Cloning, expression, and chromosomal mapping of a human ganglioside sialidase.

Here we report the cDNA sequence of a human ganglioside sialidase. The cDNA was isolated from a human brain cDNA library by screening with a 240 bp probe generated by polymerase chain reaction using primers based on the sequences of rat cytosolic and bovine membrane sialidases which we previously cloned. The 3.0 kb cDNA encodes an open reading frame of 436 amino acids containing a putative transmenbrane domain and an Arg-Ile-Pro and three Asp-box sequences characteristic of sialidases and showing overall 83% and 39% identities to the bovine and rat enzymes, respectively. Northern blot analysis revealed high expression in skeletal muscle and testis, but low level in kidney, placenta, lung, and digestive organs. Transient expression of the cDNA in COS-1 cells resulted in a 130-fold increase in sialidase activity compared to the control level, and the activity was found to be almost specific for gangliosides. Fluorescent in situ hybridization allowed the human sialidase gene localized to chromosome 11 at q 13.5.     

Nucleotide sequence of rat liver iodothyronine 5'-monodeiodinase (5' MD): its identity with the protein disulfide isomerase

We report here the isolation and sequence of a near full length cDNA clone for the 5'MD. Screening of gt11 cDNA library with a 32P-labeled partial 5'MD clone (#23) yielded two further clones (#2301 and 2302). Clone 2301 was contained entirely within clone 23 while clone 2302 contained 0.5 kb upstream of 5' end and 1.0 kb downstream 3' end of clone 23. Clone 2302 has an open reading frame of 1,447 nucleotides followed by a stop codon and 584 nucleotides of the untranslated 3' end region. The predicted amino acid sequence showed a 99% and a 95% identity with protein disulfide isomerase (PDI) and the membrane associated thyroid hormone binding protein (MTHBP), respectively. Monoclonal antibodies against human placental PDI (HP13) neutralized the 5'MD and showed only one band in western blot analysis of rat liver solubilized microsomal proteins. The results suggest that clone 2302, MTHBP and PDI may be the same protein and that it represents 5' MD.     

Identification of a cDNA clone that contains the complete coding sequence for a 140-kD rat NCAM polypeptide

Neural cell adhesion molecules (NCAMs) are cell surface glycoproteins that appear to mediate cell-cell adhesion. In vertebrates NCAMs exist in at least three different polypeptide forms of apparent molecular masses 180, 140, and 120 kD. The 180- and 140-kD forms span the plasma membrane whereas the 120-kD form lacks a transmembrane region. In this study, we report the isolation of NCAM clones from an adult rat brain cDNA library. Sequence analysis indicated that the longest isolate, pR18, contains a 2,574 nucleotide open reading frame flanked by 208 bases of 5' and 409 bases of 3' untranslated sequence. The predicted polypeptide encoded by clone pR18 contains a single membrane-spanning region and a small cytoplasmic domain (120 amino acids), suggesting that it codes for a full-length 140-kD NCAM form. In Northern analysis, probes derived from 5' sequences of pR18, which presumably code for extracellular portions of the molecule hybridized to five discrete mRNA size classes (7.4, 6.7, 5.2, 4.3, and 2.9 kb) in adult rat brain but not to liver or muscle RNA. However, the 5.2- and 2.9-kb mRNA size classes did not hybridize to either a large restriction fragment or three oligonucleotides derived from the putative transmembrane coding region and regions that lie 3' to it. The 3' probes did hybridize to the 7.4-, 6.7-, and 4.3-kb message size classes. These combined results indicate that clone pR18 is derived from either the 7.4-, 6.7-, or 4.3-kb adult rat brain RNA size class. Comparison with chicken and mouse NCAM cDNA sequences suggests that pR18 represents the amino acid coding region of the 6.7- or 4.3-kb mRNA. The isolation of pR18, the first cDNA that contains the complete coding sequence of an NCAM polypeptide, unambiguously demonstrates the predicted linear amino acid sequence of this probable rat 140-kD polypeptide. This cDNA also contains a 30-base pair segment not found in NCAM cDNAs isolated from other species. The significance of this segment and other structural features of the 140-kD form of NCAM can now be studied.     

Uracil DNA glycosylase-mediated cloning of polymerase chain reaction-amplified DNA: application to genomic and cDNA cloning.

