Degenerate Polymerase Chain Reaction Primers Research Articles

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.

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.

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).

Degenerate polymerase chain reaction strategy with DNA microarray for detection of multiple and various subtypes of virus during blood screening

The risk of transferring blood-borne infections during transfusion is continually increasing because of newly emerging and reemerging viruses. Development of a rapid screening method for emerging viruses that might be transmitted by transfusion is required to eliminate such pathogens during blood donor screening. Owing to increased use of human materials in organ transplants and cell therapy, the risk of donor-transmitted viral infections is also increasing. Although nucleic acid amplification technology (NAT) is dedicated to blood screening, a small, convenient detection system is needed at the laboratory and hospital level. We developed a new pathogen detection system that can detect multiple viruses simultaneously, using originally designed degenerate polymerase chain reaction primers to amplify a wide range of viral genotypes. Amplified samples were identified using a DNA microarray of pathogen-specific probes. We detected very low copy numbers of multiple subtypes of viruses, such as human hepatitis C virus (HCV), human hepatitis B virus (HBV), human parvovirus B19 (PVB19), and West Nile virus (WNV), using a single plate. We also detected all genotypes of human immunodeficiency virus (HIV) but sensitivity was less than for the other viruses. We developed a microarray assay using novel primers for detection of a wide range of multiple pathogens and subtypes. Our NAT system was accurate and reliable for detection of HIV, HBV, HCV, PVB19, and WNV, with respect to specificity, sensitivity, and genotype inclusivity. Our system could be customized and extended for emerging pathogens and is suitable as a future NAT system.

Degenerate Primed Polymerase Chain Reaction for Detection of Both Nucleopolyhedrovirus and Granulovirus

A technique using the polymerase chain reaction (PCR) was developed for simultaneous detection of the nucleopolyhedrovirus (NPV) and granulovirus (GV). Ninety one and 73 amino acid sequences of polyhedrin and granulin genes were compared in pairwise and multiple alignment sequences. Seven highly conserved DNA sequences within the coding region of the polyhedrin/granulin genes were identified. Four candidate regions were targeted for amplification and consequently one pair of degenerate PCR primers was designed to produce fragments of about 384 bp. The baculoviruses tested by this technique were Autographa californica (AcMNPV), Bombyx mori NPV (BmNPV), Lymantria dispar NPV (LdNPV), Spodoptera littoralis NPV (SpliNPV), S. littoralis GV (SpliGV), Pieris rapae GV (PrGV), and two local GV isolates (GVG213 and GVF115). Furthermore, four randomly chosen PCR products were cloned and sequenced. The sequencing data showed that the four PCR products were fragments of polyhedrin and granulin genes. Conclusively, this technique would be useful in monitoring the environmental fate, distribution of Baculovirus species, release of the wild type and recombinant Baculovirus and quality control studies of Baculovirus insecticides, as well.

Biotransformation of the Neonicotinoid Insecticide Thiacloprid by the Bacterium Variovorax boronicumulans Strain J1 and Mediation of the Major Metabolic Pathway by Nitrile Hydratase

A neonicotinoid insecticide thiacloprid-degrading bacterium strain J1 was isolated from soil and identified as Variovorax boronicumulans by 16S rRNA gene sequence analysis. Liquid chromatography-mass spectrometry and nuclear magnetic resonance analysis indicated the major pathway of thiacloprid (THI) metabolism by V. boronicumulans J1 involved hydrolysis of the N-cyanoimino group to form an N-carbamoylinino group containing metabolite, THI amide. Resting cells of V. boronicumulans J1 degraded 62.5% of the thiacloprid at a concentration of 200 mg/L in 60 h, and 98% of the reduced thiacloprid was converted to the final metabolite thiacloprid amide. A 2.6 kb gene cluster from V. boronicumulans J1 that includes the full length of the nitrile hydratase gene was cloned and investigated by degenerate primer polymerase chain reaction (PCR) and inverse PCR. The nitrile hydratase gene has a length of 1304 bp and codes a cobalt-type nitrile hydratase with an α-subunit of 213 amino acids and a β-subunit of 221 amino acids. The nitrile hydratase gene was recombined into plasmid pET28a and overexpressed in Escherichia coli BL21 (DE3). The resting cells of recombinant E. coli BL21 (DE3)-pET28a-NHase with overexpression of nitrile hydratase transformed thiacloprid to its amide metabolite, whereas resting cells of the control E. coli BL21 (DE3)-pET28a did not. Therefore, the major hydration pathway of thiacloprid is mediated by nitrile hydratase.

