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PubMed1995

Mapping sites of interaction of p47-phox and flavocytochrome b with random-sequence peptide phage display libraries.

During assembly of the phagocyte NADPH oxidase, cytosolic p47-phox translocates to the plasma membrane and binds to flavocytochrome b, and binding domains for p47-phox have been identified on the C-terminal tails of both flavocytochrome b subunits. In the present report, we further examine the interaction of these two oxidase components by using random-sequence peptide phage display library analysis. Screening p47-phox with the peptide libraries identified five potential sites of interaction with flavocytochrome b, including three previously reported regions of interaction and two additional regions of interaction of p47-phox with gp91-phox and p22-phox. The additional sites were mapped to a domain on the first predicted cytosolic loop of gp91-phox encompassing residues S86TRVRRQL93 and to a domain near the cytosolic C-terminal tail of gp91-phox encompassing residues F450EWFADLL457. The mapping also confirmed a previously reported binding domain on gp91-phox (E554SGPRGVHFIF564) and putative Src homology 3 domain binding sites on p22-phox (P156PRPP160 and G177GPPGGP183). To demonstrate that the additional regions identified were biologically significant, peptides mimicking the gp91-phox sequences F77LRGSSACCSTRVRRQL93 and E451WFADLLQLLESQ463 were synthesized and assayed for their ability to inhibit NADPH oxidase activity. These peptides had EC50 values of 1 microM and 230 microM, respectively, and inhibited activation when added prior to assembly but did not affect activity of the preassembled oxidase. Our data demonstrate the usefulness of phage display library analysis for the identification of biologically relevant sites of protein-protein interaction and show that the binding of p47-phox to flavocytochrome b involves multiple binding sites along the C-terminal tails of both gp91- and p22-phox and other regions of gp91-phox nearer to the N terminus.

Binding Sites↗

PubMed2003

Differential representation of sunflower ESTs in enriched organ-specific cDNA libraries in a small scale sequencing project.

BACKGROUND: Subtractive hybridization methods are valuable tools for identifying differentially regulated genes in a given tissue avoiding redundant sequencing of clones representing the same expressed genes, maximizing detection of low abundant transcripts and thus, affecting the efficiency and cost effectiveness of small scale cDNA sequencing projects aimed to the specific identification of useful genes for breeding purposes. The objective of this work is to evaluate alternative strategies to high-throughput sequencing projects for the identification of novel genes differentially expressed in sunflower as a source of organ-specific genetic markers that can be functionally associated to important traits. RESULTS: Differential organ-specific ESTs were generated from leaf, stem, root and flower bud at two developmental stages (R1 and R4). The use of different sources of RNA as tester and driver cDNA for the construction of differential libraries was evaluated as a tool for detection of rare or low abundant transcripts. Organ-specificity ranged from 75 to 100% of non-redundant sequences in the different cDNA libraries. Sequence redundancy varied according to the target and driver cDNA used in each case. The R4 flower cDNA library was the less redundant library with 62% of unique sequences. Out of a total of 919 sequences that were edited and annotated, 318 were non-redundant sequences. Comparison against sequences in public databases showed that 60% of non-redundant sequences showed significant similarity to known sequences. The number of predicted novel genes varied among the different cDNA libraries, ranging from 56% in the R4 flower to 16 % in the R1 flower bud library. Comparison with sunflower ESTs on public databases showed that 197 of non-redundant sequences (60%) did not exhibit significant similarity to previously reported sunflower ESTs. This approach helped to successfully isolate a significant number of new reported sequences putatively related to responses to important agronomic traits and key regulatory and physiological genes. CONCLUSIONS: The application of suppressed subtracted hybridization technology not only enabled the cost effective isolation of differentially expressed sequences but it also allowed the identification of novel sequences in sunflower from a relative small number of analyzed sequences when compared to major sequencing projects.

DNA, Complementary↗

PubMed1989

A strategy for high-volume sequencing of cosmid DNAs: random and directed priming with a library of oligonucleotides.

