Incomplete Open Reading Frames Research Articles

CDNA sequences of GHF9 endo-β-1,4-glucanases in terrestrial Crustacea

This study aimed to sequence and identify a glycosyl hydrolase family 9 (GHF9) endo-β-1,4-glucanase expressed in the midgut gland of the herbivorous gecarcinid land crab, Gecarcoidea natalis. Hence this would explain the gene responsible for the production of previously purified and characterised endo-β-1,4-glucanases. Three different transcripts, two complete and one partial were sequenced from cDNA and an open reading frame of 1383bp was produced. Translated, this would produce a putative protein of 460 amino acid residues, including a 16 amino acid residue signal peptide. The mature protein (without signal peptide) is predicted to have a molecular mass of 47.6–47.7kDa; this closely matches the molecular mass (47.4kDa) of one of the three endo-β-1,4-glucanase/lichenase enzymes purified previously from G. natalis. It is therefore proposed that the gene described here encodes one of the previously characterised enzymes. The presence of multiple transcripts suggests gene duplication. To confirm that the gene is widely expressed within the Crustacea, cDNA encoding a GHF9 endo-β-1,4-glucanase was also sequenced in diverse crustaceans, the deposit feeding soldier crab, Mictyris platycheles and the terrestrial hermit crabs, Coenobita purlatus and C. brevimanus. An open reading frame of 1356bp was sequence from M. platycheles, while an incomplete open reading frames of 1384 and 1523bp were respectively sequenced from Coenobita brevimanus and C. perlatus. The midgut gland of M. platycheles contained activity (0.704±0.218μmol reducing sugars produced. min−1·mg−1 tissue wet weight) of a 26.3±0.3(5) endo-β-1,4-glucanase isozyme (determined from activity staining). These species, particularly M. platycheles does not consume and digest significant amounts of plant cellulose. This implies that the ancestral enzyme is not a cellulase, but rather it may be involved in hydrolysing cellulose like polysaccharides within other organisms such as algae.

Transcriptomics-based identification of WRKY genes and characterization of a salt and hormone-responsive PgWRKY1 gene in Panax ginseng.

WRKY proteins belong to a transcription factor (TF) family and play dynamic roles in many plant processes, including plant responses to abiotic and biotic stresses, as well as secondary metabolism. However, no WRKY gene in Panax ginseng C.A. Meyer has been reported to date. In this study, a number of WRKY unigenes from methyl jasmonate (MeJA)-treated adventitious root transcriptome of this species were identified using next-generation sequencing technology. A total of 48 promising WRKY unigenes encoding WRKY proteins were obtained by eliminating wrong and incomplete open reading frame (ORF). Phylogenetic analysis reveals 48 WRKY TFs, including 11 Group I, 36 Group II, and 1 Group III. Moreover, one MeJA-responsive unigene designated as PgWRKY1 was cloned and characterized. It contains an entire ORF of 1077 bp and encodes a polypeptide of 358 amino acid residues. The PgWRKY1 protein contains a single WRKY domain consisting of a conserved amino acid sequence motif WRKYGQK and a C2H2-type zinc-finger motif belonging to WRKY subgroup II-d. Subcellular localization of PgWRKY1-GFP fusion protein in onion and tobacco epidermis cells revealed that PgWRKY1 was exclusively present in the nucleus. Quantitative real-time polymerase chain reaction analysis demonstrated that the expression of PgWRKY1 was relatively higher in roots and lateral roots compared with leaves, stems, and seeds. Importantly, PgWRKY1 expression was significantly induced by salicylic acid, abscisic acid, and NaCl, but downregulated by MeJA treatment. These results suggested that PgWRKY1 might be a multiple stress-inducible gene responding to hormones and salt stresses.

Detection of serum anti-NV-F antibodies in the convalescent phase of severe hepatitis in patients positive for tissue NV-F antigen and novel virus-like particles.

