Lambda Library Research Articles

Isolation of an Ecotropic Porcine Endogenous Retrovirus PERV-C from a Yucatan SLAD/D Inbred Miniature Swine.

Xenotransplantation may compensate the limited number of human allografts for transplantation using pigs as organ donors. Porcine endogenous retroviruses inherit infectious potential if pig cells, tissues, or organs were transplanted to immunosuppressed human recipients. Particularly, ecotropic PERV-C that could recombine with PERV-A to highly replication-competent human-tropic PERV-A/C should be excluded from pig breeds designed for xenotransplantation. Because of their low proviral background, SLAD/D (SLA, swine leukocyte antigen) haplotype pigs are potential candidates as organ donors as they do not bear replication-competent PERV-A and -B, even if they carry PERV-C. In this work, we characterized their PERV-C background isolating a full-length PERV-C proviral clone number 561 from a SLAD/D haplotype pig genome displayed in a bacteriophage lambda library. The provirus truncated in env due to cloning in lambda was complemented by PCR, and the recombinants were functionally characterized, confirming an increased infectivity in vitro compared to other PERV-C. Recombinant clone PERV-C(561) was chromosomally mapped by its 5'-proviral flanking sequences. Full-length PCR using 5'-and 3'-flanking primers specific to the PERV-C(561) locus verified that this specific SLAD/D haplotype pig harbors at least one full-length PERV-C provirus. The chromosomal location is different from that of the previously described PERV-C(1312) provirus, which was derived from the porcine cell-line MAX-T. The sequence data presented here provide further knowledge about PERV-C infectivity and contribute to targeted knockout in order to generate PERV-C-free founder animals. IMPORTANCE Yucatan SLAD/D haplotype miniature swine are candidates as organ donors for xenotransplantation. A full-length replication-competent PERV-C provirus was characterized. The provirus was chromosomally mapped in the pig genome. In vitro, the virus showed increased infectivity compared to other functional PERV-C isolates. Data may be used for targeted knockout to generate PERV-C free founder animals.

Identification and Analysis of Putative Polyhydroxyalkanoate Synthase (PhaC) in Pseudomonas fluorescens.

Pseudomonas fluorescens KLR101 was found to be capable of producing polyhydroxyalkanoate (PHA) using various sugars and fatty acids with carbon numbers ranging from 2 to 6. The PHA granules consisted mainly of a poly(3-hydroxybutyrate) homopolymer and/or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer. Genomic DNA of P. fluorescens was fractionated and cloned into a lambda library, in which a 5.8-kb fragment that hybridized to a heterologous phaC probe from Ralstonia eutropha was identified. In vivo expression in Klebsiella aerogenes KC2671 (pUMS), restriction mapping, Southern hybridization experiments, and sequencing data revealed that PHA biosynthesis by P. fluorescens relied upon a polypeptide encoded by a 1,683-bp non-operonal ORF, which was preceded by a possible -24/-12 promoter and highly similar to DNA sequences of a gene encoding PHA synthase in the genus Pseudomonas. In vivo expression of the putative PHA synthase gene (phaCPf) in a recombinant Escherichia coli strain was investigated by using glucose and decanoate as substrates. E. coli (phaCPf+, pUMS) grown in medium containing glucose accumulated PHA granules consisting mainly of 3-hydroxybutyrate, whereas only a trace amount of 3-hydroxydecanoate was detected from an E. coli fadR mutant (phaCPf+) grown in medium containing decanoate. In vitro enzymatic assessment experiments showed that 3-hydroxybutyryl-CoA was efficiently used as a substrate of purified PhaCPf, suggesting that the putative PHA synthase of P. fluorescens utilizes mainly short-chain-length PHA precursors as a substrate.

