China before 1949
China has, as a single kingdom existed since 1700 BC This is longer than any of history's other great empires [1] . The Chinese saw it as "Middle Kingdom" and others as insignificant barbarian states. Therefore, as the emperor of China as well as the entire world's sole ruler. His task, to maintain the natural harmony between man and the universe, was awarded the sky [2] . This system of a despotic emperor exists right up to 1912 [3] . The Chinese have so for thousands of years had an exalted, divine and autocratic leader.
In the Empire of China, the majority of the population respectively, while the owner and tenants. Both were subject to a neighboring landowner, who could find to charge up to 50% of the crop in taxes. While the peasants could, at any time, called for a large construction project such as. The Great Wall of China. This subclass then lived a hard life, which was exacerbated by smaller and smaller plots as a result of a doubling of population between 1750 and 1850. In contrast to this class, with a living standard far cry higher than the peasants, were the upper class, which consisted of landowners and mandarins, Chinese officials. [4] The great divide between these two classes created fertile ground for the insurgency as seen in recent Chinese history. Nianoprøret In 1853, in 1855 and 1862 Muslim rebellion respectively south and west and China in 1851 Taipingoprøret, which is one of world history's bloodiest conflicts, where many millions of people die [5] . All these rebels will be killed by the dynasty that first vanquished in 1911 by a rebellion led by Sun Yat-sen. This produces the Republic of China. Democracy is it not to, for Yuan Shih-kai, a military man who has been appointed provisional president declares Kuomintang party, which has just been substantial majority after China's first parliamentary elections in 1913, illegally. Yuan Shih-Kaiser's death in 1916, beginning at the "warlord period" in which local Generals' struggles for power splitting the country. This period ends until 22 years later [6] .
Chinese Communist Party (PPP) will, inter alia, Mao Tsu-tung, founded in 1921. From Moscow gets the Communist International (Comintern) in 1922, PPP to initiate cooperation with Kumintang party. The idea of this collaboration is to first get the imperialist powers out of China with a national revolution, then to implement a communist revolution [7] . When Chiang Kai-shek in 1925, becoming leader of Kumitang takes a lot violent swing to the right, and in 1927, shortly after he conquered Beijing and has the bulk of China under him, he breaks with PPP [8] . Communists are expelled from the cities to the countryside, where they set up a number of Soviet republics. One of these, Kiangsi, with Mao and Chu Te in Management [9] . But when Chiang Kai-shek, in 1934, initiating a violent attack, the Communists forced to abandon their newly republics. They begin the long march, which leads them to Yenan, through 10.000km of China's most inaccessible mountains. The force that starts the march will be reduced by 80-90% before they reach the goal one year after [10] . Here are Mao, who is appointed KKPs party chairman, the opportunity to test some of its political, social and economic models in practice. Meanwhile, the long march led to greater sympathy for the PPP among the population, as more farmers hear about the Communist land reform policy. The communist army proves simultaneously disciplined and honest, unlike its opponents. In 1936 becomes Chiang Kai-shek forced to call off the offensive against the Communists when he realizes that the only way he can beat back the invading Japanese, is a global offensive to both Kumitang and PPP. In those areas the Communists liberator introduce the reforms and their getting in the way much support behind him. When the war ends in 1945, many Chinese see them as liberators, and they ride with the nationalist wave that moves against China. This wide support among people looking for Communists final victory over Chiang Kai-shek and the Chinese People's Republic established in 1949 [11] .
1949 People's Republic of creation
Between 1949 and 1955 growing numbers of members of PPP with more than double to 10 million. The party will need to obtain a large number of new members to manage the large country. Many of the former officials are also on their lines to keep roughly in order. This results in many new party members who are not really communists, but just have joined to share in the benefits it carries with it. Later, by cleansing campaigns, expelled many of these members. To hold the country together waiting lot also with too many socialist reforms. [12] China's policy in the reconstruction period was to get the economy and food production in time before the initiation of major structural changes. However eliminated landowner class and the Communist land reform from the civil war years up to, will be implemented throughout China. Earth is parceled out to local farmers, who then owns their own fields. This includes but not for long, for the first five year plan (1953-1957) goes farms with agricultural collectives [13] .
PPP structure
The party is built like a pyramid [14] with the National Party Congress at the top. Each step of this pyramid consists of a party committee, chosen by a party conference or at a party meeting. The Committee selects a party secretary, but must be approved from above. The National Party Congress consists of over 1000 members who are elected in a 5 years period of the various provincial congresses. The Congress party should formally meet once a year, this will not be respected. Until the mid 1980s, there is not perish no actual discussion in this great assembly, which in reality has no power. Central Committee (which is formally elected by party congresses, consisting of around 100 members) elect the Politburo, consisting of between 12 and 24 members. This is the Politburo, which has the real power in the party and therefore in China. In 1956 created in the Politburo Standing Committee, which brings power with him. This committee consisted of Mao, four deputy chairmen of the Party and Deng Xiaoping. Later, Lin Biao also in this Committee. The decisions taken by the Politburo and the Standing Committee, implemented through the Secretariat [15] .
The first Five Year Plan
The idea of a five-year plan had the Chinese got from the Soviet Union, which had implemented such plans as far back as the 1920s. The contents of the Chinese first five year plan was indeed heavily influenced by the Stalinist development strategy. As in the Soviet would introduce a planned economy, where control of production lay with the government. It was about getting the industry started. And with Russian assistance succeeded indeed to expand production to increase by 130%. Although there are collectivisation of agriculture, increasing production here only with the average 3.8% per year between 1953 and 1957 [16] .
Let 100 flowers bloom
In 1956, Mao said "Let 100 flowers bloom, let the 100 schools of thought compete". Lot of thought is that the intellectuals should have more freedom to express their views. It is intended that artists and scientists to express their opinions on the deficiencies in the system who looked at this way can later be repaired. But the situation runs out of control, and there are demands for independence from political parties PPP. Mao's ideas are criticized, and the party is being criticized for their control over the universities' academic work. This results in anti-right campaign, initiated by the party, which stamps around. 550,000 intellectuals as "right-dissidents". Many of these dissidents belonging to the right party, and because many of them are sent to the country or moved, the campaign used by other party members to rise in the hierarchy. Among the population, many favor against the persecution of intellectuals [17] .
The great leap forward
The first five year plan had increased production in the industry significantly, but limped after agriculture. Within the party there were two different views on how this problem could be solved. One, more moderate, went out to major investments in fertilizer plants and agricultural machinery. The second, which Mao and Lin Biao backed up on, was more radical. Mao felt that the Soviet model of top-down was ideal, and he advocated greater decentralization. At the same time, he was impressed by the success of collectivisation, which had been introduced years earlier. Mao's plan was to reorganize agriculture in larger units and thereby create a larger production. At the same time he went to the industry was distributed throughout the country and that all should take part in production. [18]
Anti-right campaign, which was at its height just as the decision on which model one should choose for the future led many in the party for fear of being branded a right-dissidents, supporting up on Mao's model. And in 1958 began the decentralization of industry and a stronger collectivisation of agriculture. It was created so-called public municipalities, consisting of average 5000 households across China. In these people municipalities established nurseries, playgrounds and common canteens. Population was, after military pattern, organized in production teams. Many believed that families were a relic of the past, children were placed in institutions and schools. The great leap forward was the beginning of a communist society, where one provider within their abilities and enjoy as needed [19] .
Across China, farmers were organized to giant overrislingsprojektor and in 1958 was 100 million peasants allegedly made 7.8 million hectares of fertile farmland. Agricultural production would also appear to rise very unrealistic. The production of cereals fell from the reported 375 million tonnes by nearly half. This was all reported a much bigger production than was possible, for fear of being judged as right-dissidents [20] . Concomitant bony many Chinese, regardless of what else they worked with, with self-built blast furnaces to produce iron. This iron is found later to be of such poor quality that it can not be used for anything. The consequence of the Great Leap Forward was a massive famine, which mainly affected the rural population where about 20 million Chinese died. Mao would not admit his mistake in the Great Leap Forward and then Peng Dehuai criticized him for it, Mao accused him of being right-opportunist and had him earmarked as defense minister. Lin Biao took his place. But the situation deteriorated, and Mao's support within the party became less. In 1959, he was compelled to give the president Liu Shaoqi. He was seated as chairman of the party. [21]
Liu Shaoqi trying to get the country back on track by stopping many of the projects were introduced during the Great Leap Forward. The central administration will turn more control over development, private plots return, the many small local industrial production, as furnaces, are shelved and instead of mass mobilizations will be more emphasis on scientific methods to increase agricultural production. [22]
Cultural Revolution
In 1964 set up the party leadership with Liu Shaoqi and Deng Xiaoping in the head, pushed by Mao, known femmands group, led by Peking Mayor Peng Zhen, to coordinate efforts in the third Great Proletarian Cultural Revolution. [23] In November 1956 attack Mao, through a Shanghai journalist named Yao Wenyuan, the author Wu Han. Wu He has written a play which, after Mao's view, is a criticism of his dismissal of Defense Minister Peng Dehuai in 1959. Besides being a writer Wu He is also deputy mayor of Beijing and a close friend and colleague of the leader of the group femmands Peng Zhen. Mao's attack on Wu Han is not an isolated attack on a writer but an attack on femmands group. [24] Through the Central Committee succeeds Mao, to send out a circular which femmands group is allocated, and there is formed a new group for the continuation of the Cultural Revolution. This group, Mao-group membership includes of Mao's secretary Chen Boda, Keng Sheng and Mao's wife Jiang Qing. They have quickly taken over by the mass media, and their violent campaign of the Cultural Revolution spread to universities and schools.
In May 1966 bring a philosophy professor who sympathizes with Mao, a vægavis up at university in Beijing. The attack on the university administration. Since Deng Xiaoping and Liu Shaoqi makes "work teams" in order to stifle unrest, it has the opposite effect, and more and more radical teachers and students turn against party members. Mao sheep poster content published and unrest spread to other universities and between schools [25] . Everywhere in the college are formed so-called Red Guards, who "will defend Mao and his thought against all enemies?" [26] . Central Committee publishes in August the same year a directive with 16 points on the Cultural Revolution. This Directive fed for vigilance against those who would seek to undermine the revolution from within, but while trying to stabilize the situation by the request to show "special consideration" to the scientists and technical personnel, and to debate out of words rather than violence. But rødgardistbevægelsen grow, and in August organizes Mao huge parades in the Forbidden City in Beijing, where the young Guards can meet their "great teacher, great leader, great command and great coxswain." These gigantic mass mobilizations organized through Lin Biao, the military. At year end, the situation is unstable highest. All schools and universities are closed, and youth are invited to the destruction of old buildings, temples and artefacts and to attack teachers, school, party leaders and parents. This leads to, often innocent, accused of being feudal or reactionary and large meetings tortured until they plead guilty. Many thousands are beaten to death or commit suicide during these meetings. The meetings take place also at the highest level, and thus cleansed both Liu Shaoqi and Deng Xiaping from the lot. [27] When they are out, added Jiang Qing, Zhang Chunqiao and Yao Wenyan, which all belong to the cultural revolutionary group in the Politburo. At the same time, Lin Biao brought up the hierarchy to number two in the bureau [28] . The various rødgardistgrupper starts now fighting each other, and although Mao in 1967 trying to stop them fighting flare up again in 1968. Arsenal is sacked, and the army are conflicting orders on whom to support. The forces of Mao has been unleashed, proves not to be controlled. In some cities, which develops the full-scale war between the various rødgardistfraktioner. [29] The situation is not sustainable, and in 1968 Mao gives the order to disband rødgardistgrupperne. Rødgardisterne being sent to the countryside for part of their training, to work as peasants. Army people are now increasingly put into administration at all levels. In April 1969, at the 9th Party Congress declared the Cultural Revolution have been executed. But the cultural revolution is still in important key positions, including the Politburo and the propaganda apparatus, which they repeatedly launch campaigns. In 1971 Lin Biao suddenly disappear from newspapers and news releases after allegedly having formed a conspiracy, as in a coup in September would assassinate Mao and Lin Biao paste as new ruler of China. The plan is revealed, and Lin Biao trying to flee to the Soviet Union by plane. He rushes down over Mongolia and die. [30] Zhou Enlai, who still sits as head of government, the sheep in 1973 Deng Xiaoping rehabilitated and reinstated as deputy prime minister. And although the cultural revolutionary groups, with Jiang Qing as the frontman, still sitting on the propaganda apparatus succeeds Zhou Enlai to rehabilitate a large number of cadres. Mao himself is suffering from Parkinson's, heart attack and is in great periods sidelined. At the same time losing the group about the Cultural Revolution, more and more ground to Zhou Enlai and Deng Xiaoping, who at the 4th National People's Congress in January 1975 to publish a program which included: modernization of agriculture, industry, defense and science. On top of it adopted a new constitution, which the peasants have the right to private plots and sideline sale on the open market. In 1976 Zhou Enlai dies. Hua Guofeng, a compromise between the moderates and the radicals, will be put in as prime minister. On 9 September 1976 death of Mao, and 6 October is Jiang Qing and her group were arrested. They are called gang of four and is blamed for everything bad that has happened in the last 10 years [31] .
