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 cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4'-hydroxylase

A cytochrome P-450 (P-450) multigene family codes for several related human liver enzymes, including the P-450 responsible for (S)-mephenytoin 4'-hydroxylation. This enzyme activity has previously been shown to be associated with a genetic polymorphism. Genomic (Southern) blot analysis using non-overlapping 5' and 3' portions of a cDNA clone suggests that approximately seven related sequences are present in this gene family. In this study four cDNA clones, all nearly full-length, were isolated from a bacteriophage lambda gt11 library prepared from a single human liver. These clones can be grouped into two categories that are approximately 85% identical at the level of DNA sequence. The cDNA clones in one category (MP-4, MP-8) both match the N-terminal sequences of the P-450MP-1 and P-450MP-2 proteins, which had previously been shown to be catalytically active in (S)-mephenytoin 4'-hydroxylation. These two cDNAs, MP-4 and MP-8, differ in only two bases in the coding region but are quite distinct in their 3' noncoding regions. Another protein (P-450MP-3) was isolated on the basis of its immunochemical similarity to P-450MP-1 but was found to be catalytically inactive; amino acid sequencing of tryptic peptides of P-450MP-3 showed a correspondence to the second category of cDNA clones (MP-12, MP-20), which differ from each other in only four (nonsilent) base changes. Oligonucleotides specific for the two groups of cDNA clones were used as probes of human liver mRNAs--individual liver samples examined expressed both types of mRNAs but no correlation was observed between the abundance levels of any mRNA and catalytic activity. Further, oligonucleotide probes indicated that mRNAs corresponding to both the MP-4 and MP-8 clones were apparently present in individual liver samples. A monoclonal antibody was isolated that recognized P-450MP-1 but not P-450MP-2 or P-450MP-3; the amount of protein detected by the antibody in different liver samples was not correlated with the mephenytoin 4'-hydroxylase activity. These results indicate that several closely related P-450 genes are all expressed in individual human livers. The MP-4/MP-8 gene products are proposed to be the ones most likely involved in mephenytoin 4'-hydroxylation, and much of the variation in catalytic activity among individuals is not a result of differences in levels of P-450MP-1 or mRNA but may be due to base differences in the structural gene(s).     

Characterization of three novel human cadherin genes (CDH7, CDH19, and CDH20) clustered on chromosome 18q22-q23 and with high homology to chicken cadherin-7

Full-length coding sequences of two novel human cadherin cDNAs were obtained by sequence analysis of several EST clones and 5' and 3' rapid amplification of cDNA ends (RACE) products. Exons for a third cDNA sequence were identified in a public-domain human genomic sequence, and the coding sequence was completed by 3' RACE. One of the sequences (CDH7L1, HGMW-approved gene symbol CDH7) is so similar to chicken cadherin-7 gene that we consider it to be the human orthologue. In contrast, the published partial sequence of human cadherin-7 is identical to our second cadherin sequence (CDH7L2), for which we propose CDH19 as the new name. The third sequence (CDH7L3, HGMW-approved gene symbol CDH20) is almost identical to the mouse "cadherin-7" cDNA. According to phylogenetic analysis, this mouse cadherin-7 and its here presented human homologue are most likely the orthologues of Xenopus F-cadherin. These novel human genes, CDH7, CDH19, and CDH20, are localized on chromosome 18q22-q23, distal of both the gene CDH2 (18q11) encoding N-cadherin and the locus of the six desmosomal cadherin genes (18q12). Based on genetic linkage maps, this genomic region is close to the region to which Paget's disease was linked. Interestingly, the expression patterns of these three closely related cadherins are strikingly different.     

