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1.
J Cell Biol ; 149(2): 271-80, 2000 Apr 17.
Article in English | MEDLINE | ID: mdl-10769021

ABSTRACT

DNA replication occurs in microscopically visible complexes at discrete sites (replication foci) in the nucleus. These foci consist of DNA associated with replication machineries, i.e., large protein complexes involved in DNA replication. To study the dynamics of these nuclear replication foci in living cells, we fused proliferating cell nuclear antigen (PCNA), a central component of the replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that replication foci are heterogeneous in size and lifetime. Time-lapse studies revealed that replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses suggested that replication factories are stably anchored in the nucleus and that changes in the pattern occur through gradual, coordinated, but asynchronous, assembly and disassembly throughout S phase.


Subject(s)
Cell Cycle/physiology , Cell Nucleus/physiology , DNA Replication , Animals , COS Cells , Cell Line , Cell Nucleus/ultrastructure , Green Fluorescent Proteins , Humans , Kinetics , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Mice , Microscopy, Video/methods , Muscle, Skeletal , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Recombinant Fusion Proteins/metabolism , Transfection
2.
Crit Rev Eukaryot Gene Expr ; 9(3-4): 345-51, 1999.
Article in English | MEDLINE | ID: mdl-10651251

ABSTRACT

Over the last decades it became clear that mammalian nuclei are highly organized. Nuclear processes like DNA replication and RNA metabolism take place in distinct subnuclear foci, which are enriched for enzymes involved in the corresponding biochemical reactions. This colocalization of functions with their respective factors is often referred to as functional organization of the nucleus. This organization is achieved by assembly of different enzymes and regulatory factors into high-molecular-weight complexes that are tethered to insoluble nuclear structures. Recently, several links between nuclear structure, gene expression, DNA replication, and methylation have been described that illustrate the interrelation of higher-order structures and nuclear functions. New insights into the functional organization of the nucleus and how it could explain the high precision and overall coordination of nuclear processes are discussed.


Subject(s)
Cell Nucleus/genetics , DNA Methylation , DNA Replication , Gene Expression , Animals , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Humans , Structure-Activity Relationship
3.
J Cell Biochem Suppl ; 30-31: 243-9, 1998.
Article in English | MEDLINE | ID: mdl-9893277

ABSTRACT

Mammalian nuclei are highly organized into functional compartments. Major nuclear processes like DNA replication and RNA processing take place in distinct foci. These microscopically visible foci are formed by the assembly of, for example, DNA replication factors and associated proteins into megadalton complexes often referred to as protein machines or factories. Thus far, two proteins, DNA ligase I and DNA methyltransferase (DNA MTase), have been analyzed in greater detail. In both cases, the assembly process appears to be controlled by distinct targeting sequences that were attached to the catalytic protein core in the course of evolution and mediate the association with replication factories in mammalian cells. The dynamics of these nuclear structures throughout the cell cycle are analyzed using green fluorescent protein (GFP). Further studies are needed to elucidate the architecture, regulation, and role of these subnuclear structures.


Subject(s)
Cell Nucleus/genetics , Cell Nucleus/metabolism , DNA Replication/genetics , Animals , Cell Nucleus/chemistry , Humans
4.
J Cell Biol ; 139(3): 579-87, 1997 Nov 03.
Article in English | MEDLINE | ID: mdl-9348276

ABSTRACT

The mammalian nucleus is highly organized, and nuclear processes such as DNA replication occur in discrete nuclear foci, a phenomenon often termed "functional organization" of the nucleus. We describe the identification and characterization of a bipartite targeting sequence (amino acids 1-28 and 111-179) that is necessary and sufficient to direct DNA ligase I to nuclear replication foci during S phase. This targeting sequence is located within the regulatory, NH2-terminal domain of the protein and is dispensable for enzyme activity in vitro but is required in vivo. The targeting domain functions position independently at either the NH2 or the COOH termini of heterologous proteins. We used the targeting sequence of DNA ligase I to visualize replication foci in vivo. Chimeric proteins with DNA ligase I and the green fluorescent protein localized at replication foci in living mammalian cells and thus show that these subnuclear functional domains, previously observed in fixed cells, exist in vivo. The characteristic redistribution of these chimeric proteins makes them unique markers for cell cycle studies to directly monitor entry into S phase in living cells.


