Moss - What genes cant do - 2003 (522929), страница 34
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With the assistance of then graduatestudent Howard Temin, Rubin succeeded in establishing a cell-culturesystem for analyzing the chicken virus he acquired from “Old ManRous,” that would prove to be a springboard for the subsequent development of tumor virology. By the late 1950s several lines of evidencesuggested that the Rous sarcoma virus (RSV) was composed of RNA (asopposed to DNA), although the first actual intact isolation and characterization of RNA from the virus was not performed until 1965 (Rubin1965).
This appeared to be paradoxical in light of evidence of the high134Chapter 4radiation sensitivity of the cell’s capacity to support RSV infectionand viral replication, which suggested that like a temperate phage, RSVbecame integrated into the genome of the host cell (Rubin 1965).But how could RSV be both composed of RNA and yet become integrated into the host genome? This apparant paradox was resolved withHoward Temin’s discovery of the reverse transcriptase enzyme. RSV andother now so-called retroviruses were found to contain, in their RNA,gene-sequence information for the synthesis of an enzyme capable ofusing viral RNA as a template for the synthesis of complementary DNA,which could then be integrated into the host DNA.
In addition to solvingthe paradox of retroviral RNA composition and radiation sensitivity, thediscovery of the reverse transcriptase enzyme (for which Howard Teminalong with David Baltimore were awarded Nobel Prizes) constitutedthe addition of an invaluable tool for biotechnology. With the reversetranscriptase in hand, molecular biologists could synthesize complexsequences of DNA (so-called cDNA) using “messenger RNA” found incell cytoplasm as templates.By 1969, with experimental systems for studying tumor virology wellestablished and with the range of taxa in which tumors could be produced by retroviruses expanded from that of just chickens to include alsomice, cats, and hamsters, the time had come to bring virally induced, andnon-virally induced, causes of cancer into a common explanatory framework.
The move toward explanatory unification in the realm of oncology took the form of a series of oncogene hypotheses, the first of whichwas put forward by Huebner and Todero in 1969. Huebner and Todarosuggested that cancer, “both spontaneous cancers and those induced bychemical and physical agents,” is the result of the expression of viralgenes, i.e., “oncogenes” that become resident in the genomes of animals.The central hypothesis implies, therefore that the cells of many if not allvertebrates carry vertically transmitted (inherited) RNA tumor virus information(virogenes) which serve as an indigenous source of information (oncogenes)which transforms normal cells into tumor cells; additional phenotypic expression of viral information may or may not also occur (Huebner and Todero1969).All cancer, in this view, is the result not of any form of inherent potential gone astray but rather of the agency of an enemy lodged within.
SuchDialectics of Disorder: Normalization and Pathology as Process135agency is attributed to a kind of infectious, microscopic pathogen whichhas achieved, for reasons presumed to be explicable in terms of a logicof natural selection, the ability to pass from one generation to the nextwhile entering into tumor-forming activity typically late in the life cycleof the host. This hypothesis introduced the concept of “oncogenes.” Byconceiving of oncogenes as viral genes lodged in the germ line Heubnerand Todero endorsed a model of the cancer cell as internally determined,albeit without subscribing to a somatic mutation etiology.
Internal determination of an aberrant “autonomous” condition and somatic mutation,in the Heubner-Todero hypothesis, became uncoupled.In 1971 Howard Temin offered an alternative hypothesis whichattempted to account for the origins of the retroviruses and do so in away which recovered a somatic mutation model of carcinogenesis, albeitwith an interesting new twist.Temin theorized that normal somatic differentiation occurred througha process of sequential somatic mutations facilitated by the activity ofthe reverse transciptase enzyme. Temin’s theory placed carcinogenesisback into the context of the processes of normal development but nowwith a characteristically “informationistic” orientation.An organism needs to identify cells in a stable way, so that one cell is identifiedas a retinal cell at a particular position, and another cell is committed to makeantibody to a particular antigen.
The most stable storage place for such information appears to be the cellular DNA. RNA Æ DNA information transfer insomatic cells would provide a mechanism for stable differentiation of DNA(Temin 1971).Viruses are explained in this view as a contingent side effect of theprocesses of normal cellular differentiation. In Temin’s view, RNA originating from one cell is reverse transcribed into the DNA of another cell,resulting in somatic differentiation. Temin uses the term protovirus torefer to that normal sequence of reverse-transcribed DNA, the integration of which into a cell’s genome (thus a somatic mutation) causes itsproper differentiation.The process of protovirus transfer might work as follows.
