Cervical and breast cancer screening rates in Sioux Indian women

We cloned a novel human beta-defensin gene and determined its full-length cDNA sequence. The entire gene spanned more than 7 kb and included a large 6962-bp intron. The 362-bp cDNA encoded a prepropeptide that corresponded precisely to the recently identified human beta-defensin HBD-1, an antimicrobial peptide implicated in the resistance of epithelial surfaces to microbial colonization. By two-color fluorescence in situ hybridization on both metaphase chromosome and released chromatin fiber, HBD-1 gene (DEFB1 in HUGO/GDB nomenclature) mapped to chromosomal region 8p23.1-p23.2 in close proximity (within 100-150 kb) to the gene for the human neutrophil alpha-defensin HNP-1 (DEFA1). Thus, despite a complete lack of DNA sequence similarity and despite differences in their disulfide-pairing pattern, the alpha- and beta-families appear to have evolved from a common premammalian defensin gene.     

Avian IFN-gamma genes: sequence analysis suggests probable cross-species reactivity among galliforms

Little is known about the evolution of cytokines in non-mammalian systems. To address this problem, we attempted to clone the gene for interferon-gamma (IFN-gamma) from a variety of avian species using oligonucleotide primers based on the sequence of the chicken IFN-gamma gene. The coding sequence and partial intron sequences were determined for four species, namely guinea fowl, ring-necked pheasant, Japanese quail, and turkey. To obtain sequence information on the gene extremities, a modified 5' and 3' RACE protocol was used. The sequence information showed that the coding regions of the IFN-gamma gene are highly conserved among the species studied (93.5%-96.7% and 87.8%-97.6% at the nucleotide and peptide levels, respectively) and are more conserved at the amino-terminal region (exons 1 and 2) than the carboxyl-terminal (exons 3 and 4). This high degree of overall identity at the predicted primary amino acid sequence level of the protein, including the deduced IFN-gamma receptor binding motifs, suggests that IFN-gamma may be cross-reactive among these species. Phylogenetic analysis shows that the similarity of the avian IFN-gamma sequences parallels the presumed evolutionary relationships between the species.     

Porcine spleen deoxyribonuclease II. Covalent structure, cDNA sequence, molecular cloning, and gene expression

Porcine spleen DNase II, a lysosomal acid hydrolase, is a noncovalently linked alpha.beta heterodimer (Liao, T.-H. (1985) J. Biol. Chem. 260, 10708-10713). The alpha subunit, after disulfide cleavage, yields two chains, alpha1 and alpha2. The complete amino acid sequences of the alpha1, beta, and alpha2 chains were elucidated by protein sequencing, and the pairings of one interchain disulfide between alpha1 and alpha2 and of three intrachain disulfides in alpha2 were assigned. Six carbohydrate attachment sites, two in beta and four in alpha2, were detected by sugar analyses. The cDNA of DNase II was amplified using primers synthesized on the basis of the amino acid sequences determined. The amplified fragments shown to be a cDNA sequence of 1,292 bases. This cDNA sequence has an open reading frame encoding a 364-amino acid polypeptide containing a putative transmembrane peptide at the NH2-end, two small connecting peptides in the middle, and a peptide at the COOH terminus. These are evidently removed to form mature DNase II. Thus, all three chains in the sequence alpha1, beta, and alpha2 are coded by the same cDNA. When Chinese hamster ovary cells were transfected with a cloned plasmid with an inserted cDNA fragment encoding the entire reading frame, the expressed protein was released into the growth medium as an active form of DNase II.     

Molecular cloning and expression of an avian G protein-coupled P2Y receptor

A family of G protein-coupled P2Y receptors that are activated by adenine and uridine nucleotides has been identified recently. Degenerate primers based on conserved sequences in these P2Y receptors were used to amplify turkey DNA, which was used to isolate the complete coding sequence of a cDNA that encodes a novel G protein-coupled receptor. Stable expression of this avian cDNA in 1321N1 human astrocytoma cells resulted in the conveyance of marked inositol phosphate responses to various nucleotides. Although this cloned avian receptor exhibited its highest homology to the previously cloned mammalian P2Y4 receptor, its pharmacological selectivity was not consistent with the avian receptor's being a species homologue of the P2Y4 receptor. That is, whereas the P2Y4 receptor is selectively activated by UTP and is not activated by ATP or Ap4A, the novel avian receptor was potently activated by ATP and Ap4A as well as by UTP. Taken together, these results describe the identification of an avian phospholipase C-coupled P2Y receptor that, like the mammalian P2Y2 receptor, is activated by both adenine and uridine nucleotides.     

