Degenerate Polymerase Chain Reaction Research Articles

Improved performance of nucleic acid-based assays for genetically diverse norovirus surveillance.

Nucleic acid-based assays, such as polymerase chain reaction (PCR), that amplify and detect organism-specific genome sequences are a standard method for infectious disease surveillance. However, challenges arise for virus surveillance because of their genetic diversity. Here, we calculated the variability of nucleotides within the genomes of 10 human viral species in silico and found that endemic viruses exhibit a high percentage of variable nucleotides (e.g., 51.4% for norovirus genogroup II). This genetic diversity led to the variable probability of detection of PCR assays (the proportion of viral sequences that contain the assay's target sequences divided by the total number of viral sequences). We then experimentally confirmed that the probability of the target sequence detection is indicative of the number of mismatches between PCR assays and norovirus genomes. Next, we developed a degenerate PCR assay that detects 97% of known norovirus genogroup II genome sequences and recognized norovirus in eight clinical samples. By contrast, previously developed assays with 31% and 16% probability of detection had 1.1 and 2.5 mismatches on average, respectively, which negatively impacted RNA quantification. In addition, the two PCR assays with a lower probability of detection also resulted in false negatives for wastewater-based epidemiology. Our findings suggest that the probability of detection serves as a simple metric for evaluating nucleic acid-based assays for genetically diverse virus surveillance. IMPORTANCE Nucleic acid-based assays, such as polymerase chain reaction (PCR), that amplify and detect organism-specific genome sequences are employed widely as a standard method for infectious disease surveillance. However, challenges arise for virus surveillance because of the rapid evolution and genetic variation of viruses. The study analyzed clinical and wastewater samples using multiple PCR assays and found significant performance variation among the PCR assays for genetically diverse norovirus surveillance. This finding suggests that some PCR assays may miss detecting certain virus strains, leading to a compromise in detection sensitivity. To address this issue, we propose a metric called the probability of detection, which can be simply calculated in silico using a code developed in this study, to evaluate nucleic acid-based assays for genetically diverse virus surveillance. This new approach can help improve the sensitivity and accuracy of virus detection, which is crucial for effective infectious disease surveillance and control.

Cloning and physical localization of male-biased repetitive DNA sequences in Spinacia oleracea (Amaranthaceae)

Spinach (Spinacia oleracea Linnaeus, 1753) is an ideal material for studying molecular mechanisms of early-stage sex chromosome evolution in dioecious plants. Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) technique facilitates the retrotransposon-relevant studies by enriching specific repetitive DNA sequences from a micro-dissected single chromosome. We conducted genomic subtractive hybridization to screen sex-biased DNA sequences by using the DOP-PCR amplification products of micro-dissected spinach Y chromosome. The screening yielded 55 male-biased DNA sequences with 30 576 bp in length, of which, 32 DNA sequences (12 049 bp) contained repeat DNA sequences, including LTR/Copia, LTR/Gypsy, simple repeats, and DNA/CMC-EnSpm. Among these repetitive DNA sequences, four DNA sequences that contained a fragment of Ty3-gypsy retrotransposons (SP73, SP75, SP76, and SP77) were selected as fluorescence probes to hybridization on male and female spinach karyotypes. Fluorescence in situ hybridization (FISH) signals of SP73 and SP75 were captured mostly on the centromeres and their surrounding area for each homolog. Hybridization signals primarily appeared near the putative centromeres for each homologous chromosome pair by using SP76 and SP77 probes for FISH, and sporadic signals existed on the long arms. Results can be served as a basis to study the function of repetitive DNA sequences in sex chromosome evolution in spinach.