Previously, a non-human DNA fragment named NV-F was isolated from a patient with non-A-E fulminant hepatitis. This sequence encoded an incomplete open reading frame (the NV-F antigen). In this study, we developed a western blot assay to detect serum anti-NV-F antibodies. Serum samples from 347 patients with severe hepatitis (ALT > fivefold ULN) were analyzed to understand the prevalence and distribution of the NV-F associated virus (HnFV) infection. Of these patients, acute HnFV infection was diagnosed (by positive serum NV-F DNA) in 34 patients (9.8%). However, none of these 34 serum samples were positive for serum anti-NV-F antibodies. In the remaining patients negative for serum NV-F DNA, 62 (17.9%) were positive for serum anti-NV-F antibodies. Liver biopsy samples from 35 severe hepatitis patients were submitted for immunohistochemistry and electron microscopy examination. Of them, seven were positive for hepatic NV-F antigen expression. Electron microscopy identified a novel virus-like particle in all of the seven NV-F antigen-positive liver tissues but not in the remaining 28 NV-F antigen-negative liver tissues. Longitudinal serum sample analysis revealed transient positivity of serum NV-F DNA in three of the seven patients during the clinical courses. Seroconversion of anti-NV-F antibody from negative to positive was found in four of the seven patients and all positive anti-NV-F antibodies were detected in the convalescent phases. In conclusion, in patients with severe hepatitis, a novel hepatotropic virus, temporarily named HnFV, was found in liver tissues expressing the NV-F antigen. Serum anti-NV-F antibodies were detected in the convalescent serum samples.

MGC: a metagenomic gene caller

BackgroundComputational gene finding algorithms have proven their robustness in identifying genes in complete genomes. However, metagenomic sequencing has presented new challenges due to the incomplete and fragmented nature of the data. During the last few years, attempts have been made to extract complete and incomplete open reading frames (ORFs) directly from short reads and identify the coding ORFs, bypassing other challenging tasks such as the assembly of the metagenome.ResultsIn this paper we introduce a metagenomics gene caller (MGC) which is an improvement over the state-of-the-art prediction algorithm Orphelia. Orphelia uses a two-stage machine learning approach and computes a model that classifies extracted ORFs from fragmented sequences. We hypothesise and demonstrate evidence that sequences need separate models based on their local GC-content in order to avoid the noise introduced to a single model computed with sequences from the entire GC spectrum. We have also added two amino-acid features based on the benefit of amino-acid usage shown in our previous research. Our algorithm is able to predict genes and translation initiation sites (TIS) more accurately than Orphelia which uses a single model.ConclusionsLearning separate models for several pre-defined GC-content regions as opposed to a single model approach improves the performance of the neural network as demonstrated by the experimental results presented in this paper. The inclusion of amino-acid usage features also helps improve the overall accuracy of our algorithm. MGC's improvement sets the ground for further investigation into the use of GC-content to separate data for training models in machine learning based gene finders.

Evolution of the Phosphatase Gene Family Across Nematode Worms and Flies

Phosphatase genes have been shown to be involved in male meiosis in the nematode worm, Caenorhabditis elegans, and are expressed in the testis in the fruit fly, Drosophila melanogaster. However, the evolution of this multi-gene family among nematodes and flies had not previously been investigated. We conducted a phylogenetic analysis of all genes in the phosphatase gene family across nematodes and flies using sequences from a 6-way alignment of nematode worms and a 15- way alignment of insects, including 12 Drosophila species. We found that: 1) multiple alignments contain spurious alignments that should be filtered for quality control; 2) several gene sequences with incomplete open reading frames are highly conserved, so may actually be functional genes; and, 3) the phosphatase gene family appears to have expanded independently in the common ancestor of nematodes, and again in the common ancestor of flies (but not all insects).

