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PubMed2002

Mouse testin: complementary DNA cloning, genomic organization, and characterization of its proximal promoter region.

Testin is a secretory protein that was initially identified from rat Sertoli cell-enriched cultures and has been suggested to be a sensitive marker to monitor the integrity of Sertoli-germ cell junctions. However, the expression of the testin gene in other species and the molecular mechanisms that govern its transcription are unknown. To address these issues, we cloned and characterized the mouse testin gene. A full-length mouse testin cDNA encoding a polypeptide of 333 amino acid residues was isolated by library screening. Sequence analysis revealed that mouse testin shares 90.1%, 58.9%, 62.2%, and 64.6% identity with rat testin and cathepsin L of mouse, rat, and human, respectively, at the amino acid level. Reverse transcription-polymerase chain reaction and Southern blot analysis demonstrated that mouse testin transcripts were predominantly expressed in the gonads. The mouse testin gene spans over 21 kilobases (kb) and contains eight exons interrupted by seven introns. Primer extension analysis and 5' rapid amplification of cDNA ends identified a major transcription start site located 134 base pairs upstream from the translation initiation codon. Analysis of a 2.3-kb mouse testin 5'-flanking region revealed that it lacked TATA and CAAT boxes, and the region was not GC rich. By the use of deletion analysis, in vitro DNase I footprinting, and site-directed mutagenesis, we identified within the proximal promoter region three closely spaced putative binding sites for GATA, sex-determining factor, and steroidogenic factor 1 that are important for testin gene transcription in mouse Sertoli (MSC-1) cells. These cis-acting elements are also present in the conserved Mullerian-inhibiting substance (MIS) proximal promoters, raising a possibility that the transcriptions of testin and MIS genes are controlled by similar mechanisms.

Amino Acid Sequence↗
PubMed1996

Protein fold recognition by sequence threading: tools and assessment techniques.

Protein fold recognition has been approached by threading an amino acid sequence onto a library of folds, calculating a sequence-structure compatibility score, and ranking these scores. Due to imperfections in the empirically derived pairwise potential functions and the necessarily heuristic approach to the sequence-structure alignment problem, the method benefits from the assessment of threaded models to evaluate the most probable structures among the list of possible folds. THREADER and ANALYST, software tools available through the Internet, facilitate the alignment and assessment steps of a threading prediction. No process has been found to be universally reliable for the detection of folds related to the structure of a known input sequence, but several useful steps and approaches are discussed.

Algorithms↗
PubMedDate not supplied

LEC/chTNT-3 fusion protein for the immunotherapy of experimental solid tumors.

The human chemokine liver-expression chemokine (LEC) was originally found in an expressed sequence tag library, and later the LEC gene was located to chromosome 17q in the ML chemokine gene cluster. LEC has been shown to chemoattract monocytes, lymphocytes, and polymorphonuclear leukocytes (PMNs) by its binding to CCR1 and CCR8 chemokine receptors. Because of its potency as a chemoattractant for immune cells, LEC was used to genetically engineer a fusion protein with chTNT-3, a monoclonal antibody previously shown to target tumors by binding to DNA exposed in necrotic zones. Because the N-terminus of chemokines is important for their activity, the C-terminus of LEC was genetically linked to the chTNT-3 heavy chain variable region and, along with the light chain gene, cotransfected into NSO murine myeloma cells using the glutamine synthetase gene amplification system. The expressed LEC/chTNT-3 fusion protein was purified by tandem protein-A affinity and ion-exchange chromatography and chemotaxis and binding assays confirmed the bioactivity of the purified fusion protein. Pharmacokinetic and biodistribution studies in vivo showed that LEC/chTNT-3 had a biologic half-life of 3 hours and had good uptake in tumor (2.4% injected dose/g), which remained stable at 12 and 24 hours postinjection. Immunotherapy studies performed in three solid tumor models of the BALB/c mouse showed between 37% and 55% tumor reduction at 19 days post-implantation. Immunohistochemical studies using tumor sections obtained at different time points after the administration of control chTNT-3 and LEC/chTNT-3 showed heavy infiltration of CD4+ and CD8+ T cells, PMNs, B cells, and CD11c+CD11b+ dendritic cells in the LEC/chTNT-3 treated groups. The results of these studies demonstrate that this novel fusion protein has potent antitumor activity that is associated with the infiltration of different subpopulations of immune cells. The targeting of LEC to necrotic areas of tumors where the release of tumor antigens is prevalent may be a new approach for the immunotherapy of solid tumors.

