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PubMed1998

Expressed sequence tags of fruits, peels, and carpels and analysis of mRNA expression levels of the tagged cDNAs of fruits from the Fuji apple.

In order to understand molecular events during fruit development and provide genetic resources for molecular breeding, 430 expressed sequence tags (ESTs) were generated from randomly selected clones of cDNA libraries prepared from young fruits, peels of mature fruits, and carpels of the Fuji apple (Malus domestica Borkh.). Database comparisons of the ESTs revealed that 180 non-redundant clones showed a high similarity with previously identified genes. Among these, 138 clones exhibited a homology with previously identified plant genes and 12 were identical to genes that were previously identified from apples. The deduced amino acid sequences of 42 clones had a homology to proteins that have not been reported from plants. Eighteen cDNA clones from the young fruit library were selected for studying expression levels and patterns in reproductive organs and leaves. This study revealed that the clones can be classified into 3 different groups based on their expression levels. The first 9 clones were expressed strongly in at least one reproductive organ. Eight of these clones (vacuolar processing protease, sucrose phosphate synthase, arabinogalactan protein, UDP-glucose glucosyl transferase, major allergen D1, cystein proteinase inhibitor, lipoxygenase, and protease subunit SUG2) were highly expressed in mature flowers and young fruits, whereas one clone (z-carotene desaturase protein precursor) was preferentially expressed in mature flowers but weakly in young fruits. The second group includes 6 cDNA clones (glucose transport protein, aminomethyl transferase precursor protein, dTDP-D-glucose-4,6-dehydrogenase, 2 types of protein kinase, and selenium binding protein) that were weakly expressed. These clones were characterized by their preferential expression patterns in mature flowers and young fruits. The transcripts of 3 cDNA clones in the third group (vacuolar aminopetidase, beta-galactosidase, and EREBP-4) were detectable only by RT-PCR and they were preferentially expressed in young fruits. These results indicate that most ESTs that were isolated from young fruits are preferentially expressed in reproductive organs and thereby play important roles during reproductive organ development.

DNA, Complementary↗

PubMed2000

Probing the human natural autoantibody repertoire using an immunoscreening approach.

While an increasing number of studies report the presence of antibodies capable of recognizing self-antigens, the function of these natural autoantibodies remains elusive. A variety of concepts has been advanced ranging from evolutionarily tolerated but non-functional natural autoantibodies to autoantibodies effecting various biological functions. Known IgM, IgG, and IgA natural autoantibodies are directed against various antigens, including nuclear and cell surface proteins. To explore further autoantibodies and their autoantigens, we employed an immunological screening method called SEREX recently used to characterize tumour-expressed antigens eliciting an immune response in patients [1]. Sera from 12 individuals were used to screen a cDNA expression library prepared from a cytogenetically normal meningioma to identify antigens reactive with normal human sera from individuals without obvious disease. Nineteen reactive normal antigen clones were identified representing 15 different antigens, including nine genes with known functions, five genes with unknown functions, and one gene with a novel sequence not present in the databases. Of the 12 individual normal sera tested, 75% were reactive to one or more of the 15 different antigens with two highly reactive sera demonstrating reactivity with 33% of the antigens. When screening the same meningioma expression library with serum from the patient, eight antigens were identified that were totally different from those identified using sera from normal individuals. This SEREX immunological screening method presents a new option for probing the natural autoantibody repertoire and identifying normal antigens whose functions may provide additional insights into how natural autoantibodies effect various biological functions.

Autoantibodies↗

PubMed1998

Construction and characterization of a 10-fold genome equivalent rat P1-derived artificial chromosome library.