Direct sequencing of cosmid DNAs using a library of oligonucleotide primers of length 8, 9, or 10 is proposed. The statistics of priming indicate that a primer library sufficient for determining the sequence of the entire human genome (100,000 cosmids) would be small enough to be assembled and managed. Such a library would greatly reduce the cost and effort of high-volume sequencing: primers would be instantly available; the sequence of each cosmid DNA could be determined from a single DNA preparation without the necessity for mapping or subcloning; and, because each primer would be used repeatedly, the cost of primers would become a negligible fraction of other costs. A combination of random and directed priming could determine the sequence of a cosmid DNA in 1.2-1.5 times the minimum number of sequencing reactions required, and completely directed priming would be even more efficient. The success of this strategy requires that a considerable fraction of octamers, nonamers, or decamers be able to prime selectively in double-stranded DNAs 45,000 base pairs (bp) long; initial results indicate that this is likely to be the case. The strategy is not limited to cloned DNAs and would be useful for rapid identification and direct sequencing of viral nucleic acids.

Base Sequence↗

PubMed1996

Cloning, sequencing, and characterization of LDH-C4 from a fox testis cDNA library.

A full-length cDNA encoding the sperm-specific enzyme lactate dehydrogenase-C4 was isolated from a fox testis cDNA expression library and sequenced. The deduced translated protein sequence was shown to be 86% identical to that of human LDH-C4. In the fox testis, mRNA encoding LDH-C4 was first detected in pachytene spermatocytes. The LDH-C4 protein monomer was identified in Western blots of sperm membrane extracts as having a molecular weight of approximately 35,000, consistent with the monomeric size of this subunit previously identified in sperm from other species. The LDH-C4 protein is localized on the sperm plasma membrane overlying the principal piece of the tail. Based on the available sequence data, we were able to identify an epitope within the N-terminal region of the LDH-C4 amino-acid sequence which when administered to female foxes is antigenic and produces antibodies capable of recognizing the native protein.

Amino Acid Sequence↗

PubMed1998

The human homologue of the Drosophila tailless gene (TLX): characterization and mapping to a region of common deletion in human lymphoid leukemia on chromosome 6q21.

Deletion of the long arm of chromosome 6 (6q) is one of the most common chromosomal abnormalities in human hematological malignancies. Two distinct regions of minimal deletion have been identified by loss of heterozygosity studies at 6q25 to 6q27 (RMD-1) and at 6q21 to 6q23 (RMD-2), suggesting the presence of one or more tumor suppressor genes. We have cloned sequences within RMD-2 and screened for novel genes using a combination of direct sequencing, cDNA library screening, and exon trapping. Sequences generated from a cosmid fragment, mapping within RMD-2, showed homology to the Drosophila tailless gene (tll). The human homologue of the Drosophila tailless gene (human tlx; MGMW-approved symbol, TLX) was subsequently cloned from a fetal brain cDNA library. The gene is a member of the steroid nuclear receptor superfamily and is homologous to tll genes from other species that are involved in brain development. TLX is predominately expressed in the brain and maps to RMD-2 at 6q21 between DNA markers FYN and D6S447, in a YAC clone that also contains marker D6S246. The contributions of this gene to human B-cell leukemia and to brain development are unknown at present.

Amino Acid Sequence↗

PubMed2001

Phylogenetic diversity of bacterial and archaeal communities in the anoxic zone of the Cariaco Basin.