Subject(s)
DNA Ligases/genetics , DNA Ligases/metabolism , DNA Replication , Peptide Mapping , Amino Acid Sequence , Animals , COS Cells , Cell Cycle , Cell Nucleus/metabolism , DNA Ligase ATP , Green Fluorescent Proteins , HeLa Cells , Humans , Luminescent Proteins/genetics , Peptide Fragments/genetics , Peptide Fragments/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Deletion
5.
Cytotechnology ; 11(1): 67-75, 1993.
Article in English | MEDLINE | ID: mdl-7763638

ABSTRACT

We have investigated large scale production processes (up to 2 liters) of recombinant proteins using the baculovirus expression system in order to optimize the product yields. Experiments using cell lines of Spodoptera frugiperda (Sf9) and Mamestra brassicae (IZD-Mb0503) were performed to show the different production capacities of the cell lines. The influence of the infection at different cell densities is described. Beyond that, TC100-, IPL41- and serum-free IPL41-medium were compared to demonstrate their different capabilities of supporting cell growth and protein expression. Additionally, the inhibitory effect of FCS on the protease activity of kallikrein, which is produced in its zymogenic form, is discussed Improved production parameters are described, which enabled us to produce up to 8000 units of activated pro-kallikrein within 14 days using perfusion cultivation.


Subject(s)
Baculoviridae/metabolism , Biotechnology , Enzyme Precursors/biosynthesis , Kallikreins/biosynthesis , Animals , Cattle , Cell Count , Cells, Cultured , Culture Media , Enzyme Precursors/metabolism , Humans , Kallikreins/metabolism , Moths/cytology , Moths/metabolism , Perfusion , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Trypsin
6.
Eur J Biochem ; 206(1): 225-33, 1992 May 15.
Article in English | MEDLINE | ID: mdl-1587272

ABSTRACT

A full-length cDNA encoding human salivary-gland preprokallikrein was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in transfected Spodoptera frugiperda cells and the recombinant product secreted into the culture medium. By alternating anion-exchange chromatography and gel-filtration steps, twice repeated, prokallikrein was purified to homogeneity, which was confirmed by amino acid analysis and N-terminal sequence determination. The prepropeptide was processed correctly, including the removal of the signal peptide. The resulting proenzyme was found to be glycosylated, had a molecular mass of 35 kDa and an isoelectric point of 4.6. The yield of purified recombinant protein reached a level of 5 mg/l insect cell culture. After trypsin digestion of prokallikrein, the biological activity of the released kallikrein was demonstrated by its specific amidase, esterase and kininogenase activity. The expression and purification of prokallikrein, as described here, offers the opportunity to study the proenzyme activation through protein engineering techniques in detail.


Subject(s)
Baculoviridae/genetics , Enzyme Precursors/genetics , Prekallikrein/genetics , Salivary Glands/enzymology , Amino Acid Sequence , Animals , Blotting, Western , Cell Line , Chromatography, DEAE-Cellulose , Chromatography, Gel , DNA/genetics , DNA/isolation & purification , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Humans , Kinetics , Molecular Sequence Data , Molecular Weight , Moths , Plasmids , Prekallikrein/isolation & purification , Prekallikrein/metabolism , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Restriction Mapping , Transfection
7.
Agents Actions Suppl ; 38 ( Pt 1): 66-73, 1992.
Article in English | MEDLINE | ID: mdl-1466304

ABSTRACT

A cDNA fragment encoding human salivary-gland kallikrein, including the kallikrein-owned signal peptide, was inserted into a baculovirus vector adjacent to the polyhedrin promoter and expressed in transfected insect cells. Biologically active kallikrein was isolated to homogeneity from serum-free culture supernatant using a four-step protocol. The N-terminal amino acid sequence of the insect-derived kallikrein was identical to that of the natural proteinase, thus indicating the proper removal of the mammalian signal peptide.


Subject(s)
Kallikreins/genetics , Salivary Glands/metabolism , Amino Acid Sequence , Animals , Baculoviridae/genetics , Cell Line , Gene Expression , Genetic Vectors , Insecta , Kallikreins/isolation & purification , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
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