Use of a urethral catheter to remove an upper airway foreign body.
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Anesthesia, Inhalation↗
Biomedical subjects
A C Porter
Publications and source records attributed to A C Porter.
Gene targeting: techniques and applications to transplantation.
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Animals↗
Cloning and functional expression of guinea pig atrial alpha 2-adrenergic receptor subtypes.
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Animals↗
Construction by gene targeting in human cells of a "conditional' CDC2 mutant that rereplicates its DNA.
We describe a novel gene targeting strategy for the genetic analysis of essential genes in mammalian cells and its use to study the role of the cell cycle control gene CDC2 in human cells. A cell line (HT2-19) was generated in which endogenous CDC2 gene expression and cell viability depend on the presence of an inducer in the growth medium. In the absence of inducer, HT2-19 cells undergo extensive DNA rereplication and apoptosis. Rereplication is indicative of a role for human CDC2 in a control mechanism, previously undetected in mammalian cells, that prevents premature entry into S-phase.
Apoptosis↗
Heterologous expression of the cloned guinea pig alpha 2A, alpha 2B, and alpha 2C adrenoceptor subtypes. Radioligand binding and functional coupling to a CAMP-responsive reporter gene.
Functional studies have shown that 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4,5-tetrahydro-3- benzazepine (SKF 104078) has very low affinity for prejunctional alpha 2-adrenoceptors (alpha 2-AR) in the guinea pig atrium. In this study, we have cloned guinea pig homologues of the human alpha 2-C10, alpha 2-C4 AR subtypes and have studied them in isolation by heterologous expression in cultured mammalian cells. Oligonucleotide primers, designed from conserved areas of the human alpha 2-ARs were used in a polymerase chain reaction (PCR) with template cDNA synthesized from guinea pig atrial mRNA. Three PCR products were obtained that shared identity with the three human alpha 2-AR subtypes. A guinea pig (gp) genomic library was screened with a cDNA clone encoding a portion of the gp-alpha 2A, and genes containing the complete coding sequences of the guinea pig alpha 2A, alpha 2B, and alpha 2C AR subtypes were obtained. These guinea pig genes were subcloned into a eukaryotic expression plasmid and were expressed transiently in COS-7 cells. The binding of the alpha 2-selective antagonist [3H]MK-912 to membranes prepared from these cells was specific and of high affinity with Kd values of 810 pM for gp-alpha 2A, 2700 pM for gp-alpha 2B and 110 pM for gp-alpha 2C. Competition for the binding of [3H]MK-912 by SKF 104078 indicated that it was of moderately high affinity (approximately 100 nM) but that it was not selective for any of the guinea pig alpha 2-AR subtypes. Co-expression of guinea pig alpha 2-AR subtypes with a cyclicAMP-responsive chloramphenicol acetyltransferase (CAT) reporter gene resulted in agonist-dependent modulation of CAT activity. For the gp-alpha 2 A, a biphasic response was obtained with low concentrations of noradrenaline (NE) decreasing forskolin-stimulated CAT activity and high concentrations causing a reversal. For the gp-alpha 2B, NE produced mostly potentiation of forskolin-stimulated activity, and for the gp-alpha 2C, NE caused mainly inhibition. Overall, the pharmacology of the cloned guinea pig alpha 2-AR subtypes was in agreement with data obtained for the native guinea pig receptors and was functionally similar to that of the cloned human alpha 2-AR subtypes.
Adrenergic alpha-Antagonists↗
Characterisation of a novel minisatellite that provides multiple splice donor sites in an interferon-induced transcript.
