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PubMed1994

Identification of an Arabidopsis thaliana gene encoding a plasma membrane H(+)-ATPase whose expression is restricted to anther tissue.

A gene (aha9) for a plasma membrane H(+)-ATPase was isolated from an Arabidopsis thaliana genomic library and sequenced. Comparison of the aha9 predicted amino acid sequence with those of aha1, 2 and 3 and analogous genes from other species indicated the existence of at least two aha gene subfamilies whose divergence precedes that of the Nicotiana and Arabidopsis species. Transcript analysis in various organs revealed expression of aha9 in flower tissues only. Introduction of aha9 into Nicotiana tabacum by genetic transformation gave rise to transgenic plants which also express aha9 in flower tissues. A more detailed analysis showed that aha9 expression was restricted to anther tissues.

Amino Acid Sequence↗

PubMed1995

Genetic diversity of N2-fixing bacteria associated with rice roots by molecular evolutionary analysis of a nifD library.

The rhizosphere of wetland rice has significant N2-fixing activity. It has been suggested that N2 fixation in the rice root zone is associated with the activity of various N2-fixing heterotrophic bacteria that inhabit the rice rhizosphere. Because of the generic diversity, many different isolation media and conditions are required to count and isolate these bacteria. In an attempt to overcome any bias from culture-dependent methods we amplified nifD segments from crude rice root DNA by the polymerase chain reaction. The nifD fragments were then cloned into a pT7 Blue T-vector to construct a nifD library. Sixteen cloned nifD genes chosen at random from the library were sequenced. A comparison with published sequences indicated the presence of seven novel groups of NifD proteins, which implies the existence of at least seven components in the diazotrophic community of rice roots, dominated mainly by proteobacteria. We also observed genetic variability within the clusters, which suggests the coexistence of many closely related bacterial lineages. However, we did not find Azospirillum-like nifD clones, although many reports indicated the widespread presence of Azospirillum spp. Therefore, it remains to be clarified whether Azospirillum species are the widespread N2-fixing bacteria in rice roots.

Amino Acid Sequence↗

PubMed2004

[Construction of combined site-directed random mutation libraries of recombinant human lymphotoxin].

To construct the combined site-directed random mutation library of recombinant human Lymphotoxin (rhLT) for in vitro molecular evolution study, and to study the structure and function relationship. The random point mutations at the sites of 46,106 and 130 were individually generated by overlap PCR amplification with the random nucleotide primers. The three point mutations were combined and cloned into pMD-18T vector to construct the combined mutation library. DNA sequencing was used to evaluate the diversity and randomness of the mutation sites. The combined mutation library was re-engineered, inserted in prokaryotic expression vector pBV220, transformed and expressed in Escherichia coli strain DH5alpha. The biological activity of some of the mutants was tested in 1929 mouse fibroblast cells. As much as 1.5 x 10(5) clones were obtained, which represents 4.5 times of the complete mutation libraries at 99% confidence. Sequencing 50 clones revealed no obvious bias in the nucleotide and amino acid mutations at the sites. Among the 30 expressed samples underwent for the bioassay, 70% (21 samples) were inactive, 23.3% (7 samples) had lower activity than rhLT, the remaining 6.7% (2 samples) had higher activity than rhLT. The combined site-directed random mutation library of rhLT has been constructed successfully. In combination with phase display, the library is ready for in vitro molecular evolution study.

Amino Acid Sequence↗

PubMedDate not supplied

In vitro selection of a ligase ribozyme carrying alkylamino groups in the side chains.

A novel ligase ribozyme was in vitro selected from a random-sequence RNA library including N(6)-aminohexyl-modified adenine residues in place of natural adenine residues. This ribozyme mediated the formation of a phosphodiester bond with a DNA oligonucleotide through condensation with a 5'-triphosphate moiety on the ribozyme. Among the clones isolated from this selection, one was shown to accelerate ligation about 250-fold compared to the original random-sequence RNA library. Almost no rate acceleration was observed when the N(6)-aminohexyl-groups on adenine residues were omitted. Furthermore, ligation was dependent on the presence of a template DNA oligonucleotide that bridged the two strands.

Base Sequence↗

PubMedDate not supplied

Primary structure and characterization of the precursor to human pituitary adenylate cyclase activating polypeptide.

