The “Gene” Unraveled
In this article we’re going to look at a definition of Genetic Engineering, explore the concept of a “gene” and how that understanding has evolved over the past 150 years or so and why it is still not 100% agreed upon by everyone, as well as some fundamental mechanics of genetic engineering like “restriction enzymes”. Finally we’ll learn how the role of “junk DNA” is becoming more understood as anything but, and we’ll end with a quick look at the Human Genome Project and how that project has provided more information…and questions…on what exactly a “gene” really is.
Inside this Page
- A Definition of Genetic Engineering
- Our Evolving Understanding of the Gene and Genetic Engineering
- Genotype and Phenotype
- Evolution of the term “Gene”
- Restriction Enzymes
- Junk DNA
- The Human Genome Project
Definition of “Genetic Engineering”
In its very simplest form, genetic engineering can be defined as a technique used for the manipulation of genes. However, a more accurate and broader definition would be that genetic engineering encompasses a number of methodologies which enable new combinations of genetic material to be artificially constructed in a laboratory. If we take a closer look at the simpler defintion, it might seem that genetic engineering is a very old field because humans have been manipulating the genome of organisms using artificial and selective breeding for thousands of years. But the science of entering into a cell and manipulating its genome by adding or removing gene (s) is a very new field which has made remarkable progress in the past few decades.
Genetic engineering as a field of biology is a result of progress in many different fields including molecular biology, evolution, heredity, molecular genetics, engineering and so on. Many discoveries and discoverers have contributed to the development of this particular branch of biology. Some of the major discoveries will be highlighted here.
Evolution of the “Gene” and Genetic Engineering Concept
Genotype and Phenotype
If we take a minute and think about whats around us we will realize that there are two major types of things: living and non-living. Living organisms are made up of about 98% of basic elements which are carbon, hydrogen and oxygen, 1% nitrogen and the rest of 25 different elements. The non-living world (only our planet) is made up of 100 known elements. The biggest difference in living and non-living world is that the combination and composition of elements in the living world happens in such a way which makes a system, capable of growing and most importantly reproducing itself. How this happened is an entirely different field of biology called molecular evolution.
Living things are capable of reproducing themselves but how is it that a dog produces a dog, potato seeds grow into potato plants and humans reproduce to have a baby? The physical traits of a living organism combine to be called its phenotype. The differences in phenotype even withing the same species (whether it be potatos or people) are possible because parents can pass something called genome (entire set of genes) to their offspring. This trait of a living organism is its genetic make up referred to as genotype. Thus, the phenotype of an organism is the result of the expression of its genotype.
This is the reason why there are differences among humans, eye color, height and so on. Each individual have their own set of genotype and thus a different phenotype. By now we can realize that a gene is something that brings a phenotype into being. However, for a phenotype to manifest there are many levels of complexity arising from the interaction of various genes, their expression to form proteins, regulation of expression and so on.
Human Understanding of the Gene
The concept of “gene” has undergone a lot of changes and has become more complex compared to when it was first coined by Wilhelm Johanssen in 1909 (1). Infact, the concept is still changing and there is not a complete and clear conscience of what a “gene” really is among scientists. Johanssen coined the term gene to explain observations made by Gergor Mendel in 1866 during his work with peas. Mendel founded theory of heredity by crossing peas with different traits and studying the traits in successive generations. The factor responsible for transmission of biological traits was called “gene” (2).
In 1900, Walter Sutton came up with “Chromosomal theory of heredity” which showed that genes are located in chromosomes. His work was important because it mixed genetics (breeding experiments) with cytology (study of cell structure) (2). Then during the 1910’s gene was found to be a “distinct loucs”. T.H. Morgan came up with a model to show that genes are linearly arranged and the frequency with which the cross-over is proportional to the distance between them (3). The first genetic map was made in 1913 by Sturtevant (4) and in 1915 T.H. Morgan came up with “Mechanism of Mendelian Inheritance” describing the mechanism responsible for what Mendel had discovered and thus vindicating him (3). For geneticists of this time, gene did not have a physical existence, it was something abstract whose existence manifested only as a result of transmission of physical traits. Then in 1941, Beadle and tatum showed that mutation in a gene causes defect in steps of metabolic processes. Thus they came up with “one gene one enyme” hypothesis which was later replaced as “one gene one polypeptide” hypothesis (5). This was a more mechanistic view of gene, it meant that genes are implict information about individual molecules in a metabolic pathway. During the 1950’s it was shown that gene was not something abstract but had a physical existence. This was due to various observations. Muller showed that X-ray could cause mutation (6), Griffith showed that virulent but dead Pneumococcus could be taken up by avirulent living one and get transformed into virulent (7). Avery in 1944 showed that the unit of inheritance or gene could be destroyed by DNAse (any of several enzymes that break down the DNA molecule into its component nucleotides) (8). Finally in 1955, Hershey and Chase proved conclusively that the unit of inheritance was DNA and not protein (9). 1960s was the time when the science of gene and molecular biology sky-rocketed. Watson and Crick cracked the structure of DNA, they showed how a double stranded helical DNA could replicate itself thus solving the structural basis for inheritance (10).
