complete human β-myosin heavy chain gene

Summary The entire gene coding for the human -myosin heavy chain has been isolated from genomic EMBL3A phage libraries by chromosomal walking starting from clone gMHC-1, reported earlier (Appelhans and Vosberg 1983). gMHC-1 has been shown to carry coding information for the C-terminal two-thirds of -myosin heavy chain, which is expressed in cardiac muscle and in slow skeletal muscle fibers (Lichter et al. 1986). Three DNA clones were identified as overlapping with gMHC-1 by restriction mapping and DNA sequencing. They span a 30-kb region in the genome. About 22 kb extend from the initiation codon ATG to the poly(A) addition site. The clones include about 4 kb of 5 flanking sequences upstream of the promoter. Comparisons of - and -myosin heavy chain sequences indicate that gene duplication of the cardiac myosin heavy chain isogenes preceded the mammalian species differentiation.     

Cloning and characterization of a β-galactosidase encoding region in Lactobacillus coryniformis CECT 5711

A chromosomal DNA fragment of 7.8 kb from Lactobacillus coryniformis CECT 5711 was cloned in Escherichia coli K-12 and was found to express a functional β-galactosidase. Nucleotide sequence analysis showed that this fragment contained two partially overlapping genes, the lacL (1,881 bp) and the lacM (960 bp), that encode the subunits of a heterodimeric β-galactosidase, with estimated molecular masses of 72,129 and 35,233 Da, respectively. Other three incomplete open reading frames showing homology to another β-galactosidase, an α-galactosidase, and a galactokinase, respectively, were also found. The L. coryniformis β-galactosidase was overproduced in E. coli by using an isopropyl-β-d-thiogalactopyranoside (IPTG) expression system. Two new proteins with an estimated M _r s of approximately 72,000 and 35,000 appeared upon induction with IPTG, and extracts of the recombinant E. coli strain showed β-galactosidase activity.