Molecular characterization of β-thalassemia in Czechoslovakia

Summary We have identified different -thalassemia mutations in 93 members of 34 families of Czech or Slovakian descent using gene amplification, hybridization with specific ³²P-labeled oligonucleotide probes, sequencing of amplified DNA, and gene mapping. The GA mutation at IVS-I-1 was found in 18 families; other Mediterranean mutations were IVS-II-1 (GA), IVS-II-745 (CG), IVS-I-110 (GA), and codon 39 (CT); these were present in 9 additional families. The GT mutation at codon 121, known to cause Heinzbody -thalassemia, was present in 3 families, and the frameshift at codons 82/83 (-G), first described in the Azerbaijanian population, in 2 families. A newly discovered allele was a frameshift at codons 38/39 (-C). One -thalassemia allele was incompletely characterized. We observed in 2 families a TC mutation at position +96 UTR (untranslated region) relative to the termination codon; this mutation likely is a rare polymorphism, -Thalassemia was rare; only one person carried the -^3.7 heterozygosity, and one other had a yet to be identified -thalassemia-1, while seven had the ^{anti 3.7} triplication.     

Molecular characterization of β-thalassemia in Hungary

We have identified seven different -thalassemia mutations and one -thalassemia determinant (the Sicilian type) in 32 members of 17 Hungarian families. The most common mutation is the IVS-I-1 (GA) change; its high frequency is comparable to that observed in neighboring Czechoslovakia. Additional mutations are of Mediterranean origin. One rare mutation (initiation codonATGGTG) was identified as an independent mutation because of the absence of known polymorphisms in the -globin gene. One new frameshift at codon 51 (-C) was observed in a single individual; hematological data were as expected for a °-thalassemia heterozygosity.     

Molecular characterization of β-thalassemia in Azerbaijan

We have analyzed the -thalassemia mutations in 99 chromosomes of 49 adults with -thalassemia major and of one with Hb S--thalassemia, who are regular patients at a large hematology clinic in Bakü, Azerbaijan. A total of 20 different mutants were identified; three [frameshift at codon 8 (-AA); IVS-II-I (GA); IVS-I-110 (GA)] were present in about two-thirds of all chromosomes. Most alleles are the same as found in Mediterranean populations; a few have an Asian origin or come from Kurdistan, Lebanon, Saudi Arabia, or a black population. One mutant [frameshift at codons 82/ 83 (-G)] might be specific for the Azerbaijanian population. Nearly all patients were transfused, which made quantitation of Hb F impossible; high^G values were present in the Hb F of those patients whose -thalassemia chromosome carried the C T mutation at position — 158 in the promoter of the ^G-globin gene.     

Molecular characterization of β-thalassemia mutations in Egypt

Summary The relative frequency of different -thalassemia mutations and their association with -globin haplotypes were studied in patients from the Nile delta region, Egypt, by means of the polymerase chain reaction, oligonucleotide hybridization and restriction analysis. We found that 8 mutations account for 77% of -thalassemia chromosomes in this population, the commonest being IVS-1 nt 110, IVS-1 nt 6 and IVS-1 nt 1. Each mutation was associated with a specific haplotype, with the exception of IVS-1 nt 110, found on 3 different chromosomal backgrounds. Our data show that testing for the 8 detectable mutations makes feasible prenatal diagnosis in 65% of at risk couples and exclusion testing in an additional 25% of cases.     

Molecular basis of β-thalassemia in Thailand: analysis of β-thalassemia mutations using the polymerase chain reaction

Summary -Thalassemia mutations in 71 chromosomes of Thai patients from the northeast, the middle and the south of the country were investigated using dot blot hybridization of PCR (polymerase chain reaction)-amplified DNA with allelespecific oligonucleotide probes. Eight different known molecular defects were detected, at different frequencies. There was an amber mutation in codon 17, a C-T transversion at position 654 of IVS-2, a frameshift mutation between codons 71 and 72, an A-G transition at nucleotide -28 within the TATA box (known as Chinese mutations), a G-T transversion at position 1 of IVS-1 (an Indian mutation), a 4bp deletion in codons 41/42 and a G-C transversion at position 5 of IVS-1 (described as both Chinese and Indian mutations) and a Thai original mutation, an ochre mutation in codon 35. Analysis of the three unknown alleles by DNA sequencing of the cloned DNA fragment amplified by PCR revealed an A-G substitution at the second position of the codon for amino acid 19 (AAC-AGC). The analytic approach used in the present study and the characteristic distribution of mutations in each region of Thailand will prove useful for setting up a prenatal diagnosis program.     

