Death of the Central Dogma

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"The doctor of the future will give no medicine, but will interest his patients in the care of the human frame, in diet and in the cause and prevention of disease."
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Recommended Reading:

Bland, Jeffrey S., Genetic Nutritioneering, McGraw-Hill, 1999.

Williams, Roger, Biochemical Individuality, McGraw-Hill, 1998.

 

 

 

The Institute of Science in Society

Science Society Sustainability http://www.i-sis.org.uk

General Enquiries sam@i-sis.org.uk Website/Mailing List press-release@i-sis.org.uk ISIS Director m.w.ho@i- sis.org.uk
 

ISIS Press Release 03/09/04

Life after the Central Dogma

The biotech industry was launched on the scientific myth that organisms are hardwired in their genes, a myth thoroughly exploded by scientific findings accumulating since the mid 1970s and especially so since genome sequences have been accumulating (see Living with the Fluid Genome, by Mae-Wan Ho ).

We bring you the latest surprises that tell you why our health and environmental policies based on genetic engineering and genomics are completely misguided; and more importantly, why the new genetics demands a thoroughly ecological approach.

The series will not be circulated all at once; so do look out for the articles.

Death of the Central Dogma

It is amazing how much scientific and religious fundamentalism have in common. The late Francis Crick won the Nobel Prize jointly with James Watson and Maurice Wilkins for working out the structure of DNA; and rather like the new `Potentate' of biology, issued the "Central Dogma" to the faithful, which decreed that genetic information flows linearly from DNA to RNA to protein, and never in reverse. That was just another way of saying that organisms are hardwired in their genetic makeup, and that the environment has little if any influence on the structure and function of the genes.

The Central Dogma goes hand in glove with the other dogma of biology, the neo- Darwinian theory of evolution by natural selection, which says that the genetic material mutate at random, and individuals which happen to have good genes leave more offspring, just as individuals with bad genes are weeded out. The neo-Darwinian theory is beloved of the status quo, because it endows the rich and powerful with a certain mystique, as those who have won the race in the struggle for survival of the fittest, of being in possession of good genes (= good breeding); while the poor and dispossessed have only their bad genes to blame.

Since the mid-1970s, if not before, molecular geneticists studying the genetic material have been turning up evidence that increasingly contradicts the Central Dogma. There is an immense amount of necessary cross talk between genes and the environment in the life of the organism, which not only changes the function of the genes but also the structure of the genes and genomes. By the early 1980s, the new genetics of the "fluid genome" has emerged.

But apart from a few heretics like Barry Commoner and myself, no one dared to say a word against the Central Dogma or the neo-Darwinian theory of evolution.

Things may have changed within the past two years, thanks to the good sense and good management of the public gene sequencing consortium to insist on depositing gene sequences in a single public database, freely available to all researchers.

This database is not much use for business and drug discovery; that much is clear, as one after another `bioinformatics' company that tried to horde the data has gone out of business. But, collected in one freely accessible central database, it is very good for research that exposes the poverty of the genetic determinism ideology that has led to the creation of the database in the first place.

The evidence against the Central Dogma has piled up to such an extent that rumblings of "challenging the dogma" and "a new theory is needed to replace the central dogma" can even be heard in the mainstream scientific journals. Though Dr. Ewan Birney, who gave the Royal Society's inaugural Francis Crick Lecture in December 2003, still paid elaborate homage to the Central Dogma, with arrows pointing strictly one-way from DNA to RNA to protein, leaving out all the many more arrows that point in reverse.

What are the latest surprises that the fluid and flexible genome has in store? One area is the importance and pervasiveness of epigenetics, specifically, chemical markings on the DNA and proteins binding to the DNA in the chromosomes that determine patterns of gene expression, or which bits of the genetic text is actually read. That is overwhelmingly determined by experience. In an earlier issue (SiS 20), we showed the mother's diet and stress can affect patterns of gene expression in the embryo and foetus, which determines the individuals' health prospects much later in life.

Now, researchers are finding genes that are marked for life in rat pups, strictly by how their mothers care for them during their first week of life after birth (see "Caring mothers reduce response to stress for life", this series). It leaves one in no doubt that the environment is giving the instruction of which genes to turn on.

Only a few years ago, people were referring to the 98% or more of the genome that doesn't code for proteins as "junk DNA". Not any more. The genome has a definite `architecture' that holds up beneath the fluidity. There is a high degree of non-randomness in the parts of the genome that undergo change. While some parts are hypermutable, certain families of sequences are `homogenized' to be nearly identical (see "Keeping in concert", this series), while still others are `ultraconservative' in that they have remained absolutely unchanged in hundreds of millions of years of evolution ("Are ultraconserved elements indispensable?" this series). And when cells get into a tight corner metabolically speaking, there may even be genes that mutate to get them out of it ("To mutate or not to mutate", this series).

Most of all, there is a big treasure trove within the apparent junkyard of the genome. Many sequences that don't code for proteins are involved in regulating development and gene expression. Many of the surprises are associated with findings that indicate most of the action is not in proteins, but in the numerous species of RNA `interfering' at all levels of the `readout' of genetic information: with the DNA, with other RNA species, and with proteins (see "RNA subverting the genetic text", this series).

All of this goes against the very grain of the Central Dogma that posits linear, mechanistic control. Instead, layers upon layers of chaotic complexity are coordinated, it seems, by mutual agreement, in an incredibly elaborate, exquisite dance of life that dances itself freely and spontaneously into being.