Degenerate PCR primers for assays to track steps of nitrogen metabolism by taxonomically diverse microorganisms in a variety of environments

Steps in the global nitrogen cycle are mainly catalyzed by microorganisms. Accordingly, the activities of these microorganisms affect the health and productivity of ecosystems. Their activities are also used in wastewater treatment systems to remove reactive nitrogen compounds and prevent eutrophication events triggered by nutrient discharges. Therefore, tracking the activities of these microorganisms can provide insights into the functioning of these systems. The presence and abundance of genes encoding nitrogen-metabolizing enzymes can be traced via polymerase chain reaction (PCR); however, this requires primers that are sensitive to a heterogenous gene pool yet specific enough to the target biomarker. The ever-expanding diversity of sequences available from databases includes many sequences relevant to nitrogen metabolism that match poorly with primers previously designed to track their presence and/or abundance. This includes genes encoding ammonia monooxygenase (AMO) of ammonia oxidizing microorganisms, nitrite oxidoreductase (NXR) of nitrite oxidizing bacteria, and nitrous oxide reductase (NOS) of denitrifying bacteria. Some primers are also not designed to generate the short (~200 nucleotides) amplicons required for real-time quantitative PCR (qPCR) and reverse-transcriptase qPCR (qRT-PCR). In this study, genes collected from the Integrated Microbial Genomes database (IMG) were aligned to design PCR primers that could capture more sequence diversity than is possible using existing primers. Primers were designed to target three clades of AMO (Betaproteobacteria, Chrenarchaeota, and complete ammonia oxidizing Nitrospira), periplasmic NXR and two clades of NOS (Proteobacteria and Bacteroidetes/Firmicutes). These primers successfully amplified target sequences from two wastewater treatment plants with biological nitrogen removal (one with simultaneous nitrification/denitrification and one with distinct anoxic/oxic zones) and estuary sediment. Nucleotide sequences of the amplicons retrieved homologs when used to query GenBank by BLAST. While convincingly identified as target sequences for these primer pairs, these amplicons were divergent from each other, and quite divergent (as low as 73%) from those present in GenBank, suggesting these primers are capable of capturing a diverse range of sequences. A direct comparison showed that primers designed here are better suited to environmental samples, such as wastewater treatment facilities, by producing a greater number of amplicons from the same sample than primers currently established in literature.

Molecular basis of triterpene-based chemophenetics in ferns

A hypothesis that squalene cyclase genes are widely distributed throughout ferns was proposed. We successfully isolated a squalene cyclase pseudogene from a fern from which no triterpene hydrocarbons were detected Ferns are the most primitive vascular plants, with their locations ranging from tropical to cold temperate regions and from lowland to alpine zones. The triterpene hydrocarbons and their derivatives are characteristic fern metabolites, and are also chemophenetic markers. Recently, our biosynthetic study into fern squalene cyclases (SCs), the enzymes responsible for triterpene synthesis, gave an unexpected inconsistency between genotype (enzyme function) and chemotype (triterpene profile). This finding prompted us to propose a hypothesis that SC genes are widely distributed throughout ferns and lycophytes whether or not they produce triterpene hydrocarbons. To test this hypothesis, we employed a multifaceted approach based on phytochemical, biochemical, and phylogenetic analyses. As anticipated, we successfully isolated two SC pseudogenes from a fern from in which no or only one triterpene hydrocarbon was detected. Subsequent mutagenesis experiments resulted in the functional conversion of these pseudogenes into active SC genes. Given an auxiliary hypothesis regarding the inherent limit of the degenerate polymerase chain reaction (PCR) method, the overall dataset supported our hypothesis, although correction was required with respect to plant coverage. Not only did the corrected hypothesis outline the distribution of SC genes throughout ferns, it provided insight into the molecular basis of the triterpene-based chemophenetics in ferns, which is also discussed.