Animals↗
PubMed1992

Molecular cloning of human endothelin (ET) receptors ETA and ETB.

Complementary DNAs coding for two different endothelin (ET) receptors were isolated from a human lung cDNA library. DNA sequencing revealed that they corresponded to either the ET-1-selective receptor subtype originally identified in cows (ETA) or the nonselective subtype first found in rats (ETB). An open reading frame coding for a 427 (ETA) or a 442 (ETB) amino acid-long protein, with a transmembrane topology also found in other G protein-coupled receptors, was determined. Whereas the amino acid identity is about 90% between the characterized members of a given receptor subtype, with the best conserved parts being the hydrophobic transmembrane domains, less than 60% identity was found between the human A and B subtypes. Southern blot analysis of human genomic DNA revealed the existence of multiple hybridizing bands when using probes derived from the full-length ETA or ETB cDNAs. However, when shorter fragments were selected as probes, only one band remained detectable, which suggests the existence of a single-copy gene for each subtype of ET receptor.

Amino Acid Sequence↗
PubMed2001

Characterization of microbial consortia in a terephthalate-degrading anaerobic granular sludge system.

The microbial composition and spatial distribution in a terephthalate-degrading anaerobic granular sludge system were characterized using molecular techniques. 16S rDNA clone library and sequence analysis revealed that 78.5% of 106 bacterial clones belonged to the delta subclass of the class Proteobacteria; the remaining clones were assigned to the green non-sulfur bacteria (7.5%), Synergistes (0.9%) and unidentified divisions (13.1%). Most of the bacterial clones in the delta-Proteobacteria formed a novel group containing no known bacterial isolates. For the domain Archaea, 81.7% and 18.3% of 72 archaeal clones were affiliated with Methanosaeta and Methanospirillum, respectively. Spatial localization of microbial populations inside granules was determined by transmission electron microscopy and fluorescent in situ hybridization with oligonucleotide probes targeting the novel delta-proteobacterial group, the acetoclastic Methanosaeta, and the hydrogenotrophic Methanospirillum and members of Methanobacteriaceae. The novel group included at least two different populations with identical rod-shape morphology, which made up more than 87% of the total bacterial cells, and were closely associated with methanogenic populations to form a nonlayered granular structure. This novel group was presumed to be the primary bacterial population involved in the terephthalate degradation in the methanogenic granular consortium.

Anaerobiosis↗
PubMed2025

Automated chromatin profiling with spa-ChIP-seq uncovers the impacts of condition variations.

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is widely used to study the genomic localization of DNA-associated proteins. However, conventional protocols include multiple manual steps that can introduce inconsistency and limit scalability, thereby restricting the inclusion of appropriate replicates and controls. Although the introduction of liquid handling platforms has improved reproducibility, most existing efforts have automated only a subset of the workflow, and extending automation to efficiently map non-histone proteins, such as chromatin regulators, remains challenging. Here, we present a fully automated implementation of our previously developed single-pot ChIP-seq protocol (Texari et al. 2021), named spa-ChIP-seq, which enables scalable processing of 8 to 96 ChIP-seq samples from crosslinked cells to sequencing-ready library in approximately three days with an estimated cost of $70 per sample. Benchmarking spa-ChIP-seq against manual ChIP-seq performed in parallel demonstrates comparable signal-to-noise ratio between the two workflows. Using spa-ChIP-seq, we systematically evaluate multiple parameters including shearing and crosslinking conditions, buffer compositions, and the ratio of antibody to cell-number. We find, for the first time to our knowledge, that weaker genomic localization signals are sensitive to changing the antibody to cell-number ratio, whereas the stronger signals remain unaffected. This finding underscores the importance of maintaining consistent antibody-to-cell-number ratio for comparative studies, such as treatment responses or chromatin-QTL mapping. The spa-ChIP-seq protocol is publicly available, including deck setups, operational parameters, and scripts. We envision that this robust, cost-efficient protocol will facilitate high-throughput, reproducible ChIP-seq analyses, supporting large-scale studies of antibody validation, compound screening, population genomics, and diagnostic frameworks.

Journal Article↗
PubMed2000

Characterization of nonfunctional V1R-like pheromone receptor sequences in human.

