DNA (Mary Ann Liebert, Inc.) (DNA)

Publications in this journal

• Article: Structure of the human RD gene: a highly conserved gene in the class III region of the major histocompatibility complex.

  P W Speiser, P C White
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  ABSTRACT: We cloned and determined the DNA sequence of a novel gene (termed RD or D6S45) situated in the class III region of the human major histocompatibility complex (HLA) between the Bf and C4A complement genes. The coding region of the gene is contained in 10 exons spread over approximately 6 kb of DNA. The encoded protein is predicted to contain 371 amino acid residues with a molecular weight of 41,000. The predicted amino acid sequence is notable for a central region containing 24 consecutive pairs of alternating basic (Arg) and acidic (Asp or Glu) residues similar to, but more strictly alternating than, those seen in the 70K protein of the U1 small nuclear ribonucleoprotein (snRNP). There is a high degree of homology both at the amino acid and DNA level between the human RD gene and its murine homology. Although the central region is highly repetitious and contains a high proportion (13%) of CpG dinucleotides-both features that might predispose to frequent mutations-sequence analysis of this region in genes amplified from six individuals revealed no polymorphism.
  DNA (Mary Ann Liebert, Inc.) 01/1990; 8(10):745-51.
• Article: A vector that expresses secreted proteins on the cell surface.

  X B Wang, R Milne, Y Marcel, E Rassart
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  ABSTRACT: A new vector, CDM8PI, has been constructed. It was derived from the plasmid expression vector CDM8, which has been used in the epitope-loss mutant isolation technique to map the epitopes on cell-surface proteins. The new vector allows the production of fusion proteins between normally secreted proteins and the membrane anchor moiety from a cell-surface protein, LFA-3, thereby expressing the fusion proteins on the cell surface. The vector extends the application of the epitope-loss mutant isolation technique to secreted proteins. The vector also allows the easy recovery of mutated proteins in unfused forms after the immunoselection and characterization.
  DNA (Mary Ann Liebert, Inc.) 01/1990; 8(10):753-8.
• Article: Ultrasonic degradation of DNA.

  H I Elsner, E B Lindblad
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  ABSTRACT: Different results are obtained when DNA in aqueous solution and DNA in biological tissue are exposed to ultrasound. At intensities of ultrasound comparable to those applied clinically, ultrasonication is able to degrade purified DNA in aqueous solution, making ultrasonication a useful tool for preparing DNA fragments in vitro. Ultrasonic degradation of DNA in solution occurs by breaking hydrogen bonds and by single-strand and double-strand ruptures of the DNA helix. Two mechanisms are mainly responsible: cavitation and a thermal or mechanical effect. Stable cavitation is seen at low intensities of ultrasound. Increasing the intensity of the ultrasound above 2 W/cm2 is followed by increases in single-strand ruptures due to the creation of free radicals by transient cavitation. Following sonication, the distribution of the resulting DNA fragments approaches a lower size limit of 100-500 bp. Breaks in the DNA helix occur mainly between oxygen and carbon atoms, resulting in DNA fragments with a phosphorylated 5' end and a free alcohol at the 3' end. The relative lack of specificity in degrading the DNA helix makes ultrasonication a complementary alternative to the highly specific fragmentation obtained by restriction endonucleases.
  DNA (Mary Ann Liebert, Inc.) 01/1990; 8(10):697-701.
• Article: Differential regulation of oncogenic and cellular p185 by serine/threonine kinases.

  K Dobashi, D B Weiner, M I Greene
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  ABSTRACT: 185c-neu is a member of a family of growth factor receptors with tyrosine kinase activity. A point mutation in the transmembrane region leads to activation of the enzymatic domain. We demonstrate that TPA (phorbol-12-myristate-13-acetate) stimulates the phosphorylation of p185c-neu on serine and threonine residues coincident with the inhibition of its intrinsic tyrosine kinase and the proliferation of cells that express it. The tyrosine kinase activity as well as the phosphorylation pattern of serine and threonine residues of oncogenic p185 (p185neu) and the growth of p185neu-expressing cells are not influenced by TPA. These observations indicate that the functional activity of p185c-neu can be regulated through protein kinase C (PKC) but the transmembrane point mutation present in p185neu renders it refractory to serine/threonine kinase regulation.
  DNA (Mary Ann Liebert, Inc.) 01/1990; 8(10):723-32.
• Article: A rapid procedure for cloning genes from lambda libraries by complementation of E. coli defective mutants: application to the fabE region of the E. coli chromosome.

