Contrary to popular belief, biotechnology has been around for a while. The idea encompasses a wide range of procedures for modifying all forms of living organisms for human use. Early forms of biotechnology date back to the domestication of animals and the cultivation of plants where improvements were made through breeding programs and the implementation of artificial selection and hybridization. In the past forty years with the discovery of recombinant DNA technology, biotechnology in the form of genetic engineering has become more advanced than ever before. For the first time in history, genetic engineering (GE) is not limited to species or cultivar, but across all genomes. This paper will attempt to discuss biotechnology from an agricultural perspective, and what implications it may have on the future of food systems and the perseverance of Homo sapiens.
Current statistics reveal that one sixth of the world’s population does not have enough food to sustain a productive life. Though some will say this has to do with food speculators, corruption, and global trade, others will say that our agriculture production systems must be improved through GE. Whether an agreement is made on how to solve the problem or not, most people can agree that proper nutrition outweighs medical intervention and through translational agriculture sciences, solutions can be reached.
Agriculture Revolution: starvation ended or growth enabled?
In general the concept of agriculture has only been around 10,000 years or so (others may argue less), and is one of the most famous cultural revolutions. According to Vavilov throughout those 10,000 years food had eight centers of origin; however, these origins were different then the location of the crops’ actual genetic diversification. In short, the location of domestication was much different then the origin. As domestication occurred, the germplasm, or genetic base of a particular crop, began to grow and develop. New landraces, or traditionally selected cultivars, were created and humans continued to expand gene pools up into the 20th century when a second revolution occurred.
Norman Borlaug, the father of the Green Revolution, is hailed for his improved wheat varieties that supposedly saved 1 billion people from starvation. A plant pathologist/agronomist, Borlaug’s hybrid varieties were planted all over the world. More importantly the concept of hybridization and improved varieties was planted all over the world, and this is when the idea of biotechnology really grew (pun intended). With a tripled food supply in the last thirty years and almost tripled population growth; biotechnology would appear to be a blessing. To understand this better a bit of genetics must be reviewed.
Genetic basics: a deep understanding is paramount
In a nutshell genetic engineering is the addition of a desired gene, in a noncoding locus on the chromosome, to produce a desired phenotype. Some individuals are heterozygous, meaning a different allele at the same locus, whereas others are homozygous, or the same allele at the same locus. Often times homozygous individuals will have more success being engineered, because the genes for the trait are the same. New traits can arise from a mutation, and when successfully and permanently incorporated into the genome, or the entire genetic material in a chromosome set, natural selection has occurred. Mutations should not be confused with polymorphisms, such as eye color, which are the coexistence of two or more common phenotypes for the same genotype. Mutations are essentially nature’s form of GE, and their concept played a major roll in the discovery of recombinant DNA technology in 1972. Recombinant DNA themed research continued to advance, with multi-billion dollar companies involved in research, and in 1982 insulin, the first GE product was marketed; world food crops would soon follow.
To modify the phenotype of an individual, a person must have a strong understanding of genetics. It is one thing to successfully place a foreign gene into a chromosome and produce a phenotype; it is another thing to have an individual successfully pass the gene, and subsequent phenotype, onto its offspring. This is why understanding the genetics side of reproduction is so important. In meiosis when chromosomes are reduced from diploid to haploid, a process known as independent assortment (IA) occurs. IA is the process of random segregation and assortment of chromosomes during gametogenesis, which results in genetically unique gametes. During meiosis, a number of genetic ‘phenomena’ can occur such as crossing over or recombination, which result in a segment from one chromatid swapping locations with the same segment on the other chromatid. This leads to a crossover chromosome. The take home message here is during gametogenesis and meiosis; a certain gene assortment will take place. Not all genes from the GE parent will end up in the offspring, and a selection process to find the ‘modified’ individuals will need to occur. So where does this all begin?
DNA: structural & functional approaches
All organisms have trillions of cells that make up their body. With exception to a few organisms, each eukaryotic cell contains a nucleus with identical complements of chromosomes, each chromosome is one long DNA molecule, and the ‘functional’ regions are known as genes. At this point in history, studies will indicate that genes are transcribed to an RNA transcript, which are later packaged by nuclear proteins to leave the nucleus through a pore. After entering the cytosol, mRNA, in simplified terms, makes it way to the ribosome where it is ‘translated’ into an amino acid polymer known as a protein. There are dozens of regulatory steps along the way that could easily take up a lifetime of research and not a day spent outside of the lab.
Though the process is heavily regulated and monitored, mutations do occur. If the mutation does not lead to cancer, apoptosis (programmed cell death), or another negative outcome, the new mutation is now apart of the genome and has the potential to be passed along during reproduction. This process, whether spontaneous or induced, is known as forward genetics, where a phenotype leads to a change in the DNA. The antithesis to this process is reverse genetics, where a specific change is introduced into a specific gene. This is useful if there is no known phenotype to look for, or if genetic engineering is the goal. So how exactly is DNA created and replicated?