The vomeronasal organ (VNO) or Jacobson's organ is responsible in terrestrial vertebrates for the sensory perception of pheromones, chemicals that elicit stereotyped behaviors among individuals of the same species. Pheromone-induced behaviors and a functional VNO have been described in a number of mammals, but the existence of this sensory system in human is still debated. Recently, two nonhomologous gene families, V1R and V2R, encoding pheromone receptors have been identified in rat. These receptors belong to the seven-transmembrane domain G-protein-coupled receptor superfamily. We sought to characterize V1R-like genes in the human genome. We have identified seven different human sequences by PCR and library screening with rodent sequences. These human sequences exhibit characteristic features of V1R receptors and show 52%-59% of amino acid sequence identity with the rat sequences. Using PCR on a monochromosomal somatic cell hybrid panel and/or FISH, we demonstrate that these V1R-like sequences are distributed on chromosomes 7, 16, 20, 13, 14, 15, 21, and 22 and possibly on additional chromosomes. One sequence hybridizes to pericentromeric locations on all the acrocentric chromosomes (13, 14, 15, 21, and 22). All of the seven V1R-like sequences analyzed show interrupted reading frames, indicating that they represent nonfunctional pseudogenes. The preponderence of pseudogenes among human V1R sequences and the striking anatomical differences between rodent and human VNO raise the possibility that humans may have lost the V1R/VNO-mediated sensory functions of rodents.

Amino Acid Sequence↗
PubMed2005

Genomics of the future: identification of quantitative trait loci in the mouse.

Positional cloning of quantitative trait loci in rodents is a common approach to identify genes involved in complex phenotypes, including genes important to human disease. However, cloning the causative genes has proved to be more difficult than determining their positions. New tools such as genomic sequence, clone libraries, and new genomic-based methods offer new approaches to identify these genes. Here we review how these new tools and approaches will improve our ability to discover the genes important in complex traits.

Animals↗
PubMed2003

Kinesin superfamily proteins (KIFs) in the mouse transcriptome.

In the post genomic era where virtually all the genes and the proteins are known, an important task is to provide a comprehensive analysis of the expression of important classes of genes, such as those that are required for intracellular transport. We report the comprehensive analysis of the Kinesin Superfamily, which is the first and only large protein family whose constituents have been completely identified and confirmed in silico and at the cDNA, mRNA level. In FANTOM2, we have found 90 clones from 33 Kinesin Superfamily Protein (KIF) gene loci. The clones were analyzed in reference to sequence state, library of origin, detection methods, and alternative splicing. More than half of the representative transcriptional units (TU) were full length. The FANTOM2 library also contains novel splice variants previously unreported. We have compared and evaluated various protein classification tools and protein search methods using this data set. This report provides a foundation for future research of the intracellular transport along microtubules and proves the significance of intracellular transport protein transcripts as part of the transcriptome.

Alternative Splicing↗
PubMed2003

Photosynthetic acclimation is reflected in specific patterns of gene expression in drought-stressed loblolly pine.

Because the product of a single gene can influence many aspects of plant growth and development, it is necessary to understand how gene products act in concert and upon each other to effect adaptive changes to stressful conditions. We conducted experiments to improve our understanding of the responses of loblolly pine (Pinus taeda) to drought stress. Water was withheld from rooted plantlets of to a measured water potential of -1 MPa for mild stress and -1.5 MPa for severe stress. Net photosynthesis was measured for each level of stress. RNA was isolated from needles and used in hybridizations against a microarray consisting of 2173 cDNA clones from five pine expressed sequence tag libraries. Gene expression was estimated using a two-stage mixed linear model. Subsequently, data mining via inductive logic programming identified rules (relationships) among gene expression, treatments, and functional categories. Changes in RNA transcript profiles of loblolly pine due to drought stress were correlated with physiological data reflecting photosynthetic acclimation to mild stress or photosynthetic failure during severe stress. Analysis of transcript profiles indicated that there are distinct patterns of expression related to the two levels of stress. Genes encoding heat shock proteins, late embryogenic-abundant proteins, enzymes from the aromatic acid and flavonoid biosynthetic pathways, and from carbon metabolism showed distinctive responses associated with acclimation. Five genes shown to have different transcript levels in response to either mild or severe stress were chosen for further analysis using real-time polymerase chain reaction. The real-time polymerase chain reaction results were in good agreement with those obtained on microarrays.

Acclimatization↗
PubMed1988

Chitinase cDNA cloning and mRNA induction by fungal elicitor, wounding, and infection.

