Unit – 9A

Molecular Genetics covering

Q1) What is Molecular Genetics covering?

A1)

Molecular genetics is a sub-field of biology that addresses how difference in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the structure and/or function of genes in an organism's genome using genetic screens. The field of study is based on the merging of several sub-fields in biology: classical Mendelian inheritance, cellular biology, molecular biology, biochemistry, and biotechnology Researchers search for mutations in a gene or induce mutations in a gene to link a gene sequence to a specific phenotype. Molecular genetics is a powerful methodology for linking mutations to genetic conditions that may aid the search for treatments/cures for various genetics diseases.

Q2) What is molecular genetics technique?

A2)

Forward genetics

Forward genetic is a molecular genetics technique used to identify genes or genetic mutations that produce a certain phenotype. In a genetic screen, random mutations are generated with mutagens (chemicals or radiation) or transposons and individuals are screened for the specific phenotype. Often, a secondary assay in the form of a selection may follow mutagenesis where the desired phenotype is difficult to observe, for example in bacteria or cell cultures. The cells may be transformed using a gene for antibiotic resistance or a fluorescent reporter so that the mutants with the desired phenotype are selected from the non-mutants.

Reverse genetics

Reverse genetics is the term for molecular genetics techniques used to determine the phenotype resulting from an intentional mutation in a gene of interest. The phenotype is used to deduce the function of the un-mutated version of the gene. Mutations may be random or intentional changes to the gene of interest. Mutations may be a mis-sense mutation caused by nucleotide substitution, a nucleotide addition or deletion to induce a frameshift mutation, or a complete addition/deletion of a gene or gene segment. The deletion of a particular gene creates a gene knockout where the gene is not expressed and a loss of function results (e.g. knockout mice). Mis-sense mutations may cause total loss of function or result in partial loss of function, known as a knockdown.

Q3) Describe tools for RNA structure, functions and research?

A3)

Tools for RNA structure, function, and research

RNA structure is thought to play a central role in many cellular processes, including transcription initiation, elongation and termination, mRNA splicing, and retroviral infection of eukaryotic cells.  Elucidating the mechanistic aspects of these intricate processes will require detailed understanding of the underlying RNA structure. The structure of RNA molecules are typically comprised of single-stranded and double-stranded regions that give rise to complex three-dimensional structures. These structures are involved in the molecule's interactions with other nucleic acids, proteins, and small molecules.  We have developed an extensive portfolio of products for the synthesis and modification of RNA in order to further understand the role of RNA structure.

Enzymes for researching RNA structure and function

RNA-Grade Ribonucleases: A collection of ribonucleases (RNases A and T1) that are optimized for researchers performing RNA structure, RNA sequencing, protein footprinting and boundary experiments.

Poly(A) Polymerase: Catalyzes the addition of adenosine to the 3´ end of RNA in a sequence-independent fashion.

T7 RNA Polymerase: Catalyzes the 5´→3´ synthesis of RNA on either single-stranded DNA or double-stranded DNA downstream from its promoter.

SP6 RNA Polymerase: Catalyzes the 5´→3´ synthesis of RNA on either single-stranded DNA or double-stranded DNA downstream from its promoter and incorporates modified nucleotides.

Anza T4 Polynucleotide Kinase: Transfers the terminal phosphate of ATP to the 5´ hydroxyl terminus of DNA or RNA. Used for 5´ end-labeling of oligonucleotides and polynucleotides.

T4 RNA Ligase: Catalyzes the formation of a phosphodiester linkage between a 5´-phosphoryl-terminated ribonucleic acid and a 3´-hydroxyl-terminated ribonucleic acid.

KinaseMax 5' End Labeling Kit: For end-labeling DNA, RNA, and oligonucleotides using T4 polynucleotide kinase and [γ-32P]ATP.

Terminal Transferase: Catalyzes the addition of deoxynucleotides to the 3´ hydroxyl terminus of DNA.

CIP (Calf Intestinal Phosphatase): Phosphomonoesterase that removes 3´ and 5´ phosphates from DNA and RNA.