Dynamic Actin Gene Family Evolution in Primates

Actin is one of the most highly conserved proteins and plays crucial roles in many vital cellular functions. In most eukaryotes, it is encoded by a multigene family. Although the actin gene family has been studied a lot, few investigators focus on the comparison of actin gene family in relative species. Here, the purpose of our study is to systematically investigate characteristics and evolutionary pattern of actin gene family in primates. We identified 233 actin genes in human, chimpanzee, gorilla, orangutan, gibbon, rhesus monkey, and marmoset genomes. Phylogenetic analysis showed that actin genes in the seven species could be divided into two major types of clades: orthologous group versus complex group. Codon usages and gene expression patterns of actin gene copies were highly consistent among the groups because of basic functions needed by the organisms, but much diverged within species due to functional diversification. Besides, many great potential pseudogenes were found with incomplete open reading frames due to frameshifts or early stop codons. These results implied that actin gene family in primates went through “birth and death” model of evolution process. Under this model, actin genes experienced strong negative selection and increased the functional complexity by reproducing themselves.

Evolutionary origins of Brassicaceae specific genes in Arabidopsis thaliana

BackgroundAll sequenced genomes contain a proportion of lineage-specific genes, which exhibit no sequence similarity to any genes outside the lineage. Despite their prevalence, the origins and functions of most lineage-specific genes remain largely unknown. As more genomes are sequenced opportunities for understanding evolutionary origins and functions of lineage-specific genes are increasing.ResultsThis study provides a comprehensive analysis of the origins of lineage-specific genes (LSGs) in Arabidopsis thaliana that are restricted to the Brassicaceae family. In this study, lineage-specific genes within the nuclear (1761 genes) and mitochondrial (28 genes) genomes are identified. The evolutionary origins of two thirds of the lineage-specific genes within the Arabidopsis thaliana genome are also identified. Almost a quarter of lineage-specific genes originate from non-lineage-specific paralogs, while the origins of ~10% of lineage-specific genes are partly derived from DNA exapted from transposable elements (twice the proportion observed for non-lineage-specific genes). Lineage-specific genes are also enriched in genes that have overlapping CDS, which is consistent with such novel genes arising from overprinting. Over half of the subset of the 958 lineage-specific genes found only in Arabidopsis thaliana have alignments to intergenic regions in Arabidopsis lyrata, consistent with either de novo origination or differential gene loss and retention, with both evolutionary scenarios explaining the lineage-specific status of these genes. A smaller number of lineage-specific genes with an incomplete open reading frame across different Arabidopsis thaliana accessions are further identified as accession-specific genes, most likely of recent origin in Arabidopsis thaliana. Putative de novo origination for two of the Arabidopsis thaliana-only genes is identified via additional sequencing across accessions of Arabidopsis thaliana and closely related sister species lineages. We demonstrate that lineage-specific genes have high tissue specificity and low expression levels across multiple tissues and developmental stages. Finally, stress responsiveness is identified as a distinct feature of Brassicaceae-specific genes; where these LSGs are enriched for genes responsive to a wide range of abiotic stresses.ConclusionImproving our understanding of the origins of lineage-specific genes is key to gaining insights regarding how novel genes can arise and acquire functionality in different lineages. This study comprehensively identifies all of the Brassicaceae-specific genes in Arabidopsis thaliana and identifies how the majority of such lineage-specific genes have arisen. The analysis allows the relative importance (and prevalence) of different evolutionary routes to the genesis of novel ORFs within lineages to be assessed. Insights regarding the functional roles of lineage-specific genes are further advanced through identification of enrichment for stress responsiveness in lineage-specific genes, highlighting their likely importance for environmental adaptation strategies.