Cloning and characterization of replication‐competent ecotropic porcine endogenous retroviruses (PERV‐C) in the genome of pigs used and intended for clinical pig‐to‐human xenotransplantation

Porcine endogenous retroviruses (PERV) pose a xenozoonotic risk when applying pig organs, tissues and cells in clinical xenotransplantation. Three classes of replication‐competent PERV were described in host range and interference studies [1]. Polytropic PERV‐A and PERV‐B are able to infect not only human cells but also various cell lines in vitro using host membrane proteins as receptors [2]. Although cell tropism of ecotropic PERV‐C is mainly restricted to porcine cells, PERV‐C represents a considerable infectious risk. On the one hand, PERV‐C serves as template for recombination with PERV‐A resulting in highly infectious human‐tropic PERV‐A/C [3]. On the other hand, PERV‐C may evolve towards an infectious human‐tropic variant since the PERV‐C receptor‐binding domain could be bound to human cells and solely four amino acid exchanges in the surface unit of the envC gene are sufficient to permit receptor‐mediated membrane fusion and virus entry [4]. To guarantee retroviral safety in pig‐to‐human xenotransplantation generation of appropriate pigs free from replication‐competent PERV‐A and PERV‐B as well as ecotropic PERV‐C is required.Using PCR‐based methods and directional cloning strategies, we cloned and characterized PERV‐C. Genomic DNA was prepared from peripheral blood mononuclear cells derived from three different pig subspecies which are either already used or intended for clinical xenotransplantation. One PERV‐C clone was reconstructed using genomic DNA of an Auckland Islands pig derived from a DPF herd in New Zealand. Islet cell clusters of these PERV non‐transmitters are used in clinical trials to treat diabetes type I patients. Moreover, the potential of brain cells from these animals to treat Parkinson's and Huntington's disease is currently tested clinically. Similarly, a PERV‐C clone was reconstructed from genomic DNA of Göttingen minipig which is a PERV non‐transmitter as tested in co‐cultures with susceptible human HEK 293 cells [5]. Finally, a bacteriophage lambda library was constructed from genomic DNA of a d/d haplotype miniature pig. The individuals of this pig line show lower PERV transmission levels in vitro [6]. We isolated a λ‐clone containing PERV‐C 5′‐LTR, gag, pro/pol as well as large part of the envC gene compared to replication‐competent PERV‐C(1312) [7]. The provirus is truncated due to our cloning strategy. Nonetheless, using PCR and PERV‐C specific primers the missing part of the envC gene and 3′‐LTR was amplified and ligated to the proviral fragment present in the λ‐clone. Replication‐competence of reconstructed full‐length viruses is currently tested in susceptible ST‐Iowa cells.

Selection and Characterization of Human Serum Albumin-specific Porcine scFv Antibodies Using a Phage Display Library

A new single-chain variable fragment (scFv) antibody library was generated and human serum albumin (HSA)-specific clones were characterized to investigate the usefulness of porcine antibodies. Phage libraries were developed from pigs immunized with the model antigen HSA. The library size was 1.5 × 10(7) for kappa (VL) and 1.4 × 10(7) for lambda fragments. Eight HSA-specific clones from the kappa library and one clone from the lambda library were isolated using affinity selection. The binding specificity of these clones was confirmed using a phage enzyme-linked immunosorbent assay (ELISA). The scFvs were expressed in Escherichia coli and purified from the periplasm fraction for further investigation. Based on the results of ELISA and Western blot analysis, four scFv clones with high activity and high yield were selected and purified. The purified scFvs from four of the nine clones exhibited an approximate KD of 10(-8) M. This is the first report describing isolation of HSA-specific porcine scFv antibodies from an antibody phage library and characterization of their binding properties.