It is not so much that we need a new theory to replace the central dogma; it is more important than that. We need a new way of knowing and being organisms that will prevent us from mistaking organisms for instruments and machines. That's the real challenge


This article can be found on the I-SIS website at http://www.i-sis.org.uk/DCD.php
 
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The Institute of Science in Society

Science Society Sustainability http://www.i-sis.org.uk

General Enquiries sam@i-sis.org.uk Website/Mailing List press-release@i-sis.org.uk ISIS Director m.w.ho@i- sis.org.uk
 

Life after the Central Dogma

The biotech industry was launched on the scientific myth that organisms are hardwired in their genes, a myth thoroughly exploded by scientific findings accumulating since the mid 1970s and especially so since genome sequences have been accumulating (see Living with the Fluid Genome, by Mae-Wan Ho ).

We bring you the latest surprises that tell you why our health and environmental policies based on genetic engineering and genomics are completely misguided; and more importantly, why the new genetics demands a thoroughly ecological approach.


ISIS Press Release 07/09/04

Caring Mothers Reduce Response to Stress for Life

How a rat responds to stress depends on whether its mother cared for it properly as a pup, which marks its genes for life. Dr. Mae-Wan Ho reports

References for this article are posted on ISIS members' website. Details here.

Maternal effects in the spotlight

Maternal effects on the development of offspring are well known. But they are thought to be due to nutritional and physiological factors affecting the foetus in the womb; and within the past few years, geneticists have discovered that diet and stress can profoundly change the pattern of gene expression in the offspring, affecting their health prospects as adults (see Diet trumping genes, SiS 20).

A team of researchers from the Douglas Hospital Research Centre and McGill University in Montreal Canada, and the Molecular Medicine Centre, in Edinburgh University Western General Hospital in the UK, now report a remarkable experiment in which the behaviour of the mother nursing her pups not only affects the pups' response to stress as adults, but are correlated with changes in gene expression states in brain cells that persist into adult life. Such changes are referred to as `epigenetic' as they do not involve alterations in the base sequence of DNA in the genome, only their off and on states; but they can persist in the brain cells and are passed on to all the daughter cells.

Caring mothers reduces stress response of pups

In the nest, the mother rat licks and grooms her pups, and while nursing, arches her back to groom and lick her pups. Some mothers (high performers) tend to do these more frequently than others (low performers). As adults, the offspring of high performers are less fearful and show more modest responses to stress in the hypothalamus-pituitary-adrenal (HPA) neuro-endocrine pathway.

Cross-fostering studies showed that the biological offspring of low-performers reared by high- performers, resemble the offspring of high performers, and vice versa.

Maternal behaviour, therefore, alters the development of the HPA responses to stress. The magnitude of the HPA response is a function of the corticotropin-releasing factor (CRF) secreted by the hypothalamus, which activates the pituitary-adrenal system. This is modulated by glucocorticoid, which feeds back to inhibit CRF synthesis and secretion, thus dampening the HPA responses to stress. The adult offspring of high-versus low performer mothers show increased glucocorticoid expression the hippocampus, and enhanced sensitivity to glucocorticoid feedback. If this difference is eliminated, so is the difference in HPA responses to stress.

Maternal care and gene expression

Previous studies indicate that the maternal behaviour of licking and grooming and arching her back to do so while nursing increased the expression of glucocorticoid receptor (GR), accompanied by, among other things, an increased expression of a special transcription factor, NGF1-A, which binds to the promoter of the GR gene to increase its transcription and expression. But how could this be transmitted from the neonate to the adult?

The answer is: through the structure of chromatin (complex of protein and DNA in the chromosomes), and the methylation of DNA. DNA methylation is a stable chemical modification of the cytosine in the cytosine- guanine (CpG) dinucleotides, often associated with stable variations in gene transcription. Under-methylation of CpG dinucleotides is associated with active transcription. The researchers decided to look at the methylation state of the GR promoter around the binding region of the NGF1-A transcription factor in the hippocampus of adult offspring from high and low performers.

Sure enough, they found highly significant differences in methylation, with low methylation in offspring from high-performing mothers and high methylation in offspring from low-performing mothers, corresponding to high and low expression respectively of the GR.

Cross-fostering results in methylation patterns associated with the adoptive mother, as consistent with the change in the adult offspring's responses to stress. Moreover, these epigenetic differences due to maternal behaviour during the first week of life persisted into adulthood.

A clean slate at birth

Amazingly, the pups of both high and low-performing mothers start out life genetically the same. Just before birth, the entire region of the GR promoter was unmethylated in both groups; and day one after birth, methylation is found in the region in both groups to the same extent.

The changes in methylation pattern then develops within the first week according to the behaviour of the mother, and thereafter remain for the rest of their lives. This finding is consistent with earlier studies showing that the first week of postnatal life is a `critical period' for the effects of early experiences on hippocampus GR expression.

The hippocampus is the `emotion centre' of the brain, and is believed to be responsible for transferring memory to the rest of the brain. It is vulnerable to stress and richly supplied with receptors for the sex hormones [2, 3].

