scholarly journals Activation of a mammalian origin of replication by chromosomal rearrangement.

1992 ◽  
Vol 12 (6) ◽  
pp. 2804-2812 ◽  
Author(s):  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Dihydrofolate Reductase ◽  
Mammalian Cells ◽  
Chromosomal Rearrangement ◽  
Chinese Hamster ◽  
Regulatory Elements ◽  
Replication Initiation ◽  
Type I ◽  
Type Ii ◽  
Direct Demonstration

The methotrexate-resistant Chinese hamster cell line DC3F/A3-4K (A3/4K) contains at least two prominent dihydrofolate reductase amplicon types. The type I amplicons, constituting approximately 80% of the total, are at least 650 kb in length, but the endpoints have not yet been characterized. The type II sequences represent approximately 20% of amplicons, are 450 kb in length, and are arranged as alternating head-to-head and tail-to-tail repeats. In previous studies on the CHOC 400 line, in which the amplicons are much smaller, a replication initiation locus (ori-beta/ori-gamma) has been shown to reside downstream from the dihydrofolate reductase gene. In a more recent study on the larger amplicons of A3/4K cells, we detected an additional initiation locus (ori-alpha) lying approximately 240 kb upstream from ori-beta/ori-gamma. Interestingly, in vivo labelling experiments suggested that replication forks diverge from ori-alpha only in the downstream direction. This finding suggested either that ori-alpha is a unidirectional origin or that a terminus lies immediately upstream from ori-alpha. However, in this study, we show that ori-alpha is actually very close to the head-to-head palindromic junction sequence between the minor type II amplicons in A3/4K cells; furthermore, ori-alpha is active in the early S period in the type II amplicons but not in the larger type I sequences that lack this palindromic junction. This is the first direct demonstration in mammalian cells that a cryptic origin can be activated by chromosomal rearrangement, presumably by deleting negative regulatory elements or by creating a more favorable chromosomal milieu for initiation.

scholarly journals Activation of a mammalian origin of replication by chromosomal rearrangement

1992 ◽  
Vol 12 (6) ◽  
pp. 2804-2812
Author(s):  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Dihydrofolate Reductase ◽  
Mammalian Cells ◽  
Chromosomal Rearrangement ◽  
Chinese Hamster ◽  
Regulatory Elements ◽  
Replication Initiation ◽  
Type I ◽  
Type Ii ◽  
Direct Demonstration

The methotrexate-resistant Chinese hamster cell line DC3F/A3-4K (A3/4K) contains at least two prominent dihydrofolate reductase amplicon types. The type I amplicons, constituting approximately 80% of the total, are at least 650 kb in length, but the endpoints have not yet been characterized. The type II sequences represent approximately 20% of amplicons, are 450 kb in length, and are arranged as alternating head-to-head and tail-to-tail repeats. In previous studies on the CHOC 400 line, in which the amplicons are much smaller, a replication initiation locus (ori-beta/ori-gamma) has been shown to reside downstream from the dihydrofolate reductase gene. In a more recent study on the larger amplicons of A3/4K cells, we detected an additional initiation locus (ori-alpha) lying approximately 240 kb upstream from ori-beta/ori-gamma. Interestingly, in vivo labelling experiments suggested that replication forks diverge from ori-alpha only in the downstream direction. This finding suggested either that ori-alpha is a unidirectional origin or that a terminus lies immediately upstream from ori-alpha. However, in this study, we show that ori-alpha is actually very close to the head-to-head palindromic junction sequence between the minor type II amplicons in A3/4K cells; furthermore, ori-alpha is active in the early S period in the type II amplicons but not in the larger type I sequences that lack this palindromic junction. This is the first direct demonstration in mammalian cells that a cryptic origin can be activated by chromosomal rearrangement, presumably by deleting negative regulatory elements or by creating a more favorable chromosomal milieu for initiation.

Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line

1988 ◽  
Vol 8 (6) ◽  
pp. 2316-2327
Author(s):  
C Ma ◽  
J E Looney ◽  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Pulse Field ◽  
Ovary Cell ◽  
Type I ◽  
Type Ii ◽  
Gradient Gel Electrophoresis

We have recently isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase (dhfr) amplicon types from the methotrexate-resistant Chinese hamster ovary (CHO) cell line CHOC 400. In the work described in this report, we used pulse-field gradient gel electrophoresis to analyze large SfiI restriction fragments arising from the amplified dhfr domains. The junction between the 260-kilobase type I amplicons (which are arranged in head-to-tail configurations in the genome) has been localized, allowing the construction of a linear map of the parental dhfr locus. We also show that the 220-kilobase type II amplicons are arranged as inverted repeat structures in the CHOC 400 genome and arose from the type I sequence relatively early in the amplification process. Our data indicate that there are a number of minor amplicon types in the CHOC 400 cell line that were not detected in previous studies; however, the type II amplicons represent ca. 75% of all the amplicons in the CHOC 400 genome. Both the type I and type II amplicons are shown to be composed entirely of sequences that were present in the parental dhfr locus. Studies of less resistant cell lines show that initial amplicons can be larger than those observed in CHOC 400. Once established, a given amplicon type appears to be relatively stable throughout subsequent amplification steps. We also present a modification of an in-gel renaturation method that gives a relatively complete picture of the size and variability of amplicons in the genome.

High-resolution mapping of replication fork movement through the amplified dihydrofolate reductase domain in CHO cells by in-gel renaturation analysis

1989 ◽  
Vol 9 (2) ◽  
pp. 523-531
Author(s):  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster ◽  
Slight Modification ◽  
Single Copy ◽  
Replication Initiation ◽  
Ovary Cell ◽  
Restriction Fragments ◽  
Labeling Pattern ◽  
Reductase Domain

Utilizing an in vivo labeling method on synchronized cultures, we have previously defined a 28-kilobase (kb) replication initiation locus in the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) (N. H. Heintz and J. L. Hamlin, Proc. Natl. Acad. Sci. USA 79:4083-4087, 1982; N. H. Heintz and J. L. Hamlin, Biochemistry 22:3552-3557, 1983; N. H. Heintz, J. D. Milbrandt, K. S. Greisen, and J. L. Hamlin, Nature [London] 302:439-441, 1983). To locate the origin of replication in this 243-kb amplicon with more precision, we used an in-gel renaturation procedure (I. Roninson, Nucleic Acids Res. 11:5413-5431, 1983) to examine the labeling pattern of restriction fragments from the amplicon in the early S phase. This method eliminates background labeling from single-copy sequences and allows quantitation of the relative radioactivity in individual fragments. We used this procedure to follow the movement of replication forks through the amplicons, to roughly localize the initiation locus, and to estimate the rate of fork travel. We also used a slight modification of this method (termed hybridization enhancement) to illuminate the labeling pattern of smaller restriction fragments derived solely from the initiation locus itself, thereby increasing resolution. Our preliminary results suggest that there are actually two distinct initiation sites in the amplicon that are separated by approximately 22 kb.

scholarly journals High-resolution mapping of replication fork movement through the amplified dihydrofolate reductase domain in CHO cells by in-gel renaturation analysis.

1989 ◽  
Vol 9 (2) ◽  
pp. 523-531 ◽  
Author(s):  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster ◽  
Slight Modification ◽  
Single Copy ◽  
Replication Initiation ◽  
Ovary Cell ◽  
Restriction Fragments ◽  
Labeling Pattern ◽  
Reductase Domain

Utilizing an in vivo labeling method on synchronized cultures, we have previously defined a 28-kilobase (kb) replication initiation locus in the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) (N. H. Heintz and J. L. Hamlin, Proc. Natl. Acad. Sci. USA 79:4083-4087, 1982; N. H. Heintz and J. L. Hamlin, Biochemistry 22:3552-3557, 1983; N. H. Heintz, J. D. Milbrandt, K. S. Greisen, and J. L. Hamlin, Nature [London] 302:439-441, 1983). To locate the origin of replication in this 243-kb amplicon with more precision, we used an in-gel renaturation procedure (I. Roninson, Nucleic Acids Res. 11:5413-5431, 1983) to examine the labeling pattern of restriction fragments from the amplicon in the early S phase. This method eliminates background labeling from single-copy sequences and allows quantitation of the relative radioactivity in individual fragments. We used this procedure to follow the movement of replication forks through the amplicons, to roughly localize the initiation locus, and to estimate the rate of fork travel. We also used a slight modification of this method (termed hybridization enhancement) to illuminate the labeling pattern of smaller restriction fragments derived solely from the initiation locus itself, thereby increasing resolution. Our preliminary results suggest that there are actually two distinct initiation sites in the amplicon that are separated by approximately 22 kb.

scholarly journals Organization and genesis of dihydrofolate reductase amplicons in the genome of a methotrexate-resistant Chinese hamster ovary cell line.