Chitinase, which catalyzes the hydrolysis of beta-1,4 N-acetylglucosamine linkages of the fungal cell wall polymer chitin, is a component of the inducible defenses of plants. We show that chitinase synthesis is stimulated in bean (Phaseolus vulgaris L.) cell suspension cultures treated with fungal cell wall elicitors and in hypocotyls in response to infection with the fungus Colletotrichum lindemuthianum. Chitinase cDNA clones were isolated by antibody screening of a lambdagt11 cDNA library containing sequences complementary to poly A(+) RNA from elicited cells. The identity of these clones was confirmed by nucleotide sequence analysis and comparison of the deduced amino acid sequence with that determined for the amino-terminal sequence of bean chitinase. Elicitor causes a very rapid activation of chitinase transcription with a 10-fold stimulation after 5 minutes and 30-fold increase within 20 minutes. This leads to a marked, transient accumulation of chitinase transcripts with maximum levels 2 hours after elicitor treatment, concomitant with the phase of rapid enzyme synthesis. Chitinase transcripts also markedly accumulate in wounded and infected hypocotyls. Chitinase cDNA sequences hybridize to several genomic fragments suggesting there are several chitinase genes in the bean genome.

Journal Article↗
PubMed1992

Gene structure of Bombyx mori larval serum protein (BmLSP).

To understand the molecular mechanisms of the larval-specific transcription of Bombyx mori larval serum protein (BmLSP), we isolated a clone of the BmLSP gene from a genomic library and sequenced a 3.5-kb fragment. An intron was found in the 5' noncoding region of the BmLSP gene. A putative transcription start point was determined by primer extension analysis. Genomic Southern hybridization showed that there is one copy of the BmLSP gene in a haploid genome. A database search revealed that the BmLSP gene has presumptive repetitive sequences found in other B. mori genes, the sequence homologous to ecdysone-responsive elements and a heptamer sequence found in storage protein genes.

Amino Acid Sequence↗
PubMed1993

Isolation and analysis of a linear plasmid-located gene of Borrelia burgdorferi B29 encoding a 27 kDa surface lipoprotein (P27) and its overexpression in Escherichia coli.

Using an antiserum of a patient with cutaneous manifestations of Lyme borreliosis we have isolated the gene encoding a 27 kDa protein antigen (P27) of Borrelia burgdorferi B29 from a lambda-gt11 expression library. Nucleotide sequence analysis revealed that it is a basic protein of 248 amino acids with a typical prokaryotic leader sequence of 17 amino acid residues at the N-terminus of the proposed translation product. Biochemical investigations showed that P27 is a surface-exposed lipoprotein. From pulsed-field gel electrophoresis and subsequent Southern blot analysis it is evident that the p27 gene is located on a linear plasmid of a size of approximately 55 kb. It was overexpressed in Escherichia coli and the purified recombinant protein was used for biochemical and serological studies. Northern and Western blot analysis demonstrated that p27 is expressed in the European B. burgdorferi strain B29, but not in the American strain B31.

Amino Acid Sequence↗
PubMed1995

glmS of Thermus thermophilus HB8: an essential gene for cell-wall synthesis identified immediately upstream of the S-layer gene.

A 30 kbp chromosomal region containing the S-layer gene (slpA) from Thermus thermophilus HB8 was cloned from a lambda phage gene library. DNA sequence analysis of the region upstream to the slpA gene revealed the presence of an open reading frame (ORF) which coded for a 604-amino-acid protein highly homologous to the glucosamine-6-P synthases (EC 2.6.1.16) of both prokaryotic and eukaryotic origin. The identification of this ORF as the glucosamine-6-P synthase gene from T. thermophilus (glmSth) has been carried out using three different strategies: (i) complementation of an Escherichia coli glmS mutant; (ii) in vivo insertional inactivation of the gene; and (iii) in vitro synthesis of glucosamine-6-P at 60 degrees C by a cytoplasmic extract of an overproducing E. coli strain. The glmSth gene is transcribed divergently from slpA in a 2.0 kb mRNA which probably also includes a tryptophan tRNA gene (trpTth) identified at its 3' extreme. As the products of both the glmSth and the slpA genes are main components of the cell envelope of T. thermophilus, their unusual clustering in the chromosome could be related to the existence of specific mechanisms for their coordinate expression.

Amino Acid Sequence↗
PubMed2006

Biosynthesis of cellulose-enriched tension wood in Populus: global analysis of transcripts and metabolites identifies biochemical and developmental regulators in secondary wall biosynthesis.