Molecular characterization of a β-1,4-endoglucanase from an endophytic Bacillus pumilus strain

Endophytes comprise mainly microorganisms that colonize inner plant tissues, often living with the host in a symbiotic manner. Several ecological roles have been assigned to endophytic fungi and bacteria, such as antibiosis to phytopathogenic agents and plant growth promotion. Nowadays, endophytes are viewed as a new source of genes, proteins and biochemical compounds that may be used to improve industrial processes. In this study, the gene EglA was cloned from a citrus endophytic Bacillus strain. The EglA encodes a -1,4-endoglucanase capable of hydrolyzing cellulose under in vitro conditions. The predicted protein, EglA, has high homology to other bacterial cellulases and shows a modular structure containing a catalytic domain of the glycosyl hydrolase family 9 (GH9) and a cellulose-binding module type 3 (CBM3). The enzyme was expressed in Escherichia coli, purified to homogeneity, and characterized. EglA has an optimum pH range of 5–8, and remarkable heat stability, retaining more than 85% activity even after a 24-h incubation at pH 6–8.6. This characteristic is an important feature for further applications of this enzyme in biotechnological processes in which temperatures of 50–60°C are required over long incubation periods.     

Molecular characterization of β-fructofuranosidases from Rhizopus delemar and Amylomyces rouxii

Of the 19 strains of Rhizopus delemar deposited as Rhizopus oryzae, seven of them, NBRC 4726, NBRC 4734, NBRC 4746, NBRC 4754, NBRC 4773, NBRC 4775, and NBRC 4801, completely hydrolyzed exogenous sucrose and fructooligosaccharides. The sucrose-hydrolyzing enzyme was purified from the culture filtrate of R. delemar NBRC 4754 and classified to β-fructofuranosidase, similar to that of Amylomyces rouxii CBS 438.76. Fragments including β-fructofuranosidase genes (sucA) of seven strains of R. delemar and A. rouxii CBS 438.76 were amplified and sequenced by PCR with degenerated primers synthesized on the basis of the internal amino acid sequences of purified enzymes and successive inverse PCR. Nucleotide sequences of the obtained fragments revealed that open reading frames of 1,569 bp have no intron and encode 522 amino acids. The presumed proteins contained the typical domain of the glycoside hydrolase 32 family, including β-fructofuranosidase, inulinase, levanase, and fructosyltransferases. Amino acid sequences of SucA proteins from the seven strains of R. delemar were identical and showed 90.0 % identity with those of A. rouxii CBS 438.76. A dendrogram constructed from these amino acid sequences showed that SucA proteins are more closely related to yeast β-fructofuranosidases than to other fungal enzymes.     

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.     

Purification and characterization of a β-amylase from soya beans

1. β-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16·3%, its extinction coefficient, E^1%_1cm., at 280mμ was 17·3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5·85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean β-amylase is in the range 5–6. Pretreatment of the enzyme at pH3–5 decreases enzyme activity, whereas at pH6–9 it is not affected.     