Additional markings of the gene

Next, the researchers looked at the structure of chromatin around the GR gene, as chromatin structure determines whether a gene is transcribed or not. Chemical modification of the histones (major chromatin protein) by adding an acetyl- group is a well-established marker for `active' chromatin around transcribed genes, which makes it accessible for the transcription enzyme complex. Again, they found highly significant changes in acetylation between the two groups of pups. There was greater acetylation and threefold greater binding of the NGF1-A transcription factor to the GR promoter in the adult offspring of high- compared with low- performing mothers.

Marked for life?

Now, a critical question is, are these gene-marking changes reversible? Is the adult doomed to conditioning by the mother's behaviour towards it as a pup? The general belief is that one is marked for life. DNA methylation pattern is irreversible. However, recent data from in vitro experiments suggests that under certain circumstances, it is possible to demethylate DNA by increasing histone acetylation through a chemical inhibitor of the deacetylating enzyme, trichostatin A (TSA). The researchers, rather crudely, infused the adult brain with TSA by applying the solution into the ventricle (space inside the brain), and obtained more than 3-fold binding of the NGF1-A protein to the GR promoter in the adult offspring of low-performers, and as expected, no change in the adult offspring of high- performers. Simultaneously correlated changes in DNA methylation pattern of the GR promoter was found in the adults reared by low-performing mothers treated with TSA, but not those reared by high-performing mothers. In other words, those epigenetic changes were reversed.

The next question is, are the reversal of epigenetic changes associated with reversal in HPA responses to stress? The answer, incredibly, is yes. The TSA treatment, crude as it was, appeared to significantly decreased plasma corticosterone in the offspring of low-performer in response to stress.

This is all grist to the mill of the fluid and adaptive, adaptable genome [4] that makes nonsense of the Central Dogma.


This article can be found on the I-SIS website at http://www.i- sis.org.uk/MCDIRTS.php
 
If you like this original article from the Institute of Science in Society, and would like to continue receiving articles of this calibre, please consider making a donation or purchase on our website. ISIS is an independent, not-for-profit organisation dedicated to providing critical public information on cutting edge science, and to promoting social accountability and ecological sustainability in science.
 
 
  • If you would prefer to receive future mailings as plain text please let us know.

The Institute of Science in Society, PO Box 32097, London NW1 OXR
telephone: [44 20 8643 0681]   [44 20 7383 3376]   [44 20 7272 5636]

General Enquiries sam@i-sis.org.uk - Website/Mailing List press-release@i-sis.org.uk - ISIS Director m.w.ho@i- sis.org.uk

MATERIAL IN THIS EMAIL MAY BE REPRODUCED IN ANY FORM WITHOUT PERMISSION, ON CONDITION THAT IT IS ACCREDITED ACCORDINGLY AND CONTAINS A LINK TO http://www.i-sis.org.uk/

 
 

The Institute of Science in Society

Science Society Sustainability http://www.i-sis.org.uk

General Enquiries sam@i-sis.org.uk Website/Mailing List press-release@i-sis.org.uk ISIS Director m.w.ho@i- sis.org.uk
 

Life after the Central Dogma

The biotech industry was launched on the scientific myth that organisms are hardwired in their genes, a myth thoroughly exploded by scientific findings accumulating since the mid 1970s and especially so since genome sequences have been accumulating (see Living with the Fluid Genome, by Mae-Wan Ho ).

We bring you the latest surprises that tell you why our health and environmental policies based on genetic engineering and genomics are completely misguided; and more importantly, why the new genetics demands a thoroughly ecological approach.


ISIS Press Release 09/09/04

Subverting the Genetic Text

Dr. Mae-Wan Ho exposes the hidden intrigues in the vast RNA underworld where layers of interference and machinations subvert the chain of command from DNA to RNA to protein.

The references and diagram in Figure 1 is posted on ISIS members' website. Details here.

Updating and re-interpreting the sacred text

According to the Central Dogma, DNA, the genetic text, is read out into RNA and RNA is translated into protein. RNA is rather like the scribe copying and translating the sacred text to direct the faithful.

But geneticists are now uncovering a vast underworld of heresy to the Central Dogma where RNA agents not only decide which bits of text to copy, which copies get destroyed, which bits to delete and splice together, which copies to be transformed into a totally different message and finally, which resulting message - that may bear little resemblance to the original text - gets translated into protein. RNAs even get to decide which parts of the sacred text to rewrite or corrupt.

The whole RNA underworld also resembles an enormous espionage network in which genetic information is stolen, or gets re-routed as it is transmitted, or transformed, corrupted, destroyed, and in some cases, returned to the source file in a totally different form.

And this underworld is big, really big. The protein- coding sequence is only about 1.5% of the human genome. Yet, around 97 - 98% of the transcriptional readout of the human genome is non-protein-coding RNA. This estimate is based on the fact that intronic RNA makes up 95% of the primary protein-coding transcripts on average, and there are large numbers of non-coding RNA transcripts which may represent at least half of all transcripts. Most of the miRNAs (microRNA, see below), for example, are derived from (intergenic) regions between genes; and almost half of all transcripts from the mouse genome are non-coding RNAs. A similar estimate applies to the human genome [1].

The inescapable conclusion is that the job of mediating between DNA and protein is really the centre stage of molecular life. And who gives orders to the multitudes of RNA agents? In a sense it is everyone and no one, because the system works by perfect intercommunication. It is not the DNA, but rather, the particular environment in which the RNA agents find themselves.