Human cDNAs encoding the precursor to pituitary adenylate cyclase activating polypeptide (PACAP) were cloned from human testis and cerebral cortex cDNA libraries. Nucleotide sequencing revealed that cDNA from the testis library encoded the entire precursor for PACAP, while cDNA from the brain library represented only the carboxy-terminal half of the precursor. The predicted human PACAP precursor consisted of 176 amino acid residues and was very similar to the ovine one (82%). Both human and ovine precursors contained both PACAP and another peptide, PACAP-related peptide (PRP), having 29 amino acids. PACAP and PRP were preceded and followed by paired basic amino acids, recognized as important for post-translational processing. The PACAP precursor resembles the vasoactive intestinal peptide (VIP) precursor, which contains VIP and peptide histidine methionine/isoleucine amide (PHM/PHI). Structurally, PRP had some similarity to PHM/PHI, growth hormone-releasing hormone (GRH) and PACAP. Northern blot analysis indicated that a 3.0-kb transcript was expressed in the ovine hypothalamus. Tissue distribution of PACAP mRNA was also clarified in the rat. Southern blot analysis of human genomic DNA gives single bands with six restriction enzymes, indicating that a single copy of the PACAP gene is contained in a haploid genome. The cDNA for human PACAP precursor was expressed using COS-7 and Chinese hamster ovary (CHO) cells. Immunoreactive PACAP was secreted into the culture media of both transfected cell lines.

Amino Acid Sequence↗

PubMed2005

Characterization of bacterial community structure in rhizosphere soil of grain legumes.

Molecular techniques were used to characterize bacterial community structure, diversity (16S rDNA), and activity (16S rRNA) in rhizospheres of three grain legumes: faba beans (Vicia faba L., cv. Scirocco), peas (Pisum sativum L., cv. Duel) and white lupin (Lupinus albus L., cv. Amiga). All plants were grown in the same soil under controlled conditions in a greenhouse and sampled after fruiting. Amplified 16S rDNA and rRNA products (using universal bacterial primers) were resolved by denaturing gradient gel electrophoresis (DGGE). Distinct profiles were observed for the three legumes with most of the bands derived from RNA being a subset of those derived from DNA. Comparing the total bacterial profiles with actinomycete-specific ones (using actinomycete-specific primers) highlighted the dominance of this group in the three rhizospheres. 16S PCR and RT-PCR products were cloned to construct libraries and 100 clones from each library were sequenced. Actinomycetes and proteobacteria dominated the clone libraries with differences in the groups of proteobacteria. Absence of beta-subdivision members in pea and gamma-subdivision members of proteobacteria in faba bean rhizosphere was observed. Plant-dependent rhizosphere effects were evident from significant differences in the bacterial community structure of the legume rhizospheres under study. The study gives a detailed picture of both residing and "active" bacterial community in the three rhizospheres. The high abundance of actinomycetes in the rhizospheres of mature legumes indicates their possible role in soil enrichment after the legumes are plowed into the soil as biofertilizers.

DNA, Bacterial↗

PubMed1993

SH2 domains recognize specific phosphopeptide sequences.

A phosphopeptide library was used to determine the sequence specificity of the peptide-binding sites of SH2 domains. One group of SH2 domains (Src, Fyn, Lck, Fgr, Abl, Crk, and Nck) preferred sequences with the general motif pTyr-hydrophilic-hydrophilic-Ile/Pro while another group (SH2 domains of p85, phospholipase C-gamma, and SHPTP2) selected the general motif pTyr-hydrophobic-X-hydrophobic. Individual members of these groups selected unique sequences, except the Src subfamily (Src, Fyn, Lck, and Fgr), which all selected the sequence pTyr-Glu-Glu-Ile. The variability in SH2 domain sequences at likely sites of contact provides a structural basis for the phosphopeptide selectivity of these families. Possible in vivo binding sites of the SH2 domains are discussed.

Amino Acid Sequence↗

PubMed2005

Identification of novel low molecular weight CXCR4 antagonists by structural tuning of cyclic tetrapeptide scaffolds.

A highly potent CXCR4 antagonist, compound 2, was previously found by using two orthogonal cyclic pentapeptide libraries involving conformation-based and sequence-based libraries based on the pharmacophore of a 14-mer peptidic antagonist, 1. Herein, cyclic tetrapeptides derived from replacements of the dipeptide unit (Nal-Gly) with a gamma-amino acid and pseudopeptides cyclized by disulfide and olefin bridges were synthesized to find novel scaffold structures different from that of cyclic pentapeptides. These compounds contain a reduced number of peptide bonds compared to compound 2. Furthermore, several analogues with chemical modification of the side chain of Arg(4) in 2 were also prepared. From these, several new leads possessing high to moderate CXCR4-antagonistic activity were characterized.

