Mouse immunoglobulin A: nucleotide sequence of the structural gene for the alpha heavy chain derived from cloned cDNAs

The cDNAs complementary to mouse immunoglobulin alpha heavy chain mRNAs have been cloned into the PstI site of the plasmid vector pBR322. Recombinant plasmids have been identified by hybrid-arrested translation and purification of alpha heavy chain mRNA on DNA-DBM filters. The nucleotide sequence of the inserts encodes the constant and 3' untranslated regions of the alpha heavy chain mRNA. The CH3 domains of human and mouse alpha chains are highly homologous, including a 36 amino acid fragment not reported in the protein sequence (Robinson and Appella, 1980). As in the case of the mu secreted heavy chain, the alpha heavy chain contains a carboxy terminal piece of 20 amino acids.     

Transcripts coding for variant surface glycoproteins of Trypanosoma brucei have a short,identical exon at their 5′ end

The nucleotide sequence of the 5′ end of the mRNA for variant surface glycoprotein (VSG) 117 has been determined and compared with the sequence of the unexpressed basic copy (BC) of the VSG 117 gene. This shows the existence of an exon 35 nucleotides long at the 5′ end of the mRNA. The evidence suggests that this ‘mini-exon’ is derived from the expression site into which the VSG 117 BC is transposed during activation. The nucleotide sequence of this mini-exon is indistinguishable from that recently found for a different VSG, 118 (Van der Ploeg et al., Nucl. Acids Res. 10 (1982) 3591–3604). Analysis of the 5′ end of the mRNA for another VSG (221) whose gene is thought to be activated by a different mechanism to that of VSGs 117 and 118 indicates that the 5′- most 35 nucleotides of the VSG 221 mRNA are identical to the 117/118 mini-exon sequence. The implications of these results for the mechanism of VSG gene expression are discussed.     

A new type of EcoRI polymorphism of the human ribosomal DNA repeating unit revealed by analysis of cloned DNA fragments

Several clones of rDNA have been isolated from an adult human liver DNA Charon 4A library by using cDNA probes synthesized from human 18S and 28S rRNA. The insert of one recombinant Charon 4A clone contained, besides the already known 5.7-kb EcoRI fragment of rDNA, comprising the major portion of the 18S rRNA gene and all the external transcribed spacer (ETS), a previously unidentified EcoRI fragment of rDNA of 8.5 kb in size. DNA transfer hybridization experiments utilizing EcoRI digests of the human DNA used to construct the library and of another human DNA showed the presence of the 8.5-kb EcoRI fragment in a minority of the rDNA repeats on the 5'-end side of the 5.7-kb fragment, thus defining a hitherto unidentified type of EcoRI polymorphism of these repeats.     

Cloning of two members of the calcitonin-family receptors from stingray, Dasyatis akajei: possible physiological roles of the calcitonin family in osmoregulation

In cartilaginous fish, two cDNAs encoding calcitonin-family receptors were isolated for the first time from the stingray brain. The open reading frame of one receptor cDNA coded a 525-amino acid protein. The amino acid identity of this receptor to human calcitonin-receptor-like receptor (CRLR) is 64.5%, frog CRLR is 64.7%, and flounder CRLR is 61.2% and this was higher than to human calcitonin receptor (CTR) (46.1%), frog CTR (54.7%), and flounder CTR (48.9%). We strongly suggested that this receptor is a ray CRLR based on phylogenetic analysis. In case of the second receptor, amino acid identity among CRLRs (human 50.5%, frog 50.7%, flounder 48.0%) and CTRs (human 43.2%, frog 49.1%, flounder 41.8%) was similar. From phylogenetic analysis of both CRLRs and CTRs, we believe that this receptor is ray CTR. The expression of ray CRLR mRNA was predominantly detected in the nervous system (brain) and vascular system (atrium, ventricle, and gill), which reflects the similar localization of CGRP in the nervous and vascular systems as mammals. It was observed that the second receptor was expressed in several tissues, namely cartilage, brain, pituitary gland, gill, atrium, ventricle, pancreas, spleen, liver, gall bladder, intestine, rectal gland, kidney, testis and ovary. This localization pattern was very similar to flounder CTR. Both receptor mRNAs were strongly expressed in the gill. This suggests that the calcitonin-family members are involved in the osmoregulation of stingray as this fish is known to be euryhaline. When a stingray was transferred to diluted seawater (20% seawater), the expression of both receptors significantly decreased in the gill. Similar results were obtained in the kidney of the stingray. Thus, our cloning and isolation of both receptors in the stingray will be helpful for elucidation of their physiological role(s) such as osmoregulation including calcium metabolism of cartilaginous fish.     

