scholarly journals Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors.

1981 ◽  
Vol 1 (9) ◽  
pp. 854-864 ◽  
Author(s):  
S Subramani ◽  
R Mulligan ◽  
P Berg
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Mammalian Cells ◽  
Deoxyribonucleic Acid ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Ovary Cells ◽  
Complementary Deoxyribonucleic Acid

A mouse complementary deoxyribonucleic acid segment coding for the enzyme dihydrofolate reductase has been cloned in two general classes of vectors containing simian virus 40 deoxyribonucleic acid: (i) those that can be propagated as virions in permissive cells and (ii) those that can be introduced into and maintained stably in various mammalian cells. Both types of vectors express the mouse dihydrofolate reductase by using signals supplied by simian virus 40 deoxyribonucleic acid sequences. Moreover, plasmid vectors carrying the complementary deoxyribonucleic acid segment can complement Chinese hamster ovary cells lacking dihydrofolate reductase.

Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors

1981 ◽  
Vol 1 (9) ◽  
pp. 854-864
Author(s):  
S Subramani ◽  
R Mulligan ◽  
P Berg
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Mammalian Cells ◽  
Deoxyribonucleic Acid ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Ovary Cells ◽  
Complementary Deoxyribonucleic Acid

A mouse complementary deoxyribonucleic acid segment coding for the enzyme dihydrofolate reductase has been cloned in two general classes of vectors containing simian virus 40 deoxyribonucleic acid: (i) those that can be propagated as virions in permissive cells and (ii) those that can be introduced into and maintained stably in various mammalian cells. Both types of vectors express the mouse dihydrofolate reductase by using signals supplied by simian virus 40 deoxyribonucleic acid sequences. Moreover, plasmid vectors carrying the complementary deoxyribonucleic acid segment can complement Chinese hamster ovary cells lacking dihydrofolate reductase.

Growth-dependent expression of dihydrofolate reductase mRNA from modular cDNA genes

1983 ◽  
Vol 3 (9) ◽  
pp. 1598-1608
Author(s):  
R J Kaufman ◽  
P A Sharp
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Ovary Cells ◽  
Dividing Cells ◽  
Major Late Promoter ◽  
The Stability

Dihydrofolate reductase (DHFR) synthesis is regulated in a growth-dependent fashion. Dividing cells synthesize DHFR at a 10-fold-higher rate than do stationary cells. To study this growth-dependent synthesis. DHFR genes have been constructed from a DHFR cDNA segment, the adenovirus major late promoter, and fragments of simian virus 40 (SV40) which provide signals for polyadenylation. These genes have been introduced into Chinese hamster ovary cells. The DHFR mRNAs produced in different transformants are identical at their 5' ends, but differ in sequences in their 3' ends as different sites are utilized for polyadenylation. Three transformants that utilize either DHFR polyadenylation signals or the SV40 late polyadenylation signal exhibit growth-dependent DHFR synthesis. The level of DHFR mRNA in growing cells is approximately 10 times that in stationary cells for these transformants. This growth-dependent DHFR mRNA production probably results from posttranscriptional events. In contrast, three transformants that utilize the SV40 early polyadenylation signal and another transformant that utilizes a cellular polyadenylation signal do not exhibit growth-dependent DHFR synthesis. In these three cell lines, the fraction of mRNAs polyadenylated at different sites in a tandem array shifts between growing and stationary cells. These results suggest that the metabolic state of the cell is important in determining either the efficiency of polyadenylation at various sites or the stability of mRNA polyadenylated at various sites.

scholarly journals Mapping an origin of DNA replication at a single-copy locus in exponentially proliferating mammalian cells.

1990 ◽  
Vol 10 (9) ◽  
pp. 4685-4689 ◽  
Author(s):  
L T Vassilev ◽  
W C Burhans ◽  
M L DePamphilis
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Single Copy ◽  
Simian Virus ◽  
Metabolic Inhibitors ◽  
Ovary Cells ◽  
Cellular Origin

A general method for determining the physical location of an origin of bidirectional DNA replication has been developed recently and shown to be capable of correctly identifying the simian virus 40 origin of replication (L. Vassilev and E. M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989). The advantage of this method over others previously reported is that it avoids the use of metabolic inhibitors, the requirement for cell synchronization, and the need for multiple copies of the origin sequence. Application of this method to exponentially growing Chinese hamster ovary cells containing the nonamplified, single-copy dihydrofolate reductase gene locus revealed that DNA replication begins bidirectionally in an initiation zone approximately 2.5 kilobases long centered about 17 kilobases downstream of the DHFR gene, coinciding with previously described early replicating sequences. These results demonstrate the utility of this mapping protocol for identifying cellular origins of replication and suggest that the same cellular origin is used in both the normal and the amplified DHFR locus.

scholarly journals Growth-dependent expression of dihydrofolate reductase mRNA from modular cDNA genes.