Molecular cloning of human class IV alcohol dehydrogenase cDNA

A cDNA encoding a new type of alcohol dehydrogenase was cloned from a human stomach cDNA library. PCR amplification of 5'-stretch human stomach lambda gt11 library, using degenerate inosine-containing oligonucleotide probes compatible with peptide sequences of human sigma-ADH, resulted in a single product. Subsequently, internal non-degenerate primers were constructed according to the sequences occurring in the product. By PCR with combinations of these new primers and lambda gt11 forward and reverse primers, fragments of the cDNA containing its 5' and 3' ends were amplified. The full length cDNA sequence has 1125 nucleotides with a 72% similarity to those of human class I ADH. The polypeptide sequence, predicted from the cDNA, corresponds to 373 amino acids with a high degree of similarity (96%) to fragments of sigma-ADH previously reported. Northern hybridization analysis with the specific probe for the mRNA of this protein showed that it is expressed in the human stomach but not in the liver. These data indicate that the cDNA we cloned is that of human class IV ADH.     

Thallium poisoning presenting with abdominal colic, paresthesia, and irritability

We have determined the complete nucleotide sequence for the cDNA encoding rat eosinophil major basic protein (MBP) using the rapid amplification of cDNA ends (RACE) procedure. The deduced amino acid sequence revealed that the rat prepro-MBP has three functional domains, namely the signal peptide, the acidic peptide that contains numerous acidic amino acids, and the mature MBP, as in human and guinea pig MBP.     

Purification and characterization of a kinin-releasing and fibrinogen-clotting serine proteinase (KN-BJ) from the venom of Bothrops jararaca, and molecular cloning and sequence analysis of its cDNA

Two forms of a proteinase, KN-BJ 1 and 2, were purified to homogeneity from the venom of Bothrops jararaca. In SDS/PAGE reduced KN-BJ 1 and 2 migrated as single bands with molecular masses of 38 kDa and 39 kDa. The two enzymes have similar N-terminal amino acid sequences and specific activities on synthetic chromogenic substrates, and both release bradykinin from bovine low-molecular-mass kininogen. KN-BJ 1 and KN-BJ 2 clot fibrinogen with specific activities of 245 NIH U/mg and 219 NIH U/mg, releasing only fibrinopeptide A. The amidolytic, kinin-releasing and coagulant activities are inhibited by phenylmethylsulfonyl fluoride, demonstrating that KN-BJ is a serine proteinase. Benzamidine derivatives, which are competitive inhibitors of trypsin-like proteinases, also inhibited the amidolytic activity of KN-BJ. A cDNA clone (HS104, 2.2 kb) has been isolated from a cDNA library of B. jararaca venom glands with an ORF of 771 bp. The deduced amino acid sequence contains segments that are identical to the sequences of the N-terminus and three tryptic peptides of KN-BJ 2. Therefore, the cDNA is believed to represent the gene of KN-BJ 2. The deduced amino acid sequence indicates that KN-BJ 2 is synthesized as a prezymogen of 257 amino acids with a putative signal peptide of 18 amino acids and an activating peptide of six amino acid residues. The sequence of 233 amino acids representing the mature enzyme exhibits high similarity to sequences of serine proteinases isolated from crotalid venoms.     

Cloning and expression of human Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase

Here we report the cDNA sequence of human Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase. The cDNA was isolated from a human placental cDNA library by screening with a 150bp probe generated by PCR using degenerate primers based on the sequences found in the sialyl motif. Comparative analysis of this cDNA with the rat liver alpha 2,3-sialyltransferase sequence indicates 91% nucleotide similarity between the two sequence in the predicted coding region. On the amino acid level, the degree of conservation is 97%. Surprisingly, Northern analysis indicated that the gene is expressed at low levels in human placenta but is abundantly expressed in skeletal muscle and fetal tissues.     