Echinococcus granulosus: variability of the host-protective EG95 vaccine antigen in G6 and G7 genotypic variants

Cystic hydatid disease in humans is caused by the zoonotic parasite Echinococcus granulosus. As an aid to control transmission of the parasite, a vaccine has been produced for prevention of infection in the parasite’s natural animal intermediate hosts. The vaccine utilizes the recombinant oncosphere protein, EG95. An investigation into the genetic variability of EG95 was undertaken in this study to assess potential antigenic variability in E. granulosus with respect to this host-protective protein. Gene-specific PCR conditions were first established to preferentially amplify the EG95 vaccine-encoding gene (designated eg95-1) from the E. granulosus genome that also contains several other EG95-related genes. The optimized PCR conditions were used to amplify eg95-1 from several parasite isolates in order to determine the protein-coding sequence of the gene. An identical eg95-1 gene was amplified from parasites showing a G1 or G2 genotype of E. granulosus. However, from isolates having a G6 or G7 genotype, a gene was amplified which had substantial nucleotide substitutions (encoding amino acid substitutions) compared with the eg95 gene family members. The amino acid substitutions of EG95 in the G6/G7 genotypes may affect the antigenicity/efficacy of the EG95 recombinant antigen against parasites of these genotypes. These findings indicate that characterization of eg95 gene family members in other strains/isolates of E. granulosus may provide valuable information about the potential for the EG95 hydatid vaccine to be effective against E. granulosus strains other than the G1 genotype.     

Molecular cloning and mRNA expression analysis of ornithine decarboxylase antizyme 2 in ovarian follicles of the Sichuan white goose (Anser cygnoides)

The ornithine decarboxylase antizyme 2 (OAZ2) gene is a member of the antizyme gene family. Antizymes play pivotal roles in various cellular pathways, including polyamine anabolism and apoptosis. The molecular structure and expression profile of the OAZ2 in goose ovarian follicles have not been reported. In this study, the OAZ2 cDNA sequence of the Sichuan white goose was cloned (Anser cygnoides), and phylogenetic and structural analyses of the OAZ2 were performed. The expression profiling of OAZ2 mRNA in goose ovarian follicles was examined using quantitative real-time PCR. The sequence analysis showed that the 756 bp OAZ2 sequence contained two overlapping open reading frames (ORF). ORF1 was 99 bp in length, and encoded a 32 aa polypeptide. ORF2 was 477 bp in length, and encoded a 158 aa polypeptide. The frameshift site that initiates the translation of ORF2 was located at nucleotide position 97 in the OAZ2. The analysis of OAZ2 mRNA expression in hierarchical follicles showed that the level of OAZ2 mRNA was higher in the SWF and F2 follicular stages than that in the ovarian stroma (P < 0.05). The lowest level of OAZ2 expression was detected in the ovarian stroma. These results suggest that the highly conserved frameshift region plays an important role in sustaining the function of OAZs. Furthermore, the significantly higher level of OAZ2 mRNA in the SWF stage indicates that OAZ2 may be involved in recruiting hierarchical follicles. Our results also suggest that OAZ2 may augment the effects of OAZ1 in follicle development.     

Cloning of eukaryotic genes in single-strand phage vectors: the human interferon genes

Using oligonucleotide probes with defined sequences, we have selected clones from a human lymphocyte cDNA library which represent human leukocyte (HuIFN-α) and fibroblast (HuIFN-β) interferon gene sequences. Double-stranded f1 phage DNA was used as the vector for initial cloning of cDNA. Clones carrying interferon gene sequences were identified by hybridization with the oligonucleotide probes. The same oligonucleotide probes were used as primers for dideoxy chain termination sequencing of the clones. One HuIFN-α clone, 201, has a nucleotide sequence different from published HuIFN-α sequences. Under control of the lacUV5 promoter, the 201 gene has been used to express biologically active HuIFN-α in Escherichia coli.     

