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PubMed2006

Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding.

DNA barcoding should provide rapid, accurate and automatable species identifications by using a standardized DNA region as a tag. Based on sequences available in GenBank and sequences produced for this study, we evaluated the resolution power of the whole chloroplast trnL (UAA) intron (254-767 bp) and of a shorter fragment of this intron (the P6 loop, 10-143 bp) amplified with highly conserved primers. The main limitation of the whole trnL intron for DNA barcoding remains its relatively low resolution (67.3% of the species from GenBank unambiguously identified). The resolution of the P6 loop is lower (19.5% identified) but remains higher than those of existing alternative systems. The resolution is much higher in specific contexts such as species originating from a single ecosystem, or commonly eaten plants. Despite the relatively low resolution, the whole trnL intron and its P6 loop have many advantages: the primers are highly conserved, and the amplification system is very robust. The P6 loop can even be amplified when using highly degraded DNA from processed food or from permafrost samples, and has the potential to be extensively used in food industry, in forensic science, in diet analyses based on feces and in ancient DNA studies.

Base Sequence↗
PubMed2001

Genetic variation among chicken lines and mammalian species in specific genes.

Thirteen gene-specific primer sets provided by the U.S. Poultry Genome Coordinators were used to investigate DNA polymorphisms between two highly inbred chicken lines of Leghorn and Fayoumi origin. Nucleotide and predicted amino acid sequences were then compared among these chicken lines and the Genbank sequences of chicken, mouse, and human. The following genes were selected as candidates for immune response or transcription activation: B2M, DAD1, IAP1, IL2, IREB1, LAP18, MAFL, POU1F1, RREB1, TAD, TBP1, TCRG, and ZOV3. Total cDNA was obtained from the spleens of Leghorn and Fayoumi lines by reverse transcriptase-polymerase chain reaction (PCR) and was used as a template to PCR-amplify gene-specific products. All primers except POU1F1 and TCRG generated single PCR products of the predicted 325- to 667-bp size, confirming the efficacy of these gene-specific primers in the chicken. Three and seven of the 11 amplified gene fragments yielded line-specific nucleotide polymorphisms between the Leghorn and Fayoumi sequences and between the Leghorn and Genbank chicken sequences respectively. Similarities between inbred Leghorn and mammalian species were 36 to 86% for nucleotides and 25 to 96% for predicted amino acid sequence. The polymorphisms of some gene fragments between the Leghorn and Fayoumi lines will allow for investigation of associations of these genes with immune response and other biological traits.

Amino Acid Sequence↗
PubMed2000

Mu-class GSTs are responsible for aflatoxin B(1)-8, 9-epoxide-conjugating activity in the nonhuman primate macaca fascicularis liver.

Mice are resistant to the carcinogenic effects of the mycotoxin aflatoxin B(1) (AFB(1)) because they constitutively express an alpha-class glutathione S-transferase (mGSTA3-3) that has high (approximately 200,000 pmol/min/mg) activity toward aflatoxin B(1)-8, 9-epoxide (AFBO). Rats do not constitutively express a GST with high AFBO-conjugating activity and are sensitive to AFB(1)-induced hepatocarcinogenesis. Constitutively expressed human hepatic alpha-class GSTs (hGSTA1-1 and hGSTA2-2) possess little or no AFBO-detoxifying activity (<2 pmol/min/mg). Recently, we found that the nonhuman primate, Macaca fascicularis (Mf), exhibits significant (approximately 300 pmol/min/mg) constitutive hepatic GST activity towards AFBO. To determine which specific GST isoenzyme(s) is (are) responsible for this activity, MF: GSTs were purified from liver tissue and characterized and, Mf mu-class GST cDNAs were cloned by reverse transcriptase-coupled polymerase chain reaction (RT-PCR). Purification by glutathione agarose (GSHA) affinity chromatography yielded a protein, GSHA-GST, that exhibited relatively high AFBO-conjugating activity (239 pmol/min/mg) compared to other GST-containing peaks. Western blotting and enzymatic activity analyses revealed that GSHA-GST belongs to the mu class. Two distinct mu-class GST cDNAs, mfaGSTM1 (GenBank accession # AF200709) and mfaGSTM2 (GenBank accession # AF200710), were generated by RT-PCR. CDNA-derived amino acid sequence analysis revealed that mfaGSTM1 and mfaGSTM2 share 97% and 96% homology with the human mu-class GSTs hGSTM4 and hGSTM2, respectively. In contrast to recombinant mfaGSTM1-1, which had no detectable AFBO-conjugating activity, mfaGSTM2-2 exhibited this activity at 333 pmol/min/mg. Activity profiles for the stereoisomers exo- and endo-AFBO, and of 1-chloro-2,4-dinitrobenzene of the purified protein GSHA-GST and recombinant mfaGSTM2-2, suggested that they are two distinct enzymes. Our results indicate that, in contrast to rodents, mu-class GSTs are responsible for the majority of AFBO-conjugating activity in the liver of Macaca fascicularis.

