All articles are found at http://www.iflscience.com/
Human DNA Gives Mice Bigger Brains Than
Chimp DNA
February 20, 2015 | by Justine Alford
photo credit: Silver lab, Duke University
What makes us human is one of the most intriguing questions in biology. While it’s easy
to point out the physiological and cognitive differences that separate us from our closest
ape relatives, these are only the tip of the iceberg. What scientists are itching to divulge
is which bits of DNA are responsible for our unique characteristics—what gave us the
evolutionary shove that resulted in our success? Scientists could be one step closer to
finally teasing out the answer with the discovery of a DNA sequence that, when
introduced into mice, made their brains 12% larger than those with the same stretch
from chimps.
Fascinatingly, despite these striking differences, this sequence contains only subtle
changes between humans and chimpanzees. Furthermore, it doesn’t code for a protein,
but instead controls the activity of other genes. This finding could therefore provide us
with some long sought-after insight into the genetic mechanisms that led to our superior
brains. You can read up on the study in Current Biology.
There’s no question—our brains are pretty special. Take the neocortex, for example. This
relatively new addition to our brain, which contains some 100 billion cells, is the hub of
our higher mental functions, and it’s significantly smaller in other great ape species.
Itsexpansion throughout human evolution underlies our distinct mental abilities, but its
genetic basis has been elusive.
Although we may share some 95% of our DNA with chimpanzees, our genome contains
unique segments, called human accelerated regions (HARs), which remained largely
unaltered during mammalian evolution but then underwent rapid change after we
diverged from chimps. While they don’t encode proteins, scientists suspect that these
could be responsible for our uniquely human traits. In particular, studies have hinted that
HAR sequences which increase expression of certain genes, so called human-accelerated
regulatory enhancers (HAREs), are likely candidates, but none had been linked to a
specific trait before.
To delve deeper, scientists from Duke University began comparing the genomes of
humans and chimps with the hope of finding HAREs that are both different between the
two species and predominantly expressed in the brain. Out of 106 candidates, one in
particular —HARE5—stood out because of its close proximity to a gene called Frizzled 8,
which is known to be involved in a pathway crucial for brain development.
The team then added either the human version or the chimp version, which differed by
only16 DNA letters, into mouse embryos, alongside a reporter gene which made tissue
turn blue whenever the enhancer was switching a gene on. Not only did they find that
Frizzled 8 is likely under the control of HARE5, but embryos with the human DNA snippet
also became blue sooner and over a larger area than those with the chimp version.
Furthermore, human HARE5 caused neuronal precursor cells to divide more, resulting in
more brain cells and thus a marked—12%—difference in brain size when compared with
chimp HARE5, and the region affected was the neocortex.
“What we found is a piece of the genetic basis for why we have a bigger brain,” study
author Gregory Wray said in a statement. “This is probably only one piece—a little piece.”
[Via Duke University, Current Biology, Science and National Geographic]
Diet of Mother Can Lead to Alterations in Her Child’s
DNA
April 30, 2014 | by Justine Alford
photo credit: Wikimedia Commons. DNA Methylation.
A new study conducted by researchers from the London School of Hygiene & Tropical
Medicine, published in Nature Communications, has found that maternal diet around the
time of conception can influence certain properties of the child’s DNA. This could have
lifelong implications.
The researchers that conducted this study weren't looking at the actual DNA sequences of
the children; they wanted to see whether the diet of the mother was capable of causing
epigenetic changes. Epigenetics refers to changes in gene expression that occur without
alterations in the DNA sequence itself.
One example of epigenetic modification is DNA methylation, which involves the addition of
methyl groups to certain bits of DNA. Methyl groups can be obtained from the diet by
eating certain foods, for example those containing choline or particular vitamins such as B6
and B12.
It was demonstrated previously that maternal diet prior to conception can induce epigenetic
changes in the offspring of mice, but the same had not been investigated in humans prior to
this study.
Scientists chose to study pregnant women in rural Gambia because populations here are
dependent on foods that they have grown themselves and therefore their diets are different
between the dry and rainy seasons. 84 women that conceived at the peak of the rainy
season and 83 women that conceived at the peak of the dry season participated in the
study.
