Sequence analysis of Vicia faba highly repeated DNA: the BamHI repeated sequence families

Cleavage of Vicia faba nuclear DNA with the restriction endonuclease BamHI yielded discrete size classes of 250, 850, 900, 990, 1 150, 1 500 and 1 750 bp of highly repetitive DNA. Each of these sequence families comprised about 3% of the total genomic DNA. Some sequence members from each sequence family were cloned in pBR322 and their primary structures determined. Computer analyses of nucleotide sequences suggested the existence of about 60 bp sequence periodicity within the repeating unit of the 990 bp sequence family, though the extent of homology among the surmised shorter subrepeat units was very low. With other BamHI sequence families, however, the data did not show any clear internal sequence periodicity. The repeat units of the 850 bp and 1 750 bp sequence families contained nucleotide sequences homologous to the 250 bp family sequence. No sequence relationship between or among other sequence families was observed. There was 13–25% sequence variation among 6 cloned members of the 250 bp family and probably also among those of other BamHI repeat families. DNA sequences homologous to these V. faba BamHI repeat families were detected in Pisum sativum DNA by Southern blot hybridization. Furthermore, very weak cross-hybridization was observed with plant DNAs from Phaseolus vulgaris, Triticum aestivum, Cucumis sativus and Trillium kamtschaticum.     

The size and ratio of homologous sequence to non-homologous sequence in gene disruption cassette influences the gene targeting efficiency in <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

Targeted gene replacement via homologous recombination (HR) is a conventional approach for the analysis of gene function. However, this event is rare in Beauveria bassiana, which hampers efficient functional analysis in this widely used entomopathogenic fungus. To improve homologous recombination frequency in B. bassiana, we investigated the effect of the ratio of homologous sequence to non-homologous sequence (HS/NHS) in gene disruption cassette upon the HR frequency by two gene loci BbNtl and BbThi, using the herpes simplex virus thymidine kinase as a negative selectable marker against ectopic transformants. Our data revealed that an increase of the ratio of HS/NHS achieved by either extending homologous sequence or decreasing non-homologous sequence could improve HR frequency in B. bassiana. We determined empirically that (1) at least 700 bp of homology to both sides of a target gene was needed to get a reasonable number of disruptants, e.g., 6.7‰ to 13.3‰ in B. bassiana. (2) When the ratio of HS/NHS was above 0.8, an acceptable HR frequency could be achieved for gene replacement in B. bassiana, while when the ratio was below 0.3, few gene disrupted mutants were obtained.     

Nucleotide sequences of two contiguous and highly homologous xylanase genes xynA and xynB and characterization of XynA from Clostridium thermocellum

A 5.7-kbp region of the Clostridium thermocellum F1 DNA was sequenced and found to contain two contiguous and highly homologous xylanase genes, xynA and xynB. The xynA gene encoding the xylanase XynA consists of 2049 bp and encodes a protein of 683 amino acids with a molecular mass of 74 511 Da, and the xynB gene encoding the xylanase XynB consists of 1371 bp and encodes a protein of 457 amino acids with a molecular mass of 49 883 Da. XynA is a modular enzyme composed of a typical N-terminal signal peptide and four domains in the following order: a family-11 xylanase domain, a family-VI cellulose-binding domain, a dockerin domain, and a NodB domain. XynB exhibited extremely high overall sequence homology with XynA (identity 96.9%), while lacking the NodB domain present in the latter. These facts suggested that the xynA and xynB genes originated from a common ancestral gene through gene duplication. XynA was purified from a recombinant Escherichia coli strain and characterized. The purified enzyme was highly active toward xylan; the specific activity on oat-spelt xylan was 689 units/mg protein. Immunological and zymogram analyses suggested that XynA and XynB are components of the C. thermocellum F1 cellulosome. Received: 21 September 1998 / Received revision: 30 October 1998 / Accepted: 29 November 1998     

Phylogenetic relationships between the chlorophyll a/b binding protein (CAB) multigene family: An intra- and interspecies study

Summary The genome ofGlycine max (L.) Merr. cv. Dare contains a chlorophyll a/b binding (Cab) protein gene family consisting of 10 genes. The primary structures of two linkedCab genes (Cab 4 andCab 5) were determined. A comparison of the nucleic acid and predicted amino acid sequences ofCab 4 andCab 5 revealed a high degree of similarity (96% and 98%, respectively). Phylogenetic inferences drawn from sequence comparisons between previously characterized soybeanCab 1, 2, and 3 andCab 4 and 5 suggested that soybeanCab 3 was an evolutionarily distant member within this family. We further investigated the molecular evolution of theCab gene family by comparing nucleotide sequences from 25 differentCab genes representing diverse phylogenetic taxa including moncot and dicot species. Phylogenetic inferences from these data support existing morphological phylogenies in that all species within one family clustered together. These data suggested that the Solanaceae were more evolutionarily distant from the monocots than the Fabaceae and Brassicaceae. In addition, these data supported the theory thatCab Type I and II genes originated prior to divergence of the monocots and dicots.     