Before understanding replication, a few enzymes must be mentioned. DNA polymerase uses a DNA template to synthesize a DNA strand, RNA polymerase uses a DNA template to synthesize an RNA strand (transcription), and reverse transcriptase, commonly in viruses, uses an RNA strand to synthesize a DNA strand. These processes are also in need of other regulatory enzymes and proteins such as ligases or histones to repair and package the chromosome. Though the process functions quite well, there are always mistakes. Some mistakes are harmful to the cell and others will simply create a change in phenotype. One example of this is a single-nucleotide polymorphism (SNP). While DNA polymerase is working hard in the replication bubble, or ‘split’ duplicating region of the chromosome, it may accidently swap a single nucleotide basepare; for example, T & A instead of C & G. Though this only results in a one-basepare difference between chromosomes, the resulting change in sequence can give rise to a completely different phenotype.
Genomes: parameters & practicality
Before anyone begins modifying the genes of an individual, taking a look at the bigger picture will be helpful helpful. Again, the genome is the total genetic content, coding or non-coding, contained in a haploid set of chromosomes. This is true for eukaryotes, however for bacteria the genome refers to a single chromosome and for viruses, simply the DNA or RNA. Understanding the genome has a number of practical applications such as geographical variation based on evolution, species relationships, gene expression analysis, cDNA libraries, linkage maps, and recombination distance estimates for better GE success rates. To understand and speak about a genome, a few parameters must be used. The C-value, is expressed as the content of DNA per haploid set of chromosomes. This value is expressed in Picograms (pg or Gram x 10-12), which can later be expressed in a base pair number. Base pairs are the linkage between two nitrogenous bases (A & T, C & G) on complimentary DNA; there are kilobasepairs (Kpb), megabasepairs (Mbp), and Gigabasepairs (Gbp), each of which are a 1000x the previous. The number of basepairs can be expressed as the mass in pg x 0.978 x 109. Aren’t you glad someone else figured that out?
Now that a language has been created, a successful dialogue can take place and genomes can be better understood. This is referred to as genomics, or the study of the entire DNA sequence in an organism. There are a number of different “omics”, but fort the purpose of this paper the focus will be the genome. Plant genomes are often broken down by monocot or dicot and gymnosperm or angiosperm. These can range anywhere from 0.06pg (G. margaretae) to 153pg (P. japonica). Animal genomes are a little more complicated. Initially broken down by phylum, and later by family, animal genomes range in size from 0.02pg (plant-parasitic nematode) to 133pg (marbled lungfish). By understanding the size of the genome, what genes are where, and which sequences are introns (non coding) or exons (coding), strategies for genetic engineering can be formulated and implemented.
Until the advent of flow cytometry (FC) in the 1960’s, genome size estimation was relatively non-existent. BD Biosciences sponsored a broadcast Feb. 1, 2012, on how their new cytometer enables rapid determination of eukaryotic genome size and cell type-specific gene expression. In genetics FC is a technique used to count and examine microscopic particles, in this case chromosomes, by suspending them in a stream of fluid, blasting them with a laser until they excite and give off a wavelength of energy. Electronic detection devices pick up the wavelength, which can later be analyzed on a print out in the form of a peak. Today this technology is the basis of genome research such as genome size measurements, ploidy screening, characterization of unsuspected phenomena such as endoreduplication, as well as molecular and cellular biology of the nucleus. GE strategies begin with genome projects and if it weren’t for these new forms of FC some projects may never be completed.
Complexity of the Genome: chromatin, gene pyramiding, & synthetic chromosomes
At this point it is clear that even though a gene has found its way into a particular chromosome, the process of turning that into a viable individual can be very difficult due to the multiple areas of potential error or the many locations the gene could end up within the genome. As stated earlier, the genome contains multiple chromosomes, each of which possesses two types of chromatin, the combination of DNA and proteins within the nucleus. Chromatin is organized into two regions, euchromatin is loosely packaged and heterochromatin is densely packed. Due to the less dense nature of chromatin it is considered genetically active, and most proteins are transcribed and translated from these regions. Heterochromatin on the other hand, is broken down into two groups: ‘constitutive’ contains repetitive sequences containing mainly “junk” DNA and transposons (sequences of DNA with the ability of excising themselves from the chromosome and reinserting themselves in a new loci); whereas, ‘facultative’ is comprised of transcriptionally active or inactive regions such as the silenced X chromosome in mammals. These two types of chromatin are wound tightly around histone proteins, ultimately determining their conformation and affecting gene expression. Understanding this is paramount for GE. Different patterns of histone modification could change regulatory signals resulting in different processes such as replication, transcription or DNA repair taking place.
Gene pyramiding is a technique used to “stack” genes located at different loci on the chromosome with the ultimate goal being the accumulation of genes that have been identified in multiple parents into a single genotype. One way this is achieved is through the slow and error-laden process known as backcrossing, process of crossing a hybrid with one of its parents to achieve a genetic identity closer to the parent. This depends on a few variables such as the number of genes to be transferred, the distance between the target genes and markers, as well as the nature of the germplasm.