Q4) Brief description about Ribosomal RNA?

A4)

Ribosomal RNA is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. Ribosomal RNA is transcribed from ribosomal DNA (rDNA) and then bound to ribosomal proteins to form small and large ribosome subunits. rRNA is the physical and mechanical actor of the ribosome that forces transfer RNA (tRNA) and messenger RNA (mRNA) to process and translate the latter into proteins.Ribosomal RNA is the predominant form of RNA found in most cells; it makes up >80% of cellular RNA despite never being translated into proteins itself.

Ribosomal RNA depletion

Ribosomal depletion is a critical method in transcriptomics that allows for efficient detection of functionally relevant coding as well as non-coding transcripts through removal of highly abundant rRNA species. Use of oligo dT primer to capture the polyadenylated 3′ end of the transcripts and isolate mRNA is routine in many RNA sequencing preparations; however this method lacks the ability to handle degraded samples where most of the RNA is separated from the 3′ tail, or to isolate non-polyadenylated transcripts such as lncRNAs. Ribosomal removal methods address these issues by directly depleting the rRNA while leaving other transcripts intact.

Q5) Describe DNA structure and function in brief?

A5)

DNA is the information molecule. It stores instructions for making other large molecules, called proteins. These instructions are stored inside each of your cells, distributed among 46 long structures called chromosomes. These chromosomes are made up of thousands of shorter segments of DNA, called genes. Each gene stores the directions for making protein fragments, whole proteins, or multiple specific proteins.

DNA is well-suited to perform this biological function because of its molecular structure, and because of the development of a series of high performance enzymes that are fine-tuned to interact with this molecular structure in specific ways. The match between DNA structure and the activities of these enzymes is so effective and well-refined that DNA has become, over evolutionary time, the universal information-storage molecule for all forms of life. Nature has yet to find a better solution than DNA for storing, expressing, and passing along instructions for making proteins.

The molecular structure of DNA

In order to understand the biological function of DNA, you first need to understand its molecular structure. This requires learning the vocabulary for talking about the building blocks of DNA, and how these building blocks are assembled to make DNA molecules.

DNA molecules are polymers

Polymers are large molecules that are built up by repeatedly linking together smaller molecules, called monomers. Think of how a freight train is built by linking lots of individual boxcars together, or how this sentence is built by sticking together a specific sequence of individual letters (plus spaces and punctuation). In all three cases, the large structure—a train, a sentence, a DNA molecule—is composed of smaller structures that are linked together in non-random sequences— boxcars, letters, and, in the biological case, DNA monomers.

Q6) What is Nucleotides and how many nucleotide monomer?

A6)

Just like a sentence “polymer” is composed of letter “monomers,” a DNA polymer is composed of monomers called nucleotides. A molecule of DNA is a bunch of nucleotide monomers, joined one after another into a very long chain.

There are four nucleotide monomers

The English language has a 26 letter alphabet. In contrast, the DNA “alphabet” has only four “letters,” the four nucleotide monomers. They have short and easy to remember names: A, C, T, G. Each nucleotide monomer is built from three simple molecular parts: a sugar, a phosphate group, and a nucleobase. (Don’t confuse this use of “base” with the other one, which refers to a molecule that raises the pH of a solution; they’re two different things.)

The sugar and acid in all four monomers are the same

All four nucleotides (A, T, G and C) are made by sticking a phosphate group and a nucleobase to a sugar. The sugar in all four nucleotides is called deoxyribose. It’s a cyclical molecule—most of its atoms are arranged in a ring-structure. The ring contains one oxygen and four carbons. A fifth carbon atom is attached to the fourth carbon of the ring. Deoxyribose also contains a hydroxyl group (-OH) attached to the third carbon in the ring.

Q7) What is the difference between RNA and DNA?