  J H Alix
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  ABSTRACT: I describe a general and rapid procedure allowing the isolation of specialized lambda transducing phages from a lambda library by lysogenic complementation of defective mutants of Escherichia coli. As an example, the cloning of the E. coli fabE gene and of two other adjacent genetic determinants is presented. Subcloning and determination of its nucleotide sequence reveals that fabE codes for the biotin carboxyl carrier protein (BCCP), one of the three subunits of acetyl coenzyme A carboxylase.
  DNA (Mary Ann Liebert, Inc.) 01/1990; 8(10):779-89.
• Article: Superpolylinkers in cloning and expression vectors.

  J Brosius
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  ABSTRACT: Versatile DNA polylinkers of more than 300 bp were constructed. They contain the recognition sequences of all restriction enzymes--whether known or still to be discovered--that recognize palindromic hexamers. In addition to these 64 uninterrupted hexameric recognition sites, a number of sites containing interrupted palindromes and nonpalindromic sequences and two recognition sequences with 8 bp are present. Polylinkers (in several variants) were inserted into frequently utilized Escherichia coli cloning vectors such as pBluescript (yielding pSLJ10, pSL250, pSL260, pSL270, and pSL300), pUC18/pUC19 (yielding pSL180 and pSL190, respectively), or pUC118/pUC119 (yielding pSL1180 and pSL1190, respectively). A subtle color discrimination between presence and absence of insert in pSL300 (mid-blue to light-blue or white) was seen in a number of test ligations. The mid-blue color that is generated by pSL300 is presumably due to translational restarts. A different intergenic region for translational restarts was used in plasmids pSL251, pSL261, pSL271, and pSL301. The polylinker was also inserted into expression vector pUC120, yielding pSE1200, and into expression vector pKK233-2, yielding pSE220 and a shortened version thereof, pSE280. Finally, the polylinker was inserted into pTrc99A, resulting in pSE380, which carries a lac repressor gene. This expands the use of the expression system beyond lacIq strains to other bacterial hosts. These versatile vectors have broad applications in genetic engineering.
  DNA (Mary Ann Liebert, Inc.) 01/1990; 8(10):759-77.
• Article: Mapping contacts between unpurified human progesterone receptor and the hormone response element of mouse mammary tumor virus.

  B Kühnel, D el-Ashry, D P Edwards, S K Nordeen
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  ABSTRACT: Binding of steroid hormone receptors to specific recognition sites of hormone-inducible genes is one of the events required for hormonal regulation of gene transcription. We have employed an immunoprecipitation assay to map the interaction between unpurified human progesterone receptors from crude nuclear extracts of T47D cells and the hormone response element of the mouse mammary tumor virus (MMTV). DNase I footprints and methylation interference patterns are similar to those reported with highly purified rabbit progesterone receptors, suggesting that both human and rabbit receptors recognize similar features in the hormone response element. More importantly, these patterns suggest that if other factors are associated with unpurified nuclear receptor, they do not alter the contacts made by receptor nor do they make contacts themselves with MMTV DNA in a manner detected by DNase I or methylation interference assays. The sites of interaction of receptors bound with the clinically important progestin antagonist, RU 486, are comparable to those observed with an agonist-receptor complex. These results suggest that the antagonist prevents receptor action at a step after its recognition and binding to specific sites on a hormone-responsive enhancer element.
  DNA (Mary Ann Liebert, Inc.) 01/1990; 8(10):703-13.
• Article: Analysis of possible repressor elements in the 5'-flanking region of the human beta-globin gene.

