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PubMed1991

Cloning of the cDNA and gene for mouse mast cell protease 4. Demonstration of its late transcription in mast cell subclasses and analysis of its homology to subclass-specific neutral proteases of the mouse and rat.

Based on the amino-terminal amino acid sequence of the mature form of mouse mast cell protease 4 (MMCP-4), previously identified in peritoneal connective tissue mast cells (CTMC) and Kirsten sarcoma virus-immortalized mast cells (KiSV-MC), a 26-mer oligonucleotide probe was constructed and used to clone cDNAs for MMCP-4 from a KiSV-MC1 cDNA library. MMCP-4 is the first secretory granule serine protease of CTMC to be molecularly cloned. Using a cDNA probe derived from the 3'-untranslated portion of the MMCP-4 cDNA, the gene for MMCP-4 and a second highly related gene (mouse mast cell protease-like, MMCP-L) were cloned from a BALB/c mouse genomic DNA library and sequenced entirely, including approximately 2 kilobases of the 5'-flanking region. MMCP-4 and MMCP-L have five exons of identical length, four introns of nearly identical length, and approximately 900 base pairs of 5'-flanking DNA with sequence similarity by dot matrix analysis. By RNA blot analysis with gene-specific probes for MMCP-4 (bases 497-633 of the cDNA) and MMCP-L (bases 502-638 of the cDNA), mRNA for MMCP-4 was present in KiSV-MC5, CTMC, and the intestine of a mouse infected with the parasite Nippostrongylus brasiliensis markedly enriched for mucosal mast cells (MMC); MMCP-L mRNA was detected only in the intestine of the N. brasiliensis-infected mouse. MMCP-4 mRNA was not expressed in normal mouse intestine or in interleukin 3-dependent bone marrow-derived mast cells, which can serve as precursors to both MMC and CTMC. This finding suggests that MMCP-4 is transcribed relatively late in the development of both the CTMC and MMC subclasses and underscores the fact that mouse bone-marrow-derived mast cells are immature mast cells, rather than tissue culture equivalents of the MMC subclass.

Amino Acid Sequence↗

PubMed2005

Gene expression in pharyngeal arch 1 during human embryonic development.

Craniofacial abnormalities are one of the most common birth defects in humans, but little is known about the human genes that control these important developmental processes. To identify relevant genes, we analyzed transcription profiles of human pharyngeal arch 1 (PA1), a conserved embryonic structure that develops into the palate and jaw. Using microdissected, normal human craniofacial structures, we constructed 12 SAGE (serial analysis of gene expression) libraries and sequenced 606 532 tags. We also performed Affymetrix microarray analysis on 25 craniofacial targets. Our data revealed not only genes "enriched" or differentially expressed in PA1 during fourth and fifth week of human development, but also 6927 genes newly identified to be expressed in human PA1. Many of these genes are involved in biosynthetic processes and have binding function and catalytic activity. We compared expression profiles of human genes with those of mouse homologs to look for genes more specific to human craniofacial development and found 766 genes expressed in human PA1, but not in mouse PA1. We also identified 1408 genes that were expressed in mouse as well as human PA1 and could be useful in creating mouse models for human conditions. We confirmed conservation of some human PA1 expression patterns in mouse embryonic samples with whole mount in situ hybridization and real-time RT-PCR. This comprehensive approach to expression profiling gives insights into the early development of the craniofacial region and provides markers for developmental structures and candidate genes, including SET and CCT3, for diseases such as orofacial clefting and micrognathia.

Animals↗

PubMed1997

Carbonic anhydrase IX, MN/CA IX: analysis of stomach complementary DNA sequence and expression in human and rat alimentary tracts.

BACKGROUND & AIMS: CA IX (formerly MN protein) is a carbonic anhydrase isoenzyme whose expression is associated with human tumors. However, it has also been found in normal gastric mucosa. The aim of this study was to determine differences in complementary DNAs (cDNAs), to obtain an overview of distribution in the alimentary tract, and to obtain data on expression in tumors. METHODS: A CA9 cDNA isolated from a human stomach library was sequenced along with the cDNA derived from HeLa cells. Western blotting and immunohistochemical analyses of human and animal tissues were performed using CA IX-specific monoclonal antibody and rabbit antiserum to human CA II. RESULTS; Sequence analysis showed no differences between the stomach- and HeLa-derived cDNAs. CA IX was detected at the basolateral surface of gastric, intestinal, and gallbladder epithelia. In stomach tumor samples, expression of CA IX was lost or reduced. CONCLUSIONS: Differential distribution of CA IX in normal and tumor tissues is not associated with cDNA mutations. Evolutionary conservation in vertebrates as well as abundant expression of CA IX protein in normal human gastric mucosa, but not in derived tumors, indicate its physiological importance.