A rat PAC library was constructed in the vector pPAC4 from genomic DNA isolated from female Brown Norway rats. This library consists of 215,409 clones arrayed in 614,384-well microtiter plates. An average insert size of 143 kb was estimated from 217 randomly isolated clones, thus representing approximately 10-fold genome coverage. This coverage provides a very high probability that the library contains a unique sequence in genome screening. Tests on randomly selected clones demonstrated that they are very stable, with only 4 of 130 clones showing restriction digest fragment alterations after 80 generations of serial growth. FISH analysis using 70 randomly chosen PACs revealed no significant chimeric clones. About 7% of the clones analyzed contained repetitive sequences related to centromeric regions that hybridized to some but not all centromeres. DNA plate pools and superpools were made, and high-density filters each containing an array of 8 plates in duplicate were prepared. Library screening on these superpools and appropriate filters with 10 single-locus rat markers revealed an average of 8 positive clones, in agreement with the estimated high genomic coverage of this library and representation of the rat genome. This library provides a new resource for rat genome analysis, in particular the identification of genes involved in models of multifactorial disease. The library and high-density filters are currently available to the scientific community.

Animals↗

PubMed2006

16S rDNA directed PCR primers and detection of methanogens in the bovine rumen.

AIMS: To assess the diversity of ruminal methanogens in a grazing cow, and develop PCR primers targeting the predominant methanogens. METHODS AND RESULTS: DNA was extracted from rumen contents collected from a cow grazing pasture. Archaeal 16S rRNA genes were amplified by PCR using two pairs of archaea-specific primers, and clone libraries prepared. Selected clones were sequenced. Phylogenetic analysis revealed that for one primer pair, most sequences clustered with Methanobrevibacter spp. whereas with the other primer pair most clustered with Methanosphaera stadtmanae. One sequence belonged to the Crenarcheota. PCR primers were designed to detect Msp. stadtmanae and differentiate between Mbb. ruminantium and Mbb. smithii and successfully tested. CONCLUSIONS: The ruminal methanogens included Mbb. ruminantium, Mbb. smithii, Mbb. thaueri and methanogens similar to Msp.stadtmanae. The study showed that apparent methanogen diversity can be affected by selectivity from the archaea-specific primers used to create clone libraries. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed a greater diversity of ruminal methanogens in grazing cows than previously recognized. It also shows the need for care in interpreting methanogen diversity using PCR-based analyses. The new PCR primers will enable more information to be obtained on Msp. stadtmanae and Methanobrevibacter spp. in the rumen.

Animals↗

PubMed1991

Localization of the gene encoding steroid hydroxylase cytochrome P-450 from Rhizopus nigricans inside a HindIII fragment of genomic DNA.

The gene encoding steroid inducible cytochrome P450 of Rhizopus nigricans ATCC 6227b has been found inside a HindIII fragment of the genomic DNA by hybridization with a partial length cDNA probe. The latter was isolated by immunoscreening a cDNA library prepared in the lambda gt11 expression system and identified on the basis of inducibility and sequence analysis. The nucleotide sequence of the cDNA probe revealed a coding sequence for the heme binding segment characteristic of the P450 gene family.

Amino Acid Sequence↗

PubMed1992

Genes encoding Xenopus laevis Ig L chains. Implications for the evolution of kappa and lambda chains.

Xenopus laevis Ig contain two distinct types of L chains, designated rho or L1 and sigma or L2. We have analyzed Xenopus genomic DNA by Southern blotting with cDNA probes specific for L1 V and C regions. Many fragments hybridized to the V probe, but only one or two fragments hybridized to the C probe. Corresponding C, J, and V gene segments were identified on clones isolated from a genomic library prepared from the same DNA. One clone contains a C gene segment separated from a J gene segment by an intron of 3.4 kb. The J and C gene segments are nearly identical in sequence to cDNA clones analyzed previously. The C segment is somewhat more similar and the J segment considerably more similar in sequence to the corresponding segments of mammalian kappa chains than to those of mammalian lambda chains. Upstream of the J segment is a typical recombination signal sequence with a spacer of 23 bp, as in J kappa. A second clone from the library contains four V gene segments, separated by 2.1 to 3.6 kb. Two of these, V1 and V3, have the expected structural and regulatory features of V genes, and are very similar in sequence to each other and to mammalian V kappa. A third gene segment, V2, resembles V1 and V3 in its coding region and nearby 5'-flanking region, but diverges in sequence 5' to position -95 with loss of the octamer promoter element. The fourth V-like segment is similar to the others at the 3'-end, but upstream of codon 64 bears no resemblance in sequence to any Ig V region. All four V segments have typical recombination signal sequences with 12-bp spacers at their 3'-ends, as in V kappa. Taken together, the data suggest that Xenopus L1 L chain genes are members of the kappa gene family.