The genomic organization and evolution of the natural killer immunoglobulin-like receptor (KIR) gene cluster

Natural killer (NK) immunoglobulin-like receptors (KIRs) are a family of polymorphic receptors which interact with specific motifs on HLA class I molecules and modulate NK cytolytic activity. In this study, we analyzed a recently sequenced subgenomic region on chromosome 19q13.4 containing eight members of the KIR receptor repertoire. Six members are clustered within a 100-kb continuous sequence. These genes include a previously unpublished member of the KIR gene family 2DS6, as well as 2DL1, 2DL4, 3DL1, 2DS4, 3DL2, from centromere to telomere. Two additional KIR genes, KIRCI and 2DL3, which may be located centromeric of this cluster were also analyzed. We show that the KIR genes have undergone repeated gene duplications. Diversification between the genes has occurred postduplication primarily as a result of retroelement indels and gene truncation. Using pre- and postduplication Alu sequences identified within these genes as evolutionary molecular clocks, the evolution and duplication of this gene cluster is estimated to have occurred 30–45 million years ago, during primate evolution. A proposed model of the duplication history of the KIR gene family leading to their present organization is presented. Received: 25 November 1999 / Revised: 10 January 2000     

Utilization of the TEF1-a gene (TEF1) promoter for expression of polygalacturonase genes, pgaA and pgaB, in Aspergillus oryzae

For the development of an efficient gene expression system in a shoyu koji mold Aspergillus oryzae KBN616, the TEF1 gene, encoding translation-elongation factor 1α, was cloned from the same strain and used for expression of polygalacturonase genes. The TEF1 gene comprised 1647 bp with three introns. The TEF1-α protein consisted of 460 amino acids possessing high identity to other fungal TEF proteins. Two nucleotide sequences homologous to the upstream activation sequence, characterized for the ribosomal protein genes in Saccharomyces cerevisiae, as well as the pyrimidine-rich sequences were present in the TEF1 gene promoter region, suggesting that the A. oryzae TEF1 gene has a strong promoter activity. Two expression vectors, pTFGA300 and pTFGB200 for production of polygalacturonases A and B respectively, were constructed by using the TEF1 gene promoter. A polygalacturonase (PGB) gene cloned from the same strain comprised 1226 bp with two introns and encoded a protein of 367 amino acids with high similarity to other fungal polygalacturonases. PGA and PGB were secreted at approximately 100 mg/l in glucose medium and purified to homogeneity. PGA had a molecular mass of 41 kDa, a pH optimum of 5.0 and temperature optimum of 45 °C. PGB had a molecular mass of 39 kDa, a pH optimum of 5.0 and temperature optimum of 55 °C. Received: 28 November 1997 / Received revision: 24 February 1998 / Accepted: 6 March 1998     

Molecular cloning and characterization of a multidomain endoglucanase from Paenibacillus sp BP-23: evaluation of its performance in pulp refining

The gene celB encoding an endoglucanase from Paenibacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 4161 bp DNA fragment containing the celB gene was determined, revealing an open reading frame of 2991 nucleotides that encodes a protein of 106,927 Da. Comparison of the deduced amino acid sequence of endoglucanase B with known β-glycanase sequences showed that the encoded enzyme is a modular protein and exhibits high homology to enzymes belonging to family 9 cellulases. The celB gene product synthesized in E. coli showed high activity on carboxymethyl cellulose and lichenan while low activity was found on Avicel. Activity was enhanced in the presence of 10 mM Ca²⁺ and showed its maximum at 53 °C and pH 5.5. The effect of the cloned enzyme in modifying the physical properties of pulp and paper from Eucalyptus was tested (CelB treatment). An increase in mechanical strength of paper and a decrease in pulp dewatering properties were found, indicating that CelB treatment can be considered as a biorefining. Treatment with CelB gave rise to an improvement in paper strength similar to that obtained with 1,000 revolutions increase in mechanical refining. Comparison with the performances of recently developed endoglucanase A from the same strain and with a commercial cellulase showed that CelB produced the highest refining effect. Received: 25 February 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000     

Cloning and characterization of a novel member of human β-1,4-galactosyltransferase gene family

By using the EST strategy for identifying novel members belonging to homologous gene families, a novel fulklength cDNA encoding a protein significantly homologous to UDP-Gal: N-acetylglucosamine β-1, 4-galactosyltransferase (GalT) was isolated from a human testis cDNA library. A nucleotide sequence of 2 173 bp long was determined to contain an open reading frame of 1 032 nucleotides (344 amino acids). In view of the homology to memben of the galactosyltransferase gene family and especially the closest relationship toGallus gallus GalT type I (CK I), the predicted product of the novel cDNA was designated as human β-1,4-galactosyltransferase homolog I (HumGT-H1). Its mRNA is present in different degrees in 16 tissues examined. Southern analysis of human genomic DNA revealed its locus on chromosome 3.     

