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PubMed1999

A 210 kDa protein is located in a membrane-microtubule linker at the distal end of mature and nascent basal bodies.

A monoclonal antibody raised against purified flagellar basal apparatuses from the green flagellate Spermatozopsis similis reacted with a protein of 210 kDa (p210) in western blots. The protein was partially cloned by immunoscreening of a cDNA library. The sequence encoded a novel protein rich in alanine (25%) and proline (20%), which contained regions similar to proteins of comparable amino acid composition such as extracellular matrix components or the membrane-cytoskeletal linker synapsin. Using a polyclonal antibody (anti-p210) raised against the C-terminal part of p210, it was shown that the protein was highly enriched in the basal apparatuses. Immunogold electron microscopy of isolated cytoskeletons or whole cells revealed that p210 was located in the flagellar transition region. The protein was part of the Y-shaped fibrous linkers between the doublet microtubules and the flagellar membrane, as indicated by statistical analysis of post-labeled sections using anti-centrin and anti-tubulin as controls. In premitotic cells p210 was located in a fibrous layer at the distal end of nascent basal bodies, which was perforated by the outgrowing axoneme. During deflagellation the protein remained at the basal body but we observed changes in its distribution, indicating that p210 partially moved to the tip of the basal body. p210 can be used as a marker to determine basal body position, orientation (parallel or antiparallel) and number in S. similis by indirect immunofluorescence. We suppose that p210 is involved in linking basal bodies to the plasma membrane, which is an important step during ciliogenesis.

Algal Proteins↗
PubMed2006

RNA viral community in human feces: prevalence of plant pathogenic viruses.

The human gut is known to be a reservoir of a wide variety of microbes, including viruses. Many RNA viruses are known to be associated with gastroenteritis; however, the enteric RNA viral community present in healthy humans has not been described. Here, we present a comparative metagenomic analysis of the RNA viruses found in three fecal samples from two healthy human individuals. For this study, uncultured viruses were concentrated by tangential flow filtration, and viral RNA was extracted and cloned into shotgun viral cDNA libraries for sequencing analysis. The vast majority of the 36,769 viral sequences obtained were similar to plant pathogenic RNA viruses. The most abundant fecal virus in this study was pepper mild mottle virus (PMMV), which was found in high concentrations--up to 10(9) virions per gram of dry weight fecal matter. PMMV was also detected in 12 (66.7%) of 18 fecal samples collected from healthy individuals on two continents, indicating that this plant virus is prevalent in the human population. A number of pepper-based foods tested positive for PMMV, suggesting dietary origins for this virus. Intriguingly, the fecal PMMV was infectious to host plants, suggesting that humans might act as a vehicle for the dissemination of certain plant viruses.

Adult↗
PubMed2006

Extraction of high-quality, melanin-free RNA From Mycosphaerella fijiensis for cDNA preparation.

High-quality RNA preparations are critical for further applications such as reverse transcriptase-polymerase chain reaction (RT-PCR) transcript amplifications, and elaboration of cDNA and expressed sequence tag libraries. Melanins are phenolic compounds present in many fungi and apparently play key roles in fungi pathogenesis and survival. However, during RNA extraction these compounds constitute a significant challenge to extraction of substantial quantities of high-quality RNA, and consequently to preparation of cDNA libraries. No method currently exists for RNA extraction from Mycosphaerella fijiensis that produces high quantities of melanin-free RNA. This fungus is the most important pathogen of cultivated Musa sp. varieties. A comparison is made between results obtained from the Trizol and RNeasy protocols for RNA extraction, two commercially available methods commonly used to obtain RNA from various sources. An improved methodology is described that allows isolation of intact RNA and elimination of melanins from M. fijiensis mycelium. RNA quality is evaluated by electrophoresis in formaldehyde-agarose gels, RT into cDNAs, and subsequent PCR amplification using primers designed against actin and beta- tubulin from fungi.

Ascomycota↗
PubMedDate not supplied

Aptamers that recognize the lipid moiety of the antibiotic moenomycin A.

Moenomycin A is an amphiphilic phosphoglycolipid antibiotic that interferes with the transglycosylation step in peptidoglycan biosynthesis. The antibiotic consists of a branched pentasaccharide moiety, connected to the moenocinol lipid via a glycerophosphate linker. We have previously described the selection of aptamers that require the lipid group and the disaccharide epitopes of the oligosaccharide moiety for moenomycin binding. Here we report that the enriched moenomycin-binding library contains sequences that evolved for specific recognition of the unpolar lipid group of the antibiotic. These results suggest that the evolution of hydrophobic binding pockets in RNA molecules may be much more common than previously assumed.