Amino Acid Sequence↗

PubMed1999

Identification of Culturable Oligotrophic Bacteria within Naturally Occurring Bacterioplankton Communities of the Ligurian Sea by 16S rRNA Sequencing and Probing.

> Abstract Typical marine bacteria (i.e., obligately oligotrophic) that were numerically dominant members of naturally occurring marine communities were identified by cloning and sequencing the amplified 16S rRNA genes obtained from dilution cultures of the original samples. The data reported here refer to two different habitats of a marine pelagic environment (28 miles offshore, in the northwestern Mediterranean Sea). The samples were taken from the water column at two representative layers, i.e., the 30-m depth, corresponding to the chlorophyll maximum layer, and the 1800-m depth, representative of a deep, oligotrophic environment. Three major lineages were found in the 16S rDNA clone libraries prepared from the two samples, two of which could be assigned to the Vibrio and the Rhodobacter groups. The third lineage was a distant relative of the genus Flavobacterium, but it was not closely related to any marine isolate. Six oligonucleotide probes, either complementary to the conserved sequence domains or selectively hybridizing to the clone sequences, were designed for use as hybridization group-specific and strain-specific probes. A single-mismatch discrimination between certain probes and nontarget sequences was demonstrated by detecting the probes' specificity at different hybridization and washing conditions. The screening of the clone libraries with the obtained probes revealed that neither the 30-m sample higher dilution nor the 1800-m one were pure cultures. While some representatives of the Vibrio group were found in both the surface and the deep sample, the members of the Flavobacterium and Rhodobacter lineages were detected only in the deep and the euphotic layers, respectively. We suggest an approach for analyzing autochthonous marine bacteria able to grow in unamended seawater.

Journal Article↗

PubMed2026

Evaluation of one-step amplicon-based targeted enrichment for SARS-CoV-2 whole-genome sequencing using the Midnight amplicon scheme.

Genomic surveillance proved invaluable during the COVID-19 pandemic for tracking SARS-CoV-2 variants and guiding outbreak responses, underscoring the ongoing need to reduce whole-genome sequencing (WGS) costs and improve workflow efficiency to ensure accessibility in resource limited settings. Here, we evaluated a one-step reverse transcription polymerase chain reaction (RT-PCR) approach using the Midnight V2 primer scheme for targeted amplification of the SARS-CoV-2 genome, assessed its compatibility with Illumina sequencing, and compared its performance to a well-established two-step method. Initially, we determined optimal RT-PCR reaction conditions using the Midnight V2 primer panel for the one-step RT-PCR kit and scaled reaction volumes for both RT-PCR and library preparation. Clinical specimens (n = 53) that had undergone routine WGS for surveillance purposes using the established two-step RT-PCR method were compared using the one-step RT-PCR assay. For samples with genome completeness greater than 70%, both methods gave comparable results with similar sequence coverage and 100% concordance for lineage assignment. Further investigation revealed a higher percentage of reads aligning to the SARS-CoV-2 genome with a greater depth of coverage using the one-step method compared to the two-step method. Finally, analysis of scaled one-step and library reaction volumes revealed significant cost savings for samples undergoing WGS. Overall, the results presented here verify the accuracy and reproducibility of one-step targeted amplification and offer an efficient and cost-effective workflow for routine SARS-CoV-2 genomic surveillance.

