Recombinant DNA Era Research Articles

We'll always have RNA.

We'll always have RNA.

Escalating Obesity Rates Pose Health, Budget Threats

the recombinant DNA era. Maniatis’ work was integral to the creation of the gene cloning “toolkit,” which includes the technique of creating DNA copies of messenger RNA (called complementary DNA) that can be manipulated for study. These techniques, which he used in his studies of the genetic basis of disease, enabled a generation of emerging molecular biologists to identify, isolate, and control the expression of genes of interest. In addition to their contributions to furthering the understanding of genetics, both Brown and Maniatis have had a lasting effect on the scientific community through their support of colleagues. Maniatis was instrumental in disseminating gene cloning techniques with his book Molecular Cloning: A Laboratory Manual, which was first published in 1982 and has become an invaluable resource for molecular biology laboratories around the world. In 1980, Brown founded the Life Sciences Research Foundation, which partners with the US government and industry to fund postdoctoral fellowships for promising young scientists.

Studying gene expression for a long time

In the 1960s and 70s before the recombinant DNA era we studied the structure and expression of the repetitive Xenopus laevis ribosomal RNA genes and 5S ribosomal RNA genes. They were isolated by physical and chemical means from genomic DNA. These genes revealed transcribed and nontranscribed spacers, pseudogenes, horizontal evolution of tandem repeating genes including the conservation of functional sequences and the divergence of spacer regions. The Xenopus oocyte accumulates enormous numbers of ribosomes for a single cell. The solution for this demand for quantity differs for each component. The genes encoding the large 18S and 28S RNAs are amplified by about 1000 times. This was the first instance of specific gene amplification. The 5S RNA genes are not amplified but encoded in a huge family of about 20,000 members expressed only in the oocyte. The purified 5S RNA genes were convenient for studying gene expression in vitro revealing the DNA signals for initiation and termination of transcription. In 1990 I changed my research to a more complex problem of gene expression, amphibian metamorphosis. Essentially every cell type in each tadpole organ is targeted for change by thyroid hormone (TH). Some organs (for example the limb) complete differentiation by multiple cell autonomous programs. Others (intestine and pancreas) have TH-induced cellcell interaction. This biological problem has not yet revealed its most exciting secrets. Many scientists have contributed to my research. It was my greatest good fortune to have discovered the Carnegie Institution. I am extremely grateful to the NIGMS and the Mathers Foundation for long term funding.

A Tribute to the Xenopus laevis Oocyte and Egg

When I was asked to reflect as part of the celebration of the 100-year anniversary of the Journal of Biological Chemistry I recalled a talk by Seymour Cohen at the Federation meetings in Atlantic City about 1960. He began by thanking bacteriophage for providing him with so many wonderful research problems. I decided in the same spirit to pay homage to the Xenopus laevis egg and oocyte, two states of a cell that has played and continues to play a central role in most of the disciplines of modern biology including biochemistry and molecular biology. Many of us owe a debt of gratitude to this cell. The egg is the most important and interesting cell in the repertoire of any organism. In X. laevis this single cell has a diameter of about 1.3 mm. The oocyte is permeable to small molecules, but after it undergoes meiosis and becomes an unfertilized egg it is impermeable to these same molecules. The oocyte is an active site of RNA and protein synthesis but not DNA replication, whereas the unfertilized egg is poised to replicate every 10 min after it is fertilized. During the cleavage period the embryo is transcriptionally silent. An oocyte can be cultured for days, but once ovulated the egg degenerates rapidly if it is not fertilized. The oocyte is the largest single cell in the body yet it has many of the same structures as somatic cells. These compartments are so exaggerated in size that they are accessible to cell biologists for manipulation and visualization. The huge oocyte nucleus is called a germinal vesicle (GV). The premeiotic tetraploid chromosomes are expanded into a “lampbrush” configuration so that they can be studied with a light microscope. The X. laevis egg and its possibilities for experimental manipulation were introduced to modern biology in the late 1950s by John Gurdon (Fig. 1), then a graduate student in the laboratory of Michail Fischberg in the Department of Zoology at Oxford University. Gurdon’s thesis was to reproduce with the South African “clawed toad” X. laevis the famous nuclear transplantation experiments that Briggs and King had perfected with the American leopard frog, Rana pipiens (1). Embryonic nuclei were injected into eggs whose own nuclei had been removed or destroyed. These early cloning experiments addressed the question of whether a differentiated and specialized somatic cell nucleus was still capable of expressing its full genetic repertoire. Briggs and King found that nuclei from R. pipiens embryos older than gastrula never promoted normal development, suggesting that the genetic material might change in cells as they specialize during embryonic development. This result was challenged by John Gurdon’s demonstration that nuclei from X. laevis embryos are more permissive. Even a small fraction of nuclei derived from differentiated intestinal epithelial cells supported normal development (2). This crucial scientific question of whether irreversible changes occur in the genome of highly specialized cells was addressed more precisely by experimental manipulation than by biochemistry or genetics. The concepts behind these experiments have influenced the stem cell field. The topic of whether the genome is altered in specialized somatic cells was revisited just this year by the same strategy of nuclear transplantation. Postmitotic nuclei from olfactory neurons support the development of normal fertile mice when transplanted into enucleated mouse eggs (3). THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 44, Issue of October 29, pp. 45291–45299, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Plant molecular biology and biotechnology research in the post-recombinant DNA era.

