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At least 541 records · Page 30Linked to original sources
PubMed1990

OP-1 cDNA encodes an osteogenic protein in the TGF-beta family.

Amino acid sequences of two tryptic peptides derived from enriched bovine osteogenic protein preparations revealed considerable homology to two members of the TGF-beta (transforming growth factor beta) supergene family, DPP (decapentaplegic protein) of Drosophila and Vg-1 (vegetal protein) of Xenopus. Building upon this information we constructed a synthetic consensus gene to use as a probe to screen human genomic libraries. This resulted in the isolation of three interrelated genes. Among these were BMP-2b and BMP-3 which have recently been described by others. The third gene, termed OP-1 (osteogenic protein one), is new and was subsequently shown to encode the human homolog of a major component of bovine osteogenic protein. The genomic clones were used to isolate the corresponding complementary DNA (cDNA) clones. Sequence analysis indicates that OP-1 is a relative of the murine Vgr-1 (Vg-1 related gene). This report describes the cDNA structure and putative amino acid sequence of OP-1.

Amino Acid Sequence↗
PubMed1996

Cathepsin K: isolation and characterization of the murine cDNA and genomic sequence, the homologue of the human pycnodysostosis gene.

Cathepsin K(EC 3.4.22.38) is a lysosomal cysteine protease that is strongly implicated in bone resorption. The human cathepsin K gene is highly expressed in osteoclasts and gene mutations cause pycnodysostosis, an autosomal recessive skeletal dysplasia. To investigate the evolutionary relatedness of cathepsin K across species, the mouse cathepsin K gene was isolated. A mouse heart cDNA clone, pMCatKl, contained the 3' untranslated region, mature enzyme coding sequence, and most of the propeptide. The remainder of the gene was amplified from mouse melanocyte RNA using 5' rapid amplification of cDNA ends. The gene contained a 990-bp open reading frame, predicting a 329-amino-acid prepropolypeptide. The structure of the protein included a 15-amino-acid presignal, a 99-amino-acid proregion, and a 215-amino-acid mature enzyme. Two potential N-glycosylation sites were identified, one in the proregion and one in the mature enzyme. The 5' untranslated region was 135 bp. The 3' untranslated region was 470 bp including a 9-bp poly(A) tract and contained two polyadenylation signals. The mouse cathepsin K nucleotide and amino acid sequences were highly conserved with the human, rabbit, and chicken homologues across the proregion and mature enzyme. The mouse cathepsin K gene was isolated from an V129 genomic library, and characterization of its genomic structure and intron sizes revealed exons with the initiation ATG in exon 2 and termination TGA in exon 8, a genomic organization that was highly conserved with its human homologue. The availability of the mouse cathepsin K cDNA and genomic sequences will facilitate generation of a mouse model of cathepsin K deficiency by gene targeting.

Amino Acid Sequence↗
PubMed1995

The gene coding for the mustard trypsin inhibitor-2 is discontinuous and wound-inducible.

The gene coding for the mustard trypsin inhibitor-2 has been isolated from a genomic library and characterized. Comparison of genomic and cDNA sequences indicates that the gene is interrupted by an intron of 193 bp. The eukaryotic peculiar regulatory sequences have been detected in the 5' flanking region of the gene. In addition, a decanucleotide has been detected that is highly similar to the proposed G-box and to the ABRE motifs required for the gene expression induced by methyl jasmonate and abscissic acid. Northern blot analysis demonstrates that the gene is expressed in immature seeds as well as in wounded leaves.

Amino Acid Sequence↗
PubMed1988

Sequence and structure of the nucleolin promoter in rodents: characterization of a strikingly conserved CpG island.

