Characterization of the porcine FSCN3 gene: cDNA cloning, genomic structure, mapping and polymorphisms

Fascin 3 (FSCN3)is a testis-specific actin-bundling protein involved in spermatid development. Here we describe the molecular characterisation of the porcine FSCN3 gene. The 1,800-bp cDNA sequence contains a 1,497-bp open reading frame encoding a protein of 498 amino acids with a calculated molecular mass of 56.2 kDa and an isoelectric point of 6.82. The porcine FSCN3 protein shares high identity with other mammalian FSCN3. The FSCN3 gene contains seven exons, spans approximately 9 kb, and maps to pig chromosome 18. We also identified 24 DNA polymorphisms.     

Human arylsulfatase B: MOPAC cloning, nucleotide sequence of a full-length cDNA, and regions of amino acid identity with arylsulfatases A and C

cDNAs encoding the human lysosomal hydrolase, arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase, EC 3.1.6.1), were isolated from a hepatoma cell cDNA library using an ASB-specific oligonucleotide generated by the MOPAC (mixed oligonucleotide primed amplification of cDNA) technique. To facilitate cDNA cloning, human ASB was purified to apparent homogeneity and a total of 112 amino acid residues were microsequenced from the N-terminus and four internal tryptic peptides of the 47-kDa subunit. Based on the ASB N-terminal amino acid sequence, two oligonucleotide mixtures containing inosines to reduce the mixture complexity were constructed and used as primers to amplify an ASB-specific product from human placental cDNA by the polymerase chain reaction. DNA sequencing of this MOPAC product demonstrated colinearity with 21 N-terminal ASB amino acids. Based on this sequence and on codon usage for the adjacent conserved amino acids in human arylsulfatases A and C, a unique 66-mer was synthesized and used to screen a human hepatoma cell cDNA library. Four putative positive cDNA clones were isolated, and the largest insert (pASB-1) was sequenced in both orientations. The 1834-bp pASB-1 insert had a 1278-bp open reading frame encoding 425 amino acids that was colinear with 85 microsequenced amino acids of the purified enzyme, demonstrating its authenticity. Using the pASB-1 cDNA as a probe, a full-length cDNA clone, pASB-4, was isolated from a human testes library and sequenced in both orientations. pASB-4 had a 2811-bp insert containing a 559-bp 5' untranslated sequence, a 1602-bp open reading frame encoding 533 amino acids (six potential N-glycosylation sites), a 641-bp 3' untranslated sequence, and a 9-bp poly(A) tract. Comparison of the predicted amino acid sequences of arylsulfatases A, B, and C revealed regions of identity, particularly in their N-termini.     

Cloning, Genomic Structure, and Expression of Mouse Ring Finger Protein GeneZnf179

ZNF179,a RING finger protein encoding gene, has been mapped within the critical deletion region for Smith–Magenis syndrome (SMS), a disorder characterized by mental retardation and multiple congenital anomalies associated with del(17)(p11.2). Here we report the cloning ofZnf179,the mouse homologue ofZNF179,and characterization of its gene structure. The 3028-bp cDNA has a 1.9-kb open reading frame that contains a RING finger domain at its N-terminus and an alanine-rich and glycine-rich domain at its C-terminus.Znf179genomic sequence includes 15 introns and spans about 10 kb on mouse chromosome 11, which maintains conserved synteny with human 17p. Northern analysis indicates thatZnf179is predominantly expressed in brain and testis. Although contained within the SMS common deletion interval, FISH experiments show thatZNF179is not deleted in two SMS patients with smaller deletions.     

Molecular cloning of a cDNA encoding mouse D-aspartate oxidase and functional characterization of its recombinant proteins by site-directed mutagenesis

Summary. The cDNA encoding D-aspartate oxidase (DASPO) was cloned from mouse kidney RNA by RT–PCR. Sequence analysis showed that it contained a 1023-bp open reading frame encoding a protein of 341 amino acid residues. The protein was expressed in Escherichia coli with or without an N-terminal His-tag and had functional DASPO activity that was highly specific for D-aspartate and N-methyl-D-aspartate. To investigate the roles of the Arg-216 and Arg-237 residues of the mouse DASPO (mDASPO), we generated clones with several single amino acid substitutions of these residues in an N-terminally His-tagged mDASPO. These substitutions significantly reduced the activity of the recombinant enzyme against acidic D-amino acids and did not confer any additional specificity to other amino acids. These results suggest that the Arg-216 and Arg-237 residues of mDASPO are catalytically important for full enzyme activity.     