For the organism (organization) to survive, it needs to turnover the DNA text continuously, adapting to the realities of its environment. In the process, it keeps certain texts invariant (see "Are ultra- conserved elements indispensable?" this series), while changing others rapidly in non-random ways (see "To mutate or not to mutate", this series). It also needs to keep referring to texts that are relevant, modifying it, or updating the interpretation in keeping with the times (see "Keeping in concert" this series).

RNA interference

RNA interference (RNAi) was first discovered in the nematode worm, C. elegans in the 1990s. Researchers noticed that injecting either sense RNA (the sequence that gets read and translated into protein) or antisense RNA (the complementary sequence, which does not code for protein) into the worm led to specific silencing of the gene involved. It was later found that the phenomenon was actually caused by double-stranded RNA (dsRNA) contaminating the sense or antisense RNA. RNAi now refers to all gene-silencing induced by dsRNA.

These include a host of other phenomena discovered at around the same time [2, 3]. For example, a gene could be silenced, or `co- suppressed', simply by introducing an extra copy into the genome as a transgene, and transgenes themselves may be silenced either at or after transcription. The coat protein gene of a virus transferred into a plant may protect the plant from the virus, by silencing the virus' genes.

All these phenomena are interlinked through special pathways of RNA processing that are only just being defined (see Fig. 1). Abnormal single stranded RNA (ssRNA) is turned into a double stranded RNA (dsRNA) by an RNA-dependent RNA polymerase enzyme (RDRP). The dsRNA is then chopped up into small pieces or microRNA (miRNA) by the enzyme Dicer. The same enzyme also processes certain hairpin RNA (hpRNA) and related pre-microRNA (pre-miRNA) into miRNA. The miRNA is further processed into single-stranded RNA that's incorporated into a multiprotein complex called RNA-induced silencing complex (RISC). At this point, the single stranded RNA fragment binds to complementary part of the messenger RNA and either causes the breakdown of the mRNA or prevents its translation into protein.

Remember that all this depends on complementary base pairing, just as in DNA, so these mechanisms could potentially exist for each and every one of the now estimated 24 500 genes in the genome.

 

Figure 1. RNA interference pathways

It turns out that dsRNA is not only involved in signalling the breakdown or inactivation of specific mRNA to prevent the expression of the protein coded, it is also involved in triggering anti- viral response in mammals. And this is a major obstacle to achieving RNAi in mammals, which might be useful in silencing specific genes in gene therapy.

Double- stranded RNAs longer than 30 nt (nucleotide) activate an antiviral response that includes the production of interferon, resulting in the non-specific breakdown of RNA transcripts and a general shutdown of protein synthesis. In order to overcome this obstacle, synthetic 21nt miRNAs have been used. These are long enough to induce gene-specific suppression and short enough to evade host interferon response. However, recent work has shown that under certain conditions, even such small miRNAs can activate the interferon system. One activating signal for the interferon response appears to be the triphosphate group at the 5' end of the miRNA synthesized by a phage polymerase [4]. In addition, there are other problems, such as avoiding interfering with non-target sequences [5], especially as perfect base-pairing is not required, and matches of as few as 11 consecutive nucleotides can give non-target effects.

RNA-directed DNA read-out

The dsRNA involved in RNA interference can selectively silence genes at the read-out or transcription stage [6]; dsRNA species homologous to promoters are involved in crippling the promoter by methylation (adding methyl (-CH3) groups) in the region of sequence overlap, so no transcription can occur. In other cases, a dsRNA resulting from a bi-directional transcription of a repeat element leads to methylation of a nearby histone protein H3 in chromatin, which, too, results in gene silencing.

Transcriptional gene silencing can potentially be initiated by the dsRNA formed from pairs of transcriptional units arranged in a tail-to tail orientation (sense antisense transcription units, SATs). In humans, SATs account for most overlapping transcriptional units (70%). A recent survey estimated that there are 1 600 human SATs (or 3 200 transcription units). When both transcriptional units are active, formation of dsRNA occurs by default, leading to modification of the histone protein and gene silencing. This mechanism is involved in imprinting: the marking of genes in chromosomes to determine whether they are expressed in cell clones. Expression of the gene only occurs when the antisense promoter is methylated and inactive.

Recently, a new kind of trans-acting (acting across to different parts of the genome) RNA was identified in mouse [7]. B2 RNA originates from a short interspersed repetitive element (SINE) repeated more than 105 copies in the genome of multicellular plants and animals. They were previously thought to be molecular parasites with no function. However, the level of B2 and related RNAs have been found to increase up to 100-fold in response to environmental stresses such as heat shock. And B2 RNA is required for the concomitant inhibition of RNA polymerase II during heat shock, by interacting directly with the enzyme, preventing it from working. RNA polymerase II is involved in the transcription of all protein-coding RNA. So an inhibition of RNA polymerase II will decrease the synthesis of many proteins.

A special kind of RNA directed DNA read-out is accomplished via RNA `riboswitches' to switch genes off in response to the concentration of a metabolite in the cell, without the need for a protein repressor (see Box).

Riboswitch and other RNA regulators

A new molecular switch involves an RNA molecule with enzyme activity, a ribozyme, which can self-destruct by self-cleavage [8]. This self-cleavage is accelerated 1 000 fold in the presence of a small sugar molecule, glucosamine-6-phosphate, which is generated by the enzyme protein encoded by a portion of the mRNA downstream from the ribozyme sequence.

