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H Pannekoek

Publications and source records attributed to H Pannekoek.

At least 109 records · Page 6Linked to original sources
PubMed1986

Functional analysis of the human tissue-type plasminogen activator protein: the light chain.

A pBR322::Rous sarcoma virus(RSV)-based shuttle vector was used to insert fused genes, composed of the amino-terminal portion of the bacterial chloramphenicol-acetyltransferase gene (cat) and the entire coding region for the C-terminally derived light (L) chain of human tissue-type plasminogen activator (t-PA) cDNA. Cotransfection of rat 3Y1 cells with pRSVneo DNA and pRSVcat/t-PA DNA yielded stably integrated G418-resistant transfectants which contain unrearranged copies of pRSVcat/t-PA DNA. These transfectants synthesize cat/t-PA L-chain mRNA, apparently correctly initiated and terminated. With the help of an enzyme-linked immunosorbent assay (ELISA), it is demonstrated that these cells produce human t-PA antigen. Furthermore, pRSVcat/t-PA L-chain cDNA-containing rat 3Y1 cells synthesize a plasminogen-dependent amidolytic activity which is suppressed by specific anti-human t-PA antibodies. This activity cannot be stimulated by fibrin, a property displayed by native t-PA. It is concluded that the t-PA L-chain cDNA contains the complete genetic information for the plasminogen activator activity.

Amino Acid Sequence↗
PubMed1986

Autonomous functions of structural domains on human tissue-type plasminogen activator.

Transfected mouse Ltk- cells were employed for transient expression of recombinant human tissue-type plasminogen activator (t-PA; EC 3.4.21.31) or of recombinant-t-PA deletion proteins, encoded by SV40-pBR322-derived t-PA cDNA plasmids. The t-PA cDNA deletion mutants have two features in common, i.e., cDNA programming the signal peptide and the coding region for the light chain. Consequently, recombinant t-PA mutant proteins are efficiently secreted and display plasminogen activator activity. The gene encoding the amino-terminal heavy chain [an array of structural domains homologous to other plasma proteins (finger, epidermal growth factor, and kringle domains)] was mutated using restriction endonucleases to delete one or more structural domains. The stimulatory effect of fibrinogen fragments on the plasminogen activator activity of t-PA was demonstrated to be mediated by the kringle K2 domain and to a lesser extent by the finger/epidermal growth factor region but not by the kringle K1 domain. These data correlate well with the fibrin-binding properties of the recombinant t-PA deletion proteins, indicating that the stimulation of the activity by fibrinogen fragments is based on aligning the substrate plasminogen and t-PA on the fibrin matrix. Our results support the evolutionary concept of exon shuffling, arranging structural domains that constitute autonomous functions of the protein.

Base Sequence↗
PubMed1985

Construction of cDNA coding for human von Willebrand factor using antibody probes for colony-screening and mapping of the chromosomal gene.

Von Willebrand Factor (vWF) mRNA was identified in fractionated polyA+ RNA preparations isolated from cultured human endothelial cells. Micro-injection of specific polyA+ RNA fractions in Xenopus laevis oocytes provoked the synthesis of a vWF-like product which could be detected with an immunoradiometric assay relying on Sepharose-linked monoclonal anti-vWF IgG and different radiolabeled monoclonal anti-vWF IgGs. A vWF-mRNA-containing polyA+ RNA preparation served as substrate for a size-selected cDNA-expression library of 60 000 colonies which was screened for the synthesis of antigens related to vWF, using polyclonal anti-vWF IgG and a second antibody conjugated with peroxidase. Eight positive colonies were detected of which two reacted strongly in the enzyme-linked assay. Immunoblotting of bacterial extracts of "expression clones" with a monoclonal anti-vWF IgG revealed polypeptides which size fits within the length of the cDNA insertions. Northern blotting of human endothelial RNA, employing fragments of vWF cDNA as probes, showed specific hybridization with a mRNA of about 9000 nucleotides. DNA-sequence analysis of a vWF-cDNA insertion revealed an open reading frame followed by a translation stopcodon. It is argued that the cDNA insertions encode the carboxy-terminal part of the vWF protein. vWF-cDNA probes were employed to map the von Willebrand factor gene on chromosome 12 using a panel of 35 human-rodent somatic cell hybrids.

Animals↗
PubMed1983

In vivo transcription of the E. coli uvrB gene: both promoters are inducible by UV.

The transcriptional activity of the tandem promoters of the Escherichia coli uvrB gene was measured in vivo. Both promoters are shown to be inducible by UV irradiation. P1, the most proximal promoter, is responsible for the main part of transcription both in uninduced and induced cells. Plasmids have been constructed carrying small deletions in the lexA binding site that overlaps with P2, the distal promoter. These deletions result in constitutive transcription from P1. This indicates that the DNA region which contains P2 functions mainly as a target site for regulation of P1 transcription in vivo.

Base Sequence↗
PubMed1981

The structure and function of the regulatory elements of the Escherichia coli uvrB gene.

