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Transformation in Bacteria Transformacija u Bakterije

Griffith laid the foundation for the identification of DNA as the genetic material in 1928 with his experiments on transformation in the bacterium pneumococcus, now known as Streptococcus pneumoniae.  The wild-type organism is a spherical cell surrounded by a mucous coated capsule.  The cells form large, glistening colonies, characterized as smooth.  These cells are virulent- capable of causing lethal infections upon injection into mice.  A certain mutant strain of S. Griffith postavi temelj za identifikaciju DNA kao genetički materijal u 1928 sa svojim eksperimentima na transformacije u bakterija pneumococcus, sada poznat kao Streptococcus pneumoniae. Wild-type organizam je sferna stanica okružena sluzav obložene kapsula. Stanice form veliki, blistavim kolonije, karakteriziraju kao glatka. Te stanice su zarazan-sposoban za izazivanje smrtonosno infekcije na injekcije u miševi. određene mutant strain of S. pneumoniae has lost the ability to form a capsule.  As a result, it grows as small, rough colonies.  More importantly it is avirulent since it has no protective coat, it is engulfed by the host’s white blood cells before it can proliferate enough to do any damage. pneumoniae je izgubio sposobnost da formira kapsulu. Kao rezultat, raste kao mala, neobrađeno kolonije. Još važnije je avirulent jer nema zaštitne kaput, to je engulfed od strane iznajmljivača bijele krvne stanice prije nego što možete proliferate dovoljno činiti štetu. The key finding of Griffith’s work was that heat-killed virulent colonies of S.pneumoniae could transform avirulent cells to virulent ones.  Neither the heat-killed virulent bacteria nor the live avirulent ones by themselves could cause a lethal infection.  Together, however they were deadly.  Somehow the virulent trait passed from the dead cells to the live, avirulent ones.  Transformation was not transient; the ability to make a capsule and therefore to kill host animals, once conferred upon the avirulent bacteria, was passed to their descendents as a heritable trait.  In other words, the gene for virulence, missing in the avirulent cells, was somehow gained during transformation.  This meant that the transforming substance in the heat-killed bacteria was probably the gene for virulence itself.  The missing piece of the puzzle was the chemical nature of the transforming substance. The key finding of Griffith rada je da heat-ubijeni zarazan kolonije od S.pneumoniae mogao transformirati avirulent stanica za one otrovan. Topline Ni-ubijenih bakterija otrovan ni živjeti avirulent one koju sami mogli uzrokovati smrtonosno infekcije. Zajedno, međutim oni su bili smrtonosni. nekako je zloban potez prošao od mrtvih stanica na live, avirulent one. Preobrazba nije prijelazna; mogućnost da jedna kapsula i zato da ubije životinja domaćin, jednom conferred na avirulent bakterija, donesen do njihovih potomaka kao naslijeđen potez. Drugim riječima, gena za otrovnost, nestalo u avirulent stanice, bio je nekako stekao tijekom transformacije. To je značilo da su transformisane u tvar topline ubili bakterije je vjerojatno gena virulencije za sebe. nestalih komad slagalice bio kemijske prirode i pretvoriti tvar.

Avery, MacLeod and McCarty supplied the missing piece in 1944.  They used a transformation test similar to the one that Griffith has introduced.  First, they removed the protein from the extract with organic solvents and found that the extract still transformed.  Next, they subjected it to digestion with various enzymes.  Trypsin and Chymotrypsin, which destroy protein, had no effect on transformation.  Neither did ribonuclease, which degrades RNA.  These experiments ruled out protein or RNA as the transforming material.  On the other hand, Avery and his coworkers found that the enzyme deoxyribonuclease (DNase), which breaks down DNA, destroyed the transforming ability of the virulent cell extract.  These results suggested that the transforming substance was in fact DNA. Avery, MacLeod i McCarty opskrbljuje nestalih komad u 1944. Oni su koristili slične transformacije test na jednom Griffith koja je uvela. Prvo, oni uklanjaju proteina iz ekstrakta s organskih otapala i utvrdili da se izvuci i dalje transformirao. Zatim su podvrgnuti da se s raznim probavu enzima. Trypsin i Chymotrypsin, koji uništi protein, nije imao utjecaj na transformaciju. niti ribonuclease, koji degradirati RNA. Ovi eksperimenti isključeno protein ili RNA kao preobrazbe materijala. S druge strane, Avery i njegova coworkers utvrdio kako je enzim deoxyribonuclease (DNase), koje razbija niz DNA, uništeno je sposobnost transformacije u zarazan stanica izvuci. Ovi rezultati sugerirao da se pretvoriti tvar je zapravo DNA. Direct physical-chemical analysis supported the hypothesis that the purified transforming substance was DNA.  The analytical tools Avery and his colleagues used were as following: Direct fizičko-kemijske analize su podržane hipoteze da su očistili pretvarajući je tvar DNA. Analitičkih alata Avery i njegove kolege koristili su kao sljedeći:

1. Ultracentrifugation:  They spun the transforming substance in an ultracentrifuge (a very high-speed centrifuge) to estimate its size.  The material with transforming activity sedimented rapidly (moved rapidly toward the bottom of the centrifuge tube), suggesting a very high molecular weight, characteristic of DNA. Ultracentrifugation: Oni spun, pretvarajući tvari u jednoj Ultracentrifuga (vrlo high-speed centrifuga) na procjenu svoje veličine. Materijala s pretvarajući aktivnosti sedimented brzo (preselio ubrzano prema dnu je centrifuga tube), predlažemo vrlo visoke molekularne težine, karakterističan DNA.
2. Electrophoresis:  They placed the transforming agent in an electric field to see how rapidly it moved.  The transforming activity had a relatively high mobility, also characteristic of DNA because of its high charge/mass ratio. Elektroforeza: Oni su stavili pretvarajući agent u jednom električnog polja da biste vidjeli kako brzo ga preselio. Pretvarajući aktivnosti je relativno visoka mobilnost, također karakteristične DNA zbog visoke naplate / mass ratio.
3. Ultraviolet absorption spectrophotometry: They placed a solution of the transforming substance in an spectrophotometer to see what kind of ultraviolet light is absorbd most strongly.  Its absorption spectrum matched the DNA.  That is, the light it absorbed most strongly had a wavelength of about 260 nm, in contrast to protein, which absorbs maximally at 280 nm. Ultraljubičasti apsorpcija spektrofotometrija: Oni su stavili rješenje o transformaciji tvar u spektrofotometar vidjeti kakav je ultraljubičasto svjetlo absorbd većina snažno. Njezin absorption spectrum podudarnim DNA. To je, svjetlost se apsorbira većinu snažno je valna duljina oko 260 nm , U kontrastu protein, koji apsorbira na maksimalno 280 nm.
4. Elementary chemical analysis:  This yielded an average nitrogen/phosphorus ratio of 1.67,  about what one would expect for DNA, which is rich in both elements, but vastly lower that the value expected for protein, which is rich in nitrogen but poor in phosphorus.  Even a slight protein contamination would have raised the nitrogen/phosphorus ratio. Osnovna kemijska analiza: Ova yielded prosječna dušika / fosfor omjer 1,67, o tome što bi se očekivati za DNA, koja je bogata u oba elementa, ali da je znatno niža vrijednost očekivanih za protein koji je bogat u dušik, ali siromašni u fosfor. Čak i lakom protein kontaminacija bi podigli dušika / fosfor sistem.