Dynamics of Actin Evolution in Dinoflagellates

Dinoflagellates have unique nuclei and intriguing genome characteristics with very high DNA content making complete genome sequencing difficult. In dinoflagellates, many genes are found in multicopy gene families, but the processes involved in the establishment and maintenance of these gene families are poorly understood. Understanding the dynamics of gene family evolution in dinoflagellates requires comparisons at different evolutionary scales. Studies of closely related species provide fine-scale information relative to species divergence, whereas comparisons of more distantly related species provides broad context. We selected the actin gene family as a highly expressed conserved gene previously studied in dinoflagellates. Of the 142 sequences determined in this study, 103 were from the two closely related species, Dinophysis acuminata and D. caudata, including full length and partial cDNA sequences as well as partial genomic amplicons. For these two Dinophysis species, at least three types of sequences could be identified. Most copies (79%) were relatively similar and in nucleotide trees, the sequences formed two bushy clades corresponding to the two species. In comparisons within species, only eight to ten nucleotide differences were found between these copies. The two remaining types formed clades containing sequences from both species. One type included the most similar sequences in between-species comparisons with as few as 12 nucleotide differences between species. The second type included the most divergent sequences in comparisons between and within species with up to 93 nucleotide differences between sequences. In all the sequences, most variation occurred in synonymous sites or the 5' UnTranslated Region (UTR), although there was still limited amino acid variation between most sequences. Several potential pseudogenes were found (approximately 10% of all sequences depending on species) with incomplete open reading frames due to frameshifts or early stop codons. Overall, variation in the actin gene family fits best with the "birth and death" model of evolution based on recent duplications, pseudogenes, and incomplete lineage sorting. Divergence between species was similar to variation within species, so that actin may be too conserved to be useful for phylogenetic estimation of closely related species.

Molecular characterization of a new macluravirus from yam in Yunnan, China

Yams (Dioscorea spp.) are important in China as vegetables and as sources of traditional herbal medicine. They suffer from various viral diseases, but there has only been one small study of them in China [6]. We have investigated the viruses associated with diseased yams at two farms 5 km apart in Yongsheng county, Yunnan province. Samples 1–3 from D. zingbierensis had a combination of vein clearing, veinal necrosis and chlorotic symptoms on the expanded leaves. Samples 1 and 2 were from the same farm. Sample 4 from D. parviflora (same site as sample 3) had systemic chlorosis, mottling and mosaic symptoms that also affected the newly emerging leaves. Virus particles and inclusion bodies typical of members of the family Potyviridae were observed in infected leaves. Particles were purified [3], RNA was extracted and the 30-terminal portion of the viral genome was then amplified by RT-PCR by standard methods using universal Potyviridae primers [4]. Products were separated, purified and cloned (into pMD19-T; TaKaRa), and three independent clones were auto-sequenced in both directions using an ABI PRISM 3770 DNA Sequencer. Sequence alignments and phylogenetic analysis were done using MEGA 4.0 [7]. Fragments of 1,742–1,748 nucleotides in length were amplified and sequenced from each sample. Isolates 1, 3 and 4 were almost identical to one another (99.4–99.5% nucleotide identities) while isolate 2 had 78.2% identity to the others. The sequences consisted of an incomplete open reading frame with a stop codon at position 1531–1533 followed by a 30-UTR of 209 nucleotides (215 in sample 2). In comparisons with published sequences, the nearest matches were those for Chinese yam necrotic mosaic virus (ChYNMV, genus Macluravirus), with about 69% nt and 72–73% aa identity. Comparisons with all other macluravirus sequences suggested that the partial ORF of 510 amino acid residues in the new sequences consisted of 222 aa at the C-terminus of the NIb and a coat protein (CP) of 288 aa (32.3 kDa) with a cleavage site of INLQ/M (IDLQ/ M in sample 2). The LQ/M cleavage is typical of members of the genus Macluravirus [1]. The partial NIb gene contained the consensus motifs (T/S)GX3-TX3-N(T/S) and GDD of the RNA-dependent RNA polymerase. The N-terminus of the CP did not contain a DAG (or similar) motif that is known to be associated with aphid transmission in the genus Potyvirus. Molecular criteria for discriminating between species in the family Potyviridae indicate a threshold of 76% (nt) or 82% (aa) identity [2]. Based on these criteria, the new yam sequences belong to a single new species in the genus Macluravirus. These conclusions were confirmed by phylogenetic analysis (Fig. 1). Although isolate 2 is rather different to the others at the molecular level, it was one of the three from D. zingbierensis that had similar symptoms and is not sufficiently different to justify placement in a further new species. Interestingly, there is a similar range of differences between isolates of Cardamom mosaic virus J.-G. Wang X.-J. Zou H.-R. Chen (&) The Phytopathology Laboratory of Yunnan Province, Yunnan Agricultural University, 650201 Kunming, People’s Republic of China e-mail: hrchen44@yahoo.cn