Mouse and human CD14 (myeloid cell-specific leucine-rich glycoprotein) primary structure deduced from cDNA clones

cDNA clones complementary to MS7-4 (Setoguchi et al. (1988) Somat. Cell Mol. Genet. 14, 427-438) from a mouse macrophage cDNA library were separated. Sequence analysis of these clones demonstrated that the longest cDNA clone, MS7X, had a 1366 bp insert and high homology with that of the human CD14 gene (Ferrero and Goyert (1988) Nucleic Acids Res. 16, 4173). Using the MS7X cDNA probe, cDNA clones were separated from cDNA libraries constructed from a human macrophage cell line and macrophages. The total cDNA sequence was 1364 bp in length, with an open reading frame of 1125 nucleotides matching that of the human CD14 gene except for one nucleotide difference. The amino-acid sequence (mouse CD14), deduced from the nucleotide sequence of the MS7X insert consisted of 351 amino-acid residues with a high leucine content (17.66%) and five putative N-glycosylation sites, and in vitro translation predicted a protein of molecular mass of 37.5 kDa. Human CD14 had 356 amino-acid residues, with high leucine content (15.5%), and contained four putative N-glycosylation sites. Mouse CD14 showed 13 building blocks, of which internal nine blocks have a conserved leucine motif and significant homology with human leucine-rich alpha 2-glycoprotein.     

cDNA cloning, characterization and chromosome mapping of the gene encoding human cartilage associated protein (CRTAP)

We have recently isolated and characterized cDNA clones coding for a novel developmentally regulated avian and mouse embryo protein, CASP for Cartilage Associated Protein. Here we describe the isolation and characterization of the gene coding for the human CASP. The comparison of the putative human and mouse protein sequences with the chick sequence revealed an overall high identity (89% and 51%, respectively). Homology search with known DNA and protein sequences showed that CASPs are related to two mammalian nuclear proteins. Here we demonstrate definitively that CASPs are distinct from these nuclear proteins. However, sequence comparison analyses suggest that all of these proteins belong to a new family. In all human tissues examined two CASP mRNA species were detected, whereas a single mRNA and three mRNAs were found in chick and mouse, respectively. The human CASP gene (CRTAP) was assigned to chromosome 3p22 by fluorescence in situ hybridization.     

Human gastric intrinsic factor: characterization of cDNA and genomic clones and localization to human chromosome 11

A human gastric intrinsic factor (IF) cDNA clone was isolated using a rat cDNA clone as a probe. Comparison of the predicted amino acid sequence revealed 80% identity of human IF with rat IF. These cDNA clones were used to isolate and map two overlapping clones encoding the human IF gene. The first exon of the cloned region (exon 2) contains 30 bp of the 5' untranslated region, the signal peptide, and the first 8 amino acids of the mature protein. Exons 3-10 encode the remainder of the coding and 3' noncoding regions. Southern analysis of genomic DNA indicated the presence of a single human IF gene and also revealed the presence of strong hybridizing sequences in genomic DNA from monkey, rat, mouse, cow, and human, suggesting that the IF gene is well conserved. The IF gene was localized to human chromosome 11 by concurrent cytogenetic and cDNA probe analysis of a panel of human X mouse somatic cell hybrids. Southern analysis of genomic DNA from patients with congenital pernicious anemia (lacking intrinsic factor) revealed normal restriction fragment patterns, suggesting that a sizable gene deletion was not responsible for the deficiency.     

The human alpha 2(XI) collagen (COL11A2) chain. Molecular cloning of cDNA and genomic DNA reveals characteristics of a fibrillar collagen with differences in genomic organization

We have isolated three overlapping cDNA clones encoding the pro alpha 2(XI) collagen chain from a human chondrocyte cDNA library. Together, the cDNAs code for 257 uninterrupted Gly-X-Y triplets (almost 80% of the triple helical domain) and about 200 amino acid residues of the carboxyl telopeptide and carboxyl propeptide. The identification of the clones as pro alpha 2(XI) cDNAs was based on the complete identity between the amino acid sequences of three tryptic peptides derived from human alpha 2(XI) collagen and the cDNA-derived sequence. We have also sequenced six exons within a human genomic alpha 2(XI) cosmid clone. This sequence shows that although type XI collagen belongs to the fibril-forming class of collagens, there are substantial differences in exon sizes at the 3' end of the gene when comparing the alpha 2(XI) gene with those of human types I, II, and III collagens. Finally, pro alpha 2(XI) cDNA has been used as a probe to determine the location of the gene by in situ hybridization of chromosome spreads. The results demonstrate that the gene is located close to the region p212 on chromosome 6. Northern blot analysis shows that the gene is expressed in cartilage but not in adult liver, skin, and tendon.     