A region of DNA incell A serves as a template for synthesis of an RNA which is transferred to cellB. In cell B, a new DNA is made by an RNA dependent DNA polymerase, usingthe transferred RNA as template. This new DNA then integrates into the DNAof cell B. This integration could be next to the homologous DNA or at a136Chapter 4different place. In either case, cell B would differ from cell A, which remainsunchanged (Temin 1971).Somatic mutation, in Temin’s view, is not in itself an aberrant occurrencebut rather a basic and unavoidable feature of cellular differentiation.Viruses emerge from such processes strictly by chance.
They result whena reverse-transcribed stretch of DNA happens to become integratedinto a genome such as to result in a new string of nucleic acids (genes)that are capable of quasi-independent replication. In the vast majorityof cases, the formation of the reverse-transcribed DNA (a protovirus)does not result in a virus but only in a properly differentiated somaticcell. Cancer, according to the Temin theory, is a product of the limitedfidelity of normal somatic-cell differentiation. Cancer, for Temin, is onceagain the result of somatic-cell mutation, although only of the odd caseof it.The usual process leading to cancer could be a variation in the normal physiological evolution of the protovirus DNA, so that variants which contain information for the cancer appeared either by mutation of the base sequence or byintegration in incorrect places or both (Temin 1971).Temin’s idea of differentiation by somatic mutation, with the notableexception of the immune system, has largely not been borne out.
Interesting as it was, Temin’s hypothesis of cancer causation could not beredeemed if directed somatic mutation is not found to be operative inprocesses of cellular differentiation (other than in the immune systemwhere just such a process is well established).The kinetics of RSV-induced tumor formation had long been recognized as highly variable. Cloning and nucleic acid sequencing studies ofthe retroviral genome during the 1970s revealed that there were twoclasses of retroviruses, one which was acutely transforming—capable ofrapidly inducing tumor formation in animals—and the other which wasonly weakly transforming, that is, capable of inducing tumors but onlyafter a long latency period.
It was soon recognized that these were structurally different in only one respect. The acutely transforming viruseswere larger than the weakly transforming viruses by one stretch ofnucleic acid, that is, by one gene (Cooper 1990). This gene, whatever ithappened to be, constituted the difference between acutely transformingand only weakly transforming viruses. Owing to its apparent significanceDialectics of Disorder: Normalization and Pathology as Process137in tumor formation, whatever gene was making the difference wasawarded Huebner’s and Todaro’s designation oncogene.The first oncogene to be identified, through comparing the nucleic acidsequence composition of acutely transforming and weakly transformingRous sarcoma virus strains, was named the “src” (for sarcoma) gene(Duesberg 1983).
It was found that different so-called species of tumorcausing retroviruses differed from their less-virulent isoforms, throughsingle genes, but that all these genes were different. Sequence comparisons of other retroviruses began producing a growing catalog of putative oncogenes. The end of 1982 saw the identification of 17 other suchoncogenes from retroviruses (Bishop 1982).
An obvious question in theaftermath of both the Huebner & Todoro and Temin hypotheses was:Where did the oncogenes come from? Were they of foreign originationand akin to the oncogenes of Huebner and Todoro or the stochastic sideeffect of some otherwise normal process of somatic rearrangement, à laTemin?Further studies indicated that certain animals that had been infectedwith a weakly transforming virus eventually expressed a tumor fromwhich acutely transforming virus could then be isolated (Cooper 1990).If a weakly transforming virus enters an animal without an oncogeneand then reemerges from the animal with an oncogene it would appearthat it “picked-up” the oncogene from within the animal.
This muchseemed clear. What was left to be determined was whether the putativeoncogene picked up from the host was originally derived from a virus,and thus like the oncogenes depicted by the Heubner-Todaro model, orwas native to the host. And if the latter, then how and when did it becomean oncogene?Working in the laboratories of Harold Varmus and J. Michael Bishop,Dominique Stehlin constructed a molecular probe for the RSV src gene.Nucleic acid probes (DNA or RNA) take advantage of the same chemical features that are used by the cell in DNA replication.
Of the fourDNA building blocks—A, T, C, G—there is differential recognition andbinding between A and T and between C and G. A and T are thus complementary base pairs as are G and C. The principle of nucleic acidpolymer replication, whether in vivo or in vitro, is simply that of usinga sequence of bases as a template for forming its A-T/G-C complement.138Chapter 4A DNA probe must satisfy two requirements. It must have the correctsequence to bind to the gene of interest (and no unrelated genes) throughcomplementary base pairing with the target DNA (that has been uncoupled from its double helix complement by being separated into singlestrands). And it must also be linked to some visualizable marker in orderto enable the investigator to locate it. When a DNA probe derived fromone species is successful at locating a target sequence in another species,it suggests that the target sequence has been highly conserved over evolutionary time and is thus likely to be of much biological significance.
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