Characterization of the rrn operons in the channel catfish pathogen Edwardsiella ictaluri

To advance diagnostics and phylogenetics of Edwardsiella ictaluri by sequencing and characterizing its rrn operons. The Edw. ictaluri rrn operons were identified from a 5-7 kbp insert lambda library and from Edw. ictaluri fosmid clones. We present the complete sequences and analysis of all eight Edw. ictaluri rrn operons and unique regions located upstream and downstream. Two rrn operons were located in tandem with 169 bp separating them, which is apparently a conserved feature between Edw. ictaluri and Edwardsiella tarda. I-CeuI enzyme digestion of Edw. ictaluri genomic DNA and analysis by pulsed field gel electrophoresis indicated that rrn operon number and chromosomal locations are conserved within the species Edw. ictaluri. The rrn operons of Edw. ictaluri have similar structure and flanking regions compared with other members of the family Enterobacteriaceae; however, the presence of eight copies of the rrn operon makes Edw. ictaluri unique within the family. This research clarifies previous phylogenetic analyses of Edw. ictaluri and provides support for the Edw. ictaluri genome sequencing project. In addition, we identified a unique feature of two rrn operons that shows potential for the development of a diagnostic PCR method.

Identification of Antigenic Components of Staphylococcus epidermidis Expressed during Human Infection

A spectrum of in vivo-expressed Staphylococcus epidermidis antigens was identified by probing a bacteriophage lambda library of S. epidermidis genomic DNA with human serum from infected and uninfected individuals. This analysis resulted in identification of 53 antigen-encoding loci. Six antigenic polypeptides were expressed from these loci and purified. These polypeptides were the propeptide, mature amidase, and repeat sequence domains of the major autolysin AtlE, GehD (lipase), and two members of a conserved family of surface proteins (ScaA [AaE] and ScaB). AtlE, ScaA, and ScaB all exhibit human ligand binding capacity. Screening a bank of human serum samples revealed that there were significant increases in the amounts of reactive immunoglobulin G in infected individuals compared to the amounts in healthy individuals for the repeat sequence and mature amidase domains of AtlE, ScaB, and GehD. Vaccination of mice with recombinant antigens stimulated an immune response which in vitro opsonized S. epidermidis. In this study we identified prospective candidate antigens for prophylaxis or immunotherapy to control disease.

Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B

To construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B. The total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5' end of the RNA transcript (SMART) technique and CDS III/3' primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction (LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to lambdaTriplEx2 vector. Then lambda phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. The titers of unamplifed and amplified libraries were 1.94 x 10(6) pfu/ml and 1.49 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and 98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29%, and 0.5-1.0 kb in 35.71%. A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.

Cloning and expression of the first nonmammalian interleukin‐11 gene in rainbow trout Oncorhynchus mykiss

Interleukin (IL)-11 is a multifunctional cytokine that stimulates hematopoietic progenitor cells and exerts a series of important immunomodulatory effects. It was believed to be restricted to mammals, but here we report the first nonmammalian IL-11 gene, in rainbow trout (Oncorhynchus mykiss). A trout IL-11 cDNA clone was isolated that contains a 5'-untranslated region (UTR) of 400 bp, an open reading frame of 612 bp and a large 3'-UTR of 1924 bp. Analysis of a genomic DNA clone from a trout lambda library revealed that the trout IL-11 gene has the same five exon/four intron gene organization, as well as the same intron phase, as mammalian IL-11 genes. The 204 amino acid trout IL-11 translation has a predicted signal peptide of 26 amino acids and mature peptide of 178 amino acids, with a calculated molecular mass of 20.5 kDa and a theoretical pI of 9.83. The mature peptide contains a cysteine residue and a potential N-linked glycosylation site that are not present in mammals. Phylogenetic analysis clearly grouped trout IL-11 with IL-11 molecules from other species and separated from other members of the IL-6 family. The IL-11 gene is highly expressed in intestine and gills in healthy fish and its expression can also be detected in spleen, head kidney, brain, skin and muscle. Bacterial infection of rainbow trout markedly up-regulates IL-11 expression in liver, head kidney and spleen. IL-11 expression is also up-regulated in RTS-11 cells (a trout macrophage cell line), which constitutively expressed the lowest level of IL-11 of the four trout cell lines examined, after stimulation with bacteria, lipopolysaccharide, poly(I:C) and recombinant trout IL-1beta. Only a single transcript of 3.2 kb could be detected in lipopolysaccharide or recombinant IL-1beta-stimulated RNA samples by northern blotting. The expression results, showing that IL-11 is widely distributed and modulated by infection and other cytokines, suggest that fish IL-11 is an active player in the cytokine network and the host immune response to infection.