The team took blood samples from the mothers in order to compare differences in nutrition;
in particular they wanted to look at the levels of substances that can donate methyl groups,
and therefore possibly influence DNA methylation. When they later investigated the DNA of
the children they found that those conceived during the rainy season had higher rates of
methylation in all of the genes studied when compared with those conceived during the dry
season. They found that these changes were associated with maternal nutrient levels; in
particular two amino acids called cysteine and homocysteine. They also found that increased
maternal body mass index was associated with lower rates of infant DNA methylation.
It’s important to note that while associations were made between maternal diet and infant
DNA methylation, this study did not investigate the consequences that this may have on the
children. Although this initial study involved a small number of participants, the team believe
that the data is important and hope to progress the work with larger, more in-depth
studies.
Woolly Mammoths Suffered Major Birth
Defects Before Extinction
March 25, 2014 | by Lisa Winter
photo credit: Tracy O
Last week, it was determined that there was no genetic evidence of the Moa declining at
all before they were hunted to extinction. Unfortunately, the same does not appear to be
true for woolly mammoths. According to a new study, many mammoths suffered severe
birth defects in the form of cervical ribs as the species drew nearer to extinction. The
research was performed by Jelle Reumer of Utrecht University and was published in the
open access journal, PeerJ.
Mammoths first appeared on Earth around 5 million years ago and lasted until about
4,500 years ago. Humans are believed to have played a large role in their extinction, as
early humans targeted the animals for their meat, bones, and fur. Smaller populations are
more susceptible to succumbing to natural forces such as disease.
During fetal development, an error can occur and cause ribs to grow from cervical
vertebrae. The severity of the ribs can range from thread-like projections to an actual
full-sized rib. This can cause undue pressure on blood vessels or nerves with some pretty
serious consequences. In humans, if the embryo even survives until birth (nearly 80%
don’t), there is an 86% chance that it will die within the first year of life. Cervical ribs also
have a strong correlation with the development of childhood cancers such as leukemia.
Clearly, this is a trait that would be very harshly selected against.
Cervical ribs occur in about 1% of all human births. For Asian elephants, the closest living
relative of the mammoth, the number jumps up to 3.6%. For mammoths living near the
North Sea about 12,000 years ago, a jaw-dropping 33.3% of infants had cervical ribs. The
sample size was admittedly small, as Reumer and his team analyzed only 16 mammoth
cervical vertebrae. However, it was very striking that such a large percentage of those
vertebrae would have ribs, especially ones as pronounced as they were.
According to the researchers, this influx of birth defects could have come about in two
different ways. The genetic mutations could have arisen from inbreeding depression. As
mammoths were reduced in number, genetic diversity would have plummeted and the
number of mutations would have risen sharply. The other explanation offered states that
expecting mothers would have been under considerable stress as the population
dwindled. This prenatal stress could have had negative consequences for fetal
development.
Cervical vertebra from a mammoth with cervical rib. Credit: Joris van Alphen
Human Language Gene Speeds Up Learning
in Mice
September 16, 2014 | by Janet Fang
photo
credit: Wikimedia commons
For over two decades, scientists have suspected a link between the Foxp2 gene and the
development of speech and language in humans. Now, researchers show that introducing
the human version of this gene into mice speeds up their learning. The findings,
published inProceedings of the National Academy of Sciences this week, could help
explain the evolution of our unique ability to produce and understand speech -- which
may be the result of a gene mutation that arose more than half a million years ago.
Nicknamed the language gene, Foxp2 was first identified in a family with severe speech
difficulties; they carried only one functional copy of the gene coding for transcription
factor forkhead box P2. Since humans split from chimps, there’ve only been two key
mutations in this gene, which makes you wonder: What would happen if chimps had our
version of the gene?
For starters, a large international team led by MIT’s Ann Graybiel and Svante Pääbo from
the Max Planck Institute for Evolutionary Anthropology engineered mice to express
“humanized” Foxp2 by introducing two human-specific amino acid changes into the
gene. This change affected their striatum, a brain area essential for motor and cognitive
behaviors in humans. Different parts of the striatum are responsible for two modes of
learning: a conscious form called declarative learning and a non-conscious form called
procedural learning.
The team placed the mice through a series of maze experiments. Mice with humanized
Foxp2 performed the same as normal mice when just one type of memory was needed.