We report the isolation of the complete genes encoding nucleolin from rat and hamster. The DNA clones were obtained from partial genomic libraries by probing with a genomic DNA fragment containing the leader and promoter regions of the mouse nucleolin gene. We have determined the complete nucleotide sequence of the 5'-terminal region for the three rodent species. The sequenced regions extend over 1 kb downstream and upstream from the cap sites and include a conserved CpG island 1500 nucleotides (nt) long. The 5' end of the CpG island in each species has maintained a long alternating purine-pyrimidine sequence which could adopt a Z-DNA conformation. By sequence comparison, 42 blocks of homology are defined in the 5'-terminal region, of which 36 appear in the CpG island and contain numerous conserved CpG dinucleotides. Two blocks, 110 and 49 nt long, encompassing the cap sites and the region immediately upstream, respectively, present features characteristic of regulated genes: a possible TATA box (ATTA), two pyrimidine-rich nucleotide stretches and two inverted juxtaposed CCAAT-like boxes (GGTTGG). Furthermore, the adjacent upstream conserved region presents features characteristic of housekeeping genes: four G/C boxes, embedded in a high G + C-content sequence, among them one presenting a perfect consensus Sp 1-binding site (GCCCCGCCCC). Among unusual features, we report numerous large G + C-rich conserved sequences located in the first intron. One of these sequences contains two G/C boxes which border a sequence presenting a dyad symmetry (GCGCACGTGCTC). Our findings shed some light on the putative role of the CpG island. We show that CpG-rich sequence motifs are under strong selective pressure over the whole 5'-terminal region and are presumably involved in regulatory mechanisms.

Amino Acid Sequence↗
PubMed2002

Petunia actin-depolymerizing factor is mainly accumulated in vascular tissue and its gene expression is enhanced by the first intron.

Actin-depolymerizing factor (ADF) is one of the actin cytoskeleton-modulating proteins. We have characterized the accumulation pattern of petunia ADF proteins. PhADF proteins are accumulated in every petunia organ and their accumulation is differentially regulated by developmental signals. Their cellular localization is vascular tissue-preferential in vegetative organs, whereas somewhat different in reproductive organs. In reproductive organs, PhADFs are present in outer integument, endocarp of ovary wall, transmitting tissue of style, and epidermis and endothecium of young anther. From a petunia genomic library, we have isolated a genomic clone encoding PhADF1. Comparison to complementary DNA sequence revealed that the coding region of PhADF1 gene consists of three exons and two introns. Analysis of chimeric gene expression using beta-glucuronidase as a reporter gene in transgenic Arabidopsis revealed that PhADF1 was strongly expressed in every vegetative tissue except petal. In addition, expression of the gene was highly enhanced by its first intron. These results suggest that PhADF1 gene of petunia is mainly expressed in vascular tissues and its expression is regulated by intron-mediated enhancement mechanism.

Actin Depolymerizing Factors↗
PubMed1986

Mouse thymidylate synthase messenger RNA lacks a 3' untranslated region.

Analysis of the sequence of cDNA corresponding to mouse thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) mRNA revealed that the termination codon TAA was followed immediately by a poly(A) sequence. This raised the possibility that mouse thymidylate synthase mRNA lacks a 3' untranslated region. In the present study, we have further investigated this possibility. DNA corresponding to the 3' end of the thymidylate synthase gene was isolated from a genomic library. The sequence of the genomic DNA was identical to that of the cDNA in the coding region. However, the termination codon was TAG in the genomic sequence rather than TAA, and poly(A) was not present in the genomic DNA. Sequences flanking the site of poly(A) addition were in good agreement with polyadenylylation consensus sequences. S1 nuclease analysis revealed that approximately 80% of the thymidylate synthase mRNA molecules were polyadenylylated at the termination codon. A secondary polyadenylylation site was detected 190-200 nucleotides downstream of the primary site. We conclude that the major species of mouse thymidylate synthase mRNA lacks a 3' untranslated region and that the final A of the termination codon is added by poly(A) polymerase. It appears that a 3' untranslated region is not essential for the accumulation or translation of this mRNA.

