We have previously reported the identification of a novel activity residing in the nuclear fracti... more We have previously reported the identification of a novel activity residing in the nuclear fraction of mammalian cells that selectively binds and hydrolyses deoxyribonucleoside triphosphates. Incubation of this protein with [alpha-32P]dATP leads to the appearance of a retarded band relative to free dATP when the reaction is analysed on non-denaturing polyacrylamide gels. We now show that the retarded species comprises the product of dATP hydrolysis (dADP or dAMP) bound to an as yet unidentified species. We have termed this complex the ‘product-nucleotide binding particle’ or PNBP*. Through a combination of continuous elution polyacrylamide-gel electrophoresis and gel-filtration chromatography, we demonstrate that the hydrolytic activity (dNTPase) is distinct from the radiolabelled species detected in gel-retardation experiments. T.l.c. confirms that the labelled product does not share RF values associated with a range of mono-, di- and tri-phosphate deoxyribonucleotide standards, an...
It is the exquisite interplay of proteins and nucleic acids within the cell which gives rise to t... more It is the exquisite interplay of proteins and nucleic acids within the cell which gives rise to the controlled expression and replication of the genetic material. Our present understanding of these processes is in part the result of the successful purification and characterization of the participating macromolecules. The achievements of the early molecular biologists in obtaining active, homogeneous preparations of low-abundance gene regulatory proteins are outstanding. The strategies employed for the purification of such proteins are, however, no different in principle to those procedures used to purify high-abundance proteins. Moreover, one of the goals of molecular biologists in purifying gene regulatory proteins is to clone the corresponding gene. When this has been achieved, the gene can often be overexpressed, and the purification of the gene product becomes trivial by comparison.
The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selectio... more The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2′-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with Taq polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be sub...
Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the reaction pathway... more Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the reaction pathway, have been deployed extensively in the analysis of metabolic pathways and as candidate drugs. The inhibition of cytosine-[C5]-specific DNA methyltransferases (C5 MTases) by oligodeoxynucleotides containing 5-azadeoxycytidine (AzadC) and 5-fluorodeoxycytidine (FdC) provides a well-documented example of mechanism-based inhibition of enzymes central to nucleic acid metabolism. Here, we describe the
Summary Triple-labelled nutrient solution was used to compare the effects of seven root-infecting... more Summary Triple-labelled nutrient solution was used to compare the effects of seven root-infecting fungi on uptake of K, Ca and P by wheat. Plants grown in sand or hydroponic culture were transferred to solutions that contained42K,45Ca and32P for 24 h, then dried, ashed and digested in 6M HCl. To distinguish radiation emitted by42K,45Ca and32P plant digests were counted on two
... Author: Ben Cousins (with Rauri Alcock, Ngididi Dladla, Donna Hornby, Mphethethi Masondo, Gug... more ... Author: Ben Cousins (with Rauri Alcock, Ngididi Dladla, Donna Hornby, Mphethethi Masondo, Gugu Mbatha, Makhosi Mweli and Creina Alcock) Copy editor: Prem Egert Series editor: Rebecca Pointer All photographs ... Chapter 3: A brief history of the Mchunu and Mthembu tribes ...
... D. HORNBY 1 ,; GL BATEMAN 1 ,; RW PAYNE 1 ,; ME BROWN 1 ,; DR HENDEN 1 ,; R. CAMPBELL 2. Arti... more ... D. HORNBY 1 ,; GL BATEMAN 1 ,; RW PAYNE 1 ,; ME BROWN 1 ,; DR HENDEN 1 ,; R. CAMPBELL 2. Article first published online: 26 FEB 2008. DOI: 10.1111/j.1744-7348.1993.tb04031. x. Issue. Annals of Applied Biology. Volume 122, Issue 2, pages 253270, April 1993. ...
We have previously reported the identification of a novel activity residing in the nuclear fracti... more We have previously reported the identification of a novel activity residing in the nuclear fraction of mammalian cells that selectively binds and hydrolyses deoxyribonucleoside triphosphates. Incubation of this protein with [alpha-32P]dATP leads to the appearance of a retarded band relative to free dATP when the reaction is analysed on non-denaturing polyacrylamide gels. We now show that the retarded species comprises the product of dATP hydrolysis (dADP or dAMP) bound to an as yet unidentified species. We have termed this complex the ‘product-nucleotide binding particle’ or PNBP*. Through a combination of continuous elution polyacrylamide-gel electrophoresis and gel-filtration chromatography, we demonstrate that the hydrolytic activity (dNTPase) is distinct from the radiolabelled species detected in gel-retardation experiments. T.l.c. confirms that the labelled product does not share RF values associated with a range of mono-, di- and tri-phosphate deoxyribonucleotide standards, an...
It is the exquisite interplay of proteins and nucleic acids within the cell which gives rise to t... more It is the exquisite interplay of proteins and nucleic acids within the cell which gives rise to the controlled expression and replication of the genetic material. Our present understanding of these processes is in part the result of the successful purification and characterization of the participating macromolecules. The achievements of the early molecular biologists in obtaining active, homogeneous preparations of low-abundance gene regulatory proteins are outstanding. The strategies employed for the purification of such proteins are, however, no different in principle to those procedures used to purify high-abundance proteins. Moreover, one of the goals of molecular biologists in purifying gene regulatory proteins is to clone the corresponding gene. When this has been achieved, the gene can often be overexpressed, and the purification of the gene product becomes trivial by comparison.
The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selectio... more The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2′-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with Taq polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be sub...
Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the reaction pathway... more Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the reaction pathway, have been deployed extensively in the analysis of metabolic pathways and as candidate drugs. The inhibition of cytosine-[C5]-specific DNA methyltransferases (C5 MTases) by oligodeoxynucleotides containing 5-azadeoxycytidine (AzadC) and 5-fluorodeoxycytidine (FdC) provides a well-documented example of mechanism-based inhibition of enzymes central to nucleic acid metabolism. Here, we describe the
Summary Triple-labelled nutrient solution was used to compare the effects of seven root-infecting... more Summary Triple-labelled nutrient solution was used to compare the effects of seven root-infecting fungi on uptake of K, Ca and P by wheat. Plants grown in sand or hydroponic culture were transferred to solutions that contained42K,45Ca and32P for 24 h, then dried, ashed and digested in 6M HCl. To distinguish radiation emitted by42K,45Ca and32P plant digests were counted on two
... Author: Ben Cousins (with Rauri Alcock, Ngididi Dladla, Donna Hornby, Mphethethi Masondo, Gug... more ... Author: Ben Cousins (with Rauri Alcock, Ngididi Dladla, Donna Hornby, Mphethethi Masondo, Gugu Mbatha, Makhosi Mweli and Creina Alcock) Copy editor: Prem Egert Series editor: Rebecca Pointer All photographs ... Chapter 3: A brief history of the Mchunu and Mthembu tribes ...
... D. HORNBY 1 ,; GL BATEMAN 1 ,; RW PAYNE 1 ,; ME BROWN 1 ,; DR HENDEN 1 ,; R. CAMPBELL 2. Arti... more ... D. HORNBY 1 ,; GL BATEMAN 1 ,; RW PAYNE 1 ,; ME BROWN 1 ,; DR HENDEN 1 ,; R. CAMPBELL 2. Article first published online: 26 FEB 2008. DOI: 10.1111/j.1744-7348.1993.tb04031. x. Issue. Annals of Applied Biology. Volume 122, Issue 2, pages 253270, April 1993. ...
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