Unit – 9A
Molecular Genetics covering
Molecular genetics is the study of the molecular structure of DNA, its cellular activities (including its replication), and its influence in determining the overall makeup of an organism. Molecular genetics relies heavily on genetic engineering (recombinant DNA technology), which can be used to modify organisms by adding foreign DNA, thereby forming transgenic organisms. Since the early 1980s, these techniques have been used extensively in basic biological research and are also fundamental to the biotechnology industry, which is devoted to the manufacture of agricultural and medical products. Trans-genesis forms the basis of gene therapy, the attempt to cure genetic disease by addition of normally functioning genes from exogenous sources.
TECHNIQUES OF MOLECULAR GENESIS
1: Prelude to Molecular Genetics
Today, classical genetics is often complemented by molecular biology, to give molecular genetics, which involves the study of DNA and other macromolecules that have been isolated from an organism. Usually, molecular genetics experiments involve some combination of techniques to isolate and analyze the DNA or RNA transcribed from a particular gene.
DNA purification strategies rely on the chemical properties of DNA that distinguish it from other molecules in the cell, namely that it is a very long, negatively charged molecule. To extract purified DNA from a tissue sample, cells are broken open by grinding or lysing in a solution that contains chemicals that protect the DNA while disrupting other components of the cell (Figure 8.2). These chemicals may include detergents, which dissolve lipid membranes and denature proteins.
3: Isolating or Detecting a Specific Sequence by PCR
The Polymerase Chain Reaction (PCR) is a method of DNA replication that is performed in a test tube (i.e. in vitro). Here “polymerase” refers to a DNA polymerase enzyme extracted and purified from bacteria, and “chain reaction” refers to the ability of this technique produce millions of copies of a DNA molecule, by using each newly replicated double helix as a template to synthesize two new DNA double helices. PCR is therefore a very efficient method of amplifying DNA.
4: Cutting and Pasting DNA- Restriction Digests and DNA Ligation
Many bacteria have enzymes that recognize specific DNA sequences and then cut the double stranded DNA helix at this sequence. These enzymes are called site-specific restriction endonucleases, or more simply “restriction enzymes”, and they naturally function as part of bacterial defenses against viruses and other sources of foreign DNA. To cut DNA at known locations, researchers use restriction enzymes
5: Cloning DNA - Plasmid Vectors
Many bacteria contain extra-chromosomal DNA elements called plasmids. These are usually small (a few 1000 bp), circular, double stranded molecules that replicate independently of the chromosome and can be present in high copy numbers within a cell. In the wild, plasmids can be transferred between individuals during bacterial mating and are sometimes even transferred between different species. Plasmids often carry genes for pathogenicity and drug-resistance.
6: DNA Analysis - Gel Electrophoresis
A solution of DNA is colourless, and except for being viscous at high concentrations, is visually indistinguishable from water. Therefore, techniques such as gel electrophoresis have been developed to detect and analyze DNA.
7: DNA Analysis- Blotting and Hybridization
Bands of DNA in an electrophoretic gel form only if most of the DNA molecules are of the same size, such as following a PCR reaction, or restriction digestion of a plasmid. In other situations, such as after restriction digestion of chromosomal (genomic) DNA, there will be a large number of variable size fragments in the digest and it will appear as a continuous smear of DNA, rather than distinct bands.
Transgenic organisms contain foreign DNA that has been introduced using biotechnology. Foreign DNA (the transgene) is defined here as DNA from another species, or else recombinant DNA from the same species that has been manipulated in the laboratory then reintroduced. The terms transgenic organism and genetically modified organism (GMO) are generally synonymous.
DNA and RNA are the two most important macromolecules on Earth. These two nucleic acids are responsible for storing all of the information that is used to build an organism (including the human genome).
Both are made up of monomers called nucleotides. There are two classes of nucleotides used in nucleic acids: purines and pyrimidines.
RNA and DNA have two purines and two pyrimidines each. The difference between the five nucleotides is dependent on their attached base. A nucleotide plus a unique base is called a nucleobase.
The bases that form DNA and RNA's pyrimidines are adenine and cytosine. DNA's two purine nucleobases are thymine and guanine. RNA has one different purine nucleobase called uracil, but RNA still uses guanine.
In DNA the nucleobases of the two separate strands will point towards the middle of the double helix and bond with each other. Adenine always bonds with thymine, and cytosine always bonds with guanine.
Each purine and pyrimidine has a similar structure consisting of a 5-carbon sugar with an attached phosphate. DNA's sugar is deoxyribose, while RNA's sugar is ribose.
The phosphate emerges from this sugar ring off of the fifth carbon. This is called the "five prime end". When nucleotides are joined together to form a DNA or RNA molecule, the phosphate that comes off of the 5' (pronounced "five prime") carbon on the sugar molecule is bonded to the third carbon on the ring of another sugar molecule. This is called five prime to three prime (5' to 3').
When DNA twists into its double helix shape, it orients itself so that its two strands of nucleotides run in opposite directions. The purines and pyrimidines all point towards the middle of the helix and bond together.