Interferon treatment of chronic hepatitis C in human immunodeficiency virus infected patients]

There are no reports to date of entire gene sequences coding for chitinolytic enzymes from entomopathogenic fungi, even though these enzymes act synergistically with proteolytic enzymes to solubilize insect cuticle during the key step of host penetration, having considerable importance in the biological control of some insect pests. This paper reports the complete nucleotide sequence and analysis of the chromosomal and full-length cDNA copies of the regulated gene (chit1) coding one of the chitinases produced by the biocontrol agent Metarhizium anisopliae. Degenerated primers, encompassing conserved regions of other fungal chitinases, were used to amplify a 650-bp DNA fragment, which was used to isolate genomic and cDNA clones from M. anisopliae. Albeit at least two different chitinases are characterized in this fungus, only one chit gene was isolated. The chit1 gene is interrupted by three short typical fungal introns and has a 1,521-bp ORF, which encodes a protein of 423 amino acids with a stretch of 35 amino acid residues displaying characteristics of signal peptide. The deduced sequence of the mature protein predicts a 42-kDa protein with pI of 5.8. Southern analysis of genomic DNA indicates a single copy of chit1 in the M. anisopliae genome.     

A cDNA clone for taxadiene synthase, the diterpene cyclase that catalyzes the committed step of taxol biosynthesis

The committed step of taxol (paclitaxel) biosynthesis is catalyzed by taxa-4(5),11(12)-diene synthase, a diterpene cyclase responsible for transforming the ubiquitous isoprenoid intermediate geranylgeranyl diphosphate to the parent olefin with a taxane skeleton. To obtain the corresponding cDNA clone, a set of degenerate primers was constructed based on consensus sequences of related monoterpene, sesquiterpene, and diterpene cyclases. Two of these primers amplified a 83-base pair fragment that was cyclase-like in sequence and that was employed as a hybridization probe to screen a cDNA library constructed from poly(A)+ RNA extracted from Pacific yew (Taxus brevifolia) stems. Twelve independent clones with insert size in excess of 2 kilobase pairs were isolated and partially sequenced. One of these cDNA isolates was functionally expressed in Escherichia coli, yielding a protein that was catalytically active in converting geranylgeranyl diphosphate to a diterpene olefin that was confirmed to be taxa-4(5),11(12)-diene by combined capillary gas chromatography-mass spectrometry. The sequence specifies an open reading frame of 2586 nucleotides, and the complete deduced polypeptide, including a long presumptive plastidial targeting peptide, contains 862 amino acid residues and has a molecular weight of 98,303, compared with about 79,000 previously determined for the mature native enzyme. Sequence comparisons with monoterpene, sesquiterpene, and diterpene cyclases of plant origin indicate a significant degree of similarity between these enzymes; the taxadiene synthase most closely resembles (46% identity, 67% similarity) abietadiene synthase, a diterpene cyclase from grand fir.     

Inefficiency of upward displacement operating theatre ventilation

In the previous study the authors found at least one peptide of 39 amino acid residues of several purified tryptic-fragments of the cobra serum antitoxic protein (CSAP), whose sequence had shown certain correlation with the rat serum albumin. For further clarification of the toxin-binding activity of CSAP in relation with its corresponding structure, the cDNA gene library of cobra liver cells was established and DNA primers were designed according to the known amino acid sequences of the tryptic peptides of CSAP. The specific probes were prepared and the specific clone was screened out from the library by PCR. Finally, the CSAP cDNA was sequenced from its sub-clones. Then the complete amino acid sequence of CSAP was elucidated. It is a polypeptide chain of 614 amino acid residues with a molecular size of 69.8 KDa. In comparing the whole sequence of CSAP, especially its signal peptide, with mammalian serum albumins, the authors have come to realize that CSAP is just the cobra serum albumin (CSA).     