Anti-Bacterial Agents↗
PubMed2004

Identification of transcripts generated during the response of resistant Biomphalaria glabrata to Schistosoma mansoni infection using suppression subtractive hybridization.

Suppression subtractive hybridization (SSH) was used to construct a complementary DNA library enriched for transcripts more abundantly expressed in the resistant BS90 strain of Biomphalaria glabrata at 12 hr postinfection with Schistosoma mansoni as compared with the susceptible M-line strain under the same circumstances. One hundred and twelve clones of the library were sequenced, yielding 88 unique SSH-expressed sequence tags (ESTs). Differential expression screening indicated that 22 of the 88 unique transcripts were strong candidates for differential expression in the BS90 strain relative to the M-line strain. Analysis of a subset of 4 transcripts using quantitative reverse transcriptase-polymerase chain reaction (qPCR) substantially supports the patterns obtained using the differential expression screen. Furthermore, the qPCR results revealed that gene upregulation in resistant snails, downregulation in susceptible snails, and differences in constitutive gene expression can all account for differential expression during the defense responses of resistant and susceptible snails. The majority (71.6%) of the SSH-ESTs recovered consisted of novel sequences not identified by sequence similarity to known genes. This work complements previous efforts to elucidate the genetic components underlying a successful response to S. mansoni by B. glabrata and identifies a series of transcripts deserving additional study in comparing susceptible and resistant snails.

Analysis of Variance↗
PubMed1989

Osteocalcin: characterization and regulated expression of the rat gene.

An osteocalcin gene was isolated from a rat genomic DNA library, and sequence analysis indicated that the mRNA is represented in 953 nucleotide segment of DNA consisting of 4 exons and 3 introns. Although the introns in the rat gene are larger, its overall organization is similar to the human gene. Analysis of the 5' flanking sequences of the rat gene shows a modular organization of the promotor as reflected by the the presence of at least 3 classes of regulatory elements. These include (1) typical sequences associated with most genes transcribed by RNA polymerase II (e.g. TATA, CAAT, AP1, AP2), (2) a series of consensus sequences for cyclic nucleotide responsive elements and several hormone receptor binding-sites (estrogen, thyroid and clusters of AG-rich putative Vitamin D responsive elements); and (3) a 24 nucleotide highly conserved sequence between the rat and human gene having a CAAT motif as a central element, designated as an "osteocalcin box." Two regulatory factors of osteocalcin gene expression have been identified. First, contained within the 600 nucleotides immediately upstream from the transcription initiation site are sequences which support Vitamin D dependent transcription of the rat osteocalcin gene. 1,25(OH)2D3 increases osteocalcin mRNA by 6-20 fold increases. In contrast, up to a 200 fold increase in osteocalcin gene expression occurs with mineralization of the extracellular matrix produced by osteoblasts. We propose osteocalcin is a bone-specific marker protein of the mature osteoblast in a mineralizing matrix.

Animals↗
PubMed2026

Transcriptomic Profiling of Canine Testicular Leydig Cell Tumors Uncovers Key Upregulated Gene Pathways.

Total RNA was isolated from sections of healthy testes and Leydig cell tumors of mixed-breed dogs using TMA Master II device. The RNA-seq libraries were sequenced on the Illumina platform. Following differential expression analysis, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were applied with quality control obtained using FastQC and Trimmomatic. This analysis revealed 1500 transcripts, including 928 upregulated and 168 downregulated genes. The results demonstrated that a significant proportion of these differentially expressed genes are directly involved in the control of sex steroid production (CYP11A1, STAR, and 3β-HSD3B1) or tube formation, angiogenesis, and extracellular matrix remodeling in interstitial cells (ESM1, FGG, and VEGFA). Moreover, we identified the upregulation of transcripts responsible for neurotransmitter or neuroendocrine signaling (SLC6A4, GRIN2C, GABRB3) and cholesterol metabolism and its regulation (GPX3, MSMO1, DHCR24). These genes were strongly associated with the phosphatidylinositol-3-kinase (PI3K)-Protein Kinase B (Akt) cascade and extracellular matrix interactions, features shared with various malignancies. Alterations in estrogen and relaxin signaling appear to be distinctive, understudied mechanisms specific to canine Leydig cell tumors. Concurrently, downregulated genes (e.g., DMRTC2, SEMA3C, ALOX12) were linked with cell differentiation, signaling and immunoregulatory pathway suppression involved in tumorigenesis. A complex transcriptomic profile of canine Leydig cell tumors was developed, revealing a conserved oncogenic core shared in some aspects with human malignancies alongside unique species-specific alterations. Findings seem to be useful for identifying novel diagnostic biomarkers and targeted therapies in veterinary oncology, establishing canine reproductive tissues as a valuable comparative biomedical model for research in human.