Identification of the Tslc1 gene,a mouse orthologue of the human tumor suppressor TSLC1 gene

We have recently identified the TSLC1 gene as a novel tumor suppressor in human non-small lung cancer on chromosome 11q23.2. TSLC1 encodes a membrane glycoprotein showing significant homology with immunoglobulin superfamily molecules. Here, we report the isolation of a mouse orthologous gene, Tslc1. The Tslc1 cDNA contains a single open reading frame of 1335 bp encoding a putative protein of 445 amino acids, and its expression was detected in all tissues examined. The Tslc1 gene is mapped on mouse chromosome 9, a synteny of human chromosome 11q, and is composed of ten exons, the exon-intron junctions being highly conserved between human and mouse. The predicted amino acids of mouse Tslc1 display 98% identity with that of human TSLC1. Furthermore, data base analysis indicates that the amino acid sequences corresponding to the cytoplasmic domain of Tslc1 are identical in five mammals and highly conserved in vertebrates, suggesting an important role of Tslc1 in normal cell-cell interaction.     

Isolation and characterization of a novel RING-H2 finger gene induced in response to cold and drought in the interfertile Citrus relative Poncirus trifoliata

Many genes in different organisms encode proteins with really interesting gene (RING) finger domain(s). The RING zinc finger domain is involved in a wide variety of functions in diverse organisms. A cDNA clone showing homology with RING zinc finger genes and nine-fold induction in response to cold was previously identified during a gene expression study in the interfertile Citrus relative Poncirus trifoliata (L.) Raf. In this study, the full-length cDNA of this clone was isolated from 2-day cold-acclimated P. trifoliata by a rapid amplification of cDNA ends method using gene-specific primers. The full-length cDNA was 956 bp containing a complete open reading frame of 474 bp encoding a polypeptide of 158 amino acids. The full-length cDNA showed a high level of homology with genes encoding putative RING zinc finger proteins in plants. The deduced amino acid sequence of this gene contained a signature sequence motif for a RING zinc finger close to the C terminus of the protein. The RING zinc finger domain was significantly similar to previously characterized RING zinc finger proteins from different organisms. Additionally, it had a histidine residue at the fifth co-ordination site, indicating that this gene encodes a RING-H2 finger protein. Northern blot hybridization showed that the expression of the RING finger gene was induced in response to cold in cold-hardy P. trifoliata but not to the same extent in cold-sensitive Citrus grandis L. Osb. (pummelo). However, the gene was induced by drought stress similarly in both the species. To our knowledge, this study presents the first isolation of the full-length sequence of a RING zinc finger gene induced in response to abiotic stress in plants and the initial characterization of this gene in Citrus .     

Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans

Golgi alpha-mannosidase II (GlcNAc transferase I-dependent alpha 1,3[alpha 1,6] mannosidase, EC 3.2.1.114) catalyzes the final hydrolytic step in the N-glycan maturation pathway acting as the committed step in the conversion of high mannose to complex type structures. We have isolated overlapping clones from a murine cDNA library encoding the full length alpha-mannosidase II open reading frame and most of the 5' and 3' untranslated region. The coding sequence predicts a type II transmembrane protein with a short cytoplasmic tail (five amino acids), a single transmembrane domain (21 amino acids), and a large COOH-terminal catalytic domain (1,124 amino acids). This domain organization which is shared with the Golgi glycosyl-transferases suggests that the common structural motifs may have a functional role in Golgi enzyme function or localization. Three sets of polyadenylated clones were isolated extending 3' beyond the open reading frame by as much as 2,543 bp. Northern blots suggest that these polyadenylated clones totaling 6.1 kb in length correspond to minor message species smaller than the full length message. The largest and predominant message on Northern blots (7.5 kb) presumably extends another approximately 1.4-kb downstream beyond the longest of the isolated clones. Transient expression of the alpha-mannosidase II cDNA in COS cells resulted in 8-12-fold overexpression of enzyme activity, and the appearance of cross-reactive material in a perinuclear membrane array consistent with a Golgi localization. A region within the catalytic domain of the alpha-mannosidase II open reading frame bears a strong similarity to a corresponding sequence in the rat liver endoplasmic reticulum alpha-mannosidase and the vacuolar alpha-mannosidase of Saccharomyces cerevisiae. Partial human alpha-mannosidase II cDNA clones were also isolated and the gene was localized to human chromosome 5.     

Cloning and sequencing of the cDNA encoding tyrosinase of the Japanese pond frog, Rana nigromaculata.

We cloned and sequenced the cDNA encoding tyrosinase (TYN) of the Japanese pond frog, Rana nigromaculata. The 3511-bp cDNA contained a 54-bp 5'-noncoding region, a 1596-bp open reading frame encoding TYN of 532 amino acids (aa), and a 1861-bp 3'-noncoding region. The aa sequence of frog TYN predicted from the cDNA sequence was homologous to that of mouse and human TYNs. The aa sequence including the copper-binding domain, which is likely the active center of TYN, was highly conserved among these three species and Neurospora crassa, Streptomyces antibioticus, and S. glaucescens. The frog TYN also contains possible glycosylation sites and conserved Cys at sites similar to those in the mouse and human TYNs. There are two hydrophobic regions at the N-terminus and near the C-terminus, which are likely the signal (leader) peptide and a transmembrane domain, respectively.     

Primary structure of the embryo-expressed gene KE2 from the mouse H-2K region.