Aflatoxin B1↗
PubMed2026

Infectious Clone Development of Zucchini Green Mottle Mosaic Virus Infecting Medicinal Plant Trichosanthes kirilowii and Establishment of a Serological Assay System.

Trichosanthes kirilowii has long been cultivated for application in traditional Chinese medicine. In this study, we identified two isolates of zucchini green mottle mosaic virus (ZGMMV; species Tobamovirus cucurbitae) from T. kirilowii plants. We determined the complete genome sequences of the ZGMMV isolates named ZGMMV-GL-1 and ZGMMV-GL-2. Each ZGMMV genome was 6,517 nucleotides in length, with only a single nucleotide variation detected between two sequences. Sequence analysis revealed that the ZGMMV isolates from this study shared 88.07 to 91.62% nucleotide identity with five other ZGMMV isolates deposited in GenBank. Phylogenetic analysis indicated that ZGMMV isolates can be clustered into two distinct groups; our two isolates shared the highest sequence similarity with the ZGMMV isolate from Nanning (GenBank accession number MF066176) and clustered within Group II. The coat protein (CP) gene was cloned from ZGMMV-infected T. kirilowii samples, and the CPZGMMV was expressed using the pET28(a) vector. Specific polyclonal antiserum CPZGMMV was generated by immunizing rabbits with the purified protein, and its sensitivity was determined to be satisfactory. Leveraging the high accuracy and sensitivity of the CPZGMMV antiserum, we developed a rapid, precise, and scalable diagnostic method for ZGMMV. We then constructed the full-length cDNA clones (ZGMMV-GL-1 and ZGMMV-GL-2). Additionally, the ZGMMV cDNA infectious clones from T. kirilowii were also able to infect Nicotiana benthamiana and Cucumis sativus systemically, inducing rough-textured and curled leaves in N. benthamiana and mosaic symptoms in C. sativus and T. kirilowii. In this study, we produced an antiserum against the ZGMMV CP and developed a sensitive, rapid, and reliable diagnostic assay, which lays a technical foundation for the detection and monitoring of ZGMMV. Therefore, the establishment of the ZGMMV infectious clone facilitates further research on viral protein functions, plant-pathogen interactions, and the formulation of effective ZGMMV management strategies.

Nicotiana benthamiana↗
PubMed2005

Genes preferentially expressed in bovine oocytes revealed by subtractive and suppressive hybridization.

To isolate bovine oocyte marker genes, we performed suppressive and subtractive hybridization between oocytes and somatic tissues (i.e., intestine, lung, muscle, and cumulus cells). The subtracted library was characterized by sequencing 185 random clone inserts, representing 146 nonredundant genes. After Blast analysis within GenBank, 64% could be identified, 21% were homologous to unannotated expressed sequence tag (EST) or genomic sequences, and 15% were novel. Of 768 clone inserts submitted for differential screening by macroarray hybridization, 83% displayed a fourfold overexpression in the oocyte. The 40 most preferential nonredundant ESTs were submitted to GenBank analysis. Several well-known oocyte-specific genes were represented, including growth differentiation factor 9, bone morphogenetic protein 15, or the zona pellucida glycoprotein genes. Other ESTs were not identified. We investigated the expression profile of several candidates in the oocyte and a panel of gonadal and somatic tissues by reverse transcription-polymerase chain reaction. B-cell translocation gene 4, cullin 1, MCF.2 transforming sequence, a locus similar to snail soma ferritin, and three unidentified genes were, indeed, preferentially expressed in the oocyte, even though most were also highly expressed in testis. The transcripts were degraded throughout preimplantation development and were not compensated for by embryonic transcription after the morula stage. These profiles suggest a role in gametogenesis, fertilization, or early embryonic development.