A simple and rapid method for cloning of amplification products directly from the polymerase chain reaction (PCR) has been developed. The method is based on the addition of a 12-base dUMP-containing sequence (CUACUACUACUA) to the 5' end of PCR primers. Incorporation of these primers during PCR results in the selective placement of dUMP residues into the 5' end of amplification products. Selective degradation of the dUMP residues in the PCR products with uracil DNA glycosylase (UDG) disrupts base pairing at the termini and generates 3' overhangs. Annealing of 3' protruding termini to vector DNA containing complementary 3' ends results in chimeric molecules which can be transformed, with high efficiency, without in vitro ligation. Directional cloning of PCR products has also been accomplished by incorporating different dU-containing sequences at the end of each PCR primer. Substitution of all dT residues in PCR primers with dU eliminates cloning of aberrant "primer dimer" products and enriches cloning of genuine PCR products. The method has been applied to cloning of inter-Alu DNA sequences from human placental DNA. Using a single primer, DNA sequences between appropriately oriented Alu sequences were amplified and cloned. Cloning of cDNA for the glyceraldehyde-3'-phosphate dehydrogenase gene from rat brain RNA was also demonstrated. The 3' end region of this gene was amplified by the 3' RACE method and the amplified DNA was cloned after UDG digestion. Characterization of cloned DNAs by sequence analysis showed accurate repair of the cloning junctions. The ligase-free cloning method with UDG should prove to be a widely applicable procedure for rapid cloning of PCR-amplified DNA.     

蜡样杆菌（Bacillus cereus）M22 Mn-SOD cDNA片断的克隆

分析不同种细菌Mn SOD氨基酸序列的保守区域 ,设计一对简并引物进行RT PCR扩增 ,产物经T A载体连接转化大肠杆菌JM 1 0 9,筛选阳性克隆、测序并进行同源性分析 ,得到 4 36bpMn SODcDNA片段。根据此片段设计 5′端特异性引物 ,然后进行 5′RACE扩增 ,获得Mn SOD 5′端 387bpcDNA片断。将 2段序列进行拼接获得 5 94bpcDNA片断 ,编码 1 72个氨基酸。对推定氨基酸序列进行BLAST分析 ,结果表明其与炭疽芽孢杆菌 (Bacillusanthracis)同源性为 95 % ,且Gly77,Aly78,Phe86,Gln1 50 和Asp1 51 在已报道的Mn SOD中均存在 ,构成Mn SOD的活性中心。表明该序列为蜡样芽孢杆菌Mn SOD的cDNA序列     

The cloning and characterization of a second brain enzyme with NAAG peptidase activity

The peptide neurotransmitter N-acetylaspartylglutamate is inactivated by extracellular peptidase activity following synaptic release. It is speculated that the enzyme, glutamate carboxypeptidase II (GCPII, EC 3.14.17.21), participates in this inactivation. However, CGCPII knockout mice appear normal in standard neurological tests. We report here the cloning and characterization of a mouse enzyme (tentatively identified as glutamate carboxypeptidase III or GCPIII) that is homologous to an enzyme identified in a human lung carcinoma. The mouse peptidase was cloned from two non-overlapping EST clones and mouse brain cDNA using PCR. The sequence (GenBank, AY243507) is 85% identical to the human carcinoma enzyme and 70% homologous to mouse GCPII. GCPIII sequence analysis suggests that it too is a zinc metallopeptidase. Northern blots revealed message in mouse ovary, testes and lung, but not brain. Mouse cortical and cerebellar neurons in culture expressed GCPIII message in contrast to the glial specific expression of GCPII. Message levels of GCPIII were similar in brains obtained from wild-type mice and mice that are null mutants for GCPII. Chinese hamster ovary (CHO) cells transfected with rat GCPII or mouse GCPIII expressed membrane bound peptidase activity with similar V(max) and K(m) values (1.4 micro m and 54 pmol/min/mg; 3.5 micro m and 71 pmol/min/mg, respectively). Both enzymes are activated by a similar profile of metal ions and their activities are blocked by EDTA. GCPIII message was detected in brain and spinal cord by RT-PCR with highest levels in the cerebellum and hippocampus. These data are consistent with the hypothesis that nervous system cells express at least two differentially distributed homologous enzymes with similar pharmacological properties and affinity for NAAG.     

Restriction enzyme site-directed amplification PCR: a tool to identify regions flanking a marker DNA

An innovative combination of various recently described molecular methods was set up to efficiently identify regions flanking a marker DNA in insertional mutants of Chlamydomonas. The technique is named restriction enzyme site-directed amplification PCR (RESDA-PCR) and is based on the random distribution of frequent restriction sites in a genome and on a special design of primers. The primer design is based on the presence of a restriction site included in a low degenerated sequence at the 3' end and of a specific adapter sequence at the 5' end, with the two ends being linked by a polyinosine bridge. Specific primers of the marker DNA combined with the degenerated primers allow amplification of DNA fragments adjacent to the marker insertion by using two rounds of either short or long cycling procedures. Amplified fragments from 0.3 to 2 kb or more are routinely obtained at sufficient purity and quantity for direct sequencing. This method is fast, is reliable (87% success rate), and can be easily extrapolated to any organism and marker DNA by designing the appropriate primers. A procedure involving the PCR over enzyme digest fragments is also proposed for when, exceptionally, positive results are not obtained.     