So, this simple gene regulatory circuit involves the mRNA being translated into the enzyme, which makes the product, glucosamine-6-phospate. As the product accumulates, it binds to the special catalytic element in the mRNA, causing it to self-destruct. The region of the mRNA that can confer this regulatory activity is roughly 75 nucleotides long. When placed upstream of an un-related reporter gene, it also shuts down its expression, showing that this active RNA element is transplantable.

A particular group of ribozymes forms a pocket that binds guanosine monophosate, one of the four building blocks of RNA. A specific region of the RNA from the Human Immunodeficiency Virus (HIV) binds a derivative of the amino acid arginine. Short (<100 nucleotide) RNA aptamers (DNA or RNA molecules that bind other molecules) have been identified that specifically bind everything, from hydrophobic (water-hating) amino acids to small organic molecules and metal ions. An RNA aptamer can even distinguish the plant alkaloid theophylline from the closely related molecule caffeine.

Aptamers found within some natural mRNAs bind small molecules as part of their gene-regulatory feedback circuits. In the E. coli bacterium, coenzyme B12 binds directly to, and thereby represses translation of, the mRNA coding for the protein that transports its precursor, cobalamin. In Bacillus species, the synthesis of thiamine and riboflavin involves discrete genetic units or operons, controlled by direct binding of thiamine pyrophospate and flavin mononucleotide to leader sequences of the corresponding mRNAs, resulting in the premature termination of transcription.

Several research groups had previously engineered artificial riboswitches that accomplish exactly the same task, that is, induce ribozyme-mediated cleavage of the RNA on binding small molecules, before these were discovered in nature.

RNA splicing

It is estimated that 64% of the genes in the human genome is interrupted [9]; i.e., the coding regions exist in short stretches (exons) interrupted by long non-coding stretches (introns). After the entire sequence is transcribed into RNA, the non-coding stretches are spliced out, leaving the coding sequence. However, different exons can be spliced together, and the borders between the exons and introns can themselves be shifted. Alternative splicing multiplies the number of different proteins that can be obtained from a single gene. This is a case of extensive cutting and pasting of the genetic text to suit the occasion.

The fruitfly gene Dscam (homologue of the Down syndrome cell adhesion molecule) codes for a cell-surface protein essential for the development of the fruitfly's brain. It has so many exons that a total of 38 016 possible alternative splice forms could be generated. Geneticists from the Whitehead Institute for Biomedical Research, Cambridge, Massachusetts in the United States analysed the splice forms expressed by different cell types and by individual cells, and found that the choice of splice variants is regulated both spatially and temporally [10].

Different subtypes of photoreceptor cells express broad yet distinctive spectra of Dscam splice forms. Individual photoreceptor cells express about 14-50 splice forms chosen from the spectrum of thousands distinctive of its cell type. Thus, the repertoire of each cell is different from those of its neighbours.

The complexity does not end there. Not only are different splice variants obtained from the same primary transcript, trans-splicing between different primary transcripts can also take place [11], multiplying the combinatorial possibilities of proteins available.

There's increasing evidence that genomic variants in both coding and non-coding sequences in genes can have unexpected deleterious effects on the splicing of gene transcripts [12]. Even synonymous base substitutions (those that do not change the amino acid sequence of the encoded protein) and sequence changes within the introns can affect splicing and cause diseases.

RNA-directed rewriting of RNA

Some nucleotides are deleted during splicing and others changed by editing. Around 41 to 60% of mouse multi-exon genes generate alternatively spliced transcripts, the frequency of edited transcripts is unknown. These processes generate new sequences not found in the gene. Trypanosomes show the importance of RNA rewriting. Their survival depends on editing defective mitochondrial transcripts using trans- encoded RNA sequences to guide insertion and deletion of uridine bases. The rewriting of RNA restores the correct reading frame, allowing the production of functional gene products. RNA guides are also used to direct rewriting of RNA during editing and splicing of pre-mRNA. In some cases, editing creates splice sites and in others splicing prevents editing.

Rewriting of RNA is associated with a high turnover of transcripts. Of all the RNA transcribed in the human nucleus, only about 5% enters the cytoplasm Quality control mechanisms dispose of incompletely or improperly processes messages encoding flawed proteins.

RNA- directed rewriting of DNA

Genomes can be rewritten using reverse transcription to record elements of successful `ribotypes' (combination of RNAs). Around 45% of the human genome is derived from retrotransposition. RNA-directed rewriting of DNA also has an essential role in maintaining genome stability. Telomerase is a reverse transcriptase that uses an RNA guide to rewrite the ends of chromosomes (telomeres) and prevent their loss, which is important for maintaining the stability of the genome..

Coordination of information

In each ribotype, only specific transcripts are produced and particular mRNAs translated. These outcomes are achieved by `coRNAs' that coordinate the action of highly conserved pathways. An RNA product from one processing event may regulate a downstream event, making the second outcome contingent on the first. For example, a miRNA encoded in an intron would only be expressed when the host gene is transcribed. CoRNA may facilitate coordination of pathways by interacting with sequence motifs shared by a number of targets.

Evolution of rule sets requires creation of new coRNAs, possibly by duplication and mutation. New coRNAS would result in assembly of new regulatory complexes on conserved DNA elements, new patterns of gene expression during development.