English

Bovine papillomavirus (BPV) causes benign tumours in the mucosal and cutaneous epithelium and is characterized by the presence of warts. The present study includes the molecular identification of BPV strains in samples of warts using degenerate polymerase chain reaction (PCR) primers FAP59/64. Skin biopsies were taken from crossbred cattle from two experimental farms of Instituto de Investigacion Agropecuaria de Panama (IDIAP) during the period from July 2016 to February 2018. Fourteen samples were positive by PCR amplification and sequenced at the laboratory of the Institute of Legal Medicine and Forensic Sciences (IMELCF). The analysis of the sequences allowed the identification of strains related to seven viral types; this was the first time that this type of study was carried out in Panama. The present study showed that PCR amplification with the primers FAP59/64, which partially amplify the L1 gene, followed by direct sequencing was useful for genotyping BPV. This study possibly identified local strains of BPV2 and BBA2; however, it is necessary to carry out more studies to establish the diversity and distribution of this virus in the country. The results in this study are important for the development of prophylactic and therapeutic measures that contribute to reducing the economic losses associated with BPV in Panama. Key words: Livestock, biotechnology, molecular genetics, virology, animal health.

Cytomegalic Inclusion Disease caused by Cytomegalovirus Infection in the Salivary Glands of an African Hedgehog (Atelerix arbiventris)

Cytomegalic inclusion disease (CID) in the salivary gland of African hedgehogs (Atelerix arbiventris) has been reported before, and is suspected to reflect a cytomegalovirus infection. However, a recent ultrastructural study reported that African hedgehog CID reflected oncocytic metaplasia, mimicking a cytomegalovirus infection. We examined the submandibular and sublingual salivary glands of a 1-year-old male African hedgehog. Histologically, there were multiple foci composed of cytomegalic cells with intranuclear inclusion bodies. Ultrastructurally, viral particles (109-118 nm in diameter) were observed in the nuclei of the cytomegalic cells. There were numerous vesicles containing various numbers of enveloped viruses in the cytoplasm. We also attempted to detect viral DNA fragments by degenerate polymerase chain reaction and obtained amplicons of a predicted size. Phylogenetic analysis indicated that the virus is a betaherpesvirus, comparatively related to human and rodent cytomegaloviruses. The present study suggested that African hedgehog CIDs also include those caused by the cytomegalovirus.

Chromosome sorting for the masses?

Chromosome sorting for the masses?

Gene cloning of an efficiency oleate hydratase from Stenotrophomonas nitritireducens for polyunsaturated fatty acids and its application in the conversion of plant oils to 10-hydroxy fatty acids.

Hydroxy fatty acids are used as precursors of lactones and dicarboxylic acids, as starting materials of polymers, and as additives in coatings and paintings. Stenotrophomonas nitritireducens efficiently converts cis-9 polyunsaturated fatty acids (PUFAs) to 10-hydroxy fatty acids. However, gene encoding enzyme involved in this conversion has not been identified to date. We purified a putative fatty acid double-bond hydratase from S. nitritireducens by ultrafiltration and HiPrep DEAE FF and Resource Q ion exchange chromatographies. Peptide sequences of the purified enzyme were obtained by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Sequence of the partial gene encoding this putative fatty acid double-bond hydratase was determined by degenerate polymerase chain reaction (PCR) based on the peptide sequences. The remaining gene sequence was identified by rapid amplification of cDNA ends using cDNA of S. nitritireducens as a template, and the full-length gene was cloned subsequently. The expressed enzyme was identified as an oleate hydratase by determining its kinetic parameters toward unsaturated fatty acids. S. nitritireducens oleate hydratase showed higher activity toward PUFAs compared with other available oleate hydratases. This suggested that the enzyme could be used effectively to convert plant oils to 10-hydroxy fatty acids because these oils contained unsaturated fatty acids such as oleic acid (OA) and linoleic acid (LA) and PUFAs such as α-linolenic acid and/or γ-linolenic acid. The enzyme converted soybean oil and perilla seed oil hydrolyzates containing 10 mM total unsaturated fatty acids, including OA, LA, and ALA, to 8.87 and 8.70 mM total 10-hydroxy fatty acids, respectively, in 240 min. To our knowledge, this is the first study on the biotechnological conversion of PUFA-containing oils to hydroxy fatty acids. Biotechnol. Bioeng. 2017;114: 74-82. © 2016 Wiley Periodicals, Inc.

