Genes and Genomes

Genomic DNA is located in the cell nucleus of eukaryotes, as well as small amounts in mitochondria and chloroplasts. In prokaryotes, the DNA is held within an irregularly shaped body in the cytoplasm called the nucleoid.The genetic information in a genome is held within genes, and the complete set of this information in an organism is called its genotype. A gene is a unit of heredity and is a region of DNA that influences a particular characteristic in an organism. Genes contain an open reading frame that can be transcribed, as well as regulatory sequences such as promoters and enhancers, which control the transcription of the open reading frame.

In many species, only a small fraction of the total sequence of the genome encodes protein. For example, only about 1.5% of the human genome consists of protein-coding exons, with over 50% of human DNA consisting of non-coding repetitive sequences.The reasons for the presence of so much non-coding DNA in eukaryotic genomes and the extraordinary differences in genome size, or C-value, among species represent a long-standing puzzle known as the "C-value enigma".However, DNA sequences that do not code protein may still encode functional non-coding RNA molecules, which are involved in the regulation of gene expression.

Nucleic acid secondary structure

In biochemistry and structural biology, secondary structure is the general three-dimensional form of local segments of biopolymers such as proteins and nucleic acids (DNA/RNA). It does not, however, describe specific atomic positions in three-dimensional space, which are considered to be tertiary structure. Secondary structure is formally defined by the hydrogen bonds of the biopolymer, as observed in an atomic-resolution structure. In proteins, the secondary structure is defined by patterns of hydrogen bonds between backbone amide and carboxyl groups (sidechain-mainchain and sidechain-sidechain hydrogen bonds are irrelevant), where the DSSP definition of a hydrogen bond is used. In nucleic acids, the secondary structure is defined by the hydrogen bonding between the nitrogenous bases.

For proteins, however, the hydrogen bonding is correlated with other structural features, which has given rise to less formal definitions of secondary structure. For example, residues in protein helices generally adopt backbone dihedral angles in a particular region of the Ramachandran plot; thus, a segment of residues with such dihedral angles is often called a "helix", regardless of whether it has the correct hydrogen bonds. Many other less formal definitions have been proposed, often applying concepts from the differential geometry of curves, such as curvature and torsion. Least formally, structural biologists solving a new atomic-resolution structure will sometimes assign its secondary structure "by eye" and record their assignments in the corresponding PDB file.

The secondary structure of a nucleic acid molecule refers to the basepairing interactions within a single molecule or set of interacting molecules. The secondary structure of biological RNA's can often be uniquely decomposed into stems and loops. Frequently these elements, or combinations of them, can be further classified, for example, tetraloops, pseudoknots and stem-loops. There are many secondary structure elements of functional importance to biological RNA's; some famous examples are the Rho-independent terminator stem-loops and the tRNA cloverleaf.

Alternate DNA structures

DNA exists in many possible conformations that include A-DNA, B-DNA, and Z-DNA forms, although, only B-DNA and Z-DNA have been directly observed in functional organisms. The conformation that DNA adopts depends on the hydration level, DNA sequence, the amount and direction of supercoiling, chemical modifications of the bases, the type and concentration of metal ions, as well as the presence of polyamines in solution.

The first published reports of A-DNA X-ray diffraction patterns— and also B-DNA used analyses based on Patterson transforms that provided only a limited amount of structural information for oriented fibers of DNA. An alternate analysis was then proposed by Wilkins et al., in 1953, for the in vivo B-DNA X-ray diffraction/scattering patterns of highly hydrated DNA fibers in terms of squares of Bessel functions. In the same journal, James D. Watson and Francis Crick presented their molecular modeling analysis of the DNA X-ray diffraction patterns to suggest that the structure was a double-helix.

Although the `B-DNA form' is most common under the conditions found in cells, it is not a well-defined conformation but a family of related DNA conformations that occur at the high hydration levels present in living cells. Their corresponding X-ray diffraction and scattering patterns are characteristic of molecular paracrystals with a significant degree of disorder.

Protein quantification

For genes encoding proteins the expression level can be directly assessed by a number of means with some clear analogies to the techniques for mRNA quantification.

The most commonly used method is to perform a Western blot against the protein of interest - this gives information on the size of the protein in addition to its identity. A sample (often cellular lysate) is separated on a polyacrylamide gel, transferred to a membrane and then probed with an antibody to the protein of interest. The antibody can either be conjugated to a fluorophore or to horseradish peroxidase for imaging and/or quantification. The gel-based nature of this assay makes quantification less accurate but it has the advantage of being able to identify later modifications to the protein, for example proteolysis or ubiquitination, from changes in size.

By replacing the gene with a new version fused a green fluorescent protein (or similar) marker expression may be directly quantified in live cells. This is done by imaging using a fluorescence microscope. It is very difficult to clone a GFP-fused protein into its native location in the genome without affecting expression levels so this method often cannot be used to measure endogenous gene expression. It is, however, widely used to measure the expression of a gene artificially introduced into the cell, for example via an expression vector. It is important to note that by fusing a target protein to a fluorescent reporter the protein's behavior, including its cellular localization and expression level, can be significantly changed.

The enzyme-linked immunosorbent assay works by using antibodies immobilised on a microtiter plate to capture proteins of interest from samples added to the well. Using a detection antibody conjugated to an enzyme or fluorophore the quantity of bound protein can be accurately measured by fluorometric or colourimetric detection. The detection process is very similar to that of a Western blot, but by avoiding the gel steps more accurate quantification can be achieved.

Bioinformatics

Bioinformatics involves the manipulation, searching, and data mining of DNA sequence data. The development of techniques to store and search DNA sequences have led to widely applied advances in computer science, especially string searching algorithms, machine learning and database theory. String searching or matching algorithms, which find an occurrence of a sequence of letters inside a larger sequence of letters, were developed to search for specific sequences of nucleotides. In other applications such as text editors, even simple algorithms for this problem usually suffice, but DNA sequences cause these algorithms to exhibit near-worst-case behaviour due to their small number of distinct characters. The related problem of sequence alignment aims to identify homologous sequences and locate the specific mutations that make them distinct. These techniques, especially multiple sequence alignment, are used in studying phylogenetic relationships and protein function. Data sets representing entire genomes' worth of DNA sequences, such as those produced by the Human Genome Project, are difficult to use without annotations, which label the locations of genes and regulatory elements on each chromosome. Regions of DNA sequence that have the characteristic patterns associated with protein- or RNA-coding genes can be identified by gene finding algorithms, which allow researchers to predict the presence of particular gene products in an organism even before they have been isolated experimentally.