Alkenes↗

PubMed2003

Gene trap mutagenesis of hnRNP A2/B1: a cryptic 3' splice site in the neomycin resistance gene allows continued expression of the disrupted cellular gene.

BACKGROUND: Tagged sequence mutagenesis is a process for constructing libraries of sequenced insertion mutations in embryonic stem cells that can be transmitted into the mouse germline. To better predict the functional consequences of gene entrapment on cellular gene expression, the present study characterized the effects of a U3Neo gene trap retrovirus inserted into an intron of the hnRNP A2/B1 gene. The mutation was selected for analysis because it occurred in a highly expressed gene and yet did not produce obvious phenotypes following germline transmission. RESULTS: Sequences flanking the integrated gene trap vector in 1B4 cells were used to isolate a full-length cDNA whose predicted amino acid sequence is identical to the human A2 protein at all but one of 341 amino acid residues. hnRNP A2/B1 transcripts extending into the provirus utilize a cryptic 3' splice site located 28 nucleotides downstream of the neomycin phosphotransferase start codon. The inserted Neo sequence and proviral poly(A) site function as an 3' terminal exon that is utilized to produce hnRNP A2/B1-Neo fusion transcripts, or skipped to produce wild-type hnRNP A2/B1 transcripts. This results in only a modest disruption of hnRNPA2/B1 gene expression. CONCLUSIONS: Expression of the occupied hnRNP A2/B1 gene and utilization of the viral poly(A) site are consistent with an exon definition model of pre-mRNA splicing. These results reveal a mechanism by which U3 gene trap vectors can be expressed without disrupting cellular gene expression, thus suggesting ways to improve these vectors for gene trap mutagenesis.

3' Untranslated Regions↗

PubMed2001

Construction and characterization of a bacterial artificial chromosome library from chili pepper.

A library of the bacterial artificial chromosome (BAC) that consisted of a total of 78,336 clones with an average insert size of 80 kb was constructed from Capsicum annuum, 'CM334', which is resistant to Phytophthora capsici and PVY. Based on a haploid genome size of pepper of 2,702 Mbp/C, the BAC library was estimated to contain approximately three genome equivalents and represented at least 90% of the pepper genome. In order to determine the percentage of BAC clones that contained mitochondrial DNAs, the entire library was screened with probes of chili pepper mitochondrial DNAs. The result showed that only twenty-five clones, which is 0.03% of the total BAC clones, were hybridized to mitochondrial gene probes. This indicates that the library is comprised predominantly of the nuclear sequences. The library was also tested for isolating specific clones by screening with a few known genes from the chili pepper, phytoene synthase gene, and two MADS genes--HpMADS1 and HpMADS3. The result showed that the three clones for phytoene synthase and the two clones for each MADS gene were positively hybridized to the specific probes. This indicates that the library is highly reliable and represents a resource for initiating map-based cloning and contig mapping in chili pepper.

Alkyl and Aryl Transferases↗

PubMed2000

Comparison of the oral bacterial flora in saliva from a healthy subject and two periodontitis patients by sequence analysis of 16S rDNA libraries.

The oral bacterial flora in the saliva from two patients with periodontitis and from a periodontally healthy subject were compared using a sequence analysis of 16S rDNA libraries without cultivation. 16S rDNAs were amplified from salivary DNA by PCR and cloned. Randomly selected clones were partially sequenced. On the basis of sequence similarities, the clones were classified into several clusters corresponding to the major phylum of the domain Bacteria. The major phylum in the libraries was the low G+C Gram-positive bacteria. There was no clonal sequence affiliated with periodontopathic bacteria in the salivary sample from the healthy subject, while a number of periodontal pathogens such as Campylobacter rectus, Prevotella intermedia, Porphyromonas gingivalis and Treponema socranskii were detected in the salivary samples from the patients with periodontitis. In addition, a number of previously uncharacterized and uncultured microorganisms were recognized. These organisms may have some role in periodontal disease. This study reveals some potential for a molecular-biological technique to analyze the oral microflora associated with periodontal disease, including previously uncharacterized and uncultured microorganisms, without cultivation.

Adult↗

PubMed2003

Transcriptional program of the endocrine pancreas in mice and humans.

