CBG.
02: Genomes
ENCODE PROJECT WRITE EULOGY
FOR JUNK DNA
- human DNA has 3 billion bases
- instead of the expected 100,000 genes, the
initial analysis found about 35,000 and that
number has since been whittled down to about
21,000
- 80% of the human genome serves some
purpose, biochemically speaking:
- specify landing spots for proteins that influence
gene activity
- strands of RNA with myriad roles
- places where chemical modifications serve to
silence stretches of our chromosomes
- a genes regulation is far more complex than
previously thought, being influenced by multiple
stretches of regulatory DNA located both near
and far from the gene itself and by strands of
RNA not translated into proteins (=noncoding
RNA)
- 11,224 DNA stretches are classified as
pseudogenes, dead genes now known to be
active in some cell types or individuals
- there are many genes out there in which DNA
codes for RNA, not a protein, as the end product
- various cell genes home in on different cell
compartments, as if they have fixed addresses
where they operate: some go to the nucleus,
some to the nucleolus and some to the
cytoplasm
GINGERAS: the fundamental unit of the genome
and the basic unit of heredity should be the
transcript the piece of RNA decoded from DNA-
and not the gene
- 5% of the human genome is conserved across
mammals
- DNAs bases function in gene regulation through
their interactions with transcription factors and
other proteins;
- 8% of the genome falls within a transcription
factor binding site, a percentage that is expected
to double once more transcription factors have
been tested
ON THE IMMORALITY OF TELEVISION
- from an evolutionary viewpoint, a function can be
SETS: FUNCTION IN THE HUMAN
GENOME ACCORDING TO THE
EVOLUTION-FREE GOSPEL OF
ENCODE (Dan Graur)
- the fact that sometimes it is difficult to identify
- less than 10% of the genome is evolutionarily
conserved through purifying selection
- according to ENCODE, a biological function can be
maintained indefinitely without selection, which -
implies that at least 80-10=70% of the genome is
perfectly invulnerable to deleterious mutations,
either because no mutation can ever occur in these
functional regions or because no mutation in
these regions can be deleterious
- problems in ENCODE logic:
o seldom used causal role definition of biological
function and then applying it inconsistently to
different biochemical properties
o logical fallacy affirming the consequent
o failing to appreciate the crucial difference
between junk DNA and garbage DNA
o using analytical methods that yield biased errors
and inflate estimates of functionality
o favouring statistical sensitivity over specificity
o emphasizing statistical significance rather than
the magnitude of the effect
- in biology, there are 2 main concepts of function:
o selected effect: function of a trait is
the effect for which it was selected, or
by which it is maintained
o causal role: historical and non-
evolutionary: for a trait Q to have a
causal role function, G, it is
necessary and sufficient that Q
performs G
(ex:) TATAAA maintained by natural selection to
bind a transcription factor; a mutated sequence,
resembling this one, also binds the transcription
factor, but does not result in transcription (no
adaptive or maladaptive consequence); hence, the
second sequence has no selected effect function,
but its causal role function is to bind a transcription
factor
assigned to a DNA sequence if and only if it is
possible to destroy it; unless a genomic
functionality is actively protected by selection, it will
accumulate deleterious mutations and will cease to
be functional
selection should never be used as a justification to
ignore selection altogether in assigning functionality
to parts of the human genome
the surest indicator of the existence of a genomic
function is that losing it has some phenotypic
consequence for the organism
- functional regions of the genome should evolve
more slowly and be more conserved among species
than non-functional ones
- Ward and Kellis confirmed that approx.. 5% of the
genome is interspecifically conserved and an
additional 4% of the human genome in under - For the last decade, geneticists have run a
selection
- According to ENCODE:
o 74.7% of the genome is transcribed
o 56.1% is associated with modified
histones
o 15.2% is found in open-chromatin
areas - The ENCODE team have mappedallof these to
o 8.5% binds transcription factors
o 4.6% consists of methylated CpG
dinucleotides
- transcription is fundamentally a stochastic process
- classes of sequences that are known to be
abundantly transcribed, but are typically devoid of
function:
- pseudogenes (up to 1/10 transcribed; lack coding
potential due to the presence of disruptive
mutations, evolve very rapidly and are mostly
subject to no functional constraint),
- introns (some human introns harbour regulatory
sequences TISHKOFF, 2006 as well as sequences
that produce small RNA molecules (HIROSE, 2003, -
ZHOU, 2004))
- mobile elements
- less than 2% of the histone modifications may have
something to do with function
- nematode Caenorhabditis elegans has 20,517
protein-coding genes
- misconceptions in common objections to junk DNA
o a lack of knowledge of he original and-
correct sense of the term
o the belief that evolution can always
can rid of the nunfunctional DNA
o the belief that future potential
constitutes a function
- in the majority of known bacterial species, selection
against excess genome is extremely efficient due to
the enormous effective population sizes, and the
fact that replication time and, hence, generation -
time are correlated with genome size
BRENNER: differentiated between junk DNA and
garbage DNA; the excess DNA in our genome is
junk and it is there because it is harmless, as well as
being useless, and because the molecular process
generating extra DNA outpaces those getting rid of
it;
- indifferent DNA refers to DNA sites that are
functional, but show no evidence of selection
against point mutations; deletion of these sites,
however, is deleterious, and is subject to purifying
selection
PURPOSE IS THE ONLY THING EVOLUTION CANNOT
PROVIDE - although only 5% of the human genome is
A SLIGHTLY DIFFERENT RESPONSE
TO TODAYS ENCODE HYPE
ENCODE: THE ROUGH GUIDE TO THE
HUMAN GENOME
seemingly endless stream of genomewide
associationstudies (GWAS), attempting to
understand the genetic basis of disease. They
have thrown up a long list of SNPs variants at
specific DNA lettersthat correlate with the risk
of different conditions.