There was the discovery of messenger RNA which is a transcription product of DNA. The genetic code was discovered which explained how mRNA dictated the sequence of incorporation of amino acids in a chain to form protein. Thus the flow of information from DNA to RNA to protein was clear, this was termed “The Central Dogma” by Francis Crick (11). Gene was now a code that gave rise to a functional product. However, there were many exceptions: it was known that many genes do not make proteins. For e.g. some genes make tRNA, rRNA but these are never translated to make proteins. By now it was clear that gene is a stretch of DNA and it produces a functional product which somehow brings a trait into being.
The landmark discovery which lead to the foundation of genetic engineering was the discovery of restriction enzymes (restriction endonucleases) by Herbery Boyert and Stanley Cohen in 1973. Restriction enzymes are produced by bacteria and function as a simple immune system. They protect bacteria from possible invasaion of viruses. They recognize a certain sequence in DNA or RNA and chop it. HindII was the first discovered restriction enzyme but thousands were dicovered later. The property which has made restriction enzymes workhorses of molecular biology is that each resriction enzyme recognizes and cuts a specific, short nucleotide sequence not found in the bacteria. Since restriction enzymes can recognize and cleave that particular sequence not only in viruses but any other DNA/RNA containing that sequence, their use was immediately identified. It meant that now it was possible to cut a piece of DNA from one source and possibly ligate the piece into DNA from another source cut with the same enzyme (using enzymes called ligases) (12).
Junk DNA. Not!
Now that it was possible to move a particular gene around, it became easier to study how genes behaved. It was found that over 98% of the genome in humans do not code for proteins,instead they were labelled “junk DNA”. However, it is slowly being discovered that these so called junk DNA are important not only to regulate the level of expression of the genes but also play an important role in evolution, as they contain clues to what direction did evolution take. Similarly, a whole myraid of small RNA molecules were discovered which were termed as non-coding RNA. Examples include: micro RNA, small interfering RNAs and so on. These small RNAs have been shown to be important in regulating gene expression at post-transcriptional level. Similarly, many genetic genetic disorders have been found to correlate with abnormal levels or activities of these small non-coding RNAs.
The Human Genome Project
The completion of the Human Genome Project was a landmark in the history of science. However, there was a shock among biologists when it was discovered that the human genome contains only 25-30000 genes but about 100000 proteins. If gene is a stretch of DNA making functional product (protein), how can it be that the number of observed genes are so much less than the number of proteins. This is due to the enormous complexity of the nature of molecular genetics in humans. Phenomenon like splicing, can make two mRNA from the same DNA stretch, thus making 2 different proteins.
There are so many other processes and structures in the human genome which together help and maintain a homeostasis in the level of gene expression, both spatial and temporal. Although there is no one clear definition that scientists accept, there are a few working definitions which make it easier for scientists to commnunicate without making things complicated (13). A gene was defined by the Human Genome Nomenclature Organization as “a DNA segment that contributes to phenotype/function. In the absence of demonstrated function a gene may be characterized by sequence, transcription or homology” (14). The Sequence Ontology Consortium reportedly called the gene a “locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions and/or other functional sequence regions” (15). Although these are widely accepted there are problems with these views of the gene because it is a more “sequence based” view. There are phenomenon like frameshifting, splicing which can produce mutiple protein products. The protein coding sequence of DNA is usually present between non coding ones forming an open reading frame. This causes a problem when genes are defined as “locatable region of genomic sequence”. There are distinct regulatory sequences which are directly involved in transcription e.g. enhancers, silencers but these sequences do not code for any functional product. There are mobile elements in the genome which enable genetic elements to appear in a new location over generations. There are processes of epigenetics which change the phenotype without changing the sequence of DNA and many such mechanisms exist which do not agree totally with the “sequence-based” definition of a gene.
