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 Report 20/09/04
How to Keep in Concert
One of the biggest puzzles of the fluid genome is why multiple copies
of a gene scattered throughout the genome can be kept so nearly identical,
which may be good for the organism. But the mechanism responsible has its
downside in converting healthy genes to defective ones when cells are stressed.
Dr. Mae-Wan Ho reports
A
longer article
with sources is posted on ISIS members’ website.
Details here.
The mystery of perfect copies
‘Multigene’ families are families of genes that serve the same
function and are almost identical copies of one another. Multigene families
exist in the genome of all living organisms, and are present either in blocks
of repeats, or in single copies dispersed throughout the genome.
One question that has preoccupied geneticists right from the first is
how the multiple copies of gene sequence remains so uniform within a
species, which is out of all proportion to expectations based on the rate of
random mutations that strike most other parts of the species’ genome, and
much more so compared to the same gene sequence present between species.
The members of multigene families seem to evolve ‘in concert’ within
a species.
The ribosomal RNA (rRNA) genes - required for protein synthesis in the
ribosomes within the cell - are the best-studied examples of
‘concerted’ evolution in eukaryotes (organisms including human
beings, whose genomes are enclosed within a nucleus); and gene conversion has
been proposed as a mechanism, especially for genes that are dispersed
throughout the genome. Gene conversion is a process whereby the sequence of one
gene converts that of another in the genome, so the end result is a closer
resemblance between them.
Large numbers of rRNA genes are present in eukaryote, typically more
than 100, and in some cases more than 1 000; and the size of the repeated unit
also tends to be very big. This makes precise analysis very difficult. The
repeat unit of human rRNA genes, for example, is about 43kb; and there are five
blocks of tandem repeats of about 100, each on a different chromosome.
A closer look
Microbiologist Liao Daiqing of the University of Sherbrooke in Quebec,
Canada, compared the sequences of multiple rRNA genes within the genome of 12
bacteria that have multiple copies of the rRNA genes. The genes for the three
rRNA molecules (23S, 15S and 5S) found in the ribosome are typically linked
together and transcribed in a single unit called an operon in
prokaryotes. The length of these three rRNA genes is ~2 900bp (23S), ~1 500bp
(16S), and ~120 bp (5S), and their sizes as well as sequences are well
conserved between different prokaryotic species. The multiple rRNA operons are
generally dispersed throughout the prokaryotic genome. Liao analysed the rRNA
genes and their immediate flanking sequences in 19 completely sequenced
genomes, but seven of the genomes surveyed contain only one copy of each rRNA
gene.
He found striking sequence homogeneity of each individual rRNA gene
family within a species, in contrast to the divergence of gene sequences
between species.
Within a genome, evidence of gene conversion was found throughout the
entire length of each individual rRNA genes and their immediate flanking
regions. Individual conversion events, however, convert only a short sequence
tract, and the conversion partner can be any gene within the gene family in the
genome. He confirmed that gene sequences undergo much slower divergence than
their flanking sequences, and any homogeneous flanking regions that exist may
have been incidental co-conversion with the gene sequence.
The average divergence (difference) among the seven 16S rRNA genes
present in E. coli is 0.0055 per site, whereas the average divergence
between the 16S RNA genes in E. coli and its close relative H.
influenze is 0.1325, or 24 times greater. The same applies to the 23S and
5S rRNA genes. No sequence heterogeneity was detected for multiple copies of
23S, 16S or 5S in Aquifex aeolicus, Chlamydia trachomatis,
Haemophilus influenze, Helicobacter pylori, Methanobacterium
thermoautotrophicum and Synechocystis PCC6803. Five of these six
species have only two rRNA operons, whereas there are six operons in H.
influenzae. There are 10 and 7 rRNA operons in B. subtilis and E.
coli, but the rRNA genes in these two species also display remarkable
sequence homogeneity.
Obvious sequence heterogeneity was found for the intergenic
‘spacer’ sequences between 16S and 23S genes in B. subtilis,
E. coli, H. influenzae and T. pallidum. This is mainly due
to the presence or absence of tRNA (transfer RNA) genes or the presence of
different tRNA genes in this intergenic region. The contrast of homogeneity in
the gene sequences to heterogeneity in the intergenic spacers implies that
concerted evolution does not reflect gross replacement of one operon with
another; rather it is a gradual, region-by-region homogenisation process.
Individual conversion tracts appear to be short, apparently less than
500bp, similar to those observed in other organisms.
How genes may convert
Several mechanisms can lead to sequence conversion. The first is via
reverse transcriptase (RT) of a rRNA sequence into complementary rDNA, which is
then inserted in place of other rRNA genes in the genome. The second mechanism
involves recombination between different rRNA genes during DNA replication, so
they end up with the same sequence or more similar sequences. The third
mechanism involves the ‘invasion’ of one gene by the single stranded
DNA of another gene to form a hybrid duplex, followed by DNA repair to remove
the mismatch.