Mao's intentions with the Cultural Revolution
After the failure of the Great Leap Forward was Mao's policy of driving out on a siding. Mass mobilization, decentralization, public municipalities, collective farming and common canteens were slowly abandoned, and the things that private plots were re-introduced. While Mao still has something to say, his role is now mostly as a figurehead for the regime, power is now in the Deng Xiaoping, Liu Shaoqi and Zhou Enlai. Mao's official view of the cultural revolution was to realize a revolution in what Marxist theory calls the superstructure. The Revolution of 1956 was implemented in the economy, which calls the theory base, but now it was then extended to the superstructure, which is culture and politics. Mao believed that a socialist society does not just occur because a Communist Party has arisen. Old values and traditions will continue to exist. Mao stresses the revolutionary development between two lines, he respectively calls the centralist, bureaucratic and revisionist line, which places emphasis on excellence and economic growth, and the revolutionary mass line which is about the masses initiative and political awareness [32]. He believes that after the Great Leap Forward emphasis has been on the first, and it is some of what he would do away with the Cultural Revolution. Mao's age may also have played in - in 1966 he turns 73 and may have thought that if he should come to do something before his death, it was high time.
Mao knew that he had the Chinese people and, through Lin Biao, the army with him. Years up to his popularity has grown enormously, almost to the divine. He was described as "red, red sun in our hearts [33] and in a typical Chinese village, Chen, was to think so far as to hold a kind of worship before each meal, in honor of Mao. Here recited down this little prayer:
"We respectfully expressing our desire to continue living in the reddest sun in our hearts, the great leader Chairman Mao. And just as Vice-Chairman Lin Biao: he had to maintain good health forever. We have been liberated by the land reforms and will never forget the Communist Party. We will always follow Chairman Mao on the revolution come! [34] "
The people therefore believe that Mao is a form of human and infallible. This is Mao user, through the Cultural Revolution, to have eliminated its opponents, implemented its policy and put himself in the power center. Mao's actions suggest that he had no qualms about using such large masses in his political struggles. And although many were tortured and killed in the chaos created by rødgardisterne, I think that Mao looked back on the Cultural Revolution as a victory. Since the Cultural Revolution in 1969 declared completed, sits Mao and his followers in the leading positions in the party.
Consequences of the Cultural Revolution
From 1966 until Mao's death in 1976, we now define the years of the Cultural Revolution, died 3rd million people as a consequence of Mao's actions. Additional 100 million, or one ninth of the population were exposed to suffering in one form or another [35]. They could be accused of being reactionary and be publicly humiliated and tortured for offenses like being educated or otherwise meeting "better" than others.
A month after Mao's death arrested the "Gang of Four", which consisted of Jiang Qing and three of her closest supporters. And in the coming years, more and more politicians who were condemned during the Cultural Revolution, rehabilitated. Together with four gang are many other politicians who rose in the hierarchy during the Cultural Revolution, pushed away by people like Deng Xiaoping. Over the next year, many of the reforms which occurred during the Cultural Revolution, dropped, and a new policy which abandoned the collectivization of agriculture, gave factory managers more autonomy and liberalized culture, introduced [36].
In the so-called program of the Cultural Revolution, which was issued by the Central Committee, declares, inter alia, Education and culture:
"... Currently, it is our goal to struggle against and crush those persons in authority who have embarked on the capitalist road, to criticize and refute the reactionary bourgeois, academic "authorities" and the bourgeoisie and all other yield class ideology and to transform education, literature and art and all other parts of the superstructure that do not correspond to the socialist economic base .. "- Central Committee Decision on the Cultural Revolution, 8th August 1966 [37]
In the 10 years the Cultural Revolution lasted were almost all educational institutions closed and many teachers and intellectuals sent to labor camps. Since Mao get stopped rødgardistbevægelsen, he sends while almost all of China's youth away from schools to the countryside. This results in that we now have a generation in China, where almost no one has an education.
Under the slogan to attack the four "old" (old customs, old habits, old culture and old thinking), presented rødgardisterne historic buildings in ruins. Had it not been for the army, the Forbidden City would probably not exist today. Along with buildings, were quantities of art objects, books, records and anything else that could symbolize the past, destroyed. Large parts of the Chinese irreplaceable heritage went under in those years.
In 1980 initiated a trial of four gang accused of having "directed against suspects and prosecuted 729,511 people" and persecuted 34,800 "leading to death." [38] All are found guilty. In this way you can protect Mao's personality by giving the blame for everything bad that has happened, to a large gang of four persons. Even today, China's official attitude towards Mao that 70% of what he did was good and 30% poor [39]. With the 70% refers to the great work he did up to the People's Republic of creation in 1949. While the 30% refers to his role in the Great Leap Forward and Cultural Revolution. This breakdown makes a lot of time to criticize the Cultural Revolution and Mao shelf. Official Chinese history also shows this division of Mao's life. If you visit the Mao museum in Shaoshan, you will be introduced to every little detail until the year 1963, when then nothing until his death [40]. The same will you discover if you look at a Chinese school textbook in history. Here is the same terrible things that happened in China under Mao, also failed [41]. The reason for the party in this way suppresses the terrible things that happened under Mao, it may be that without him, the government loses its power. If the party accepts Mao's ideology was wrong, they can not avoid also admit that the basis for their existence is wrong. Therefore, it is so smart with the division into two "Maoer" a good that created the ideology and founded the republic and a less good, as did some stupid things in 60-70s and otherwise suppressed. That is why Mao's cult worship, which still exists in China, and that is why you, if you visit the Tiananmen Square in Beijing, will still see a large painting of Chairman Mao and a queue of Chinese who are waiting to see his embalmed body.
Cultural Revolution as seen from the West
During the Cultural Revolution ends Chinese foreign policy and diplomacy to function normally. Foreign Ministry in Beijing was occupied by rødgardister and is in great periods of paralysis. Embassies around the world are now used most to disseminate propaganda about the new socialist China [42]. Meanwhile devastate Vietnam War, which China supports the Viet Cong, not far away. Propaganda and the great disgust of U.S. involvement in Vietnam will have many in the West, mostly on the left to look at China in a positive light. Here is a country that chooses a different path than the capitalist and seems to do well. World Health Organization and the World Bank says are both positive on China's achievements in 1973 and expresses the Danish foreign minister, after an official visit to China, admiration towards the country's social progress. [43] It is "in" keeping with the Chinese. In Denmark, shooting associations such as the "Association Friendship with China" and "Society for Cultural Relations with China" (the later friendship association) up. All and solidarity with the Cultural Revolution and China. On the left wing in Denmark creates two poles: the Maoist and Soviet communist. While attention to the war in Vietnam grows more and more begin to see the Maoist model of armed revolution, more relevant than the Soviet Communist idea of peaceful coexistence [44].
During the Cultural Revolution will be released in Denmark many travelogues. Here describes the Danes, who with myself have seen the revolution, their experiences. Most of these books idylliserer China strong, and it may be several reasons for this. China during this period a very closed country, and it is very difficult to get admission to it. Therefore, Chinese are very precise control who they will let in and who must be away. In this way the group of people who already have expressed criticism of the regime. At the same time by the persons entering the country know that if they later publish anything that expresses itself negatively on what they have experienced, they will simply not be able to visit the country again. When visitors arrived in the country, they were presented to the grand operas, were served sumptuous banquets and stayed at the finest hotels. Throughout their visit was carefully planned, and there was no room for anything impromptu. It was planted in a bus and had looked through a hectic program of kindergartens and public kitchens. The institutions, one was presented, rose high above the standard, but was often used in recent reports as a general picture of China. These trip reports are one of the main sources of information the general Danish population's cultural revolution, and since these descriptions are so positive about the regime, is the general attitude equally [45]. One expression of this can be seen as the famous Svanemøllen Collective in Hellerup in 1970 changes its name to Mao's desire. Another example of the idealization that is about Mao and the Cultural Revolution, is because in a three-page obituary of Mao in the information, surrounded by a rim is sure: "Now Mao's work completed, perhaps the mightiest of our century, [46 ].
Only with Lin Biaos decline in 1971 and particularly with the arrest of four gang 1976, many Westerners to be able to see that the dreams and the ideology of their perception of China was based, has very little to do with reality.
[1] K.2 p.8 [2] K.3 p.5 [3] K.2 p.8 [4] K.2 p.10 [5] K.3 says approx. 20 million. (p.7) while K.5 say up to 30th million. [6] K.3 p.10 [7] K.2 p.13 [8] K.2 p.14 [9] K.5 p.7 [10] K.5 (p.7) and k. 3 (p.15) says about 20% while the K6 (s.442) says 11% [11] K.5 p.7-8 [12] K.2 p.18 [13] P. K.3 16 [14] See Appendix 1 [15] K.2 p.19 [16] The entire section K.2 p.23-24 & K.5 p.9 [17] The entire section K.2 p.28 [18] Entire Section K.2 p. 39-40 [19] the whole section (before) K.2 p.40-41 and K.5 p.10 [20] K.6 s.600 [21] The last K.5 p.10 and K2 p.42-45 [22] The entire section K.5 p.10 [23] K.2 p. 58 & 60 [24] K.2 p.61 and K.6 s.623 [25] Eventually. K.2 p.61 and K.6 p.39-40 [26] K.2 p.61 [27] The last K.6 s.626-627 [28] at the end. K.2 p.62 [29] From last K.2 p. 64-65 [30] For the last K2 p.65-67 and K6 s.636 [31] For the last K2 p. 68 -69 [32] All this with Mao's theoretical views K.1 p.1 & K.2 s.58.59 [33] K.7 p.14 [34] K.6 s.635 [35] K.8 P. 621 [36] K.2 s.95-96 [37] K.2 p.84-85 [38] K9 s.226 [39] K.10 [40] K.10 [41] K.11 [42] From the start K.2 s.77.78 [43] K.11 p.1 [44] K.12 and K.11 p.1 [45] the concrete details of the whole section: p.1-K.11 22 [46] K.14
lørdag den 11. juni 2011
Article Presentation
Presentation of the article "Identification of Yersinia
enterocolitica genes affecting survival in an animal
host using signature-tagged transposon Mutagenesis. "
Andrew J. Darwin & Virginia L. Miller, 1999.
Introduction
Here I will review the main points from
The above article from the reputable
journal Molecular Microbiology.
First I give the background to
survey was conducted, what the authors
purpose and hypothesis was how they did what
they found out and finally I will comment
implications of the findings.
Background
Three species of the genus Yersinia, a Gram-negative
rod-shaped facultative anaerobic Enterobacteriaceae,
pathogenic to humans. These three species are Y.
pestis, Y. pseudotuberculosis and Y. enterocolitica
and are associated with both local and systemic
infections. It is the last of these bacteria
authors here deal with, but
results are nevertheless significant for all three species. Mon
have previously identified a 70KB virulensplasmid
with Yersinia, called pYV that can be found in all three
species. Genes on this plasmid codes for many
virulence factors such as type III secreting
(TTSS) (discussed later) and effektorproteinerne
to secretion. However, it is shown that pYV is not alone
on virulence, but there are also chromosomal
genes involved. Mix Secondly, genes
coding for adhesion and penetration into the host cell,
binding of iron (siderophorersyntese) and
synthesis of lipopolysakkarid O-antigen.
Purpose and hypothesis
As the sequencing of entire genomes is
easier, it has been genetically mapped several
pathogenic bacteria. However, shortcomings still
much insight into which genes coding for the
proteins, and particularly what genes are
involved in bacterial virulensmønster.
Due to difficulties with the need to test each
single mutants virulensegenskaber and associated
genes in a testdyr had it at that
date not earlier been possible to make
Large-scale trials that would identify this particular
for the whole genomes. We can now through
signature-tagged Mutagenesis (STM). Here,
authors make a variety of mutants, and a part
these will be with impaired virulensegenskaber,
simultaneously test multiple strains in a
single host, and then find out which genes
in the given case is disturbed.
They expect from study start to a series of
their mutants will be mutated in pYV and that these
would have sharply reduced virulence. In addition,
they hope to discover the genes on the chromosome which
the virulence and survival in the host.
Method
A number of signature-tagged mutants were
manufactured by PUT vector containing
mini-Tn5 km2 transposons were inserted into E. coli
strain CC118, and then transferred to Y.
enterocolitica strain JB580v via conjugation.
These mutants were screened for insertion of the
said transposon by ampicilin Selection
(Different colonization pattern with and without
insert) and were performed only at 18 Southern
random samples which were screened for
kanamycinresistens who sat on transposons.
Preliminary studies showed that the best
infection method on test animals (6-7 weeks old
BALB / c mice) were intraperitoneal (IP) injection
(Direct injection into abdomen), with approx. 107 colonyforming
units (cfu), followed by capture from
spleen after 48 hours.
In order to avoid injecting the weakened auxotrofe
bacteria were selected transposonmutanter
grown on minimal medium.