Animals↗
PubMed1999

Identification of progesterone-dependent messenger ribonucleic acid regulatory patterns in the rhesus monkey endometrium by differential-display reverse transcription-polymerase chain reaction.

We used differential-display reverse transcription-polymerase chain reaction (DDRT-PCR) to identify different patterns of progesterone (P4)-dependent gene regulation in rhesus monkey endometria. Complementary DNA populations representing the proliferative phase (estrogen dominant, EcDNA) and an inadequate secretory phase (low level of P4, IcDNA) were compared with a cDNA population representing an adequate secretory phase (normal level of P4, PcDNA). We were able to distinguish four different levels of mRNA regulation: 1) up-regulation by P4 during an adequate secretory phase, 2) autologous down-regulation (IcDNA versus PcDNA), 3) lower abundance in IcDNA compared to PcDNA, and 4) P4-dependent inhibition of EcDNA gene expression. We isolated and sequenced 16 fragments representing these different levels of P4 regulation. The sequence of three fragments that were autologously down-regulated (I1, I2, I4) matched previously entered GenBank mRNAs: I1 encodes serine/threonine protein phosphatase A; I2 encodes oxobutanoate dehydrogenase E1b-beta; and I4 encodes line-1 reverse transcriptase homologue. Six other fragments exhibited homology to uncharacterized expressed sequence tags, sequence site tags, and cosmid clones. The remaining seven fragments exhibited no significant homology to GenBank entries at this time. The various patterns of P4-dependent gene regulation identified in the present study are likely to play roles in the temporal orchestration of events that lead to proper maturation of the endometrium.

Animals↗
PubMed2000

Identification of a novel testis-specific leucine-rich protein in humans and mice.

A novel testis-specific protein, termed LRTP, was identified by screening both human and mouse testis and mouse pachytene spermatocyte cDNA libraries. Sequence analyses (GenBank accession number: AF092208) revealed that LRTP contains an amino terminus leucine-rich repeat domain. There are several acidic regions rich in glutamic acid in the C-terminus. The sequence, by GenBank search, shows similarities to LANP and SDS22+, leucine-rich repeat proteins localized to the nucleus and involved in the regulation of protein phosphatases. In mouse, the mRNA is first detected at about Day 14 postpartum, presumably when mid-pachytene spermatocytes are first seen. In situ hybridization confirmed the expression of the LRTP mRNA at this stage of spermatogenesis. Immunohistochemical analysis revealed that the protein is most abundant in the cytoplasm of pachytene and diplotene cells, corresponding to late prophase of meiosis I. Immunohistochemical localization is markedly reduced in secondary spermatocytes, suggesting a functional association of LRTP with meiosis. An LRTP cDNA probe did not bind to mouse ovary RNA in a dot blot assay.

Amino Acid Sequence↗
PubMed2003

Genetic diversity and phylogenetic analysis of glycoprotein 5 of European-type porcine reproductive and respiratory virus strains in Spain.

The gene encoding glycoprotein 5 (ORF5) of 21 porcine reproductive and respiratory syndrome virus (PRRSV) isolates from Spain and two European-type vaccines currently available in that country were analysed using RT-PCR and sequencing. Sequences were then compared with other European-type sequences available through GenBank. Results showed percentages of similarity to Lelystad virus (LV), which, in most cases, were below 90 %. In contrast, two strains were very similar (>99 %) to a PRRSV variant from the Czech Republic. Evolutionary trees showed three types of strains: one grouped old Spanish sequences; a second grouped isolates from this study together with two Czech variant strains; and the third comprised other GenBank sequences. Regarding the predicted protein sequences, some isolates from this study showed a low degree of similarity to LV (below 50 %) and most of the strains examined had additional N-linked glycosylation sites compared to LV. These results provide evidence of the existence of variant PRRSV strains in Spain with characteristics that may be advantageous for immune evasion.