Animals↗
PubMed1986

Genomic and cDNA clones for maize phosphoenolpyruvate carboxylase and pyruvate,orthophosphate dikinase: Expression of different gene-family members in leaves and roots.

We have isolated cDNA clones for the maize leaf enzymes phosphoenolpyruvate (P-ePrv) carboxylase [orthophosphate:oxaloacetate carboxy-lyase (phosphorylating) EC 4.1.1.31] and pyruvate,orthophosphate (Prv,P(i)) dikinase (ATP:pyruvate,orthophosphate phosphotransferase, EC 2.7.9.1) by exploiting the light-inducibility and large size of the mRNAs (3.5 kilobases) that encode the two enzymes. The clones were identified by hybrid-selection and immunoprecipitation assays. From a maize genomic library, two different types of genomic clones were screened with both the P-ePrv carboxylase and the Prv,P(i) dikinase cDNA clones. Information from these genomic clones and genome blots indicates that the P-ePrv carboxylase gene family has at least three members and the Prv,P(i) dikinase family at least two. Transcripts for both enzymes were detected in green leaves, etiolated leaves, and roots. The results show that the P-ePrv carboxylase mRNAs in green leaves and roots are encoded by different genes. Whereas the P-ePrv carboxylase mRNAs in all three tissues appear to be the same size, the Prv,P(i) dikinase mRNA in green leaves is about 0.5 kilobases longer than the Prv,P(i) dikinase mRNAs in etiolated leaves and roots. It is possible that all these Prv,P(i) dikinase transcripts are encoded by one gene, and the size differences may correspond to the presence or absence of a sequence encoding a chloroplast transit peptide.

Journal Article↗
PubMed1986

The chicken ubiquitin gene contains a heat shock promoter and expresses an unstable mRNA in heat-shocked cells.

A chicken genomic library was screened to obtain genomic clones for ubiquitin genes. Two genes that differ in their genomic location and organization were identified. One gene, designated Ub I, contains four copies of the protein-coding sequence arranged in tandem, while the second gene, Ub II, contains three. The origin of the two major mRNAs that are induced after heat shock in chicken embryo fibroblasts was determined by generating DNA probes from the 5'-and 3'-noncoding regions of the two genes. Both mRNAs are transcribed from Ub I, the larger being the unspliced precursor of the smaller. A 674-base-pair intron was located within the 5'-noncoding region of Ub I. The second gene, Ub II, does not appear to code for an RNA species in normal or heat-shocked chicken embryo fibroblasts. The expression of ubiquitin mRNA during heat shock and recovery was examined. Addition of actinomycin D before heat shock completely abolished the response of ubiquitin mRNA to the stress. Analysis of the stability of the mRNA during recovery revealed that the mRNA accumulated during the heat shock is rapidly degraded with a half-life of approximately 1.5 h, suggesting a specialized but transient role for ubiquitin during heat shock.

Animals↗
PubMed1988

Cloning and characterization of a core histone gene tandem repeat in Urechis caupo.

A Urechis caupo histone gene tandem repeat has been isolated from a 5.0-kilobase EcoRI genomic library in lambda gtWES.lambda B. Genomic reconstruction experiments indicate that the cloned sequence is repeated approximately 100 times per haploid genome. Unique restriction fragments from the cloned sequence hybridize with individual core histone genes from a histone gene tandem repeat of the sea urchin, Strongylocentrotus purpuratus. No hybridization is detected when restriction digests are probed with a sea urchin H1 histone gene. Hybrid selection and in vitro translation of embryo mRNAs demonstrate that the clone contains sequences complementary to all four core histones; however, no H1 histone is detected among the translation products. Based on a restriction site map of the clone and the subcloned sequences which hybridize to the histone mRNAs, the order of the core histone genes in the clone is shown to be H3 H2A H2B H4. S1 nuclease hybrid protection mapping is used to locate the coding regions and to determine the transcript lengths of the core histone mRNAs. The transcript lengths of H2A, H2B, H3, and H4 mRNAs are approximately 464, 438, 494, and 397 bases, respectively. The S1 nuclease mapping also demonstrates that H2A and H4 are transcribed from one DNA strand while H2B and H3 are transcribed from the other strand. In the tandem repeat, the genes are organized so that transcription of the H2A-H2B and H3-H4 gene pairs is divergent.