After the beginning of the recombinant DNA era in the mid-1970s, researchers in India started to make use of the new technology to understand the structure of plant genes and regulation of their expression. The outcome started to appear in print in early the 1980s and genes for histones, tubulin, photosynthetic membrane proteins, phototransduction components, organelles and those regulated differentially by developmental and extrinsic signals were sequenced and characterized. Some genes of biotechnological importance like those encoding an interesting seed protein and the enzyme glyoxalase were also isolated. While work on the characterization of genome structure and organization was started quite early, it remained largely focused on the identification of DNA markers and genetic variability. In this context, the work on mustard, rice and wheat is worth mentioning. In the year 2000, India became a member of the international consortium to sequence entire rice genome. Several laboratories have also given attention to regulated expression of plastid and nuclear genes as well as to isolate target-specific promoters or design promoters with improved potential. Simultaneously, transgenic systems for crops like mustard, rice, wheat, cotton, legumes and several vegetables have been established. More recently, genes of agronomic importance like those for insect resistance, abiotic stress tolerance, nutritional improvement and male sterility, isolated in India or abroad, have been utilized for raising transgenics for crop improvement. Some of these transgenics have already shown their potential in containment facility or limited field trials conducted under the stipulated guidelines. Plant molecular biology and biotechnology are thus clearly poised to make an impact on research in basic biology and agriculture in the near future.

Human Genome Project: Discovery of New Drugs

Genomics will surpass the recombinant DNA era for generating therapeutic product opportunities. It will also be a key core technological competency for all therapeutic area. To determine biological functions of novel genes and to develop the therapeutic drugs for human diseases, we have applied various techniques and discovered the potential drugs. In my talk, I will give two examples how we determined the biological functions of the novels genes. Osteoprotegerin (OPG) is a novel molecule isolated from the human genome (EST) project. This molecule shares homology with tumor necrosis factor receptor. In vivo, over expression of OPG in the transgenic mice results in a profond osteopetrosis, coincident with a decrease in later stage of osteoclast differentiation. These same effects are observed upon administration of recombinant OPG into normal mice. In vitro, osteoclast differentiation from precursor cells is blocked in a dose-dependent manner by recombinant OPG. Furthermore, OPG blocks ovariectomy-associated bone loss in rat. These data show that OPG can act as a soluble factor in the regulation of bone loss and imply a utility for OPG in the treatment of osteoporosis. Currently, OPG has been in the phase III clinical trial for osteoporosis. NNT-1/BSF-3 is another novel molecule isolated from cDNA library of the activated T lymphoma cells. It is novel member of the IL-6 cytokine family. Over expression of the gene in transgenic mice induced proliferation of lymphoid tissue particularly in the speen and lymph node. It also increased blood B cells number in transgenic mice. In vitro study showed that NNT-1/BSF-3 also supported differentiation of chicken embryonic motor and sympathetic neurons. In vivo, it potentiated the induction by IL-1 of corticosterone and IL-6. Administration of the recombinant protein to the mice also induced b cells hyperplasia with serum IgG and IgM increase showing the same effect as in the transgenic mice. Currently, we are in the process of isolating several other novel growth factors and cytokine receptors. These molecules may have some relation with cancer biology, bone formation or wound healing. Determination of their biological functions and further development of potential drug are in progress. In summary, these studies demonstrate that human genome project is a good source for the discovery of potential drugs. Functional genomics will not only give us a revolutionary influence in understanding the biology of the novel genes but also greatly facilitate the development of new drugs for therapeutics in the 21th century.

Some genes were isolated and their structure studied before the recombinant DNA era.

Some genes were isolated and their structure studied before the recombinant DNA era.

Gene Therapy: Possibilities and Promise

Gene Therapy: Possibilities and Promise

Proceed with Caution: Predicting Genetic Risks in the Recombinant DNA Era

Proceed with Caution: Predicting Genetic Risks in the Recombinant DNA Era

Proceed with Caution. Predicting Genetic Risks in the Recombinant DNA Era. Neil A. Holtzman

<i>Proceed with Caution. Predicting Genetic Risks in the Recombinant DNA Era</i>. Neil A. Holtzman

Proceed With Caution: Predicting Genetic Risks in the Recombinant DNA Era

Medical genetics is in its golden age. Discoveries that elucidate a variety of genetic diseases are occurring rapidly. The definition of the basic defect in cystic fibrosis in 1989 is a spectacular example that is likely to lead to extensive genetic testing for the carrier state. A book that deals with the social and medical impact of genetic screening is therefore timely and welcome. Dr Holtzman is eminently qualified to write such a book, having spent most of his efforts over the last few years studying and pondering these issues. He starts out by providing the biologic background and explains how modern gene technology can be used in the diagnosis of genetic disease. While the molecular diagnosis of a given genetic defect is usually clear-cut, the resultant clinical manifestations often show variability in age of onset and severity. This axiom is particularly true for common diseases that depend upon interaction

Dangerous Curves Ahead Proceed with Caution: Predicting Genetic Risks in the Recombinant DNA Era Neil A. Holtzman

Dangerous Curves Ahead Proceed with Caution: Predicting Genetic Risks in the Recombinant DNA Era Neil A. Holtzman

Proceed with caution: predicting genetic risks in the recombinant DNA era

Proceed with caution: predicting genetic risks in the recombinant DNA era

Book ReviewProceed with Caution: Predicting Genetic Risks In The Recombinant DNA Era

Book ReviewProceed with Caution: Predicting Genetic Risks In The Recombinant DNA Era