Leydig cell tumor↗
PubMed2006

Identification of differentially expressed genes in mouse hepatocarcinoma ascites cell line with low potential of lymphogenous metastasis.

AIM: To identify genes differentially expressed in mouse hepatocarcinoma ascites cell line with low potential of lymphogenous metastasis. METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with low potential of lympho-genous metastasis Hca-P and its synogenetic cell line Hca-F with high metastatic potential was constructed by suppression subtracted hybridization (SSH) method. The screened clones of the subtracted library were sequenced and GenBank homology search was performed. RESULTS: Fifteen differentially expressed cDNA fragments of Hca-P were obtained which revealed 8 known genes, 4 expressed sequence tags (ESTs) and 3 cDNAs showed no homology. CONCLUSION: Tumor metastasis is an incident involving multiple genes. SSH is a useful technique to detect differentially expressed genes and an effective method to clone novel genes.

Animals↗
PubMed1999

Isolation, characterization, molecular cloning and molecular modelling of two lectins of different specificities from bluebell (Scilla campanulata) bulbs.

Two lectins have been isolated from bluebell (Scilla campanulata) bulbs. From their isolation by affinity chromatography, they are characterized as a mannose-binding lectin (SCAman) and a fetuin-binding lectin (SCAfet). SCAman preferentially binds oligosaccharides with alpha(1,3)- and alpha(1,6)-linked mannopyranosides. It is a tetramer of four identical protomers of approx. 13 kDa containing 119 amino acid residues; it is not glycosylated. The fetuin-binding lectin (SCAfet), which is not inhibited by any simple sugars, is also unglycosylated. It is a tetramer of four identical subunits of approx. 28 kDa containing 244 residues. Each 28 kDa subunit is composed of two 14 kDa domains. Both lectins have been cloned from a cDNA library and sequenced. X-ray crystallographic analysis and molecular modelling studies have demonstrated close relationships in sequence and structure between these lectins and other monocot mannose-binding lectins. A refined model of the molecular evolution of the monocot mannose-binding lectins is proposed.

Amino Acid Sequence↗
PubMed1999

CLAR1, a novel gene that exhibits enhanced expression in advanced human prostate cancer.

The molecular events involved in prostate cancer progression are, at present, poorly understood. Using a differential display technique, we identified a cDNA fragment that is present in greater abundance in stage D prostate tumors compared to stage B tumors. Northern analysis was used to confirm that transcripts for this gene are expressed at higher levels in prostate tumors of later pathological stage and higher Gleason grade compared to tumors of earlier stage and lower grade. These transcripts were also expressed at high levels in all four human prostate cancer cell lines, the neonatal prostate cell line FNC 267beta1, and in a variety of other normal human adult and fetal tissues. The cDNA fragment obtained by differential display was used as a probe to clone the full-length cDNA for this gene from a human heart cDNA library. DNA sequence analysis confirmed that the cDNA was novel, and we have named this gene CLAR1. The gene displays two transcripts of 2.6 and 2.0 kb in all tissues examined. CLAR1 maps to chromosome 19q13.3 and appears highly conserved among mammals. The deduced amino acid sequence of CLAR1 encodes a proline-rich protein that contains several SH3-binding domains and a serine phosphorylation site. The presence of these motifs suggests a possible role for CLAR1 in one or more signal transduction pathways. The enhanced expression of this novel gene in more advanced forms of prostate cancer and its potential role in signal transduction both argue that this gene should be further investigated.

Adaptor Proteins, Signal Transducing↗
PubMed1999

[Identification and mapping on human chromosome 19 of chromosome-specific repeated element preferentially binding with nuclear matrix].

From a library of sequences binding preferentially to nuclear matrix (matrix attachment regions, MARs), a fragment of about 300 bp in length (CEA (carcinoembryonic antigen)-MAR) was isolated and characterized. The CEA-MAR sequence was found in more than ten loci of chromosome 19 containing elements similar to genes of the CEA family. No sequences of this group were found on other human chromosomes. Two CEA-MAR-containing loci were sequenced, and sequences for another seven loci were found in GenBank. A comparative analysis of CEA-MARs and the flanking sequences is reported. Based on the sequence of the CEA-containing chromosome 19 loci, a hypothetical model of the domain structure of a 2-Mb chromosome region was constructed and the mutual arrangement of CEA-MARs and genes of CEA family was elucidated. The CEA-MARs were located 5-20 kb downstream of the CEA genes. These results suggest that the duplication unit of the CEA family may coincide with chromatin domains containing these genes.