DNA
DNA stands for deoxyribonucleic acid. It is found in the nucleus of eukaryotic cells, and free-floating in prokaryotic cells. DNA consists of deoxyribonucleotides which are made of a pentose (five-carbon) sugar, a phosphate group, and a nitrogenous base. In DNA the four nitrogenous bases are adenine, cytosine, guanine, and thymine (abbreviated as A, C, G, and T). In DNA the pentose sugar has one less oxygen than the pentose sugar in RNA.
The deoxyribonucleotides in DNA are linked together by phosphodiester bonds. These bonds connect nucleotides that are next to each other at the phosphate group and the free 3' hydroxyl group. This creates a single strand of DNA.
DNA is a double-stranded molecule. This means that two strands of bonded deoxyribonucleotides interact through hydrogen bonding with another complimentary, anti-parallel strand of DNA. In DNA, the complimentary base pairing rules state that A pairs with T, and G pairs with C.
RNA
RNA stands for ribonucleic acid. RNA consists of ribonucleotides made up of a pentose (5-carbon) sugar, a phosphate group, and a nitrogenous base. In RNA the four nitrogenous bases are adenine, cytosine, guanine, and uracil (abbreviated as A, C, G, and U). Complimentary base pairing rules for RNA states that A pairs with U, and G pairs with C. In RNA, the pentose sugar (called ribose sugar) has one more oxygen than the sugar in DNA.
The ribonucleotides in RNA are linked together by phosphodiester bonds. These bonds connect nucleotides that are next to each other at the phosphate group and the free 3' hydroxyl group. This creates a single strand of RNA.
Unlike DNA, RNA is a single-stranded molecule. There are also a few different types of RNA that have specific functions. Messenger RNA is used in transcription, transfer RNA is used in translation, and ribosomal RNA helps make ribosomes.
GENES
A gene is the basic physical and functional unit of heredity. Genes are made up of DNA. Some genes act as instructions to make molecules called proteins. However, many genes do not code for proteins. In humans, genes vary in size from a few hundred DNA bases to more than 2 million bases.
Every person has two copies of each gene, one inherited from each parent. Most genes are the same in all people, but a small number of genes (less than 1 percent of the total) are slightly different between people. Alleles are forms of the same gene with small differences in their sequence of DNA bases. These small differences contribute to each person’s unique physical features.
The concept of the gene is and has always been a continuously evolving one. In order to provide a structure for understanding the concept, its history is divided into classical, neoclassical, and modern periods. The classical view prevailed into the 1930s, and conceived the gene as an indivisible unit of genetic transmission, recombination, mutation, and function. The discovery of intragenic recombination in the early 1940s and the establishment of DNA as the physical basis of inheritance led to the neoclassical concept of the gene, which prevailed until the 1970s. In this view the gene (or cistron, as it was called then) was subdivided into its constituent parts, mutons and recons, identified as nucleotides. Each cistron was believed to be responsible for the synthesis of a single mRNA and hence for one polypeptide. This co-linearity hypothesis prevailed from 1955 to the 1970s. Starting from the early 1970s, DNA technologies have led to the modern period of gene conceptualization, wherein none of the classical or neoclassical criteria are sufficient to define a gene. Modern discoveries include those of repeated genes, split genes and alternative splicing, assembled genes, overlapping genes, transposable genes, complex promoters, multiple polyadenylation sites, polyprotein genes, editing of the primary transcript, and nested genes.
GENE REGULATION
Gene regulation is the process of turning genes on and off. During early development, cells begin to take on specific functions. Gene regulation ensures that the appropriate genes are expressed at the proper times. Gene regulation can also help an organism respond to its environment. Gene regulation is accomplished by a variety of mechanisms including chemically modifying genes and using regulatory proteins to turn genes on or off.
In the human genome, there are a little less than 20,000 genes. In some cells, many genes are active--say, 10,000--and the other 10,000 would be inactive. In other kinds of cells, maybe the other 10,000 would be active and the first 10,000 would be inactive. And so, gene regulation is the process by which the cell determines which genes will be active and which genes will not be active. And gene regulation is at the bottom of what makes a cell decide to become a red blood cell, or a neuron, or a hepatocyte in the liver, or a muscle cell. So, different gene regulation will give you a different program of genes and different genes expressed. There are several different kinds of gene regulation. Some genes, called housekeeping genes, are expressed in almost every cell. And these require a regulatory network or machinery that keeps them on in almost every cell, so these are the enzymes that help make DNA, and perform glycolysis, and burn sugar, and things like that. There are other genes that are called tissue-specific genes. These are genes that, say, would only be expressed in a red blood cell or a neuron. Very often, these genes have transcription factors, which are proteins that bind to DNA, near these genes. And those transcription factors actually help the RNA machinery get there and transcribe that gene in those cells, and those tissues, transcription factors, rather, are expressed specifically in those tissues. There are also factors expressed in those tissues that will be suppressors that can turn a gene off. And then there are genes that are regulated during development. Sometimes they're expressed in fetal life and then turned off in adults, and sometimes it's vice versa. So there are very complex different ways that genes are regulated. I kind of look at it as playing music: You have chords on a guitar, or you play with a right and a left hand on the piano. It depends what strings you push down and what strings you strum, or what keys are up and what keys are down, that determine what the profile of the gene expression will be or the sound that you hear.