Humans↗

PubMed1989

Cloning of cDNA coding for connective tissue activating peptide III from a human platelet-derived lambda gt11 expression library.

We report here the cloning of the cDNA coding for platelet connective tissue-activating peptide-III (CTAP-III) from a lambda gt11 expression library prepared using messenger RNA (mRNA) isolated from human platelets. The open reading frame of the clone coded for a protein with 128 amino acid residues. Since the precursor of CTAP-III, platelet basic protein (PBP is 94 amino acids long, the 5'-translated region of the cDNA codes for a leader sequence 34 amino acids long. This leader sequence, like the sequence of mature CTAP-III, shows significant homology to the sequence of platelet factor 4 (PF4), the only other platelet specific alpha-granule protein cloned until now, from a human erythroleukemic (HEL) cell line-derived cDNA library. These leader sequences are probably critical for targeting such proteins to the alpha-granule. Northern blot hybridization with platelet and megakaryocyte mRNA shows a single species mRNA of approximately 0.8 kb, suggesting that the corresponding cDNA is full length. The cloning of platelet specific CTAP-III provides additional evidence for the platelet specificity of the cDNA library used.

Amino Acid Sequence↗

PubMed2006

Quality enhancement of the non-immune phage scFv library to isolate effective antibodies.

The non-immune phage antibody library system is one of the most attractive technologies available to current therapeutic, diagnostic and basic scientific research. This system allows the rapid isolation of antibodies of interest that could subsequently be applied directly to drug delivery systems and antibody therapy. Previously, we reported the primer sets to encompass the antibody repertoire and thus improve library quality. However, a wide number of varying primer sets cause to decrease the amplification efficiency of antibody genes. In the present study, we re-generated the library primer sets newly and constructed an improved library from non-immune mice that was far superior in terms of variety and quality. This new library contained 2.4 billion independent clones. In addition, we optimized the selection step from this library to isolate high-affinity antibodies. The optimization of an affinity panning protocol by the incorporation of an automated Microfluidics instrument led to the successful isolation of three different monoclonal antibodies for human vascular endothelial growth factor receptor 2 (KDR). These antibodies were demonstrated to exhibit high specificity and were able to detect a mere 0.6 fmol of KDR by dot blot analysis. Previously reported antibodies for luciferase were also isolated successfully from this library. Our results clearly demonstrate the importance of the improved protocol for the library preparation of antibodies and the resulting isolation of antibodies for clinical and research applications.

Amino Acid Sequence↗

PubMed2000

Differential display analysis of gene expression during the induction of mucosal immunity.

One approach to understanding the physiologically relevant events during the induction of an immune response is to identify genes that are expressed when the immune system first encounters antigen. Such an investigation requires a naive but fully functional immune system, and the fetal lamb provides these conditions during the last trimester of gestation. 'Intestinal segments,' containing a jejunal Peyer's patch, were surgically prepared in fetal lambs (>120 days gestation) and individual 'intestinal segments' were injected with either culture medium or infectious bovine rotavirus. Peyer's patch tissue was collected 18 h postinfection. Histology and virus culture confirmed that bovine rotavirus had infected the mucosal epithelium. RNA was extracted from jejunal Peyer's patch tissue and mRNA differential display was used to identify genes expressed following rotavirus infection. Ten cDNAs were identified by differential display and these cDNAs were isolated, cloned, and sequenced. One of the cDNAs sequenced, displayed homology to the gene encoding the sperm surface protein Sp17. Differential expression of this gene in antigen-exposed jejunal Peyer's patches was confirmed by Northern blot and RT-PCR. The complete sequence for sheep Sp17 mRNA was obtained from a lambda cDNA library, prepared from the jejunal Peyer's patch of a young lamb. Sp17 expression was detected by RT-PCR in a variety of mucosa-associated lymphoid tissues but not in primary or other secondary lymphoid tissues. Thus, the fetal lamb model may be appropriate for identifying genes relevant to mucosal immunity.