Microbial community samples were collected from the anoxic zone of the Cariaco Basin at depths of 320, 500, and 1,310 m on a November 1996 cruise and were used to construct 16S ribosomal DNA libraries. Of 60 nonchimeric sequences in the 320-m library, 56 belonged to the epsilon subdivision of the Proteobacteria (epsilon-Proteobacteria) and 53 were closely related to ectosymbionts of Rimicaris exoculata and Alvinella pompejana, which are referred to here as epsilon symbiont relatives (ESR). The 500-m library contained sequences affiliated with the fibrobacteria, the Flexibacter-Cytophaga-Bacteroides division, the division Verrucomicrobia, the division Proteobacteria, and the OP3 candidate division. The Proteobacteria included members of the gamma, delta, epsilon and new candidate subdivisions, and gamma-proteobacterial sequences were dominant (25.6%) among the proteobacterial sequences. As in the 320-m library, the majority of the epsilon-proteobacteria belonged to the ESR group. The genus Fibrobacter and its relatives were the second largest group in the library (23.6%), followed by the delta-proteobacteria and the epsilon-proteobacteria. The 1,310-m library had the greatest diversity; 59 nonchimeric clones in the library contained 30 unique sequences belonging to the planctomycetes, the fibrobacteria, the Flexibacter-Cytophaga-Bacteroides division, the Proteobacteria, and the OP3 and OP8 candidate divisions. The proteobacteria included members of new candidate subdivisions and the beta, gamma, delta, and epsilon-subdivisions. ESR sequences were still present in the 1,310-m library but in a much lower proportion (8.5%). One archaeal sequence was present in the 500-m library (2% of all microorganisms in the library), and eight archaeal sequences were present in the 1,310-m library (13.6%). All archaeal sequences fell into two groups; two clones in the 1,310-m library belonged to the kingdom Crenarchaeota and the remaining sequences in both libraries belonged to the kingdom Euryarchaeota. The latter group appears to be related to the Eel-TA1f2 sequence, which belongs to an archaeon suggested to be able to oxidize methane anaerobically. Based on phylogenetic inferences and measurements of dark CO(2) fixation, we hypothesized that (i) the ESR are autotrophic anaerobic sulfide oxidizers, (ii) sulfate reduction and fermentative metabolism may be carried out by a large number of bacteria in the 500- and 1,310-m libraries, and (iii) members of the Euryarchaeota found in relatively large numbers in the 1,310-m library may be involved in anaerobic methane oxidation. Overall, the composition of microbial communities from the Cariaco Basin resembles the compositions of communities from several anaerobic sediments, supporting the hypothesis that the Cariaco Basin water column is similar to anaerobic sediments.

Anaerobiosis↗

PubMed1997

Molecular cloning and characterisation of a putative aspartate proteinase associated with a gut membrane protein complex from adult Haemonchus contortus.

A cDNA was isolated from an adult Haemonchus contortus cDNA expression library the deduced amino acid sequence of which showed significant homology to mammalian pepsinogen sequences. The library was screened with antisera raised against Haemonchus galactose-containing glycoprotein complex, a gut membrane protein complex with aspartyl proteinase activity which has shown considerable potential as a protective antigen. The amino acid sequence obtained corresponded very closely in part to the N-terminal amino acid sequences of two polypetides within the complex. The enzyme was shown to be almost exclusively expressed by the blood-feeding parasite stages. The cDNA was expressed in E. coli, and antibody produced to the recombinant protein bound to the luminal surface of the gut in the adult parasite. The proteinase may play a central role in digesting the blood meal and is considered a potential sub-unit vaccine candidate.

Amino Acid Sequence↗

PubMed2002

Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites.

The development of a set of synthetic mammalian promoters with different specific activities is described. The library is based on a synthetic promoter, JeT, constructed as a 200 bp chimeric promoter built from fragments of the viral SV40 early promoter and the human beta-actin and ubiquitin C promoters. The JeT promoter was made by separating the included consensus boxes by the same distances in base pairs as found in the wild-type promoters, thus preserving transcription factor interaction. The resulting promoter was shown to drive reporter expression to high levels in enhanced green fluorescent protein and secreted alkaline phosphatase reporter assays. By replacing sequences separating the transcription factor binding sites with randomized sequences of the same length, sets of new promoters with different strengths, spanning a 10-fold range of transcriptional activity in cell culture, was obtained. The measured activity of each promoter in the library was highly specific and reproducible when tested in HiB5 and ARPE-19 cell culture.

Actins↗

PubMed1999

The SBASE protein domain library, release 6.0: a collection of annotated protein sequence segments.