  J M Krakowsky, E S Panke, R F Lee, J McNeish, S S Potter, J B Lingrel
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  ABSTRACT: Human beta-globin gene expression is confined predominantly to the adult with little or no expression of this gene occurring during embryonic or fetal life. The lack of expression of this gene in embryonic and fetal erythroid tissue could be due to the absence of required positive regulatory factors in these cells or the presence of negative regulatory factors which prevent expression of the adult globin gene. To test the repressor model, we have used a gel electrophoretic mobility shift assay to identify regions in the human beta-globin gene which bind proteins found in K562 cells, a cell line that expresses embryonic and fetal globins but not adult beta-globin. DNA fragments comprising the entire human beta-globin gene were assayed using nuclear proteins from K562 cells, and four regions were found that bind proteins. These are located within the 5'-flanking region, within the first and second introns, and at the 3'-flanking region of the gene. Previous studies have suggested the presence of potential repressor sites 5' of exon 2. For this reason, we examined whether the lack of the binding regions in the 5'-flanking sequence allow expression of the human beta-globin gene in transgenic mice during embryonic life. beta-globin gene expression was confined to adult life, indicating that if a transcriptional repressor is responsible for inactivating this gene in embryonic tissue, it is not regulated solely by sequences upstream from -122 bp in the 5'-flanking region of the human beta-globin gene.
  DNA (Mary Ann Liebert, Inc.) 01/1990; 8(10):715-21.
• Article: The primary structure of human liver type phosphofructokinase and its comparison with other types of PFK.

  D Levanon, E Danciger, N Dafni, Y Bernstein, A Elson, W Moens, M Brandeis, Y Groner
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  ABSTRACT: The complete mRNA sequence of the human liver-type phosphofructokinase (hPFKL) was determined. The sequence included 55 nucleotides of 5' and 515 of 3' noncoding regions, as well as 2,337 nucleotides encoding the 779 amino acids of the hPFKL. Extensive similarity (approximately 90%) in the coding region was observed between the hPFKL and the mouse PFKL, whereas the degree of similarity between different types of PFK, i.e., hPFKL and human muscle-type PFK (hPFKM), was merely 68%. Nevertheless, striking similarity between these different types of PFK was noticed when the amino acid residues creating the various active sites of the enzyme were compared. Human PFK L- and M-specific probes were constructed and used to quantitate the mRNA levels in fetal and adult brains and fetal liver. It was found that while relative amount of PFKL mRNA in adult brain was one-fourth of that detected in fetal brain the level of PFKM mRNA in adult brain was slightly higher than in fetal tissue, suggesting that PFK expression might be controlled at the transcriptional level.
  DNA (Mary Ann Liebert, Inc.) 01/1990; 8(10):733-43.
• Article: The major dopamine D2 receptor: molecular analysis of the human D2A subtype.

  L A Selbie, G Hayes, J Shine
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  ABSTRACT: The structural diversity of the human D2 dopamine receptor was examined at the nucleic acid level. Sequence analysis of receptor cDNA clones isolated from human brain and pituitary libraries and polymerase chain reaction (PCR) analysis of rat brain RNA and human genomic DNA demonstrate the presence of a predominant D2 subtype, D2A. The D2A subtype differs from the D2B subtype, previously described in rat brain RNA, in that an additional 29 amino acids are present in the putative third cytoplasmic domain, a region thought to be important for coupling to different G-proteins. The demonstration of intron sequences flanking the DNA encoding the 29-amino-acid insertion suggests that the generation of two distinct D2 dopamine receptor subtypes may arise from alternative splicing of a common genomic sequence.
  DNA (Mary Ann Liebert, Inc.) 12/1989; 8(9):683-9.
• Article: Characterization and sequence analysis of the human ornithine decarboxylase gene.