Animals↗
PubMed2002

ViewGene: a graphical tool for polymorphism visualization and characterization.

The human genome project is producing an enormous amount of sequence data, based on which single base changes between individuals can be identified. Unfortunately, computer tools that were adequate for sequence assembly are less than ideal for the characterization of polymorphism data [single nucleotide (snp) or insertion/deletion (indel)] and other sequence features, and their relationship to each other. We have developed viewGene as a flexible tool that takes input from a number of sequence formats and analysis programs (Genbank, FASTA, RepeatMasker, Cross match, BLAST, user-defined data) to construct a sequence reference scaffold that can be viewed through a simple graphical interface. polymorphisms generated from many sources can be added to this scaffold through the same sequence formats, with a variety of options to control what is displayed. Large amounts of polymorphism data can be organized so that patterns and haplotypes can be readily discerned. In our laboratory, viewGene has been used to view annotated genbank records, find nonrepetitive sequence fragments for polymorphism detection, and visualize similarity search results. Manipulation, cross-referencing, and haplotype viewing of snp data are essential for quality assessment and identification of variants associated with genetic disease, and viewGene provides all three of these important functions.

Amino Acid Sequence↗
PubMed2002

Large-scale protein annotation through gene ontology.

Recent progress in genomic sequencing, computational biology, and ontology development has presented an opportunity to investigate biological systems from a unique perspective, that is, examining genomes and transcriptomes through the multiple and hierarchical structure of Gene Ontology (GO). We report here our development of GO Engine, a computational platform for GO annotation, and analysis of the resultant GO annotations of human proteins. Protein annotation was centered on sequence homology with GO-annotated proteins and protein domain analysis. Text information analysis and a multiparameter cellular localization predictive tool were also used to increase the annotation accuracy, and to predict novel annotations. The majority of proteins corresponding to full-length mRNA in GenBank, and the majority of proteins in the NR database (nonredundant database of proteins) were annotated with one or more GO nodes in each of the three GO categories. The annotations of GenBank and SWISS-PROT proteins are available to the public at the GO Consortium web site.

Animals↗
PubMed2003

Purification and characterization of four beta-expansins (Zea m 1 isoforms) from maize pollen.

Four proteins with wall extension activity on grass cell walls were purified from maize (Zea mays) pollen by conventional column chromatography and high-performance liquid chromatography. Each is a basic glycoprotein (isoelectric point = 9.1-9.5) of approximately 28 kD and was identified by immunoblot analysis as an isoform of Zea m 1, the major group 1 allergen of maize pollen and member of the beta-expansin family. Four distinctive cDNAs for Zea m 1 were identified by cDNA library screening and by GenBank analysis. One pair (GenBank accession nos. AY104999 and AY104125) was much closer in sequence to well-characterized allergens such as Lol p 1 and Phl p 1 from ryegrass (Lolium perenne) and Phleum pretense, whereas a second pair was much more divergent. The N-terminal sequence and mass spectrometry fingerprint of the most abundant isoform (Zea m 1d) matched that predicted for AY197353, whereas N-terminal sequences of the other isoforms matched or nearly matched AY104999 and AY104125. Highly purified Zea m 1d induced extension of a variety of grass walls but not dicot walls. Wall extension activity of Zea m 1d was biphasic with respect to protein concentration, had a broad pH optimum between 5 and 6, required more than 50 micro g mL(-1) for high activity, and led to cell wall breakage after only approximately 10% extension. These characteristics differ from those of alpha-expansins. Some of the distinctive properties of Zea m 1 may not be typical of beta-expansins as a class but may relate to the specialized function of this beta-expansin in pollen function.

Allergens↗
PubMed2000

Purification, enzymatic characterization, and nucleotide sequence of a high-isoelectric-point alpha-glucosidase from barley malt.

