Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1994-09-19
2004-03-30
Horlick, Kenneth R. (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024310, C536S024320
Reexamination Certificate
active
06713619
ABSTRACT:
GOVERNMENT SUPPORT
The work described herein was supported by grants from the National Institutes of Health, the National Cancer Institute and the American Business Cancer Research Foundation.
TECHNICAL FIELD
This invention is in the field of molecular biology and more specifically relates to isolating discrete genes from mammalian DNA and to defining differences between mutant alleles and their corresponding wild type alleles, particularly oncogenes and proto-oncogenes, and to assays which take advantage of such differences.
BACKGROUND ART
Previous work relating to chemical carcinogenesis has demonstrated that carcinogenic potency of a compound often correlates with its mutagenic power. See McCann, J., Choi, E., Yamasaki, E. and Ames, B. N.
Proc. Natl. Acad. Sci. USA
72: 5135-5139 (1975); McCann, J. and Ames, B. N.
Proc. Natl. Acad. Sci. USA
73: 950-954 (1976); Bridges, B. A.
Nature
261: 195-200 (1976); and, Bouck, N. and diMayorca, G.
Nature
264: 722-727 (1976). This suggests that DNA is the ultimate target of carcinogenic activation. Because of this, researchers have attempted to identify and study DNA segments in tumor cells, often referred to as “oncogenes,” whose alteration is critically important for oncogenic conversion.
The molecular basis of malignant transformation leading to cancer is poorly understood. It is generally thought that transformation begins with damage to DNA, although the exact nature of the damage is in dispute. Presumably, the alteration of critical genes, sometimes called oncogenes, which are carried in the DNA is an essential step in a conversion of a normal cell to one that is capable of causing cancer. Such oncogenes could be activated by carcinogens of various kinds known to cause cancer.
There has been a tremendous amount of research effort directed toward identifying, and presumably subsequently isolating, such oncogenes. Despite the amount of effort which has been undertaken, little is understood about activation and activity of oncogenes.
DISCLOSURE OF THE INVENTION
This invention relates to a method for isolating a discrete, transmissible gene from DNA of mammalian origin and to an investigation of the differences between oncogenes of mammalian origin and their corresponding proto-oncogenes. In particular, it relates to definition of differences between the EJ oncogene, previously shown to cause human bladder cancer, and its proto-oncogene and to assessment of cellular DNAs to determine whether they include lesions or alterations in a neu gene which result in activation of the proto-oncogene and conversion of the proto-oncogene to an oncogene. The procedures involved in defining the differences in these proto-oncogene/oncogene pairs can be used to define differences between any mutant allele and its corresponding wild type allele; the procedures are particularly useful in defining the differences between oncogenes and their corresponding proto-oncogenes.
Identifying and isolating oncogenes has many desirable consequences. The oncogene isolated can be compared with closely related sequences in normal DNA and such comparison should lead to an understanding of what alterations occur to lead to the creation of an active oncogene; in fact, such comparison has made it possible to demonstrate that a proto-oncogene can be converted to an oncogene by a single nucleotide alteration or point mutation. This has made it possible to produce oligonucleotide probes which are specific for nucleotide sequences of the region in which the activation mutation(s) resides. Such probes can be used to determine the presence or absence of an oncogene which differs from its proto-oncogene. Methods of detecting an oncogene using such probes are described herein. As described in detail in the following sections, investigation of the activity and characteristics of oncogenes and their corresponding proto-oncogenes entailed discovery of a method for isolating a discrete, transmissible gene of mammalian origin, initial assessment of the basis of functional differences between the two (oncogene and proto-oncogene) and subsequent determination of the means by which a proto-oncogene is converted or activated to its oncogene form.
Discovery of a Method for Isolating a Discrete, Transmissible Gene
As described in application Ser. No. 379,721, filed May 19, 1982, now abandoned, the concept of a discrete, definable oncogene has been directly demonstrated by molecular isolation of discrete transforming genes from the EJ human bladder carcinoma cell line. That such a discrete, definable oncogene exists was demonstrated using a method described in the co-pending application.
In the method described, mammalian DNA from a donor is fragmented into a multiplicity of fragments, at least one of which contains a discrete, transmissible gene of interest. A “marker” is provided on the fragment containing the gene of interest, if such a marker is not already present. The multiplicity of fragments are then transmitted into recipient cells which are capable of phenotypically expressing the presence of the discrete transmissible gene, i.e., the phenotype of the gene can be scored by cells carried in culture. These recipient cells are then cultured under conditions which allow phenotypic expression by the gene of interest, and cells showing such phenotypic expression are selected. Because of the phenotypic expression, the selected recipient cells are known to contain the gene of interest; they may also contain additional DNA sequences on the donor fragment transmitted into the recipient cells, as well as their own endogenous DNA. The selected recipient cells' DNA is then recovered and used in the aforementioned series of steps in place of the original donor DNA. These steps are repeated until the recipient cells selected in the last step have acquired essentially only the discrete, transmissible, mammalian gene and its associated marker. At this point, the discrete, transmissible gene is recovered from the cells selected employing its associated marker.
In one embodiment of this invention which has actually been experimentally performed, an oncogene for human bladder cancer has been isolated from DNA obtained from a human bladder cancer cell line. The original DNA was serially passed by transfection into mouse fibroblast cells using the above method until a mouse fibroblast cell containing essentially only the human bladder cancer oncogene and a marker was selected. In this case, the marker was an Alu DNA sequence which is repeated about 300,000 times in a human DNA molecule but is not present in mouse fibroblast cells. The interspecies transfection thus resulted in the ultimate selection of a cell containing the oncogene of interest and its associated marker. All of the DNA from the transfected cell was employed in the creation of a genomic library in a lambdaphage and the appropriate chimeric lambdaphage was then selected using a probe specific for the human Alu marker.
A sub-cloned insert of 6.6 kb which carried transforming activity was amplified in the plasmid vector pBR322. The sub-cloned oncogene has been used as a sequence probe in Southern blot analyses. The oncogene appears to derive from sequences present in normal cellular DNA. Structural analysis has so far failed to reveal differences between the oncogene and its normal cellular homolog. The oncogene is unrelated to transforming sequences detected in a variety of other types of human cell lines of colonic, lung, and neuroblastoma origin. In contrast, the human bladder oncogene isolated from one cell line appears closely related to oncogenes active in other human bladder carcinoma cell lines.
Assessment of the Basis of Functional Differences Between Oncogenes and Their Corresponding Proto-oncogenes
Initially, experiments were performed to determine whether the dramatic functional difference between the EJ oncogene and its proto-oncogene were due to a regulation mechanism or to one of sequence differences. These experiments provide data indicating that upregulation of this gene was not responsible for cellular transfor
Bradley Scott M.
Tabin Clifford J.
Weinberg Robert A.
Cooper & Dunham LLP
Horlick Kenneth R.
Massachusetts Institute of Technology
White John P.
LandOfFree
Oncogenes and methods for their detection does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Oncogenes and methods for their detection, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Oncogenes and methods for their detection will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3204260