Characterization of two β-glucosidase genes fromClostridium thermocellum

Summary Two -glucosidase genes, designatedbglA andbglB, were isolated from a gene bank ofClostridium thermocellum DSM 1237. The coding sequences forbglA andbglB were located on non-homologous DNA fragments of 3.2– and 3.4-kb, respectively. Both genes direct inEscherichia coli the synthesis of cytoplasmic -glucosidases, which differ with respect to substrate specificity and temperature profile. The properties of thebglA-encoded -glucosidase A closely resemble that of a -glucosidase previously isolated fromC. thermocellum cultures.     

Analysis of β-galactosidase production and their genes of two strains of <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis>

A bacterial β-galactosidase delivery system is a potential therapy for lactose intolerance. Currently, two Lactobacillus bulgaricus strains with different biological characteristics are under consideration as potential sources. However, differences in these β-galactosidase genes and their resulting production levels are poorly characterized. The β-galactosidase ORF of L. bulgaricus yogurt isolate had high variability and was terminated at site 1924 due to a stop codon. However, the full 114 kDa β-galactosidase band was still resolved by SDS-PAGE, which may indicate that the interrupted ORF was translated into more than one peptide, and they together were folded into the complete enzyme protein that showed much higher β-galactosidase activity (6.2 U/mg protein) than the enzyme generated from L. bulgaricus reference strain (2.5 U/mg protein).     

Simultaneous production of endoglucanase and β-glucosidase using synthetic two cistron genes

Summary Simultaneous production of endoglucanase and -glucosidase by using a synthetic two cistron system inEscherichia coli was attempted as a possible way of reducing production cost. The first cistron in this system we constructed is an endoglucanase gene fused to a tac promoter that provides for efficient expression. The second cistron is a -glucosidase structural gene. A ribosome binding site sequence of 33-base was inserted between the two cistron genes.E. coli cells transformed with the system produced 12.4 units/mg protein of endoglucanase and 327 units/mg protein of -glucosidase, which represent 15% and 22% of total cellular protein, respectively, in L medium within three hours after induction with IPTG.     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.     

Differential RNA accumulation of two β-tubulin genes in arbuscular mycorrhizal fungi

RNA was isolated from spores of different arbuscular mycorrhizal (AM) fungi and used for RT-PCR with degenerate primers for beta-tubulin genes. PCR products were cloned and the sequence of several clones was analysed for each fragment. Comparison of sequences identified two loci for beta-tubulin genes with different GC content and codon usage. Btub1 sequences were most similar to beta-tubulin genes from the Oomycota, while Btub2 sequences showed highest similarity to sequences from the Zygomycota. RT-PCR experiments were carried out to monitor RNA accumulation patterns of Btub1 and Btub2 in asymbiotic germinating spores and in symbiotic extraradical hyphae of three different AM fungi. This indicated that Btub1 is constitutively expressed in Gigaspora rosea, but down-regulated during symbiosis in Glomus mosseae and Glomus intraradices. In contrast, Btub2 showed constitutive expression in the two Glomus species, but down-regulation in G. rosea. Further analysis of different fungi indicated that Btub2 primers could be used to specifically monitor RNA accumulation of AM fungi in environmental samples.     

Characterization of the two H1°-encoding genes from Xenopus laevis

We have analyzed the promoter and the coding sequences of the two homologous histone H1°-encoding genes from Xenopus laevis, here termed H1°-1 and H1°-2. Both genes encode proteins of 193 amino acids and differ at just 16 amino-acid residues. Putative regulatory sequences identified in the promoter region are the same and are highly conserved. However, significant differences exist in the 5′ untranslated regions (UTR) of the transcribed sequences of these two genes, such as several deletions in the 5′-UTR of the H1°-2 gene in comparison with the H1°-1 gene 5′-UTR. The 3′-UTR is a short sequence of about 200 bp which is unexpected compared with the long 3′-UTR of mammalian H1° mRNA, but it is in the same size range as in avian H5 mRNA. Thus, the main differences between these two genes are observed in sequences potentially involved in the regulation of the H1° gene expression such as the 5′-UTR. The two genes are expressed during embryogenesis and in several adult tissues. We discuss these findings in terms of the evolution of histone H1° genes in vertebrates and the appearance of histone H5 in avian species.© 1997 Elsevier Science B.V. All rights reserved.     