Subtractive hybridization and random arbitrarily primed PCR analyses of a benzoate-assimilating bacterium, Desulfotignum balticum

Subtractive hybridization (SH) and random arbitrarily primed PCR (RAP-PCR) were used to detect genes involved in anaerobic benzoate degradation by Desulfotignum balticum. Through SH, we obtained 121 DNA sequences specific for D. balticum but not for D. phosphitoxidans (a non-benzoate-assimilating species). Furthermore, RAP-PCR analysis showed that a 651-bp DNA fragment, having 55% homology with the solute-binding protein of the ABC transporter system in Methanosarcina barkeri, was expressed when D. balticum was grown on benzoate, but not on pyruvate. By shotgun sequencing of the fosmid clone (38,071 bp) containing the DNA fragment, 33 open reading frames (ORFs) and two incomplete ORFs were annotated, and several genes within this region corresponded to the DNA fragments obtained by SH. An 11.3-kb gene cluster (ORF10-17) revealed through reverse transcription-PCR showed homology with the ABC transporter system and TonB-dependent receptors, both of which are presumably involved in the uptake of siderophore/heme/vitamin B(12), and was expressed in response to growth on benzoate.

Systematic analysis of mRNA 5' coding sequence incompleteness in Danio rerio: an automated EST-based approach

BackgroundAll standard methods for cDNA cloning are affected by a potential inability to effectively clone the 5' region of mRNA. The aim of this work was to estimate mRNA open reading frame (ORF) 5' region sequence completeness in the model organism Danio rerio (zebrafish).ResultsWe implemented a novel automated approach (5'_ORF_Extender) that systematically compares available expressed sequence tags (ESTs) with all the zebrafish experimentally determined mRNA sequences, identifies additional sequence stretches at 5' region and scans for the presence of all conditions needed to define a new, extended putative ORF. Our software was able to identify 285 (3.3%) mRNAs with putatively incomplete ORFs at 5' region and, in three example cases selected (selt1a, unc119.2, nppa), the extended coding region at 5' end was cloned by reverse transcription-polymerase chain reaction (RT-PCR).ConclusionThe implemented method, which could also be useful for the analysis of other genomes, allowed us to describe the relevance of the "5' end mRNA artifact" problem for genomic annotation and functional genomic experiment design in zebrafish.Open peer reviewThis article was reviewed by Alexey V. Kochetov (nominated by Mikhail Gelfand), Shamil Sunyaev, and Gáspár Jékely. For the full reviews, please go to the Reviewers' Comments section.

Characterization of an Unusual Cytoplasmic Chimera Detected in Bolting Garlic Clones