Replication of ribotypes

Both genetic modification, involving changes in DNA, and epigenetic modifications, such as DNA methylation and histone acetylation, can be inherited. For example, imprinting is determined by the parent of origin of a chromosome, which means that at some point maternal and paternal chromosomes are marked so that they can be distinguished during embryonic development. Methylation may undergo variable erasure during primordial germ cell development, producing epigenetic mosaic individuals. The persistence of such epigenetic marks is relevant to the origin of complex diseases. Here, the susceptibility of offspring to disease can depend on whether there is maternal or paternal history of disease as well as ethnicity.

Transmission of ribotypes also occurs more directly. The embryo receives RNA from the mother that is important in specifying cells fate. The foetus is also exposed to the maternal environment, which can influence the foetal phenotype. For example, pregnant female mice fed a diet rich in methyl donors have litters with fewer yellow-coloured agouti Avy offspring, reflecting enhanced silencing of the retroviral promoter in this allele (see "Diet trumping genes", SiS 20). In other cases, integration of signals received from maternal hormones may trigger epigenetic modifications that alter long-term phenotypic development by modulating RNA co- regulatory networks. Low birth weight, for example, has been shown to correlate with lifetime risk of cardiovascular disease and diabetes mellitus.

Recently, it has been demonstrated that the plasma of pregnant women contains circulating mRNA originating from the foetus [13], which is rapidly cleared after delivery. This raises the question of whether coRNAs secreted by various somatic tissues are also used to transmit information from mother to foetus, a serious case of the inheritance of acquired characteristics not coded in the genome.


This article can be found on the I-SIS website at http://www.i- sis.org.uk/RNASTGT.php
 
If you like this original article from the Institute of Science in Society, and would like to continue receiving articles of this calibre, please consider making a donation or purchase on our website. ISIS is an independent, not-for-profit organisation dedicated to providing critical public information on cutting edge science, and to promoting social accountability and ecological sustainability in science.
 
 
  • If you would prefer to receive future mailings as plain text please let us know.

The Institute of Science in Society, PO Box 32097, London NW1 OXR
telephone: [44 20 8643 0681]   [44 20 7383 3376]   [44 20 7272 5636]

General Enquiries sam@i-sis.org.uk - Website/Mailing List press-release@i-sis.org.uk - ISIS Director m.w.ho@i- sis.org.uk

MATERIAL IN THIS EMAIL MAY BE REPRODUCED IN ANY FORM WITHOUT PERMISSION, ON CONDITION THAT IT IS ACCREDITED ACCORDINGLY AND CONTAINS A LINK TO http://www.i-sis.org.uk/

 
 

The Institute of Science in Society

Science Society Sustainability http://www.i-sis.org.uk

General Enquiries sam@i-sis.org.uk Website/Mailing List press-release@i-sis.org.uk ISIS Director m.w.ho@i- sis.org.uk
 

Life after the Central Dogma

The biotech industry was launched on the scientific myth that organisms are hardwired in their genes, a myth thoroughly exploded by scientific findings accumulating since the mid 1970s and especially so since genome sequences have been accumulating (see Living with the Fluid Genome, by Mae-Wan Ho ).

We bring you the latest surprises that tell you why our health and environmental policies based on genetic engineering and genomics are completely misguided; and more importantly, why the new genetics demands a thoroughly ecological approach.


ISIS Press Release 15/09/04

To Mutate or Not to Mutate

Contrary to views widely held not so long ago, genes do not as a rule mutate at random, and cells may choose what, or at least, when to mutate. Dr. Mae-Wan Ho reports

A fully referenced version of this paper is posted on ISIS members' website. Details here.

Non-random `adaptive' mutations?

The backbone of modern genetics and the neo-Darwinian theory of evolution by natural selection is that gene mutations occur at random, independently of the environment in which the organisms find themselves. Those mutations that happen to be `adaptive' to the environment are `selected', while those that are deleterious are weeded out.

The idea that genes do not mutate at random, but `adaptively', as though `directed' by the environment in which the organisms find themselves, is so heretical that most biologists simply dismiss it out of hand; or try their utmost to explain away the observations that give life to the idea.

Microbiologist Max Delbrück first used the term `adaptive mutations' in1946 to refer to mutations formed in response to an environment in which the mutations are selected. The term was adopted more than 40 years later by a research team investigating gene amplification in rat cells. They distinguished between mutations that pre-exist at the time a cell is exposed to a selective environment from those `adaptive' mutations formed after exposure to the environment.

Other workers have followed the same definition. These `adaptive' mutations arise in non-growing or slowly growing cells after the cells were exposed to conditions that favour the mutants, preferentially, though not exclusively, in those genes that could allow growth if mutated. Unselected mutations also accumulated in most studies, to varying degrees, so the mutations are not strictly `directed'. Instead, the cells appear to activate a number of different mechanisms that target mutations to genes, the end result of which is to enable them to grow, which they otherwise would not be able to do.

The archetypal experiment

John Cairns and Patricia Foster created an E. coli strain defective in the lac gene that leaves the cells unable to grow on lactose. They plated out the bacteria on a minimal medium with lactose, and looked for mutants that revert back to normal. As the cells used up the small amount of nutrient they stopped growing. But after some time, mutants began to appear that could grow on lactose. However, the mutations are not strictly directed to the gene in which mutations could be advantageous, as unselected mutations also accumulated. In fact, the mechanisms look like "inducible genetic chaos" according to a reviewer.