But when both declarative and procedural forms of learning were engaged, mice with
humanized Foxp2 learned “stimulus-response associations” much faster than regular
mice. For example, knowing whether to turn left or right at a T-shaped junction -- based
on the texture of the maze floor and visible lab furniture -- to earn a tasty treat.
Turns out, humanized Foxp2 gene makes it easier to transform new experiences and
mindful actions into behavioral routine procedures. The engineered mice learned the
route within a week, while regular mice did it in 11.
“This really is an important brick in the wall saying that the form of the gene that
allowed us to speak may have something to do with a special kind of learning, which
takes us from having to make conscious associations in order to act to a nearly
automatic-pilot way of acting based on the cues around us,” Graybiel says in a news
release.
By turning other genes on and off, Foxp2 helped tune the brain, adapting it to speech
and language acquisition. Speech is often seen as requiring a leap in conscious thoughtprocessing abilities, but it’s also dependent on complex movements of the lips and
tongue becoming automatic, Graybiel explains to New Scientist. When we first learn to
talk as infants, Foxp2 may have provided us unconscious control over our lips and
tongue. Perhaps the gene also helped with the emergence of speech in early humans,
the team suggests.
Loss of Y Chromosome Increases Risk for
Cancer and Early Death
April 29, 2014 | by Lisa Winter
photo credit: Theis Kofoed Hjorth used via CC BY-SA
4.0
Though overall life expectancy varies around the globe, it is true for pretty much any
country you look at that females live longer than males. There are many factors assumed
to play into this, such as a later onset of cardiovascular disease for females compared to
males, but a new study from a team of researchers led by Jan Dumanski of Uppsala
University has found a correlation between loss of the Y chromosome and early death, in
addition to an increased risk of cancer. Their findings were published in Nature Genetics.
As a person ages, the genome alters slightly through random mutations over a lifetime
of DNA replication for cell division. This natural process has been attributed to an
increase in risk for diseases like cancer or diabetes later in life, but not much was known
about the causal mechanism. Dumanski’s team sought to find if there were any common
trends about which parts of the genome were changing. Blood samples were taken from
over 1,600 elderly men and their health was tracked until their deaths.
The most common place to identify the age-related loss of the Y chromosome (LOY) was
in the white blood cells, which play a role in tumor suppression. One cohort found that
about 8.2% of the men with non-hematological cancer had LOY, and those individuals
lived an average of 5.5 shorter than those with the Y chromosome in tact. An
independent cohort found that about one in five men had LOY and died earlier,
regardless of the cause.
"Men who had lost the Y chromosome in a large proportion of their blood cells had a
lower survival, irrespective of cause of death,” said Lars Forsberg, lead author on the
study, in a press release. “We could also detect a correlation between loss of the Y
chromosome and risk of cancer mortality."
The researchers note that this indicator could be used as a biomarker for physicians to
use in explaining the risk carcinogenesis to their patients. This may also indicate that the
Y chromosome, which contains the SRY gene responsible for sex determination, may also
have other functions that are not currently well-understood.
“You have probably heard before that the Y chromosome is small, insignificant and
contains very little genetic information. This is not true,” Dumanski explained in the press
release. “Our results indicate that the Y chromosome has a role in tumor suppression and
they might explain why men get cancer more often than women.”
New Study Suggests Only 8.2% Of Our
Genome Is Functional
July 25, 2014 | by Justine Alford
photo credit: ynse, "DNA Rendering," via Flickr. CC BY-SA 2.0
In contrast to earlier estimates that suggested as much as 80% of our DNA has some
function, University of Oxford scientists have found that a mere 8.2% of the human
genome is presently functional.
Our DNA is made up of 3.2 billion base pairs- the chemical building blocks found in
chromosomes that are strung together to form our genome. It’s a pretty impressive
number, but how much of this DNA is functional? That has been a subject of great
interest recently given revelations about the vast amount of “junk” DNA, or DNA that
does not encode proteins, that seems to be present. In fact, almost 99% of the human
genome does not encode proteins.
Back in 2012, scientists from the ENCODE (Encyclopedia of DNA Elements) project
claimed that 80% of our DNA has some biochemical function. However, many scientists
were not satisfied with this assertion given that the word “function” is hazy and too
broad. In particular, DNA activity does not necessarily have a functional consequence.
Researchers therefore needed to demonstrate that the activity is important.