Molecular characterization of β-lactam-resistant Escherichia coli isolated from Fu River, China

The present study aims to demonstrate the β-lactam resistance phenotypes and genotypes of Escherichia coli isolates from the Fu River in Chengdu, southwestern China. We obtained 108 E. coli isolates from nine sampling sites during May and December 2010. The total bacterial count varied from 79 colony-forming units (CFU)/ml to 14,550 CFU/ml, and coliform group number from 13 to 1,435 MPN/ml. Among the 108 isolates, 0-31.48% were resistant to β-lactams and β-lactam inhibitors, 1.85-7.40% to aminoglycoside, 1-20% to fluoroquinolone, and 50% to trimethoprim- sulfamethoxazole. The total bacterial count and antimicrobial resistance of different sites were significantly correlated (P < 0.05). Among the 34 ampicillin-resistant isolates, polymerase chain reaction (PCR) amplification and DNA sequencing showed that bla (TEM), bla (SHV), and bla (CTX-M) were detected in 85.29% (n = 29), 41.18% (n = 14), and 5.88% (n = 2) of the isolates, respectively, whereas bla (KPC) and bla (GES) were not observed in any of the isolates. Enterobacterial repetitive intergenic consensus-PCR patterns revealed that the 34 ampicillin-resistant E. coli isolates belonged to three distinct groups. Plasmid DNAs from the 14 SHV producer isolates yielded one to five bands of ca. 0.15-40 kb. To our knowledge, the current study is the first to describe the phenotypic and genetic characterizations of β-lactam resistance in E. coli isolates of river water origin from the Fu River, Chengdu, southwestern China. Results of the present study suggest that the river water may be considered as a reservoir for antibiotic resistance genes.     

Purification and characterization of β-galactosidase from a strain of Bacillus coagulans

-Galactosidase from B. coagulans strain L4 is produced constitutively, has a mol. wt. of 4.3×10⁵ and an optimal temperature of 55°C. The optimal pH at 30°C is 6.0 whereas at 55°C it is 6.5. The energy of activation of enzyme activity is 41.9 kJ/mol (10 kcal/mol). No cations are required. The K_m with ONPG as substrate is 4.2–5.6mm and with lactose is 50mm. The K_i for inhibition by galactose is 11.7–13.4mm and for dextrose is 50mm. Galactose inhibited competitively while dextrose inhibited noncompetitively. The purified and unprotected enzyme is 70% destroyed in 30 min at 55°C whereas in the presence of 2 mg/ml of BSA 42% of the activity is destroyed in 30 min at 55°C. An overall purification of 75.3-fold was achieved.     

Molecular basis of β-thalassemia in Turkey: detection of rare mutations by direct sequencing

Summary Using restriction endonuclease analysis, oligonucleotide hybridization, and direct sequencing of amplified genomic DNA, we characterized 11 different mutations in the DNA of 26 patients from Turkey homozygous for -thalassemia. We found that mutations IVS-1 nt110, IVS-1 nt6, and the frameshift at codon 8 were the most frequent. By direct sequencing we characterized two very rare mutations not previously reported in the Turkish population: a frameshift +1 at codons 9/10 and a nonsense mutation at codon 15.     

Molecular and Biochemical Characterization of a β-Fructofuranosidase from Xanthophyllomyces dendrorhous

