Q J Med 2004; 97: 243-244
QJM vol. 97 no. 4 (c) Association of Physicians 2004; all rights reserved.
Biologic |
Junk, bricolage or ‘objets de virtue’?
It is an intriguing fact that while the complexity of organisms does not correlate with the number of protein coding genes they possess (Drosophila has less than a nematode, rice has more than Man), the amount of non-coding DNA scales with complexity. Protein coding sequences account for around only 2% of human chromosomal DNA, although a considerable further chunk produces RNA only. These latter ‘genes’ do not have stop and start codons, and vary so much that they cannot be picked up by computer programmes, so quantification is difficult.
As we have seen before, comparisons are helpful in deciding about the significance of genetic information. Computer-driven comparisons of twelve species, including Man, found 1194 segments of the genome that appeared with only minor changes in several species, of which only 244 were within a protein-coding sequence. Roughly two-thirds of these sequences are within introns and the rest are scattered in what has been called ‘junk’. This is a major piece of conservation. I remember thinking some time ago that it was inconceivable that introns were there by mistake (in 1990, at a Pathological Society meeting, I wrote a note about it in the abstract book that I still have—it was finding the note when throwing a lot of things out that provoked me into writing this article).
Pseudogenes (PGs, defective copies of real genes) were thought to be non-functional, as they failed to produce effective protein, but the demonstration that insertion of extra sequences into a pseudogene (makorin1) was lethal in mice demonstrated that the RNA from the PG can control the expression of the ‘true’ copy. In the same way, the assumption that the anti-sense chain of a gene was never expressed is wrong: at least 2000 human genes normally produce anti-sense RNA. This may affect expression by a kind of competitive inhibition, but there is also a true RNA interference machinery. Double-stranded RNA can be separated within cells, and the one fragment may then bind to other messages—a common form of defence against viruses, but also an effective silencing methodology.
MicroRNAs, made up of around 22 non-coding nucleotides, that target the messages of protein-coding genes for cleavage or repression of translation, are found in many plants and animals. Man has around 250 of these genes (say 1% of the protein coding total) and shares half of these with the puffer fish, who ceased to be a close relative around 400 million years ago. There is recent evidence (in the mouse) that microRNAs are critical in the determination of haematopoietic lineages.1 Selective silencing appears to be the mechanism whereby a number of cell lineages become restricted during development, certainly in the central nervous system.
Riboswitches are RNA sequences that act by presenting a non-coding region as a binding site for a target that, when bound, causes the coding sequence to change shape. In this way, the functional protein is produced only when a non-genetic signal operates. The sequences that produce these switches are found in intergenic DNA, and in bacteria encode housekeeping genes concerned in fundamental cellular processes.
And then we come to epigenesis. It seems that the difficulties of interfering with DNA, in terms of therapeutic endeavour, has lead to an increasing awareness of the mechanisms that may allow opportunities for interventions ‘downstream’ of DNA/RNA interactions.
DNA needs to be packed carefully; its high axial ratio makes it a vulnerable protein. It is packed in association with octamers of histone proteins (nucleosomes). These histone proteins, when acetylated, make access to chromatin difficult. The introduction of variant histone proteins into nucleosomes, and adenosine-triphosphate-dependent remodelling of histones by protein complexes, which silence the chromatin, are other methods of transcriptional control.
Of these, imprinting is the example that may be the most familiar to QJM readers. In the most obvious example, the inactivated X, there is hypermethylation and down-regulation of expression of most genes by a regulatory gene, XIST (X-inactive specific transcript). It is interesting how this phenomenon may interact with others to produce surprising results. In Oklahoma (where the wind comes sweeping down the plain), a sheep was born with a very large rump and was called variously ‘Solid Gold’ by farmers and callipyge (ca) by geneticists. Offspring of this mutant were unsurprisingly 50% normal and 50% ca, whether male or female. However normal male/ca female crosses produced all normal sheep, although some had the ca mutation, and normal-appearing ca male/normal ewes produced 50% callipyge rear ends. The gene was thus effective only when inherited from the paternal germ line—it looked like classical imprinting. Rams homozygous for the mutation (ca/ca) produced normal offspring.
Ten years of work by Georges et al.2 unravelled the problem. A single nuclotide mutation (A-G) tens of kilobases from any gene increases the expression of protein in muscle, producing the big rear end, and activates two RNA-only genes. This effect does not override the effects of imprinting. The two active RNA-producing genes are only effective on the maternal chromosomes. In homozygously mutated sheep, the paternal mutation overactivates muscle-producing genes and the A-G mutation in the female increases the output of RNA-producing genes. These block the overproduction, so the animals appear normal.
There is ample scope for errors in imprinting to develop. In Man, most imprinting is removed early in zygotic differentiation, and is then re-established (established in more than 170 genes so far) by mid-gestation. A number of defects in development (Beckwith-Wiederman syndrome, Prader-Willi and Angelman syndromes) are due to failed imprinting, and where there is twin discordance in this type of defect, it is often the case that imprinting had failed in the affected twin.
The large amount of DNA we have acquired form viruses is mostly inactivated by methylation. Randy Jirtle has shown that feeding agouti mice a diet that encouraged methylation changed the coat colour from yellow to brown—though not to full black—as a result of increased methylation of DNA in the agouti transposon. Others have also found that under-methylation promotes genetic instability, and altered methylation is thought to be a major factor in the failure of many cloning experiments—the adult cells used may give the wrong information. Methylation plays a part in Lyonization of the X chromosome; RNA produced by the gene Xist binds to the X to be suppressed, and antisense RNA is produced from the expressed X to prevent this binding. DNA becomes methylated in the repressed X, and histone packing is affected by loss of acetyl groups. The non-expressed chromosome becomes the Barr body, by collapse into a mass of DNA/RNA and imperfectly used packing proteins.
But there are other ways of affecting proteins post-translation. Glycosylation, phosphorylation, ubiquitination are common; glutathonyalation hydroxylation, transglutamination, sulphation and epimerization are comparatively rare. All of these, and the other mechanisms described above, have their part to play in the production of effectively functioning biological systems. It's not all in the genes.
References
1. Chen CZ, Li L, Lodish HF, Bartel DP. MicroRNAs modulate hemopoietic lineage differentiation. Science 2004; 303:83–6.
2. Georges M, Charlier C, Cockette N. The Callipyge Locus; Evidence for the trans interaction of reciprocally imprinted genes. Trends Genet 2003; 19:248–52.[CrossRef][Web of Science][Medline]
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