To do this, Oxford researchers looked at which parts of our genome have avoided
accumulating mutations over the last 130 million years. This is because slow rates of
genomic evolution are an indication that a sequence is important, i.e. it has a certain
function that needs to be retained. In particular, they were looking for insertions or
deletions of DNA sequences within various different mammalian species, from humans
and horses to guinea pigs and dogs. While this can occur randomly throughout the
sequence, the researchers would not expect this to happen to such an extent in stretches
that natural selection is acting to preserve.
The researchers found that 8.2% of our DNA is presently functional; the rest is leftover
material that has been subjected to large losses or gains over time. However, they also
note that not all of this 8.2% is equally important. As mentioned, only 1% of our DNA
encodes the proteins that make up our bodies and play critical roles in biological
processes.
It’s believed that the remaining 7% plays regulatory roles, switching genes on and off in
response to environmental factors.
“The proteins produced are virtually the same in every cell in our body when we are born
to when we die,” lead author Chris Rands said in a news-release. “Which of them are
switched on, where in the body and at what point in time, needs to be controlled—and it
is the 7% that is doing this job.”
Another interesting finding was that while the protein-coding genes were well conserved
across the different mammalian species investigated, the regulatory regions experienced
a high turnover, with pieces of DNA being added and lost frequently over time. While
this dynamic evolution was unexpected, the majority of changes in the genome occurred
within the so-called “junk” DNA.
Intriguingly, it was discovered that only 2.2% of our genome is functional and shared
with mice. But according to the researchers, that doesn’t necessarily mean we are that
different and it’s difficult to tell what explains our differences as species.
“We are not so special. Our fundamental biology is very similar,” said co-author Chris
Ponting. “Every mammal has approximately the same amount of functional DNA, and
approximately the same distribution of functional DNA that is highly important and less
important."
[Via PLOS GENETICS and University of Oxford]
Check Out These GLOWING Fish!
May 22, 2014 | by Lisa Winter
photo credit: Ruby Jylin via YouTube
Many organisms have been genetically engineered to
glow: cats, mice, pigs, monkeys,dogs, and even plants. When a new glowing organism is
announced, many people don’t seem to understand or appreciate why it has been done.
While there is the inherent “cool” factor with a glowing organism, the technique wasn’t
developed just for fun.
Biologists utilize genetic engineering for a variety of purposes. They can learn more
about the function of a gene if they suppress or amplify its expression. Some genes are
pleiotropic, in that they influence many phenotypes, even if they seem unrelated. For this
reason, it is incredibly important to understand where a gene is being expressed, but that
is slightly more difficult. For that reason, reporter genes for green fluorescent protein
(GFP; which gives certain jellyfish their natural bioluminescence) can be inserted into the
genome and produce a visible signal when the desired gene is expressed. This allows the
researchers to get visual confirmation that the genetic engineering was successfully
completed in the desired location.
But how do they actually do it?
The gene that encodes GFP is replicated through polymerase chain reaction (PCR), which
essentially works like a Xerox machine for DNA. The environmental conditions coax the
DNA into replicating until there is an adequate supply. The DNA is then altered to over
express the GFP, which will make it easier to see in the animal model. The GFP gene is
combined with promotor and enhancer sequences to ensure the finished product will be
inserted into the desired location. The engineered DNA is then inserted into a newlyfertilized egg and researchers will screen the organisms later to select the ones that had
successfully taken in and expressed the new gene. The success cases will be mated in
order to create a reliable strain of organisms that is homozygous for the transgene and
useful for study.
FDA Considering Releasing GeneticallyModified Mosquitos In Florida
January 26, 2015 | by Lisa Winter
photo credit: Oxitec
Mosquito-borne diseases such as malaria, Chikungunya, Dengue Fever, and Yellow Fever
claim the lives of over 1 million people each year. In an effort to quell mosquito
populations, researchers at the British biotech company Oxitec have been developing
genetically-altered mosquitos to be released into the wild. Even though tests with wild
mosquito populations in locations such as Brazil and the Cayman Islands have been
successful, public misunderstanding about genetic modification might prevent the U.S.
Food and Drug Administration (FDA) from releasing the same mosquitos in the Florida
Keys.