Flow sorting and microcloning of maize chromosome 1.

Flow sorting maize chromosome 1 and construction of the first chromosome 1 DNA Lambda library are described. Maize metaphase chromosome suspensions were prepared from synchronized seedling root tip cells of the maize hybrid line Seneca 60 and stained with propidium iodide for flow karyotyping and sorting. The observed flow karyotype was very similar to the predicted flow karyotype constructed based on published values for the relative chromosome sizes of Seneca 60. The estimated size of chromosomes from the peak for the chromosome 1 matched the expected size of maize chromosome 1. The peak for the chromosome 1 was well resolved from other peaks on the flow karyotype. An average of 7 x 10(3) chromosomes of chromosome 1 could be produced from 10 root tips. About 0.6 million chromosomes of maize chromosome 1 were sorted and pooled based on the cytogram of fluorescent pulse area Vs fluorescent pulse width and stored at -20 degrees C in the freezer. DNA isolated from sorted chromosomes was good quality of more than 100 kb in size. Chromosome 1 DNA was partially digested with BamHI, dephosphorylated and ligated with arms of BamHI digested Lambda Dash vector. A total of 1.2 x 10(5) independent recombinants with the average insert size 12.6 kb was obtained. This library covered approximately 90% of maize chromosome 1. Hybridization of cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. PCR (polymerase chain reaction) using chromosome-specific primers confirmed the specificity of this library. The individual chromosome library is useful in plant genome mapping and gene isolation.

Characterization of germline porcine endogenous retroviruses from Large White pig.

Porcine endogenous retroviruses (PERV) are of concern when the microbiological safety aspects of xenotransplantation are considered. Four unique isolates of PERV B have been identified previously from a lambda library constructed from genomic DNA from a Large White pig. This study shows that none of these isolates are replication competent when transfected into permissive human or pig cells in vitro, and the removal of flanking genomic sequences does not confer a human tropic replication competent (HTRC) phenotype on these PERV proviruses. Analysis of the envelope sequences revealed that PERV B demonstrated high similarity to the envelope sequences derived from replication-competent PERV, indicating that lack of replication competence does not appear to be attributable to this region of the provirus. These data complement recent findings that HTRC PERV are recombinants between the PERV A and PERV C subgroups, and that these recombinants are not present in the germline of miniature swine. Together, these results indicate that these individual PERV B proviruses are unlikely to give rise to HTRC PERV.

Cloning and partial characterization of zwittermicin A resistance gene cluster from Bacillus thuringiensis subsp. kurstaki strain HD1.

The study seeks to shed light on the aminopolyol, broad-spectrum antibiotic zwittermicin A gene cluster of Bacillus thuringiensis subsp. kurstaki HD1 and to identify any new uncharacterized genes with an eventual goal to establish a better understanding of the resistance gene cluster. We screened 51 serovars of B. thuringiensis by PCR and identified 12 zmaR-positive strains. The zmaR-positive B. thuringiensis subsp. kurstaki HD1 strain displayed inhibition zones against indicator fungal strain Phytophthora meadii and bacterial strain Erwinia herbicola as well as against Rhizopus sp., Xanthomonas campestris and B. thuringiensis subsp. finitimus. The zmaR gene cluster of strain HD1 was partially cloned using a lambda library and was extensively characterized based on the information available from a study performed on a similar group of genes in Bacillus cereus. Three of the five genes in the zwittermicin gene cluster, including the zmaR gene, had counterparts in B. cereus, and the other two were new members of the B. thuringiensis zmaR gene cluster. The two new genes were extensively analysed and the data is presented. Understanding antifungal activity of B. thuringiensis may help us to design suitable Cry toxin delivery agents with antifungal activity as well as enhanced insecticidal activity.