  M C Fitzgerald, M A Flanagan
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  ABSTRACT: This report describes the characterization and complete sequence of the human ornithine decarboxylase (ODC) gene. Genomic Southern blot analysis shows only a single gene hybridizing at high stringency, in contrast to the murine multigene family. A Pst I restriction fragment length polymorphism was identified and an allele of the human ODC gene containing the polymorphic Pst I site was cloned and sequenced. The ODC gene is divided into 12 exons and spans 8 kb. Comparison of the human, rat, and mouse ODC genes shows striking conservation of genomic organization, as well as 82% identity in the first 148 bp of the 5'-flanking region. This region contains a TATA box, cAMP-responsive element, CCAAT box, and AP-2 binding site and is consistent with induction of ODC gene expression by both the cAMP and protein kinase C-mediated signaling pathways. The first intron of the human gene is 2,849 bp in length, and contains two putative Sp1 binding sites, as well as an Ap1 binding site, suggesting a role for the first intron in transcriptional regulation. The 5' noncoding region of the predicted mRNA contains regions of virtual identity with that of mouse and rat ODC mRNA, suggesting sequences involved in translational regulation. In addition, it was found that the exon segments corresponding to the amino and carboxyl termini of Saccharomyces cerevisiae and Trypanosoma b. brucei are unrelated to their mammalian counterparts, whereas the middle segments of the protein are conserved. These differences may influence the difference in protein half-life seen between T. b. brucei and mammalian ODC.
  DNA (Mary Ann Liebert, Inc.) 12/1989; 8(9):623-34.
• Article: Isolation and sequence of a cDNA clone for human calcineurin B, the Ca2+-binding subunit of the Ca2+/calmodulin-stimulated protein phosphatase.

  D Guerini, M H Krinks, J M Sikela, W E Hahn, C B Klee
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  ABSTRACT: We have identified and cloned human cDNA for the Ca2+-binding subunit of calcineurin, the brain isozyme of the Ca2+/calmodulin-stimulated protein phosphatase. The 2.5-kb cDNA has an open reading frame of 510 bp, a leader sequence of at least 500 bp, and a 1,277-bp 3'-noncoding sequence. The deduced sequence of the human protein differs from bovine brain calcineurin B by an additional valine at the carboxyl terminus and substitution of Met-11 and Ser-153 by cysteine. A partial clone of the mouse protein corresponding to amino acids 75-150 was also isolated. This portion of the human and mouse protein sequence is identical, with the DNA sequences showing 94% identity. The respective mRNAs in human and mouse are also of similar size. As was observed with protein levels, mRNA abundance in brain is 20-60 times that found in other tissues with the exception of HeLa cells which, like brain, contain abundant calcineurin B mRNA.
  DNA (Mary Ann Liebert, Inc.) 12/1989; 8(9):675-82.
• Article: Human immunodeficiency virus type 1 rev protein as a negative trans-regulator.

  M R Sadaie, Z N Benaissa, B R Cullen, F Wong-Staal
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  ABSTRACT: Even though the rev gene of the human immunodeficiency virus type 1 (HIV-1) is essential for viral replication, high levels of rev also downregulate viral gene expression. As the degree of rev protein expression exceeds expression of wild-type virus, a gradient of decreasing viral mRNA synthesis becomes evident. The target sequence for this downregulation resides outside of trans-activating region (TAR) and upstream from the enhancer sequences in the long terminal repeat (LTR), suggesting that regulation is at a transcriptional level.
  DNA (Mary Ann Liebert, Inc.) 12/1989; 8(9):669-74.
• Article: Cloning of ovine insulin-like growth factor-I cDNAs: heterogeneity in the mRNA population.

  E A Wong, S M Ohlsen, J A Godfredson, D M Dean, J E Wheaton
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  ABSTRACT: We have isolated and characterized lamb liver cDNAs encoding ovine insulin-like growth factor-I (oIGF-I) precursor polypeptide to study IGF-I gene expression in ruminants. Four cDNA clones were sequenced revealing two different exon 1 sequences (designated 1A and 1B) and four different putative poly(A) adenylation sites. cDNAs containing exon 1A or exon 1B encode precursor polypeptides of 138 or 154 amino acids, respectively. A 130-amino-acid peptide is encoded by all cDNAs examined. These precursors include a hydrophobic leader peptide of varying lengths, the 70-amino-acid oIGF-I, and a 35-amino-acid carboxyl terminal extension peptide. The predicted amino acid sequence of the oIGF-I peptide differs from the human, bovine, and porcine IGF-Is at a single amino acid (at position 66, alanine is substituted for proline) and differs from rat and mouse IGF-Is at 4 and 5 positions, respectively. Both the amino- and carboxy-terminal extension peptides showed regions of extensive sequence homology. Ovine IGF-I amino-terminal peptides are 1 amino acid longer than other mammalian IGFs due to the presence of an extra amino acid (glutamine) present at the proposed boundary of exon 1 and exon 2. Northern blot analysis revealed multiple oIGF-I transcripts in a broad band at 800-1,100 nucleotides and other transcripts of higher molecular weight in liver. There was no detectable expression in either spleen or brain.
  DNA (Mary Ann Liebert, Inc.) 12/1989; 8(9):649-57.
• Article: Alkaline phosphatase fusions to the respiratory syncytial virus F protein as an approach to analyze its membrane topology.