STM
Method to screen for attenuated bacteria
was referred done with InDesign tours-tagged
Mutagenesis (STM). STM is a negative
selection method where this experiment uses
their pool of mutants (21 pools of 96 mutants)
to infect groups of three mice. What is special about
STM is that the inserted DNA has a special
signature trademark, a signature tag that is a little
familiar sequence which can subsequently be found by
Southern blot hybridicering. During the initial
screening infected mice and bacteria recovered
from spleen. These are mixed and transferred to 96
chamber micro titter plate. Then screener Mon
with proper original bacteria (input - all
original mutants) and those who were found in the spleen
(Output - the survivors). That way you can
find the attenuated bacteria by their lack
presence in the spleen accumulated
bacteria (negative selection), which is seen as
missing or weak hybridicering compared to the
from input (the first part of Fig. 1 from the article). Then
was taken to the mutants that demonstrated a lack
hybridicering - so attenuated virulence, and drove
secondary screening with the same procedure as
before but with two conditions (2x3 mice).
This should help reduce the risk to take
mutants actually was not attenuated (second part
fig. One from the article).
Subsequently, attenuated mutants studied
and classified phenotypic and by cloning
followed by sequencing and was performed
competition and vækstassays.
Results
Based on the two initial STM screening
they found up to 68 mutants with attenuated
virulence. As expected, a proportion of these being
mutated in pYV and it was indeed found that 29
were pYV mutants. This was examined by
growth pattern on LBMOX plates and kanamycinsensitivitet.
This means that out of the original 68
mutants were 39 who had mutations in
chromosomal virulensgener. Out of these were in
16 cases observed the same phenotype
(Aggregation in liquid medium, and turbid growth
LB agar plates) due to a defect in LPS
synthesis and thus synthesis of O antigen, which
is essential for Y. enterocolitica virulence. So
there were now 23 mutants back where mutation
sat somewhere else than in pYV or LPS synthesis
genes. To be confirmed whether they were really
weakened, it was checked every 68 in a competition
assay in mice, which measured about bacteria
fared worse than wild-type Y.
Enterocolitica. For this test it was found that 10 of
the 23 were actually weakened and were overall 55 of the
68 fact impaired. This in their eyes, good result
attribute the dual STM screenings
procedure.
By pYV mutants was especially type III
sekretionsgenerne that the insert was to identify and
especially in lcrV and yscL, this could indicate a
'Hot spot' for insertion of transposons mini-Tn5
Km2 just here (Fig. 2 from the article).
Of the 16 with suspected mutation in LPS / Oantigen
synthesis apparatus, this was verified
by sequencing (Fig. 2 from the article).
The remaining 10 chromosomal mutants were also
by cloning and sequencing studied. Here was
Mon insert, and thus the disruption of genes
coded outer membrane synthesis (yifH and nlpD)
nutrient uptake / acquisition (pstC and irp1) and stress
response (dnaJ). The last five had mutations in
genes homologous to E. coli genes coding for DNA
topoisomerase I (topaz), phage shock protein
operon (pspC) and two were found with
mutations in yibP, a gene of unknown function in E.
coli. Finally found a mutation in a sequence
there was inconclusive in Genbank database.
(Fig. 2 from the article).
NB. The article makes the reconstruction of pspC
mutant for further study. This, I
explain in question 3 later.
Discussion
They were in this study demonstrated that STM technique can
used to identify a number of genes in Y.
enterocolitica with more or less important
for its virulence. They were reidentificeret a series
Essential virulensgener in pYV (type III
secreting) and in LPS synthesis genes in
chromosome. The results from the chromosomal
mutants recalled earlier STM results
the pathogenic Gram-negative bacteria Salmonella
typhimurium and Vibrio cholera which also
found that bacteria with mutations in genes
involved in O-anti-reunion theory, stress response and
nutrient uptake / acquisition had weakened
virulence. The most sensational they found out
of was to pspC gene, homologous to the gene in E.
coli is part of the phage shock protein operon,
had significance for Y. enterocolitica's virulence.
This mutant exhibited very low virulensgrad in
test animal, but grew well in vitro. This gene has
in E. coli no known function, but we see an increased
expression by overexpression of sekretinproteiner,
inter alia used in Type III secreting. So
a suspected link to important virulensgener.
There must be investigated further in this
context for understanding the importance of pspC
gene and its virulensfunktion.
The article has the sense that it shows that a long
number virulensgener be identified quite
fast and relatively simple in STM method.
This means that other pathogenic bacteria, Gram-positive
as well as Gram-negative, rapidly
screened virulensgener, which can have significant
important for disease treatment, drug development
and our general knowledge of
pathogenic bacteria.
It is also useful in connection with that
more and more organisms (particularly bacteria) genome
sequences, and the need to understand
importance of individual genes in the studied
organism.
General works article vellavet over
trials building, reproducibility and
general structure of the article.
Specific questions
Question 1:
Signature-Tagged Mutagenesis (STM) is a technique
used to examine genes function in
vivo. You make a transposon with a reputation
signature sequence, which then transferred to the
organism to be studied mostly by E. coli.
Transposons, with signature tag, sits a
random location in the genome or a plasmid, and will
thus very likely disturb and
destroy the gene and thus the translated
protein. In STM, each transposon its own
unique signature, and can subsequently
found by DNA probehybridicering (Southern
only). Unlike ordinary random transposon
Mutagenesis (RTM) where each
mutation need a testdyr to negative control
here you can use a whole pot (in case the article
using the 96 per. testdyr) and find a specific
attenuated mutant. In the original design of the STM as
was designed by Hensel a al.1 used, as in
experiment here hybridiseringsprober, now a
Another method of PCR detection, developed by
Lehoux a al.2 used. Here is the idea that each
roof amplified with tag-specific primers.
The presence of a PCR product from a roof in
input pool and the absence of the same PCR product from
output pool shows the loss of the specific mutant
during the negative selection screening.
The advantages of STM with both signature-tag
detection methods are so clear, since it both
saves time (and thus money) and testdyr. Outright
disadvantages compared to RTM, I have a hard time
find.
Question 2:
The article dobbeltscreener their authors
mutants. This is done by making probes are
designed to hybridize to the inserted transposon
(Base pairing principle). When one acquires bacteria
from mouse spleen, the bacteria are finding that,
be those who are able to infect the mouse, so
the bacteria we do not want to investigate. But if
found the bacteria there, so to speak, is missing
can you find the gene is disrupted by
look at the input pool. This makes the order that the
primarily attenuated bacteria they inject, and
Following this double screening obtains the a
result which states that 55 of the original 68
mutants were effectively attenuated, which corresponds to
81%. This is an improvement over previous
results, where only 45% of those screened
mutants were effectively attenuated (here screened Mon
Vibrio cholera).
1 Hensel M, Shea JE, Gleeson C, Jones MD, Dalton E, Holden DW:
Simultaneous identification of bacterial virulence genes by negative
selection. Science 1995, 269:400-403.
2 Lehoux DE, Sanschagrin F, Levesque RC: Defined oligonucleotide tag
pools and PCR screening in signaturetagged Mutagenesis of essentialism
genes from bacteria. Biotechniques 1999, 26:473-478, 480th
Question 3:
The trial will determine whether the phenotypic
changes caused by transposon mutagenesis or
whether there is a spontaneous mutation. In all of
near a single mutant, they may exclude
spontaneous mutation. The exception is gay gallery to
E. coli gene pspC. Therefore, the authors construct
three new pspC mutants in the same strain of Y.
enterocolitica. One new mutant would be a
copy of the mutant from the original trial
while the other two would be mutants with a
insertion-deletion in the gene, with a kanamycinresistens
cassette. The copy was made by the
inserted fragment from the original was propagated
by PCR and equality into a vector and transferred to Y.
enterocolitica by conjugation. The other two were
made by kanamycin resistance cassette was
inserted in two different orientations of the ring. Of
these three new bacteria were again conducted a
konkurrenceassay. The results of this assay
with the reconstructed mutants found in Table 3 in
article and can be seen in comparison with pspC
results from the first konkurrenceassay (Table
2) the results are very similar - mutants
infect mice poorly, but grows well in
Vitro. The finds therefore that mutations in pspC
gay gallery Y. enterocolitica substantially weakens
its virulence.
Question 4:
Type III secreting (TTSS) is a complex
trans-membrane protein structure found in many
Gram-negative bacteria, pathogens and nonpatogene.
In the pathogenic used it mainly to
secrete proteins that help bacteria to
infect its host, and therefore there is strong
correlation between virulence and the presence
of TTSS. Hall Marketplace in the system is the needle.
It sits anchored only to the inner membrane in
the inner membrane ring, via a connector over
peptidoglykanlaget to the outer membrane ring
after which it has crossed the entire bacterial
wall system. The actual needle hole with a diameter
around. 3 nm, which means that most effector
proteins to be secreted in the unfolded state. (See fig.
1 below)
Figure 1 Schematic drawing of the TTSS. From Wikimedia Commons
Previously it was thought that the needle was able to
perforating the host cells, but this picture is
now changed. Now the theory that it emits
proteins called translocators, which form a pore
in the host cell membrane through which other
effectors can flow.
The article will transposons in one of the mutants
inserted into a sycT-yopM gene region, and this
suppose they can affectation genes involved
in TTSS. They examine whether it is due to errors in
secretion of proteins called yop (Yersinia
outer proteins), which helps to perforate
host cells that are the cause of the weakened
virulence. Therefore examined the protein composition
in growth medium for the said
mutants, and compared with wild type (Fig. 2).
In the experiments, they found no visible difference in
protein picture for the mutants and wild type.
Question 5:
There are many bacteria use TTSS in
their virulence. Of these species may
include Shigella (bacillary dysentery), Salmonella
(Typhoid fever), Escherichia coli (food poisoning)
Burkholderia (glanders), Yersinia (plague), Chlamydia
(Chlamydia) and Pseudomonas. The high prevalence
of TTSS of pathogens may be due to TTSS
cassette can be transferred horizontally between species.
As an example I will use the bacterium Shigella.
The most common cause of shigellainfektion is
contamination of food with faecal matter. After you have
eating infected food or water will bacterium
invade its host through epithelial cells of the colon
through its TTSS. This injects the IPAD
protein into epithelial cells, which triggers it to
incorporate the bacterium. This vakuole breaking down
of the divisional IpaB and IPAC proteins. Here may
now divide, and furthermore it may by
ICSA protein "take over" epithelial actin
polymeriseringsapparat which it uses to
be shot around the cell, and into other
neighboring cells, which then becomes infected. Total causes
Shigella approx. 165 million annual cases of
dysentery, and approx. A million deaths - almost
exclusively in developing countries
enterocolitica genes affecting survival in an animal
host using signature-tagged transposon Mutagenesis. "
Andrew J. Darwin & Virginia L. Miller, 1999.
Introduction
Here I will review the main points from
The above article from the reputable
journal Molecular Microbiology.
First I give the background to
survey was conducted, what the authors
purpose and hypothesis was how they did what
they found out and finally I will comment
implications of the findings.
Background
Three species of the genus Yersinia, a Gram-negative
rod-shaped facultative anaerobic Enterobacteriaceae,
pathogenic to humans. These three species are Y.
pestis, Y. pseudotuberculosis and Y. enterocolitica
and are associated with both local and systemic
infections. It is the last of these bacteria
authors here deal with, but
results are nevertheless significant for all three species. Mon
have previously identified a 70KB virulensplasmid
with Yersinia, called pYV that can be found in all three
species. Genes on this plasmid codes for many
virulence factors such as type III secreting
(TTSS) (discussed later) and effektorproteinerne
to secretion. However, it is shown that pYV is not alone
on virulence, but there are also chromosomal
genes involved. Mix Secondly, genes
coding for adhesion and penetration into the host cell,
binding of iron (siderophorersyntese) and
synthesis of lipopolysakkarid O-antigen.
Purpose and hypothesis
As the sequencing of entire genomes is
easier, it has been genetically mapped several
pathogenic bacteria. However, shortcomings still
much insight into which genes coding for the
proteins, and particularly what genes are
involved in bacterial virulensmønster.
Due to difficulties with the need to test each
single mutants virulensegenskaber and associated
genes in a testdyr had it at that
date not earlier been possible to make
Large-scale trials that would identify this particular
for the whole genomes. We can now through
signature-tagged Mutagenesis (STM). Here,
authors make a variety of mutants, and a part
these will be with impaired virulensegenskaber,
simultaneously test multiple strains in a
single host, and then find out which genes
in the given case is disturbed.
They expect from study start to a series of
their mutants will be mutated in pYV and that these
would have sharply reduced virulence. In addition,
they hope to discover the genes on the chromosome which
the virulence and survival in the host.
Method
A number of signature-tagged mutants were
manufactured by PUT vector containing
mini-Tn5 km2 transposons were inserted into E. coli
strain CC118, and then transferred to Y.
enterocolitica strain JB580v via conjugation.
These mutants were screened for insertion of the
said transposon by ampicilin Selection
(Different colonization pattern with and without
insert) and were performed only at 18 Southern
random samples which were screened for
kanamycinresistens who sat on transposons.
Preliminary studies showed that the best
infection method on test animals (6-7 weeks old
BALB / c mice) were intraperitoneal (IP) injection
(Direct injection into abdomen), with approx. 107 colonyforming
units (cfu), followed by capture from
spleen after 48 hours.