Animals↗
PubMed1984

Molecular structures of human argininosuccinate synthetase pseudogenes. Evolutionary and mechanistic implications.

In the human genome there is one expressed gene for argininosuccinate synthetase and 14 pseudogenes. A cDNA coding for human argininosuccinate synthetase was used to screen a human genomic library. Twenty-five unique genomic clones were isolated and extensively characterized. At least seven clones represented processed argininosuccinate synthetase pseudogenes that lost the introns in the expressed gene. Restriction mapping demonstrated that these processed pseudogenes were located in distinct regions of the human genome. Complete nucleotide sequences of two processed pseudogenes, psi AS-1 and psi AS-3, and a partial sequence of psi AS-7 were determined. Both psi AS-1 and psi AS-3 had an adenine-rich region at their 3' end and were flanked by distinct imperfect direct repeats. A comparison of these pseudogene sequences to that of the cDNA demonstrated that psi AS-1 and psi AS-3 were 93% homologous to the cDNA, whereas psi AS-7 was 89% homologous to the cDNA. Therefore, it is estimated that psi AS-1 and psi AS-3 were created 10-11 million years ago, whereas psi AS-7 arose approximately 21 million years ago. We have estimated the evolutionary rate for the expressed argininosuccinate synthetase gene based on the sequences of psi AS-1 and psi AS-3. These data indicate that the expressed argininosuccinate synthetase gene is evolving at a rate similar to that of the beta-globin gene and much faster than the alpha-tubulin gene. Furthermore, a comparison of the sequences of psi AS-1 and psi AS-3 suggests the possibility that these pseudogenes arose from a common intermediate.

Argininosuccinate Synthase↗
PubMed1992

Construction and characterization of a region-specific microdissection library from human chromosome 2q35-q37.

A region-specific genomic library for human chromosome 2q35-q37 has been constructed using the microdissection and polymerase chain reaction-mediated linker-adaptor microcloning method. Twenty fragments from the chromosome region 2q35-q37 were dissected and a library consisting of 20,000 recombinant microclones was obtained. The insert size ranged between 50 and 800 bp, with a mean of approximately 270 bp. About 50-60% of the microclones contained unique sequences. The microdissection library has been demonstrated to derive from the dissected region 2q35-q37 by chromosome painting using the fluorescence in situ hybridization (FISH) technique. Southern blot analysis of the unique sequence microclones from the library showed that 54% (26/48) of the clones are of human origin and chromosome 2 specific. Four of these microclones have been further mapped to the 2q37 region by using a cell hybrid containing only 2q37. The unique sequence microclones have also been characterized for their insert size and the hybridizing genomic fragments cleaved with HindIII. As shown previously, these microclones will be useful in isolating corresponding yeast artificial chromosome (YAC) clones with large inserts for high-resolution physical mapping and also in screening cDNA libraries to isolate expressed gene sequences as candidate genes to facilitate search for the crucial genes underlying genetic diseases and specific forms of cancer assigned to the region.

Blotting, Southern↗
PubMed1989

Cloning of the bovine major histocompatibility complex class II genes.

Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library. The library was constructed in the bacteriophage lambda vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome. Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes. Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis. The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail. These analyses showed that these 37 clones were derived from five different A and 10 different B genes. The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes. Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle.

Animals↗
PubMed1993

Organization of a rainbow trout estrogen receptor gene.