Molecular characterization and expression analysis of nine cotton GhEF1A genes encoding translation elongation factor 1A

The translation elongation factor 1A, eEF1A, plays an important role in protein synthesis, catalyzing the binding of aminoacyl-tRNA to the A-site of the ribosome by a GTP-dependent mechanism. To investigate the role of eEF1A for protein synthesis in cotton fiber development, nine different cDNA clones encoding eukaryotic translation elongation factor 1A were isolated from cotton (Gossypium hirsutum) fiber cDNA libraries. The isolated genes (cDNAs) were designated cotton elongation factor 1A gene GhEF1A1, GhEF1A2, GhEF1A3, GhEF1A4, GhEF1A5, GhEF1A6, GhEF1A7, GhEF1A8, GhEF1A9, respectively. They share high sequence homology at nucleotide level (71-99% identity) in the coding region and at amino acid level (96-99% identity) among each other. Phylogenetic analysis demonstrated that the nine GhEF1A genes can be divided into 5-6 subfamilies, indicating the divergence occurred in structures of the genes as well as the deduced proteins during evolution. Real-time quantitative RT-PCR analysis revealed that GhEF1A genes are differentially expressed in different tissues/organs. Of the nine GhEF1A genes, five are expressed at relatively high levels in young fibers. Further analysis indicated that expressions of the GhEF1As in fiber are highly developmental-regulated, suggesting that protein biosynthesis is very active at the early fiber elongation.     

Molecular cloning of cDNA from foot-and-mouth disease virus C1-Santa Pau (C-S8). Sequence of protein-VP1-coding segment

cDNA segments copied from the RNA of foot-and-mouth disease virus (FMDV) C₁-Santa Pau (isolate C-S8) have been cloned in plasmid pBR322. A 998-bp DNA fragment, that includes the region coding for capsid protein VP1, the carboxy terminus of VP3, and the amino terminus of precursor protein p52 has been sequenced. Comparison of the nucleotide sequence with those from FMDV O₁K, A₁₀61, a₁₂ and C₃ Indaial (Kurz et al., Nucl. Acids Res. 9 (1981) 1919–1931; Kleid et al., Science 214 (1981) 1125–1129; Boothroyd et al., Gene 17 (1982) 153–161; Makoff et al., Nucl. Acids Res. 10 (1982) 8285–8295) indicates extensive variability between the corresponding gene segments, including short insertions and deletions. Base transversions are more frequent than transitions within the VP1 coding segment, but not in the sequence coding for the amino-terminal end of p52. The nucleotide sequence divergence is reflected in variability in both the primary and the predicted higher-order structures of the encoded VP1s.     

Identification and characterization of rad51 of Pneumocystis

Rad51, a eukaryotic homolog of RecA, is an important protein involved in DNA recombination and repair. We have characterized rad51 of Pneumocystis carinii and Pneumocystis murina. rad51 is a single copy gene that encodes a 1.2 kb mRNA, which contains an open reading frame encoding 343 amino acids. Rad51 from Pneumocystis showed high homology to those from yeast. ATP binding motifs GEFRTGKS and LLIVD, similar to those of Saccharomyces cerevisiae and Schizosaccharomyces pombe, are conserved in Pneumocystis Rad51. The recombinant protein when expressed in E. coli showed DNA-dependent ATPase activity. Since Rad51 is a key enzyme in DNA repair and recombination, it potentially plays an important role in the recombination process leading to antigenic variation and thereby resistance to host immune responses in Pneumocystis.     

Recombination following transformation of Escherichia coli by heteroduplex plasmid DNA molecules

Circular heteroduplex DNA molecules introduced into Escherichia coli-competent cells are converted to new recombinant plasmids as a result of enzymatic actions in vivo. A pair of plasmids with partial sequence homology were each linearized at a different position with restriction enzymes, and the termini were made flush with the single-strand-specific S1 nuclease. Duplex molecules were then formed by melting and annealing these plasmid DNAs together. In contrast to linear homoduplex molecules, heteroduplexes circularize and therefore transform E. coli efficiently. Unique DNA sequences on each of the parental strands in the transforming heteroduplexes can be selectively incorporated or deleted as a result of in vivo enzymatic activities in transformed cells. This method permits the generation of new recombinant sequences in vivo without relying solely on the presence of convenient restriction sites for manipulation of DNA fragments in vitro.     