An extracellular β-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70°C) and thermostability (with a T₅₀ in the range 66 to 71°C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-β-(2→1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the k_cat/K_m ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial β-fructofuranosidases, the enzyme from X. dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Using a 41% (wt/vol) sucrose solution, the maximum fructooligosaccharide concentration reached was 65.9 g liter⁻¹. In addition, we isolated and sequenced the X. dendrorhous β-fructofuranosidase gene (Xd-INV), showing that it encodes a putative mature polypeptide of 595 amino acids and that it shares significant identity with other fungal, yeast, and plant β-fructofuranosidases, all members of family 32 of the glycosyl-hydrolases. We demonstrate that the Xd-INV could functionally complement the suc2 mutation of Saccharomyces cerevisiae and, finally, a structural model of the new enzyme based on the homologous invertase from Arabidopsis thaliana has also been obtained.The basidiomycetous yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) produces astaxanthin (3-3′-dihydroxy-β,β-carotene-4,4 dione [17, 25]). Different industries have displayed great interest in this carotenoid pigment due to its attractive red-orange color and antioxidant properties, which has intensified the molecular and genetic study of this yeast. As a result, several genes involved in the astaxanthin biosynthetic pathway have been cloned and/or characterized, as well as some other genes such as those encoding actin (60), glyceraldehyde-3-phosphate dehydrogenase (56), endo-β-1,3-glucanase, and aspartic protease (4). In terms of the use of carbon sources, a β-amylase (9), and an α-glucosidase (33) with glucosyltransferase activity (12), as well as a yeast cell-associated invertase (41), have also been reported.Invertases or β-fructofuranosidases (EC 3.2.1.26) catalyze the release of β-fructose from the nonreducing termini of various β-d-fructofuranoside substrates. Yeast β-fructofuranosidases have been widely studied, including that of Saccharomyces cerevisiae (11, 14, 45, 46), Schizosaccharomyces pombe (36), Pichia anomala (40, 49), Candida utilis (5, 8), or Schwanniomyces occidentalis (2). They generally exhibit strong similarities where sequences are available, and they have been classified within family 32 of the glycosyl-hydrolases (GH) on the basis of their amino acid sequences. The catalytic mechanism proposed for the S. cerevisiae enzyme implies that an aspartate close to the N terminus (Asp-23) acts as a nucleophile, and a glutamate (Glu-204) acts as the acid/base catalyst (46). In addition, the three-dimensional structures of some enzymes in this family have been resolved, such as that of an exoinulinase from Aspergillus niger (var. awamori; 37) and the invertase from Arabidopsis thaliana (55).As well as hydrolyzing sucrose, β-fructofuranosidases from microorganisms may also catalyze the synthesis of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked to the sucrose skeleton by different glycosidic bonds depending on the source of the enzyme (3, 52). FOS are one of the most promising ingredients for functional foods since they act as prebiotics (44), and they exert a beneficial effect on human health, participating in the prevention of cardiovascular diseases, colon cancer, or osteoporosis (28). Currently, Aspergillus fructosyltransferase is the main industrial producer of FOS (15, 52), producing a mixture of FOS with an inulin-type structure, containing β-(2→1)-linked fructose-oligomers (¹F-FOS: 1-kestose, nystose, or ¹F-fructofuranosylnystose). However, there is certain interest in the development of novel molecules that may have better prebiotic and physiological properties. In this context, β-(2→6)-linked FOS, where this link exits between two fructose units (⁶F-FOS: 6-kestose) or between fructose and the glucosyl moiety (⁶G-FOS: neokestose, neonystose, and neofructofuranosylnystose), may have enhanced prebiotic properties compared to commercial FOS (29, 34, 54). The enzymatic synthesis of 6-kestose and other related β-(2→6)-linked fructosyl oligomers has already been reported in yeasts such as S. cerevisiae (11) or Schwanniomyces occidentalis (2) and in fungi such as Thermoascus aurantiacus (26) or Sporotrichum thermophile (27). However, the production of FOS included in the ⁶G-FOS series has not been widely reported in microorganisms, probably because they are not generally produced (2, 15) or because they represent only a minor biosynthetic product (e.g., with baker''s yeast invertase) (11). Most research into neo-FOS production has been carried out with Penicillium citrinum cells (19, 31, 32, 39). In this context, neokestose is the main transglycosylation product accumulated by whole X. dendrorhous cells from sucrose (30), although the enzyme responsible for this reaction remained uncharacterized.Here, we describe the molecular, phylogenetic, and biochemical characterization of an extracellular β-fructofuranosidase from X. dendrorhous. Kinetic studies of its hydrolytic activity were performed using different substrates, and we investigated its fructosyltransferase capacity. The functionality of the gene analyzed was verified through its heterologous expression, and a structural model of this enzyme based on the homologous invertase from A. thaliana has also been obtained.     

Plasma lipids and lipoproteins in children and young adults with major β-thalassemia from western Iran: influence of genotype

To determine the plasma lipid and lipoprotein profiles and their possible association with the type of β-thalassemia mutation we studied 103 major β-thalassemia patients including 71 children and 32 young adults compared to 102 healthy subjects consisted of 90 children and 12 young healthy adults. The plasma lipid and lipoprotein levels were measured by conventional methods. Considering all of the patients the levels of total cholesterol (TC), LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C) were significantly lower compared to controls. However, the level of TG was significantly higher in cases than controls. Comparing thalassemic patients homozygous for a β⁰ type of mutation with those homozygous for a β⁺ type of mutation (IVSI.110 G:A) indicated that the levels of LDL-C, TC were significantly increased and TG concentration tended to be higher in the latter patients. In conclusion, our study indicates that hemolytic stress results in hypocholesterolemia in major β-thalassemia patients and the presence of more severe genotype in patients is correlated with more reduction in TG, TC, and LDL-C levels.     