The defective lac gene in the E. coli strain was in fact a frameshift mutant, in which a small deletion or addition of a nucleotide shifted the whole reading frame of the gene, so it became translated into a totally different enzyme that has little or no ability to break down lactose. This defective lac gene was carried in an F' plasmid involved in bacterial conjugation. Two types of adaptive genetic change are now known to occur in the lac frameshift system: point mutations involving changes in base sequence of the DNA, and gene amplification involving the generation of multiple copies of the defective gene so that large amounts of defective enzyme can still function to metabolise enough lactose to allow the cells to grow.

The point mutation mechanisms are highly diverse, and includes DNA breakage, recombination break repair, genome-wide hypermutation in a subpopulation of cells that give rise to some or all of the adaptive mutants, a special inducible mutation-generating DNA polymerase (polIV or DinB) that has homologues in all three domains of life. There are now many bacterial and yeast assay systems in which adaptive and stationary-phase mutations have been reported, but the mechanisms are largely unknown.

Some of the mechanisms that underlie adaptive genetic change bear similarities to genetic instability in yeast and in some cancers and to somatic hypermutation in the immune system. They might also be important in bacterial evolution to antibiotic resistance, and the evolution of phase-variable pathogens, which evade the host immune system by frequent variation of their surface components.

In the experiment, Lac+ mutants that existed before exposure to the lactose plates form visible colonies by about two days. The colonies that emerged after 2 days fall into two classes. Most of the Lac+ colonies (~160 /108 cells at 10 days) are adaptive point mutants, which occur by a recombination dependent mechanism and produce compensatory frameshift mutations. On later days (from ~4), an increasing fraction (up to ~35 out of a total of ~160 on day 10) of the colonies are not point mutants but amplifications (20-50 direct repeats) of a 7-40kb region of DNA that contains the lac frameshift gene, which provides sufficient gene activity to allow growth on lactose medium. The number of E. coli cells does not increase during the first five days.

A profusion of mechanisms

There are many ways to generate adaptive mutations.

Interestingly, adaptive point mutations in the lac system requires homologous recombination proteins of the E. coli RecBCD double-strand break-repair system which is widely involved in gene conversion and recombination (see "How to keep in concert", this series). Double-strand ends could be generated during DNA replication by a number of different mechanisms.

The adaptive Lac+ point mutations that revert a framewhift allele are nearly all -1 deletions (deletion of a single nucleotide) in small mononucleotide repeats, whereas the pre-existing (non- adaptive) Lac+ reversions are heterogeneous. Mononcleotide repeat instability is thought to reflect DNA polymerase errors, which is consistent with the requirement of a special error-prone DNA polymerase (polIV) for adaptive mutations.

The `SOS response' is the bacteria's response to DNA damage or the inhibition of DNA replication. It involves de-repression of at least 42 genes that carry out DNA repair, recombination, mutation, translesion DNA synthesis (synthesis across non-repaired or damaged DNA) and prevent cell division.

Global hypermutation is thought to occur in a subpopulation of the cells. This is because the frequencies of unselected mutations are about two orders of magnitude higher among Lac+ mutants than in the main population of Lac- starved cells. These results mean that stationary-phase mutations in this system are not directed exclusively to the lac gene, and both adaptive and neutral mutations are formed. Some or all of the adaptive mutants arise in a subpopulation that is hypermutable relative to the main population.

The subpopulation of cells that are transiently mutable is estimated to be between 10-3 and 10-4 of all cells. Despite that, the frequency per unit length of DNA in the genome is markedly uneven, with definite hotspots and coldspots, perhaps depending on the proximity to double strand breaks (DSBs) in DNA that are generated.

Gene amplification is `adaptive' in the sense that it only occurs in response to the selective environment. Cells carrying the amplification are not hypermutated in unselected genes, and neither the SOS response nor polIV is required. Dependence on homologous recombination is implied in that adaptive Lac+ colonies do not appear in the absence of RecA and RecBCD enzyme, and RuvAB and C recombination proteins.

Similar findings in bacteria isolated from the wild

Until 2003, the phenomenon of adaptive mutations has been observed only in laboratory strains. But researchers from the University of Paris, France, and the National University of Mexico (UNAM) reported similar stress-inducible mutagenesis in stationary-phase bacterial colonies grown from strains culled from the wild. This provides evidence that most natural isolates of E. coli from diverse habitats worldwide increase their mutation rates in response to the stress of starvation.

A total of 787 E. coli isolates were collected from habits including air, water and sediments, and the guts of a variety of host organisms. Colonies formed during the exponential growth phase were subjected to starvation during a prolonged stationary phase, and the production of mutants was monitored in the starved aging colonies. The vast majority of colonies showed an increased number of mutants. In a sample of colonies, the authors were able to link the increased mutagensis to starvation and oxidative stress by showing that either additional sugar or anaerobic incubation could block the increased mutagenesis.

The bacteria were highly variable in their inducible mutator activity. The frequency of mutations conferring resistance to rifampicin (RifR) in day 1 (D1) and day 7 (D7) was measured. For all strains, the median values of RifR mutations were 5.8 x 10-9 on day 1, and 4.03 x 10-8 on day 7, an increase of 7 fold, while the median number of colony-forming units increased 1.2-fold. In comparison, the E. coli K12 MG1655 lab strain showed a 5.5-fold increase in frequency of RifR and a 1.7 fold increase in colony forming units. Constitutive mutator strains having a D1 mutation frequencies >10-fold or >100-fold higher than the median D1 frequency of all the strains represented 3.3% and 1.4% of isolates respectively. The D7/D1 mutation frequency ratio showed that 45% of strains had more than a 10-fold, and 13% more than a 100-fold increase in mutagenesis over 7 days. Interestingly, constitutive mutagenesis and MAC (mutagenesis in aging cells) showed a negative correlation.