There are various forms of gene regulation, that is, mechanisms for controlling which genes get expressed and at what levels. However, a lot of gene regulation occurs at the level of transcription.
Bacteria have specific regulatory molecules that control whether a particular gene will be transcribed into mRNA. Often, these molecules act by binding to DNA near the gene and helping or blocking the transcription enzyme, RNA polymerase. Let's take a closer look at how genes are regulated in bacteria.
OPERONS
In bacteria, related genes are often found in a cluster on the chromosome, where they are transcribed from one promoter (RNA polymerase binding site) as a single unit. Such a cluster of genes under control of a single promoter is known as an operon. Operons are common in bacteria, but they are rare in eukaryotes such as humans.
In general, an operon will contain genes that function in the same process. For instance, a well-studied operon called the lac operon contains genes that encode proteins involved in uptake and metabolism of a particular sugar, lactose. Operons allow the cell to efficiently express sets of genes whose products are needed at the same time.
Anatomy of an Operon
Operons aren't just made up of the coding sequences of genes. Instead, they also contain regulatory DNA sequences that control transcription of the operon. Typically, these sequences are binding sites for regulatory proteins, which control how much the operon is transcribed. The promoter, or site where RNA polymerase binds, is one example of a regulatory DNA sequence.
The promoter is found in the DNA of the operon, upstream of (before) the genes. When the RNA polymerase binds to the promoter, it transcribes the operon and makes some mRNAs.
Most operons have other regulatory DNA sequences in addition to the promoter. These sequences are binding sites for regulatory proteins that turn expression of the operon "up" or "down."
Diagram illustrating how a repressor works. A repressor protein binds to a site called on the operator. In this case (and many other cases), the operator is a region of DNA that overlaps with or lies just downstream of the RNA polymerase binding site (promoter). That is, it is in between the promoter and the genes of the operon. When the repressor binds to the operator, it prevents RNA polymerase from binding to the promoter and/or transcribing the operon. When the repressor is bound to the operator, no transcription occurs and no mRNA is made.
References:
1. Žiga Turk (2014), Global Challenges and the Role of Civil Engineering, Chapter 3 in: Fischinger M. (eds) Performance-Based Seismic Engineering: Vision for an Earthquake Resilient Society. Geotechnical, Geological and Earthquake Engineering, Vol. 32. Springer, Dordrecht
2. Brito, Ciampi, Vasconcelos, Amarol, Barros (2013) Engineering impacting Social, Economical and Working Environment, 120th ASEE Annual Conference and Exposition
3. NAE Grand Challenges for Engineering (2006), Engineering for the Developing World, The Bridge, Vol 34, No.2, Summer 2004.
4. Allen M. (2008) Cleansing the city. Ohio University Press. Athens Ohio.
5. Ashley R., Stovin V., Moore S., Hurley L., Lewis L., Saul A. (2010). London Tideway Tunnels Programme – Thames Tunnel Project Needs Report – Potential source control and SUDS applications: Land use and retrofit options
6. Ashley R M., Nowell R., Gersonius B., Walker L. (2011). Surface Water Management and Urban Green Infrastructure. Review of Current Knowledge. Foundation for Water Research FR/R0014
7. Barry M. (2003) Corporate social responsibility – unworkable paradox or sustainable paradigm? Proc ICE Engineering Sustainability 156. Sept Issue ES3 paper 13550. p 129-130
8. Blackmore J M., Plant R A J. (2008). Risk and resilience to enhance sustainability with application to urban water systems. J. Water Resources Planning and Management. ASCE. Vol. 134, No. 3, May.
9. Bogle D. (2010) UK’s engineering Council guidance on sustainability. Proc ICE Engineering Sustainability 163. June Issue ES2 p61-63
10. Brown R R., Ashley R M., Farrelly M. (2011). Political and Professional Agency Entrapment: An Agenda for Urban Water Research. Water Resources Management. Vol. 23, No.4. European Water Resources Association (EWRA) ISSN 0920-4741.
11. Brugnach M., Dewulf A., Pahl-Wostl C., Taillieu T. (2008) Toward a relational concept of uncertainty: about knowing too little, knowing too differently and accepting not to know. Ecology and Society 13 (2): 30
12. Butler D., Davies J. (2011). Urban Drainage. Spon. 3rd Ed.
13. Cavill S., Sohail M. (2003) Accountability in the provision of urban services. Proc. ICE. Municipal Engineer 156. Issue ME4 paper 13445, p235-244.
14. Charles J A. (2009) Robert Rawlinson and the UK public health revolution. Proc ICE Eng History and Heritage. 162 Nov. Issue EH4. p 199-206