Targeting clusters of transferred genes in Thermotoga maritima.

We screened a Thermotoga sp. strain RQ2 lambda library for genes present in that strain but absent from the closely related completely sequenced relative Thermotoga maritima strain MSB8, by using probes generated in an earlier genomic subtraction study. Five lambda insert fragments were sequenced, containing, respectively, an archaeal type ATPase operon, rhamnose biosynthetic genes, ORFs with similarity to an arabinosidase, a Thermotoga sp. strain RQ2-specific alcohol dehydrogenase and a novel archaeal Mut-S homologue. All but one of these fragments contained additional Thermotoga sp. strain RQ2-specific sequences not screened for, suggesting that many such strain-specific genes will be found clustered in the genome. Moreover, phylogenetic analyses, phylogenetic distribution and/or G + C content suggests that all the Thermotoga sp. strain RQ2 specific sequences in the sequenced lambda clones have been acquired by lateral gene transfer. We suggest that the use of strain-specific small insert clones obtained by subtractive hybridization to target larger inserts for sequencing is an efficient, economical way to identify environmentally (or clinically) relevant interstrain differences and novel gene clusters, and will be invaluable in comparative genomics.

Delimitation of the fertility restorer locus Rfk1 to a 43-kb contig in Kosena radish (Raphanus sativus L.).

We are pursuing a positional cloning strategy to isolate the fertility restoration gene Rfk1 from radish. Random polymorphic DNA-sequence-tagged site (RAPD-STS) markers tightly linked to the gene in radish were isolated, and a RAPD map surrounding the Rfk1 locus was constructed. We surveyed 948 F2 plants with adjacent RAPD-STS markers to isolate recombinants for bulk segregant analysis. This analysis was effective in isolating tightly linked amplification fragment length polymorphism (AFLP) markers surrounding the gene of interest. Ten tightly linked AFLP markers were obtained and used to construct a high-resolution map of the region. The closest AFLP-STS markers flanking Rfk1 were 0.1 cM and 0.2 cM away. Using the four adjacent AFLP markers, we screened lambda and cosmid libraries. The lambda and cosmid clones were aligned by examination of end sequences and restriction fragment length polymorphism (RFLP) patterns for each clone, and by hybridization to the DNA isolated from recombinants. Finally, we constructed a 198-kb contig encompassing the Rfk1 gene and comprising 20 lambda and two cosmid clones. By analysis of the breakpoints in recombinants with the rfk1/rfk1 or Rfk1/- genotype, the Rfk1 locus could be assigned to a 43-kb region comprising four lambda clones and one cosmid clone. This pinpoint localization in the radish genome has made it possible for us to identify the gene by sequence analysis and genetic transformation of cytoplasmic male-sterile Brassica napus plants.

Genetic organization of genes encoding phenol hydroxylase, benzoate 1,2-dioxygenase alpha subunit and its regulatory proteins in Acinetobacter calcoaceticus PHEA-2.

Acinetobacter calcoaceticus PHEA-2 is a phenol-degrading bacterium isolated from the wastewater from an oil refinery. A 10-kb XhoI fragment consisting of nine complete Open Reading Frames (ORFs) and one partial ORF was screened from a lambda library of PHEA-2 by Southern hybridization. The sequence analyses revealed that ORF2-ORF7, designated mphKLMNOP, are homologous to dmpKLMNOP of Pseudomonas sp. CF600 and mopKLMNOP of Acinetobacter calcoaceticus NCIB8250, sharing 38%-72% and 58.5%-93.5% respectively. The products encoded by dmp and mop genes convert phenol to catechol. The mph-operon and downstream ORFs, ORF9 and ORF10, sharing high identities to benM and benA, which encode ben-operon regulatory protein and benzoate 1,2-dioxygenase alpha subunit respectively, are separated by ORF8, whose function is unknown. The organization of the mph and ben operons is different from that described previously.