Identification of the chitinase genes from the diamondback moth, Plutella xylostella.

Chitinases have an indispensable function in chitin metabolism and are well characterized in numerous insect species. Although the diamondback moth (DBM) Plutella xylostella, which has a high reproductive potential, short generation time, and characteristic adaptation to adverse environments, has become one of the most serious pests of cruciferous plants worldwide, the information on the chitinases of the moth is presently limited. In the present study, using degenerated polymerase chain reaction (PCR) and rapid amplification of cDNA ends-PCR strategies, four chitinase genes of P. xylostella were cloned, and an exhaustive search was conducted for chitinase-like sequences from the P. xylostella genome and transcriptomic database. Based on the domain analysis of the deduced amino acid sequences and the phylogenetic analysis of the catalytic domain sequences, we identified 15 chitinase genes from P. xylostella. Two of the gut-specific chitinases did not cluster with any of the known phylogenetic groups of chitinases and might be in a new group of the chitinase family. Moreover, in our study, group VIII chitinase was not identified. The structures, classifications and expression patterns of the chitinases of P. xylostella were further delineated, and with this information, further investigations on the functions of chitinase genes in DBM could be facilitated.

Virus Detection and Elimination in Cocoa ( Theobroma cacao L.) Through Somatic Embryogenesis

Cacao swollen shoot virus (CSSV) is a major pathogen that has seriously constrained cocoa production in West Africa, particularly Ghana and Nigeria. The objective of this study was to assess the efficacy of cocoa somatic embryogenesis to produce virus-free clonal propagation material both for replanting and to facilitate the safe international exchange of germplasm. Polymerase Chain Reaction (PCR)-based screening, is employed in this study because of its capacity for CSSV detection prior to the appearance of visual symptoms. Degenerate PCR primers were developed in order to improve the CSSV-strain dependence of earlier tests. The degenerate primers were capable of detecting 37 out of a putative 56 CSSV strains, four more than the sequence specific primers. For tissue culture studies, cocoa staminodes cultures were established from flowers of CSSV-infected cocoa genotypes CL 19/10 strain 1A and Amelonado Plant 2 to produce callus, primary and secondary somatic embryos, with genotype AMAZ 15 used as a virus-free control. PCR-based CSSV detection proved that virus could be detected at callus, primary somatic embryos and secondary somatic embryo stages, indicating that the progress of the virus was progressively impeded. These findings support the use of somatic embryogenesis as a mean of improving CSSV-free clonal propagation of cocoa. Somatic embryogenesis is indeed effective for virus elimination in cocoa and it has been demonstrated to function for a range of cocoa genotypes. This also means that a likely mechanism for the interruption of CSSV movement has been identified.

Human papillomavirus genotypes distribution among Gabonese women with normal cytology and cervical abnormalities.

BackgroundCervical cancer is one of the most common tumors affecting women with a disproportionate mortality occurring in developing countries. Despite the high prevalence of cervical cancer and cervical neoplasia in Gabon, few studies have been performed to evaluate the prevalence and determinants of HPV infection in this country. The aim of this study was to determine the HPV prevalence and distribution in a population of Gabonese women with normal cytology and cervical abnormalities.MethodsA total of 200 cervical samples collected in the “Departement d’Anatomie et de Cytologie Pathologiques” of the “Faculté de Medecine et des Sciences de la Santé” in Libreville, Gabonwere analyzed. Cytological status was classified according to Bethesda 2001. Nested polymerase chain reaction (PCR) using consensus degenerate PCR primers (MY09/11 and GP5+/6+) was performed for the detection of HPV DNA and HPV typing was done by DNA sequencing.ResultsCytological analysis showed that 87 % of women had normal cytology (n = 174/200). Among the 26 women with cytological abnormalities, predominance (61.5 %; 16/26) of low grade squamous intraepithelial lesion (LSIL) was found and no cervical cancer case was detected. Overall, HPV DNA was detected in 60 % of women (120/200). With respect to the cytological status, HPV DNA was found in 57.5 % of women with normal cervix and 76.9 % of women with abnormal cytology. HPV genotyping was performed on 114 HPV positive cases and revealed the presence of 11 distinct genotypes: 16, 18, 33, 31, 56, 6, 66, 70, 35, 45 and 81. The high risk type HPV 16 was the most common genotype found in all cytological categories. Six HPV positive samples could not be typed by DNA sequencing, probably due to multiple HPV infection. Evaluation of possible risk factors showed that HPV infection was related positively with number of sexual partners (≥3, OR = 2.3; 95 % CI, 1.3–4.3), history of sexually transmitted infection (Chlamydia, OR = 1.9; 95 % CI, 1.01–3.4) and marital status (single, OR = 2.0; 95 % CI, 1.1–3.5).ConclusionThe prevalence of HPV infection among Gabonese women is high. Our findings highlight the need to set up a national program to fight cervical cancer, combining Pap smear test and HPV testing, to improve cervical cancer prevention in Gabon.