The Endocrine Pancreas Consortium was formed in late 1999 to derive and sequence cDNA libraries enriched for rare transcripts expressed in the mammalian endocrine pancreas. Over the past 3 years, the Consortium has generated 20 cDNA libraries from mouse and human pancreatic tissues and deposited >150,000 sequences into the public expressed sequence tag databases. A special effort was made to enrich for cDNAs from the endocrine pancreas by constructing libraries from isolated islets. In addition, we constructed a library in which fetal pancreas from Neurogenin 3 null mice, which consists of only exocrine and duct cells, was subtracted from fetal wild-type pancreas to enrich for the transcripts from the endocrine compartment. Sequence analysis showed that these clones cluster into 9,464 assembly groups (approximating unique transcripts) for the mouse and 13,910 for the human sequences. Of these, >4,300 were unique to Consortium libraries. We have assembled a core clone set containing one cDNA for each assembly group for the mouse and have constructed the corresponding microarray, termed "PancChip 4.0," which contains >9,000 nonredundant elements. We show that this PancChip is highly enriched for genes expressed in the endocrine pancreas. The mouse and human clone sets and corresponding arrays will be important resources for diabetes research.

Animals↗

PubMed2002

Selection of v-abl tyrosine kinase substrate sequences from randomized peptide and cellular proteomic libraries using mRNA display.

Methodologies for rapidly identifying cellular protein interactions resulting in posttranslational modification of one of the partners are lacking. Here, we select for substrates of the v-abl tyrosine kinase from two protein display libraries in which the protein is covalently linked to its encoding mRNA. Successive selection cycles from a randomized peptide library identified a consensus sequence closely matching that previously reported for the v-abl tyrosine kinase. Selections from a proteomic library derived from cellular mRNA identified several novel targets of v-abl, including a new member of a class of SH2 domain-containing adaptor proteins. Upon modification, several of the substrates obtained in these selections were found to be effective inhibitors of v-abl kinase activity in vitro. These experiments establish a novel method for identifying the substrates of tyrosine kinases from synthetic and cellular protein libraries.

Amino Acid Sequence↗

PubMed1999

Complexity and expression of the glutamine synthetase multigene family in the amphidiploid crop Brassica napus.

In the amphidiploid genome of oilseed rape (Brassica napus) the diploid ancestral genomes of B. campestris and B. oleracea have been merged. As a result of this crossing event, all gene loci, gene families, or multigene families of the A and C genome types encoding a certain protein are now combined in one plant genome. In the case of the multigene family for glutamine synthetase, the key enzyme of nitrogen assimilation, six different cDNA sequences were isolated from leaf and root specific libraries. One sequence pair (BnGSL1/BnGSL2) was characterized by the presence of amino-terminal transit peptides, a typical feature of all nuclear encoded chloroplast proteins. Two other cDNA pairs (BnGSR1-1/BnGSR1-2 and BnGSR2-1/BnGSR2-2) with very high homology between each other were found in a root specific cDNA library and represent protein subunits for cytosolic glutamine synthetase isoforms. Comparative PCR amplifications of genomic DNA isolated from B. napus, B. campestris and B. oleracea followed by sequence-specific restriction analyses of the PCR products permitted the assignment of the cDNA sequences to either the A genome type (BnGSL1/BnGSR1-1/BnGSR2-1) or the C genome type (BnGSL2/BnGSR1-2/BnGSR2-2). Consequently, the ancestral GS genes of B. campestris and B. oleracea are expressed simultaneously in oilseed rape. This result was also confirmed by RFLP (restriction fragment length polymorphism) analysis of RT-PCR products. In addition, the different GS genes showed tissue specific expression patterns which are correlated with the state of development of the plant material. Especially for the GS genes encoding the cytosolic GS isoform BnGSR2, a marked increase of expression could be observed after the onset of leaf senescence.

Blotting, Northern↗

PubMed1998

Fast-painting of human metaphase spreads using a chromosome-specific, repeat-depleted DNA library probe.