The organization of genes tightly linked to the Ha locus in Aegilops tauschii, the D-genome donor to wheat.

The grain hardness locus, Ha, is located at the distal end of the short arm of chromosome 5D in wheat. Three polypeptides, puroindoline-a, puroindoline-b, and grain softness protein (GSP-1), have been identified as components of friabilin, a biochemical marker for grain softness, and the genes for these polypeptides are known to be tightly linked to the Ha locus. However, this region of the chromosome 5D has not been well characterized and the physical distance between the markers is not known. Separate lambda clones containing the puroindoline-a gene and the puroindoline-b gene have been isolated from an Aegilops tauschii (the donor of the D genome to wheat) genomic lambda library and investigated. Considerable variation appears to exist in the organization of the region upstream of the gene for puroindoline-b among species closely related to wheat. Using in situ hybridization the genes for puroindoline-a, -b, and GSP-1 were demonstrated to be physically located at the tip of the short arm of chromosome 5 of A. tauschii. Four overlapping clones were isolated from a large-insert BAC library constructed from A. tauschii and of these one contained genes for all of puroindoline-a, puroindoline-b, and GSP-1. The gene for puroindoline-a is located between the other two genes at a distance no greater than approximately 30 kb from either gene. The BAC clone containing all three known genes was used to screen a cDNA library constructed from hexaploid wheat and cDNAs that could encode novel polypeptides were isolated.

The Lambda Library: unnamed functions in C++

AbstractThe Lambda Library (LL) adds a form of lambda functions to C++, which are common in functional programming languages. The LL is implemented as a template library using standard C++; thus no language extensions or preprocessing is required. The LL consists of a rich set of tools for defining unnamed functions. In particular these unnamed functions work seamlessly with the generic algorithms in the C++ Standard Library. The LL offers significant improvements, in terms of generality and ease of use, compared to the current tools in the C++ Standard Library. Copyright © 2003 John Wiley & Sons, Ltd.

The genome sequence of Haemophilus influenzae.

Publication of the complete 1.83-Mb genome sequence of the bacterium Haemophilus influenzae strain Rd in 1995 () heralded a revolution in the study of microorganisms. The availability of a complete microbial genome sequence enormously facilitates computer-based and experimental investigations of the respective organism by providing complete lists of genes, their genetic contexts, and their predicted functions. The significance of the H. influenzae sequence was not just that it presented for the first time a complete genome from a free-living organism, but that it proved that complete genomes could be sequenced rapidly and effectively at a low cost. This was achieved through the novel approach taken by Venter and his colleagues at The Institute for Genomic Research (TIGR) to sequence the H. influenzae genome. Randomly generated small chromosomal fragments, 1–2 kb in length, were cloned into a high-copy-number plasmid vector and then both ends of the clone were sequenced using plasmid specific primers. The vast amount of DNA sequence obtained from the shotgun clones was assembled into large contiguous sequences by computers using specifically developed software. The equivalent to sequencing the entire genome 6 times was required for sufficient accuracy and coverage of the majority of the chromosome. To obtain a final contiguous sequence, gaps between contigs (tracts of contiguous sequence) were closed using homology comparisons between end sequences, the polymerase chain reaction, and sequencing of larger DNA inserts from lambda libraries. The shotgun method was rapid, as it circumvented the need for construction of a physical map of the genome, a prerequisite of the ordered approach taken to other bacterial sequencing projects.

Preparing lambda libraries for expression of proteins in prokaryotes or eukaryotes.

