Cloning of a complementary DNA encoding an 80 kilodalton nuclear cap binding protein.

It has been shown that the monomethylated cap structure plays important roles in nuclear events. The cap structure has been implicated in the enhancement of pre-mRNA splicing. More recently, this structure has also been suggested to facilitate RNA transport from the nucleus to the cytoplasm. We have previously identified and purified an 80kD Nuclear Cap Binding Protein (NCBP) from a HeLa cell nuclear extract, which could possibly mediate these nuclear activities. In this report, we describe cloning of complementary DNA (cDNA) encoding NCBP. The partial protein sequences of NCBP were determined, and the full-length cDNA of NCBP was isolated from HeLa cDNA libraries. This cDNA encoded an open reading frame of 790 amino acids with a calculated molecular mass of 91,734 daltons, which contained most of the determined protein sequences. However, the protein sequence had no significant homology to any known proteins. Transfection experiments demonstrated that the epitope-tagged NCBP, transiently expressed in HeLa cells, was localized exclusively in the nucleoplasm. Similar experiments using a truncated NCBP cDNA indicated that this nuclear localization activity is conferred by the N-terminal 70 amino-acid region.     

Cloning and characterization of human CAGLP gene encoding a novel EF-hand protein.

The EF-hand proteins, containing conserved Ca2+ binding motifs, play important roles in many biological processes. Through data mining, a novel human gene, CAGLP (calglandulin-like protein) was predicted and subsequently isolated from human skeleton muscle. The open reading frame of CAGLP is 543 bp in length, coding a putative Ca2+ binding protein with four EF-hand motifs. The deduced amino acid sequence of CAGLP displays high similarity with Bothrops insularis snake protein calglandulin (80%). The results of PCR amplification using cDNA from 17 human tissues indicated that human CAGLP is expressed in prostate, thymus, heart, skeleton muscle, bone marrow and ovary. Functional CAGLP::EGFP (enhanced green fluorescent protein) fusion protein revealed that CAGLP accumulated through-out Hela cells. Western blot using anti-EGFP antibodies indicated that the CAGLP protein has a molecular weight of about 19 kD. A phylogenetic tree showed that CAGLP and calglandulin may be orthologous proteins representing a distinct group in the EF-hand proteins.     

Isolation of partial complementary DNA encoding human thromboxane synthase

Thromboxane synthase catalyzes the biosynthesis of thromboxane A2 which plays a key role in the proaggregatory and vasoconstrictive processes. In this communication, we reported the successful cloning of thromboxane synthase cDNA from a human lung cDNA library. Oligonucleotides were synthesized according to the direct amino acid sequence of 2 peptides derived from purified human thromboxane synthase. Polymerase chain reaction was carried out using these oligonucleotides as primers to isolate a complementary DNA from human lung cDNA library. The longest cDNA thus obtained was 687 base pairs in length. Amino acid sequences deduced from the cDNA contained all three peptide sequences reported, confirming the authenticity of the cDNA clone.     

Cloning of rat brain protein kinase C complementary DNA

Four peptides derived from rat brain protein kinase C were partially sequenced. Using synthetic oligonucleotides deduced from the amino acid sequences as probes, a clone of complementary DNA (cDNA) was isolated from a cDNA library prepared from the same tissue. The nucleotide sequence of this cDNA clone revealed the primary structure of the carboxyl-terminal region as having 224 amino acids, with significant sequence homology with cyclic AMP-dependent and cyclic GMP-dependent protein kinases.     

Cloning and characterization of DNA complementary to rat liver UDP-glucuronosyltransferase mRNA

UDP-glucuronosyltransferase (transferase) clones were isolated from a cDNA bank constructed in pBR322 using transferase-enriched mRNA from the livers of phenobarbital-treated rats. The enrichment of mRNA was accomplished by polysome immunoadsorption with antibody to purified mouse liver transferase. This antibody was shown to bind specifically to rat transferase by Ouchterlony double diffusion analysis, immunoadsorption of glucuronidating activities, and selective inhibition of the immunoadsorption of in vitro synthesized enzyme by purified rat liver transferase. The isolated clones were verified to contain DNA complementary to transferase mRNA by hybrid translation-selection. Three classes of transferase cDNAs were characterized by restriction endonuclease mapping, and the largest insert-containing clone of each class was designated pUDPGTr-1, pUDPGTr-2, and pUDPGTr-3. Their insert sizes were approximately 2,400, 2,000, and 2,000 bp, respectively. All three cDNAs hybridized with a 2,300 +/- 150 bp mRNA, and each selected the translation of a 52,000-dalton polypeptide. Immunoadsorption of the 35S-labeled translation product could be competitively inhibited in each case by the addition of purified rat liver transferase. pUDPGTr-1 and pUDPGTr-3 inserts shared extensive sequence homology. This was demonstrated by Southern blot analysis using purified inserts and electron microscopic heteroduplex analysis. Southern blot analysis revealed that these cDNAs hybridized to overlapping genomic fragments. pUDPGTr-2 shared less sequence homology with the other two classes of cDNAs, based on the above criteria. In addition, mRNA corresponding to pUDPGTr-2 was elevated 5-fold by phenobarbital treatment, whereas the other mRNAs levels were unaffected. These studies demonstrate that in rat liver there are a minimum of three distinct transferase mRNAs, two of which may be associated with a common gene or gene family.     

