Principles Of Gene Manipulation And Genomics

Composition of the Human Genome

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Discuss About The Principles Gene Manipulation And Genomics?

The human genome comprises of nucleic acid sequences that is encoded as DNA present within chromosome pairs (23) within the cell nuclei. The regions that are present inside the human genome comprises of protein coding and non-coding DNA genes, telomeres and centromeres. The coding DNA comprises of sequences that can go for transcription into mRNA and translated to form proteins during the life cycle in humans, although it comprises of only <2% (small fraction) of the whole genome and non-coding genome comprises of 98% of the human genome that are not used for encoding proteins. Transfer RNA (tRNA) and ribosomal RNA (rRNA) are non-coding RNAs that perform crucial biological functions (ENCODE Project Consortium, 2012).

Protein-coding sequences participate in human protein production through biological processes like alternative pre-mRNA and DNA arrangements. This region is contained with in economics comprising of DNA sequences that are coded by exons that are translated into proteins having 20,000 proteins. Through the eukaryotic evolution, a mutational load that occurred from deleterious mutations has placed an upper limit of 40,000 functional loci that comprises of functional non-coding and coding genes. Examples of protein-coding genes are cytochrome b, dystrophin (largest protein-coding gene), Histone H1A, Titin, Breast cancer type 2 susceptibility protein and Cystic fibrosis transmembrane conductance regulator. RBFOX1 (RNA binding protein, fox-1 homolog 1 is the largest protein-coding gene of total 2.47 MB (Lek et al. 2016).

The non-coding protein comprises of rRNA and tRNA, introns and 5’ and 3’ mRNA untranslated regions. Introns comprises of 26% of the human genome. Pseudogenes are the inactive protein-coding genetic copies that evolved through gene duplication and became non-functional through inactivating mutations accumulation comprising of 13,000. The process of gene duplication is an essential mechanism where generation of new genetic material takes place during the molecular evolution. Olfactory receptor gene is the best-documented pseudogene and comprises of more than 60% of the genes (Guttman and Rinn 2012).

The forces of mutation, recombination, genetic drift and selection are the evolutionary mechanisms that have modified the eukaryotic genome. The new genetic variants arise from mutation that spread and maintained in the human population through the process of natural selection or genetic drift. As the mutations are rare, they are accumulated through generations very slowly. The mutation rate per generation, differences in nucleotide numbers between the two sequences and divergence times are effectively estimated through molecular clock leading to eukaryotic evolution (Jobling, Hurles and Tyler-Smith 2013).

Centromeres are satellite DNA sequences that are not conserved broadly through evolution. Villasante, Abad and Méndez-Lago (2007) published a paper that proposed an evolutionary scenario for centromere evolution from telomeres. The ancestral circular genophore breakage activated the transposition of DNA ends retroelements. This allowed telomere formation by a mechanism of recombination-dependent replication. The tubulin-based cytoskeleton modification allowed specific subtelomeric repeats that gave rise to first centromere. This genophore to tubulin transition contributed to chromosomal segregation, breakage and instability that generated telocentric chromosomes and eventually, holocentric or metacentric chromosomes.  

Protein Coding and Non-Coding Genes

Telomeres are repetitive DNA sequences that are found at the end of chromosomes that play an important role in cellular proliferation regulation and shorten in size with increasing age of human tissues proliferation. The age-related telomere shortening takes place in early life and decreases with increasing age. The proliferation of cells is limited by shortening of telomeres through the process of natural selection. Energetic constraints limit telomere length and therefore, short telomeres leads

Numerous attempts have been used to identify the regulatory domains in the human genome. The approaches include both experimental and computational methods like detection of transcription factor binding-site motifs in co-expressed genes. These strategies give data about the genomic regions over which the gene regulation is likely to be involves, but they do not provide information about the functions of this region. Khambata-Ford et al. (2003) have employed functional selection for the promoter regions in the human genome via employing retroviral plasmid library-based system. This approach helps in the detection of the promoter region of the isolated DNA fragments under in-vitro cell culture assay. By employing this method, Khambata-Ford et al. (psychology) have discovered the regulatory or promoter region of known or predicted genes. It also helped in the elucidation of putative promoter regions based on the presence of CpG islands. This promising method is extremely useful in the domain of genome-wide function-based approach, which can complement existing methods for the detection of promoter region (Khambata-Ford et al. 2003).

Other popularly used approach used for the identification of the promoter region of DNA or gene include use of bioinformatics approach via making use of the electronic database. Meyer et al. (2012) have utilized University of California Santa Cruz (UCSC) Genome Browser in order to detect the promoter region of the gene. de Wit and de Laat (2012) have cited the use of 3D modeling for the detection of the promoter region of DNA via providing detailed insight about the nuclear organization.