High-isoelectric-point (pI) alpha-glucosidase was purified 7, 300-fold from an extract of barley (Hordeum vulgare) malt by ammonium sulfate fractionation, ion-exchange, and butyl-Sepharose chromatography. The enzyme had high activity toward maltose (k(cat) = 25 s(-1)), with an optimum at pH 4.5, and catalyzed the hydrolysis by a retaining mechanism, as shown by nuclear magnetic resonance. Acarbose was a strong inhibitor (K(i) = 1.5 microM). Molecular recognition revealed that all OH-groups in the non-reducing ring and OH-3 in the reducing ring of maltose formed important hydrogen bonds to the enzyme in the transition state complex. Mass spectrometry of tryptic fragments assigned the 92-kD protein to a barley cDNA (GenBank accession no. U22450) that appears to encode an alpha-glucosidase. A corresponding sequence (HvAgl97; GenBank accession no. AF118226) was isolated from a genomic phage library using a cDNA fragment from a barley cDNA library. HvAgl97 encodes a putative 96.6-kD protein of 879 amino acids with 93.8% identity to the protein deduced from U22450. The sequence contains two active site motifs of glycoside hydrolase family 31. Three introns of 86 to 4,286 bp interrupt the coding region. The four exons vary from 218 to 1,529 bp. Gene expression analysis showed that transcription reached a maximum 48 h after the start of germination.

Amino Acid Sequence↗
PubMed1998

Detection of verotoxin-producing Escherichia coli O157:H7 by multiplex polymerase chain reaction.

We constructed primers for multiplex polymerase chain reaction (PCR) to detect verotoxin-producing Escherichia coli (VTEC) O157:H7. The multiplex PCR primers were designed from the sequence of the flagellin structural gene of Escherichia coli flagellar type H7 (GenBank under accession number L07388), and from the sequence of the rfbE gene of Escherichia coli O157:H7 (GenBank under accession number S83460). In addition to these primers, we used a primer pair reported by Karch and Meyer (J. Clin. Microbiol. 27: 2751-2757, 1989) to amplify various VT genes from VTEC. All of the examined specimens (18 isolates) of VT-producing E. coli O157:H7 showed a positive result by the multiplex PCR test with the three sets of primers. The sensitivity of detection for VT-producing E. coli O157:H7 was shown to be at least 3,000 cells per PCR tube.

Animals↗
PubMed1992

The 32.6 kb Indian delta beta-thalassaemia deletion ends in a 3.4 kb L1 element downstream of the beta-globin gene.

The Indian delta beta-thalassaemia, with elevated fetal gamma globin gene expression, was previously found to have a large deletion beginning 1 kb 3' of the (A) gamma globin gene at GenBank HUMHBB coordinate 42151, and extending into a new L1 sequence. We have now determined the 3' breakpoint of this deletion, and in doing so we have extended the known beta-globin gene cluster DNA sequence from its end at 73326 to projected GenBank coordinate 79016. These data show that the deletion is 32.6 kb long, terminating 11 kb 3' of the beta-globin gene. This 3' breakpoint is at 74772, within a 3.4 kb partial L1 repeat at 74263-77665; the Black ((A) gamma delta beta)(0)-thalassaemia also terminates in this L1, at 76508. In addition, two Alu sequences were found, at 73692-73816 and 78171-78441. Among the protein-binding DNA sequence motifs 3' to the Indian delta beta-thalassaemia breakpoint, at 76581/76607 there is a TGATAA/ACACCC pair that binds the erythroid-specific GATA-1 and ubiquitous CACCC-box binding proteins. We hypothesize that elevated fetal haemoglobin may be due to an enhancer or enhancers 3' to the deletion breakpoints and may involve the TGATAA/ACACCC pair.

Base Sequence↗
PubMed2006

Cloning and characterization of protease inhibitor-like cDNAs from the Hessian fly mayetiola destructor (SAY).