The complete coding region of the estrogen receptor gene was isolated from a rainbow trout genomic library. This gene is divided into ten exons spanning at least 30 kb of genomic DNA. With two exceptions, intron positions are identical to those of the human estrogen receptor gene. The 5' end of the gene, including 1.7 kb of the promoter region, was sequenced. This region exhibits several putative regulatory elements. Localization of a potential estrogen responsive element to the first exon suggests that this gene is autoregulated. This 5' end region was also shown to be able to drive the expression of a CAT reporter gene in Xenopus laevis oocytes.

Amino Acid Sequence↗
PubMed1998

The construction of cDNA libraries from human single preimplantation embryos and their use in the study of gene expression during development.

PURPOSE: The construction and application of polymerase chain reaction (PCR)-based cDNA libraries from unfertilized human oocytes and single preimplantation-stage embryos are described. The purpose of these studies is to provide a readily available resource for the study of gene expression during human preimplantation development. METHODS: Rapid, reproducible, and efficient procedures for the construction of PCR-based cDNA libraries from fewer than 10 cells were first developed in small populations of fibroblast cells. We then constructed cDNA libraries from eight unfertilized oocytes and single two-cell, -4-cell, -7-cell, and blastocyst-stage embryos. Differential display PCR using the libraries as template allows the analysis of stage-specific expression of embryonic genes. Genomic libraries are also prepared from parental samples (cumulus cells and sperm) corresponding to the individual embryo generating the cDNA library. RESULTS: The complexities (between 10(5) and 10(6) clones) of the human embryo libraries indicate that they may represent the entire active gene population at these early stages of human development. Nucleotide sequence analyses of random clones showed the presence of a variety of transcripts, such as the human transposable element. LINE-1, Alu repeat sequences, housekeeping genes, and tissue-specific genes, (e.g., alpha-globin, FMR-1, and interleukin-10). Also present at the expected frequency are the ubiquitous cytoskeletal elements, beta-actin, keratin-18, and alpha-tubulin. In addition to cDNAs corresponding to known expressed sequence tags (ESTs) in the GenBank and dbEST databases, a high proportion of novel sequences was also detected. Several cDNAs were detected only at specific stages of preimplantation development by the differential display analysis. CONCLUSIONS: PCR-based cDNA libraries from single human preimplantation embryos provide a new and important resource for the identification and study of novel genes or gene families. As such, they will increase our basic understanding of the molecular control of human development. In the clinical context, the libraries identify the time of onset of specific genes, and hence the diseases resulting from mutation of these genes, and provide information about new methods of preimplantation diagnosis. The molecular analysis of early gene transcription in human embryogenesis may be expected to lead to advances in contraception, assisted reproduction, and preimplantation genetic diagnosis.

Blastocyst↗
PubMed1994

Molecular evolution of a Y-chromosomal repetitive sequence family in the genus Mus.

A 522-base-long Y-chromosomal sequence was isolated from a BALB/c genomic library and was designated "BF046." It is repeated about 200 times in the male genome, and a difference was detected between the Mus musculus musculus and the M. m. domesticus type Y chromosomes. BF046-related sequences were present over the entire length of the Y chromosome as visualized by in situ hybridization. Southern blot analysis against DNAs isolated from eight species in the genus Mus showed that BF046-related sequences were amplified in the Y chromosomes of three closely related species: M. musculus, M. spicilegus, and M. spretus. To gain insight into the stability of the BF046 sequence family, we isolated 18 additional clones from these three mouse species and compared their sequences. The M. musculus sequences differed from the M. spicilegus and M. spretus sequences by two indels. The remaining parts of the sequences were very similar, but both parsimony and distance-based analytical methods divided the sequences into the same four subgroups, with each species having its own subgroup(s). Thus, the Y chromosomes of M. musculus, M. spicilegus, and M. spretus can be distinguished from one another.

Animals↗
PubMed2006

Extensive genomic plasticity in Pseudomonas aeruginosa revealed by identification and distribution studies of novel genes among clinical isolates.

