Gene Complexity

Each region of DNA that codes for a single RNA or protein is called a gene, and the entire set of genes in a cell, organelle or virus forms its genome. Cells and organelles may contain more than one copy of their genome. Genomic DNA from nearly all prokaryotic and eukaryotic organisms is also complexed with protein and termed chromosomal DNA. Each gene is located at a particular position along the chromosome, termed the locus, whilst the particular form of the gene is termed the allele. In mammalian DNA, each gene is present in two allelic forms that may be identical (homozygous) or that may vary (heterozygous). There are approximately 27 000 genes present in the human genome, giving rise to a conservatively estimated ~60 000 known transcripts. Not all will be expressed in a given cell at the same time and it is more common for a particular cell type to express only a limited repertoire of genes. The occurrence of different alleles at the same site in the genome is termed polymorphism. In general, the more complex an organism the larger its genome, although this is not always the case, since many higher organisms have non-coding sequences, some of which are repeated numerous times and termed repetitive DNA. In mammalian DNA, repetitive sequences may be divided into low-copy-number and high-copy-number DNA. The latter is composed of repeat sequences that are dispersed throughout the genome and those that are clustered together. The repeat cluster DNA may be defined into so-called classical satellite DNA, mini satellite and microsatellite DNA (Table 1), the last being mainly composed of dinucleotide repeats. These sequences are termed polymorphic, collectively termed polymorphisms, and vary between individuals; they also form the basis of genetic fingerprinting.

Table1. Repetitive satellite sequences found in DNA and their characteristics

Single Nucleotide Polymorphisms (SNPs)

 A further important source of polymorphic diversity known to be present in genomes is termed single nucleotide polymorphisms or SNPs (pronounced snips). SNPs are substitutions of one base at a precise location within the genome. Those that occur in coding regions are termed cSNPs. Estimates indicate that a SNP occurs every once in every 300 bases, which based on the size of the human genome (3 × 10⁹ bases) would give 10 million SNPs. However, results from the 1000 Genomes Project suggest an average of 3.6 million SNPs per individual. Interest in SNPs lies in the fact that these polymorphisms may account for the differences in disease susceptibility, drug metabolism and response to environmental factors between individuals. Indeed, there are now a number of initiatives to identify SNPs and produce genomic SNP maps. One initiative is the international HapMap project, which is the production of a haplotype map of common sources of variations from groups of associated SNPs. This will potentially allow a set of so-called tag SNPs to be identified and potentially provide an association between the haplotype and a disease.

Chromosomes and Karyotypes

 Higher organisms may be identified by using the size and shape of their genetic material at a particular point in the cell division cycle, termed the metaphase. At this point, DNA condenses to form a number of very distinct chromosome structures. Various morphological characteristics of chromosomes may be identified at this stage, including the centromere and the telomere. The array of chromosomes from a given organism may also be stained with dyes such as Giemsa stain and subsequently ana lysed by light microscopy. The complete array of chromosomes in an organism is termed the karyotype. In certain genetic disorders aberrations in the size, shape and number of chromosomes may occur and thus the karyotype may be used as an indicator of the disorder. Perhaps the most well-known example of such a correlation is the Down syndrome where three copies of chromosome 21 (trisomy 21) exist rather than two as in the normal state.

Renaturation Kinetics and Genome Complexity

 When preparations of double-stranded DNA are denatured and allowed to renature, measurement of the rate of renaturation can give valuable information about the complexity of the DNA, i.e. how much information it contains (measured in base pairs). The complexity of a molecule may be much less than its total length if some sequences are repetitive, but complexity will equal total length if all sequences are unique, appearing only once in the genome. In practice, the DNA is first cut randomly into fragments about 1 kb in length, and is then completely denatured by heating above its T m. Renaturation at a temperature about 10 °C below the T m is monitored either by decrease in absorbance at 260 nm (the hypochromic effect), or by passing samples at intervals through a column of hydroxylapatite, which will adsorb only double-stranded DNA, and measuring how much of the sample is bound. The degree of renaturation after a given time will depend on the concentration of double-stranded DNA prior to denaturation ( c 0 ; in nucleotides per unit volume), and t , the duration of the renaturation (in seconds).

For a given c 0 , it should be evident that a preparation of bacteriophage λ DNA (genome size 49 kb) will contain many more copies of the same sequence per unit volume than a preparation of human DNA (haploid genome size 3 × 10⁶ kb), and will therefore renature far more rapidly, since there will be more molecules complementary to each other per unit volume in the case of λ DNA, and therefore more chance of two complementary strands colliding with each other. In order to compare the rates of renaturation of different DNA samples, one usually measures c 0 and the time taken for renaturation to proceed halfway to completion, t _½ , and then multiplies these values together to give a c 0 × t_½ value. The larger this value, the greater the complexity of the DNA; hence λ DNA has a far lower c 0 × t_½ than does human DNA.

