Major Achievements in Biology
Researchers and scientists are always intrigued by biological processes and activities, and strive to utilize them for benefit of mankind. Antibiotics, monoclonal antibodies, vaccines and several therapeutic agents, high yielding as well as disease tolerant plants and animals are all results of extensive research in the fields of cell and molecular biology, immunology, biotechnology and genetic engineering. Treatment to several diseases, even genetic disorders are available today only due to breakthrough discoveries in biology. Three such important discoveries are revelation of structure of ribosomes through X-ray crystallography, identification of tumor specific antigens (TSA) expressed on the surface of cancer cells, and discovery of plasmids that are used as valuable tools in recombinant DNA technology. These discoveries and their significance to society, health, and the culture of modern life are discussed in this paper.
Ribosome Structure Revelation through X-ray Crystallography
X-ray crystallographic studies on ribosomes have been reported since two decades (Poehlsgaard and Stephen, 2005), and they have facilitated in identifying the key role of ribosomes in protein synthesis as well as the sites targeted by antibiotics. Ribosomes consist of r-protein and ribonucleic acid (rRNA), and function as an enzyme driving the translation process. In protein synthesis after transcription of DNA into mRNA, ribosomes act as the entire machinery for assembling the amino acids in sequence. Through X-ray crystallographic studies it has been revealed that the smaller subunit involves in codon and anticodon base pairing between the mRNA and the specific amino acid harbored on the tRNA (Poehlsgaard and Stephen, 2005). The peptide bond is formed in the larger subunit. But, only through crystallographic studies it was revealed that there is no role of ribosomal protein in the bond formation, and the rRNA itself purely drives it (Poehlsgaard and Stephen, 2005).
There are differences in structure of prokaryotic and eukaryotic ribosomes. While prokaryotic ribosomes are composed of 30S and 50S subunits, eukaryotic ribosomes have 40S and 60S subunits. Hence, several antibiotics such as tetracycline, streptomycin, erythromycin and chloramphenicol that target 30S and 50S ribosomal subunits were developed to specifically target, and inhibit protein synthesis in pathogenic microbes without affecting the host human cells. The binding sites of various antibiotics in the 30S and 50S units are shown in Fig 1. Though few of these sites could be guessed by biochemical and genetic techniques, exact location and details of the binding could be revealed only by X-ray crystallography. Also, microorganisms have developed resistance to ribosome targeting antibiotics by several mechanisms. Only crystallographic revelations can explain antibiotic resistance, drug inhibition as well as combinatorial effects clearly (Poehlsgaard and Stephen, 2005).
Antibiotic resistance is developed either by nucleic acid substitution or methylation in the rRNA, or amino acid substitution in the ribosomal protein (Lambert, 2012). While these modifications enable the pathogen to survive in a particular antibiotic containing medium or host, the overall rate of protein synthesis, growth rate, and ability to thrive in a antibiotic less medium is greatly affected. The nucleic acid and amino acid mutations accumulating in an antibiotic resistant microbe (compensatory mutations) might eventually affect its survival due to restricted biological pathways in the pathogen (Criswell, 2004). According to Levin, Perrot and Walker (2000), in a medium devoid of the specific antibiotic, wild type non-resistant microbes survive better compared to the resistant strains, which have undergone several mutations. Thus, antibiotic resistance can be overcome in future if this aspect of compensatory mutations in ribosomal RNA and protein is studied in detail. To study these aspects of antibiotic resistance X-ray crystallography is a valuable tool.
Further, novel drugs can be developed, or existing ones can be improved based on the specific binding site in the target ribosomal units in the pathogen (Poehlsgaard and Stephen, 2005). Thus, the crystallography technique is a boon to mankind, as it has helped us understand the significance of the “dynamic machine” i.e. ribosome, and the mechanism of protein synthesis, which is key to all living processes. Further, a whole new generation of antimicrobials can be developed using the ribosomal crystal structures, despite microbes acquiring resistance through various means. Thus, drug resistance problem can be addressed with better clarity with further research in the area of ribosome targeting drugs.
Expression of Tumor Specific Antigens on Cancer Cells
For the past 25 years discoveries about cancer cells, and the immune system’s response towards them have lead to development of several anti-cancer drugs and therapies. Human immune system detects tumor specific antigens (TSA) expressed exclusively on the surface of tumor cells, as well as tumor-associated antigens (TAA) that can be expressed on normal and cancer cells, and initiates response to them (Finn, 2008). But, cancer cells suppress these immune responses, and establish themselves. So, triggering or amplifying the immune response can cure the tumor. A major breakthrough in TSA/TAA research happened when tumor specific T cells could be isolated from cancer patients and propagated in the laboratory (Finn, 2008). MAGE-1, a melanoma TAA that triggered T cell response was thus detected (Finn, 2008), and several other TSAs are still being identified.
Cell mediated immune response to cancer cells is initiated by presentation of the TSA or TAA to cytotoxic T cells. TSAs and TAAs are usually proteins synthesized by altered normal cells, products of gene mutations or viral infection (Finn, 2008). These altered, mutated or foreign viral proteins, are broken-down by proteasomes, and Major Histocompatability Complex (MHC) molecules present these peptides to cytotoxic CD8 T-cells (Finn, 2008). Thus, TSA’s trigger the immune response.