The distributed genome hypothesis (DGH) states that each strain within a bacterial species receives a unique distribution of genes from a population-based supragenome that is many times larger than the genome of any given strain. The observations that natural infecting populations are often polyclonal and that most chronic bacterial pathogens have highly developed mechanisms for horizontal gene transfer suggested the DGH and provided the means and the mechanisms to explain how chronic infections persist in the face of a mammalian host's adaptive defense mechanisms. Having previously established the validity of the DGH for obligate pathogens, we wished to evaluate its applicability to an opportunistic bacterial pathogen. This was accomplished by construction and analysis of a highly redundant pooled genomic library containing approximately 216,000 functional clones that was constructed from 12 low-passage clinical isolates of Pseudomonas aeruginosa, 6 otorrheic isolates and 6 from other body sites. Sequence analysis of 3,214 randomly picked clones (mean insert size, approximately 1.4 kb) from this library demonstrated that 348 (10.8%) of the clones were unique with respect to all genomic sequences of the P. aeruginosa prototype strain, PAO1. Hypothetical translations of the open reading frames within these unique sequences demonstrated protein homologies to a number of bacterial virulence factors and other proteins not previously identified in P. aeruginosa. PCR and reverse transcription-PCR-based assays were performed to analyze the distribution and expression patterns of a 70-open reading frame subset of these sequences among 11 of the clinical strains. These sequences were unevenly distributed among the clinical isolates, with nearly half (34/70) of the novel sequences being present in only one or two of the individual strains. Expression profiling revealed that a vast majority of these sequences are expressed, strongly suggesting they encode functional proteins.

Bacteriophages↗
PubMed2002

Isolation of A/D and C genome specific dispersed and clustered repetitive DNA sequences from Avena sativa.

DNA gel-blot and in situ hybridization with genome-specific repeated sequences have proven to be valuable tools in analyzing genome structure and relationships in species with complex allopolyploid genomes such as hexaploid oat (Avena sativa L., 2n = 6x = 42; AACCDD genome). In this report, we describe a systematic approach for isolating genome-, chromosome-, and region-specific repeated and low-copy DNA sequences from oat that can presumably be applied to any complex genome species. Genome-specific DNA sequences were first identified in a random set of A. sativa genomic DNA cosmid clones by gel-blot hybridization using labeled genomic DNA from different Avena species. Because no repetitive sequences were identified that could distinguish between the A and D gneomes, sequences specific to these two genomes are refereed to as A/D genome specific. A/D or C genome specific DNA subfragments were used as screening probes to identify additional genome-specific cosmid clones in the A. sativa genomic library. We identified clustered and dispersed repetitive DNA elements for the A/D and C genomes that could be used as cytogenetic markers for discrimination of the various oat chromosomes. Some analyzed cosmids appeared to be composed entirely of genome-specific elements, whereas others represented regions with genome- and non-specific repeated sequences with interspersed low-copy DNA sequences. Thus, genome-specific hybridization analysis of restriction digests of random and selected A. sativa cosmids also provides insight into the sequence organization of the oat genome.

Avena↗
PubMed2000

Human secretin (SCT): gene structure, chromosome location, and distribution of mRNA.

Secretin is an endocrine hormone that stimulates the secretion of bicarbonate-rich pancreatic fluids. Recently, it has been discussed that secretin deficiency may be implicated in autistic syndrome, suggesting that the hormone could have a neuroendocrine function in addition to its role in digestion. In the present study, the human secretin gene (SCT) was isolated from a bacterial artificial chromosome genomic library. SCT contains four exons, with the protein coding regions spanning 713 bp of genomic DNA. Human SCT is similar structurally to the secretin genes of other species. Amino acid conservation, however, is most pronounced within the exon encoding the biologically active mature peptide. Northern blot analysis shows that human SCT transcripts are located in the spleen, intestinal tract, and brain. Radiation hybrid mapping places the SCT locus on chromosome 11p15.5.

Amino Acid Sequence↗