Nucleotide (nt) sequence of the KE2 wild-type (wt) cDNA revealed a novel 669-bp open reading frame encoding a putative hydrophilic protein of 127 amino acids, pI 6.17. Comparison of the wt to the genomic nt sequence from the tw5 mutant shows the KE2 gene is conserved and is probably a functional gene unrelated to the tw5 lethality.     

Molecular cloning of LMO41, a new human LIM domain gene

We have identified a new human LIM domain gene by isolating an autoantigenic cDNA clone from a human breast tumor cDNA library. The predicted amino acid sequence of the cDNA clone's 495 bp open reading frame contains two tandem LIM domain motifs, and within the LIM domain region there is 62% identity with the analogous region of the LIM-only gene LMO1. The homology to LMO1 is restricted to the 360 bp region encoding the tandemly repeated LIM domains, the rest of the open reading frame as well as the extensive, GC-rich 5' untranslated region, and 3' region of the 2 kb cDNA sequence are unrelated to any known genes. This gene has been designated LMO4.     

Cloning of rat parkin cDNA and distribution of parkin in rat brain

The rat parkin cDNA sequence was characterized after screening a rat hypothalamus cDNA library with a 32P-labeled probe containing the entire open reading frame of the human parkin cDNA. This sequence encompasses 1,576 bp and contains a single open reading frame that encodes a 465-amino acid protein. The rat parkin amino acid sequence exhibits a very striking homology to the human and mouse parkin, with 85 and 95% identity, respectively. Both the N-terminal ubiquitin and the ring-IBR (in between ring)-ring finger domains appear to be highly conserved among rat, human, and mouse parkin. An affinity-purified polyclonal antibody (ASP5p) was generated with a synthetic peptide corresponding to amino acids 295-311 of the parkin sequence, which is identical in the three species. Western blotting revealed that ASP5p recognizes a single 52-kDa band, which corresponds to the molecular mass of the parkin protein. Immunostaining with ASP5p showed that parkin is principally located in the cytoplasm of neurons that are widely distributed in the rat brain. Parkin-immunoreactive neurons abound in structures that are specifically targeted in Parkinson's disease, e.g., subtantia nigra, but are also present in unaffected structures, e.g., cerebellum. Furthermore, parkin-enriched glial cells can be detected in various nuclei of the rat brain. Thus, the role of parkin may be much more global than previously thought on the basis of genetic findings gathered in cases of early-onset parkinsonism.     

Molecular characterization of an arachnid sodium channel gene from the varroa mite (Varroa destructor)

Voltage-gated sodium channels are essential for the generation and propagation of action potentials in most excitable cells. They are the target sites of several classes of insecticides and acaricides. Isolation of full-length sodium channel cDNA is a critical and often difficult step toward an understanding of insecticide and acaricide resistance. We previously cloned and sequenced two overlapping cDNA clones covering segment 3 of domain II (IIS3) to segment 6 of domain IV (IVS6) of an arachnid sodium channel gene (named VmNa) from the varroa mite (Varroa destructor) (J. Apicultureal Res. 40 (2002) 5.). In this study, we isolated three more overlapping cDNA clones and revealed the entire coding region of VmNa (Genbank accession number: AY259834), thus providing the first complete cDNA sequence of an arachnid sodium channel gene. The composite VmNa cDNA contains 6645 nucleotides with an open reading frame encoding 2215 amino acids. The deduced amino acid sequence of VmNa shares a 51% overall identity with Drosophila Para and a 41% identity with the mammalian sodium channel alpha-subunit Na(v)1.2. All hallmarks of sodium channel proteins are conserved in the VmNa protein. Three optional exons and one retained intron were identified in VmNa. The precise position and size of only one exon is conserved in three insect sodium channel genes and mammalian Na(v)1.6 genes in human, mouse and fish, whereas the other three are novel. Interestingly, one of the novel exons is located in the C-terminus, where no alternative exons have been identified in any other sodium channel gene.     

Characterization of the cDNA coding for mouse plasminogen and localization of the gene to mouse chromosome 17

A full-length cDNA coding for mouse plasminogen has been isolated and characterized. The cDNA is 2720 bp in length (excluding the poly(A) tail) and contains a 24-bp 5' noncoding region, an open reading frame of 2436 bp, and a 3' noncoding region of 257 bp. The open reading frame codes for 812 amino acids and includes a signal peptide that is likely 19 amino acids in length and the mature protein of 793 amino acids. The calculated Mr of mouse plasminogen is 88,706 excluding carbohydrate. There are two potential N-linked carbohydrate addition sites; one of which is glycosylated in human, bovine, and porcine plasminogens. Mouse plasminogen was found to contain two additional amino acids compared to the human protein. In addition, mouse and human plasminogens were found to be 79 and 76% identical at the protein and DNA levels, respectively. Analysis of the segregation of two allelic forms, Plgb and Plgd, of plasminogen DNA in three sets of recombinant inbred strains has allowed the localization of the mouse plasminogen gene to the proximal end of mouse chromosome 17 within the t complex and close to the locus D17Rp17. The Plg gene is deleted in the semidominant deletion mutant, hair-pintail (Thp).