Vitamin B12 biosynthesis gene diversity in the Ross Sea: the identification of a new group of putative polar B12 biosynthesizers

Vitamin B₁₂, a cobalt-containing micronutrient, has been shown to limit phytoplankton growth in the Ross Sea of the Southern Ocean. However, B₁₂ biosynthesis potential in this environment remains uncharacterized. Select bacteria and archaea synthesize B₁₂ while many phytoplankton require it for growth. Low ratios of bacterial biomass production to primary productivity and high concentrations of labile cobalt in Antarctic surface water suggest that factors controlling bacterial growth rather than cobalt availability may determine vitamin production rates here. In order to assess B₁₂ biosynthesis potential, degenerate polymerase chain reaction primers were designed to target the genetic locus cbiA/cobB, encoding cobyrinic acid a,c-diamide synthase, a B₁₂ biosynthesis protein. Sequencing the DNA compliment of Ross Sea 16S rRNA (see Supporting information) allowed targeting of cbiA/cobB probes to dominant bacterial groups. CbiA/cobB DNA sequences were successfully identified in clone libraries from the Ross Sea. To our knowledge, this study represents the first targeted molecular characterization of environmental B₁₂ biosynthesis potential. A newly identified group of cbiA/cobB sequences dominated the diversity of the sequences retrieved; their expression was confirmed via mass spectrometry-based peptide detection. These sequences seem to have originated from a previously undescribed group of bacteria that could dominate the B₁₂ biosynthesizing community in polar systems.

Novel phycodnavirus genes amplified from Canadian freshwater environments

Extant and newly designed primers for the polymerase chain reaction (PCR) were used to amplify phycodnavirus DNA polymerase (polB) gene fragments from numerous samples collected at different times of the year from 3 freshwater environments in Ontario, Canada. Overall, a total of 143 cloned PCR fragments were sequenced and 106 putative phycodnavirus polB gene fragments were identified. Although most of these 106 gene fragments were very closely related (i.e. >97% identical) to polB sequences from chloroviruses, or environmental sequences related to prasi- noviruses, 16 represented 2 new types of phycodnavirus polB genes. More specifically, polB frag- ments that formed a new clade of chloroviruses were amplified from Lake Ontario using newly de- signed Chlorovirus-specific PCR primers, and a polB sequence most closely related to genes from the prymnesioviruses PgV-03T and CbV-PW1 was amplified from a pond sample from Mississauga, On- tario, using the degenerate algal virus-specific PCR primers AVS1 and AVS2. Thus, the results of the present study provide evidence for a new type of Chlorovirus, and the first observation of polB se- quences from freshwater phycodnaviruses that are presumed to infect algae other than chlorophytes.

Multiple receptor tyrosine kinases are expressed in adult rat retinal ganglion cells as revealed by single-cell degenerate primer polymerase chain reaction