their data. They found that just 12 percent of the
SNPs lie within protein-coding areas. They also
showed that compared to random SNPs, the
disease-associated ones are 60 percent more
likely to lie within functional, non-coding regions,
especially in promoters and enhancers. This
suggests that many of these variants are
controlling the activity of different genes, and
provides many fresh leads for understanding how
they affect our risk of disease. It was one of
those too good to be true moments, says Birney.
Literally, I was in the room [when they got the
result] and I went: Yes!
Imagine a massive table. Down the left side are
all the diseases that people have done GWAS
studies for. Across the top are all the possible cell
types and transcription factors (proteins that
control how genes are activated) in the ENCODE
study. Are there hotspots? Are there SNPs that
correspond to both? Yes. Lots, and many of them
are new.
Take Crohns disease, a type of bowel disorder.
The team found five SNPs that increase the risk
of Crohns, and that are recognised by a group of
transcription factors called GATA2. That wasnt
something that the Crohns disease biologists
had on their radar, says Birney. Suddenly weve
made an unbiased association between a disease
and a piece of basic biology. In other words, its
a new lead to follow up on.
Were now working with lots of different disease
biologists looking at their data sets, says Birney.
In some sense, ENCODE is working form the
genome out, while GWAS studies are working
from disease in. Where they meet, there is
interest. So far, the team have identified 400
such hotspots that are worth looking into. Of
these, between 50 and 100 were predictable.
Some of the rest make intuitive sense. Others are
head-scratchers.
EVIDENCE OF ABUNDANT PURIFYING
SELECTION IN HUMANS FOR
RECENTLY ACQUIRED REGULATORTY
FUNCTIONS
conserved across mammals, a substantially
larger portion is biochemically active, raising the
question of whether the additional elements
 evolve neutrally or confer a lineage-specific
fitness advantage
- mammalian conservation suggests that aprox.
5% of the human genome is conserved due to
noncoding and regulatory roles, but more than
80% is transcribed, bound by a regulator, or
associated with chromatin states suggestive of
regulatory functions
- human constraint correlates with mammalian
conservation, mRNA splice sites and regulatory
elements; similar selective pressures act in
humans and across mammals
- a substantial factor of human constraint lies
outside mammalian-conserved regions
- regions that do not overlap with active ENCODE
elements and inactive chromatin states show
lower constraint than ancestral repeats,
suggesting that they may provide a more
accurate neutral reference than repeats that can
have exapted functions
- mammalian conserved regions lacking ENCODE
activity show reduced human constraint relative
to active regions, suggesting recent loss in
function and activity;
- these also show higher primate divergence
relative to active regions, suggesting that some
loss of constraint predates human-macaque
divergence
- almost half of human constraint lies outside
mammalian-conserved regions, even though the
strength of human constraint is higher in
conserved elements
- protein-coding constraint occurs primarily in
conserved regions, whereas regulatory constraint
is primarily lineage-specific, as proposed during
mammalian radiation
- genome-wide association studies suggest that
85% of disease-associated variants are
noncoding, a fraction similar to the proportion of
human constraint that we estimate lies outside
protein-coding regions; this suggests that
mutations outside conserved elements play
important roles in both human evolution and
disease
MEGABASE DELETIONS OF GENE
DESERTS RESULT IN VIABLE MICE
- the functional importance of the roughly 98% of
the mammalian genomes not corresponding to
protein coding sequences remains largely
undetermined
- some large-scale deletions of the non-coding
DNA (=gene deserts) can be well tolerated by
any organism
- although gene inactivation can sometimes fail to
result in detectable phenotype, this is usually
related to the removal of genes with redundancy
elsewhere in the genome
- deletion of a gene desert mapping to mouse
chromosome 3 and in chromosome 19 (with no
evidence of transcription) contain 1,243
human-mouse conserved non-coding elements
- the boundaries for the deletions permitted
proximate regulatory elements nearby the
flanking genes to remain intact
- the heterozygous mice appeared phenotypically
normal
- the homozygous deletion mice for both deletions
were viable
- the deletions werent lethal in embryons because
of approx. rate 1:2:1 (wild-type : heterozygous :
mutant homozygous)
- phenotypic parameters measures in the
homozygous deletion mice, compared with
controls:
o post-natal survival rates for 25 weeks
o measurable growth retardation
o clinical chemistry tests (general and
specific plasma parameters)
o morphological abnormalities
o abnormal growth
o tissue degeneration
o organ mass was similar in both groups
of deletion mice and their wild-type
littermates
- molecular level impact: only 2 out of the 108
quantitative assays revealed detectable
alterations in levels of expression: Prkacb
reduced in the heart and Rpp30 reduced in the
brain