Amino Acid Sequence↗

PubMed2005

Eclair--a web service for unravelling species origin of sequences sampled from mixed host interfaces.

The identification of the genes that participate at the biological interface of two species remains critical to our understanding of the mechanisms of disease resistance, disease susceptibility and symbiosis. The sequencing of complementary DNA (cDNA) libraries prepared from the biological interface between two organisms provides an inexpensive way to identify the novel genes that may be expressed as a cause or consequence of compatible or incompatible interactions. Sequence classification and annotation of species origin typically use an orthology-based approach and require access to large portions of either genome, or a close relative. Novel species- or clade-specific sequences may have no counterpart within existing databases and remain ambiguous features. Here we present a web-service, Eclair, which utilizes support vector machines for the classification of the origin of expressed sequence tags stemming from mixed host cDNA libraries. In addition to providing an interface for the classification of sequences, users are presented with the opportunity to train a model to suit their preferred species pair. Eclair is freely available at http://eclair.btk.fi.

Artificial Intelligence↗

PubMed1994

Release of GPI-anchored membrane proteins by a cell-associated GPI-specific phospholipase D.

Although many glycosylphosphatidylinositol (GPI)-anchored proteins have been observed as soluble forms, the mechanisms by which they are released from the cell surface have not been demonstrated. We show here that a cell-associated GPI-specific phospholipase D (GPI-PLD) releases the GPI-anchored, complement regulatory protein decay-accelerating factor (DAF) from HeLa cells, as well as the basic fibroblast growth factor-binding heparan sulfate proteoglycan from bone marrow stromal cells. DAF found in the HeLa cell culture supernatants contained both [3H]ethanolamine and [3H]inositol, but not [3H]palmitic acid, whereas the soluble heparan sulfate proteoglycan present in bone marrow stromal cell culture supernatants contained [3H]ethanolamine. 125I-labeled GPI-DAF incorporated into the plasma membranes of these two cell types was released in a soluble form lacking the fatty acid GPI-anchor component. GPI-PLD activity was detected in lysates of both HeLa and bone marrow stromal cells. Treatment of HeLa cells with 1,10-phenanthroline, an inhibitor of GPI-PLD, reduced the release of [3H]ethanolamine-DAF by 70%. The hydrolysis of these GPI-anchored molecules is likely to be mediated by an endogenous GPI-PLD because [3H]ethanolamine DAF is constitutively released from HeLa cells maintained in serum-free medium. Furthermore, using PCR, a GPI-PLD mRNA has been identified in cDNA libraries prepared from both cell types. These studies are the first demonstration of the physiologically relevant release of GPI-anchored proteins from cells by a GPI-PLD.

Antigens, CD↗

PubMed1996

Generation of a rat bronchiolar epithelial cell cDNA library: isolation of a proline rich protein highly enriched in bronchiolar epithelial cells.

As a tool for the identification of novel gene products expressed in rat bronchiolar epithelial (BE) cells we generated a cDNA library prepared from BE cells, enriched to contain > 40% Clara cells. Using a simple differential screening strategy we isolated and partially characterized 47 clones, 29 of which corresponded to gene products known to be expressed within the bronchiolar epithelium. A further 15 clones contained sequences which were not present in the EMBL database and therefore represented potential novel BE cell enriched clones. One of these clones, CC-4, was characterized further and shown by northern blotting to be highly enriched in the BE cell population. The deduced protein product of CC-4 is a proline rich protein of 29.5 kd. These studies suggest that this cDNA library may be a useful tool for the identification of novel lung restricted gene products.

Amino Acid Sequence↗

PubMed1990

A cDNA clone homologous to Drosophila F elements from an imaginal discs poly(A)+ library.