A7)

Function

DNA encodes all genetic information, and is the blueprint from which all biological life is created. And that’s only in the short-term. In the long-term, DNA is a storage device, a biological flash drive that allows the blueprint of life to be passed between generations. RNA functions as the reader that decodes this flash drive. This reading process is multi-step and there are specialized RNAs for each of these steps. Below, we look in more detail at the three most important types of RNA. 

What are the three types of RNA?

Messenger RNA (mRNA) copies portions of genetic code, a process called transcription, and transports these copies to ribosomes, which are the cellular factories that facilitate the production of proteins from this code.  

Transfer RNA (tRNA) is responsible for bringing amino acids, basic protein building blocks, to these protein factories, in response to the coded instructions introduced by the mRNA. This protein-building process is called translation. 

Finally, Ribosomal RNA (rRNA) is a component of the ribosome factory itself without which protein production would not be occur.

Sugar

Both DNA and RNA are built with a sugar backbone, but whereas the sugar in DNA is called deoxyribose (left in image), the sugar in RNA is called simply ribose (right in image). The ‘deoxy’ prefix denotes that, whilst RNA has two hydroxyl (-OH) groups attached to its carbon backbone, DNA has only one, and has a lone hydrogen atom attached instead. RNA’s extra hydroxyl group proves useful in the process of converting genetic code into mRNAs that can be made into proteins, whilst the deoxyribose sugar gives DNA more stability.

Bases

The nitrogen bases in DNA are the basic units of genetic code, and their correct ordering and pairing is essential to biological function. The four bases that make up this code are adenine (A), thymine (T), guanine (G) and cytosine (C). Bases pair off together in a double helix structure, these pairs being A and T, and C and G.  RNA doesn’t contain thymine bases, replacing them with uracil bases (U), which pair to adenine.

Structure

Whilst the ubiquity of Francis Crick and James Watson’s (or should that be Rosalind Franklin’s) DNA double helix means that the two-stranded structure of DNA structure is common knowledge, RNA’s single stranded format is not as well known. RNA can form into double-stranded structures, such as during translation, when mRNA and tRNA molecules pair. DNA polymers are also much longer than RNA polymers; the 2.3m long human genome consists of 46 chromosomes, each of which is a single, long DNA molecule. RNA molecules, by comparison, are much shorter.

Q8) What is Gene in molecular genetics?

A8)

Gene is a unit of hereditary information that occupies a fixed position (locus) on a chromosome. Genes achieve their effects by directing the synthesis of proteins.

Genes are made up of promoter regions and alternating regions of introns (noncoding sequences) and exons (coding sequences). The production of a functional protein involves the transcription of the gene from DNA into RNA, the removal of introns and splicing together of exons, the translation of the spliced RNA sequences into a chain of amino acids, and the posttranslational modification of the protein molecule.

Eukaryotes (such as animals, plants, and fungi), genes are contained within the cell nucleus. The mitochondria (in animals) and the chloroplasts (in plants) also contain small subsets of genes distinct from the genes found in the nucleus. In prokaryotes (organisms lacking a distinct nucleus, such as bacteria), genes are contained in a single chromosome that is free-floating in the cell cytoplasm. Many bacteria also contain plasmids—extrachromosomal genetic elements with a small number of genes.

The number of genes in an organism’s genome (the entire set of chromosomes) varies significantly between species. For example, whereas the human genome contains an estimated 20,000 to 25,000 genes, the genome of the bacterium Escherichia coli O157:H7 houses precisely 5,416 genes. Arabidopsis thaliana—the first plant for which a complete genomic sequence was recovered—has roughly 25,500 genes; its genome is one of the smallest known to plants. Among extant independently replicating organisms, the bacterium Mycoplasma genitalium has the fewest number of genes, just 517.

Q9) How to define chemical structure of gene and Gene Transcription and Translation?