1983 ◽  
Vol 3 (9) ◽  
pp. 1598-1608 ◽  
Author(s):  
R J Kaufman ◽  
P A Sharp
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Ovary Cells ◽  
Dividing Cells ◽  
Major Late Promoter ◽  
The Stability

Dihydrofolate reductase (DHFR) synthesis is regulated in a growth-dependent fashion. Dividing cells synthesize DHFR at a 10-fold-higher rate than do stationary cells. To study this growth-dependent synthesis. DHFR genes have been constructed from a DHFR cDNA segment, the adenovirus major late promoter, and fragments of simian virus 40 (SV40) which provide signals for polyadenylation. These genes have been introduced into Chinese hamster ovary cells. The DHFR mRNAs produced in different transformants are identical at their 5' ends, but differ in sequences in their 3' ends as different sites are utilized for polyadenylation. Three transformants that utilize either DHFR polyadenylation signals or the SV40 late polyadenylation signal exhibit growth-dependent DHFR synthesis. The level of DHFR mRNA in growing cells is approximately 10 times that in stationary cells for these transformants. This growth-dependent DHFR mRNA production probably results from posttranscriptional events. In contrast, three transformants that utilize the SV40 early polyadenylation signal and another transformant that utilizes a cellular polyadenylation signal do not exhibit growth-dependent DHFR synthesis. In these three cell lines, the fraction of mRNAs polyadenylated at different sites in a tandem array shifts between growing and stationary cells. These results suggest that the metabolic state of the cell is important in determining either the efficiency of polyadenylation at various sites or the stability of mRNA polyadenylated at various sites.

Mapping an origin of DNA replication at a single-copy locus in exponentially proliferating mammalian cells

1990 ◽  
Vol 10 (9) ◽  
pp. 4685-4689
Author(s):  
L T Vassilev ◽  
W C Burhans ◽  
M L DePamphilis
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Single Copy ◽  
Simian Virus ◽  
Metabolic Inhibitors ◽  
Ovary Cells ◽  
Cellular Origin

A general method for determining the physical location of an origin of bidirectional DNA replication has been developed recently and shown to be capable of correctly identifying the simian virus 40 origin of replication (L. Vassilev and E. M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989). The advantage of this method over others previously reported is that it avoids the use of metabolic inhibitors, the requirement for cell synchronization, and the need for multiple copies of the origin sequence. Application of this method to exponentially growing Chinese hamster ovary cells containing the nonamplified, single-copy dihydrofolate reductase gene locus revealed that DNA replication begins bidirectionally in an initiation zone approximately 2.5 kilobases long centered about 17 kilobases downstream of the DHFR gene, coinciding with previously described early replicating sequences. These results demonstrate the utility of this mapping protocol for identifying cellular origins of replication and suggest that the same cellular origin is used in both the normal and the amplified DHFR locus.

scholarly journals A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

1995 ◽  
Vol 6 (2) ◽  
pp. 135-150 ◽  
Author(s):  
N T Ktistakis ◽  
C Y Kao ◽  
R H Wang ◽  
M G Roth
Keyword(s):  
Chinese Hamster Ovary ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Secretory Activity ◽  
Specific Marker ◽  
Ovary Cells ◽  
Fluorescent Lipid

The use of reporter proteins to study the regulation of secretion has often been complicated by posttranslational processing events that influence the secretion of certain proteins, but are not part of the cellular mechanisms that specifically regulate secretion. This has been a particular limitation for the isolation of mammalian secretion mutants, which has typically been a slow process. To provide a reporter of secretory activity independent of protein processing events, cells were labeled with the fluorescent lipid analogue C5-DMB-ceramide (ceramide coupled to the fluorophore boron dipyrromethene difluoride) and its secretion was followed by fluorescence microscopy and fluorescence-activated cell sorting. Brefeldin A, which severely inhibits secretion in Chinese hamster ovary cells, blocked secretion of C5-DMB-ceramide. At high temperature, export of C5-DMB-ceramide was inhibited in HRP-1 cells, which have a conditional defect in secretion. Using C5-DMB-ceramide as a reporter of secretory activity, several different pulse-chase protocols were designed that selected mutant Chinese hamster ovary cells that were resistant to the drug brefeldin A and others that were defective in the transport of glycoproteins to the cell surface. Mutant cells of either type were identified in a mutagenized population at a frequency of 10(-6). Thus, the fluorescent lipid C5-DMB-ceramide can be used as a specific marker of secretory activity, providing an efficient, general approach for isolating mammalian cells with defects in the secretory pathway.

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