Background. To achieve a better understanding of the repertoire of receptor tyrosine kinases (RTKs) in adult retinal ganglion cells (RGCs) we performed polymerase chain reaction (PCR), using degenerate primers directed towards conserved sequences in the tyrosine kinase domain, on cDNA from isolated single RGCs univocally identified by retrograde tracing from the superior colliculi.Results. All the PCR-amplified fragments of the expected sizes were sequenced, and 25% of them contained a tyrosine kinase domain. These were: Axl, Csf-1R, Eph A4, Pdgfrβ, Ptk7, Ret, Ros, Sky, TrkB, TrkC, Vegfr-2, and Vegfr-3. Non-RTK sequences were Jak1 and 2. Retinal expression of Axl, Csf-1R, Pdgfrβ, Ret, Sky, TrkB, TrkC, Vegfr-2, and Vegfr-3, as well as Jak1 and 2, was confirmed by PCR on total retina cDNA. Immunodetection of Csf-1R, Pdgfrα/β, Ret, Sky, TrkB, and Vegfr-2 on retrogradely traced retinas demonstrated that they were expressed by RGCs. Co-localization of Vegfr-2 and Csf-1R, of Vegfr-2 and TrkB, and of Csf-1R and Ret in retrogradely labelled RGCs was shown. The effect of optic nerve transection on the mRNA level of Pdgfrβ, Csf-1R, Vegfr-2, Sky, and Axl, and of the Axl ligands Gas6 and ProteinS, was analysed. These analyses show transection-induced changes in Axl and ProteinS mRNA levels.Conclusions. The repertoire of RTKs expressed by RGCs is more extensive than previously anticipated. Several of the receptors found in this study, including Pdgfrβ, Csf-1R, Vegfr-2, Sky, and Axl, and their ligands, have not previously been primarily associated with retinal ganglion cells.

Universal Primer for Early and Rapid Detection of Nucleopolyhedroviruses of Multiple Species Using Polymerase Chain Reaction

A technique using the polymerase chain reaction (PCR) was developed for detection of the nucleopolyhedrovirus (NPV) polyhedrin gene. 152 nucleotide sequences of polyhedrin gene were compared in pairwise and multiple alignment sequences. Eleven highly conserved DNA sequences within the coding region of the polyhedrin gene were identified. Two candidate regions were targeted for amplification and consequently one pair of degenerate PCR primers was designed to produce fragments of about 355 bp. The NPVs tested by this technique were Autographa californica (AcMNPV), Bombyx mori NPV (BmNPV), Hyphantria cunea NPV (HcNPV), Lymantria dispar NPV (LdNPV), Spodoptera exigua NPV (SeNPV), S. litura NPV (SlNPV), Spodoptera littoralis NPV (SpliNPV) and nine local NPV isolates. Furthermore, three randomly chosen PCR products were cloned and sequenced. The sequencing data showed that the three PCR products were fragments of polyhedrin gene. Conclusively, this technique would be useful in monitoring the environmental fate, distribution of NPVs, release of the wild type and recombinant NPVs and quality control studies of baculoviral insecticides as well.

Actinorhodopsin genes discovered in diverse freshwater habitats and among cultivated freshwater Actinobacteria

Microbial rhodopsins are membrane proteins that utilize a retinal chromophore to harvest sunlight for energetic and photosensory functions. Recently, a group of novel rhodopsin sequences named 'actinorhodopsins' (ActRs) was hypothesized to exist among uncultured planktonic Actinobacteria. ActRs were discovered by mining metagenomic data obtained during the Venter Institute's Global Ocean Sampling expedition, from a hypersaline lagoon, two estuaries and a freshwater lake. On the basis of these findings, and many studies that show Actinobacteria are common inhabitants of lakes, we predicted that ActR genes would likely be present in other freshwater habitats and among the genomes of cultivated Actinobacteria. Using degenerate polymerase chain reaction primers, we discovered an ActR gene present in an actinobacterial isolate of the family Microbacteriaceae. Isolate MWH-Uga1 was cultivated prior to this study from a freshwater pond in Uganda and belongs to a group of Actinobacteria previously identified in freshwater ecosystems. ActR genes were also discovered present in numerous mixed cultures containing freshwater Actinobacteria and among environmental DNA samples obtained from three freshwater sources; a small woodland pond and the Laurentian Great Lakes Superior and Erie. An analysis of small subunit ribosomal RNA genes from metagenomic DNA samples harboring ActR genes suggests that organisms belonging to the acI lineage, an uncultured group of Actinobacteria commonly present in fresh waters, may utilize rhodopsins. The co-occurrence of an acI organism with a specific ActR variant in a mixed culture supports our hypothesis.