1988 ◽  
Vol 8 (6) ◽  
pp. 2316-2327 ◽  
Author(s):  
C Ma ◽  
J E Looney ◽  
T H Leu ◽  
J L Hamlin
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Pulse Field ◽  
Ovary Cell ◽  
Type I ◽  
Type Ii ◽  
Gradient Gel Electrophoresis

We have recently isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase (dhfr) amplicon types from the methotrexate-resistant Chinese hamster ovary (CHO) cell line CHOC 400. In the work described in this report, we used pulse-field gradient gel electrophoresis to analyze large SfiI restriction fragments arising from the amplified dhfr domains. The junction between the 260-kilobase type I amplicons (which are arranged in head-to-tail configurations in the genome) has been localized, allowing the construction of a linear map of the parental dhfr locus. We also show that the 220-kilobase type II amplicons are arranged as inverted repeat structures in the CHOC 400 genome and arose from the type I sequence relatively early in the amplification process. Our data indicate that there are a number of minor amplicon types in the CHOC 400 cell line that were not detected in previous studies; however, the type II amplicons represent ca. 75% of all the amplicons in the CHOC 400 genome. Both the type I and type II amplicons are shown to be composed entirely of sequences that were present in the parental dhfr locus. Studies of less resistant cell lines show that initial amplicons can be larger than those observed in CHOC 400. Once established, a given amplicon type appears to be relatively stable throughout subsequent amplification steps. We also present a modification of an in-gel renaturation method that gives a relatively complete picture of the size and variability of amplicons in the genome.

IN VIVO SERIAL CHANGES IN BONE INDUCTION BY E. COLI-DERIVED RECOMBINANT HUMAN BMP-2

2002 ◽  
Vol 06 (01) ◽  
pp. 1-7 ◽  
Author(s):  
Kazuhisa Bessho ◽  
Junya Sinobe ◽  
Shinji Kaihara ◽  
Mariko Kawai ◽  
Yasunori Okubo ◽  
...  
Keyword(s):  
High Activity ◽  
Mammalian Cells ◽  
Type I Collagen ◽  
Expression System ◽  
Chinese Hamster ◽  
Bacterial Expression ◽  
Ovary Cell ◽  
Type I ◽  
Bacterial Expression System

At present, several recombinant human bone morphogenetic proteins (rhBMPs) can be produced in mammalian cells. If rhBMPs with a high activity could be produced in bacteria, the bacterial expression system is very useful in clinic. We examined in vivo serial changes in the bone inducing ability of an Escherichia coli-derived rhBMP-2 (ErhBMP-2) variant with N-terminal sequence. Five μg of ErhBMP-2 was mixed with 3 mg of atelopeptide type I collagen (CL) as the carrier, and specimens were implanted into calf muscle pouches of Wistar rats (n = 20). After 3, 7, 14 and 21 days, 5 specimens each were examined. New cartilage was observed 7 days after implantation of ErhBMP-2 with CL. Induced bone was found on the outermost edge of the implant after 14 days. After 21 days, bone formation was associated with much fatty marrow. ALP activity and calcium content gradually increased with time. These changes were similar to those in a study using Chinese hamster ovary cell-derived rhBMP-2. However, these increases were slightly higher than those in the rhBMP-2 study. From these findings, ErhBMP-2 appears to be completely renatured while maintaining its biological activity. ErhBMP-2 with CL may expand by absorbing body fluid in vivo to greater extent than rhBMP-2 with CL, because it lacks heparin-binding sites. A variant ErhBMP-2 showing high activity in vivo, obtained by the bacterial expression system is an important finding, as it should be possible to produce large quantities of ErhBMP-2 at a low-cost for clinical use, in the near future.

scholarly journals Isolation of the amplified dihydrofolate reductase domain from methotrexate-resistant Chinese hamster ovary cells.

1987 ◽  
Vol 7 (2) ◽  
pp. 569-577 ◽  
Author(s):  
J E Looney ◽  
J L Hamlin
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Type I ◽  
Type Ii ◽  
Mammalian Cell Line ◽  
Cho Cell ◽  
Ovary Cells ◽  
Reductase Domain

We isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase amplicon types from the methotrexate-resistant CHO cell line CHOC400. The type I amplicons are 260 kilobases long, are arranged in head-to-tail fashion, and represent 10 to 15% of the amplicons in the CHOC400 genome. The type II amplicons are 220 kilobases long, are arranged in head-to-head and tail-to-tail configurations, and constituted the majority of the remaining amplicons in CHOC400 cells. The type II amplicon sequences are represented entirely within the type I unit. These are the first complete amplicons to be cloned from a mammalian cell line.