Identification and Sequencing of Candida krusei Aconitate Hydratase Gene Using Rapid Amplification of cDNA Ends Method and Phylogenetic Analysis.

Background:The production and development of an effective fungicidal drug requires the identification of an essential fungal protein as a drug target. Aconitase (ACO) is a mitochondrial protein that plays a vital role in tricarboxylic acid (TCA) cycle and thus production of energy within the cell.Objectives:The current study aimed to sequence Candida krusei ACO gene and determine any amino acid residue differences between human and fungal aconitases to obtain selective inhibition.Materials and Methods:Candida krusei (ATCC: 6258) aconitase gene was determined by 5’Rapid Amplification of cDNA Ends (RACE) method and degenerate Polymerase Chain Reaction (PCR) and analyzed using bioinformatics softwares.Results:One thousand-four hundred-nineteen nucleotide of C. krusei aconitase gene were clarified and submitted in Genbank as a partial sequence and then taxonomic location of C. krusei was determined by nucleotide and amino acid sequences of this gene. The comparison of nucleotide and amino acid sequences of Candida species ACO genes showed that C. krusei possessed characteristic sequences. No significant differences were observed between C. krusei and human aconitases within the active site amino acid residues.Conclusions:Results of the current study indicated that aconitase was not a suitable target to design new anti-fungal drugs that selectively block this enzyme.

Isolation and characterization of a tonoplast Na<sup>+</sup>/H<sup>+</sup> antiporter from the halophyte Nitraria sibirica

Na+/H+ exchanger (NHX)-mediated Na+ and H+ antiport is an important mechanism for salt tolerance in plants. In this study, an Na+/H+ antiporter gene, referred to as NsNHX1, was isolated from the halophyte Nitraria sibirica Pall. using degenerate polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE). The resulting 2 182 bp NsNHX1 cDNA contained a 1 635 bp open reading frame (ORF) that encoded 544 amino acids and showed striking sequence similarity to tonoplast-localized NHXs from other plants. Subcellular localization analysis confirmed NsNHX1 to be a tonoplast-localized protein. Cis-elements described as being responsive to biotic and abiotic stresses were present in the NsNHX1 promoter region, and reverse transcription (RT)-PCR analysis confirmed that NsNHX1 expression was induced by exogenous abscisic acid (ABA), cold, and NaCl. Transcription of NsNHX1 increased sharply 3 h after treatment with 200 mM NaCl revealing that NsNHX1 responded rapidly to the salt stress. Overexpression of NsNHX1 enhanced salt tolerance in transgenic Arabidopsis thalliana L. suggesting that NsNHX1-mediated Na+ compartmentalization played an important role in enhancing plant salt tolerance.

Isolation of the Phosphoribosyl Anthranilate Isomerase Gene (TRP1) from Starch-Utilizing Yeast Saccharomycopsis fibuligera.