Identification of new GH 10 and GH 11 xylanase genes from Aspergillus versicolor MKU3 by genome-walking PCR

Xylanases randomly clear the backbone of xylans, which are hemicelluloses representing a considerable source of fixed carbon in nature. Consequently, these enzymes have important industrial applications. To characterize the genes responsible for producing these enzymes, we cloned xylanase genes belonging to the GH11 and GH10 families from Aspergillus versicolor MKU3 using a 2-step polymerase chain reaction (PCR) protocol involving degenerate PCR and genome-walking PCR (GWPCR). We amplified a family 10 xylanase consensus fragment using degenerate PCR primers exhibiting specificity for conserved motifs within fungal family 10 xylanase genes. We identified a single family 10 xylanase gene (xynv10) and determined its entire gene sequence during the second step of GWPCR, which was used to amplify genomic DNA fragments upstream and downstream of xynv10. The xynv10 sequence contains a 1,378-bp open reading frame separated by 8 introns with an average size of 49 bp. We also amplified a partial GH11 xylanase gene sequence (xynv11) using degenerate PCR and genome-walking methods. Amplification of the C-terminal region of xynv11 using a degenerate primer designed from sequences revealed strong homology with the partial GH11 xylanase gene of A. versicolor MKU3. The structural region in xynv11 was approximately 680 bp and has one intron that is approximately 64 bp in length. Further expression and characterization of these genes will give better understanding of the role of these genes in xylan degradation by A. versicolor.

A biomarker for the identification of four Phaeoacremonium species using the β-tubulin gene as the target sequence

A real-time polymerase chain reaction (PCR) was developed for the rapid detection and identification of Phaeoacremonium species, the fungi associated with severe diseases in grapevines. A degenerate primer pair (F2bt-R1bt) with homology to the beta-tubulin gene was designed to be used in the amplification of 11 species of Phaeoacremonium. Four species-specific probes labelled with three different fluorescent dyes were designed to be used with the degenerate primers in a real-time PCR for the identification of Phaeoacremonium aleophilum, P. parasiticum, P. viticola and P. mortoniae. Combinations of two probes in a duplex real-time PCR allowed to detect and identify a mixture of Phaeoacremonium species and cross-amplifications were not detected. This method was applied to detect Phaeoacremonium species in eight wood fragments from grapevine plants naturally infected, and results were compared with those obtained with nested PCR and culturing on growth media. Real-time PCR detected Phaeoacremonium in 100% of the analysed fragments, whereas nested PCR did only in the 62% of them and requiring subsequent restriction fragment-length polymorphism analysis to identify the species. This method is a sensitive tool to detect and identify Phaeoacremonium species in infected grapevine wood. Real-time PCR assay defined here can be used in a plant nursery program to identify pathogen-free plants in order to manage Petri disease of grapevines.

Group‐specific primers for DNA‐based detection of springtails (Hexapoda: Collembola) within predator gut contents

Group-specific, degenerate polymerase chain reaction primers for DNA-based detection of springtails (Hexapoda: Collembola) within predator gut contents have been developed for the first time. Primers were designed from 18S rDNA and amplified fragments of 272 bp and 177bp from 17 springtail species collected in agricultural habitats. Specificity tests against 41 nontarget species revealed no cross-reactivity. Group-specific polymerase chain reaction is advantageous when working in species-rich habitats and these primers could facilitate studies of trophic links between springtails and generalist arthropod predators worldwide.

Identification of Resistance Gene Analogs and Verticillium Wilt Resistance-like Sequences in Mentha longifolia

Resistance gene analog (RGA) sequences were obtained from four Mentha longifolia (L.) Huds. accessions using degenerate polymerase chain reaction (PCR) primers targeting the conserved nucleotide binding site domain found in many plant disease resistance genes. Seven distinct RGA families were identified. All M. longifolia RGAs showed similarity to sequences of the non-toll-interleukin 1 receptor R gene class. In addition, degenerate PCR primers based on the tomato (Solanum lycopersicum L.) verticillium wilt resistance (Ve) genes were used to PCR-amplify a 445-base pair (bp) Ve-like sequence from M. longifolia that had ≈57% predicted amino acid identity with Ve. Mint-specific primers based on the original mint Ve sequence were used to obtain mint-specific Ve sequences from four M. longifolia accessions and from peppermint (Mentha ×piperita L.) cultivar ‘Black Mitcham’ that had 95% to 100% predicted amino acid identity to the original mint Ve sequence. Inverse PCR was then used to obtain flanking mint Ve sequence from one M. longifolia accession extending the mint Ve sequence to 1077 bp. This is the first report of RGA sequences in the Lamiaceae and the first report of Ve-like sequences obtained with degenerate PCR primers.

