A Double Image of the Double Helix: The Recombinant DNA Debate.Clifford Grobstein

The scientific and organizational activities of the worldwide known scientist in the field of molecular biology James Dewey Watson were described in this article. 55 years ago James Watson and Francis Crick made one of the key discoveries of the twentieth century. They have found that DNA has a double helix structure. This discovery was based on the X-rays patterns obtained by Maurice Wilkson and Rosalind Franklin. Subsequently, this DNA model had been proved, and J. Watson and F. Crick were awarded with the Nobel Prize in Physiology or Medicine in 1962. Since, our knowledge of the main molecule of life has been greatly expanded. A significant flowering of molecular genetics has began: synthesis of RNA and DNA in vitro, decoding of genetic code, recombinant DNA technology, genetic engineering, sequencing of genomes and post genomic technologies. James Watson is one of the authors of the cell biology classic textbook “Molecular Biology of the Cell”. In addition, he has developed the current areas of molecular biology such as molecular oncology and molecular neurobiology. Today genomes of different animals and humans have been decoded and the functions of many genes have been determined. But at present still unknown how the DNA starts and how it affects the work of the organs and the organism as a system. Keywords: James Watson, DNA structure, Nobel Prize in physiology or medicine, Molecular Biology of the Cell.

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You Shall Be As Gods

Worldview ◽

10.1017/s0084255900029533 ◽

1977 ◽

Vol 20 (9) ◽

pp. 45-47

Author(s):

Tabitha M. Powledge

Keyword(s):

Living Cell ◽

Deoxyribonucleic Acid ◽

Recombinant Dna ◽

Double Helix ◽

Genetic Material ◽

Living Thing

Already the term "recombinant DNA" comes easily to the lips of legislators and television commentators. A few of them can even manage "deoxyribonucleic acid," which is what DNA stands for.DNA is the genetic material, the miraculous selfreplicatirig double helix of Watson and Crick, the marrow, soul, and substance of all earthly life. Molecular biologists have been tinkering with it for a couple of decades now, and only recently have they learned to slice it up and then stick it back together in new ways: hence recombinant DNA. Scientists have also discovered ways to transfer it from one living thing to another: to put genes from one kind of animal into a different kind, from bacteria into plants, or to put the newly spliced genes into just about any living cell.

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A double image of the double helix

Trends in Biochemical Sciences ◽

10.1016/0968-0004(80)90080-8 ◽

1980 ◽

Vol 5 (3) ◽

pp. XVIII-XIX

Author(s):

Waclaw Szybalski

Keyword(s):

Double Helix ◽

Double Image

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Protamine-DNA interaction

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081863 ◽

1978 ◽

Vol 36 (2) ◽

pp. 200-201

Author(s):

D.P. Bazett-Jones ◽

F.P. Ottensmeyer

Keyword(s):

Small Volume ◽

Double Helix ◽

Dna Interaction ◽

Dark Field ◽

Sperm Head ◽

Nuclear Proteins ◽

Field Electron Microscopy ◽

Dark Field Electron ◽

Dna Double Helix ◽

Positively Charged

Dark field electron microscopy has been used for the study of the structure of individual macromolecules with a resolution to at least the 5Å level. The use of this technique has been extended to the investigation of structure of interacting molecules, particularly the interaction between DNA and fish protamine, a class of basic nuclear proteins of molecular weight 4,000 daltons.Protamine, which is synthesized during spermatogenesis, binds to chromatin, displaces the somatic histones and wraps up the DNA to fit into the small volume of the sperm head. It has been proposed that protamine, existing as an extended polypeptide, winds around the minor groove of the DNA double helix, with protamine's positively-charged arginines lining up with the negatively-charged phosphates of DNA. However, viewing protamine as an extended protein is inconsistent with the results obtained in our laboratory.

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mRNA localization by Electron microscopic in situ hybridization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100086453 ◽

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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Electron microscopic assays of restriction endonuclease cutting, exonuclease digestion, and hybridization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100113317 ◽

1984 ◽

Vol 42 ◽

pp. 686-687

Author(s):

Mark Hannibal ◽

Jacob Varkey ◽

Michael Beer

Keyword(s):

Electron Microscope ◽

Low Temperature ◽

Restriction Endonuclease ◽

Double Helix ◽

Test Material ◽

Electron Microscopic ◽

Exonuclease Iii ◽

Dna Molecule ◽

Linear Molecules ◽

Exonuclease Digestion

Workman and Langmore have recently proposed a procedure for isolating particular chromatin fragments. The method requires restriction endonuclease cutting of the chromatin and a probe, their digestion with two exonucleases which leave complimentary single strand termini and low temperature hybridization of these. We here report simple electron microscopic monitoring of the four reactions involved.Our test material was ϕX-174 RF DNA which is cut once by restriction endonuclease Xho I. The conversion of circles to linear molecules was followed in Kleinschmidt spreads. Plate I shows a circular and a linear DNA molecule. The rate of cutting is shown in Figure 1.After completion of the endonuclease cutting, one portion of the DNA was treated with exonuclease III, an enzyme known to digest the 3' terminals of double helical DNA. Aliquots when examined in the electron microscope reveal a decreasing length of double helix and increasing bushes at the ends.

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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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