Production of a visible flower stalk, or bolting, has been used as a major trait to categorize garlic (Allium sativum L.) clones. Analysis of mitochondrial genome variation with polymerase chain reaction (PCR) revealed differences between bolting and nonbolting clones of garlic. Screening 333 garlic accessions from diverse geographic origins revealed a 1403-bp mitochondrial DNA marker associated with bolting that the authors call “Bolt Marker” (BltM). Bolt Marker did not amplify in any of the 131 nonbolting clones, whereas amplification of this marker was observed in 127 of 130 (97.7%) garlic clones that bolted completely in Wisconsin. Seventy-two garlic clones bolted incompletely (clones in which some but not all of the plants bolted), and this marker was not amplified in 69 (95.8%) of these clones. Because of the significant association of BltM with bolting, this PCR-based marker can be used to discriminate complete-bolting garlic clones reliably from nonbolting and incomplete-bolting ones. Sequence characterization of this marker revealed that BltM is a chimera involving both mitochondrial and chloroplast DNA. The DNA sequences including and flanking both the 5′ and 3′ ends of this marker are consistent with an ≈4.8-kbp chloroplast DNA fragment having been inserted into the mitochondrial genome downstream from the mitochondrial cox3 gene. Sequence alignment of the chloroplast genes in this chimeric region with the homologous sequences in GenBank indicate the presence of deletions, insertions, and single nucleotide polymorphisms in the coding sequences, resulting in putative, incomplete open reading frames or frame shift mutations. Hence, the authors speculate that this insertion may have occurred long ago in the evolution of garlic.

Structural characterization of the bglH gene encoding a beta-glucosidase-like enzyme in an endophytic Bacillus pumilus strain

Abstract A beta-glucosidase-like enzyme-encoding gene (bglH) of an endophytic Bacillus pumilusstrain (CL16) was clonedusing a shotgun genomic library constructed in Escherichia coli. The nucleotide sequence of the entire cloned frag-ment (2484 bp) was determined and characterized. An incomplete open reading frame (ORF) of 534 bp (ORF1) des-ignated bglP and a complete ORF of 1419 bp (ORF2) designated bglH, located in the fragment, are organized in anoperon. The protein deduced from 1419 bp (ORF2) had 472 amino acid residues without a characteristic signal pep-tide sequence, suggesting that the enzyme is localized in the cytoplasm. The amino acid sequence deduced frombglH gene had high similarity with β-glucosidases from the glycosyl hydrolase family 1. Over-expression of the B.pumilus bglH gene in E. coli showed a 54 kDa protein whose identity was confirmed by mass spectrometry(MALDI-TOF). Key words: Bacillus pumilus , bgl H, glucosidase, glycosyl hydrolase 1, PTS.Received: November 28, 2005; Accepted: August 17, 2006.

Expression of pqq Genes from Serratia marcescens W1 in Escherichia coli Inhibits the Growth of Phytopathogenic Fungi

Serratia marcescens W1, isolated from cucumber-cultivated soil in Suwon, Korea, evidenced profound antifungal activity and produced the extracellular hydrolytic enzymes, chitinase and protease. In order to isolate the antifungal genes from S. marcescens W1, a cosmid genomic library was constructed and expressed in Escherichia coli. Transformants exhibiting chitinase and protease expression were selected, as well as those transformants evidencing antifungal effects against the rice blast fungus, Magnaporthe grisea, and the cucumber leaf spot fungus, Cercospora citrullina. Cosmid clones expressing chitinase or protease exerted no inhibitory effects against the growth of fungal pathogens. However, two cosmid clones evidencing profound antifungal activities were selected for further characterization. An 8.2 kb HindIII fragment from these clones conditioned the expression of antagonistic activity, and harbored seven predicted complete open reading frames(ORFs) and two incomplete ORFs. The deduced amino acid sequences indicated that six ORFs were highly homologous with genes from S. marcescens generating pyrroloquinoline quinone(PQQ). Only subclones harboring the full set of pqq genes were shown to solubilize insoluble phosphate and inhibit fungal pathogen growth. The results of this study indicate that the functional expression of the pqq genes of S. marcescens W1 in E. coli may be involved in antifungal activity, via as-yet unknown mechanisms.