Animals↗

PubMed1986

GENEUS, a computer system for DNA and protein sequence analysis containing an information retrieval system for the EMBL data library.

We describe a comprehensive computer system, GENEUS, for extensive DNA, RNA and protein sequence analysis. The analysis system is developed for the DEC VAX/VMS computer and uses the EMBL Nucleic Acid Sequence Data Library. Help information is available on-line on terminal screen. To speed up system handling, a qualifier oriented user communication is employed. All results are stored on files making them accessible to the computer editor. An information retrieval system for the EMBL Nucleotide Sequence Data Library is also described. A defined data-base interface allows connection to other analysis programs.+

Amino Acid Sequence↗

PubMed1999

TWIK-2, a new weak inward rectifying member of the tandem pore domain potassium channel family.

Potassium channels are found in all mammalian cell types, and they perform many distinct functions in both excitable and non-excitable cells. These functions are subserved by several different families of potassium channels distinguishable by primary sequence features as well as by physiological characteristics. Of these families, the tandem pore domain potassium channels are a new and distinct class, primarily distinguished by the presence of two pore-forming domains within a single polypeptide chain. We have cloned a new member of this family, TWIK-2, from a human brain cDNA library. Primary sequence analysis of TWIK-2 shows that it is most closely related to TWIK-1, especially in the pore-forming domains. Northern blot analysis reveals the expression of TWIK-2 in all human tissues assayed except skeletal muscle. Human TWIK-2 expressed heterologously in Xenopus oocytes is a non-inactivating weak inward rectifier with channel properties similar to TWIK-1. Pharmacologically, TWIK-2 channels are distinct from TWIK-1 channels in their response to quinidine, quinine, and barium. TWIK-2 is inhibited by intracellular, but not extracellular, acidification. This new clone reveals the existence of a subfamily in the tandem pore domain potassium channel family with weak inward rectification properties.

Amino Acid Sequence↗

PubMed1994

The endogenous vascular elastase that governs development and progression of monocrotaline-induced pulmonary hypertension in rats is a novel enzyme related to the serine proteinase adipsin.

We showed previously a cause and effect relationship between increased activity of an endogenous vascular elastase (EVE) and experimentally induced pulmonary hypertension in rats. We now report the isolation and characterization of EVE. Degenerate oligonucleotides synthesized to homologous sequences in serine elastases were used in a PCR with rat pulmonary artery (PA) cDNA. The PCR product hybridized to a 1.2-kb mRNA and the intensity of hybridization was threefold increased in RNA from rat hypertensive PA at a timepoint when EVE activity was increased. The PCR product was used to screen a cDNA library and sequences obtained encoded rat adipsin. We then used immunoaffinity to purify EVE. An antibody to the elastin-binding protein was used to remove this competitor of elastase from the PA extract and the elastolytic activity increased 100-fold. The enzyme was purified using an antibody that recognizes NH2-terminal sequences of serine proteinases and the eluate was further purified using an antibody raised against recombinant adipsin. A single band at 20 kD immunoreactive with the adipsin antibody was resolved as an active enzyme on an elastin substrate gel. Immunogold labeling with an antibody to an adipsin peptide sequence localized EVE to PA smooth muscle cells. This is the first isolation of EVE; it appears to be a novel enzyme related to the serine proteinase adipsin originally found in adipose tissue.

Amino Acid Sequence↗

PubMed2001

In-vitro selection of highly stabilized protein variants with optimized surface.