In order to avoid injecting the weakened auxotrofe
bacteria were selected transposonmutanter
grown on minimal medium.
STM
Method to screen for attenuated bacteria
was referred done with InDesign tours-tagged
Mutagenesis (STM). STM is a negative
selection method where this experiment uses
their pool of mutants (21 pools of 96 mutants)
to infect groups of three mice. What is special about
STM is that the inserted DNA has a special
signature trademark, a signature tag that is a little
familiar sequence which can subsequently be found by
Southern blot hybridicering. During the initial
screening infected mice and bacteria recovered
from spleen. These are mixed and transferred to 96
chamber micro titter plate. Then screener Mon
with proper original bacteria (input - all
original mutants) and those who were found in the spleen
(Output - the survivors). That way you can
find the attenuated bacteria by their lack
presence in the spleen accumulated
bacteria (negative selection), which is seen as
missing or weak hybridicering compared to the
from input (the first part of Fig. 1 from the article). Then
was taken to the mutants that demonstrated a lack
hybridicering - so attenuated virulence, and drove
secondary screening with the same procedure as
before but with two conditions (2x3 mice).
This should help reduce the risk to take
mutants actually was not attenuated (second part
fig. One from the article).
Subsequently, attenuated mutants studied
and classified phenotypic and by cloning
followed by sequencing and was performed
competition and vækstassays.
Results
Based on the two initial STM screening
they found up to 68 mutants with attenuated
virulence. As expected, a proportion of these being
mutated in pYV and it was indeed found that 29
were pYV mutants. This was examined by
growth pattern on LBMOX plates and kanamycinsensitivitet.
This means that out of the original 68
mutants were 39 who had mutations in
chromosomal virulensgener. Out of these were in
16 cases observed the same phenotype
(Aggregation in liquid medium, and turbid growth
LB agar plates) due to a defect in LPS
synthesis and thus synthesis of O antigen, which
is essential for Y. enterocolitica virulence. So
there were now 23 mutants back where mutation
sat somewhere else than in pYV or LPS synthesis
genes. To be confirmed whether they were really
weakened, it was checked every 68 in a competition
assay in mice, which measured about bacteria
fared worse than wild-type Y.
Enterocolitica. For this test it was found that 10 of
the 23 were actually weakened and were overall 55 of the
68 fact impaired. This in their eyes, good result
attribute the dual STM screenings
procedure.
By pYV mutants was especially type III
sekretionsgenerne that the insert was to identify and
especially in lcrV and yscL, this could indicate a
'Hot spot' for insertion of transposons mini-Tn5
Km2 just here (Fig. 2 from the article).
Of the 16 with suspected mutation in LPS / Oantigen
synthesis apparatus, this was verified
by sequencing (Fig. 2 from the article).
The remaining 10 chromosomal mutants were also
by cloning and sequencing studied. Here was
Mon insert, and thus the disruption of genes
coded outer membrane synthesis (yifH and nlpD)
nutrient uptake / acquisition (pstC and irp1) and stress
response (dnaJ). The last five had mutations in
genes homologous to E. coli genes coding for DNA
topoisomerase I (topaz), phage shock protein
operon (pspC) and two were found with
mutations in yibP, a gene of unknown function in E.
coli. Finally found a mutation in a sequence
there was inconclusive in Genbank database.
(Fig. 2 from the article).
NB. The article makes the reconstruction of pspC
mutant for further study. This, I
explain in question 3 later.
Discussion
They were in this study demonstrated that STM technique can
used to identify a number of genes in Y.
enterocolitica with more or less important
for its virulence. They were reidentificeret a series
Essential virulensgener in pYV (type III
secreting) and in LPS synthesis genes in
chromosome. The results from the chromosomal
mutants recalled earlier STM results
the pathogenic Gram-negative bacteria Salmonella
typhimurium and Vibrio cholera which also
found that bacteria with mutations in genes
involved in O-anti-reunion theory, stress response and
nutrient uptake / acquisition had weakened
virulence. The most sensational they found out
of was to pspC gene, homologous to the gene in E.
coli is part of the phage shock protein operon,
had significance for Y. enterocolitica's virulence.
This mutant exhibited very low virulensgrad in
test animal, but grew well in vitro. This gene has
in E. coli no known function, but we see an increased
expression by overexpression of sekretinproteiner,
inter alia used in Type III secreting. So
a suspected link to important virulensgener.
There must be investigated further in this
context for understanding the importance of pspC
gene and its virulensfunktion.
The article has the sense that it shows that a long
number virulensgener be identified quite
fast and relatively simple in STM method.
This means that other pathogenic bacteria, Gram-positive
as well as Gram-negative, rapidly
screened virulensgener, which can have significant
important for disease treatment, drug development
and our general knowledge of
pathogenic bacteria.
It is also useful in connection with that
more and more organisms (particularly bacteria) genome
sequences, and the need to understand
importance of individual genes in the studied
organism.
General works article vellavet over
trials building, reproducibility and
general structure of the article.
Specific questions
Question 1:
Signature-Tagged Mutagenesis (STM) is a technique
used to examine genes function in
vivo. You make a transposon with a reputation
signature sequence, which then transferred to the
organism to be studied mostly by E. coli.
Transposons, with signature tag, sits a
random location in the genome or a plasmid, and will
thus very likely disturb and
destroy the gene and thus the translated
protein. In STM, each transposon its own
unique signature, and can subsequently
found by DNA probehybridicering (Southern
only). Unlike ordinary random transposon
Mutagenesis (RTM) where each
mutation need a testdyr to negative control
here you can use a whole pot (in case the article
using the 96 per. testdyr) and find a specific
attenuated mutant. In the original design of the STM as
was designed by Hensel a al.1 used, as in
experiment here hybridiseringsprober, now a
Another method of PCR detection, developed by
Lehoux a al.2 used. Here is the idea that each
roof amplified with tag-specific primers.
The presence of a PCR product from a roof in
input pool and the absence of the same PCR product from
output pool shows the loss of the specific mutant
during the negative selection screening.
The advantages of STM with both signature-tag
detection methods are so clear, since it both
saves time (and thus money) and testdyr. Outright
disadvantages compared to RTM, I have a hard time
find.
Question 2:
The article dobbeltscreener their authors
mutants. This is done by making probes are
designed to hybridize to the inserted transposon
(Base pairing principle). When one acquires bacteria
from mouse spleen, the bacteria are finding that,
be those who are able to infect the mouse, so
the bacteria we do not want to investigate. But if
found the bacteria there, so to speak, is missing
can you find the gene is disrupted by
look at the input pool. This makes the order that the
primarily attenuated bacteria they inject, and
Following this double screening obtains the a
result which states that 55 of the original 68
mutants were effectively attenuated, which corresponds to
81%. This is an improvement over previous
results, where only 45% of those screened
mutants were effectively attenuated (here screened Mon
Vibrio cholera).
1 Hensel M, Shea JE, Gleeson C, Jones MD, Dalton E, Holden DW:
Simultaneous identification of bacterial virulence genes by negative
selection. Science 1995, 269:400-403.
2 Lehoux DE, Sanschagrin F, Levesque RC: Defined oligonucleotide tag
pools and PCR screening in signaturetagged Mutagenesis of essentialism
genes from bacteria. Biotechniques 1999, 26:473-478, 480th
Question 3:
The trial will determine whether the phenotypic
changes caused by transposon mutagenesis or
whether there is a spontaneous mutation. In all of
near a single mutant, they may exclude
spontaneous mutation. The exception is gay gallery to
E. coli gene pspC. Therefore, the authors construct
three new pspC mutants in the same strain of Y.
enterocolitica. One new mutant would be a
copy of the mutant from the original trial
while the other two would be mutants with a
insertion-deletion in the gene, with a kanamycinresistens
cassette. The copy was made by the
inserted fragment from the original was propagated
by PCR and equality into a vector and transferred to Y.
enterocolitica by conjugation. The other two were
made by kanamycin resistance cassette was
inserted in two different orientations of the ring. Of
these three new bacteria were again conducted a
konkurrenceassay. The results of this assay
with the reconstructed mutants found in Table 3 in
article and can be seen in comparison with pspC
results from the first konkurrenceassay (Table
2) the results are very similar - mutants
infect mice poorly, but grows well in
Vitro. The finds therefore that mutations in pspC
gay gallery Y. enterocolitica substantially weakens
its virulence.
Question 4:
Type III secreting (TTSS) is a complex
trans-membrane protein structure found in many
Gram-negative bacteria, pathogens and nonpatogene.
In the pathogenic used it mainly to
secrete proteins that help bacteria to
infect its host, and therefore there is strong
correlation between virulence and the presence
of TTSS. Hall Marketplace in the system is the needle.
It sits anchored only to the inner membrane in
the inner membrane ring, via a connector over
peptidoglykanlaget to the outer membrane ring
after which it has crossed the entire bacterial
wall system. The actual needle hole with a diameter
around. 3 nm, which means that most effector
proteins to be secreted in the unfolded state. (See fig.
1 below)
Figure 1 Schematic drawing of the TTSS. From Wikimedia Commons
Previously it was thought that the needle was able to
perforating the host cells, but this picture is
now changed. Now the theory that it emits
proteins called translocators, which form a pore
in the host cell membrane through which other
effectors can flow.
The article will transposons in one of the mutants
inserted into a sycT-yopM gene region, and this
suppose they can affectation genes involved
in TTSS. They examine whether it is due to errors in
secretion of proteins called yop (Yersinia
outer proteins), which helps to perforate
host cells that are the cause of the weakened
virulence. Therefore examined the protein composition
in growth medium for the said
mutants, and compared with wild type (Fig. 2).
In the experiments, they found no visible difference in
protein picture for the mutants and wild type.
Question 5:
There are many bacteria use TTSS in
their virulence. Of these species may
include Shigella (bacillary dysentery), Salmonella
(Typhoid fever), Escherichia coli (food poisoning)
Burkholderia (glanders), Yersinia (plague), Chlamydia
(Chlamydia) and Pseudomonas. The high prevalence
of TTSS of pathogens may be due to TTSS
cassette can be transferred horizontally between species.
As an example I will use the bacterium Shigella.
The most common cause of shigellainfektion is
contamination of food with faecal matter. After you have
eating infected food or water will bacterium
invade its host through epithelial cells of the colon
through its TTSS. This injects the IPAD
protein into epithelial cells, which triggers it to
incorporate the bacterium. This vakuole breaking down
of the divisional IpaB and IPAC proteins. Here may
now divide, and furthermore it may by
ICSA protein "take over" epithelial actin
polymeriseringsapparat which it uses to
be shot around the cell, and into other
neighboring cells, which then becomes infected. Total causes
Shigella approx. 165 million annual cases of
dysentery, and approx. A million deaths - almost
exclusively in developing countries
Obituary: A wonderful bird is forever dead, Madagascar snipe
The extremely rare bird Madagascar alaotra-Grebe are experts has been declared extinct. Yes dear reader, you heard right, extinct. It is a fact we ornithologist, obituaries are quite happy, now can we write a little text. But now it is a shame for us all. Rest in peace dear Madagascar alaotra-Grebe, Tachybaptus rufolavatus.
Ornithologist-obituary Tøger Torkel, cand. cons.
Ornithologist-obituary Tøger Torkel, cand. cons.
Acid-base titration. Setting the hydrochloric
Purpose
Determination of the concentration of hydrochloric acid in an unknown solution by setting opposite a urtiter substance (primary standard).
Method
A known amount of Tris, a known concentration titrated with the unknown solution of HCl.
Theory
One mole of hydrochloric acid reacts with a mole Tris. Analyses with hydrochloric acid has a molaritet between 0.08 and 0.12 M are examined, we calculated the approximate folder point to be at 16.5 ml added HCl (from the information that the relationship between drug volume of Tris and HCl is 1:1 and hydrochloric acid concentration on average is 0.1 M).
Reaction Equations
Fabric Equation:
(HOCH2) 3C-NH2 (aq) + HCl (aq) ® (HOCH2) 3C-NH 3 + (aq) + Cl-(aq)
Ionligning:
R-NH2 (aq) + H + (aq) ® R-NH3 + (aq)
Results
We chose the unknown acid # 4
Grovtitrering:
- Mass of Tris: 1.5448 g - 1.3251 g (tray weight + residue) = 0.2197 g
- We titrated 15.5 ml HCl
1st accurate titration:
- Mass of Tris: 1.5109 g - 1.3251 g (tray weight + residue) = 0.1858 g
- We titrated 12.8 ml HCl
2nd accurate titration:
- Mass of Tris: 1.5615 g - 1.3197 g (tray weight + residue) = 0.2418 g
- We titrated 16.9 ml HCl
Calculations
We calculate the concentration of the unknown acid solution:
Grovtitrering:
We calculate the first n (Tris):
As mentioned earlier reacting hydrochloric acid and tris 1:1, so
n (Tris) = n (HCl) = 0.0018 mol
So the concentration of hydrochloric acid is then:
1st accurate titration:
We calculate the first n (Tris):
So the concentration of hydrochloric acid is then:
2nd accurate titration:
We calculate the first n (Tris):
So the concentration of hydrochloric acid is then:
Average of the three provisions:
Discussion
Our tests were very successful. Grovtitreringen indicated that we had a hydrochloric acid concentration of 0.12 M. And after making two accurate titrations, we were unsubstantiated assumption, and we conclude therefore that our unknown acid (IV), the concentration 0.12 M.