Annealing control primer system for improving specificity of PCR amplification

A novel primer designed to improve the specificity of PCR amplification, called the annealing control primer (ACP), comprises a tripartite structure with a polydeoxyinosine [poly(dI)] linker between the 3' end target core sequence and the 5' end nontarget universal sequence. We show that this ACP linker prevents annealing of the 5' end nontarget sequence to the template and facilitates primer hybridization at the 3' end to the target sequence at specific temperatures, resulting in a dramatic improvement of annealing specificity. The effect of this linker is demonstrated by the incorporation of ACP sequences as primers during the amplification of target nucleotide sequence and as hybridization probes in the genotyping of single nucleotide polymorphisms. This is the first report to show that a poly(dI) linker between two different sequences of ACP forms a bubble-like structure and disrupts or destabilizes DNA duplex formation at certain annealing temperatures.     

Cloning, Tissue Expression Pattern, and Chromosome Location of a Novel Human Gene BRI3BP

A novel cDNA fragment was identified from a human fetal brain cDNA library by using the coding sequence of human BRI3 gene (Accession No. NM015379) as bait in a yeast two-hybrid screening. Then by 5'-RACE (rapid amplification of cDNA end) and electronic hybridization, we obtained a 1.9 kb contig which consists of a novel gene. It was designated as BRI3BP by the HUGO Nomenclature Committee. It contains an open reading frame encoding 251 amino acids. The calculated molecular weight of the deduced protein is 27.8 kU. The predicted isoelectric point is 9.48. Northern hybridization showed its mRNA was highly expressed in brain, kidney, and liver. By RH mapping, the BRI3BP gene was mapped to human chromosome 12q24.2-qter     

Human 2-5A synthetase: characterization of a novel cDNA and corresponding gene structure.

The enzyme 2-5A synthetase is induced in cultured cells in response to interferon (IFN) treatment. A lambda gt10 cDNA library of mRNA from IFN-induced Daudi lymphoblastoid cells was screened with oligonucleotide probes. Several overlapping cDNAs were isolated and shown to be derived from the human synthetase gene using filter selection and oocyte microinjection assays. The nucleotide sequence of one of these, cDNA 8-2, extended the 2-5A synthetase sequence already described 72 bp in the 5' direction but was found to differ significantly in coding sequence at the 3' end. The longest cDNA isolated (6-2) was approximately 1.4 kb. By Northern hybridization analysis single mRNAs of 1.7 kb were detected in Daudi and T98G (glioblastoma) cells. However, in HeLa cells, four mRNAs ranging in size from 1.5 to 3.5 kb were found, one of which differed at the 3' end. Analysis of both phage and cosmid genomic clones and comparison with genomic DNA indicate that there is a single gene for 2-5A synthetase, comprising at least six exons and five introns, which can undergo a novel form of alternative RNA processing depending on cell type.     

Structural and functional characterizations of the 5'-flanking region of the mouse glucagon receptor gene: comparison with the rat gene

A putative proximal promoter was defined previously for the mouse glucagon receptor (GR) gene. In the present study, a distal promoter was characterized upstream from a novel non-coding exon revealed by the 5'-rapid amplification of cDNA ends from mouse liver tissue. The 5'-flanking region of the mouse GR gene was cloned up to 6 kb and the structural organization was compared to the 5' untranslated region of the rat gene cloned up to 7 kb. The novel exon, separated by an intron of 3.8 kb from the first coding exon, displayed a high homology (80%) with the most distal of the two untranslated exons found in the 5' region of the rat GR gene. The mouse distal promoter region, extending up to -1 kb from the novel exon, displayed 85% identity with the rat promoter. Both contain a highly GC-rich sequence with five putative binding sites for Sp1, but no consensus TATA or CAAT elements. To evaluate basal promoter activities, 5'-flanking sequences of mouse or rat GR genes were fused to a luciferase reporter gene and transiently expressed in a mouse and in a rat cell line, respectively or in rat hepatocytes. Both mouse and rat distal promoter regions directed a high level of reporter gene activity. Deletion of the Sp1 binding sites region or mutation of the second proximal Sp1 sequence markedly reduced the distal promoter activity of the reporter gene. The mouse proximal promoter activity was 2- to 3-fold less than the distal promoter, for which no functional counterpart was observed in the similar region of the rat gene.     

Construction and characterization of cDNA clones encoding the 5' end of the chicken pro alpha 1(I) collagen mRNA

As a first step in isolating the 5' end of the chicken pro alpha 1(I) collagen gene, we constructed cDNA clones complementary to the 5' end of the pro alpha 1(I) mRNA using synthetic oligodeoxynucleotides complementary to a conserved region within the N-terminal telopeptide as primers. cDNA clones corresponding to the 5'-untranslated region, signal peptide, N-propeptide and telopeptide were identified based on homology with the human pro alpha 1(I) collagen protein sequence, and on hybridization to pro alpha 1(I) mRNA on Northern blots. A comparison of the nucleotide sequence of these clones with the sequence of the 5' end of the pro alpha 2(I) collagen mRNA confirms that there is 84% homology in a 49-bp region surrounding the translation start point, and shows that there is 70% homology in the nucleotide sequences encoding the N-propeptide triple helical region of the two type-I collagen chains.