Base Sequence↗
PubMed2001

Cloning, expression, and complementation test of the RNA lariat debranching enzyme cDNA from mouse.

The RNA lariat debranching enzyme of mouse (mDBR1) was cloned by screening a NIH/3T3 cDNA library. The sequence of full-length mDBR1 cDNA contained a single 515 amino acid open reading frame of 58 kDa protein. Comparison of the amino acid sequence of mDBR1 to other DBR proteins showed 40%, 44%, 43%, 42%, and 80% identity to Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, and human debranching enzymes, respectively. The mDBR1 cDNA was shown to be functional in an interspecies specific complementation experiment, and an in vitro debranching enzyme assay. Mouse DBR1 could complement the intron accumulation phenotype of a S. cerevisiae dbrl null mutant strain. However, the level of complementation depended on the copy number of the mDBR1 cDNA. The integration of the mDBR1 cDNA in the chromosome of S. pombe also complemented both intron accumulation and slow growth phenotypes of the S. pombe dbr1 knock-out mutant strain.

3T3 Cells↗
PubMed2001

In vitro protein display in drug discovery.

Nucleic acid-encoded libraries have been used at different stages of the drug discovery process for the identification of polypeptide ligands and for target identification. Traditionally, phage display screening systems have been used to explore large libraries of peptides and proteins. Lately, novel protein selection technologies have been developed that work entirely in vitro and use the polymerase chain reaction (PCR) rather than cells to amplify genetic material. The simplicity of the linkage between the protein and its encoding nucleic acid leads to several advantages, including the use of larger libraries without the biases of cell-based amplification, greater control over binding conditions and the ease with which PCR-based mutagenesis and recombination can be incorporated. This review focuses on the latest improvements in this new generation of in vitro protein display techniques and discusses their applications to the drug discovery process.

Animals↗
PubMed2001

Molecular cloning and characterization of Mycobacterium bovis BCG pcp gene encoding pyrrolidone carboxyl peptidase.

The Mycobacterium bovis bacilli Calmette-Guerin (BCG) pcp gene that encodes the pyrrolidone carboxyl peptidase (Pcp) was cloned from a lambdagtll genomic library and sequenced. The nucleotide sequence contains a 669 bp open reading frame coding for a protein of 222 amino acid residues with a calculated molecular mass of 23,209 Da. The deduced amino acid sequence is highly homologous to the Pcps from Bacillus amyloliquefaciens, Pseudomonas fluorescens, Bacillus subtilis, Streptococcus pyogenes, and Staphylococcus aureus. A multiple sequence alignment revealed highly conserved domains. The BCG pcp gene was overexpressed in Escherichia coli. The Pcp was purified to homogeneity. The recombinant protein was further confirmed by an enzymatic assay.

Amino Acid Sequence↗
PubMed2002

Identification of metastasis-associated genes in early stage non-small cell lung cancer by subtractive hybridization.

Non-small cell lung cancer (NSCLC) is a leading cause of death and a substantial fraction of patients with surgically resected disease ultimately dies due to distant metastasis. To identify gene expression differences in early stage adenocarcinoma that either did or did not metastasize within a 5-year period, we employed a subtractive hybridization strategy of pooled RNA from primary adenocarcinomas (stage I) of the lung. Individual clones (n=225) of the subtracted cDNA library were sequenced. Further analyses of mRNA expression levels in a cohort of 70 NSCLC patients (stage I to IIIA) showed that the metastasis association of the identified genes was stage and histology specific. Cox regression analyses identified two genes (EIF4A1, MALA1) to be independent prognostic parameters for patients' survival in stage I and II disease. These findings could help to identify early-stage NSCLC patients at high risk for the development of distant metastasis.

Biomarkers, Tumor↗
PubMed2000

Cloning and Preliminary Characterization of Regulatory Domain of Mouse Nestin Gene.

Mouse nestin genomic DNA(23.3 kb)was obtained after genomic library screening. Sequence comparison of the cDNA and genomic DNA revealed three introns in this gene. The positions and length of these introns are conserved among rat and human. The effect of the second intron of mouse nestin gene on the expression of luciferase reporter gene was investigated in vitro. Transient transfection of the second intron and its series deletions of mouse nestin gene showed that the second intron of mouse nestin gene could enhance reporter gene expression and the activity was contributed by a fragment about 800 bp long of its 3' end.

Journal Article↗