Oxitec’s research has surrounded Aedes aegypti mosquitos, which serve as a vector for
several diseases. The mosquitos have a modified version of a gene that will kill their
progeny as larvae, before they are able to fly. As only female mosquitos bite, the aim is
to only release modified males. When these males breed with wild females, the next
generation will reduce considerably. During a six-month-long timespan in 2012, Oxitec
released a total of 3.3 million modified males in the Cayman Islands and successfully
reduced the native mosquito population in the area by 96%.
The initiative to release the modified mosquitos in Florida is a cooperative effort between
Oxitec and Florida Keys Mosquito Control District. Though mosquito-borne diseases in
Florida are relatively rare compared to other tropical locations, the mosquitos are
becoming increasingly resistant to insecticides and other traditional methods of keeping
the population down. The Oxitec mosquitos would be released in Key Haven in Key West,
a neighborhood of 444 homes.
Despite the previous success of the Oxitec mosquitos, the FDA is thoroughly
investigating the implications of the experiment before allowing it to proceed. In addition
to the scientific attributes, they will also need to consider public opinion, which may or
may not be influenced by misinformation about genetic engineering. Opponents of the
program are concerned about residents being bit by modified mosquitos and altered
DNA entering a human’s bloodstream.
Only female mosquitos bite, and Oxitec has said that all females are removed before
release. Since the offspring with the altered DNA dies before they are old enough to fly,
there should be no way of getting bit by a genetically-engineered female. Oxitec also
states that in the very unlikely scenario that a mutated female bites and their DNA does
enter the human bloodstream, there’s no indication that it would cause any adverse
effects. The mutated gene may be lethal to mosquitos, but it has been shown so far to
be harmless in lab animals.
Even with these assurances, detractors claim that Oxitec can’t guarantee that a couple of
females won’t get overlooked or that the DNA doesn’t pose a threat to humans. A
number of scientists who agree with the low risk cited by Oxitec and the general safety
of the programtold the Associated Press the company has more work to do to
demonstrate safety. Failing to do so could cause more harm than good for public
perception for this experiment and genetic engineering in general.
Discussion about reducing or eliminating a species like what is proposed here invariably
brings up the question of how it would affect the ecosystem, as mosquitos pollinate and
make up part of the food chain. However, ecologists agree that any hole created by
removing mosquitos would be quickly filled by something else, with not much
widespread effect. However, knowing what species that would be, and whether it is
better or worse than mosquitos, is not as straightforward.
Scientists Design Biological Safety Switch
For GMOs
January 22, 2015 | by Justine Alford
photo credit: anyaivanova, via
Shutterstock.
In a bid to reduce the risk of genetically modified organisms escaping into the
environment and disturbing natural ecosystems, two groups of scientists in the U.S. have
rewritten the genetic code of bacteria to produce strains that are totally dependent on
unnatural substances to grow.
Not only do these organisms die without their synthetic food, but they are also resistant
to viruses and unable to exchange their engineered DNA with natural counterparts.
Although the techniques have only been used on bacteria so far, the researchers believe
it may be possible to extend them for more complex organisms, such as plants. Both of
the studiescan be found in the journal Nature.
With advances in the fields of synthetic and molecular biology, genetically modified
organisms (GMOs) have rapidly emerged as valuable solutions in a variety of clinical,
industrial and environmental settings. They’re already widely used to improve agriculture,
produce biofuels and pharmaceuticals, and to help clean up the environment. But while
they are proving to be invaluable tools to today’s society, it is well recognized that these
organisms could pose a risk to natural ecosystems if they escape and proliferate
uncontrollably.
In order to prevent this from happening, and to address public concern, scientists need
to develop robust biocontainment strategies. One method that researchers have been
exploring involves tweaking bacteria so that they are unable to produce certain nutrients
necessary for growth. The problem with this is that the organisms tend to be able to
scavenge these foods from the environment, or evolve pathways to synthesize them.
Alternatively, they could exchange genes with others and acquire the ability to make
them. Scientists therefore need to come up with novel techniques that circumvent these
issues, and two new studies demonstrate that significant progress has now been made
toward “safer” GMOs.
Although the studies were independent, both went for the same strategy, but they
adopted different methods to produce their organisms. The work builds on a previous
study in which the researchers radically altered the genome of the bacterium E. coli,
producing the world’s first genomically recoded organism. Both groups then went on to
further engineer this microbe so that it is totally reliant on an amino acid—a building
block of proteins—that is not found in nature. Without this synthetic supplement, the
organisms can no longer synthesize everything they need to live.