AbstractComplementary DNA (cDNA) libraries represent the information encoded in the messenger RNA (mRNA) of a particular tissue or organism. RNA molecules are exceptionally labile and difficult to amplify in their natural form. For this reason, a cDNA library is created by converting the information encoded by the RNA into a stable DNA duplex (cDNA) and then inserting it into a self-replicating lambda or plasmid vector. Individual cDNA representing the original genetic information can be isolated from the cDNA library and examined with relative ease. KeywordsPhagemid VectorEcoRI AdapterPackaging ExtractPhage StockPrimary LibraryThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Molecular cloning and characterization of a cDNA encoding a ryegrass ( Lolium perenne) ENOD40 homologue

The ENOD40 gene, found in several leguminous and non-leguminous plant species is expressed in the pericycle of legume roots early in the nodulation process, adjacent to the protoxylem poles, but before the cortical cells divide to form the nodule itself. The ENOD40 transcript contains only short open reading frames which give rise to short peptides with a signaling function. The current work reports the cloning and analysis of ENOD40 genes from perennial ryegrass (Lolium perenne) and barley (Hordeum vulgare). Specific polymerase chain reaction (PCR) techniques lead to the isolation of a 659 bp cDNA encoding an ENOD40 homologue, designated LpE-NOD40, from a Lolium perenne stem cDNA library. In addition, a partial ENOD40 cDNA of 384 bp was isolated from barley (Hordeum vulgare) by RT-PCR cloning. The LpENOD40 transcript encodes a putative dodecapeptide, similar to that identified in ENOD40s from leguminous plants and other dicots, and also to ENOD40s from monocots. The coding sequences of ryegrass and barley ENOD40 are represented by a short open reading frame of 12 amino acids. These show a high degree of similarity to each other and to other ENOD40 sequences from monocots. The corresponding genomic DNA from a genomic ryegrass lambda library revealed that the LpENOD40 gene contains no introns. Southern blot analysis shows that the ryegrass genome contains a single copy, possibly two copies, of the gene. Alignment of the ENOD40 cDNA sequences from ryegrass and barley revealed high (77%) nucleotide homology. The ENOD40 peptides are highly conserved, not only among monocots but also on comparison with the dicot peptides. The amino acid identity of region I from ryegrass with its counterparts in maize, barley and rice is 92, 83 and 75% respectively. Expression analysis by RT-PCR demonstrates that a high level of LpENOD40 gene transcript was found expressed in stem tissue, while a lower level was detected in leaves and only a very low expression in flowers of perennial ryegrass. No LpENOD40 transcript was detected in roots.

Identification and Molecular Cloning of a Heparosan Synthase fromPasteurella multocida Type D

Pasteurella multocida Type D, a causative agent of atrophic rhinitis in swine and pasteurellosis in other domestic animals, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported previously that the capsule was removed by treating microbes with heparin lyase III. We molecularly cloned a 617-residue enzyme, pmHS, which is a heparosan (nonsulfated, unepimerized heparin) synthase. Recombinant Escherichia coli-derived pmHS catalyzes the polymerization of the monosaccharides from UDP-GlcNAc and UDP-GlcUA. Other structurally related sugar nucleotides did not substitute. Synthase activity was stimulated about 7-25-fold by the addition of an exogenous polymer acceptor. Molecules composed of approximately 500-3,000 sugar residues were produced in vitro. The polysaccharide was sensitive to the action of heparin lyase III but resistant to hyaluronan lyase. The sequence of the pmHS enzyme is not very similar to the vertebrate heparin/heparan sulfate glycosyltransferases, EXT1 and 2, or to other Pasteurella glycosaminoglycan synthases that produce hyaluronan or chondroitin. The pmHS enzyme is the first microbial dual-action glycosyltransferase to be described that forms a polysaccharide composed of beta4GlcUA-alpha4GlcNAc disaccharide repeats. In contrast, heparosan biosynthesis in E. coli K5 requires at least two separate polypeptides, KfiA and KfiC, to catalyze the same polymerization reaction.