  A Martin-Gallardo, R A Deich, K A Fien, B J Metcalf, A Anilionis, P R Paradiso
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  ABSTRACT: Manoil and Beckwith (1985) have constructed a transposon, TnphoA, that permits the generation of hybrid proteins composed of alkaline phosphatase (AP) lacking its signal peptide fused to amino-terminal sequences of other proteins. This transposon has been used to localize export signals and analyze membrane topology of bacterial proteins. We have applied this approach to the membrane fusion protein (F) of respiratory syncytial virus (RSV). The transposon TnphoA and a plasmid directing bacterial expression of the F gene were used to construct F-AP hybrids. These hybrids yielded AP activity, indicating the presence of viral sequences that promoted protein transport through the cytoplasmic membrane. Sequence analysis showed that TnphoA was inserted at four different positions within the F1 subunit. Deletion of the hydrophobic F1 amino-terminus (fusion-related domain) resulted in AP transport to the periplasm, suggesting that the hydrophobic amino-terminus of the F2 subunit is sufficient to promote protein export. Some hybrids were apparently cleaved at or near the F2/F1 junction. The periplasmic localization of an uncleaved hybrid strongly suggested that the fusion-related domain of the F protein, when in the uncleaved F0 precursor, can be moved across the bacterial cytoplasmic membrane. Although these results apply to the recombinant F protein, they agree with the presumed signal sequence and membrane topology of the native F glycoprotein. Thus, this method may be useful in determining membrane topology and in localizing important domains of viral proteins.
  DNA (Mary Ann Liebert, Inc.) 12/1989; 8(9):659-67.
• Article: Cloning and characterization of a hemolysin gene from Actinobacillus (Haemophilus) pleuropneumoniae.

  Y F Chang, R Young, D K Struck
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  ABSTRACT: Neutralizing antisera to the leukotoxin secreted by Pasteurella haemolytica neutralized the hemolysin of Actinobacillus pleuropneumoniae and recognized a 110-kD antigen in cell-free culture supernatants from this organism. A series of nine overlapping recombinant phage clones carrying the gene for this 110-kD antigen were identified using affinity-purified anti-hemolysin antibody and a DNA probe containing sequences from the P. haemolytica lktCA genes. Eight of the nine clones expressed a 110-kD protein recognized by both anti-leukotoxin and anti-hemolysin antisera. The remaining clone expressed a truncated 80-kD antigen which was also recognized by both antisera. Sequence analysis of a region of the cloned DNA revealed two open reading frames encoding proteins with predicted masses of 18.5 and 102.5 kD. These genes, which we designate appC and appA, respectively, are similar in sequence to the hlyCA genes of Escherichia coli and the lktCA genes of P. haemolytica. Hemolytic activity could be detected in lysates of E. coli harboring plasmids containing the appCa genes.
  DNA (Mary Ann Liebert, Inc.) 12/1989; 8(9):635-47.
• Article: Determination of exon-intron structure: a novel application of the polymerase chain reaction technique.