Promote is the region of the gene that acts as the binding side of the enzyme (Rittmann and McCarty 2012). For example, RNA polymerase 1 promote acts as the binding site of RNA polymerase and thereby promoting transcription and translation. Mammalian pre-rRNA gene promote apart from acting as the binding site for RNA polymerase also facilitates bipartite transcription control region. The core elements within this promoter include transcription start site and upstream control region (UCE) (Rittmann and McCarty 2012).

A landmark discovery in the field of molecular biology is Sanger sequencing. It was first discovered by Fred Sanger and his colleagues (1982).  Sanger method of DNA sequencing requires single-stranded DNA as the starting material.  The chain-terminator or dideoxy procedure for DNA sequencing highlights two properties of DNA polymerase that is their ability to dedicatedly synthesize a complimentary copy of ss-DNA and their ability to use 2'-3' dideoxynucleotide as substrates. Once the analogue is incorporated at the growing region of the DNA chain, 3' end lacks a hydro-oxy group and is no longer used as a substrate for the chain elongation. Thus the growing DNA chain is terminated. Initiation of DNA synthesis is done by chemically synthesized oligonucleotide (primer) annealed with the sequence that is required to be analysed (Primrose and Twyman 2013). The DNA sequencing is done in the presence of 4 deoxynucleoside triphosphate, which is labelled with ³²P. Thus each reaction has a population of partially synthesized radioactive DNA with common 5'end but different base pair length at 3'end. After incubation period, DNA in mixture is denatured and then electrophoresed in the sequencing gel. Thus ease of access of the reagents and easily replicable results make this process highly popular among the DNA sequencing methods (Primrose and Twyman 2013).

Initially Klenow fragment of DNA polymerase is used as it is devoid of 5' to 3' exonulcease activity that is associated with the intact enzyme. However, numerous technical improvements have been made in the domain of Sanger's sequencing and this includes replacement of Klenow fragment of Escherichia coli DNA polymerase I with the DNA polymerase of thermophilic bacterium (Thermus acquaticus - Taq DNA polymerase) and T7 DNA polymerase. T7 DNA polymerase is more productive in comparison to Klenow polymerase and thus capable of polymerizing large sequence of nucleotides before releasing them from the template. With Taq DNA polymerase, the reaction can be carried out at high temperatures and this minimizes the chain termination caused by the artefacts of the secondary structure of DNA (Primrose and Twyman 2013).

In order to improve the nature of the autoradiographic images ³²P is replaced ³³P or ³⁵S which are of much lower energy than ³²P. In case of ³⁵S, α- ³⁵S0deoxynucleoside triphosphate is used (Primrose and Twyman 2013)

References

de Wit, E. and de Laat, W., 2012. A decade of 3C technologies: insights into nuclear organization. Genes & development, 26(1), pp.11-24.

Eisenberg, D.T., 2011. An evolutionary review of human telomere biology: the thrifty telomere hypothesis and notes on potential adaptive paternal effects. American Journal of Human Biology, 23(2), pp.149-167.

ENCODE Project Consortium, 2012. An integrated encyclopedia of DNA elements in the human genome. Nature, 489(7414), p.57.

Guttman, M. and Rinn, J.L., 2012. Modular regulatory principles of large non-coding RNAs. Nature, 482(7385), p.339.

Jobling, M., Hurles, M. and Tyler-Smith, C., 2013. Human evolutionary genetics: origins, peoples & disease. Garland Science.

Khambata-Ford, S., Liu, Y., Gleason, C., Dickson, M., Altman, R. B., Batzoglou, S., & Myers, R. M. (2003). Identification of promoter regions in the human genome by using a retroviral plasmid library-based reporter gene assay. Genome research, 13(7), 1765-1774.

Lek, M., Karczewski, K.J., Minikel, E.V., Samocha, K.E., Banks, E., Fennell, T., O’Donnell-Luria, A.H., Ware, J.S., Hill, A.J., Cummings, B.B. and Tukiainen, T., 2016. Analysis of protein-coding genetic variation in 60,706 humans. Nature, 536(7616), p.285.

Meyer, L.R., Zweig, A.S., Hinrichs, A.S., Karolchik, D., Kuhn, R.M., Wong, M., Sloan, C.A., Rosenbloom, K.R., Roe, G., Rhead, B. and Raney, B.J., 2012. The UCSC Genome Browser database: extensions and updates 2013. Nucleic acids research, 41(D1), pp.D64-D69.

Primrose, S.B. and Twyman, R., 2013. Principles of gene manipulation and genomics. John Wiley & Sons.

Rittmann, B. E., & McCarty, P. L. (2012). Environmental biotechnology: principles and applications. Tata McGraw-Hill Education.

Villasante, A., Abad, J.P. and Méndez-Lago, M., 2007. Centromeres were derived from telomeres during the evolution of the economics chromosome. Proceedings of the National Academy of Sciences, 104(25), pp.10542-10547.