Antimicrobial Resistance in NontyphoidalSalmonellafrom Food Sources in Colombia: Evidence for an Unusual Plasmid-Localized Class 1 Integron in Serotypes Typhimurium and Anatum

Seventy-two isolates representing 18 serotypes recovered from various food samples collected in Colombia were tested for antimicrobial susceptibilities. The collection was further characterized for extended-spectrum cephalosporin, aminoglycoside, and tetracycline resistance markers. Multidrug resistant (MDR) isolates were further investigated for class 1 integrons and were evaluated for the presence of conjugative plasmids along with a determination of the incompatibility group by polymerase chain reaction (PCR). Antibiogram analysis showed that the incidence rate of ceftiofur resistance was moderately high (15%). A similar level of resistance to neomycin and oxytetracycline (11% and 10%, respectively) was also observed. There was a high prevalence of gene cassettes as part of one or more class 1 integrons (61%), many of which contained determinants that contributed to the resistance profile. Class 1 integrons identified in MDR Salmonella enterica serotypes Typhimurium and Anatum isolates were characterized. Sequencing identified several incomplete open reading frames (ORFs) as part of a gene cassette (bla-( imp-13 ), dfr7, blr1088, and aac8) along with a complete gene cassette (bla-(oxa2)) in each case. A mosaic of gene cassettes was identical in the two Salmonella serotypes. These integrons were located to a conjugative replicon. Plasmid profiling and incompatibility typing identified three plasmids belonging to Inc groups A/C, P, and W. Our study highlights the role of integrons, contributing to a MDR phenotype that is capable of dissemination to other bacteria.

Cloning and sequence analysis of the leucine-rich repeats region of cereal cyst nematode resistance gene

AbstractAccording to the sequence of Rccn4, which is highly similar to the nucleotide-binding site (NBS) coding region of the cereal cyst nematode resistance gene, Cre3, three 3′ nested primers were designed to amplify its 3′ flanking region through single oligonucleotide nested polymerase chain reaction (SON-PCR). One 1264 bp band, Rccn-L, was amplified from E-10, a wheat–Aegilops variabilis translocation line containing the cereal cyst nematode resistance gene from Ae. variabilis. Sequence analysis showed that Rccn-L possesses the 3′ flanking sequence of Rccn4 and contains a 55 bp-sized consensus sequence with Rccn4. The coding region was 1026 bp, consisting of an incomplete open reading frame, a terminator codon and no initiation codon and intron; it encoded a peptide of 342 amino acid residues and shared 86% nucleotide sequence identity with Cre3. The peptide had a conserved leucine-rich repeat (LRR) domain, containing the imperfect repeats, XXLXXLXXL, comprising 17% leucine residues, and shares, respectively, 89% nucleotide sequence and 78% amino acid sequence identity with the LRR sequence of the Cre3 locus. In the present study, SON-PCR was used successfully, for the first time, in plant genome research and proved to be a valuable method in plant gene cloning. The acquirement of Rccn-L established the foundation for obtaining the complete Rccn gene and further structural and functional investigations.

DNA transposon fossils present on the conditionally dispensable chromosome controlling AF-toxin biosynthesis and pathogenicity of Alternaria alternata

The strawberry pathotype of Alternaria alternata produces host-specific AF-toxin and causes Alternaria black spot of strawberry. Previously, we isolated cosmid clones pcAFT-1 and pcAFT-2 from strain NAF8 of the strawberry pathotype that contain AF-toxin biosynthetic genes, named AFT genes. In a molecular characterization here of pcAFT-1 and pcAFT-2, 11 AFT genes and five transposon-like sequences, named TLS-S1 to TLS-S5, were detected. The nucleotide sequences of TLS-S1 and TLS-S4 share high homology, and their putative products have similarity to transposases of the hAT family transposons. Thus, TLS-S1 and TLS-S4 were renamed TLS-S1-1 and TLS-S1-2, respectively. Amino acid sequences deduced from TLS-S2, TLS-S3, and TLS-S5 have similarity to transposases of the Fot1/Pogo family transposons, but they are significantly different. All five sequences have incomplete open reading frames (ORFs) for transposases owing to deletions, termination codons, and/or frameshifts, indicating that they are inactivated elements. Analysis of genomic distribution of these sequences revealed that they are specifically distributed on a 1.05-Mb chromosome of NAF8, which has been identified as a conditionally dispensable (CD) chromosome encoding AFT genes. The presence of three, four, and three copies of TLS-S1, TLS-S2, and TLS-S3, respectively, and a single copy of TLS-S5 on the CD chromosome were estimated by DNA gel blot analysis. The remaining copy of TLS-S1 and the three copies of TLS-S2 were isolated and identified to also encode incomplete ORFs. Thus, it appears that all copies of the transposon-like sequences identified are inactivated elements (fossils) unique to the CD chromosome in the genome of the strawberry pathotype.