Lactone-ring-cleaving enzyme: genetic analysis, novel RNA editing, and evolutionary implications

A lactonohydrolase from Fusarium oxysporum AKU 3702 is an enzyme catalyzing the hydrolysis of aldonate lactones to the corresponding aldonic acids. The amino acid sequences of the NH2 terminus and internal peptide fragments of the enzyme were determined to prepare synthetic oligonucleotides as primers for the PCR. An approximate 1, 000-base genomic DNA fragment thus amplified was used as the probe to clone both genomic DNA and cDNA for the enzyme. The lactonohydrolase genomic gene consists of six exons separated by five short introns. A novel type of RNA editing, in which lactonohydrolase mRNA included the insertion of guanosine and cytidine residues, was observed. The predicted amino acid sequence of the cloned lactonohydrolase cDNA showed significant similarity to those of the gluconolactonase from Zymomonas mobilis, and paraoxonases from human and rabbit, forming a unique superfamily consisting of C-O cleaving enzymes and P-O cleaving enzymes. Lactonohydrolase was expressed under the control of the lac promoter in Escherichia coli.     

Identification and cloning of prs a 1, a 32-kDa endochitinase and major allergen of avocado, and its expression in the yeast Pichia pastoris

Avocado, the fruit of the tropical tree Persea americana, is a source of allergens that can elicit diverse IgE-mediated reactions including anaphylaxis in sensitized individuals. We characterized a 32-kDa major avocado allergen, Prs a 1, which is recognized by 15 out of 20 avocado- and/or latex-allergic patients. Natural Prs a 1 was purified, and its N-terminal and two tryptic peptide sequences were determined. We isolated the Prs a 1 encoding cDNA by PCR using degenerate primers and 5'-rapid amplification of cDNA ends. The Prs a 1 cDNA coded for an endochitinase of 326 amino acids with a leader peptide of 25 amino acids. We expressed Prs a 1 in the yeast Pichia pastoris at 50 mg/liter of culture medium. The recombinant Prs a 1 showed endochitinase activity, inhibited growth and branching of Fusarium oxysporum hyphae, and possessed IgE binding capacity. IgE cross-reactivity with latex proteins including a 20-kDa allergen, most likely prohevein, was demonstrated, providing an explanation for the commonly observed cross-sensitization between avocado and latex proteins. Sequence comparison showed that Prs a 1 and prohevein had 70% similarity in their chitin-binding domains. Characterization of chitinases as allergens has implications for engineering transgenic crops with increased levels of chitinases.     

Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes

Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.     

Erythropoietin structure-function relationships: high degree of sequence homology among mammals

To investigate structure-function relationships of erythropoietin (Epo), we have obtained cDNA sequences that encode the mature Epo protein of a variety of mammals. A first set of primers, corresponding to conserved nucleotide sequences between mouse and human DNAs, allowed us to amplify by polymerase chain reaction (PCR) intron 1/exon 2 fragments from genomic DNA of the hamster, cat, lion, dog, horse, sheep, dolphin, and pig. Sequencing of these fragments permitted the design of a second generation of species-specific primers. RNA was prepared from anemic kidneys and reverse-transcribed. Using our battery of species-specific 5' primers, we were able to successfully PCR-amplify Epo cDNA from Rhesus monkey, rat, sheep, dog, cat, and pig. Deduced amino acid sequences of mature Epo proteins from these animals, in combination with known sequences for human, Cynomolgus monkey, and mouse, showed a high degree of homology, which explains the biologic and immunological cross-reactivity that has been observed in a number of species. Human Epo is 91% identical to monkey Epo, 85% to cat and dog Epo, and 80% to 82% to pig, sheep, mouse, and rat Epos. There was full conservation of (1) the disulfide bridge linking the NH2 and COOH termini; (2) N-glycosylation sites; and (3) predicted amphipathic alpha-helices. In contrast, the short disulfide bridge (C29/C33 in humans) is not invariant. Cys33 was replaced by a Pro in rodents. Most of the amino acid replacements were conservative. The C-terminal part of the loop between the C and D helices showed the most variation, with several amino acid substitutions, deletions, and/or insertions. Calculations of maximum parsimony for intron 1/exon 2 sequences as well as coding sequences enabled the construction of cladograms that are in good agreement with known phylogenetic relationships.