As mentioned earlier, the success of this can be determined by the level of gene density and whether or not the sequences are tandem or interspersed. Another method of gene pyramiding is the insertion of a manufactured chromosome into the nucleus. This process uses a tiny chromosome discovered in maize known as the B-chromosome. Scientists can insert as many genes as needed into the modified B-chromosome. This chromosome serves as a vector that can deliver multiple genes known as a gene stack. This is much faster then inserting one gene at a time, and the benefits are reliable inheritance through multiple generations, highly predictable gene expression, and a stable DNA structure. With the technology in place to deliver desired genes,
Centromeres & Telomeres
In general centromeres and telomeres are two essential features to all eukaryotic chromosomes. Each provides a unique function that is absolutely necessary for the stability of the chromosome. Centromeres, are located at the center of the chromosome, and are responsible for the ‘pinched’ look that they all tend to have. They are responsible for the segregation of sister chromatids during meiosis and mitosis. Telomeres provide terminal stability to the chromosome and ensure its survival. Other than stability why do these sequences really matter? They matter because they contain repeat sequences that help create the ‘map’ of the chromosome. The ultimate goal is to map or ‘fingerprint’ the genes on the chromosome, which leads to a deeper understanding and better decision-making. This is where tandem repeats come in.
Fingerprinting & Tandem Repeats: mapping out the chromosome
The first type of DNA fingerprinting (FP) is known as ‘single locus’. This form implements a specific probe or specific PCR primers. This is used when the single loci are known and the ultimate result is a DNA genotype. The other form is known as ‘multilocus’ where polymorphisms at multiple loci are identified simultaneously. The first type of ‘multilocus’ FP involves a mixture of single locus probes; whereas, the second uses a single probe that identifies multiple similar sequence polymorphisms. This form is detecting unidentified fragments of DNA, thus the result is a DNA phenotype, not to be confused with genotype. Each method is useful in certain applications, and there are number of repeat sequences that can be taken advantage of.
Variable nucleotide tandem repeats (VNTRs), also known as Minisatellite DNA, are non-coding sequences composed of arrays of short repeats (2-6 bp). Arrays range from 10 – 100+ bp. They are variable number sequences at different chromosomal positions and do not exist in every individual of a species. This method is highly polymorphic, producing a large number of different-sized fragments making it useful as a polymorphic marker in fingerprinting. The products can be amplified by PCR and labeled for easy identification. Repeats can also be harmful. If the copy number of the repeat increases to high, for instance 50 AGCs vs. 15 AGCs, some harmful genetic disorders can result, such as Fragile X syndrome. This issue is compounded over generation because the repeats can expand as they are passed down from parent to child. Good or bad, the discovery of VNTRs opened the door for multiple genetic applications.
Transposable Elements: mystery or miracle?
Transposable Elements (TEs) are unique sequences of DNA that have the ability to excise from the chromosome and reinsert in a new location. Like tandem repeat sequences, (TEs) are sequences located in all genomes. They range in size from 50bp-10kb, and have played a role in mutations, natural selection, and evolution as a whole. Each TE has its own ‘instruction’ for transposition, the term referring to excision and reinsertion. Once excised, the transposon relies on enzymes for insertion. Bacteria use transposase, and eukaryotes have reverse transcriptase (Class 1) and transposase (Class 2). Overall there are two types of TEs: Autonomous, capable of self-transposition and Nonautonomous who transpose only in the presence of autonomous elements. Autonomous elements code for their ‘own’ reverse transcriptase or transposase, which enables the transposition of themselves and related non-autonomous elements through an RNA intermediate.
Most TEs are flanked by repeating sequences, making them easy to identify and label. Class 1 are known as retrotransposons, and there are those with Long terminal repeats (LTR) and non-LTR labeled LINEs and SINEs. Class II are DNA transposons and there is also a Class III. TEs are labeled by discovery, species, function, or phenotypic effect. When these elements are transpositioned, they can cause a fully or partially active allele, or a variant or defective allele. If this occurs before cell division, the subsequent tissue will contain the mutation, whereas the previous cell line will not. This is incredibly useful for gene identification.
Transposon tagging uses the DNA of TEs for transformation. Since TEs have known sequences and methods, they can be implemented by transpositioning themselves at certain sequences within the chromosome. This helps manipulate or better understand gene expression, recombination, genome rearrangements and break repairs. A good example of this is the Sleeping Beauty transposon (SB). It is used in insertional mutagenesis as a ‘knockout’ transposon. This TE can essentially silence an unknown gene of choice, ultimately identifying the phenotype it is responsible for. TEs are also used to insert sequences into the plasmid of a bacteria. This is the basis of GE and when a desired gene is inserted into the plasmid of a bacterial cell, that new gene is one step closer to modifying the phenotype of a new individual.
Now that a basic language has been created, Part II will begin to tell the story of how genetic engineering is actually implemented, and final conclusions will be drawn.