The sixth release of the SBASE protein domain library sequences contains 130 703 annotated and crossreferenced entries corresponding to structural, functional, ligand-binding and topogenic segments of proteins. The entries were grouped based on standard names (2312 groups) and futher classified on the basis of the BLAST similarity (2463 clusters). Automated searching with BLAST and a new sequence-plot representation of local domain similarities are available at the WWW-server http://www.icgeb.trieste.it/sbase. A mirror site is at http://sbase.abc.hu/sbase. The database is freely available by anonymous 'ftp' file transfer from ftp.icgeb.trieste.it

Amino Acid Sequence↗

PubMed2005

Steady-state transposon mutagenesis in inbred maize.

We implement a novel strategy for harnessing the power of high-copy transposons for functional analysis of the maize genome, and report behavioral features of the Mutator system in a uniform inbred background. The unique UniformMu population and database facilitate high-throughput molecular analysis of Mu-tagged mutants and gene knockouts. Key features of the population include: (i) high mutation frequencies (7% independent seed mutations) and moderation of copy number (approximately 57 total Mu elements; 1-2 MuDR copies per plant) were maintained by continuous back-crossing into a phenotypically uniform inbred background; (ii) a bz1-mum9 marker enabled selection of stable lines (loss of MuDR), inhibiting further transpositions in lines selected for molecular analysis; (iii) build-up of mutation load was prevented by screening Mu-active parents to exclude plants carrying pre-existing seed mutations. To create a database of genomic sequences flanking Mu insertions, selected mutant lines were analyzed by sequencing of MuTAIL PCR clone libraries. These sequences were annotated and clustered to facilitate bioinformatic subtraction of ancestral elements and identification of insertions unique to mutant lines. New insertions targeted low-copy, gene-rich sequences, and in silico mapping revealed a random distribution of insertions over the genome. Our results indicate that Mu populations differ markedly in the occurrence of Mu insertion hotspots and the frequency of suppressible mutations. We suggest that controlled MuDR copy number in UniformMu lines is a key determinant of these differences. The public database (http://uniformmu.org; http://endosperm.info) includes pedigree and phenotypic data for over 2000 independent seed mutants selected from a population of 31 548 F2 lines and integrated with analyses of 34 255 MuTAIL sequences.

Breeding↗

PubMed1991

Cloning and direct sequencing from lambda cDNA libraries using the polymerase chain reaction: suppressin and the vasopressin receptor as models.

A strategy using the polymerase chain reaction (PCR) to screen a lambda gt11 pituitary cDNA library for cDNAs encoding suppressin, a putative anti-proliferative protein, and a putative vasopressin receptor is described. The use of this technique will facilitate the demonstration of e.g. the presence of "neuropeptide receptors" on cells of the lymphoid system, confirming the concept of "shared ligands and receptors" by the neuroendocrine and the immune system. Neither of the genes encoding the proteins of the present study have previously been cloned. The PCR-screening procedure requires sequence information from the gene of interest which permits the generation of complementary primers. These primers are then used in combination with lambda phage primers complementary to regions flanking the cloning site in a PCR to amplify cDNAs derived from the gene of interest. This novel screening procedure yields cDNA related to the gene of interest, including the largest clone present in the library. To confirm the utility of this technique for cDNA libraries, the library was also screened using traditional cDNA hybridization techniques. The largest clone obtained by screening the cDNA library with PCR was the same as that obtained by the conventional technique. Thus, the results of these studies show that the PCR method can be used instead of more conventional means to screen cDNA libraries. Lastly, we describe a protocol for directly sequencing PCR-amplified DNA using the same primers that are used for amplification. The combined use of these two strategies permits cloning and sequencing of cDNAs from lambda cDNA libraries in a fraction of the time required using traditional screening techniques, but with identical results.

Animals↗

PubMed2004

PCR-based screening BAC library and direct end sequencing of BAC clones.