Isolation and characterization of complementary DNAs encoding human manganese-containing superoxide dismutase

cDNAs coding for human manganese-containing superoxide dismutase (Mn SOD) have been isolated from a human liver and a dibutyryl cyclic AMP differentiated U937 cDNA library constructed in vector lambda gtll. The nucleotide sequences of the insert cDNAs had an opening reading frame coding for 222 amino acid residues. The first 24 amino acids of the primarily translated polypeptide might constitute the leader peptide for transport of the precursors to the mitochondria. Differentiation of the U937 cells with dibutyryl cyclic AMP resulted in a 70% decrease in Mn SOD mRNA. The amino acid sequences of the mature Mn SODs of human, rat and mouse are highly conserved, while the sequences of the leader peptides of these species are moderately conserved.     

Cloning and characterization of a cDNA encoding bovine mannan-binding protein

To identify the bovine mannan-binding protein (MBP), a search for the cDNA homologue of human MBP was carried out. cDNA clones encoding bovine MBP were isolated from a bovine liver cDNA library using a cDNA fragment encoding a short collagen region, neck domain and carbohydrate recognition domain of human MBP. The cDNA carried an insert of 747 bp encoding a protein of 249 amino acid (aa) residues with a signal peptide of 19 aa. The mannan-binding protein fraction of bovine serum that eluted with 100 mM mannose from a mannan-Sepharose column was analyzed under reducing conditions by SDS-PAGE. The major band of 33 kDa obtained reacted with anti-human MBP rabbit serum. The partial aa sequence of the purified 33-kDa protein was identical to the aa sequence deduced from the obtained cDNA. Results of the passive hemolysis experiment using sheep erythrocytes coated with yeast mannan suggest that this MBP has the ability to activate complement. Northern blot analysis showed a 1.8-kb mRNA that was expressed only in the liver. Based on results of genomic analysis, this bovine MBP is likely to be a homologue of human MBP and to also have homology to rat and mouse MBP-C which are localized in liver cells rather than to rat and mouse MBP-A found in serum. Alignments of bovine collectins show that bovine MBP cannot be included among the other bovine collectins, such as bovine SP-D, conglutinin and CL-43. Finally, these genomic and biological analyses indicate that the cDNA obtained here encoded a bovine serum MBP.     

Cloning and sequence analysis of complementary DNA encoding a precursor for chicken natriuretic peptide

Chicken -natriuretic peptide (-chNP) has been identified in chicken heart, which showed higher homology to brain natriuretic peptide (BNP) than to atrial natriuretic peptide (ANP) [1]. Complementary DNA (cDNA) clone encoding a chNP precursor (pre-chNP) precursor (pre-chNP) was isolated from cardiac cDNA library and sequenced. Pre-chNP was 140-residue signal peptide at the N-terminus and -chNP at the C-terminus, and did not exhibit high homology to poreine BNP except for the C-terminal region. However, a characteristic AT-rich nucleotide sequence commonly found in mammalian BNPs was also present in the 3′-untranslated region. Thus, chNP is concluded to be classified into the BNP-type     

Molecular cloning and characterization of oppo 1: a haploid germ cell-specific complementary DNA encoding sperm tail protein

We isolated a cDNA clone specifically expressed during spermatogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1085 nucleotides and had an open reading frame of 870 nucleotides encoding a putative protein of 290 amino acid residues. Northern blot analysis revealed a 1.2-kilobase mRNA exclusively expressed in the testis in adult mice; the mRNA was first detected late pachytene stage, and expression increased as the animals matured. The protein encoded by the mRNA had a molecular weight of approximately 33 kDa by Western blot analysis, and was localized to occupy the flagella from the connecting piece through the principal piece. We named this newly isolated gene oppo 1, and we suggest that it plays an important role in sperm tail structure and/or sperm movement.     