Nucleotide sequence features of the human interferon-inducible gene 6-16 are described and include, within a CpG island, a partially expressed minisatellite consisting of 26 tandemly repeated dodecanucleotides. The repeat unit consensus sequence (CAGGTAAGGGTG) is similar to the mammalian splice donor consensus sequence [(A/C)AGGT(A/G)AGT]. The splice donor site of exon 2, as determined previously, forms part of the most upstream of the repeat units. We show that the two neighbouring repeat units also provide functional splice donor sites effectively extending exon 2 by 12 or 24 nt and inserting four or eight amino acids respectively into the predicted gene product. A similar pattern of differently spliced transcripts is detected in several human cell types. Both the number of repeat units per allele and the nucleotide sequence itself show limited polymorphism within the human population. Similar minisatellites from nonhuman primates are described and also appear to modulate splicing of a 6-16 transcript. The 6-16 minisatellite is therefore an example of tandemly repeated DNA that has a role in gene expression and may provide a useful in vivo system for the analysis of 5' splice site choice and minisatellite biology.
Amino Acid Sequence↗
The novel alpha-2 adrenergic radioligand [3H]-MK912 is alpha-2C selective among human alpha-2A, alpha-2B and alpha-2C adrenoceptors.
We have determined the binding affinities of the novel alpha-2 adrenoceptor antagonist radioligand [3H]-MK912 for the cloned human alpha-2A, alpha-2B and alpha-2C adrenoceptors. The KD-values were 1.25 nM, 1.36 nM and 0.086 nM for the alpha-2A, alpha-2B and alpha-2C subtypes, respectively. Thus, the selectivity of [3H]-MK912 for the human alpha-2C adrenoceptor vs. the human alpha-2A and alpha-2B adrenoceptors is 14-fold and 16-fold, respectively. The alpha-2C selectivity, and the very high affinity of [3H]-MK912 for the alpha-2C adrenoceptor subtype (KD = 86 pM) makes this radioligand a promising tool for studying the role of alpha-2C adrenoceptors in the human. A selection of antagonists useful for differentiating between the human alpha-2A, alpha-2B and alpha-2C adrenoceptor subtypes is discussed.
Adrenergic alpha-Antagonists↗
Gene targeting in human somatic cells. Complete inactivation of an interferon-inducible gene.
The role, if any, of the human interferon-inducible 6-16 gene in the establishment of a cellular antiviral state is unknown. To address this problem, and as part of a wider investigation of homologous recombination (HR) and its applications in somatic cells, we have been using HR to disrupt the 6-16 gene in human cell lines [Itzhaki, J. E. & Porter, A. C. G. (1991) Nucleic Acids Res. 19, 3835-3842.] We describe here the design and use of insertion and replacement-type targeting constructs based on a promoterless bacterial gpt gene that is activated by HR with the 6-16 gene. In HeLa cells, both targeting constructs underwent extrachromosomal HR with a cotransfected plasmid carrying the 6-16 gene. In a previously targeted clone derived from the fibrosarcoma cell line HT1080, the replacement construct underwent HR with either the modified or the unmodified 6-16 allele. The latter events generated doubly disrupted (6-16-/-) clones that failed to express any detectable 6-16 messenger RNA in response to interferon. Plaque assays of infected 6-16-/- cells showed that expression of the 6-16 gene was not required for the induction by interferon of an antiviral state against encephalomyocarditis virus, semliki forest virus or cocal virus.
Base Sequence↗
Telomere directed fragmentation of mammalian chromosomes.
Cloned human telomeric DNA can integrate into mammalian chromosomes and seed the formation of new telomeres. This process occurs efficiently in three established human cell lines and in a mouse embryonic stem cell line. The newly seeded telomeres appear to be healed by telomerase. The seeding of new telomeres by cloned telomeric DNA is either undetectable or very inefficient in non-tumourigenic mouse or human somatic cell lines. The cytogenetic consequences of the seeding of new telomeres include large chromosome truncations but most of the telomere seeding events occur close to the pre-existing ends of natural chromosomes.
Animals↗
Targeted breakage of a human chromosome mediated by cloned human telomeric DNA.