A cDNA clone (F-id1) homologous to Drosophila F elements has been isolated from a cDNA library prepared from poly(A)+ RNA derived from imaginal discs. The F-id1 insert is identical to the 3' end terminus of Fw, an element inserted at the white locus upon irradiation. The isolation of F-id1 provides the first evidence that F elements are expressed in the fruit fly life.

Animals↗

PubMed1991

Detection and characterization of chimeric yeast artificial-chromosome clones.

Methods for the construction of yeast artificial-chromosome (YAC) clones have been designed to isolate single, large (100-1000 kb) segments of chromosomal DNA. It is apparent from early experience with this cloning system that the major artifact in YAC clones involves the formation of YACs that contain two or more unrelated pieces of DNA. Such "chimeric" YACs are not easily recognized, particularly in libraries constructed from the total DNA of an organism. In some libraries, they have been found to constitute a major fraction of the clones. Here we discuss some of our experiences with chimeric YACs, with particular emphasis on the approaches that we have employed to detect such aberrant clones. In addition, we describe the detailed characterization of one chimeric YAC isolated from a library prepared from total human DNA. The organization of this clone indicates that it formed by in vivo recombination, presumably in yeast, between two Alu sequences located on unrelated segments of human DNA.

Animals↗

PubMed2005

[Phylogenetic diversity analyse of rumen bacteria using culture independent method].

Culture independent method was used to study the diversity of rumen bacteria. Molecular diversity of rumen bacteria was analyzed by PCR amplification and sequencing of 16S rDNA clone libraries prepared from the rumen content of Holstein cows. The total DNA directly extracted from rumen fluid was used as PCR template. Bacteria universal primer 27F and 1492R was used as primer. Random clones, containing almost full size 16S rDNA sequences (about 1.5 kb long), were sequenced and subjected to an on line similarity search. The 16S rDNA sequence analysis indicated that more than half of the sequences belonged to the not-yet-cultured groups. The 16S rDNA similarity levels with cultured species was less than 90%. The bacterial community structure was also revealed by phylogenetic tree of known sequences and selected sequence. In the library from the rumen fluid, the sequences were mainly affiliated with the following major phyla: low G + C Gram-positive bacteria, Cytophaga-Flexibacter-Bacteroides, and the remaining sequences were placed within not-yet-uncultured groups that had an uncertain affiliation. These several sequences are likely to represent novel taxonomic groupings. The nucleotide sequences have been submitted to the GenBank/EMBL /DDBJ databases under accession numbers AY986777-AY986791.

Animals↗

PubMed2005

PLAUF is a novel P. lividus sea urchin RNA-binding protein.

Preliminary results have shown that various proteins bind long 3'UTR of the transcript for Paracentrotus lividus sea urchin H3.3 histone variant and are probably implicated in mRNA instability. In order to identify these RNA-binding proteins, we screened a lambda-ZAPII cDNA expression library prepared from poly(A) mRNA extracted from sea urchin embryos at blastula stage. We isolated a cDNA that codes for a novel RNA-binding protein homologous to rat and human AUF1 family proteins and we refer to it as PLAUF. Proteins present in the whole lysate of the phages expressing PLAUF bound specifically in vitro the 3'UTR of the H3.3 histone transcript. Northern blot analysis revealed three PLAUF transcripts that are already present in unfertilized eggs; during development their amount increased starting from 4-blastomere embryos and reached the plateau at blastula stage. While the transcription start point was unique, longer 3'UTRs were revealed by 3'RACE approach and further cDNA library screening. Moreover RT-PCR showed the presence of at least one alternative spliced mRNA that codes for a protein with different COOH terminus. The structure of the PLAUF gene was determined by screening a P. lividus sea urchin genomic library with the PLAUF cDNA as probe. Analysis of the positive clones showed that the PLAUF gene is split in 10 exons and 9 introns spanning a distance of about 10 kb. Moreover we demonstrated that the exon 9 was alternative spliced during mRNA processing.

3' Untranslated Regions↗