What are molecular genetics?

Identity and Structure of Genetic Material

Molecular genetics is the branch of genetics that deals with the identity of the molecules of heredity, their structure and organization, how these molecules are copied and transmitted, how the information encrypted in them is decoded, and how the information can change from generation to generation. In the late 1940s and early 1950s, scientists realized that the materials of heredity were nucleic acids. DNA was implicated as the substance extracted from a deadly strain of pneumococcal bacteria that could transform a mild strain into a lethal one and as the substance injected into bacteria by viruses as they start an infection. RNA was shown to be the component of a virus that determined what kind of symptoms of infection appeared on tobacco leaves.

The nucleic acids are made up of nucleotides
linked end to end to produce very long molecules. Each nucleotide has sugar and phosphate parts and a nitrogen-rich part called a base. Four bases are commonly found in each DNA and RNA. Three—adenine (A), guanine (G), and cytosine (C)—are found in both DNA and RNA, while thymine (T) is normally found only in DNA and uracil (U) only in RNA. In the double-helical DNA molecule, two strands are helically intertwined in opposite directions. The nucleotide strands are held together in part by interactions specific to the bases, which “pair” perpendicularly to the sugar-phosphate strands. The structure can be envisioned as a ladder. The A and T bases pair with each other, and G and C bases pair with each other, forming “rungs”; the sugar-phosphates, joined end to end, form the “sides” of the ladder. The entire molecule twists and bends in on itself to form a compact whole. An RNA molecule is essentially “half” of this ladder, split down the middle. RNA molecules generally adopt less regular structures but may also require pairing between bases.

DNA and RNA, in various forms, serve as the molecules of heredity. RNA is the genetic material that some viruses package in viral particles. One or several molecules of RNA may make up the viral information. The genetic material of most bacteria is a single circle of double-helical DNA, the circle consisting of from slightly more than 500,000 to about 5 million nucleotide pairs. In eukaryotes such as humans, the DNA genetic material is organized into multiple linear DNA molecules, each one the essence of a morphologically recognizable and genetically identifiable structure called a chromosome.

In each organism, the DNA is closely associated with proteins. Proteins are made of one or more polypeptides. Polypeptides are linear polymers, like nucleic acids, but the units linked end to end are amino acids rather than nucleotides. More than twenty kinds of amino acids make up polypeptides. Proteins are generally smaller than DNA molecules and assume a variety of shapes. Proteins contribute to the biological characteristics of an organism in many ways: They are major components of structures both inside (membranes and fibers) and outside(hair and nails) the cell; as enzymes, they initiate the thousands of chemical reactions that cells use to get energy and build new cells; and they regulate the activities of cells. Histone proteins pack eukaryotic nuclear DNA into tight bundles called nucleosomes. Further coiling and looping of nucleosomes results in the compact structure of chromosomes. These can be seen with help of a microscope. The complex of DNA and protein is called chromatin.

The term “genome” denotes the roster of genes and other DNA of an organism. Most eukaryotes have more than one genome. The principal genome is the genome of the nucleus that controls most of the activities of cells. Two organelles, the mitochondria (which produce energy by oxidizing chemicals) and the plastids (such as chloroplasts, which convert light to chemical energy in photosynthesis) have their own genomes. The organelle genomes have only some of the genes needed for their functioning. The others are present in the nuclear genome. Nuclear genomes have many copies of some genes. Some repeated sequences are organized tandemly, one after the other, while others are interspersed with unique sequences. Some repeated sequences are genes present in many copies, while others are DNAs of unknown function.

Copying and Transmission of Genetic Nucleic Acids

James Watson and Francis Crick’s double-helical structure for DNA suggested to them how a faithful copy of a DNA could be made. The strands would pull apart. One by one, the new nucleotide units would then arrange themselves by pairing with the correct base on the exposed strands. When zipped together, the new units make a new strand of DNA. The process, called DNA replication, makes two double-helical DNAs from one original one. Each daughter double-helical DNA has one old and one new strand. This kind of replication, called semiconservative replication, was confirmed by an experiment by Matthew Meselson and Franklin Stahl.

Enzymes cannot copy DNA of eukaryotic chromosomes completely to each end of the DNA strands. This is not a problem for bacteria, whose circular genomes do not have ends. To keep the ends from getting shorter with each cycle of replication, eukaryotic chromosomes have special structures called telomeres at their ends that are targets of a special DNA synthesis enzyme.