A9)

Chemical Structure of Genes

Genes are composed of deoxyribonucleic acid (DNA), except in some viruses, which have genes consisting of a closely related compound called ribonucleic acid (RNA). A DNA molecule is composed of two chains of nucleotides that wind about each other to resemble a twisted ladder. The sides of the ladder are made up of sugars and phosphates, and the rungs are formed by bonded pairs of nitrogenous bases. These bases are adenine (A), guanine (G), cytosine (C), and thymine (T). An A on one chain bonds to a T on the other (thus forming an A–T ladder rung); similarly, a C on one chain bonds to a G on the other. If the bonds between the bases are broken, the two chains unwind, and free nucleotides within the cell attach themselves to the exposed bases of the now-separated chains. The free nucleotides line up along each chain according to the base-pairing rule—A bonds to T, C bonds to G. This process results in the creation of two identical DNA molecules from one original and is the method by which hereditary information is passed from one generation of cells to the next.

Gene Transcription and Translation

The sequence of bases along a strand of DNA determines the genetic code. When the product of a particular gene is needed, the portion of the DNA molecule that contains that gene will split. Through the process of transcription, a strand of RNA with bases complementary to those of the gene is created from the free nucleotides in the cell. (RNA has the base uracil [U] instead of thymine, so A and U form base pairs during RNA synthesis.) This single chain of RNA, called messenger RNA (mRNA), then passes to the organelles called ribosomes, where the process of translation, or protein synthesis, takes place. During translation, a second type of RNA, transfer RNA (tRNA), matches up the nucleotides on mRNA with specific amino acids. Each set of three nucleotides codes for one amino acid. The series of amino acids built according to the sequence of nucleotides forms a polypeptide chain; all proteins are made from one or more linked polypeptide chains.

Experiments conducted in the 1940s indicated one gene being responsible for the assembly of one enzyme, or one polypeptide chain. This is known as the one gene–one enzyme hypothesis. However, since this discovery, it has been realized that not all genes encode an enzyme and that some enzymes are made up of several short polypeptides encoded by two or more genes.

Q10) What is Gene Regulation and gene mutation describe in brief?

A10)

Gene Regulation

Many of the genes within the cells of organisms are inactive much or even all of the time. Thus, at any time, in both eukaryotes and prokaryotes, it seems that a gene can be switched on or off. The regulation of genes between eukaryotes and prokaryotes differs in important ways.

The process by which genes are activated and deactivated in bacteria is well characterized. Bacteria have three types of genes: structural, operator, and regulator. Structural genes code for the synthesis of specific polypeptides. Operator genes contain the code necessary to begin the process of transcribing the DNA message of one or more structural genes into mRNA. Thus, structural genes are linked to an operator gene in a functional unit called an operon. Ultimately, the activity of the operon is controlled by a regulator gene, which produces a small protein molecule called a repressor. The repressor binds to the operator gene and prevents it from initiating the synthesis of the protein called for by the operon. The presence or absence of certain repressor molecules determines whether the operon is off or on. As mentioned, this model applies to bacteria.

The genes of eukaryotes, which do not have operons, are regulated independently. The series of events associated with gene expression in higher organisms involves multiple levels of regulation and is often influenced by the presence or absence of molecules called transcription factors. These factors influence the fundamental level of gene control, which is the rate of transcription, and may function as activators or enhancers. Specific transcription factors regulate the production of RNA from genes at certain times and in certain types of cells. Transcription factors often bind to the promoter, or regulatory region, found in the genes of higher organisms. Following transcription, introns (noncoding nucleotide sequences) are excised from the primary transcript through processes known as editing and splicing. The result of these processes is a functional strand of mRNA. For most genes this is a routine step in the production of mRNA, but in some genes there are multiple ways to splice the primary transcript, resulting in different mRNAs, which in turn result in different proteins. Some genes also are controlled at the translational and posttranslational levels.

Gene Mutations

Mutations occur when the number or order of bases in a gene is disrupted. Nucleotides can be deleted, doubled, rearranged, or replaced, each alteration having a particular effect. Mutation generally has little or no effect, but, when it does alter an organism, the change may be lethal or cause disease. A beneficial mutation will rise in frequency within a population until it becomes the norm.

For more information on the influence of genetic mutations in humans and other organisms, see human genetic disease and evolution.