Once TSA’s are identified, monoclonal antibodies can be developed specific to the antigens, and used to treat the cancer, or the cell mediated immune response could also be triggered. Tumor associated antigens for which therapeutic monoclonal antibodies have been developed include haematopoietic differentiation antigens such as CD20, CD30, growth factor and growth factor receptors such as epidermal growth factor receptor (EGFR), insulin-like growth factor 1 receptor (IGF1R), vascular endothelial growth factor (VEGF) and receptor (VEGFR), as well as tumor stromal and extracellular matrix antigens such as fibroblast activation protein (FAP) (Scott, Wolchok and Old, 2012). A list of monoclonal antibodies approved by FDA for cancer therapy, and their target TAAs/TSAs are shown in Table 1.
Different kinds of monoclonal antibodies to the TAAs/TSAs are used in cancer treatment. These include naked monoclonal antibodies (mAbs), conjugated i.e. radiolabelled or chemolabelled mAbs, and bispecific mAbs. Naked mAbs such as Alemtuzumab attach to the CD52 antigen on lymphocytic leukemia cells and mark them for destruction by the immune system (American Cancer Society, 2015). Trastuzumab naked mAbs on the other hand binds to HER2 protein that help the breast cancer cells grow and spread (American Cancer Society, 2015). In conjugated monoclonal antibodies the mAbs function as a vehicle to deliver a particular drug or radioactive substance specifically to the cancer cells. Ibritumomab tiuxetan is a radiolabelled antibody used to destroy cancerous B-lymphocytes that express CD20 surface antigen (American Cancer Society, 2015). Similarly, mAb Brentuximab vedotin, is used to deliver a chemotherapeutic drug to cancerous lymphocytes expressing CD30 antigen, and is used to treat Hodgkin Lymphoma (American Cancer Society, 2015). Bispecifc mAbs attach through one portion to the cancerous cell surface antigen, and through another portion to the protein on the surface immune cells such as T cells (American Cancer Society, 2015). So, the immune cells easily destroy the cancerous cells.
Thus, cancer treatment techniques have developed to a large extent only due to the discovery of tumor specific and tumor associated antigens. Several side effects arise due to destruction of normal cells that express antigens similar to the cancer cells. Thus, researchers are trying to find out more specific antigenic markers for cancers to develop more efficacious drugs. These novel cancer therapies would eradicate the fear of cancers as life threatening diseases.
Discovery of Plasmids
Plasmid is a small fragment of double stranded DNA that can exist and replicate independent of the chromosome. Plasmids may be linear or circular, and they have only few genes that code for some special properties such as antibiotic resistance, or fertility genes required for bacterial conjugation, etc. Jushua Lederberg coined the term plasmid in 1952, after discovering that fertility genes present in an “extra-chromosomal heredity element” are essential for bacterial conjugation (WhatIsBiotechnology.org). In recombinant DNA (rDNA) technology the key steps are to identify a gene of interest, isolate it and insert it into a vector, incorporate the vector insert into a host cell’s chromosome, and express the recombinant gene or replicate it to yield multiple copies. Plasmids act as excellent vectors or vehicles that help in expressing or cloning the rDNA (Lodish et al, 2000). Plasmids have only less number of genes, hence the rDNA can be easily inserted into a selectable site, and usually plasmids have markers such as antibiotic resistance genes to verify gene insertion. So, the bacterial cell that has been successfully engineered with the recombinant plasmid containing ampicillin resistant marker will be resistant to ampicillin antibiotic. In a medium containing ampicillin antibiotic, these engineered cells multiply to yield several copies of the rDNA. This process of cloning with plasmid vector is depicted in Fig 2.
A bacterial plasmid can take up a DNA insert ranging from 6-12 kilo bases (Elfarash), and its most important feature is the presence of a multiple cloning site (MCS) or polylinker. The MCS has unique sites for restriction endonucleases that do not occur anywhere else in the plasmid (Elfarash). So, the desired DNA fragment can be easily inserted into the MCS space. Thus, plasmids help in obtaining multiple copies of a desired gene as well as a recombinant gene product. Though polymerase chain reactors (PCRs) are used as a quicker alternative to plasmid based in-vivo cloning in laboratory studies, the technique is still popular among plant biotechnologists to develop recombinant varieties of plants. Ti plasmid of Agrobacterium tumefaciens is responsible for inducing crown gall’s disease, a form of tumor in plants. But, instead of the tumor causing genes, a desired gene insert is transferred using the Ti plasmid to plant cells (Vikram, 2005). The recombinant plant cells are then selected and cultured in the laboratory prior to soil transfer (Vikram, 2005). Several insect resistant, stress tolerant and high yielding varieties of genetically modified plants have been developed using this method.
Thus plasmids and genetic engineering techniques have led to a new era of genetically modified organisms GMOs, including plants as well as animals with desirable traits. These GMOs can be used to feed the future population, in a world threatened by adverse climatic conditions and droughts. Developing countries can especially benefit from high yielding and nutrient enriched GM plants, to secure food supply and manage malnutrition related diseases. Thus plasmid discovery has highly beneficial impacts on the human society as a whole.
Conclusion
All three discoveries structure of ribosome, surface antigens expressed on cancer cells and plasmids discussed above have impacts extending beyond the medical field. While ribosomal structure revelation can lead to development of new drugs, they can also help us combat the problem of antibiotic resistance. Further research in the area of Tumor specific antigens will help in development of better drugs for treating cancer, which is considered a life threatening disease. As diseases affect people from all economic backgrounds research and development in biology can only help in developing cheaper drugs in large scale. Thus better treatment options will be available to all sections of the society. Finally, discoveries in rDNA technology such as the plasmid vectors have provided us a valuable tool to address the food scarcity problem. On the whole, future sustainability of mankind depends only on such novel discoveries, and their practical applications.
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