Importantly, the researchers targeted genes that are critical for survival, and also made a
large number of alterations, meaning it would be very difficult for the organism to evolve
mechanisms to live without the artificial food. Furthermore, because the genetic code is
so vastly different to that of natural counterparts, the risk of exchanging DNA with other
organisms in the environment is virtually eliminated.
The microbes were so dependent on the unnatural amino acid that even after growing
billions of the cells in numerous experiments, the researchers couldn’t detect a single
organism that was capable of surviving without the synthetic food. “Our strains, to the
extent that we can test them, won’t escape,” says study author Dan Mandell.
While the researchers have only tested out this technique on bacteria, it may be possible
to modify it for use in other organisms, such as plants and animals.
GM Potatoes With Health Benefits
Approved By USDA
November 11, 2014 | by Justine Alford
photo credit: United Soybean Board,
"Potatoes," via Flickr. CC BY 2.0
The US Department of Agriculture (USDA) has just given the go ahead for farmers to
start commercially growing several different genetically modified potatoes, the New York
Times reports. The potatoes, which come in Russet Burbank, Ranger Russet and Atlantic
Varieties, have been engineered to produce less of an ingredient that can turn into a
cancer-causing agent when fried. The potatoes also resist bruising, a common occurrence
in harvesting and transport which can reduce their value or even render them unsellable.
The new varieties, which have been dubbed “Innate” potatoes, were developed by Idahobased biotech company JR Simplot. The potatoes are joining a new generation of GM
foods that are designed to benefit both the farmers and the consumers, rather than just
the growers as, for example, herbicide or pesticide resistant varieties would. Several GM
apple varieties, for instance, were recently created which take longer to brown when
sliced, although these “Arctic apples” have yet to receive approval.
To achieve the improved qualities, Simplot scientists added desirable genes to the tubers
that are naturally found in other cultivated and wild potatoes. The genes encode a
system that results in decreased production of an amino acid (the building block of
proteins) calledasparagine. Although asparagine is found in many foods, it’s produced in
high concentrations in some varieties of potatoes. When heated to high-temperatures,
for example during frying or baking, it can form a chemical called acrylamide if the right
sugar molecules are present. French fries and potato chips have been found to contain
particularly high levels of acrylamide when compared with other foods.
Lab investigations found that the Innate potatoes produced between 50 to 75% less
acrylamide when fried than non-engineered varieties, but overall the levels of other
nutrients were unaffected. Although it’s known that acrylamide is a toxic chemical, the
benefits of these potatoes to consumers are hazy at this stage.
While acrylamide is listed as a “probable human carcinogen,” at the moment it
is unclearwhether eating foods with a higher acrylamide content can actually increase the
risk of developing cancer. The World Health Organization and Food and Agriculture
Organization have also stated that the levels of acrylamide in foods pose a major health
concern, but they call for further investigation as the risk of dietary exposure to the
chemical has yet to be determined. So if we don’t know how much, or how little,
acrylamide we have to eat for it to be bad for our health, we can’t be sure that reducing
it in foods is going to have any positive effects. That being said, reducing the likelihood
of bruising will definitely benefit growers.
Because the Innate varieties were created by adding in genes from other potatoes, rather
than different organisms, Simplot are hopeful that consumers will be more welcoming of
the crops. However, realistically it’s unlikely that this will sway anti-GMO advocates, and
some have already complained that the technology has not been adequately regulated
and thus approval should not have been granted this early. One group has also pressed
McDonald’s to not use the potatoes, despite the fact that Simplot have been a major
supplier of frozen French fries to the chain since the 1960s.
Name _____________________
Date_____________
After selecting 2 articles you should answer the following questions:
Questions:
1. Name 3 biological facts that you learned in class that helped you understand
this article? Cite the quotes you are referring to.
a. Explain the facts in your own words
2. What was you opinion about this article? Explain with 2 facts. If it is “amazing”
then say what was amazing in the article
3. What are three vocabulary words you did not know and the meanings you
looked up.
Experimental design (use only 1 of the articles to complete this section)
4. What is the hypothesis?
5. What is independent variable? (Make sure units are included.)
6. What is the dependent Variable? (Make sure units are included.)
7. What was the control?
8. What are two constants (variables that are being controlled)?
9. What particular evidence supported the hypothesis?