The first two mechanisms are considered unlikely in prokaryotes.
Although RT-mediated gene conversion appears to occur in the eukaryote yeast,
RT activity cannot be detected in many different types of cells including E.
coli. Unequal reciprocal recombination can in principle account for
homogenisation of tandemly repeated genes. However, that could not
satisfactorily explain the remarkable heterogeneity of sequences flanking the
rRNA genes. Furthermore, ectopic recombination between repetitive sequences in
different parts of the genome can result in sequence deletion, inversion or
translocation and such drastic genomic changes lead to genome instability.
So that leaves gene conversion via heteroduplex formation, probably
mediated by the complex bacterial enzyme RecBCD that controls recombination at
particular ‘Chi’ (pronounced "Kye") recombination hotspots, with the
sequence GCTGGTGG (see box). The Chi element is one of the most abundant
repeated sequences in the E. coli genome. Chi-like sequences are
frequently found within the 16S and 23S rRNA genes and their vicinities. For
example, the sequence stretch GCTGGCGG near the 5’ end of the 16S rRNA
gene differs from Chi by only one nucleotide, and this change does not appear
to affect its function. This Chi sequence is conserved in all bacterial 16S
rRNA genes. Although RecBCD/Chi system may not operate in all the species,
similar recombination machinery may be responsible.
Gene conversion in health & disease
Evidence for gene conversion via heteroduplex formation has emerged in
other bacteria and in yeast. Analysis of the RNU2 gene in various human
populations reveals that repeats within an individual tandemly repeated array
are more homogeneous than between different arrays, while the intergenic
flanking regions are not homogeneous, suggesting that gene conversion is
involved instead.
Chi-like sequences have been found in many eukaryotic genomes and are
suspected to be involved in gene conversion events, for example, within the MHC
(Major Histocompatibility Complex), a complex of around 100 gene in
vertebrates, include the extremely polymorphic (variable) cell surface proteins
called HLA in humans and H-2 in mice, which provide immunological markers for
‘self’, and are involved in immune response against
‘nonself’, including transplants.
Alec Jeffreys and Celia May at Leicester University examined human
sperm for evidence of gene conversion. The formation of germ cells – egg
and sperm - during meiosis is the usual point in the life-cycle of higher
organisms when chromosomes pair up, ‘cross-over’ and exchange parts,
thereby shuffling the genes they inherit from each parent. But it appears that
instead of an equal exchanging of parts at the cross over points, there is an
unequal conversion of one allele by the other.
Jeffreys and May first concentrated on a recombination hotspot DNA3
located in the MHC, which is surrounded by single nucleotide polymorphism
(SNP), with many men heterozygous for multiple SNPs. They found evidence of
gene conversion - 1.3-3.4 x 10-3 per sperm – that was two to
three times higher than the rate of crossover. All conversions involve the
transfer of short stretches of DNA (300bp to 1091 bp). Conversion rates
declined rapidly with distance and defined a very steep gradient extending in
each direction from the centre of the hotspot.
Another crossover hotspot DMB2 in the MHC was much less active than
DNA3, but the pattern of gene conversion was very similar. A third crossover
hotspot is the gene SHOX in the pseudo-autosomal pairing region PAR1 on the sex
chromosomes. The crossover rate is much higher (3.7x10-3) per sperm,
although the density of SNP is low. Again, there is evidence of gene conversion
involving short tracts of DNA.
The mean length of conversion tracts probably lies in the range of
55-290bp. They estimate that somewhere between 80% and 94% of recombinations at
hotspot DNA3 are gene conversions rather than reciprocal cross-overs.
Similar results have been observed earlier in mice. The number of
crossovers during meiosis is tightly regulated to one to two per pair of
chromosomes in mice, and their distribution is not random, there are
recombination hot and cold regions. Researchers in the Institute of Human
genetics, Montpellier, France, found a high frequency of gene conversion in the
region of highest crossover density. They found 16 gene conversion events among
6 000 molecules of sperm DNA, corresponding to a frequency of 2.7 x
10-3. Most of the gene conversion events involve less than 540bp
tracts.
Gene conversion is increasingly implicated in human disease, in which
the disease-causing mutations appear to be copied from a closely related
pseudogene (a mutated gene that is no longer functional) in the genome. Cases
attributed to Chi sequences include the T4 cationic trypsinogen gene associated
with pancreatitis, the b-crystallin gene
CRYBB2 in a dominant form of
cataracts, the CYP21B gene responsible for steroid 21-hydroxylase
deficiency and congenital adrenal hyperplasia and Von Willebrand disease (VWD),
the commonest inherited bleeding disorder. Such pathological gene conversions
may be linked to stress, and resemble the controversial phenomenon of
‘directed’ mutations found in stressed and starving bacterial cells
(see "To mutate or not to
mutate", this series).
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