Exercise 3b: Synthesis of iron (II) sulfate heptahydrate
Purpose
Production of iron (II) sulfate heptahydrate FeSO4 × 7H2O.
Method
Iron nails are dissolved in sulfuric acid under hydrogen development and formation of iron (II) sulfate in solution.
Theory
Iron is less electro-negative than hydrogen, and therefore will be formed iron (II) ions in the solution with sulfuric acid. We use an excess of iron, which causes precipitation of metals which are more electronegative than iron, and prevents oxidation of Fe2 + to Fe3 +.
Reaction Equations
Ionligning:
Fe (s) + 2 H + (aq) → Fe2 + (aq) + H2 (g)
Fabric Equation:
Fe (s) + H2SO4 (aq) + 7 H2O (l) → FeSO4 × 7 H2O (s) + H2; (g)
The following reaction will proceed by oxidation with atmospheric oxygen:
2 H2O (l) + 4 Fe2 + (aq) + O2 (g) + 8 OH-(aq) → 4 Fe (OH) 3 (s)
Results
The mass of jernsøm: 4.95 g
Quantity added sulfuric acid: 4 ml
Yield of iron (II) sulfate heptahydrate: 12.88 g
Calculations
Fe (s)
+
H2SO4 (aq)
+
7 H2O (l)
→
FeSO4 × 7 H2O (s)
+
H2 (g)
m
4.95 g
20.02 g
M
55.85 g / mol
278.022 g / mol
n
0.0886 mol
0.072 mol
0072
V
4 ml
c
18 M
As you can see, sulfuric acid is the limiting factor.
The theoretical yield is: 20.02 g.
Dividend rate:
Discussion
As seen, we've got a relatively small yield. This is mainly due to the reaction failed to run to completion, because when we stopped cooking there were still large hydrogen development around the seams, and we believe that if we eg. gave the half to full hour more, then there was formed a lot more FeSO4 × 7H2O. Another small source of error is that during the trial (especially filtration) may lose a little of the substance.
Exercise 3c: In vitro experiments
1st attempt
- 4 M NH4Cl:
pH read about. 5th
Reaction Equations:
NH4Cl (aq) → NH4 + (aq) + Cl-(aq)
NH4 + (aq) + H2O (aq) ⇌ NH3 (aq) + H3O + (aq)
pH is calculated to:
As seen, there is no large deviation between measured and calculated pH value.
- 2 M NH3:
pH read about. 11th
Reaction Equation:
NH3 (aq) + H2O (aq) ⇌ NH4 + (aq) + OH-(aq)
pH is calculated to:
pH values are very close, considered the highest we could measure with the indicator paper was the 11th
2nd attempt
We saw no color change or precipitate, but there was a gas production, and we measured the pH of the gas to 11
Ionligning:
NH4 + (aq) + OH-(aq) → NH3 (g) + H2O (l)
Fabric Equation:
NH4Cl (aq) + NaOH (aq) → NH3 (g) + H2O (l) + NaCl (aq)
When we measure the gas has a pH of approx. 11, it fits quite well with the ammonia in gaseous form is being formed.
3rd attempt
Change Color: clear blue → Blue (cloudy / cloudy) → dark blue (clear)
Precipitate: a drop of NH3 resulting dark blue precipitate, and 6 drops dissolve the precipitate.
Reaction:
When copper (II) sulfate dissolved. Kobberionen is (bright) blue in water:
CuSO4 (s) ® CU2 + (aq) + SO42-(aq)
Ammonia is a weak base:
NH3 (aq) + H2O (l) ⇌ NH4 + (aq) + OH-(aq)
Addition of ammonia to copper (II) sulfate gives first a precipitate of blue copper (II) hydroxide:
CU2 + (aq) + 2 OH-(aq) ⇌ Cu (OH) 2 (s) (*)
In excess of ammonia formed by deep mørkebå kompleksion tetraamminkobber (II) ion:
CU2 + (aq) + 4 NH3 (aq) ⇌ Cu (NH3) 42 + (aq)
Doing shift equation (*) to the left, copper (II) hydroxide is dissolved, and obtained a very dark blue solution:
Cu (OH) 2 + 4 NH3 (aq) ⇌ Cu (NH3) 42 + + 2 OH
4th attempt
Change Color: clear milky → → clear
Precipitate: a drop of NH3 resulting white precipitate, and 7 drops leads to precipitate dissolved.
When zinc sulphate is dissolved:
ZnSO4 (s) ® Zn2 + (aq) + SO42-(aq)
Addition of ammonia to zinc sulphate gives first a precipitate of white zinc hydroxide:
Zn2 + (aq) + 2 OH-(aq) ⇌ Zn (OH) 2 (s) (*)
In excess of ammonia formed the complex ion tetraamminzink (II) ion:
Zn2 + (aq) + 4 NH3 (aq) ⇌ Zn (NH3) 42 + (aq)
Doing shift equation (*) left, zinc hydroxide is dissolved, and obtained a clear solution:
Zn (OH) 2 (s) + 4 NH3 (aq) ⇌ Zn (NH3) 42 + + 2 OH-(aq)
Determination of the concentration of hydrochloric acid in an unknown solution by setting opposite a urtiter substance (primary standard).
Method
A known amount of Tris, a known concentration titrated with the unknown solution of HCl.
Theory
One mole of hydrochloric acid reacts with a mole Tris. Analyses with hydrochloric acid has a molaritet between 0.08 and 0.12 M are examined, we calculated the approximate folder point to be at 16.5 ml added HCl (from the information that the relationship between drug volume of Tris and HCl is 1:1 and hydrochloric acid concentration on average is 0.1 M).
Reaction Equations
Fabric Equation:
(HOCH2) 3C-NH2 (aq) + HCl (aq) ® (HOCH2) 3C-NH 3 + (aq) + Cl-(aq)
Ionligning:
R-NH2 (aq) + H + (aq) ® R-NH3 + (aq)
Results
We chose the unknown acid # 4
Grovtitrering:
- Mass of Tris: 1.5448 g - 1.3251 g (tray weight + residue) = 0.2197 g
- We titrated 15.5 ml HCl
1st accurate titration:
- Mass of Tris: 1.5109 g - 1.3251 g (tray weight + residue) = 0.1858 g
- We titrated 12.8 ml HCl
2nd accurate titration:
- Mass of Tris: 1.5615 g - 1.3197 g (tray weight + residue) = 0.2418 g
- We titrated 16.9 ml HCl
Calculations
We calculate the concentration of the unknown acid solution:
Grovtitrering:
We calculate the first n (Tris):
As mentioned earlier reacting hydrochloric acid and tris 1:1, so
n (Tris) = n (HCl) = 0.0018 mol
So the concentration of hydrochloric acid is then:
1st accurate titration:
We calculate the first n (Tris):
So the concentration of hydrochloric acid is then:
2nd accurate titration:
We calculate the first n (Tris):
So the concentration of hydrochloric acid is then:
Average of the three provisions:
Discussion
Our tests were very successful. Grovtitreringen indicated that we had a hydrochloric acid concentration of 0.12 M. And after making two accurate titrations, we were unsubstantiated assumption, and we conclude therefore that our unknown acid (IV), the concentration 0.12 M.
Exercise 3b: Synthesis of iron (II) sulfate heptahydrate
Purpose
Production of iron (II) sulfate heptahydrate FeSO4 × 7H2O.
Method
Iron nails are dissolved in sulfuric acid under hydrogen development and formation of iron (II) sulfate in solution.
Theory
Iron is less electro-negative than hydrogen, and therefore will be formed iron (II) ions in the solution with sulfuric acid. We use an excess of iron, which causes precipitation of metals which are more electronegative than iron, and prevents oxidation of Fe2 + to Fe3 +.
Reaction Equations
Ionligning:
Fe (s) + 2 H + (aq) → Fe2 + (aq) + H2 (g)
Fabric Equation:
Fe (s) + H2SO4 (aq) + 7 H2O (l) → FeSO4 × 7 H2O (s) + H2; (g)
The following reaction will proceed by oxidation with atmospheric oxygen:
2 H2O (l) + 4 Fe2 + (aq) + O2 (g) + 8 OH-(aq) → 4 Fe (OH) 3 (s)
Results
The mass of jernsøm: 4.95 g
Quantity added sulfuric acid: 4 ml
Yield of iron (II) sulfate heptahydrate: 12.88 g
Calculations
Fe (s)
+
H2SO4 (aq)
+
7 H2O (l)
→
FeSO4 × 7 H2O (s)
+
H2 (g)
m
4.95 g
20.02 g
M
55.85 g / mol
278.022 g / mol
n
0.0886 mol
0.072 mol
0072
V
4 ml
c
18 M
As you can see, sulfuric acid is the limiting factor.
The theoretical yield is: 20.02 g.
Dividend rate:
Discussion
As seen, we've got a relatively small yield. This is mainly due to the reaction failed to run to completion, because when we stopped cooking there were still large hydrogen development around the seams, and we believe that if we eg. gave the half to full hour more, then there was formed a lot more FeSO4 × 7H2O. Another small source of error is that during the trial (especially filtration) may lose a little of the substance.
Exercise 3c: In vitro experiments
1st attempt
- 4 M NH4Cl:
pH read about. 5th
Reaction Equations:
NH4Cl (aq) → NH4 + (aq) + Cl-(aq)
NH4 + (aq) + H2O (aq) ⇌ NH3 (aq) + H3O + (aq)
pH is calculated to:
As seen, there is no large deviation between measured and calculated pH value.
- 2 M NH3:
pH read about. 11th
Reaction Equation:
NH3 (aq) + H2O (aq) ⇌ NH4 + (aq) + OH-(aq)
pH is calculated to:
pH values are very close, considered the highest we could measure with the indicator paper was the 11th
2nd attempt
We saw no color change or precipitate, but there was a gas production, and we measured the pH of the gas to 11
Ionligning:
NH4 + (aq) + OH-(aq) → NH3 (g) + H2O (l)
Fabric Equation:
NH4Cl (aq) + NaOH (aq) → NH3 (g) + H2O (l) + NaCl (aq)
When we measure the gas has a pH of approx. 11, it fits quite well with the ammonia in gaseous form is being formed.
3rd attempt
Change Color: clear blue → Blue (cloudy / cloudy) → dark blue (clear)
Precipitate: a drop of NH3 resulting dark blue precipitate, and 6 drops dissolve the precipitate.
Reaction:
When copper (II) sulfate dissolved. Kobberionen is (bright) blue in water:
CuSO4 (s) ® CU2 + (aq) + SO42-(aq)
Ammonia is a weak base:
NH3 (aq) + H2O (l) ⇌ NH4 + (aq) + OH-(aq)
Addition of ammonia to copper (II) sulfate gives first a precipitate of blue copper (II) hydroxide:
CU2 + (aq) + 2 OH-(aq) ⇌ Cu (OH) 2 (s) (*)
In excess of ammonia formed by deep mørkebå kompleksion tetraamminkobber (II) ion:
CU2 + (aq) + 4 NH3 (aq) ⇌ Cu (NH3) 42 + (aq)
Doing shift equation (*) to the left, copper (II) hydroxide is dissolved, and obtained a very dark blue solution:
Cu (OH) 2 + 4 NH3 (aq) ⇌ Cu (NH3) 42 + + 2 OH
4th attempt
Change Color: clear milky → → clear
Precipitate: a drop of NH3 resulting white precipitate, and 7 drops leads to precipitate dissolved.
When zinc sulphate is dissolved:
ZnSO4 (s) ® Zn2 + (aq) + SO42-(aq)
Addition of ammonia to zinc sulphate gives first a precipitate of white zinc hydroxide:
Zn2 + (aq) + 2 OH-(aq) ⇌ Zn (OH) 2 (s) (*)
In excess of ammonia formed the complex ion tetraamminzink (II) ion:
Zn2 + (aq) + 4 NH3 (aq) ⇌ Zn (NH3) 42 + (aq)
Doing shift equation (*) left, zinc hydroxide is dissolved, and obtained a clear solution:
Zn (OH) 2 (s) + 4 NH3 (aq) ⇌ Zn (NH3) 42 + + 2 OH-(aq)
Freshwater turtles in Denmark - biology report
Purpose
There have been sighted European freshwater turtles (Emys orbicularis) in Denmark, modalities by Salten Å (we look at two here) and Igelsø. The question which arises is, whence comes this? We were presented with two theories, but a third exists:
1st A natural teacher 's belief that swamp turtle has survived in central Jutland, and is not extinct 2000 years ago, as we thought it would. They were after his testimony to a small population of the original Danish (North European) subspecies. This survival was achieved without genuine described observations.
2nd That it is purchased pet is either put out a few copies, or that they have been systematically deployed from the genus would then be of those from the Balkans, namely Serbia and Ukraine, as it is these you can buy in pet shops.