Molecular characterization of a novel β-1,3-exoglucanase related to mycoparasitism of Trichoderma harzianum

Trichoderma harzianum, a soil-borne filamentous fungus, is capable of parasitizing several plant pathogenic fungi. Secretion of lytic enzymes, mainly glucanases and chitinases, is considered the most crucial step of the mycoparasitic process. The lytic enzymes degrade the cell walls of the pathogenic fungi, enabling Trichoderma to utilize both their cell walls and cellular contents for nutrition. We have purified a 110 kDa novel extracellular β-1,3-exoglucanase from T. harzianum, grown with laminarin or in dual cultures with host fungi. The corresponding gene, lam1.3, and its cDNA were isolated and their nucleotide sequences determined. The deduced amino-acid sequence predicted a molecular mass of 110.7 kDa of a mature protein excluding a signal peptide. LAM1.3 showed high homology to EXG1, a β-1,3-exoglucanase of the phytopathogenic fungus Cochliobolus carbonum, and a lower homology to BGN13.1, a β-1,3-endoglucanase isolated from T. harzianum. However, it contains a unique C-terminal embodying cysteine motifs. The expression of lam1.3 in growth with laminarin, but not with glucose, was found to be a result of differential accumulation of the corresponding mRNA.     

Molecular characterization of four β-tubulin genes from dinitroaniline susceptible and resistant biotypes of Eleusine indica

Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, resistant biotypes of goosegrass (Eleusine indica) developed in previously susceptible wild-type populations. We have previously reported that -tubulin missense mutations correlate with dinitroaniline response phenotypes (Drp) (Plant Cell 10: 297–308, 1998). In order to ascertain associations of other tubulins with dinitroaniline resistance, four -tubulin cDNA classes (designated TUB1, TUB2, TUB3, and TUB4) were isolated from dinitroaniline-susceptible and -resistant biotypes. Sequence analysis of the four -tubulin cDNA classes identified no missense mutations. Identified nucleotide substitutions did not result in amino acid replacements. These results suggest that the molecular basis of dinitroaniline resistance in goosegrass differs from those of colchicine/dinitroaniline cross-resistant Chlamydomonas reinhardtii and benzimidazole-resistant fungi and yeast. Expression of the four -tubulins was highest in inflorescences. This is in contrast to -tubulin TUA1 that is expressed predominantly in roots. Collectively, these results imply that -tubulin genes are not associated with dinitroaniline resistance in goosegrass. Phylogenetic analysis of the four -tubulins, together with three -tubulins, suggests that the resistant biotype developed independently in multiple locations rather than spreading from one location.     

Molecular modelling of a psychrophilic β-galactosidase

An Antarctic strain of bacteria was isolated from the digestive tract of the crustacean Thysanoessa macrura and classified as Pseudoalteromonas sp. 22b based on 16SrRNA gene sequence and physiological as well as biochemical properties. This bacterium turned out to be a good producer of a cold-adapted β-galactosidase. The enzyme displays high catalytic and molecular adaptation to low temperatures. Here we present a homology model of the psychrophilic β-galactosidase based on the structural template of the mesophilic β-galactosidase from Escherichia coli (PDB code: 1JZ7, resolution 1.5 Å). Our aim was to identify and characterize potential cold-adaptational features of the target psychrophilic β-galactosidase at the level of the three-dimensional structure rather than solely from the analysis of the amino acid sequence. We report the results of comparisons between the psychrophilic and mesophilic β-galactosidases and point out similarities and differences in the catalytic site and in other parts of the structure. The model allowed us to pinpoint a number of characteristics that are frequently observed in psychrophilic enzymes and allowed interpretation of the results of immunochemical and biochemical analyses.