-  Author Nils Roll-Hansen
- Title The crucial experiment of Wilhelm Johannsen
- Publication Biology and Philosophy
- Date July 1989
- Volume/Section/Page Volume 4, Issue 3, pp 303-329
- Web Address http://link.springer.com/article/10.1007%2FBF02426630
-  Author Mark B. Gerstein et al.
- Title What is a gene, post-ENCODE? History and updated definition
- Publication Genome Res
- Date 2007
- Volume/Section/Page Volume 17: 669-681
- Web Address http://genome.cshlp.org/content/17/6/669
-  Author T.H. Morgan et al.
- Title The Mechanisms of Mendelian Heredity
- Publication Science
- Date 1916
- Volume/section/page Volume 44, Issue 1137, Pages 536-543
-  Author A.H. Sturtevant
- Title The linear arrangement of sx sex-linked factors in Drosophila, as shown by their mode of association
- Publication Journal of experimental zoology
- Date January 1913
- Volume/section/page Volume 14, Issue 1, Pages 43-59
-  Author Beadle, GW. and Tatum E.L.
- Title Genetic control of biochemical reactions in Neurospora
- Publication PNAS
- Date 1941
- Volume/section/page Volume 27, Pages 499-506
-  Author Muller H.J.
- Title Artificial transmutation of the gene
- Publication Science
- Date 1927
- Volume/section/page Volume 46, Pages 84-87
-  Author Griffith, F.
- Title The significance of pneumococcal types
- Publication J. Hyg. (London)
- Date 1928
- Volume/section/page Volume 27, Pages 113-159
-  Author Avery, O.T. et al.
- Title Studies on the chemical nature of the substance inducing transformation of pneumococcal types
- Publication J. Exp. Med.
- Date 1944
- Volume/section/page Volume 79, PAges 137-158
- Web addess http://www.ncbi.nlm.nih.gov/pubmed/20474956?dopt=Abstract
-  Author Hershey, A.D., Chase, M.
- Title An upper limit to the protein content of the germline substance of bacteriophage T2
- Publication Virology
- Date 1955
- Volume/section/page Volume 1, Pages 108-127
-  Author Watson, J.D. and Crick, F.H.C
- Title Genetic implications of the structure of deoxyribonucleic acid
- Publication Nature
- Date 1953
- Volume/section/page Volume 171, Pages 964-967
- Web addess http://www.ncbi.nlm.nih.gov/pubmed/13063483?dopt=Abstract
-  Author Crick, F.H.C
- Title On protein synthesis
- Publication Symp. Soc. Exp. Biol.
- Date 1958
- Volume/section/page Volume XII, Pages 138-163
-  Web addess http://www.genomenewsnetwork.org/resources/timeline/1973_Boyer.php
-  Web addess http://www.genome.gov/10001772
-  Author Hester M. Wain et al.
- Title Guidelines for Human Gene Nomenclature
- Publication Genomics
- Date April 2002
- Volume/section/page Volume 79, Issue 4, Pages 464-470
- Web addess http://www.sciencedirect.com/science/article/pii/S0888754302967480
-  Author Pearson H.
- Title Genetics: What is a gene?
- Publication Nature
- Date May 25, 2006
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- Web addess http://www.ncbi.nlm.nih.gov/pubmed/16724031?dopt=Abstract
- http://www.jbc.org/content/279/39/40247.full Robert Sinsheimer’s personal account of what he witnessed for the past 60 years as molecular biology exploded as a branch of biology.
- Reference 2 provides a nice overview of the “gene” concept and how genes are viewed in today’s biology. It also provides resources if you want to explore. http://genome.cshlp.org/content/17/6/669
- “A History of Molecular Biology” written by Michael Morange is one of the well wrtitten books which introduces molecular basis of life in a step-wise and clear manner. ISBN: 0674001699
- www.genome.gov Official website of the NIH’s, National Human Genome Research Institute provides a lot of interesting informations in lots areas.