Analysis of transcriptomes from the salivary glands and midgut of Hessian fly larvae Mayetiola destructor (say) identified a set of diverse cDNAs that encode proteins with a relatively high percentage (over 10%) of cysteinyl residues. Structural comparison of these putative proteins with known sequences in GenBank revealed that the positions of the cysteinyl residues in the identified proteins were highly conserved within a family of proteinase inhibitors despite very little overall sequence similarity. Phylogenetic analysis sorted this set of cDNAs into five different groups. To determine if these cDNAs indeed encode proteinase inhibitors, recombinant proteins were generated with two cDNAs from two different groups. Biochemical analysis of the recombinant proteins against commercial and insect gut proteinases demonstrated that the recombinant proteins are strong proteinase inhibitors with different specificities. Northern blot and real-time PCR analysis revealed that the different genes were expressed at different developmental stages and in different tissues. The overall results indicated that M. destructor contains a complex of genes that code for proteinase inhibitors which may regulate proteinase activities in different regulatory pathways. The GenBank accession numbers for the cDNAs in this paper were DQ232690 to DQ232718.

Amino Acid Sequence↗
PubMed2006

Analysis of mutations in the pbp genes of penicillin-non-susceptible pneumococci from Turkey.

Sequence analysis of the pbp genes from 20 Streptococcus pneumoniae isolates from Turkey (eight with high-level penicillin-resistance, nine with low-level penicillin-resistance, and three that were penicillin-susceptible) was performed and phylogenetic trees were constructed. Most isolates clustered together within a single branch that was distinct from sequences deposited previously in GenBank, which suggests that these isolates have probably evolved following new recombination events. The most prominent active-site mutations, which have also been associated previously with resistance, were T371A in PBP1a, E481G followed by T451A in PBP2b, and T338A in PBP2x. All isolates also possessed a (570)SVES/TK(574) block in the PBP2b sequence, instead of the QLQPT sequence of R6, which is fairly uncommon in GenBank sequences. This is the first study to analyse alterations in the pbp sequences of pneumococci isolated in Turkey.

Bacterial Proteins↗
PubMedDate not supplied

Strombidinopsis jeokjo n. sp. (ciliophora: choreotrichida) from the coastal waters off western Korea: morphology and small subunit ribosomal DNA gene sequence.

The planktonic ciliate Strombidinopsis jeokjo n. sp. is described from Quantitative Protargol-Stained (QPS) preparations, and the sequence of the small subunit rDNA (SSU rDNA) from cultured cells is reported. This species is ovoid and bluntly tapered towards the posterior. The ranges (and mean +/- standard deviation, n = 31) of cell length, cell width, and oral diameter of the QPS-stained specimens were 100-190 microm (149 +/- 25), 60-105 microm (79 +/- 13), and 55-80 microm (64 +/- 5), respectively. Fifteen to seventeen external oral polykinetids had oral membranelle cilia 20-35 microm long. Twenty-six to twenty-eight somatic kineties were equally spaced around the cell body and extended from the oral to the posterior regions with 23-44 dikinetids per kinety. Both kinetosomes of each kinetid bore cilia 3-7 microm long. Strombidinopsis jeokjo had two ovoid macronuclei of 25-38 microm x 12-15 microm. When properly aligned, the sequence of the SSU rDNA of S. jeokjo (GenBank Accession No. AJ628250) was approximately 2% different from that of an unidentified Strombidinopsis species (GenBank Accession No. AF399132-AF399135), the closest species in the SSU rDNA sequence.

Animals↗
PubMed1999

Sample sequencing of a Salmonella typhimurium LT2 lambda library: comparison to the Escherichia coli K12 genome.

As part of the ongoing sequencing of the complete Salmonella typhimurium LT2 genome, a partly ordered set of 416 lambda clones has been developed, representing over 90% of the genome. The average insert size is 17 kb. Sequences were obtained from both ends of each clone in this set. A total of over 600 kb of sequence has been deposited in the genome survey sequence section of GenBank. This resource of clones is available from the Salmonella Genome Stock Center. A preliminary comparison with the Escherichia coli K12 genome indicates that there are likely to be many hundred insertion deletion events, encompassing more than one gene, that distinguish these genomes. Fully 30% of the S. typhimurium sequences have no close homologs in the GenBank database.

Adult↗