When a cell divides, each daughter cell must get one and only one complete copy of the mother cell’s DNA. In most bacterial chromosomes, this DNA synthesis starts at only one place, and that starting point is controlled so that the number of starts equals the number of cell fissions. In eukaryotes, DNA synthesis begins at multiple sites, and each site, once it has begun synthesis, does not begin another round until after cell division. When DNA has been completely copied, the chromosomes line up for distribution to the daughter cells. Protein complexes called kinetochores bind to a special region of each chromosome’s DNA called the “centromere.” Kinetochores attach to microtubules, fibers that provide the tracks along which the chromosomes move during their segregation into daughter cells.

Gene Expression, Transcription, and Translation

DNA is often dubbed the blueprint of life. It is more accurate to describe DNA as the computer tape of life’s instructions because the DNA information is a linear, one-dimensional series of units rather than a two-dimensional diagram. In the flow of information from the DNA tape to what is recognized as life, two steps require the decoding of nucleotide sequence information. The first step, the copying of the DNA information into RNA, is called transcription, an analogy to medieval monks sitting in their cells copying, letter by letter, old Latin manuscripts. The letters and words in the new version are the same as in the old but are written with a different hand and thus have a slightly different appearance. The second step, in which amino acids are polymerized in response to the RNA information, is called translation. Here, the monks take the Latin words and find English, German, or French equivalents. The product is not in the nucleotide language but in the language of polypeptide sequences. The RNAs that direct the order of
amino acids are called messenger RNAs (mRNAs) because they bring instructions from the DNA to the ribosome, the site of translation.

Multicellular organisms consist of a variety of cells, each with a particular function. Cells also respond to changes in their environment. The differences among cell types and among cells in different environmental conditions are caused by the synthesis of different proteins. For the most part, regulation of which proteins are synthesized and which are not occurs by controlling the synthesis of the mRNAs for these proteins. Genes can have their transcription switched on or switched off by the binding of protein factors to a segment of the gene that determines whether transcription will start or not. An important part of this gene segment is the promoter. It tells the transcription apparatus to start RNA synthesis only at a particular point in the gene.

Not all RNAs are ready to function the moment their synthesis is over. Many RNA transcripts have alternating exon and intron segments. The intron segments are taken out with splicing of the end of one exon to the beginning of the next. Other transcripts are cut at several specific places so that several functional RNAs arise from one transcript. Eukaryotic mRNAs get poly-A tails (about two hundred nucleotide units in which every base is an A) added after transcription. A few RNAs are edited after transcription, some extensively by adding or removing U nucleotides in the middle of the RNA, others by changing specific bases.

Translation occurs on particles called ribosomes and converts the sequence of nucleotide residues in mRNA into the sequence of amino acid residues in a polypeptide. Since protein is created as a consequence of translation, the process is also called protein synthesis. The mRNA carries the code for the order of insertion of amino acids in three-nucleotide units called codons. Failure of the ribosome to read nucleotides three at a time leads to shifts in the frame of reading the mRNA message. The frame of reading mRNA is set by starting translation only at a special codon.

Transfer RNA (tRNA) molecules actually do the translating. There is at least one tRNA for each of the twenty common amino acids. Anticodon regions of the tRNAs each specifically pair with only a specific subset of mRNA codons. For each amino acid there is at least one enzyme that attaches the amino acid to the correct tRNA. These enzymes are thus at the center of translation, recognizing both amino acid and nucleotide residues.

The ribosomes have sites for binding of mRNA, tRNA, and a variety of protein factors. Ribosomes also catalyze the joining of amino acids to the growing polypeptide chain. The protein factors, usually loosely bound to ribosomes, assist in the proper initiation of polypeptide chains, in the binding of amino acid-bearing tRNA to the ribosome, and in moving the ribosome relative to the mRNA after each additional step. Three steps in translation use biochemical energy: attaching the amino acid to the tRNA, binding the amino acyl tRNA to the ribosome-mRNA complex, and moving the ribosome relative to the mRNA.

Small RNAs

An additional level of control of gene expression is achieved via the presence of two classes of small RNAs, the microRNAs (miRNAs) and the small interfering RNAs (siRNAs). In 1993, Victor Ambrose and his coworkers discovered that in
Caenorhabditis elegans
, lin-4, a small 22-nucleotide noncoding RNA, was able to negatively regulate the translation of lin-14, which is involved in C. elegans larval development. Since then, these small RNAs have been found in plants, green algae, viruses, and animals. These small RNAs function as gene-silencers by binding to target mRNA sequences and preventing their translation or targeting the mRNAs for degradation in a process known as RNA interference (RNAi).

The pathway by which the small RNAs’ are processed has been intensively studied. After transcription and processing in the nucleus, small RNAs’ precursors are exported into the cytoplasm, where they undergo further processing by an enzyme called Dicer, which produces a single-stranded 21-23-nucleotide RNA. This small RNA attaches to an RNA-induced silencing complex (RISC) and is directed to a specific mRNA to which it shares base pair complementarity. In the case of miRNA, slight imperfections in the match between the miRNA and its target lead to a bulge in the duplex, which blocks translation. In contrast, the perfect binding of the siRNA with its target mRNA forms a duplex, which is targeted for degradation by endonucleases.