The MAC was genome wide in a large fraction of natural isolates. There was no significant correlation between MAC and phylogeny. The host's nutrition might explain some of the variation of MAC. For example, bacteria from the guts of omnivorous species like human beings have weaker stress-inducible mutator activities than those from carnivores.

The mechanisms for generating mutations looked even more diverse than in the laboratory strains.

Wider significance of adaptive mutations

Amplification is an important manifestation of chromosomal instability prevalent in many human cancers, and DSBs in DNA are also involved. Induction of mammalian amplification by selective agents is correlated with the ability of those agents to produce chromosomal breaks.

The adaptive point mutation mechanism at lac might be relevant to microbial evolution, particularly of pathogenic bacteria. Many phase variable pathogens have simple repeated sequences that flank genes that they regulate by frameshift mutation.

These `contingency genes' used under stress provide phase variations that allow evasions of the immune system. Two of them, Neisseria meningitides and N. gonorrhoeae, have one or more genes homologous to dinB. For many pathogenic bacteria, antibiotic resistance is also achieved by point mutation mechanisms and could be induced adaptively. Even antibiotics that cause lethality can be merely bacteriostatic at lower concentrations, such that stress-promoted mutation mechanisms might be significant in the development of resistance in clinical environments.

In multicellular eukarytoes, parallels between adaptive mutation and cancer have been noted, the key being that acquisition of mutations in growth-limited state (stress) allows cells to proliferate.

Humans have three E.coli polIV homologues of unknown function, in the DinB/UmuDC/Rad30/Rev1 superfamily of DNA polymerises, as well as a homologue known to carry out translesion synthesis (the tumour suppressor protein XP-V). DinB1 or polk, a true DinB orthologue, is found in germline and lymphoid cells. More and more geneticists now think that mutation is regulated, or at any rate, provoked, and highly non-random.

Indeed, in one study on 12 long-term E coli lines, 36 genes were chosen at random, and 500 bp regions sequenced in four clones from each line and their ancestors. Several mutations were found in a few lines that evolved mutator phenotypes, but no mutations were found in any of the 8 lines that retained functional DNA repair throughout the 20 000 generations experiment. This confirms the low level of `spontaneous' or unprovoked mutation.


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Please be aware that my email address is changing to alison@helloworld.com This is my new video email account and I will be following this newsletter with video email hello for those of you who have enjoyed these letters for years, but not know the face behind these health tip newsletters.
 
Food Has Always Been and Will Always Be Your Best Medicine!
 
A friend brought to my attention last week that the featured article in Newsweek magazine of January 17th is titled "Diet and Genes." I just finished reading this article today and I advise that you buy the magazine and read the article. It supports scientifically the fact that "health is determined by the interplay of nutrients and genes" (p.42).

This is not new information. It has been around and I was aware of it a few years ago when I read a book called Genetic Nutritioneering, by Jeffrey S. Bland. It is very exciting for me to start seeing this information coming out in popular magazines.

Just like this week's article in Newsweek, Jeffrey Bland's book synthesizes the amazing scientific information that describes how diet, lifestyle and environment influence genetic expression and health as we age. Scientists involved with the Human Genome Project and affiliated research are learning that such age-related diseases as heart disease, adult-onset diabetes, arthritis, digestive disorders, loss of mental acuity and certain forms of cancer are not inevitable consequences of aging. They develop because of a poor match between the genetic needs of the individual and the choices he or she makes regarding overall diet, specific nutrient intake, lifestyle and environment.

It is true that genetic inheritance does play an important role in defining our risks to most age-related diseases, but healthy aging is more controlled by how we communicate with our genes through our diet and lifestyle. Through the Human Genome Project, we now know that if your uncles and your grandfather all died prematurely of heart disease or cancer, this does not mean that you will suffer the same fate. We know that our genes do not in and of themselves give rise to disease. Rather, in most cases, disease results when the individual elects a lifestyle or diet that alters the expression of the genes in such a way that the weakness or uniqueness of inheritance factors results in a disease.

In terms of your health or disease state as an adult, your health is determined by the way you have treated your genes throughout your life. What you have eaten or drank, inhaled, surrounded yourself with in your environment, endured as stresses, participated in as activities or suffered as injury, infection or inflammation-all of these factors alter the expression of your genes and contribute in a major way to your state of health or disease.

So, if you chose to read the Newsweek article or Jeffrey Bland's book, you will come up with the same conclusion as I did, we are our own best doctors. We simply need to eat quality foods (organic and raw whole foods), and get some additional help from wild and organic whole food supplements. A simple and logical advice for healthy aging is and will always be that "Food is our best medicine".

References:

Adler, Jerry. (Jan. 17, 2005). "Diet and Genes." Newsweek. pp. 40-48.

Bland, Jerrey. (1999). Genetic Nutritioneering. Los Angeles, CA: Keats Publishing.

www.health-hope-opportunity.net 

 

***

Recommended Reading:

Bland, Jeffrey S., Genetic Nutritioneering, McGraw-Hill, 1999.

Williams, Roger, Biochemical Individuality, McGraw-Hill, 1998.

 

 

 

 

 


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