Biochemical characterization of two novel β-glucosidase genes by metagenome expression cloning

Two novel β-glucosidase genes designated as bgl1D and bgl1E, which encode 172- and 151-aa peptides, respectively, were cloned by function-based screening of a metagenomic library from uncultured soil microorganisms. Sequence analyses indicated that Bgl1D and Bgl1E exhibited lower similarities with some putative β-glucosidases. Functional characterization through high-performance liquid chromatography demonstrated that purified recombinant Bgl1D and Bgl1E proteins hydrolyzed D-glucosyl-β-(1-4)-D-glucose to glucose. Using p-nitrophenyl-β-D-glucoside as substrate, K(m) was 0.54 and 2.11 mM, and k(cat)/K(m) was 1489 and 787 mM(-1) min(-1) for Bgl1D and Bgl1E, respectively. The optimum pH and temperature for Bgl1D was pH 10.0 and 30°C, while the optimum values for Bgl1E were pH 10.0 and 25°C. Bgl1D exhibited habitat-specific characteristics, including higher activity in lower temperature and at high concentrations of AlCl(3) and LiCl. Bgl1D also displayed remarkable activity across a broad pH range (5.5-10.5), making it a potential candidate for industrial applications.     

α-amylase genes of wheat are two multigene families which are differentially expressed

Plant Molecular Biology - The α-Amy1 and α-Amy2 genes of wheat produce distinct subsets of α-amylase isozymes which show different patterns of expression in wheat aleurone cells and...     

Genetic polymorphism detection of two α-Casein genes in three Egyptian sheep breeds

Sheep milk is an excellent raw material for the milk processing industry especially in cheese production. The protein content and composition of sheep milk are important in the cheese manufacturing. The casein fraction of ruminant milk proteins consists of four caseins, namely αs1, αs2, β and κ-Casein. Casein genetic polymorphisms are important due to their effects on quantitative traits and technological properties of milk.This study aimed to detect the genetic polymorphism of αs1- and αs2-Casein genes in three native Egyptian sheep breeds; Rahmani, Barki and Ossimi. PCR-SSCP and PCR-RFLP were used to detect the genetic polymorphism of αs1-CN and αs2-CN genes, respectively.A 223-bp fragment of αs1-CN gene was amplified by PCR and SSCP results recorded the presence of three different patterns; TT, TC and CC; in 87 tested sheep animals. The sequence analysis of two homologous patterns showed a single nucleotide polymorphism (SNP) (T → C) at position 170. The frequencies of three patterns in the tested sheep breeds were 43.33%, 50.00%, and 6.67% in Rahmani; 83.33%, 13.33%, and 3.33% in Ossimi and 74.07%, 22.22%, and 3.70% in Barki, respectively. Our nucleotide sequences of αs1-CN T and C alleles were submitted to GenBank with the accession numbers KF018339 and KF018340, respectively.The restriction digestion of αs2-CN PCR product (1300-bp) by Tru1I endonuclease revealed three different genotypes; AA, AG and GG with frequencies of 66.67%, 30.00%, and 3.33% in Rahmani; 96.67%, 3.33%, and 0.00% in Ossimi and 96.15%, 3.85%, and 0.00% in Barki, respectively. The sequence analysis revealed the presence of a single nucleotide polymorphism (A → G) in intron 6 of αs2-CN gene. Our nucleotide sequence of αs2-CN gene was submitted to GenBank with the accession number JX080380.     