A PCR based strategy was developed which required four steps to identify positive BAC clones from barley BAC (bacterial artificial chromosome) library. In the protocol, two levels of BAC DNA pools (super-pool and pool) were prepared for analysis. One pool is made of one plate DNAs and one super-pool is made of mixing ten consecutive 1/100 diluted pool DNAs (1 approximately 10, 11 approximately 20 ect). First,super-pool DNAs were analysed and then 10 pool DNAs contained in every positive super-pool were analysed. Once positive BAC plates were identified,the bacterial cultures were dipped into PCR mixtures and reaction is made to identify positive BAC clones. The BAC clones identified by each marker were grouped into contigs. BAC-end sequence was obtained from BACs within each contig and primers were designed for the next step chromosome walking. In case of the BAC ends belong to repetitive sequence, the primers were designed based on the subcloned unique band in the contig (identified by Hind III digestion pattern). This method allows us to construct the BAC contig without the costly and time-consuming efforts, and no radioactivity harmful to the body.

Chromosomes, Artificial, Bacterial↗

PubMed1984

The sup-7(st5) X gene of Caenorhabditis elegans encodes a tRNATrpUAG amber suppressor.

In earlier studies, we identified in Caenorhabditis elegans two informational suppressors sup-5 III and sup-7 X and recently showed that these suppressors acted via an altered tRNA to suppress translational termination at amber (UAG) stop codons. We now show that the sup-7 (st5) suppressor is a tRNATrpUAG amber suppressor. These studies utilized a radiolabeled purified tRNA fraction to identify hybridizing genomic sequences in a phage genomic library. DNA sequence analysis of the hybridizing segment of one clone showed that the probe recognized a tRNATrpUGG sequence. The sup-7 gene was shown to be one of an 11 or 12 member tRNATrp family by Southern blot analysis, taking advantage of an Xba I restriction site induced in the anticodon sequence by the mutational event to suppressor. Sequence analysis of a recombinant lambda clone containing sup-7 gene proved that sup-7(st5) is a tRNATrpUAG. This conclusive proof of the nature of sup7(st5) will permit unambiguous interpretation in genetic applications, and the availability of the cloned sequences may allow the sup-7 gene to be used to select for the reintroduction of DNA into C. elegans.

Animals↗

PubMed1987

A novel human nonviral retroposon derived from an endogenous retrovirus.

In a human genome, we found dispersed repetitive sequences homologous to part of a human endogenous retrovirus termed HERV-K which resembled mouse mammary tumor virus. For elucidation of their structure and organization, we cloned some of these sequences from a human gene library. The sequence common to the cloned DNA was ca. 630 base-pairs (bp) in length with an A-rich tail at the 3' end and was found to be a SINE (short interspersed repeated sequence) type nonviral retroposon. In this retroposon, the 5' end had multiple copies of a 40 bp direct repeat very rich in GC content and about the next 510 nucleotides were homologous to the 3' long terminal repeat and its upstream flanking region of the HERV-K genome. This retroposon was thus given the name, SINE-R element since most of it derived from a retrovirus. SINE-R elements were present at 4,000 to 5,000 copies per haploid human genome. The nucleotide sequence was ca. 90% homologous among the cloned elements.

Base Sequence↗

PubMed2001

A novel technique for the identification of CpG islands exhibiting altered methylation patterns (ICEAMP).

Aberrant CpG methylation changes occurring during tumour progression include the loss (hypomethylation) and gain (hypermethylation) of methyl groups. Techniques currently available for examining such changes either require selection of a region, then examination of methylation changes, or utilise methylation-sensitive restriction enzymes to identify an alteration. We describe here a novel method that identifies genomic regions as a consequence of altered methylation during tumourigenesis. A methyl-CpG binding domain column isolates methylated GC-rich sequences from both tumours and surrounding normal tissue. Subsequent subtractive hybridisation removes sequences common to both, leaving only methylated sequences unique to the tumour. Libraries of sequences generated using DNA derived from a breast tumour (histological grade; poorly differentiated) as 'tester' and from matched normal tissue as 'driver' were examined; 26% of clones had the sequence criteria of a CpG island (CGI). Analysis using the bisulfite technique revealed that a number of these sequences were methylated in tumour DNA relative to the normal control. We have therefore demonstrated the ability of this technique, the identification of CGI exhibiting altered methylation patterns (ICEAMP), to isolate tumour-specific methylated GC-rich sequences. This will allow a comprehensive identification of methylation changes during tumourigenesis and will lead to a better understanding of the processes involved.