Thermostable proteins are of prime importance in protein science, but it has remained difficult to develop general strategies for stabilizing a protein. Site-directed mutagenesis based on comparisons with thermophilic homologs is rarely successful because the sequence differences are too numerous and dominated by neutral mutations. Here we used a method of directed evolution to increase the stability of a mesophilic protein, the cold shock protein Bs-CspB from Bacillus subtilis. It differs from its thermophilic counterpart Bc-Csp from Bacillus caldolyticus at 12 surface-exposed positions. To elucidate the stabilizing potential of exposed amino acid residues, six of these variant positions were randomized by saturation mutagenesis, the corresponding library of sequences was inserted into the gene-3-protein of the filamentous phage fd, and stabilized variants were selected by the Proside technique. Proside links the increased protease resistance of stabilized protein variants with the infectivity of the phage. Many strongly stabilized variants of Bs-CspB were identified in two selections, one in the presence of a denaturant and the other at elevated temperature. Several of them are significantly more stable than the naturally thermostable homolog Bc-Csp, and the best variant reaches Tm-Csp (the homolog from the hyperthermophile Thermotoga maritima) in stability. Remarkably, this variant differs from Tm-Csp at five and from Bc-Csp at all six randomized positions. This indicates that proteins can be strongly stabilized by many different sets of surface mutations, and Proside selects them efficiently from large libraries. The course of the selection could be directed by the conditions. In an ionic denaturant non-polar surface interactions were optimized, whereas at elevated temperature variants with improved electrostatics were selected, pointing to two different strategies for stabilization at protein surfaces.

Amino Acid Sequence↗

PubMed2006

Construction of a bacterial artificial chromosome library and characterization of hrp/hrc gene cluster of Pseudomonas syringae pathovar tagetis LMG5090.

Pseudomonas syringae pv. tagetis causes apical chlorosis of several plant species in the Asteraceae, including marigold. As a means to facilitate the isolation of pathogenicity genes and to characterize the genome of this bacterium, we have constructed a bacterial artificial chromosome library of P. syringae pv. tagetis strain LMG5090. The library consists of 1,536 clones with insert size ranging from 30 to 160 kb and an average size of 86 kb. Based upon colony hybridization, the BAC clone 420E23 containing the hrp/hrc gene cluster encoding the type III secretion system was identified from this library and subsequently shotgun sequenced. The hrp/hrc gene cluster of P. syringae pv. tagetis has a 23 kb sequence which contains 27 open reading frames. Comparative analysis of the hrp/hrc gene cluster of P. syringae pv. tagetis LMG5090, P. syringae pv. tomato DC3000, P. syringae pv. syringae B728a, and P. syringae pv. phaseolicola 1448A revealed that the entire hrp/hrc gene cluster of P. syringae pv. tagetis is conserved and identically arranged in all four pathovars.

Bacterial Outer Membrane Proteins↗

PubMed1988

Structure and neural expression of a zebrafish homeobox sequence.

A genomic library of zebrafish was constructed and screened with homeobox-containing probes. One of the positive clones contains a transcribed region which shares extensive sequence homology with the murine Hox-1.4 and Hox-2.6 genes and the human HHO.c13 gene. Characterization of this zebrafish homologue (ZF-13) with respect to expression demonstrated that it is transcribed during embryogenesis where a major RNA species of 2.5 kb and a minor transcript of 4.6 kb are detected. The highest concentration of both transcripts was found in embryos at the stage of somite formation. By in situ hybridization the spatial localization of expression was analysed in hatching embryos. Hybridization signals were mainly detected throughout the neural tube and in the brain. A small amount of RNA derived from ZF-13 was localized in differentiated muscle cells. Our results suggest that homeobox genes of distantly related vertebrate species are very similar with respect to structure and function.

Animals↗

PubMed1993

Cloning and characterization of a Golgi-associated GTP-binding protein homologue from Leishmania major.