Isolation and characterization of complementary DNAs encoding human pregnancy-specific beta 1-glycoprotein

Pregnancy-specific beta 1-glycoprotein (PS beta G) isolated from human placenta consists of a set of at least three glycoproteins with apparent molecular masses of 72, 64, and 54 kDa, respectively. This heterogeneity is confirmed by the detection of three nonglycosylated polypeptides of 50, 48, and 36 kDa, which can be immunoprecipitated by antiserum to placental PS beta G obtained by in vitro translation of placental poly(A)+ RNA. To examine the structural relationships between these proteins, two cDNA clones of 1912 base pairs (PSG16) and 2131 base pairs (PSG93) encoding human PS beta Gs were isolated from a human placental lambda gt11 cDNA library. The sequenced portions of these two cDNAs are identical with the exception that clone PSG93 contains an additional 86 base pairs at the end of the common 3'-coding region. This insertion could result in the generation of a PS beta G species of 419 amino acid residues instead of the 417 amino acid residues predicted by the sequence of clone PSG16. The calculated molecular masses of the two polypeptides encoded by PSG16 and PSG93 are 46.9 and 47.2 kDa, close to the size of the major nonglycosylated PS beta G of 48 kDa. The identity of proteins coded for by these cDNA clones was confirmed by comparing the predicted amino acid sequences to sequences determined from endoproteinase Lys-C peptides obtained from human placental PS beta G. Two placental PS beta G mRNAs of 2200 bases (major) and 1700 bases (minor) have been detected by Northern hybridization analysis. Primer extension and S1 nuclease mapping experiments demonstrated that PS beta G mRNAs have heterogeneous 5' termini.     

Cloning and characterization of the gene encoding the bovine BOULE protein

The Deleted in Azoospermia (DAZ) genes encode potential RNA-binding proteins that are expressed exclusively in the germ-line. The bovine Deleted in Azoospermia-like gene is a strong candidate for male cattle-yak infertility. In this work, with the goe goal to further reveal the genetic cause of male cattle-yak sterility, another bovine DAZ family gene, b-boule, was isolated and characterized. The b-boule gene is predicted to encode a polypeptide of 295 amino acids with an RNP-type RNA recognition domain. Tertiary structure analysis shows that b-boule binds specifically to polypyrimidine RNAs and might act as a nuclear ribonucleoprotein particle auxiliary factor during germ cell formation and morphological changes of germ cells. RT-PCR assays revealed that b-boule was expressed specifically in the adult testis. However, an extremely low level of expression was detected in the testis of sterile male cattle-yaks. Microstructure of the testes from sterile males showed that type A spermatogonia were the only germ cells present and that few germ cells developed further than the stage of pachytene spermatocytes. These results suggest that b-boule may function in bovine spermatogenesis, and that low levels of b-boule expression might lead to male sterility in cattle-yaks.     

Cloning of genomic and complementary DNA encoding insect pheromone binding proteins: evidence for microdiversity

Genomic DNA from the silk moth Antheraea pernyi bearing the gene of a pheromone binding protein has been isolated from a partial genomic library using specific cDNA probes. The DNA spans 3.5 kilobases, contains three exons and two intervening sequences that interrupt the protein coding region of the gene. A DNA fragment of a second gene was isolated and the complete primary structure of a corresponding cDNA clone was unravelled. The expression of two different genes, giving rise to different pheromone binding proteins, implies a more specific function of these proteins than was hitherto assumed.     

Cloning and characterization of the complementary DNA for the B chain of normal human serum C1q

Normal human C1q is a serum glycoprotein of 460 kDa containing 18 polypeptide chains (6A, 6B, 6C) each 226 amino acids long and each containing an N-terminal collagen-like domain and a C-terminal globular domain. Two unusual forms of C1q have been described: a genetically defective form, which has a molecular mass of approximately 160 kDa and is found in the sera of homozygotes for the defect who show a marked susceptibility to immune complex related disease; a fibroblast form, shown to be synthesized and secreted, in vitro, with a molecular mass of about 800 kDa and with chains approximately 16 kDa greater than those of normal C1q. A higher than normal molecular mass form of C1q has also been described in human colostrum and a form of C1q has been claimed to represent one of the types of Fc receptor on guinea-pig macrophages. To initiate studies, at the genomic level, on these various forms of C1q, and to investigate the possible relation between the C1q genes and the procollagen genes, the complementary DNA corresponding to the B chain of normal C1q has been cloned and characterized.