The construction and properties of recombinant plasmids carrying the Escherichia coli uvrB gene, including its transcriptional- and translational regulatory elements, is reported. The DNA sequence of the region, which governs the expression of the uvrB gene, has been determined. Within this sequence two non-overlapping DNA segments match the model sequence for Escherichia coli promoters (1). The '-10 regions' and the '-35 regions' of the proposed uvrB promoters are, respectively, 5'TAAAAT (P1), 5'TATAAT (P2) and 5'TTGGCA (P1), 5'GTGATG (P2). The existence and the position of these promoters has been established by elimination of one promoter (P2), using molecular cloning procedures, by length measurements of in vitro synthesized 'run-off' transcripts and by protection of the uvrB regulatory region for S1 nuclease digestion using in vivo made RNA. Potential sites of interaction within the uvrB regulatory region with regulatory proteins, such as the LexA protein (2) and the UvrC protein (3) are discussed.

Amino Acid Sequence↗
PubMed1980

Structure of a promotor on plasmid pMB9 derived from plasmid pSC101.

The DNA sequence of a 354 basepair EcoRI-HindIII fragment of plasmid pMB9 which has originally been derived from plasmid pSC101 has been resolved. This fragment contains a promoter for transcription directed towards the EcoRI site. Escherichia coli RNA polymerase binds to a region within the EcoRI-HindIII fragment which contains the heptamer 5' TATGGTG (132-126) and the duodecamer 5' TGATGAACATCA (158-147). Based on commonalities with other promotors these DNA sequences probably function as, respectively, "binding site" and "recognition site". Furthermore, this fragment harbours a translation reading frame free of nonsense codons and at about 25 basepairs from the indicated heptamer a nucleotide sequence which meets with the requirements for initiation of translation. By heteroduplex mapping it was shown that the EcoRI-HindIII fragment has been derived from a region near or within the origin of replication of pSC101. The copynumber of plasmids containing the EcoRI-HindIII fragment is two-fold lower than that of plasmids lacking this fragment. This effect might be related to the original function of this fragment on plasmid pSC101.

Base Sequence↗
PubMed1980

Relief of polarity caused by transposon Tn5: application in mapping a cloned region of the Escherichia coli uvrB locus essential for UV resistance.

The structure and function of recombinant plasmid pNP5, which consists of vector pMB9 and a 2.5 kb EcoRI fragment harbouring the Escherichia coli uvrB gene, has been investigated. Insertional inactivation with the transposons Tn1 (Apr) or Tn5 (Kmr) has been used to determine the region on pNP5 DNA that is essential for UV resistance in uvrB deletion strains. This region spans approx. 1.8 kb and is separated by at least 280 bp from the pMB9 promoter to which it has been fused. Furthermore, a procedure is described to eliminate the polarity exerted by the transposon Tn5. A combination of in vitro digestion of pNP5::Tn5 DNA with restriction endonuclease XHoI, followed by ligation and subsequent in vivo propagation of the resulting plasmid DNA yields predominantly pNP5 molecules with a site-specific nonpolar mutation. The method allows an investigation of cloned complex genetic units, such as operons.

Chromosome Mapping↗
PubMed1979

Expression of the cloned uvrB gene of Escherichia coli: dependency on nonsense suppressors.

Recombinant plasmid pNP5, consisting of plasmid pMB9 on which the uvrB gene is cloned, fully complements for the defects due to chromosomal uvrB mutations in the presence of the amber suppressor sup-6 or supF. Correndonuclease II activity was also completely restored in in UvrB strains containing both plasmid pNP5 and amber suppressor sup-6, as compared with the parental UvrB+ strain. It is shown that the amber mutation which interferes with the expression of the cloned uvrB gene is located outside this gene. Apparently, the amber mutation exerts a polar effect on uvrB expression that is relieved by sup-6 or supF. Introduction of a rho mutation into suppressor-free UvrB strains, harboring pNP5, did not relieve the polarity caused by the amber mutation.

Endonucleases↗
PubMed1979

Expression of the cloned uvrB gene of Escherichia coli: mode of transcription and orientation.

The Escherichia coli uvrB gene, located on a 1.5-megadalton EcoRI (fragment F, derived from transducing phage lambda b2att2 [lambda b2cI857intam6 delta (bioAB)bio-FCD+uvrB+], has been cloned in the unique EcoRI site of several "relaxed" plasmids, i.e., pMB9, pBR322, and pBH20 (= ;BR322, including the lac regulatory elements [K. Itakura, T. Hirose, R. Crea, A. D. Riggs, H. L. Heyneker, F. Bolivar, and H. W. Boyer, Science 198:1056--1063, 1977]y. Expression of the uvrB gene, both on pMB9 and on pBH20, occurs only when fragment F has one particular orientation. Cloning of this fragment on pBR322 in either orientation does not allow expression of the uvrB gene. Transcription of this gene on pNP5 ( = pMB9 uvrB) is shown to be dependent on a pMB9 promotor that is located on a 0.22-megadalton EcoRI-HindIII fragment. Using plasmid pBH20 as a vector, we could demonstrate that expression of the uvrB gene is under control of the lac promotor-operator region. From deoxyribonucleic acid-deoxyribonucleic acid hybridization experiments with lambda pgal8 deoxyribonucleic acid and restriction fragments of pNP5 deoxyribonucleic acid it could be shown that the uvrB gene is transcribed clockwise on the chromosome.