This paper describes the cloning of a Golgi-associated GTP-binding protein homologue from Leishmania major. The gene was isolated using degenerate oligonucleotides to conserved sequences amongst the small GTP-binding proteins in a polymerase chain reaction on genomic DNA of the L. major cloned line V121. The reading frame of one clone showed high similarity to the rab/YPT subfamily of small GTP-binding proteins. A full length copy of the gene was isolated from a lambda gt10 V121 genomic library and sequenced. At the amino acid level the gene showed highest similarity to the human/rat rab1 A gene and the mouse/yeast YPT gene and was named LmYPT. The LmYPT gene was present as a single copy gene in both the L. major and L. donovani genomes. Karyotype analysis localized the LmYPT gene to chromosome band 18 in V121, but it was located on a larger chromosome in the different L. major isolate L119. The LmYPT gene was transcribed as a 3.9-kb transcript in both the promastigote and amastigote forms of the parasite. Western blot analysis, using a polyclonal rabbit antiserum raised against an Escherichia coli expressed portion of the molecule, identified a doublet at 20 and 23 kDa in total promastigote protein. Immunoelectron microscopy in combination with peroxidase staining localized the LmYPT molecule to the Leishmania Golgi apparatus.

Amino Acid Sequence↗

PubMed1998

Characterization of the human neurocan gene, CSPG3.

Neurocan is a chondroitin sulfate proteoglycan thought to be involved in the modulation of cell adhesion and migration. Its sequence has been determined previously in rat and mouse (Rauch et al., 1992. Cloning and primary structure of neurocan, a developmentally regulated, aggregating, chondroitin sulfate proteoglycan of the brain. J. Biol. Chem. 267, 19536-19547; Rauch et al., 1995. Structure and chromosomal location of the mouse neurocan gene. Genomics 28, 405-410). We describe here the complete coding sequence of the human neurocan mRNA, known as CSPG3, as well as mapping data, expression analysis, and genomic structure. A cDNA known as CP-1 was initially sequenced as part of a gene discovery project focused on characterizing chromosome 19-specific cDNAs. Sequence homology searches indicated close homology to the mouse and rat proteoglycan, neurocan (GenBank accession Nos X84727 and M97161). Northern analysis identified a brain-specific transcript of approx. 7.5kb. A longer cDNA clone, GT-5, was obtained, fine-mapped to the physical map of chromosome 19 by hybridization to a chromosome-specific cosmid library, and sequenced. Full coding sequence of the mRNA indicates a 3963bp open reading frame corresponding to a 1321 amino acid protein, similar to the protein length found in mouse and rat. The amino acid sequence of human neurocan shows 63% identity with both the mouse and rat sequences. Finally, genomic sequencing of a cosmid containing the complete neurocan gene was performed to determine the genomic structure of the gene, which spans approx. 41kb, and is transcribed in the telomere to centromere orientation.

Amino Acid Sequence↗

PubMed2003

Discovery of the breast cancer gene BASE using a molecular approach to enrich for genes encoding membrane and secreted proteins.

To identify unknown membrane proteins that could be used as targets for breast and prostate cancer immunotherapies and secreted proteins to be used as diagnostic markers, a cDNA library was generated from membrane-associated polyribosomal RNA derived from four breast cancer cell lines, one normal breast cell line, and a prostate cancer cell line. The membrane-associated polyribosomal cDNA library was subtracted with RNA from normal brain, liver, lung, kidney, and muscle. Of the 15,581 clones sequenced from the subtracted cDNA library, sequences from 10,506 clones map to known genes, but 5,075 sequences, representing 3,181 unique transcripts, are not associated with known genes. As one example, we experimentally investigated expression of a previously uncharacterized breast cancer gene that encodes a secreted protein designated BASE (breast cancer and salivary gland expression). BASE is expressed in many breast cancers but not in essential normal tissues including the five organs used for subtraction. Further analysis of this library should yield additional gene products of use in the diagnosis or treatment of breast or prostate cancer.

Blotting, Northern↗

PubMed1996

Primary sequence and developmental expression pattern of mRNAs and protein for an alpha1 subunit of the sodium pump cloned from the neural plate of Xenopus laevis.