The discovery of miRNAs and siRNAs has had important scientific and clinical implications. miRNAs have been demonstrated to play a role in several human cancers and infectious diseases. In addition, researchers have been using RNAi both as a possible therapeutic and as a tool in research to manipulate gene expression.

Protein Processing and DNA Mutation

The completed polypeptide chain is processed in one or more ways before it assumes its role as a mature protein. The linear string of amino acid units folds into a complex, three-dimensional structure, sometimes with the help of other proteins. Signals in some proteins’ amino acid sequences direct them to their proper destinations after they leave the ribosomes. Some signals are removable, while others remain part of the protein. Some newly synthesized proteins are called polyproteins because they are snipped at specific sites, giving several proteins from one translation product. Finally, individual amino acid units may get other groups attached to them or be modified in other ways.

The DNA information can be corrupted by reaction with certain chemicals, some of which are naturally occurring while others are present in the environment. Ultraviolet and ionizing radiation can also damage DNA. In addition, the apparatus that replicates DNA will make a mistake at low frequency and insert the wrong nucleotide.

Collectively, these changes in DNA are called DNA damage. When DNA damage goes unrepaired before the next round of copying of the DNA, mutations (inherited changes in nucleotide sequence) result. Mutations may be substitutions, in which one base replaces another. They may also be insertions or deletions of one or more nucleotides. Mutations may be beneficial, neutral, or harmful. They are the targets of the natural selection that drives evolution. Since some mutations are harmful, survival of the species requires that they be kept to a low level.

Systems that repair DNA are thus very important for the accurate transmission of the DNA information tape. Several kinds of systems have evolved to repair damaged DNA before it can be copied. In one, enzymes directly reverse the damage to DNA. In a second, the damaged base is removed, and the nucleotide chain is split to allow its repair by a limited resynthesis. In a third, a protein complex recognizes the DNA damage, which results in incisions in the DNA backbone on both sides of the damage. The segment containing the damage is removed, and the gap is filled by a limited resynthesis. In still another, mismatched base pairs, such as those that result from errors in replication, are recognized, and an incision is made some distance away from the mismatch. The entire stretch from the incision point to past the mismatch is then resynthesized. Finally, the molecular machinery that exchanges DNA segments, the recombination machinery, may be mobilized to repair damage that cannot be handled by the other
systems.

Invasion and Amplification of Genes

Mutation is only one way that genomes change from generation to generation. Another way is via the invasion of an organism’s genome by other genomes or genome segments. Bacteria have evolved restriction
modification systems to protect themselves from such invasions. The gene for restriction encodes an enzyme that cleaves DNA whenever a particular short sequence of nucleotides is present. It does not recognize that sequence when it has been modified with a methyl group on one of its bases. The gene for modification encodes the enzyme that adds the methyl group. Thus the bacterium’s own DNA is protected. However, DNA that enters the cell from outside, such as by phage infection or by direct DNA uptake, is not so protected and will be targeted for degradation by the restriction enzyme. Despite restriction, transfer of genes from one species to another (horizontal, or lateral, gene transfer) has occurred.

As far as is known, restriction modification systems are unique to bacteria. Gene transfer from bacteria to plants occurs naturally in diseases caused by bacteria of the Agrobacterium
genus. As part of the infection process, these bacteria transfer a part of their DNA containing genes, active only in plants, into the plant genome. Studies with fungi and higher plants suggest that eukaryotes cope with gene invasion by inactivating the genes (gene silencing) or their transcripts (cosuppression).

Another way that genomes change is by duplications of gene-sized DNA segments. When the environment is such that the extra copy is advantageous, the cell with the duplication survives better than one without the duplication. Thus genes can be amplified under selective pressure. In some tissues, such as salivary glands of dipteran insects and parts of higher plant embryos, there is replication of large segments of chromosomes without cell division. Monster chromosomes result.

Genomes also change because of movable genetic elements. Inversions of genome segments occur in bacteria and eukaryotes. Other segments can move from one location in the genome to another. Some of these movements appear to be rare, random events. Others serve particular functions and are programmed to occur under certain conditions. One kind of mobile element, the retrotransposon, moves into new locations via an RNA intermediate. The element encodes an enzyme that makes a DNA copy of the element’s RNA transcript. That copy inserts itself into other genome locations. The process is similar to that used by retroviruses to establish infection in cells. Other mobile elements, called transposons or transposable elements, encode a transposase enzyme that inserts the element sequence, or a copy of it, into a new location. When that new location is in or near a gene, normal functioning of that gene is disturbed.