  C J Bruzdzinski, T D Gelehrter
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  ABSTRACT: We describe a novel application of the polymerase chain reaction (PCR) technique of DNA amplification to study the exon-intron structure of the rat plasminogen activator inhibitor (PAI-1) gene. This technique is relatively simple and also allows the isolation of introns for sequencing. Primers were selected based on a knowledge of the cDNA sequences of human and rat PAI-1 and of the gene structure of human PAI-1. However, knowledge of a cDNA sequence and/or the structure of a gene in another species is not a prerequisite. Sequences selected from positions along the cDNA of interest could be used to amplify the DNA either from an isolated but uncharacterized gene or directly from genomic DNA, making this technique generally applicable. Thus, this method is a useful advance in the study of gene structure and evolution.
  DNA (Mary Ann Liebert, Inc.) 12/1989; 8(9):691-6.
• Article: Isolation and characterization of two homologous cDNA clones from Torpedo electromotor neurons.

  J K Ngsee, R H Scheller
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  ABSTRACT: Two homologous cDNA clones were isolated from a Torpedo california electric lobe lambda gt11 expression library using a polyclonal antiserum directed against proteins associated with synaptic vesicles. Northern blotting reveals an 8- to 9-kb transcript in the electric lobe and the spinal cord, but not in the brain or other non-neuronal tissues. Antibodies generated against a fusion protein synthesized in Escherichia coli reacted with a 85- to 90-kD species in the neurons of the electric lobe. The immunoreactivity is associated with microsomal membranes and can be extracted readily with high salt. Immunohistochemical studies demonstrated a sparse punctate staining pattern in the cell body which colocalized with a subpopulation of post-Golgi vesicles.
  DNA (Mary Ann Liebert, Inc.) 11/1989; 8(8):555-61.
• Article: cDNA sequence, gene organization, and progesterone induction of mRNA for uteroferrin, a porcine uterine iron transport protein.

  R C Simmen, V Srinivas, R M Roberts
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  ABSTRACT: The complete nucleotide sequence of porcine uteroferrin mRNA was determined by analysis of overlapping cDNA and genomic clones. The uteroferrin mRNA is 1,424 nucleotides in length and encodes a precursor protein of 338 amino acids, of which 20 residues subsequently are cleaved to form the mature peptide. The uteroferrin gene spans 3.5 kb and consists of three exons and two introns. The first intron separates the 5' untranslated sequences from the translation initiation codon ATG while the other intron interrupts the coding region of the mature protein. Primer extension analysis localized the presumptive transcription initiation site of the mRNA 94 nucleotides 5' of the ATG. No canonical TATA or CAAT sequences were apparent upstream from the mRNA cap site. However, sequences within the 5'-flanking region of the gene exhibit similarities to defined regulatory sequences for iron- and steroid hormone-responsive genes. The steady-state level of uteroferrin mRNA is enhanced by progesterone but not by estrogen alone, although the extent of progesterone induction is lower than at midgestation. The simple organization of the uteroferrin gene, which contrasts with those of the transferrin gene family, and the progesterone induction of uteroferrin mRNA expression suggest that, although this protein may have evolved in a manner distinct from other iron binding proteins, its regulation by steroid hormones may be similar.
  DNA (Mary Ann Liebert, Inc.) 11/1989; 8(8):543-54.
• Article: The cDNA structure of rat plasma kallikrein.

  N G Seidah, R Ladenheim, M Mbikay, J Hamelin, G Lutfalla, F Rougeon, C Lazure, M Chrétien
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  ABSTRACT: From a liver cDNA library we have isolated and characterized the cDNA encoding rat plasma kallikrein. The cDNA structure contains 2,456 nucleotides with a 2,082-nucleotide-long open reading frame. Protein sequence data suggest that the signal peptide is 19 amino acids long. This results in a mature plasma prekallikrein containing 619 amino acids. Determination of tissue distributions using Northern blot analysis (3.0-kb transcript) and the polymerase chain-reaction methodology on RNA preparations demonstrated that in the rat the liver is the main source of this enzyme. Southern blots suggested the presence of a single gene coding for rat plasma kallikrein. Finally, although Southern blots revealed a homologous gene in mouse, the mRNA corresponding to the mouse hepatic proteinase is barely detectable on Northern blots, suggesting inefficient transcription or high turnover of the mRNA in this species.
  DNA (Mary Ann Liebert, Inc.) 11/1989; 8(8):563-74.