In fact, the human genome does not renature in a uniform fashion. If the extent of renaturation is plotted against lg {( c 0 × t_½ )/[( c 0 × t_½ )]} – this is known as a cot curve – it is seen that part of the DNA renatures quite rapidly, whilst the remainder is very slow to renature (Figure 1). This indicates that some sequences have a higher concentration than others; in other words, part of the genome consists of repetitive sequences. These repetitive sequences can be separated from the single-copy DNA by passing the renaturing sample through a hydroxylapatite column early in the renaturation process, at a time which gives a low value of c 0 ×  t_½ . At this stage, only the rapidly renaturing sequences will be double-stranded, and they will therefore be the only ones able to bind to the column.

Fig1. Cot curve of human DNA. DNA was allowed to renature at 60 °C after being completely dissociated by heat. Samples were taken at intervals and passed through a hydroxylapatite column to determine the percentage of double-stranded DNA present. This percentage was plotted against lg {( c 0 ×  t ½ )/[( c 0 ×  t ½ )]}. (Because the argument of a logarithm cannot include units, ( c 0 ×  t ½ ) needs to be divided by its units, denoted as [( c 0 ×  t ½ )].)

The Nature of the Genetic Code

DNA encodes the primary sequence of a protein by utilising sets of three nucleotides, termed a codon or triplet, to encode a particular amino acid. The four bases (A, C, G and T) present in DNA allow a possible 64 triplet combinations; however, since there are only 20 naturally occurring amino acids, more than one codon may encode an amino acid. This phenomenon is termed the degeneracy of the genetic code. With the exception of a limited number of differences found in mitochondrial DNA and one or two other species, the genetic code appears to be universal. In addition to coding for amino acids, particular triplet sequences also indicate the beginning (Start) and the end (Stop) of a particular gene. Only one start codon exists (ATG), which also codes for the amino acid methionine, whereas three dedicated stop codons are available (TAT, TAG and TGA). A sequence flanked by a start and a stop codon containing a number of codons that may be read in-frame to represent a continuous protein sequence is termed an open reading frame (ORF).

Epigenetics and the Nature of DNA

It has been well recognised for some time that DNA as a structure may be chemically modified without the underlying DNA sequence being altered. The most important modification is the addition of a methyl (CH 3) group to the 5′-carbon on cytosine to give methylcytosine (5mC) and results in what some describe as the fifth base in DNA. This is termed DNA methylation and catalysed by DNA methyltransferases. The DNA methyltransferases invariantly modify the cytosine in the pattern CpG (C-phosphate-G) and approximately 75% of CpGs in the human genome are methylated, which equates to around 1.5% of human DNA (termed the epigenome).

The next most common DNA modifications are generated by the Ten-eleven trans location (Tet) enzymes that catalyse the oxidation of 5mC to 5hmC (5-hydroxymethyl cytosine). Further modification of 5hmC is achieved in a stepwise fashion to generate 5fC (5-formylcytosine) and 5caC (5-carboxycytosine), reflecting an active process of demethylation. However, rather than merely adducts produced in the pathway to demethylation, evidence is growing that these additional DNA modifications can also perform specific regulatory functions and global loss of 5hmC is a common hallmark of cancer cells. CpG sites are not evenly distributed in the genome; a background CpG level of 1/100 can be compared to 1/10 nucleotides in regions known as CpG islands. These high CpG density regions occur at 40% of gene promoter regions and methylation of these sites inhibits gene expression. This feature of gene expression control is termed epigenetics and is a complex process which, in addition to DNA methylation, includes the modification of histone proteins and some small RNA molecules involved in gene expression control. Importantly, epigenetics appears to play a role in differentiation, disease states such as cancers (hyper- and hypomethylation) and certain neurological diseases which may lead to a new means of future treatment.

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مواضيع ذات صلة

Molecular Analysis of Nucleic Acid Sequences

Structure of Nucleic Acids

Genomics: Genome-Wide Analysis of Gene Structure and Function

Chromosomal Organization of Genes and Noncoding DNA

RNA Interference Causes Gene Inactivation by Destroying the Corresponding mRNA

Linkage Studies Can Map Disease Genes with a Resolution of About 1 Centimorgan

DNA Polymorphisms Are Used as Markers for Linkage Mapping of Human Mutations

The Two- Hit Theory of Tumor Suppressor Gene Inactivation in Cancer

DNA Microarrays Can Be Used to Evaluate the Expression of Many Genes at One Time

Hybridization Techniques Permit Detection of Specific DNA Fragments and mRNAs

Cloned DNA Molecules Can Be Sequenced Rapidly by Methods Based on PCR

Cellular and Molecular Mechanisms in Development: Gene Regulation by Transcription Factors