3rd A third theory, which we do not get directly out, it is that it is a new subspecies or race of the vulnerable in the years which should have put animals out (which can not be more than 100 years, if at all) should have evolution to a sub-race. This would occur only if the individual mutated, this is likely, but the probability is said to be so small that this theory can be readily dismissed.
You could say much about the first theory, if I should come with a comment, I would immediately call it pop-biology, as this theory attract much media interest but not directly by the large in itself. Since that Nature which we are presented with does not come with any concrete evidence but just a lot of circumstantial evidence and postulates. However, one can say that it has been widely accepted, for example may be mentioned that in a otherwise very informative online dictionary as wikipedia, you can read, "European Pond Turtle (Emys orbicularis) was rediscovered in Denmark, Jutland in 1997." Here we must say that they indicate that it is not just a few exposed specimens, but an actual population.
Our aim with this exercise is to determine where these turtles observed really from. We have DNA from Salten Å, Poland, Ukraine, Greece and Serbia. If their DNA matches with those from the Balkan regions, we must conclude that they are exposed, whereas, they are different, one can say that they are surviving copies of the 2000 years old Midtjyske subspecies.
Theory
There is some theoretical substance which must be here, so I chose to break it down into these four sub-themes:
· European pond turtle:
The European pond turtle, Emys orbicularis is a turtle found in South and Central Europe, West Asia and North Africa, but since this has such widespread areas describing often Europe as a undeart named Emys orbicularis orbicularis to differentiate between it and another also widespread subspecies Emys orbicularis orientalis.
It lives in slow running water, and hibernation for up to seven months. It is eggs, as many other reptiles and for them to be able to hatch, the warmest summer month's average temperature exceed 18 ° C.
· Electrophoresis and restriction enzymes:
An electrophoresis is a kind of purification of DNA pieces. But before we can proceed with electrophoresis, you should have cut its DNA into pieces. To this user Mon restriction enzymes that are specifically designed to cut after a special code. The enzymes we used is called ECO R1 and R1 PST, and these rocks, respectively, by G Eftf. of AATTC and CTGCA Eftf. of G, this looks like this:
When you have cut the DNA into smaller pieces add, a heavy marker buffer so that later in the experiment can see how much DNA piece is horizontal.
The gel, called the sieve is made of agarose, deionized water and gel buffer. Agarose is as the name suggests a sugar (carbohydrate), extracted from Japanese red algae. The volume of agarose determines how close the filter is, you have small sequences of DNA wants a dense filter, and you add therefore very agarose. When we were producing the dissolved 0.5 grams we agarose in 50 mL of TAE buffer (see below) and heat it in microwave oven so that the two substances melted together. Then pour the still liquid gel into the bathtub where it solidifies.
When one has devoted his DNA material in the gel sets Mon flow to each end of the bath. At one end is the anode (positive electrode) and the other is the cathode (negative electrode). It has reversed the gel such that the end was allocated mix of facing away from the anode. This is because DNA molecules are negatively charged, so they will migrate towards the anode. Travel through the gel takes place at different speeds depending on molecule size. The larger molecules are the slower they move through the gel. The reason we have added markørbufferen is that it will always move faster than some of the DNA molecules, so when the cursor has come down to the other end off the Mon stream.
· PCR, color marker and TAE buffer:
One critical problem in this study is the amount of DNA material to do a proper electrophoresis, which will give a realistic picture, you need very DNA material. If you do not, you can use a molecular biological reproduction method called PCR, Polymerase Chain Reaction (polymerase chain reaction) which were invented as early as 1983 by Kary Mullis, who 10 years later received the Nobel Chemistry Prize for precisely this discovery. As the name suggests, it is an artificially produced replikationsmetode. It is relatively simple, I have tried to illustrate in the figure below:
· Step 1: DNA is poured into a container, together with polymerase and free nucleotides.
· Step 2: The entire substance is heated to slightly above 65 ° C (at BIO RAD machine is more than 90 ° C) at this temperature break hydrogen bonds between nucleotides in the DNA for. And there is no risk of damage to nucleotides since they first melts at +300 ° C
· Step 3: The substance cooled again to below 65 ° C, after which free nucleotides are committed to the free seats in the DNA, and in this way has now produced two identical DNA strands, this sequence can be done again, and each time doubled the number of DNA pieces.
The color marker I mentioned in the theory section on electrophoresis, which must say indicate how far the DNA pieces is reached in the gel, they would not want to walk completely through it. This implies that the pieces of the cursor is less than the smallest pieces of DNA. But it is also made such in our experiments, so the cursor has a speed of 500bp corresponding to a sequence of 500 base pairs. The highlights of this marker are doing will be visible to the naked eye, so that as said on when to turn off.
TAE Buffer (Tri-Acetate-x) key role is to keep DNA stable at pH 7.6. This must be done because a pH value of 7.6 ensures that the DNA remains negatively charged. The image on the right shows AMP Adenosine monophosphate, a nucleotide, and one can see the negative end with O-.
· Size marker:
In this study, it is important to compare the size of the cut DNA pieces, so to have a reference using a so-called size marker, which in this case is a DNA size marker. The DNA we used came from a virus and we must assume that it is a virus that scientists have determined its entire genome, and therefore all base sequences. So when we know the size of all fragments, measuring at how far they have walked in the gel, we can make a graph in a Semi-ordinates. This shows the number of base pairs up the second axis and number wandered mm. in the gel out of the first axis. The results seen in the section entitled "Results".
Method
see Exercise Guide.
Error Sources
During the experiment we came to pour TAE buffer into the tube at the end. For this reason, our DNA samples slightly smudged. But this did not alter the outcome, which ultimately was the best, compared to the other groups. A fault is it, but maybe an ameliorative one of its kind.
Another possible source of error, although not documented, it could be that gel is not completely homogeneous but the concentration of sucrose could be just a fraction higher in some areas of the gel, this would then lead to variations in travel.
Results
I have chosen to use the manufacturer's (BIO-RAD) image of the gel, as this will give the most accurate results.
1: Size marker
2: Salten 1
3: Salten 2
4: Poland
5: Ukraine
6: Greece
7: Serbia
Here are the results from the regression of our viral DNA:
The blue line as seen, exponential regression, and the function you get out of it is drawn with thin dotted line.
I then made a rule so you can calculate the number of base pairs from the number of migrated mm. However, I have chosen to exclude the first point with 23,130 base pairs, since it differs from the otherwise straight lines in the Semi-coordinates, but with the other results are the rule to look like this: where f (x) is the number of base pairs, and x is the number of migrated mm. From this model and the measured distances from the gel, you can run a table showing the different band base pairs for the various turtles DNA:
Salten 1
Salten 2
Poland
Ukraine
Greece
Serbia
3648
2820
2970
3821
3189
2969
2969
1112
1,773
3078
2620
2070
740
862
1117
740
1061
1116
Discussion
Looking at the table of turtles and their base pair sizes, you can relatively easily determine that the turtle "Salten a" face, and that it originated from the Ukrainian subspecies. The biggest difference in base pairs between the two, is at 173 base pairs. One must say that it is relatively little when 173 base pairs almost nothing is, and we found that there was an uncertainty marker around. 100-200 base pairs. When we look at Salten 2 see it immediately worse. It has no immediate similarities with any of the others. This is a strong indication that the strains from the original northern European subspecies, although they were counted as extinct 2000 years ago. But you must tell the result's defense that if only the population is small enough, it will have been very difficult to spot in the wild. Turtle overwinters as I said in seven months, and there are otherwise here vegetation around lakes and rivers are partially gone, so that one could easily spot it. It should also be said that it is a nocturnal animal, and she will feel even remotely threatened, it glides quietly and silently into the water.
Another fact which you can see from the gel, is that they are all very closely related, however, we knew this beforehand, but it is clearly illustrated in the table in it that they all have a DNA sequence approximately 3000 base pairs. More specifically, the average of the band of 3016, and the largest deviation from this is Greece with 173 base pairs, and again this is within the uncertainty of the cursor.
We were initially told of that, "... if this summer's hottest month average temperature is below 18 °, their eggs do not hatch" This fact, I examined and considered from DMI's website a depiction of Midtjyllands climate from 1961 to 1990:
And, as evidenced by the dark line indicating the mean temperature has gennemsnitstemperatu-clean in the hottest months (July to an average of 15.4 ° C) was not near the required 18 ° C, so it provides a dilemma. Either we must wholly reject our results, which otherwise strongly suggesting that they originated from the original Danish subspecies, or may choose to ignore the fact that in a number of years at 29 years, there has been a temperature of 18 ° C.
If our restriction enzymes had not worked our gel would have given us a very bad image to work with, for in that there would not be cut anywhere in the DNA, it would become one very long base sequence that would move very slightly in the gel because agarosemængden adapted very small pieces. Therefore we would only see one big line straight out of the well, and we would not be able to use this result to something.
Conclusion
Now that I have come to the conclusion that I should have one definitive answer. This is just not true. I have two contradictory arguments: If you choose only to look at the gel, I find that swamp turtle Salten 1 is an exposed "poor" because the composition of DNA sequences recalls hitting a lot about it from Ukraine, which also is available for purchase in Danish animal dealers. And Salten 2 is a surviving subspecies, who has lived in semi-hiding in 2000 years.
If you choose the other hand also to look at the graph from DMI, we see here the contradictory argument. There have in the period 1961-1990 was no one in July with an average temperature of the required 18 °. This indicates that it is not a survivor and childbearing population, as it simply would not have a month which is warm enough to hatch its eggs.
However, I would say that should this trial be successful, we must throw away the argument with the necessary 18 °, as this completely spoils the otherwise fine performance; that there is a surviving wild swamp tortoise in Denmark. Or should we turn it on and find a solution to this problem. And a possible theoretical solution to the problem could be that the turtle has been exposed to a selective pressure that would penalize the ability to lay eggs which can hatch at lower temperatures, so that there simply has been an evolution in relation to the freshwater turtles in southern Europe. This would give very good sense.
There have been sighted European freshwater turtles (Emys orbicularis) in Denmark, modalities by Salten Å (we look at two here) and Igelsø. The question which arises is, whence comes this? We were presented with two theories, but a third exists:
1st A natural teacher 's belief that swamp turtle has survived in central Jutland, and is not extinct 2000 years ago, as we thought it would. They were after his testimony to a small population of the original Danish (North European) subspecies. This survival was achieved without genuine described observations.
2nd That it is purchased pet is either put out a few copies, or that they have been systematically deployed from the genus would then be of those from the Balkans, namely Serbia and Ukraine, as it is these you can buy in pet shops.
3rd A third theory, which we do not get directly out, it is that it is a new subspecies or race of the vulnerable in the years which should have put animals out (which can not be more than 100 years, if at all) should have evolution to a sub-race. This would occur only if the individual mutated, this is likely, but the probability is said to be so small that this theory can be readily dismissed.
You could say much about the first theory, if I should come with a comment, I would immediately call it pop-biology, as this theory attract much media interest but not directly by the large in itself. Since that Nature which we are presented with does not come with any concrete evidence but just a lot of circumstantial evidence and postulates. However, one can say that it has been widely accepted, for example may be mentioned that in a otherwise very informative online dictionary as wikipedia, you can read, "European Pond Turtle (Emys orbicularis) was rediscovered in Denmark, Jutland in 1997." Here we must say that they indicate that it is not just a few exposed specimens, but an actual population.
Our aim with this exercise is to determine where these turtles observed really from. We have DNA from Salten Å, Poland, Ukraine, Greece and Serbia. If their DNA matches with those from the Balkan regions, we must conclude that they are exposed, whereas, they are different, one can say that they are surviving copies of the 2000 years old Midtjyske subspecies.
Theory
There is some theoretical substance which must be here, so I chose to break it down into these four sub-themes:
· European pond turtle:
The European pond turtle, Emys orbicularis is a turtle found in South and Central Europe, West Asia and North Africa, but since this has such widespread areas describing often Europe as a undeart named Emys orbicularis orbicularis to differentiate between it and another also widespread subspecies Emys orbicularis orientalis.
It lives in slow running water, and hibernation for up to seven months. It is eggs, as many other reptiles and for them to be able to hatch, the warmest summer month's average temperature exceed 18 ° C.
· Electrophoresis and restriction enzymes:
An electrophoresis is a kind of purification of DNA pieces. But before we can proceed with electrophoresis, you should have cut its DNA into pieces. To this user Mon restriction enzymes that are specifically designed to cut after a special code. The enzymes we used is called ECO R1 and R1 PST, and these rocks, respectively, by G Eftf. of AATTC and CTGCA Eftf. of G, this looks like this:
When you have cut the DNA into smaller pieces add, a heavy marker buffer so that later in the experiment can see how much DNA piece is horizontal.
The gel, called the sieve is made of agarose, deionized water and gel buffer. Agarose is as the name suggests a sugar (carbohydrate), extracted from Japanese red algae. The volume of agarose determines how close the filter is, you have small sequences of DNA wants a dense filter, and you add therefore very agarose. When we were producing the dissolved 0.5 grams we agarose in 50 mL of TAE buffer (see below) and heat it in microwave oven so that the two substances melted together. Then pour the still liquid gel into the bathtub where it solidifies.