For chromosome painting, in situ suppression of repetitive DNA sequences has been well established. Such standard protocols usually require large amounts of Cot-I DNA. Recently, it has become possible to deplete repetitive DNA sequences from library probes by magnetic purification and PCR-assisted affinity chromatography. These "repeat-depleted library probes" appear to be extremely useful for Fast-FISH, a technique that omits denaturing chemical agents such as formamide in the hybridization buffer, resulting in a substantial acceleration and simplification of the complete protocol. Shown here is the application of Fast-FISH to a repeat-depleted, directly fluorochrome-labeled library probe of the q-arm of chromosome 15 (Fast-Painting) for human lymphocyte metaphase spreads. Following painting without Cot-I DNA and without formamide, visual inspection revealed sufficient chromosome painting after a few hours of hybridization. The fluorescence signals of the labeling sites were analyzed after hybridization times of 1 and 2 h (in one case, 4 h) using digital fluorescence microscopy. The painting efficiency expressed in values of relative fluorescence signal ratios was quantitatively evaluated by image analysis using line-scan procedures and area-morphometry of mean luminance. Two preparation protocols (ethanol dehydration without and with RNase A treatment followed by pepsin digestion for four different exposure times) were compared. These results indicated that RNase A treatment and pepsin digestion are steps that can be omitted.

Adult↗

PubMed2002

ASTRAL compendium enhancements.

The ASTRAL compendium provides several databases and tools to aid in the analysis of protein structures, particularly through the use of their sequences. It is partially derived from the SCOP database of protein domains, and it includes sequences for each domain as well as other resources useful for studying these sequences and domain structures. Several major improvements have been made to the ASTRAL compendium since its initial release 2 years ago. The number of protein domain sequences included has doubled from 15 190 to 30 867, and additional databases have been added. The Rapid Access Format (RAF) database contains manually curated mappings linking the biological amino acid sequences described in the SEQRES records of PDB entries to the amino acid sequences structurally observed (provided in the ATOM records) in a format designed for rapid access by automated tools. This information is used to derive sequences for protein domains in the SCOP database. In cases where a SCOP domain spans several protein chains, all of which can be traced back to a single genetic source, a 'genetic domain' sequence is created by concatenating the sequences of each chain in the order found in the original gene sequence. Both the original-style library of SCOP sequences and a new library including genetic domain sequences are available. Selected representative subsets of each of these libraries, based on multiple criteria and degrees of similarity, are also included. ASTRAL may be accessed at http://astral.stanford.edu/.

Amino Acid Sequence↗

PubMed1996

DNA sequencing by capillary electrophoresis using short oligonucleotide primer libraries.

Two strategies for DNA sequencing by primer walking using short oligonucleotide primer libraries have been successfully employed along with capillary electrophoresis using replaceable polymer solutions of linear polyacrylamide and fluorescence detection. A 3.5-kb stretch of the single-stranded M13mp18 template was sequenced with T7 PRISM dye-terminator/Sequenase chemistry. An in-house base-calling program offered read lengths of roughly 450 bases with an average of 97.8% accuracy.

Base Sequence↗

PubMed2001

Abundance, distribution, and transcriptional activity of repetitive elements in the maize genome.

Long terminal repeat (LTR) retrotransposons have been shown to make up much of the maize genome. Although these elements are known to be prevalent in plant genomes of a middle-to-large size, little information is available on the relative proportions composed by specific families of elements in a single genome. We sequenced a library of randomly sheared genomic DNA from maize to characterize this genome. BLAST analysis of these sequences demonstrated that the maize genome is composed of diverse sequences that represent numerous families of retrotransposons. The largest families contain the previously described elements Huck, Ji, and Opie. Approximately 5% of the sequences are predicted to encode proteins. The genomic abundance of 16 families of elements was estimated by hybridization to an array of 10,752 maize bacterial artificial chromosome (BAC) clones. Comparisons of the number of elements present on individual BACs indicated that retrotransposons are in general randomly distributed across the maize genome. A second library was constructed that was selected to contain sequences hypomethylated in the maize genome. Sequence analysis of this library indicated that retroelements abundant in the genome are poorly represented in hypomethylated regions. Fifty-six retroelement sequences corresponding to the integrase and reverse transcriptase domains were isolated from approximately 407,000 maize expressed sequence tags (ESTs). Phylogenetic analysis of these and the genomic retroelement sequences indicated that elements most abundant in the genome are less abundant at the transcript level than are more rare retrotransposons. Additional phylogenies also demonstrated that rice and maize retrotransposon families are frequently more closely related to each other than to families within the same species. An analysis of the GC content of the maize genomic library and that of maize ESTs did not support recently published data that the gene space in maize is found within a narrow GC range, but does indicate that genic sequences have a higher GC content than intergenic sequences (52% vs. 47% GC).

Chromosomes, Artificial, Bacterial↗