cDNA Cloning and the Construction of Recombinant DNA

1991 ◽

Vol 49 ◽

pp. 430-431

Author(s):

J. A. Pollock ◽

M. Martone ◽

T. Deerinck ◽

M. H. Ellisman

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Nucleic Acids ◽

Recombinant Dna ◽

Light And Electron Microscopy ◽

Electron Microscopic ◽

High Voltage Electron Microscopy ◽

Functional Sites ◽

Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

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Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128900 ◽

1987 ◽

Vol 45 ◽

pp. 924-925

Author(s):

F. A. Durum ◽

R. G. Goldman ◽

T. J. Bolling ◽

M. F. Miller

Keyword(s):

Inclusion Bodies ◽

Large Scale ◽

Recombinant Dna ◽

Test System ◽

Cell Protein ◽

Gram Negative Bacteria ◽

Cytoplasmic Inclusions ◽

Isolation And Purification ◽

E Coli ◽

Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

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cDNA cloning and expression of a gamma-aminobutyric acidA receptor epsilon-subunit in rat brain

European Journal of Neuroscience ◽

10.1046/j.1460-9568.2000.01343.x ◽

2000 ◽

Vol 12 (12) ◽

pp. 4318-4330 ◽

Cited By ~ 7

Author(s):

Nathalie Moragues ◽

Philippe Ciofi ◽

Pierrette Lafon ◽

Marie-Francoise Odessa ◽

Gerard Tramu ◽

...

Keyword(s):

Rat Brain ◽

Cdna Cloning ◽

Cloning And Expression ◽

Epsilon Subunit

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Murine CD70: cDNA cloning and gene structure

Immunology Letters ◽

10.1016/s0165-2478(97)87049-6 ◽

1997 ◽

Vol 56 (1-3) ◽

pp. 56

Author(s):

K Tesselaar

Keyword(s):

Cdna Cloning ◽

Gene Structure

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A Multicenter Study of Recombinant Factor VIII (RecombinateTM) in Previously Treated Patients with Hemophilia A

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656030 ◽

1997 ◽

Vol 77 (04) ◽

pp. 660-667 ◽

Cited By ~ 110

Author(s):

G C White ◽

S Courter ◽

G L Bray ◽

M Lee ◽

E D Gomperts ◽

...

Keyword(s):

Hemophilia A ◽

Recombinant Dna ◽

Home Treatment ◽

Pharmacokinetic Parameters ◽

Pharmacokinetic Studies ◽

Open Label ◽

Treat Ment ◽

Previously Treated Patients ◽

Bleeding Episodes

SummaryA prospective, open-label multicenter investigation has been conducted to compare pharmacokinetic parameters of recombinant DNA-derived FVIII (rFVIII) and plasma-derived FVIII concentrate (pdFVIII) and to assess safety and efficacy of long-term home-treat- ment with rFVIII for subjects with hemophilia A. Following comparative in vivo pharmacokinetic studies, 69 patients with severe (n = 67) or moderate (n = 2) hemophilia A commenced a program of home treatment using rFVIII exclusively for prophylaxis and treatment of all bleeding episodes for a period of 1.0 to 5.7 years (median 3.7 years). The mean in vivo half-lives of rFVIII and pdFVIII were both 14.7 h. In vivo incremental recoveries at baseline were 2.40%/IU/kg and 2.47%/IU/kg, respectively (p = 0.59). The response to home treatment with rFVIII was categorized as good or excellent in 3,195 (91.2%) of 3,481 evaluated bleeding episodes. Thirteen patients received rFVIII for prophylaxis for twenty-four surgical procedures. In all cases, hemostasis was excellent. Adverse reactions were observed in only 13 of 13,591 (0.096%) infusions of rFVIII; none was serious. No patient developed an inhibitor to r FVIII.

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