When one has devoted his DNA material in the gel sets Mon flow to each end of the bath. At one end is the anode (positive electrode) and the other is the cathode (negative electrode). It has reversed the gel such that the end was allocated mix of facing away from the anode. This is because DNA molecules are negatively charged, so they will migrate towards the anode. Travel through the gel takes place at different speeds depending on molecule size. The larger molecules are the slower they move through the gel. The reason we have added markørbufferen is that it will always move faster than some of the DNA molecules, so when the cursor has come down to the other end off the Mon stream.
· PCR, color marker and TAE buffer:
One critical problem in this study is the amount of DNA material to do a proper electrophoresis, which will give a realistic picture, you need very DNA material. If you do not, you can use a molecular biological reproduction method called PCR, Polymerase Chain Reaction (polymerase chain reaction) which were invented as early as 1983 by Kary Mullis, who 10 years later received the Nobel Chemistry Prize for precisely this discovery. As the name suggests, it is an artificially produced replikationsmetode. It is relatively simple, I have tried to illustrate in the figure below:
· Step 1: DNA is poured into a container, together with polymerase and free nucleotides.
· Step 2: The entire substance is heated to slightly above 65 ° C (at BIO RAD machine is more than 90 ° C) at this temperature break hydrogen bonds between nucleotides in the DNA for. And there is no risk of damage to nucleotides since they first melts at +300 ° C
· Step 3: The substance cooled again to below 65 ° C, after which free nucleotides are committed to the free seats in the DNA, and in this way has now produced two identical DNA strands, this sequence can be done again, and each time doubled the number of DNA pieces.
The color marker I mentioned in the theory section on electrophoresis, which must say indicate how far the DNA pieces is reached in the gel, they would not want to walk completely through it. This implies that the pieces of the cursor is less than the smallest pieces of DNA. But it is also made such in our experiments, so the cursor has a speed of 500bp corresponding to a sequence of 500 base pairs. The highlights of this marker are doing will be visible to the naked eye, so that as said on when to turn off.
TAE Buffer (Tri-Acetate-x) key role is to keep DNA stable at pH 7.6. This must be done because a pH value of 7.6 ensures that the DNA remains negatively charged. The image on the right shows AMP Adenosine monophosphate, a nucleotide, and one can see the negative end with O-.
· Size marker:
In this study, it is important to compare the size of the cut DNA pieces, so to have a reference using a so-called size marker, which in this case is a DNA size marker. The DNA we used came from a virus and we must assume that it is a virus that scientists have determined its entire genome, and therefore all base sequences. So when we know the size of all fragments, measuring at how far they have walked in the gel, we can make a graph in a Semi-ordinates. This shows the number of base pairs up the second axis and number wandered mm. in the gel out of the first axis. The results seen in the section entitled "Results".
Method
see Exercise Guide.
Error Sources
During the experiment we came to pour TAE buffer into the tube at the end. For this reason, our DNA samples slightly smudged. But this did not alter the outcome, which ultimately was the best, compared to the other groups. A fault is it, but maybe an ameliorative one of its kind.
Another possible source of error, although not documented, it could be that gel is not completely homogeneous but the concentration of sucrose could be just a fraction higher in some areas of the gel, this would then lead to variations in travel.
Results
I have chosen to use the manufacturer's (BIO-RAD) image of the gel, as this will give the most accurate results.
1: Size marker
2: Salten 1
3: Salten 2
4: Poland
5: Ukraine
6: Greece
7: Serbia
Here are the results from the regression of our viral DNA:
The blue line as seen, exponential regression, and the function you get out of it is drawn with thin dotted line.
I then made a rule so you can calculate the number of base pairs from the number of migrated mm. However, I have chosen to exclude the first point with 23,130 base pairs, since it differs from the otherwise straight lines in the Semi-coordinates, but with the other results are the rule to look like this: where f (x) is the number of base pairs, and x is the number of migrated mm. From this model and the measured distances from the gel, you can run a table showing the different band base pairs for the various turtles DNA:
Salten 1
Salten 2
Poland
Ukraine
Greece
Serbia
3648
2820
2970
3821
3189
2969
2969
1112
1,773
3078
2620
2070
740
862
1117
740
1061
1116
Discussion
Looking at the table of turtles and their base pair sizes, you can relatively easily determine that the turtle "Salten a" face, and that it originated from the Ukrainian subspecies. The biggest difference in base pairs between the two, is at 173 base pairs. One must say that it is relatively little when 173 base pairs almost nothing is, and we found that there was an uncertainty marker around. 100-200 base pairs. When we look at Salten 2 see it immediately worse. It has no immediate similarities with any of the others. This is a strong indication that the strains from the original northern European subspecies, although they were counted as extinct 2000 years ago. But you must tell the result's defense that if only the population is small enough, it will have been very difficult to spot in the wild. Turtle overwinters as I said in seven months, and there are otherwise here vegetation around lakes and rivers are partially gone, so that one could easily spot it. It should also be said that it is a nocturnal animal, and she will feel even remotely threatened, it glides quietly and silently into the water.
Another fact which you can see from the gel, is that they are all very closely related, however, we knew this beforehand, but it is clearly illustrated in the table in it that they all have a DNA sequence approximately 3000 base pairs. More specifically, the average of the band of 3016, and the largest deviation from this is Greece with 173 base pairs, and again this is within the uncertainty of the cursor.
We were initially told of that, "... if this summer's hottest month average temperature is below 18 °, their eggs do not hatch" This fact, I examined and considered from DMI's website a depiction of Midtjyllands climate from 1961 to 1990:
And, as evidenced by the dark line indicating the mean temperature has gennemsnitstemperatu-clean in the hottest months (July to an average of 15.4 ° C) was not near the required 18 ° C, so it provides a dilemma. Either we must wholly reject our results, which otherwise strongly suggesting that they originated from the original Danish subspecies, or may choose to ignore the fact that in a number of years at 29 years, there has been a temperature of 18 ° C.
If our restriction enzymes had not worked our gel would have given us a very bad image to work with, for in that there would not be cut anywhere in the DNA, it would become one very long base sequence that would move very slightly in the gel because agarosemængden adapted very small pieces. Therefore we would only see one big line straight out of the well, and we would not be able to use this result to something.
Conclusion
Now that I have come to the conclusion that I should have one definitive answer. This is just not true. I have two contradictory arguments: If you choose only to look at the gel, I find that swamp turtle Salten 1 is an exposed "poor" because the composition of DNA sequences recalls hitting a lot about it from Ukraine, which also is available for purchase in Danish animal dealers. And Salten 2 is a surviving subspecies, who has lived in semi-hiding in 2000 years.
If you choose the other hand also to look at the graph from DMI, we see here the contradictory argument. There have in the period 1961-1990 was no one in July with an average temperature of the required 18 °. This indicates that it is not a survivor and childbearing population, as it simply would not have a month which is warm enough to hatch its eggs.
However, I would say that should this trial be successful, we must throw away the argument with the necessary 18 °, as this completely spoils the otherwise fine performance; that there is a surviving wild swamp tortoise in Denmark. Or should we turn it on and find a solution to this problem. And a possible theoretical solution to the problem could be that the turtle has been exposed to a selective pressure that would penalize the ability to lay eggs which can hatch at lower temperatures, so that there simply has been an evolution in relation to the freshwater turtles in southern Europe. This would give very good sense.
Nature in Design
If we look at an area such as a piece of pristine forest, everyone can agree on (if they do not believe that a giant spaghetti monster created the earth) that everything is happening is because of evolution. The reason that trees are not a twenty centimeters high, and squirrels are not neon green is that over millions of years, due to the natural selection, fairly hit what may be the optimum. If a squirrel was born with a glowing neon green color, it would not be able to hide from predators quickly be eaten, have no kids and the luminescent property would stop there.
But is evolution and natural selection did not also elsewhere than in the pristine nature? For instance, in design. Let's say I made a mug. When I make a mug, I spend much of the experience I already have to judge the mug, which is ergonomically nice to hold in your hand - the right volume, etc., etc. The experience I have mainly from the other mugs I have had, or maybe just one I've seen in a book. I am aware that you can get inspiration from many other places than any other mug, but to this thought experiment to work, you probably need to cut edges extra hard up, and other cups would otherwise, in most cases be a great inspiration. Let us therefore say that my redesigned cup will be the sum of all the good characteristics as smallpox, which has crossed my path holds. If my cup ends up meeting the criteria that makes it a good cup, it will probably be a success. So it will be produced in large numbers and it will come in books, internet etc. This way, a lot of people learn this cup to know, and some of them may be designers who designs a second cup, which takes some elements from my cup. If my cup is found not to function and becomes a flop, it will not be produced in particularly high numbers, not get in books and on the Internet and will as neon green squirrel does not conduct its properties further.
So you could say that natural selection also applies to designs. If we as human beings and culture as a part of nature, that would perhaps work saved. In that case, all among people, as indeed is the "nature", could be explained by natural selection. But many see this divide between nature and culture, it drops us maybe did not come naturally to this conclusion.
But is evolution and natural selection did not also elsewhere than in the pristine nature? For instance, in design. Let's say I made a mug. When I make a mug, I spend much of the experience I already have to judge the mug, which is ergonomically nice to hold in your hand - the right volume, etc., etc. The experience I have mainly from the other mugs I have had, or maybe just one I've seen in a book. I am aware that you can get inspiration from many other places than any other mug, but to this thought experiment to work, you probably need to cut edges extra hard up, and other cups would otherwise, in most cases be a great inspiration. Let us therefore say that my redesigned cup will be the sum of all the good characteristics as smallpox, which has crossed my path holds. If my cup ends up meeting the criteria that makes it a good cup, it will probably be a success. So it will be produced in large numbers and it will come in books, internet etc. This way, a lot of people learn this cup to know, and some of them may be designers who designs a second cup, which takes some elements from my cup. If my cup is found not to function and becomes a flop, it will not be produced in particularly high numbers, not get in books and on the Internet and will as neon green squirrel does not conduct its properties further.
So you could say that natural selection also applies to designs. If we as human beings and culture as a part of nature, that would perhaps work saved. In that case, all among people, as indeed is the "nature", could be explained by natural selection. But many see this divide between nature and culture, it drops us maybe did not come naturally to this conclusion.
family tree for fish
Purpose
The purpose of this study and corresponding results is to determine the kinship of a number of fish and shellfish. This must be done first to gain insight into the method, which is electrophoresis of proteins instead of nuclear material, but also partly to do some evolutionary considerations to ultimately come up with a family tree.
Theory
Map of evolution and genetics
If one considers the composition and diversity of animals on earth, from a Darwinian point of view, one is the view of all animals are created from the same ancestor, which here would be a little procaryote organisms. This organism would have to have multiplied to a lot of new species, which in turn would have evolved into new species and subspecies. The reason that so today is so rich in wildlife with birds, fish and land animals is that all these individuals have been selection pressure for different make from his companions so that they were better able to exploit a specific niche. As an example of this, we giraffe, Giraffa camelopardalis, the liver primarily from the leaves of the tall acacia, which nobody else can reach. It originates from Okapi which then has been a selection pressure to isolate themselves, niche differentiation, the Okapi, so that it could reach the high leaves, and you have looked at the way for a long period of time, creating two species. This example of the giraffe must not be viewed as Jean Baptist Lamarc did it with that through giraffe's life, it will have longer necks, but the emergence of spontaneous mutations that make it a longer neck, and that his offspring will have better survival prospects as a result of more food, causing gene / property with them.
Looking at this from a molecular viewpoint, one can use it to study their origin, one can look at what proteins different individuals have, and then among other things find out how old the protein is and what animals are in family with whom. This whole branch of biology, so the doctrine of heredity, or genetics is thus the study of living organisms originate their development and inherited properties through the study of genes. In other words, the importance of inheritance and variation when an organism created and developed. And it was primarily originated when people began to be interested in, from which human strains.
Analog and homologous development
When they began to classify animals on earth, in an attempt to create a universal family tree, one would need to know which animals were in families with whom, and how close the family they were, so it looked on their similarities. And you looked at the similarities among animals from two different criteria: homologous, which is a manifestation that they have the same ancestor, and then a common heritage. So is a property similar in two animals, because their common ancestor had the property. Analog is the opposite, here looks the same, not because they have the same basic form, but because the evolutionary process has developed the same characteristics, this type of development is also called convergent development. Another disadvantage of this type of development is called divergent development, and is an expression of two animals that are homologous in the same class (same basic form) but are different because they have evolved in two different directions.
Species protein composition
The exciting thing about species protein composition is that from this, can see from where they originated, one can also quite accurately dating the protein age, because if two animals, such as fish, have the same protein, so it is the same protein, understood such that the two proteins in principle could arise from mutations independently, but the likelihood of this is very close to zero. So if you find the same protein in two species, one can say with certainty that they have the same ancestor, but only on the comparison of proteins, one can not necessarily say anything more about when they have evolved away from their common progenitor. For example, we have in the experiment included an actin-myosin protein solution, and exciting about this is that it is one of the key muscle proteins by human muscle tissue is constructed. Has the fish this too, one can say a lot about protein age, for it is as I said, really long time ago that the human stem form, and fish, did differ from one another.