Stems and branches of angiosperm trees form tension wood (TW) when exposed to a gravitational stimulus. One of the main characteristics of TW, which distinguishes it from normal wood, is the formation of fibers with a thick inner gelatinous cell wall layer mainly composed of crystalline cellulose. Hence TW is enriched in cellulose, and deficient in lignin and hemicelluloses. An expressed sequence tag library made from TW-forming tissues in Populus tremula (L.) x tremuloides (Michx.) and data from transcript profiling using microarray and metabolite analysis were obtained during TW formation in Populus tremula (L.) in two growing seasons. The data were examined with the aim of identifying the genes responsible for the change in carbon (C) flow into various cell wall components, and the mechanisms important for the formation of the gelatinous cell wall layer (G-layer). A specific effort was made to identify carbohydrate-active enzymes with a putative function in cell wall biosynthesis. An increased C flux to cellulose was suggested by a higher abundance of sucrose synthase transcripts. However, genes related to the cellulose biosynthetic machinery were not generally affected, although the expression of secondary wall-specific CesA genes was modified in both directions. Other pathways for which the data suggested increased activity included lipid and glucosamine biosynthesis and the pectin degradation machinery. In addition, transcripts encoding fasciclin-like arabinogalactan proteins were particularly increased and found to lack true Arabidopsis orthologs. Major pathways for which the transcriptome and metabolome analysis suggested decreased activity were the pathway for C flux through guanosine 5'-diphosphate (GDP) sugars to mannans, the pentose phosphate pathway, lignin biosynthesis, and biosynthesis of cell wall matrix carbohydrates. Several differentially expressed auxin- and ethylene-related genes and transcription factors were also identified.

Cell Wall↗
PubMed1988

The structure of brain-specific rat aldolase C mRNA and the evolution of aldolase isozyme genes.

The cDNA clones for rat aldolase C mRNA having the nearly complete length were isolated from a rat brain cDNA library and sequenced. The nucleotide sequence of pRAC2-1, a cDNA clone having the largest cDNA insert, indicates that the cDNA is composed of a 105-base-pair 5'-noncoding sequence, a 1089-base-pair coding-sequence and a 382-base-pair 3'-noncoding sequence. The amino acid sequence of aldolase C deduced from a possible open reading frame was composed of 362 residues having a relative molecular mass of 39,164 excluding the initiating methionine, one amino acid shorter than aldolases A and B. The length of aldolase c mRNA was 1750 residues, somewhat longer than that of the aldolase A and B transcripts. The aldolase C mRNA was distributed mainly in the brain, some in ascites hepatoma and fetal liver. Comparison of the amino acid sequences of rat aldolase C with those for rat aldolase A and B [Joh et al. (1985) Gene 39, 17-24; Tsutsumi et al. (1984) J. Biol. Chem. 259, 14572-14575], which have been determined previously, shows the existence of highly conserved stretches of amino acid among the three isozymic forms throughout their sequences. The extent of the homology between aldolases A and C is 81%, while those between aldolases A and B, and B and C are 70%, respectively. The analysis of amino acid substitution among aldolases A, B and C from several species suggests that the isozyme genes diverged much earlier than animal species appeared and that the aldolase C gene has evolved from the aldolase A gene after aldolase A and B genes diverged.

Amino Acid Sequence↗
PubMed1990

Isolation, characterization and chromosomal localization of cDNA clones for the E1 beta subunit of the pyruvate dehydrogenase complex.

A full-length cDNA clone for the E1 beta subunit of the human pyruvate dehydrogenase (PyrDH) complex was isolated from a human skin fibroblast cDNA library. When sequenced, it showed differences from the nucleotide sequence already published [Koike, K., Ohta, S., Urata, Y., Kagawa, Y. & Koike, M. (1988) Proc. Natl Acad. Sci. USA 85, 41-45], such that 19 amino acids were different in the translated open reading frame. Northern blotting of human fibroblast cell lines revealed a major mRNA species of 1.6 kb and a weaker band of 5.5 kb. In a series of nine PyrDH-complex-deficient cell lines from patients with this deficiency, no patients had severely reduced amounts of mRNA, but there was one patient cell line with an increased amount of abnormal-size mRNA. Chromosome localization carried out with DNA blots from man-mouse hybrid cell lines indicated that the E1 beta subunit of pyruvate dehydrogenase is located on chromosome 3. A motif AXGXXXXGL(R/K)X15(D/E)Q was found in common with a variety of other oxo-acid oxidoreductases, but its function is not known.

Amino Acid Sequence↗