Defective pre-mRNA splicing in PKD1 due to presumed missense and synonymous mutations causing autosomal dominant polycystic disease

Autosomal dominant polycystic kidney disease is the most common human monogenic disorder and is caused by mutations in the PKD1 or PKD2 genes. Most patients with the disease present mutations in PKD1, and a considerable number of these alterations are single base substitutions within the coding sequence that are usually predicted to lead to missense or synonymous mutations. There is growing evidence that some of these mutations can be detrimental by affecting the pre-mRNA splicing process. The aim of our study was to test PKD1 mutations, described as missense or synonymous in the literature or databases, for their effects on exon inclusion. Bioinformatics tools were used to select mutations with a potential effect on pre-mRNA splicing. Mutations were experimentally tested using minigene assays. Exons and adjacent intronic sequences were PCR-amplified and cloned in the splicing reporter minigene, and selected mutations were introduced by site-directed mutagenesis. Minigenes were transfected into kidney derived cell lines. RNA from cultured cells was analyzed by RT-PCR and DNA sequencing. Analysis of thirty-three PKD1 exonic mutations revealed three mutations that induce splicing defects. The substitution c.11156G > A, previously predicted as missense mutation p.R3719Q, abolished the donor splice site of intron 38 and resulted in the incorporation of exon 38 with 117 bp of intron 38 and skipping of exon 39. Two synonymous variants, c.327A > T (p.G109G) and c.11257C > A (p.R3753R), generated strong donor splice sites within exons 3 and 39 respectively, resulting in incorporation of incomplete exons. These three nucleotide substitutions represent the first PKD1 exonic mutations that induce aberrant mRNAs. Our results strengthen the importance to evaluate the consequences of presumed missense and synonymous mutations at the mRNA level.     

A novel histidine kinase gene, ZmHK9, mediate drought tolerance through the regulation of stomatal development in Arabidopsis

Plants have developed complex signaling networks to regulate biochemical and physiological acclimation, environmental signals were perceived and transmitted to cellular machinery to activate adaptive responses. Here, a novel drought responsive histidine kinase gene was identified and designated as ZmHK9. Under normal conditions, ZmHK9 was predominantly expressed in roots, and the roots of ZmHK9-OX transgenic lines are markedly hypersensitive to ABA and ethylene, as compare to wild type. Consistent with its expression induced by PEG and exogenous ABA treatment, promoter sequence of this gene possessed drought and ABA responsive element. Moreover, the transgenic plants were much less affected by drought stress and recovered quickly after rewatering, stomatal complex size and stomatal density in the transgenic plants are significantly smaller and lower than those of the wild-type plants. In addition, ABA induced stomatal closure and the stomatal aperture of ZmHK9-OX lines was smaller than that of wild type. Collectively, it can be concluded that ZmHK9 regulates root elongation, stomatal development and drought tolerance through ABA dependent signaling pathway in Arabidopsis.     

A novel big protein TPRBK possessing 25 units of TPR motif is essential for the progress of mitosis and cytokinesis

Through the comprehensive analysis of the genomic DNA sequence of human chromosome 22, we identified a novel gene of 702 kb encoding a big protein of 2481 amino acid residues, and named it as TPRBK (TPR containing big gene cloned at Keio). A novel protein TPRBK possesses 25 units of the TPR motif, which has been known to associate with a diverse range of biological functions. Orthologous genes of human TPRBK were found widely in animal species, from insecta to mammal, but not found in plants, fungi and nematoda. Northern blotting and RT-PCR analyses revealed that TPRBK gene is expressed ubiquitously in the human and mouse fetal tissues and various cell lines of human, monkey and mouse. Immunofluorescent staining of the synchronized monkey COS-7 cells with several relevant antibodies indicated that TPRBK changes its subcellular localization during the cell cycle: at interphase TPRBK locates on the centrosomes, during mitosis it translocates from spindle poles to mitotic spindles then to spindle midzone, and through a period of cytokinesis it stays on the midbody. Co-immunoprecipitation assay and immunofluorescent staining with adequate antibodies revealed that TPRBK binds to Aurora B, and those proteins together translocate throughout mitosis and cytokinesis. Treatments of cells with two drugs (Blebbistatin and Y-27632), that are known to inhibit the contractility of actin–myosin, disturbed the proper intracellular localization of TPRBK. Moreover, the knockdown of TPRBK expression by small interfering RNA (siRNA) suppressed the bundling of spindle midzone microtubules and disrupted the midbody formation, arresting the cells at G₂ + M phase. These observations indicated that a novel big protein TPRBK is essential for the formation and integrity of the midbody, hence we postulated that TPRBK plays a critical role in the progress of mitosis and cytokinesis during mammalian cell cycle.