Discovery of new human beta-defensins using a genomics-based approach

Epithelial beta-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a <1 Mb region of chromosome 8p22-p23. To identify new defensin genes, the DNA sequence from a contig of large-insert genomic clones from the region containing human beta-defensin-2 (HBD-2) was analyzed for the presence of defensin genes. This sequence survey identified a novel beta-defensin, termed HBD-3. The HBD-3 gene contains two exons, is located 13 kb upstream from the HBD-2 gene, and it is transcribed in the same direction. A partial HBD-3 cDNA clone was amplified from cDNA derived from IL-1beta induced fetal lung tissue. The cDNA sequence encodes for a 67 amino acid peptide that is approximately 43% identical to HBD-2 and shares the beta-defensin six cysteine motif. By PCR analysis of two commercial cDNA panels, HBD-3 expression was detected in adult heart, skeletal muscle, placenta and in fetal thymus. From RT-PCR experiments, HBD-3 expression was observed in skin, esophagus, gingival keratinocytes, placenta and trachea. Furthermore, in fetal lung explants and gingival keratinocytes, HBD-3 mRNA expression was induced by IL-1beta. Additional sequence analysis identified the HE2 (human epididymis secretory protein) gene 17 kb upstream from the HBD-3 gene. One splice variant of this gene (HE2beta1) encodes a beta-defensin consensus cysteine motif, suggesting it represents a defensin gene product. HE2beta1 mRNA expression was detected in gingival keratinocytes and bronchial epithelia using RT-PCR analysis. The discovery of these novel beta-defensin genes may allow further understanding of the role of defensins in host immunity at mucosal surfaces.     

Molecular cloning of cDNAs derived from a novel human intestinal mucin gene

A human small intestinal lambda gt11 cDNA library was screened with antibodies to deglycosylated small intestinal mucin. Four partial cDNA clones were isolated that define a novel human mucin gene. These include two partial cDNA clones, SIB 124 and SIB 139, that contain 51 nucleotide tandem repeats which encode a seventeen amino acid repetitive peptide with a consensus sequence of HSTPSFTSSITTTETTS. SIB 139 hybridized to messages produced by small intestine, colon, colonic tumors and also by high mucin variant LS174T colon cancer cells. The gene from which cDNAs SIB 124 and SIB 139 are derived (proposed name MUC 3) maps to chromosome 7, distinct from other known human mucin genes.     

Identification of a putative gamma-aminobutyric acid (GABA) receptor subunit rho2 cDNA and colocalization of the genes encoding rho2 (GABRR2) and rho1 (GABRR1) to human chromosome 6q14-q21 and mouse chromosome 4.

Screening of a genomic DNA library with a portion of the cDNA encoding the gamma-aminobutyric acid (GABA) receptor subunit rho1 identified two distinct clones. DNA sequencing revealed that one clone contained a single exon from the rho1 gene (GABBR1) while the second clone encompassed an exon with 96% identity to the rho1 gene. Screening of a human retina cDNA library with oligonucleotides specific for the exon in the second clone identified a 3-kb cDNA with an open reading frame of 1395 bp. The predicted amino acid sequence of this cDNA demonstrates 30 to 38% similarity to alpha, beta, gamma, and delta GABA receptor subunits and 74% similarity to the GABA rho1 subunit suggesting that the newly isolated cDNA encodes a new member of the rho subunit family, tentatively named GABA rho2. Polymerase chain reaction (PCR) amplification of rho1 and rho2 gene sequences from DNA of three somatic cell hybrid panels maps both genes to human chromosome 6, bands q14 to q21. Tight linkage was also demonstrated between restriction fragment length variants (RFLVs) from each rho gene and the Tsha locus on mouse chromosome 4, which is homologous to the CGA locus on human chromosome 6q12-q21. These two lines of evidence confirm that GABRR1 and newly identified GABRR2 map to the same region on human chromosome 6. This close physical association and high degree of sequence similarity raises the possibility that one rho gene arose from the other by duplication.     

