laboratório de análise da regulação da expressão gênica desenvolvimento: a historia natural...

Laboratório de Análise da Regulação da Expressão Gênica

DESENVOLVIMENTO: A HISTORIA NATURAL DOS GENES

Paulo Arruda

10-03-2011

BG - 581


O QUE SABEMOS SOBRE GENES E DESENVOLVIMENTO

(1) Tipos celulares diferentes transcrevem genes diferentes. Celulas citologicamente indistinguíveis podem ter padrões elaborados de expressão genica temporal e espacial.

(2) Embora alterações no DNA são utilizadas para determinar a ação diferencial dos genes, o controle da transcrição é a forma mais comum de regulação da expressão gênica.

(3) A explosão transiente da ação de fatores de transcrição ou sinalização leva a alterações permanentes no fenótipo celular, que embora, muitas vezes reversíveis, podem perpetuar decisões regulatórias precoces.

(4) Moléculas de RNA localizadas criam diferenças no citoplasma do óvulo ou entre celulas, criando divisões celulares assimétricas. Divisão celular assimétrica é crucial para a diferenciação de células tronco.

(5) Fatores de transcrição regulam baterias de genes. Combinações de reguladores governam a maioria dos eventos de diferenciação. O resultado depende da ação em concerto ou em oposição de reguladores.

(6) Os embriões são flexíveis em se adaptar ao número de células, a condições ambientais e alterações na ação gênica. As células são sensíveis à qualidade das células vizinhas e tendem a morrer ou mudar se as células vizinhas não são apropriadas. Elas são tambem sensíveis ao número de células vizinhas “efeito da comunidade”.

Matthew P. Scott (2000) Development: The Natural History of Genes. Cell, 100: 27-40



(7) Muitos sinais agem a curta distancia, talvez refletindo suasw origens no organismo com poucas células.

(8) Sistemas de checagem controlam a açõa de sinalizadores. Antagonistas intra- e extra- celulares limitam a atuação de sinalizadores sendo que alguns são induzidos pelos seus sinalizadores proporcionam um mecanismo de retroalimentação.

(9) As células são responsivas a sinalizadores ou fatores de transcrição em algumas condições e não em outras, criando a propriedade denominada de competencia. As células respondem diferencialmente a um sinalizador particular, dependendo da sua história. A historia pode afetar a efetividade, tornando certos genes responsívos a sinalizadores atraves da localização de moléculas dentro das células.

(10) A regulação temporal e espacial do ciclo celular é crucial para a formação de tecidos organizados,permitindo a restauração e manut6enção das células tronco.

(11) A morte celular e o envelhecimento são processsos governados geneticamente atraves de “scripts” precisos durante o desenvolvimento normal e alterados em condições de doença e injúria.

(12) A maioria das proteínas que regulam o desenvolvimento foram conservadas durante a evolução. Muitas vezes, genes conservados tem funções imilares numa vasta gama de diferentes animais.



Expressão espacial altamente precisa do gene patched1 regulado por Hedgehog. Hedgehog indus a expressão de patched1 e outros genes alvo num padrão espacial e temporal preciso, mostrado aqui pela inserção do gene lacZ no locus patched1. A regulação da diferenciação por Hedgehog é amplamente conservada em animais.


DEVELOPMENT OF EYE COLORS IN DROSOPHILA: SOMEPROPERTIES OF THE HORIVIONES CONCERNED

Abstract

Diffusible substances showing hormone-like action in modifying the development of eye color are known in Drosophila and in certain other insects. These substances are active when injected into, or fed to, appropriate test larvae. The so called v+ substance causes a modification of the eye color of vermilion brown (v bw) animals from a pale pink towards brown. Similarly, cn+ substance induces a modification of cinnabar brown (cn bw) eye color from colorless towards brown.

This paper reports some of the further information which has been obtained primarily in regard to the properties, chemical nature, and possible methods of purification of the v+ hormone.

Tatum EL and Beadle GW (1938) The journal of general physiology. X: 239-253



Método:

The v+ hormone used for this work was obtained from 24-48 hour old wild type pupae. The pupae were washed and dried at 100°C. for 1 hour. This served both to inactivate the enzymes and to dry the material. The dried pupae were kept under reduced pressure until needed. The hormone was extracted from finely ground dried pupae by procedures to be described later in this paper. Small aUquots of the extracts to be tested were dried under reduced pressure at 100°C weighed, and dissolved in Ringer's solution and, if necessary, the pH adjusted to 6.0. The solutions prepared in this way were sealed in small glass tubes and immersed in boiling water for 10 minutes. This was found to be necessary to prevent bacterial growth and consequent toxicity for the test animals. Solutions prepared in this way retained their activity indefinitely. The activity of the various solutions was determined by injecting them into the body cavities of v bw larvae shortly before puparium formation. The effect manifested itself in a modification of the eye color from neatly colorless towards brown. Ten to fifteen larvae were injected with each solution to be tested,


EFEITO DO HORMONIO v+ NA COR DO OLHO DE MOSCAS v bw


EXTRAÇÃO DO HORMONIO v+ DAS PUPAS

The ground dried pupae were extracted several times with boiling chloroform, and this inactive extract was discarded. The hormone was then extracted by repeated treatments with hot 95 per cent ethyl alcohol. The alcohol was removed from the yellow extract under reduced pressure and the residue taken up in distilled water. The water-insoluble portion of this mixture was removed by centrifuging. The final product was a fairly clear yellow solution. The results of a number of extractions are summarized in Table II. Approximately 5 per cent of the original weight of the pupae were contained in the final extract. Disregarding a few inexplicable cases in which extracts of quite low activity were obtained, the average activity was about 280,000 units per gram extract. This means that from 1 gm. of dried pupae around 17,500 units were extracted.