Cloning and characterization of a laccase gene from the white-rot basidiomycete <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

 The gene lccK encoding a laccase of the white-rot basidiomycete Pleurotus ostreatus wild-type strain collected in Japan has been cloned, sequenced, and characterized. The isolated gene consists of 2929 bp with the coding region interrupted by 19 introns and flanked by an upstream region in which putative CAAT and TATA elements were identified. Two putative N-glycosylation sites and four putative copper-binding sites found in other fungal laccase are conserved in lccK. The cDNA contains an open reading frame of 1599 bp and the gene encodes 533 amino acids preceded by a signal peptide of 23 amino acids. The nucleotide sequence of the lccK cDNA showed high homology with those of laccases of other basidiomycetes. Received: August 22, 2002 / Accepted: October 9, 2002 Present address: Faculty of Bioresource Sciences, Akita Prefectural University, Shimoshinjo-nakano, Akita 010-0195, Japan Correspondence to:K. Okamoto     

The gp49A gene has extensive sequence conservation with the gp49B gene and provides gp49A protein, a unique member of a large family of activating and inhibitory receptors of the immunoglobulin superfamily

 Members of the gp49-related family of mouse and human immunoglobulin (Ig) superfamily receptors have significant amino acid sequence homology in their C2-type, Ig-like domains and include the killer cell Ig-like receptors (KIRs) for major histocompatibility complex class I molecules. We now report the cloning, complete sequence, and organization of the mouse gp49A gene that encodes the only member of this newly-appreciated family without either of two mutually exclusive functional motifs, namely, immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or a charged transmembrane amino acid for heterodimerization with activation molecules. The gp49A and gp49B genes are 94% identical over 5.6 kilobases, the 5′ flanking regions are 94% identical over 1900 nucleotides, and the 3′ flanking regions are 97% identical for 121 nucleotides and then diverge completely; the gp49B gene encodes gp49B1 bearing two ITIMs. As measured by flow cytometry with specific antibody, gp49A is expressed on immature bone-marrow-derived mast cells, mature serosal mast cells, and several mouse mast cell lines. The substantial sequence identity of the introns of the gp49A and gp49B genes is comparable to that of the exons, establishing the gene pair as the most homologous of the gp49-related family and suggesting that the gp49A and gp49B genes arose by duplication with relatively little subsequent mutation. The findings also represent the first demonstration that gp49A is expressed on mast cells in tandem with inhibitory gp49B1, and establish that the gp49A gene is not a pseudogene, but rather encodes a protein product with characteristics different from the other family members. Received: 28 April 1999 / Accepted: 28 June 1999     

Search for nucleotide diversity patterns of local adaptation in dehydrins and other cold-related candidate genes in Scots pine (Pinus sylvestris L.)

Nucleotide variation at several cold candidate genes including seven members of the dehydrin gene family was surveyed in haplotypes of Scots pine (Pinus sylvestris) sampled in populations showing divergence for cold tolerance in Europe. Patterns of nucleotide diversity, linkage disequilibrium, and frequency spectrum of alleles were compared between north and south populations to search for signs of directional selection potentially underlying adaptation to cold. Significant differentiation between populations in allelic frequency or haplotype structure was detected at dhn1, dhn3, and abaH loci. Allelic dimorphism with no evidence of haplotype clustering by geographical distribution was found at dhn9. An excess of fixed non-synonymous mutations as compared to the outgroup P. pinaster pine species was found at dhn1. Differences in nucleotide polymorphisms were found between the members of the Kn class of dehydrin upregulated during cold acclimation (average π_sil = 0.004) as compared to the SKn class (average π_sil = 0.024). The multilocus nucleotide diversity at silent sites (θ _W = 0.009) was moderate compared to other conifer species, but higher than previous estimates for Scots pine. There was an excess of rare and high frequency derived variants as revealed by significantly negative multilocus value of Tajima’s D (D = −0.72, P < 0.01) and negative mean value of Fay and Wu H statistics (H = −0.50). The level of linkage disequilibrium decayed rapidly with an average expected r ² of 0.2 at about 200 bp. Overall, there was a positive correlation between polymorphism and divergence at ten loci when outgroup sequence was available. The discovered polymorphism will be used for further evaluation of the adaptive role of genes through association mapping studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular Cloning and Characterization of the Myostatin Gene in Croceine Croaker, Pseudosciaena crocea