Base Composition↗

PubMed2004

MEROPS: the peptidase database.

Peptidases (proteolytic enzymes) are of great relevance to biology, medicine and biotechnology. This practical importance creates a need for an integrated source of information about them, and also about their natural inhibitors. The MEROPS database (http://merops.sanger.ac.uk) aims to fill this need. The organizational principle of the database is a hierarchical classification in which homologous sets of the proteins of interest are grouped in families and the homologous families are grouped in clans. Each peptidase, family and clan has a unique identifier. The database has recently been expanded to include the protein inhibitors of peptidases, and these are classified in much the same way as the peptidases. Forms of information recently added include new links to other databases, summary alignments for peptidase clans, displays to show the distribution of peptidases and inhibitors among organisms, substrate cleavage sites and indexes for expressed sequence tag libraries containing peptidases. A new way of making hyperlinks to the database has been devised and a BlastP search of our library of peptidase and inhibitor sequences has been added.

Amino Acid Sequence↗

PubMed2003

Community structure of ammonia-oxidizing bacteria within anoxic marine sediments.

The potential for oxidation of ammonia in anoxic marine sediments exists through anaerobic oxidation by Nitrosomonas-like organisms, utilizing nitrogen dioxide, coupling of nitrification, manganese reduction, and anaerobic oxidation of ammonium by planctomycetes (the Anammox process). Here we describe the presence of microbial communities with the potential to carry out these processes in a natural marine sediment system (Loch Duich, Scotland). Natural microbial communities of Planctomycetales-Verrucomicrobia and beta- and gamma-proteobacterial ammonia-oxidizing bacteria were characterized by analysis of 16S rRNA genes amplified using group-specific primers by PCR- and reverse transcription-PCR amplification of 16S rDNA and RNA, respectively. Amplification products were analyzed by sequencing of clones and by denaturant gradient gel electrophoresis (DGGE). Amplification of primers specific for Planctomycetales-Verrucomicrobia and beta-proteobacterial ammonia-oxidizing bacteria generated products at all sampling sites and depths, but no product was generated using primers specific for gamma-proteobacterial ammonia-oxidizing bacteria. 16S rDNA DGGE banding patterns indicated complex communities of beta-proteobacterial ammonia-oxidizing bacteria in anoxic marine sediments. Phylogenetic analysis of sequences from clones and those excised from DGGE gels suggests dominance of Nitrosospira cluster 1-like organisms and of strains belonging to a novel cluster represented in dominant bands in 16S rRNA DGGE banding patterns. Their presence indicates a group of organisms closely related to recognized beta-proteobacterial ammonia-oxidizing bacteria that may be selected in anoxic environments and may be capable of anoxic ammonia oxidation. Sequence analysis of planctomycete clone libraries and sequences excised from DGGE gels also demonstrated a diverse microbial community and suggested the presence of new subdivisions, but no sequence related to recognized Anammox organisms was detected.

Ammonia↗

PubMed2004

Topochemical exploration of potent compounds using retro-enantiomer libraries of cyclic pentapeptides.

Cyclic pentapeptides have been adopted as conformationally restricted peptide templates to dispose pharmacophores of bioactive peptides. In our recent study, use of two orthogonal cyclic pentapeptide libraries involving conformation-based and sequence-based libraries containing critical residues of a bioactive peptide led to the discovery of potent downsized peptides that possess activity comparable to that of the parent peptide. The present study demonstrates that a third library consisting of retro-enantiomers (retro-inverso peptides) that possess not only all residues with the opposite configuration to those in the corresponding original peptide but also amino acid sequences with reversed arrangement, is important as an alternative library for rationally finding active compounds.

Amino Acid Sequence↗