Expression of a catalytic alpha subunit of the sodium pump was followed in early Xenopus embryos for correlation with physiological experiments showing that the sodium pump controls cavity expansion and the differentiation of neurones from the neural plate. Two cDNAs (one full length, one partial) for alpha1 subunit isoforms were cloned from a neural plate stage Xenopus library and sequenced. Other isoforms were not detected. Temporal and spatial expression patterns for alpha1 subunit transcripts and protein revealed extensive developmental regulation. At all stages, cells involved in cavity generation (outer ectoderm and cells lining the archenteron) expressed alpha1, transcripts with protein confined to the lateral and basal membranes. Before gastrulation, transcript levels were low and predominantly in animal cells. During gastrulation, alpha1 mRNAs rose significantly. Transcripts and protein were down-regulated in future outer neural plate cells as the mesoderm invaginated. Protein appeared at the blastopore on apical surfaces of lip cells and apposing surfaces of invaginating cells, suggesting that the Na pump opposes entry of fluid. In early neurulae, alpha1 mRNAs rose sharply. Transcript expression remained low in outer neural plate cells and increased in the endoderm, and protein appeared in the notochord. In midneurulae, transcripts returned in outer neural plate cells. Protein expression appeared on basal surfaces of deep neural plate cells and the floor plate, matching physiological observations. After neural tube closure, transcripts were detected in all dorsal structures. Protein was retained in the notochord and floor plate, was eliminated from the outer layer of the neural tube, and appeared on ependymal cells. The results are discussed in relation to previous physiological observations.

Amino Acid Sequence↗

PubMed1990

Characterization of a sperm-specific nuclear autoantigenic protein. I. Complete sequence and homology with the Xenopus protein, N1/N2.

In our studies on specific sperm proteins that function in fertilization, an autoantigenic, postacrosomal sperm protein has been found to originate in the testis as a nuclear-associated protein. This nuclear autoantigenic sperm protein (NASP) contains a C-terminal nuclear translocation signal and has structural similarities to the lamins and other nuclear proteins; and its 2.5 kb mRNA is apparently tissue-, but not species-, specific. DNA clones from a rabbit testis cDNA library and a rabbit genomic library were sequenced in order to characterize NASP. The polyadenylated mRNA has 39 bases of 5' untranslated sequence, an open reading frame of 2043 bases encoding 680 amino acids, and a 104 base 3' untranslated region (2,186). The encoded polypeptide has a calculated molecular weight of 73,533 and a pI = 4.06, containing 25% acidic residues. One clone (R1.2) expressing the C-terminal 446 amino acids was used to express a fusion protein. The expressed R1.2/beta-galactosidase fusion protein was found to be autoantigenic. Secondary structure predictions for NASP showed that 69% of the molecule had a high probability of forming alpha-helices and that several alpha-helical regions had a characteristic repeating heptad pattern that in the intermediate filaments and nuclear lamins is involved in coiled-coil interactions with other molecules. In addition to the nuclear translocation signal common to many nuclear proteins, NASP also showed homology with the Xenopus histone-binding protein, N1/N2.

Amino Acid Sequence↗

PubMed2003

Isolation of Resistance Gene Candidates (RGCs) and characterization of an RGC cluster in cassava.

Plant disease resistance genes (R genes) show significant similarity amongst themselves in terms of both their DNA sequences and structural motifs present in their protein products. Oligonucleotide primers designed from NBS (Nucleotide Binding Site) domains encoded by several R-genes have been used to amplify NBS sequences from the genomic DNA of various plant species, which have been called Resistance Gene Analogues (RGAs) or Resistance Gene Candidates (RGCs). Using specific primers from the NBS and TIR (Toll/Interleukin-1 Receptor) regions, we identified twelve classes of RGCs in cassava (Manihot esculenta Crantz). Two classes were obtained from the PCR-amplification of the TIR domain. The other 10 classes correspond to the NBS sequences and were grouped into two subfamilies. Classes RCa1 to RCa5 are part of the first subfamily and were linked to a TIR domain in the N terminus. Classes RCa6 to RCa10 corresponded to non-TIR NBS-LRR encoding sequences. BAC library screening with the 12 RGC classes as probes allowed the identification of 42 BAC clones that were assembled into 10 contigs and 19 singletons. Members of the two TIR and non-TIR NBS-LRR subfamilies occurred together within individual BAC clones. The BAC screening and Southern hybridization analyses showed that all RGCs were single copy sequences except RCa6 that represented a large and diverse gene family. One BAC contained five NBS sequences and sequence analysis allowed the identification of two complete RGCs encoding two highly similar proteins. This BAC was located on linkage group J with three other RGC-containing BACs. At least one of these genes, RGC2, is expressed constitutively in cassava tissues.