Myostatin (MSTN) is a negative regulator of skeletal muscle mass and has a potential application in aquaculture. We reported the characterization of the myostatin gene and its expression in the croceine croaker, Pseudosciaena crocea. The myostatin gene had three exons encoding 376 amino acids. The cDNA was 1,906 bp long with a 5′-UTR and 3′-UTR of 108 bp and 667 bp, respectively. A microsatellite sequence, CA₃₀ and CA₂₆ separated by TA, existed in the 3′-UTR. Intron I and II were 343 bp and 758 bp in length, respectively. The deduced amino acid sequence was highly conserved, and had more than 90% identical to shi drum, gilthead seabream, striped sea-bass, white perch, and white bass proteins. The myostatin of croceine croaker had a putative amino terminal signal sequence (residues 1–22), a transforming growth factor-beta (TGF-β) propeptide domain (residues 41–256), a RXXR proteolytic processing site (RARR, residues 264–267, matching the RXXR consensus site), and a TGF-β domain (residues 282–376). There were 13 conserved cysteine residues in croceine croaker myostatin, nine of which are common to all TGF-β superfamily members. The most conserved region of vertebrate myostatins is the TGF-β domain, which was the mature bioactive domain of the myostatin protein. The myostatin gene was expressed not only in the skeletal muscle, but also in the other tissues.     

Crassostrea angulata Bindin Gene and the Divergence of Fucose-Binding Lectin Repeats Among Three Species of Crassostrea

Bindin is a major protein for species-specific recognition between sperm and congenetic egg in many free-spawning marine invertebrates. We cloned a novel bindin gene from the oyster Crassostrea angulata by 3′ and 5′ rapid amplification of cDNA ends. The full-length bindin cDNA was 1,049 bp with a 771-bp open reading frame encoding 257 amino acids. The deduced amino acid sequence contained a putative signal peptide of 24 amino acids. The length of the bindin genomic DNA was 8,508 bp containing four exons and three introns. Three haplotypes of F-lectin repeat were detected from seven sequences of F-lectin repeat of six male oysters. Both neighbor-joining and minimum-evolution phylogenetic trees show that haplotype an1 was close to Crassostrea gigas while an2 and an3 were close to Crassostrea sikamea. Intron-4 in the middle of F-lectin repeat is highly variable in both size and sequence. We classified intron-4 into three types according to their size and the F-lectin repeat they were located in. Intron-4 may play an important role in recombination. We compared the number of nonsynonymous substitutions (Dn) and synonymous substitutions (Ds) per nucleotide site among 19 F-lectin haplotypes of the three species. Dn/Ds ratios suggested that positive selection occurred between C. gigas and C. sikamea and between C. gigas and C. angulata. Nine positive selected positions (p > 90%) are identified among 19 haplotypes of three species. They are located on the F-lectin binding face around the three recognition motif residues. We assume that these nine clustered amino acids are related with species-specific recognition.     

Sequence of the Corynebacterium glutamicum pyruvate carboxylase gene

Pyruvate carboxylase is an important anaplerotic enzyme replenishing oxaloacetate consumed for biosynthesis during growth, or lysine and glutamic acid production in industrial fermentations. We used regions of homology from pyruvate carboxylase sequences of 12 different species (corresponding to the ATP- and pyruvate-binding sites), to design polymerase chain reaction (PCR) primers for amplifying a fragment of the pyruvate carboxylase (pc) gene from C. glutamicum genomic DNA. This 850-base-pair fragment was used to probe a C. glutamicum cosmid library and four candidate pc cosmids were identified. The fragment was sequenced and the sequence of the complete gene was obtained by several rounds of primer synthesis, PCR on one of the positive cosmids, and sequencing. The C. glutamicumpc sequence shows 64% homology with the pc gene of Mycobacterium tuberculosis and 44% homology with the human pc gene. Regions of ATP, pyruvate and biotin binding have also been identified. Received: 16 December 1997 / Received revision: 31 March 1998 / Accepted: 19 April 1998     

Molecular cloning,purification and characterization of two endo-1,4-β-glucanases from Aspergillus oryzae KBN616

Two endo-1,4-β-glucanase genes, designated celA and celB, from a shoyu koji mold Aspergillus oryzae KBN616, were cloned and characterized. The celA gene comprised 877 bp with two introns. The CelA protein consisted of 239 amino acids and was assigned to the cellulase family H. The celB gene comprised 1248 bp with no introns. The CelB protein consisted of 416 amino acids and was assigned to the cellulase family C. Both genes were overexpressed under the promoter of the A. oryzae taka-amylase A gene for purification and enzymatic characterization of CelA and CelB. CelA had a molecular mass of 31 kDa, a pH optimum of 5.0 and temperature optimum of 55 °C, whereas CelB had a molecular mass of 53 kDa, a pH optimum of 4.0 and temperature optimum of 45 °C. Received: 3 July 1996 / Accepted: 15 July 1996