Novel approaches to the structural and functional analysis of mammalian chromosomes would be possible if the gross structure of the chromosomes in living cells could be engineered. Controlled modifications can be engineered by conventional targeting techniques based on homologous recombination. Large but uncontrolled modifications can be made by the integration of cloned human telomeric DNA. We describe here the combined use of gene targeting and telomere-mediated chromosome breakage to generate a defined truncation of a human chromosome. Telomeric DNA was targeted to the 6-16 gene on the short arm of chromosome 1 in a human cell line. Molecular and cytogenetic analyses showed that, of eight targeted clones that were isolated, one clone had the predicted truncation of chromosome 1.
Cell Line↗
Targeted disruption of a human interferon-inducible gene detected by secretion of human growth hormone.
A new method is described for the sib-selection of 'targeted' mammalian cells that have undergone homologous recombination (HR) with a transfected DNA construct. This method has been used to disrupt the 6-16 gene, an interferon (IFN)-inducible gene of unknown function, in two different human cell lines. Disruption was caused by integration of a targeting construct containing a promoterless gene for human growth hormone (hGH) which was expressed after HR with the 6-16 gene. Homologous recombinants were detected in pools of non-homologous recombinants by the appearance of hGH in the growth medium after the addition of IFN. Secondary and tertiary rounds of hGH assays were used to sib-select 9 homologous recombinants that were shown to have 1, 2 or 3 copies of the targeting construct integrated at the 6-16 locus. The method, which should be applicable to other transcribed targets, provides an alternative to selection methods, and offers advantages over other screening methods in being simple, rapid, sensitive and reliable.
Base Sequence↗
Differential response of the human 6-16 and 9-27 genes to alpha and gamma interferons.
9-27 mRNA is expressed to a high level in response to both alpha and gamma interferons. In contrast, 6-16 mRNA is expressed well in response to alpha but very poorly in response to gamma interferon in human cells. The factors governing these different levels of expression were investigated. For both genes the major effect of both interferons is on transcription. A transcriptional bias in the 6-16 promoter/enhancer accounts in large part for the differential response of 6-16 to the two interferons. No single DNA element appears responsible; the smaller the 5' region analysed the lower the absolute activity and the smaller the differential response to alpha and gamma interferons observed. Both the 6-16 and 9-27 mRNAs are very stable and no effect of the interferons on stability was detected. Nor was any direct evidence obtained for preferential processing of the 9-27 mRNA. Nevertheless, differentials between the transcription and accumulation of mature mRNAs, particularly for 6-16 mRNA in response to gamma interferon, suggest that post-transcriptional control(s) must additionally operate. The 9-27 5' promoter/enhancer is much less active than that from 6-16 when placed 5' of a marker gene, despite the similar response of the two genes to alpha interferon.
Blotting, Northern↗
A single DNA response element can confer inducibility by both alpha- and gamma-interferons.
Genomic and cDNA clones corresponding to 9-27, a member of the human 1-8 gene family highly inducible by alpha- and gamma-interferons (IFNs), have been isolated and characterized. A 1.7-kilobase genomic clone contains a complete functional gene with two exons, encoding a 125-amino acid polypeptide of unknown function. The 5' flanking region of the gene contains a 13-base-pair IFN-stimulable response element (ISRE), homologous to the ISREs of the 6-16, ISG 15, and ISG 54 genes, which are predominantly inducible by IFN-alpha, beta. Analysis of constructs containing native and mutated ISREs suggests that this motif is essential for the response of 9-27 to IFN-gamma as well as IFN-alpha. Furthermore, the 9-27 (GGAAATAGAAACT) and 6-16 (GGGAAAATGAAACT) ISREs can each confer a response to both types of IFN when placed on the 5' side of a marker gene. Since the 6-16 gene does not normally respond to IFN-gamma, the context of the ISRE must determine the specificity of the response.
Amino Acid Sequence↗
Lymphokine production in allografts--analysis of RNA by northern blotting.
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Animals↗
Changes in drinking activity, urine volume and urinary electrolyte excretion after intracerebroventricular administration of diisopropylfluorophosphate.