Pax-6 origins – implications from the structure of two coral Pax genes

 Vertebrate Pax-6 and its Drosophila homolog eyeless play central roles in eye specification, although it is not clear if this represents the ancestral role of this gene class. As the most ”primitive” animals with true nervous systems, the Cnidaria may be informative in terms of the evolution of the Pax gene family. For this reason we surveyed the Pax gene complement of a representative of the basal cnidarian class (the Anthozoa), the coral Acropora millepora. cDNAs encoding two coral Pax proteins were isolated. Pax-Aam encoded a protein containing only a paired domain, whereas Pax-Cam also contained a homeodomain clearly related to those in the Pax-6 family. The paired domains in both proteins most resembled the vertebrate Pax-2/5/8 class, but shared several distinctive substitutions. As in most Pax-6 homologs and orthologs, an intron was present in the Pax-Cam locus at a position corresponding to residues 46/47 in the homeodomain. We propose a model for evolution of the Pax family, in which the ancestor of all of the vertebrate Pax genes most resembled Pax-6, and arose via fusion of a Pax-Aam-like gene (encoding only a paired domain) with an anteriorly-expressed homeobox gene resembling the paired -like class. Received: 25 February 1998/Accepted: 23 March 1998     

Molecular characterization of two types of 22 kilodalton α-zein genes in a gene cluster in maize

Summary Five genes of the -zein subfamily four (SF4) are located in a 56 kb genomic region of the maize inbred line W22. Their nucleotide and deduced amino acid sequences have been determined. The sequences define two types of -zein SF4 genes — type 1 (T1) and type 2 (T2). The single T1 -zein SF4 gene codes for an -zein protein with a M_r of about 22 000. This is the first -zein SF4 gene sequenced that contains no early in-frame stop codons in its coding sequence. The four T2 -zein SF4 genes in this cluster contain one or two early in-frame stop codons. In addition, our T1 and T2 genes differ markedly in the base sequences of their distal 5 non-translated flanking regions. The nucleotide and the deduced amino acid sequences of these two types of -zein SF4 genes are similar ( > 90 %) to one another and to all known -zein SF4 genes and cDNAs. Of the known W22 -zein SF4 genes, only one in six does not contain an early in-frame stop codon. If the number of -zein SF4 genes is 15–20, then we estimate that only about 4 of the W22 -zein SF4 genes are without in-frame early stop codons.     

The 3′ ends of two genes in the Balbiani ring c locus ofChironomus thummi

Summary The 3-end sequences of two nonallelic genes derived from the Balbiani ring c (BRc) locus ofChironomus thummi are described. Only one of the genes appears to be transcribed abundantly in normal late larval salivary glands. The two sequences are highly similar, even in the 3 untranslated regions, but sharply diverge beyond the polyadenylation site. Together with evidence from the 3 ends of BR1 and BR2 genes ofC. pallidivittatus andC. tentans, independently characterized by others, this result suggests the existence of a sequence-homogenization mechanism that operates across the 3 ends of all BR genes characterized to date. The 3-terminal coding region of each BRc gene is divided into two portions by a short intron. The upstream portion is homologous to and continuous with the tandem repeats that make up the internal core of each BR gene; however, that portion is variant in sequence relative to the core, and apparently is not subject to the homogenization process that operates on the core repeats. The portion downstream of the intron encodes a unique, 111-residue polypeptide highly different from the rest of the BRc product. The evolution of the various segments of the BRc genes is discussed.     

Specific cleavage of chicken αA-globin and human c-Ha-ras genes by two molecular forms of calf thymus topoisomerase I

Summary Two molecular forms of topoisomerase I differing in size and sensitivity to camptothecin were isolated from calf thymus. Mapping of topo I cleavage sites of the cloned chicken ^A-globin and human c-Ha-ras genes was carried out. Camptothecin was shown to affect site specificity of the topoisomerases.