Something else that proteins course also shows the composition of DNA, since it is DNA which ultimately codes for the proteins a given cell can produce.
Method
see Exercise Guide.
The seafood we used for our experiments were as follows:
1st Shrimp (saltwater) - Prawn
2nd Edible crab - Cancer pagurus
3rd Cod - Gadus morhua
4th Pollock - Pollachius virens
5th Herring - Glupea harengus
6th Weevers - Trachinidae
7th Halibut - Reinhardtius hippoglossoides
8th Plaice - Pleuronectes platessa
Hypothesis
Before you had access to advanced molecular biology tools we have today made the stem of trees from other criteria. It looked primarily at appearance and habitat, I also thought, as a hypothesis to create a family tree based on these criteria, then later to see how accurate it is compared with the molecular biological method.
To provide a fairly realistic family tree, we need to explore different living conditions for the various fish and shellfish. The first thing we see is that they are all saltwater fish, this is not so significant compared with muscle proteins, but helps to understand their origins. One could thus imagine that even in ancient times, has been a split between the fish living in freshwater, and those who lived in salt water.
Secondly, we can see that they all have gills, this is obviously also a characteristic that is unique to just fish and shellfish, a simple observation, but it shows that they all have the same ancestor, but again far back. But that's just not all marine animals which have gills, such as turtles, saltwater crocodiles, whales and seals.
Results
Below is a scan of the gel, as it looked after electrophoresis. We had two pictures to choose from, this was the strongest pure color regularly, and I chose it for exactly this reason. For the second gel were tapes very weak, and it was therefore difficult to distinguish between the various individual bands. I think we can improve on this.
Wells contain the following:
1: Kaleidoscope standard 2: myosin-actin standard 30-10: fish (see method for sequence).
You can order now determine a protein approximated from this model (Fig. 5).. If you know its weight, one can see how much it should have horizontal and vice versa, one can find its weight by looking at migration. This we can use to find out which proteins in the fish (see liste1).
A protein that only shrimp and taskekrappen have seen as the band has been running about 8 mm, thus 5 kD, this corresponds to thymosin. Shrimp and taskekrappen thus both the protein, which in turn by the other fish have, this gives rise to a common ancestor that has evolved away from the fish. A band that almost all fish and shellfish have and which is horizontally about 4.6 mm can be nebulin. It is that they all have a band at this point, talks about protein here must be very old because it has been present in the different species now common ancestor, the protein is also working in collaboration with actin, as I will later show that they all have, so therefore, you have one, it was often the second, otherwise protein (in this case actin) does not function optimally if it is because the head would work. One of the first to get noticed, is that they all have a tape which has horizontal 4 mm, this is according to our model of myosin. This is like an old actin protein as it occurs in all species. Something else which is also unique in myosin-actin formation in all the fish is that it is they are the same muscle proteins, as we humans and most animals possess. This tells us that is, it is an incredibly ancient protein of ours, birds and fish common ancestor have had it, and this ancestor is many millions of years ago. This also says something about the protein status. They have been very essential and important for the animals, and not only that, they have also apparently functioned optimally in as many years, because had it not what would a second, more muscle protein have replaced it reasonably quickly, and with the second, more protein would have a better chance of survival. The protein actin, which is also a very essential protein, we find a little farther down, at about 5.3 mm. However, it is a bit unclear to see how many people have this protein, but ummidelbart I would think that it was most. The two proteins work together namely, one can not function without the other and vice versa.
If we now go in and look at what fish are in families with each other, we then quickly the cod and Pollock are very similar not only in appearance, but especially in protein composition, but also herring, many homologous bands with the two. That is why, from this view, safe to say that the plaice and cod are family and that they have a homologue development, herring, however, has evolved into a second recess, and through selective pressure and mutation, the so and say changed direction compared to the cod and Pollock.
Halibut and plaice is also evident from the protein composition to be in the family, and thus a homologue development, they still resemble each other much. However, one can say that rock disc is an earlier species, because its eyes more like a "regular" fish that have eyes on both sides, whereas flounder has both eyes on the same page. No mug turns out to also be the family of the two because they have very similar bands. One can however look at my previous family tree that I have placed them in the same species group, but it's also made from a purely appearance viewpoint. No face has changed considerably since torn between it and plaice. This phenomenon that a fish does not resemble anything from nature it really belongs, but that it rather resembles a second group is called a polyfyletisk group (say it as it looks like it belongs to). Shrimp and taskekrappen is looking very different, but they have some homologous bands, as none of the others. (See opg 2).
Discussion
There was only one real source of error, but this was also of much importance. Namely that our electrophoresis were not allowed to run long enough. This means that bands should not have walked so far and that therefore there is so much to spread. The spread between the tapes would otherwise have done two task considerably easier. It is therefore difficult to determine which fish have the proteins. Ribbon density has also affected the determination of kinship among the fish. However, it is also important to state here that although the two bands have walked the same length, it is not necessarily the same protein. For proteins composed as said by various amino acids and composition of molecules differ, and hence also their weight. So you can say well have two different proteins with the same molecular weight. The method to determine slægsskab I think ummidelbart, with my limited knowledge of other methods are fine enough, if so just experiment is well made.
The purpose of this study and corresponding results is to determine the kinship of a number of fish and shellfish. This must be done first to gain insight into the method, which is electrophoresis of proteins instead of nuclear material, but also partly to do some evolutionary considerations to ultimately come up with a family tree.
Theory
Map of evolution and genetics
If one considers the composition and diversity of animals on earth, from a Darwinian point of view, one is the view of all animals are created from the same ancestor, which here would be a little procaryote organisms. This organism would have to have multiplied to a lot of new species, which in turn would have evolved into new species and subspecies. The reason that so today is so rich in wildlife with birds, fish and land animals is that all these individuals have been selection pressure for different make from his companions so that they were better able to exploit a specific niche. As an example of this, we giraffe, Giraffa camelopardalis, the liver primarily from the leaves of the tall acacia, which nobody else can reach. It originates from Okapi which then has been a selection pressure to isolate themselves, niche differentiation, the Okapi, so that it could reach the high leaves, and you have looked at the way for a long period of time, creating two species. This example of the giraffe must not be viewed as Jean Baptist Lamarc did it with that through giraffe's life, it will have longer necks, but the emergence of spontaneous mutations that make it a longer neck, and that his offspring will have better survival prospects as a result of more food, causing gene / property with them.
Looking at this from a molecular viewpoint, one can use it to study their origin, one can look at what proteins different individuals have, and then among other things find out how old the protein is and what animals are in family with whom. This whole branch of biology, so the doctrine of heredity, or genetics is thus the study of living organisms originate their development and inherited properties through the study of genes. In other words, the importance of inheritance and variation when an organism created and developed. And it was primarily originated when people began to be interested in, from which human strains.
Analog and homologous development
When they began to classify animals on earth, in an attempt to create a universal family tree, one would need to know which animals were in families with whom, and how close the family they were, so it looked on their similarities. And you looked at the similarities among animals from two different criteria: homologous, which is a manifestation that they have the same ancestor, and then a common heritage. So is a property similar in two animals, because their common ancestor had the property. Analog is the opposite, here looks the same, not because they have the same basic form, but because the evolutionary process has developed the same characteristics, this type of development is also called convergent development. Another disadvantage of this type of development is called divergent development, and is an expression of two animals that are homologous in the same class (same basic form) but are different because they have evolved in two different directions.
Species protein composition
The exciting thing about species protein composition is that from this, can see from where they originated, one can also quite accurately dating the protein age, because if two animals, such as fish, have the same protein, so it is the same protein, understood such that the two proteins in principle could arise from mutations independently, but the likelihood of this is very close to zero. So if you find the same protein in two species, one can say with certainty that they have the same ancestor, but only on the comparison of proteins, one can not necessarily say anything more about when they have evolved away from their common progenitor. For example, we have in the experiment included an actin-myosin protein solution, and exciting about this is that it is one of the key muscle proteins by human muscle tissue is constructed. Has the fish this too, one can say a lot about protein age, for it is as I said, really long time ago that the human stem form, and fish, did differ from one another.
Something else that proteins course also shows the composition of DNA, since it is DNA which ultimately codes for the proteins a given cell can produce.
Method
see Exercise Guide.
The seafood we used for our experiments were as follows:
1st Shrimp (saltwater) - Prawn
2nd Edible crab - Cancer pagurus
3rd Cod - Gadus morhua
4th Pollock - Pollachius virens
5th Herring - Glupea harengus
6th Weevers - Trachinidae
7th Halibut - Reinhardtius hippoglossoides
8th Plaice - Pleuronectes platessa
Hypothesis
Before you had access to advanced molecular biology tools we have today made the stem of trees from other criteria. It looked primarily at appearance and habitat, I also thought, as a hypothesis to create a family tree based on these criteria, then later to see how accurate it is compared with the molecular biological method.
To provide a fairly realistic family tree, we need to explore different living conditions for the various fish and shellfish. The first thing we see is that they are all saltwater fish, this is not so significant compared with muscle proteins, but helps to understand their origins. One could thus imagine that even in ancient times, has been a split between the fish living in freshwater, and those who lived in salt water.
Secondly, we can see that they all have gills, this is obviously also a characteristic that is unique to just fish and shellfish, a simple observation, but it shows that they all have the same ancestor, but again far back. But that's just not all marine animals which have gills, such as turtles, saltwater crocodiles, whales and seals.
Results
Below is a scan of the gel, as it looked after electrophoresis. We had two pictures to choose from, this was the strongest pure color regularly, and I chose it for exactly this reason. For the second gel were tapes very weak, and it was therefore difficult to distinguish between the various individual bands. I think we can improve on this.
Wells contain the following:
1: Kaleidoscope standard 2: myosin-actin standard 30-10: fish (see method for sequence).
You can order now determine a protein approximated from this model (Fig. 5).. If you know its weight, one can see how much it should have horizontal and vice versa, one can find its weight by looking at migration. This we can use to find out which proteins in the fish (see liste1).
A protein that only shrimp and taskekrappen have seen as the band has been running about 8 mm, thus 5 kD, this corresponds to thymosin. Shrimp and taskekrappen thus both the protein, which in turn by the other fish have, this gives rise to a common ancestor that has evolved away from the fish. A band that almost all fish and shellfish have and which is horizontally about 4.6 mm can be nebulin. It is that they all have a band at this point, talks about protein here must be very old because it has been present in the different species now common ancestor, the protein is also working in collaboration with actin, as I will later show that they all have, so therefore, you have one, it was often the second, otherwise protein (in this case actin) does not function optimally if it is because the head would work. One of the first to get noticed, is that they all have a tape which has horizontal 4 mm, this is according to our model of myosin. This is like an old actin protein as it occurs in all species. Something else which is also unique in myosin-actin formation in all the fish is that it is they are the same muscle proteins, as we humans and most animals possess. This tells us that is, it is an incredibly ancient protein of ours, birds and fish common ancestor have had it, and this ancestor is many millions of years ago. This also says something about the protein status. They have been very essential and important for the animals, and not only that, they have also apparently functioned optimally in as many years, because had it not what would a second, more muscle protein have replaced it reasonably quickly, and with the second, more protein would have a better chance of survival. The protein actin, which is also a very essential protein, we find a little farther down, at about 5.3 mm. However, it is a bit unclear to see how many people have this protein, but ummidelbart I would think that it was most. The two proteins work together namely, one can not function without the other and vice versa.
If we now go in and look at what fish are in families with each other, we then quickly the cod and Pollock are very similar not only in appearance, but especially in protein composition, but also herring, many homologous bands with the two. That is why, from this view, safe to say that the plaice and cod are family and that they have a homologue development, herring, however, has evolved into a second recess, and through selective pressure and mutation, the so and say changed direction compared to the cod and Pollock.
Halibut and plaice is also evident from the protein composition to be in the family, and thus a homologue development, they still resemble each other much. However, one can say that rock disc is an earlier species, because its eyes more like a "regular" fish that have eyes on both sides, whereas flounder has both eyes on the same page. No mug turns out to also be the family of the two because they have very similar bands. One can however look at my previous family tree that I have placed them in the same species group, but it's also made from a purely appearance viewpoint. No face has changed considerably since torn between it and plaice. This phenomenon that a fish does not resemble anything from nature it really belongs, but that it rather resembles a second group is called a polyfyletisk group (say it as it looks like it belongs to). Shrimp and taskekrappen is looking very different, but they have some homologous bands, as none of the others. (See opg 2).
Discussion
There was only one real source of error, but this was also of much importance. Namely that our electrophoresis were not allowed to run long enough. This means that bands should not have walked so far and that therefore there is so much to spread. The spread between the tapes would otherwise have done two task considerably easier. It is therefore difficult to determine which fish have the proteins. Ribbon density has also affected the determination of kinship among the fish. However, it is also important to state here that although the two bands have walked the same length, it is not necessarily the same protein. For proteins composed as said by various amino acids and composition of molecules differ, and hence also their weight. So you can say well have two different proteins with the same molecular weight. The method to determine slægsskab I think ummidelbart, with my limited knowledge of other methods are fine enough, if so just experiment is well made.
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