The production of genes for antibodies (an important part of a human’s immune defense system) is a biological function that requires gene rearrangements. Antibody molecules consist of two polypeptides called light and heavy chains. In most cells in the body, the genes for light chains are in two separated segments, and those for heavy chains are in three. During the maturation of cells that make antibodies, the genes are rearranged, bringing these segments together. The joining of segments is not precise. The imprecision contributes to the diversity of possible antibody molecules.

Cells of baker’s or brewer’s yeast (Saccharomyces cerevisiae) have genes specifying their sex, or mating type, in three locations. The information at one location, the expression locus, is the one that determines the mating type of the cell. A copy of this information is in one of the other two sites, while the third has the information specifying the opposite mating type. Yeast cells switch mating types by replacing the information at the expression locus with information from a storage locus. Mating-type switching and antibody gene maturation are only two examples of programmed gene rearrangements known to occur in a variety of organisms.

Genetic Recombination

Recombination occurs when DNA information from one chromosome becomes attached to the DNA of another. When participating chromosomes are equivalent, the recombination is called homologous. Homologous recombination in bacteria mainly serves a repair function for extreme DNA damage. In many eukaryotes, recombination is essential for the segregation of chromosomes into gamete cells during meiosis. Nevertheless, aspects of the process are common between bacteria and eukaryotes. Starting recombination requires a break in at least one strand of the double-helical DNA. In the well-studied yeast cells, a double-strand break is required. Free DNA ends generated by breaks invade the double-helical DNA of the homologous chromosome. Further invasion and DNA synthesis result in a structure in which the chromosomes are linked to one another. This structure, called a half-chiasma, is
recognized and resolved by an enzyme system. Resolution can result in exchange so that one end of one chromosome is linked to the other end of the other chromosome and vice versa. Resolution can also result in restoration of the original linkage. In the latter case, the DNA around the exchange point may be that of the other DNA. This is known as gene conversion.

Impact and Applications

Molecular genetics is at the heart of biotechnology, or genetic engineering. Its fundamental investigation of biological processes has provided tools for biotechnologists. Molecular cloning and gene manipulation in the test tube rely heavily on restriction enzymes, other nucleic-acid-modifying enzymes, and extrachromosomal DNA, all discovered during molecular genetic investigation. The development of nucleic acid hybridization, which allows the identification of specific molecular clones in a pool of others, required an understanding of DNA structure and dynamics. The widely used polymerase chain reaction (PCR), which can amplify minute quantities of DNA, would not have been possible without discoveries in DNA replication. Genetic mapping, a prelude to the isolation of many genes, was sped along by molecular markers detectable with restriction enzymes or the PCR. Transposable elements and the transferred DNA of Agrobacterium, because they often inactivate genes when they insert in them, were used to isolate the genes they inactivate. The inserted elements served as tags or handles by which the modified genes were pulled out of a collection of genes.

The knowledge of the molecular workings of genes gained by curious scientists has allowed other scientists to intervene in many disease situations, provide effective therapies, and improve biological production. Late twentieth century scientists rapidly developed an understanding of the infection process of the acquired immunodeficiency syndrome (AIDS) virus. The understanding, built on the skeleton of existing knowledge, has helped combat this debilitating disease. Molecular genetics has also led to the safe and less expensive production of proteins of industrial, agricultural, and pharmacological importance. The transfer of DNA from Agrobacterium to plants has been exploited in the creation of transgenic plants. These plants offer a new form of pest protection that provides an alternative to objectionable pesticidal sprays and protects against pathogens for which no other protection is available. Recombinant insulin and recombinant growth hormone are routinely given to those whose conditions demand them. Through molecular genetics, doctors have diagnostic kits that can, with greater rapidity, greater specificity, and lower cost,
determine whether a pathogen is present. Finally, molecular genetics has been used to identify genes responsible for many inherited diseases of humankind. Someday medicine may correct some of these diseases by providing a good copy of the gene, a strategy called gene therapy.

Key terms



DNA

:

deoxyribonucleic acid, a long-chain macromolecule, made of units called nucleotides and structured as a double helix joined by weak hydrogen bonds, which forms genetic material for most organisms




genome

:

the assemblage of the genetic information of an organism or of one of its organelles




replication

:

the process by which one DNA molecule is converted to two DNA molecules identical to the first




RNA

:

ribonucleic acid, the macromolecule in the cell that acts as an intermediary between the genetic information stored as DNA and the manifestation of that genetic information as proteins




transcription

:

the process of forming an RNA molecule according to instructions contained in DNA




translation

:

the process of forming proteins according to instructions contained in an RNA molecule





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