Molecular cloning and chromosomal localization of a novel human tracheo-bronchial mucin cDNA containing tandemly repeated sequences of 48 base pairs.

A lambda gt11 cDNA library constructed from human tracheo-bronchial mucosa was screened with a polyclonal antiserum raised to chemically deglycosylated pronase glycopeptides from human bronchial mucins. Out of 20 positives clones, one partial cDNA clone was isolated and allowed to map a novel human tracheo-bronchial mucin gene. It contains 48 nucleotide tandem repeats quite perfectly identical which encodes a protein containing about 50% of hydroxy amino-acids. This clone hybridized to polydisperse messages produced by human tracheo-bronchial and human colonic mucosae. The gene (proposed name MUC 4) from which cDNA is derived maps to chromosome 3.     

人IL35-IgG4(Fc)融合蛋白在CHO/DG44细胞中的稳定表达

本研究利用基因重组技术构建人IL35-IgG4(Fc)融合基因真核表达载体, 稳定转染CHO/DG44细胞并检测重组蛋白的表达。主要采用聚合酶链式反应(PCR)从脂多糖(Lipopolysaccharides, LPS)诱导的人髓性白血病细胞株KG-I cDNA文库中克隆EBI3和IL-12p35 cDNA, 重叠PCR法连接2个片段, 并克隆到IgG4(Fc)- pOptiVEC?-TOPO?载体上,对新构建的IL-35-IgG4 (Fc) pOptiVEC?-TOPO?真核表达载体并进行酶切、测序、PCR鉴定; 脂质体法转染CHO/DG44细胞; RT-PCR检测转染结果, 采用a-MEM-培养基筛选实验组细胞, 对筛选的阳性克隆细胞再进行氨甲喋呤(Methotrexate, MTX)的加压筛选, ProteinG-Agarose纯化阳性克隆培养上清, 免疫印迹检测目的蛋白表达。结果显示IL-35-IgG4 (Fc) pOptiVEC?-TOPO?表达载体稳定转染CHO/DG44细胞并获得阳性克隆; SDS-PAGE电泳得到一条与预期相对分子质量大小相符的蛋白条带; 该蛋白能与羊抗人IgG4抗体特异结合。本实验获得了能够稳定表达具有稳定结构的IL35-IgG4(Fc)融合蛋白的CHO/DG44细胞株。     

A novel human Gal-3-O-sulfotransferase: molecular cloning, characterization, and its implications in biosynthesis of (SO(4)-3)Galbeta1-4(Fucalpha1-3)GlcNAc

Based on sequence homology with the previously cloned human cerebroside sulfotransferase (CST) cDNA, a novel sulfotransferase was cloned by screening a human fetal brain cDNA library. The novel sulfotransferase gene was present on human chromosome 11q13; the location was different from human CST and from that of the recently cloned human beta-Gal 3'-sulfotransferase (GP3ST). The isolated cDNA contained an open reading frame that encoded a predicted protein of 431 amino acid residues with type II transmembrane topology. The amino acid sequence showed 33% identity with that of human CST and 38% with that of human GP3ST. The recombinant enzyme expressed in Chinese hamster ovary cells catalyzed transfer of sulfate to position 3 of non-reducing beta-galactosyl residues in Galbeta1-4GlcNAc. Type 2 chains served as good acceptors, whereas type 1 chains served as poor acceptors, and intermediate activity was found toward Galbeta1-3GalNAc. Therefore, the substrate specificity was different from that of GP3ST. CST activity was not detected in the newly cloned enzyme. Northern blotting analysis showed that the sulfotransferase mRNA was strongly expressed in the thyroid and moderately expressed in the brain, heart, kidney, and spinal cord. Co-transfection of the enzyme cDNA and fucosyltransferase III into COS-7 cells resulted in expression of (SO(4)-3)Galbeta1-4(Fucalpha1-3)GlcNAc and a small amount of (SO(4)-3)Galbeta1-3(Fucalpha1-4)GlcNAc. These results indicated that the newly cloned enzyme is a novel Gal-3-O-sulfotransferase and is involved in biosynthesis of the (SO(4)-3)Galbeta1-4(Fucalpha1-3)GlcNAc structure.