The nucleotide sequence of the TRP1 gene encoding phosphoribosyl anthranilate isomerase in yeast Saccharomycopsis fibuligera was determined by degenerate polymerase chain reaction and genome walking. Sequence analysis revealed the presence of an uninterrupted open-reading frame of 759 bp, including the stop codon, encoding a 252 amino acid residue. The deduced amino acid sequence of Trp1 in S. fibuligera was 43.5% homologous to that of Komagataella pastoris. The cloned TRP1 gene (SfTRP1) complemented the trp1 mutation in Saccharomyces cerevisiae, suggesting that it encodes a functional TRP1 in S. fibuligera. A new auxotrophic marker to engineer starch-degrading yeast S. fibuligera is now available. The GenBank Accession No. for SfTRP1 is KR078268.

Cloning and expression of fumarylacetoacetate hydrolase derived from marine yeast Rhodosporidium diobovatum.

Fumarylacetoacetate hydrolase (FAH) catalyzes the hydrolytic cleavage of a carbon-carbon bond in fumarylacetoacetate to yield acetoacetate and fumarate as the final step in tyrosine degradation. In this study, the FAH genomic DNA and cDNA of Rhodosporidium. diobovatum were cloned by using the methods of degenerate polymerase chain reaction and RACE method. The results showed that the gene contained six exons, and that the encoded polypeptide held a sequence of 308 amino acid residues, containing a 75 residue N-terminal prosequence, a 216 residue proteinase domain, and a 17 residue C-terminal domain. Then, the expression vectors pET28a-FAH were constructed and expressed in Escherichia coli. The enzymatic activity of FAH protein was determined by monitoring the decrease in optical density at 330 nm of the substrate fumarylacetoacetate. The values for Km and Vmax of the purified enzyme was 1.4 µM and 2.3 µmol · min(-1) · mg(-1), respectively.

Isolation of an arsenate-respiring bacterium from a redox front in an arsenic-polluted aquifer in West Bengal, Bengal Basin.

Natural pollution of groundwater by arsenic adversely affects the health of tens of millions of people worldwide, with the deltaic aquifers of SE Asia being particularly polluted. The pollution is caused primarily by, or as a side reaction of, the microbial reduction of sedimentary Fe(III)-oxyhydroxides, but the organism(s) responsible for As release have not been isolated. Here we report the first isolation of a dissimilatory arsenate reducer from sediments of the Bengal Basin in West Bengal. The bacterium, here designated WB3, respires soluble arsenate and couples its reduction to the oxidation of acetate; WB3 is therefore implicated in the process of arsenic pollution of groundwater, which is largely by arsenite. The bacterium WB3 is also capable of reducing dissolved Fe(III) citrate, solid Fe(III)-oxyhydroxide, and elemental sulfur, using acetate as the electron donor. It is a member of the Desulfuromonas genus and possesses a dissimilatory arsenate reductase that was identified using degenerate polymerase chain reaction primers. The sediment from which WB3 was isolated was brown, Pleistocene sand at a depth of 35.2 m below ground level (mbgl). This level was some 3 cm below the boundary between the brown sands and overlying reduced, gray, Holocene aquifer sands. The color boundary is interpreted to be a reduction front that releases As for resorption downflow, yielding a high load of labile As sorbed to the sediment at a depth of 35.8 mbgl and concentrations of As in groundwater that reach >1000 μg/L.

Phylogenetic characterization and novelty of organic sulphur metabolizing genes of Rhodococcus spp. (Eu-32).