The effects of i.c.v. administration of diisopropylfluorophosphate on drinking activity, urine volume and urinary electrolyte excretion were examined in the conscious, unrestrained rat. An i.c.v. dose of 5 micrograms of diisopropylfluorophosphate was found to inhibit acetylcholinesterase activity in five nuclei in the anterior, dorsal portion of the hypothalamus. This dose of diisopropylfluorophosphate also resulted in a dipsogenic episode, a diuresis and a biphasic effect on urinary sodium and potassium excretion. Each response was independent of the other two responses. The responses were mediated by muscarinic cholinergic receptors other than nicotinic receptors as they were blocked by both atropine and pirenzepine but not mecamylamine. The results suggest that the muscarinic receptors responsible are probably of muscarinic receptor M1 class.
Animals↗
Interferon response element of the human gene 6-16.
1046 base-pairs (bp) of genomic DNA spanning the first exon of the human alpha/beta-interferon (IFN)-inducible gene 6-16 have been analysed for their role in induction. The whole gene or 5'-flanking deletion derivatives of it were assayed for inducibility in populations of stably transfected mouse cells. 5'-Flanking DNA fragments were assayed for their ability to confer inducibility on a reporter gene in stably and transiently transfected mouse and human cells. The data suggest that a 39 bp sequence is sufficient to confer transcriptional inducibility and can account in large part for the response of 6-16. Two copies of this sequence, one of which contains a dinucleotide insert, are located in tandem 88 bp upstream of the 6-16 transcriptional initiation site. For at least one of the repeat units the 5' limit of a subregion required for induction lies in the sequence GGGAAAAT. The motif GGAAA occurs in several well characterized enhancers. Furthermore, one residue 3' of the GGAAA there is a second motif, TGAAACT, which is conserved in the regulatory regions of other IFN-induced genes. In gel retardation assays the oligonucleotide GGGAAAATGAAACT competes with the repeat element for binding to IFN-modulated protein(s) but a mutated oligonucleotide, GGGAAAATGACACT does not. These results identify an alpha/beta IFN response element partially homologous to those described previously for the genes of the MHC complexes.
Animals↗
Characterization of a human gene inducible by alpha- and beta-interferons and its expression in mouse cells.
An intact interferon-inducible gene has been isolated from a cosmid library of human genomic DNA. The gene (designated 6-16) encodes a mRNA of approximately 1 kb which is induced well by alpha- and beta- but poorly by gamma-interferons. Genomic and cDNA sequences indicate that the gene contains five exons, and that the mRNA encodes a hydrophobic polypeptide of 130 amino acids with a putative NH2-terminal signal sequence. The 5' end has been identified by primer extension. The corresponding genomic DNA contains a TATA box 20 nucleotides upstream of the putative transcription initiation site. After transfection of the human genomic cosmid into mouse Ltk- cells, human 6-16 mRNA is expressed in response to mouse alpha- and beta- but not gamma-interferons with the same kinetics and dose-response as in the human cells. No such expression is observed in response to human interferons. It can be concluded that the human cosmid DNA contains all of the sequences necessary for alpha- and beta-interferon-induced gene expression and that the mechanisms governing such expression are conserved between murine and human cells.
Amino Acid Sequence↗
Role of the b subunit of the Escherichia coli proton-translocating ATPase. A mutagenic analysis.
A plasmid that carries the uncF gene of Escherichia coli, which codes for the b subunit of the proton-translocating ATPase (F1F0), was mutagenized with hydroxylamine and ethyl methanesulfonate. Mutated plasmids were characterized by complementation analysis and by in vitro transcription/translation. Eleven of the plasmids mutated specifically in uncF were studied in detail: the nucleotide sequences of their uncF genes were determined and their effects on the F1F0 were measured. The results suggest that a C-terminal portion of the b subunit that involves the glycine residue at position 131, is required for the formation of a functional proton pore as well as for the binding of F1 to F0. A mutation in the N-terminal portion of the b subunit, in the glycine residue at position 9, also prevented the formation of a functional proton pore, but had only a small effect on the binding of F1 to F0.
Amino Acid Sequence↗