Amino Acid Sequence↗

PubMed2001

Novel activator protein-2alpha splice-variants function as transactivators of the ovine placental lactogen gene.

Activator protein-2 (AP-2) has been implicated as a transactivator of the human and ovine placental lactogen (oPL) genes. Transcriptional enhancement through an AP-2 cis-acting element has been described for other genes expressed in the placenta, but the AP-2 isoform enhancing expression is species dependent. Transactivation of the oPL minimal promoter (-124 bp to +16 bp) by AP-2 was confirmed by mutational analysis in transiently transfected human choriocarcinoma cells (BeWo). AP-2alpha was localized in ovine chorionic epithelial cells by immunohistochemistry and a 3-kb transcript was identified by Northern hybridization. Four nearly full-length AP-2 cDNAs were isolated from an ovine placenta cDNA library. Nucleotide sequencing these cDNAs revealed that the AP-2 mRNA expressed in the ovine placenta shares identity with human AP-2alpha, but variations in the predicted N-terminus were observed, and three unique AP-2alpha splice-variants were identified. Expression of AP-2alpha variants in HepG2 cells, devoid of endogenous AP-2, indicates that enhancement through the AP-2 element in the oPL gene minimal promoter was variant dependent. RNA transcripts for all of the ovine AP-2alpha splice-variants were confirmed in ovine placenta by RT-PCR, and homologs for two variants were found in human placenta. However, only one AP-2alpha transcript, which shares identity to Xenopus AP-2alpha, was expressed in BeWo cells. Immunoblot analysis confirmed AP-2alpha variants in ovine chorionic binucleate cell nuclear extracts, one of which migrates similar to the AP-2alpha variant identified in BeWo cell nuclear extracts. These data indicate the presence of new mammalian AP-2alpha splice-variants that augment transactivation of the oPL gene in ovine chorionic binucleate cells.

Alternative Splicing↗

PubMed1988

Human P450PCN1: sequence, chromosome localization, and direct evidence through cDNA expression that P450PCN1 is nifedipine oxidase.

P450PCN protein levels and nifedipine oxidase activities were quantitated in 12 human livers and were shown to be highly correlated. Antibody against rat P450PCN1 completely inhibited all nifedipine oxidase activity in three human liver samples. These results suggest that a human P450 related to rat P450PCN1 is the major form of P450 catalyzing nifedipine oxidation. The cDNA for a human P450, designated phPCN1, was isolated from a human liver lambda gt11 cDNA library, and sequenced completely. The deduced amino acid sequence is 77% similar to rat P450PCN1. By use of the adenovirus- and SV40-based expression vecotr p91023(B), the phP450PCN1 cDNA was expressed in COS cells and had high nifedipine oxidase activity, providing conclusive evidence that this P450 is the primary enzyme associated with metabolism and inactivation of this important drug. Using somatic cell hybrids, the P450PCN gene was localized to human chromosome 7.

Amino Acid Sequence↗

PubMed1991

Sequencing of bovine enamelin ("tuftelin") a novel acidic enamel protein.

Enamelins are a major group of 28-70-kDa acidic proteins rich in aspartic acid, glutamic acid, serine, and glycine found in developing and mature extracellular enamel; a unique and highly mineralized ectodermal tissue covering vertebrate teeth. They have been associated with the mineralization and structural organization of this tissue. In an attempt to elucidate the primary structure of enamelin, a 2674-base pair cDNA isolated from a bovine ameloblast-enriched, lambda Zap 2 expression library, was sequenced. The identity and localization of the deduced protein was confirmed by amino acid composition, enzyme-linked immunosorbent assay, Western blotting, indirect immunohistochemistry, and high resolution protein-A gold immunocytochemistry. The immunological techniques were employed using antibodies directed against synthetic peptides corresponding to the protein sequence deduced from the cloned cDNA sequence. The results reveal the deduced protein to be a novel acidic enamel protein. It contains 389 amino acids and has a calculated molecular weight of 43,814. Its amino acid composition is similar to that of "tuft" proteins (enamel matrix protein fragments remaining in the mature tissue). It contains one potential N-glycosylation site and 5 cysteine residues. Southern hybridization of the cloned cDNA with genomic bovine DNA indicated the existence of a single gene with one or more introns.

Amino Acid Sequence↗