Rhodococcus spp. (Eu-32) has the unique ability to metabolize organic sulphur containing compounds like dibenzothiophene through an extended sulphur specific pathway (Akhtar et al., in FEMS Microbiol Lett 301:95-102, 2009). Efforts were made to isolate and characterize the presumed desulphurizing genes (dszABC) involved in the sulphur specific pathway of isolate Eu-32 by employing standard and degenerate polymerase chain reaction primers. The partial dszA gene sequence of isolate Eu-32 showed 92% sequence identity with a putative FMNH-2 dependent monooxygenase of Rhodococcus erythropolis PR4. The dszC gene sequence showed 99% homology with the dibenzothiophene monooxygenase desulphurizing enzyme of another Rhodococcus species. The dszB gene was not unambiguously identified. A phylogenetic analysis by maximum likelihood method of the 16S rRNA gene and deduced DszA and C amino acid sequences suggest that horizontal gene transfer events might have taken place during the evolution of desulphurizing genes of Rhodococcus spp. (Eu-32).

Isolation and expression analysis of Cu/Zn superoxide dismutase genes from three Caragana species

Caragana is a native desert shrub with high forage values and stress tolerance, as well as sand-fixing capabilities. Some Caragana species, including C. korshinskii, C. microphylla, and C. intermedia, are commonly used for vegetation restoration programs in the Loess Plateau region of northwestern China and are known to have ecological benefits and high commercial value. In this study, full-length sequences of Cu/Zn-SOD genes were isolated from three Caragana species using degenerate polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) techniques, and their expression under drought stress conditions were investigated. The cloned SOD cDNAs contained a predicted open reading frame (ORF) of 459 bp encoding a polypeptide of 152 amino acids with a theoretical molecular weight of 15.2 kD. Cu/Zn-SOD cDNA of C. korshinskii and C. intermedia shared 100% sequence identity, implying a close relationship. A 24-bp specific sequence was found in the 3-UTR region of C. microphylla Cu/Zn-SOD cDNA, and reverse transcription RT-PCR and genomic PCR confirmed the feasibility of the 24-bp sequence as a DNA marker for rapid variety identification of C. microphylla. RT-PCR revealed that the expression of the Caragana Cu/Zn SOD genes was induced by PEG-simulated drought stress and ABA. The three Caragana Cu/Zn-SOD genes showed similar expression patterns, and no significant differences in transcriptional level were observed among the three genes. These results increase our understanding of the molecular mechanism of drought tolerance and can be used to improve vegetation restoration programs of Caragana plants.

Cloning of the transketolase gene from erythritol-producing yeast Candida magnoliae.

The entire nucleotide sequence of the TKL1 gene encoding transketolase (TKL) in an erythritolproducing yeast of Candida magnoliae was determined by degenerate polymerase chain reaction and genome walking. Sequence analysis revealed an open reading frame of C. magnoliae TKL1 (CmTKL1) that spans 2,088 bp and encodes 696 amino acids, sharing 61.7% amino acid identity to Kluyveromyces lactis TKL. Functional analysis showed that CmTKL1 complemented a Saccharomyces cerevisiae tkl1 tkl2 double mutant for growth in the absence of aromatic amino acids and restored transketolase activity in this mutant. An enzyme activity assay and RT-PCR revealed that the expression of CmTKL1 is induced by fructose, H2O2, and KCl. The GenBank accession number for C. magnoliae TKL1 is KF751756.

Identification of a novel papillomavirus in a Northern Fulmar (Fulmarus glacialis) with viral production in cartilage.

We report the identification of a novel papillomavirus, Fulmarus glacialis papillomavirus 1 (FgPV1), present within an interdigital foot mass of a Northern Fulmar (Fulmarus glacialis). The mass of interest was composed of normal stratified and keratinized epithelium and dense mesenchymal cells with central cartilaginous islands. Within the nuclei of many chondrocytes were loose aggregates or paracrystalline arrays of virions approximately 50 nm in size. Degenerate polymerase chain reaction was used to identify the virus as a putative papillomavirus, and the entire viral genome of 8132 base pairs was subsequently amplified and sequenced. Analysis revealed canonical papillomavirus architecture, including the early open reading frames E6, E7, E1, and E2 and the 2 late proteins L1 and L2. FgPV1 is most closely related to a cluster of avian and reptilian papillomaviruses as visualized by phylogenetic trees. This observation suggests that papillomavirus virion production can occur in mesenchymal cells.