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 beta-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 beta-galactosidase, with estimated molecular masses of 72,129 and 35,233 Da, respectively. Other three incomplete open reading frames showing homology to another beta-galactosidase, an alpha-galactosidase, and a galactokinase, respectively, were also found. The L. coryniformis beta-galactosidase was overproduced in E. coli by using an isopropyl-beta-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 beta-galactosidase activity.

A gene cluster of the ochratoxin A biosynthetic genes inPenicillium

A putative ochratoxin A (OTA) biosynthetic gene cluster inP. nordicum has been identified. The first part of the gene cluster is located on a DNA fragment of 10 kb in length and harbours three genes. A gene with high homology to an alkaline serine protease gene (accession number AY557343), which represents the upstream border of the cluster. Furthermore the fragment carries a large part (about 2 kb) of the 5' end of a polyketide synthase (otapksPN, accession number AY196315) and a complete non-ribosomal peptide synthetase (otanpsPN, accession number AY534879). The second part of the cluster is located on a 4.3 kb fragment that harbours three open reading frames (ORFs) encoding putative OTA biosynthetic proteins: one incomplete ORF at the 5' end of the fragment demonstrated homology to an organic anion transporter from rat kidneys (otatraPN). This transporter has been described to be responsible for the transport of toxic OTA out of the cell. One complete ORF of 951 nucleotides is also located on this fragment. This gene has limited homology to a chloroperoxidase fromGluconobacter oxidans. At the 3' end of this DNA fragment is an incomplete open reading frame of a potential nitrate transporter. The transcription of all putative OTA biosynthetic genes is increased under OTA conducive conditions. The expression kinetics of the genes resembles that of secondary metabolite biosynthetic genes, in which these genes are co-ordinately expressed during the late growth phase.PCR analysis demonstrated that the gene cluster is only present in the two ochratoxin A producingPenicillium species,P. verrucosum andP. nordicum. P. nalgiovense, a species occurring in the same habitat asP. nordicum carries inactive homologues of the genes. All other species proved to be negative for the genes. This was also true for OTA producing Aspergilli.

Isolation and partial characterization of a cryptic polyene gene cluster in Pseudonocardia autotrophica

The polyene antibiotics, a category that includes nystatin, pimaricin, amphotericin, and candicidin, comprise a family of very promising antifungal polyketide compounds and are typically produced by soil actinomycetes. The biosynthetic gene clusters for these polyenes have been previously investigated, revealing the presence of highly similar cytochrome P450 hydroxylase (CYP) genes. Using polyene CYP-specific PCR screening with several actinomycete genomic DNAs, Pseudonocardia autotrophica was determined to contain a unique polyene-specific CYP gene. Genomic DNA library screening using the polyene-specific CYP gene probe identified a positive cosmid clone, which contained a DNA fragment of approximately 34.5 kb. The complete sequencing of this DNA fragment revealed a total of seven complete and two incomplete open reading frames, which were found to be highly similar, but still unique, when compared to previously known polyene biosynthetic genes. These results suggest that the polyene-specific screening approach may constitute an efficient method for the isolation of potentially valuable cryptic polyene biosynthetic gene clusters from various rare actinomycetes.