EXTRAÇÃO DO HORMONIO v+ DAS PUPAS


PROPRIEDADES DO HORMONIO v+


INATIVAÇÃO ENZIMÁTICA DO HORMONIO v+

Since the hormone had been shown to be relatively stable towards oxidation, it was thought advisable to investigate its inactivation by the enzymes present in the larvae and pupae. Juice obtained by crushing or grinding fresh pupae or larvae and filtering, was mixed with concentrated active extract. The solutions were allowed to stand for 15-20 hours on watch glasses at 30°C., then diluted with Ringer's solution to known volumes, sealed in glass tubes, heated, centrifuged, and tested by injection.


INATIVAÇÃO ENZIMÁTICA DO HORMONIO v+

In no case using juice from crushed pupae or larvae did inactivation result. However, the juice from wild type or vermilion larvae and wild type pupae obtained by grinding the material thoroughly with finely powdered silica, almost completely inactivated the v+ hormone


O QUE É O HORMONIO v+?

Although all the data indicate the amino acid nature of the hormone, it seems probable that it is not a simple amino acid. The substance may be destroyed or inactivated by many treatments which would not affect simple amino acids. The instabillty of the hormone to heat, and to treatment with acid and alkali illustrate this point. Furthermore, the high molecular weight of 400 to 600 as determined by the diffusion method also eliminates the common simple amino acids from consideration. the exact chemical nature of the hormone. However, there seem to be three distinct possibilities. It may prove to be a complex single amino acid, a peptide of several amino acids, or it may be a compound of one or more amino acids with other substances. In any case it is probably not a normal constituent of tissue, since its occurrence seems to be rather strictly limited to certain insects. Khouvine and Ephrussi were unable to demonstrate any hormone activity in nitrogenous extracts of sheep brain, while similarly prepared extracts of CaP liphora were very active. Although yeast is generally supposed to contain most known nitrogenous substances, we have had no indication that it contains this particular amino acid-like hormone.


GENETIC CONTROL OF BIOCHEMICAL REACTIONS INNEUROSPORA

Summary.

-A procedure is outlined by which, using Neurospora, one can discover and maintain x-ray induced mutant strains which are characterized by their inability to carry out specific biochemical processes. Following this method, three mutant strains have been established. In one of these the ability to synthesize vitamin B6 has been wholly or largely lost. In a second the ability to synthesize the thiazole half of the vitamin B1 molecule is absent, and in the third para-aminobenzoic acid is not synthesized. It is therefore clear that all of these substances are essential growth factors for Neurospora-11Growth of the pyridoxinless mutant (a mutant unable to synthesize vitamin B6) is a function of the B6 content of the medium on which it is grown. A method is described for measuring the growth by following linear progression of the mycelia along a horizontal tube half filled with an agar medium. Inability to synthesize vitamin B6 is apparently differentiated by a single gene from the ability of the organism to elaborate this essential growth substance.

Beadle GW and Tatum EL (1941) PNAS


GENETIC CONTROL OF BIOCHEMICAL REACTIONS IN NEUROSPORA:AN "AMINOBENZOICLESS" MUTANT

Summary

An x-ray induced mutant strain of Neurospora crassa has been obtained which requires p-aminobenzoic acid for growth. Its growth is a function of the amount of p-aminobenzoic acid supplied, and it is indistinguishable from normal when adequate amounts of p-aminobenzoic acid are available. The mutant differs from normal by a single gene, which must therefore control an essential step in the synthesis of p-aminobenzoic acid, and which is presumably primarily concerned only with the synthesis of p-aminobenzoic acid. Sulfanilamide inhibits the growth of both the normal and mutant strains, and in both cases the inhibition is overcome by an excess of p-aminobenzoic acid. A number of substances related to p-aminobenzoic acid are able to replace it, but their activities are much less than that of p-aminobenzoic acid itself. The addition of benzoic or p-OH-benzoic acids or tyrosine did not increase the amount of p-aminobenzoic acid produced by the normal strain. It is concluded that none of the compounds tested is concerned with the normal synthesis of p-aminobenzoic acid, and that this biosynthesis probably does not involve the introduction of an amino group into a preformed benzene ring.

By E. L. TATUM AND G. W. BEADLE (1942) PNAS

George Wells Beadle Edward Lawrie Tatum Joshua Lederberg

1/4 of the prize 1/4 of the prize 1/2 of the prize

California Institute of Technology (Caltech) Pasadena, CA, USA

Rockefeller Institute for Medical Research New York, NY, USA

University of Wisconsin Madison, WI, USA

b. 1903d. 1989

b. 1909d. 1975

b. 1925d. 2008

"for their discovery that genes act by regulating definite chemical events"

"for his discoveries concerning genetic recombination and the organization of the genetic material of bacteria"

THE NOBEL PRIZE IN PHYSIOLOGY OR MEDICINE 1958

laboratório de análise da regulação da expressão gênica desenvolvimento: a historia natural...

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