Chromosomes, Bacterial↗
PubMed1978

Expression of the uvrB gene of Escherichia coli: in vitro construction of a pMB9 uvrB plasmid.

Bacteriophage lambdab2att2 [lambdab2cI857intam6(deltabioAB)bioFCD+uvrB+phr+] codes for a function(s) that confers UV resistance (Uvr+) and reactivation of irradiated phage (Hcr+) to an Uvr-Hcr-Escherichia coli strain. It was demonstrated that these functions are expressed under the control of bacterial regulatory elements located on lambdab2att2 DNA. The location of the E. coli uvrB gene on the DNA of this transducing phage was established by heteroduplex and restriction-enzyme analyses. Recombinant DNA molecules were constructed in vitro from plasmid pMB9 (Tcr), as the vector, and an EcoRI fragment (Eco-RI-F) of lambdab2att2 DNA. The resulting plasmid, designated pNP5, has a molecular weight of 5.1 X 10(6) and replicates in a relaxed fashion. Transformation of E. coli uvrB with plasmid pNP5 resulted in clones that are Uvr+ Tcr. Irradiation of bacteria transformed with plasmid pNP5 with low UV doses revealed a complete restoratation of the Uvr+ phenotype by the presence of the cloned EcoRI-F DNA, while only a partial restoration was observed after irradiation with high UV doses. Likewise, the Hcr+ character was also partially restored due to the presence of pNP5. No correlation was found between the acquired Uvr+, Hcr+ properties, and the presence of correndonuclease II activity in an extract of bacteria that harbor plasmid pNP5.

Chromosome Mapping↗
PubMed1976

A transcriptional barrier in the regulatory region of the tryptophan operon of Escherichia coli: its role in the regulation of repressor-independent RNA synthesis.

A study was made of the influence of the growth rate of Escherichia coli bacteria on the transcription of the tryptophan (trp) operon, in various trp repressor negative strains. Selective measurement of the levels of RNA transcribed from the regulatory region (reg) of this operon and from the structural genes, respectively, has revealed that the increase of the rate of trpRNA synthesis with bacterial growth rate (Rose and Yanofsky, 1972) is due to a decrease of the frequency of termination of transcription at the transcriptional barrier in the regulatory region of the operon. In a mutant strain of E. coli with a deletion covering the promotor distal part of the regulatory region of the trp operon where the barrier is located, the rate of trpRNA synthesis is not affected by the growth rate. In suA- strains, in which Rho factor activity is reduced the bacterial growth rate does not affect the rate of synthesis of trpRNA. This result suggests that in wild type bacteria Rho factor contributes to the control of the transcription of the trp operon. In bacteria with a mutation rendering Tryptophanyl-tRNA synthetase (TRSase) inactive (trpS- strains) the rate of trpRNA synthesis is affected by the growth rate in the same way as in the isogenic wild type bacteria. This result indicates that TRSase plays no obligatory role in the control of trpRNA synthesis through a mechanism of termination and anti-termination of transcription, at least not in the studied strains, which carried a relA mutation.

Binding, Competitive↗
PubMed1975

Punctuation of transcription in vitro of the tryptophan operon of Escherichia coli. A novel type of control of transcription.

RNA transcribed in vitro from DNA of a tryptophan (trp) transducing strain of bacteriophage phi80 which contains the trp regulatory elements consists of a polycistronic messenger transcribed from the structural genes, and possibly the regulatory region, and a separate RNA species (called trp regRNA) which is transcribed from the regulatory region. This conclusion is based on hybridization experiments with trp RNA synthesized in vitro and the separate DNA strands of trp transducing strains of lambda with and without the trp regulatory elements. The length of trp regRNA determined by filtration on Sephadex G-200 is 110-180 nucleotides. From the amount and the length of trp regRNA we have calculated that 8-20 copies of trp regRNA are synthesized per copy of polycistronic trp mRNA. We conclude that during transcription of the trp operon RNA polymerase frequently is rejected at a specific site ahead of the first structural gene, trpE. The termination factor Rho is not involved in this process. A different protein fraction, which specifically stimulates the synthesis of trp enzymes in an in vitro protein-synthesizing system (Pouwels and Van Rotterdam, 1975), was found to antagonize the abortive synthesis of trp mRNA. A model is proposed for the control of transcription of the trp genes, which operates through a mechanism of punctuation of RNA synthesis at a specific site on the DNA template and anti-termination of RNA synthesis by means of a positive control factor.

Coliphages↗