Identification of a novel circovirus in Australian ravens (Corvus coronoides) with feather disease

The complete genome of a novel Circovirus isolated from an Australian raven (Corvus coronoides) with feather lesions similar to those that occur in psittacine beak and feather disease is reported. Degenerate polymerase chain reaction primers were designed to amplify and sequence novel Circovirus DNA from affected feathers. Sequence analysis indicated that the tentatively named raven circovirus (RaCV) was 1898 nucleotides in size with two major open reading frames synonymous with other avian circoviruses, ORF C1 and ORF V1, likely to encode a putative capsid protein (Cap) and replicase-associated protein (Rep), respectively. In common with other circoviruses was the conservation of several nucleotide structures and amino acid motifs implicated in virus replication. Comparison with other members of the Circoviridae demonstrated that RaCV shares the greatest sequence homology with canary circovirus (CaCV) and pigeon circovirus (PiCV) and was more distantly related to the beak and feather disease virus, goose circovirus, duck circovirus and the two porcine circoviruses, PCV1 and PCV2. Phylogenetic analysis of the genome and the putative Cap and Rep proteins provided further evidence of the close relationship of RaCV with CaCV and PiCV.

Conservation of Arabidopsis flowering genes in model legumes.

The model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) have provided a wealth of information about genes and genetic pathways controlling the flowering process, but little is known about the corresponding pathways in legumes. The garden pea (Pisum sativum) has been used for several decades as a model system for physiological genetics of flowering, but the lack of molecular information about pea flowering genes has prevented direct comparison with other systems. To address this problem, we have searched expressed sequence tag and genome sequence databases to identify flowering-gene-related sequences from Medicago truncatula, soybean (Glycine max), and Lotus japonicus, and isolated corresponding sequences from pea by degenerate-primer polymerase chain reaction and library screening. We found that the majority of Arabidopsis flowering genes are represented in pea and in legume sequence databases, although several gene families, including the MADS-box, CONSTANS, and FLOWERING LOCUS T/TERMINAL FLOWER1 families, appear to have undergone differential expansion, and several important Arabidopsis genes, including FRIGIDA and members of the FLOWERING LOCUS C clade, are conspicuously absent. In several cases, pea and Medicago orthologs are shown to map to conserved map positions, emphasizing the closely syntenic relationship between these two species. These results demonstrate the potential benefit of parallel model systems for an understanding of flowering phenology in crop and model legume species.

Distribution of human papillomavirus genotypes in plucked eyebrow hairs from Slovenian males with genital warts

In the present study, the presence and distribution of human papillomaviruses (HPVs) in the plucked eyebrow hairs obtained from 49 Slovenian male patients with genital warts were investigated. Using polymerase chain reaction (PCR) with three sets of degenerate primers targeting all known HPV genotypes, HPV DNA was found in 31 (63.3%) of 49 eyebrow hair samples. Epidermodysplasia verruciformis (EV) associated HPV specific M a/H a nested PCR system detected HPVs in 27 (55.1%) and CPI/CPIIs primers that amplify the majority of cutaneous/EV HPV genotypes in 20 (40.8%) of 49 samples tested. The CPI/CPIIg PCR specific for E1 open reading frame of genital HPVs showed the presence of HPV DNA in 10 (20.4%) of 49 specimens. Direct sequencing of the H a PCR products showed the presence of three putative new HPV genotypes, named SIBX1, SIBX2 and SIBX3. Similarly, three potential new HPV genotypes, SIBX4, SIBX5 and SIBX6, were detected by sequencing CPI/CPIIs PCR products. In total, at least 24 different HPV genotypes were detected in 31 HPV DNA positive samples of plucked eyebrow hairs. The results of our study showed that the use of a combined degenerate primer PCR approach considerably improves the HPV DNA detection over individual primer sets and greatly improves the detection of different HPV genotypes in the plucked eyebrow hairs.