- in MMU3 desert, beta-galactosidase expression
promoter
NUCLEOSOME
- each of our cells (or more correctly, nearly all of
our cells) contain a copy of this genome,
encoded in 3 billion bp of DNA
- a collection of repar enzymes corrects chemical
changes inflicted on the strands by
environmental insults
- nucleosomes protect the delicate strands from
physical damage
- the job of the
nucleosome is
paradoxical, requiring it to perform 2
opposite functions simultaneously: on -
one hand, nucleosome must be stable,
forming tight, sheltering structures that compact
the DNA and keep it from harm; on the other
hand, nucleosome must be labile enough to allow
the information in the DNA to be used;
polymerases must be allowed access to the DNA,
both to transcribe mRNA for building new
proteins and to replicate the DNA when the cell
divides; the method by which nucleosomes solve
these opposed needs is not well understood, but
may involve a partial unfolding of the DNA from
around the nucleosome, one loop at a time, as
the information in the DNA is read
- nucleosomes also modify the activity of the
genes that they store: each nucleosome is
composed of 8 histone proteins bundled tightly
together at the centre, encircled by 2 loops of
DNA; the histone proteins, however, are not
completely globular like most other proteins
they have long tails, which comprises nearly a
quarter of their length; the tails extend outward
from the compact nucleosome, reaching out to
neighbouring nucleosomes and binding them
tightly together; the nucleus contains regulatory
enzymes that chemically modifies these tails to
weaken their interactions; in this way, the cell
makes particular genes more accessible to
polymerases, allowing their particular
information to be copied and used to build new
proteins
- the histone proteins are perfectly designed for
their jobs, so much so that histones are nearly
identical in all non-bacterial organisms; even
slight modifications can be lethal;
- the surface of the octamer is decorated with
positively charged AA, that interact strongly with
the negatively-charged phosphate groups of the
DNA; this serves to glue the DNA strand to the
protein core;
HUMAN GENOME PROJECT
(TO KNOW OURSELEVES)
genetic linkage map: based on careful analyses
of human inheritance patterns; it indicates for
each chromosome the whereabouts of genes or
other heritable markers, with distances
measured in centimorgans (=measure of
recombination frequency)
the closer 2 genes are on a single
chromosome the less likely they are to get split
up during genetic recombination
when they are close enough that the chances
of being separated are only 1/100 they are said
to be separated by a distance of 1 centimorgan
physical maps: distances between features are
measured not in genetic terms, but in real
physical units (base pairs)
- some regions of the genome resist cloning in
YACs and others are prone to rearrangements
restriction enzyme: cleave dsDNA molecules at
specific recognition sites, usually 4 or 6
nucleotides long
- when digested with a particular restriction
enzyme, then, identical segments of human DNA
yield identical sets of restriction fragments; on
the other hand, DNA from the same genomic
region of 2 different people, with their subtly
different genomic sequences, can yield dissimilar
sets of fragments, which then produce different
patterns when sorted according to size
- a third necessary tool is some means of DNA
amplification. The classic example is the cloning
vector, which may be circular DNA mole- cules derived
from bacteria or from bacteriophages (viruslike
parasites of bacteria), or artificial chro-
- mosomes constructed from yeast or bacterial genomic
DNA. The characteristic all these vectors share is that
fragments of foreign DNA can be inserted into them,
whereby the inserted DNA is replicated along with the
rest of the vector as the host reproduces itself. A yeast
artificial chromo- some, or YAC, for instance, is
constructed by assembling the essential functional
parts of a nat- ural yeast chromosomeDNA
sequences that initiate replication, sequences that
mark the ends of the chromosomes, and sequences
required for chromosome separa-
- tion during cell divisionthen splicing in a frag- ment
of human DNA. This engineered chromo- some is then
reinserted into a yeast cell, which reproduces the YAC
during cell division, as if it were part of the yeasts
normal complement of chromosomes. The result is a
colony of yeast cells, each containing a copy, or clone,
of the same fragment of human DNA
 - the human genome is not so very
different from that of chimpanzees or
mice, and it even shares many common
elements with the genome of lowly fruit
fly

(PRIMER ON MOLECULAR GENETICS)

- each chromosome is a physically separated
molecule of DNA that ranges in length from
about 50 million to 250 million bp
- the average gene is 3,000 bp
- largest known human gene is dystrophin
2.4 mil. Bp
- repeat sequences are thought to have no
direct functions, but they shed light on
chromosome structure and dynamics
- chromosome 1 has the most genes (3,168)
and Y chromosome has the fewest (344)
- single nucleotide polymorphism (SNP) are
sites in a genome where individuals differ in
their DNA sequence, often by a single
base ; scientists believe that human
genome has at least 10 mil SNPs