Isolation of the amplified dihydrofolate reductase domain from methotrexate-resistant Chinese hamster ovary cells

1987 ◽  
Vol 7 (2) ◽  
pp. 569-577
Author(s):  
J E Looney ◽  
J L Hamlin
Keyword(s):  
Cell Line ◽  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Type I ◽  
Type Ii ◽  
Mammalian Cell Line ◽  
Cho Cell ◽  
Ovary Cells ◽  
Reductase Domain

We isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase amplicon types from the methotrexate-resistant CHO cell line CHOC400. The type I amplicons are 260 kilobases long, are arranged in head-to-tail fashion, and represent 10 to 15% of the amplicons in the CHOC400 genome. The type II amplicons are 220 kilobases long, are arranged in head-to-head and tail-to-tail configurations, and constituted the majority of the remaining amplicons in CHOC400 cells. The type II amplicon sequences are represented entirely within the type I unit. These are the first complete amplicons to be cloned from a mammalian cell line.

scholarly journals Dynamics of keratin assembly: exogenous type I keratin rapidly associates with type II keratin in vivo

1993 ◽  
Vol 122 (1) ◽  
pp. 123-135 ◽  
Author(s):  
RK Miller ◽  
S Khuon ◽  
RD Goldman
Keyword(s):  
Mammalian Cells ◽  
Close Association ◽  
Immunogold Electron Microscopy ◽  
Type I ◽  
Post Injection ◽  
Type Ii ◽  
Rapid Formation ◽  
Double Label ◽  
Keratin Intermediate Filaments

Keratin intermediate filaments (IF) are obligate heteropolymers containing equal amounts of type I and type II keratin. We have previously shown that microinjected biotinylated type I keratin is rapidly incorporated into endogenous bundles of keratin IF (tonofilaments) of PtK2 cells. In this study we show that the earliest steps in the assembly of keratin subunits into tonofilaments involve the extremely rapid formation of discrete aggregates of microinjected keratin. These are seen as fluorescent spots containing both type I and type II keratins within 1 min post-injection as determined by double label immunofluorescence. These observations suggest that endogenous type II keratin subunits can be rapidly mobilized from their endogenous state to form complexes with the injected type I protein. Furthermore, confocal microscopy and immunogold electron microscopy suggest that the type I-type II keratin spots from in close association with the endogenous keratin IF network. When the biotinylated protein is injected at concentrations of 0.3-0.5 mg/ml, the organization of the endogenous network of tonofilaments remains undisturbed during incorporation into tonofilaments. However, microinjection of 1.5-2.0 mg/ml of biotinylated type I results in significant alterations in the organization and assembly state of the endogenous keratin IF network soon after microinjection. The results of this study are consistent with the existence of a state of equilibrium between keratin subunits and polymerized keratin IF in epithelial cells, and provide further proof that IF are dynamic elements of the cytoskeleton of mammalian cells.

scholarly journals Autophagy as a modulator of cell death machinery

2020 ◽  
Vol 11 (7) ◽  
Author(s):  
Masayuki Noguchi ◽  
Noriyuki Hirata ◽  
Tsutomu Tanaka ◽  
Futoshi Suizu ◽  
Hiroshi Nakajima ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Survival ◽  
Mammalian Cells ◽  
Critical Parameter ◽  
Type I ◽  
Type Ii ◽  
Critical Question ◽  
A Cell ◽  
Underlying Mechanisms

Abstract The balance between cell death and survival is a critical parameter in the regulation of cells and the maintenance of homeostasis in vivo. Three major mechanisms for cell death have been identified in mammalian cells: apoptosis (type I), autophagic cell death (type II), and necrosis (type III). These three mechanisms have been suggested to engage in cross talk with each other. Among them, autophagy was originally characterized as a cell survival mechanism for amino acid recycling during starvation. Whether autophagy functions primarily in cell survival or cell death is a critical question yet to be answered. Here, we present a comprehensive review of the cell death-related events that take place during autophagy and their underlying mechanisms in cancer and autoimmune disease development.

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