Gene amplification and analysis最新文献

We have described how DNA methylases may be used to enhance the apparent specificities of restriction endonucleases [9-11, 17, 18] to generate DNA fragments averaging 6000 to 270,000,000 base pairs on random DNA. Taking into account the non-random arrangement of natural DNA, we have predicted the rarity of certain recognition sequences in the genomes of several species.

We have described the development of a selective system useful for the identification and characterization of onc genes capable of conferring growth factor independence. The use of defined, serum-free media allows us to select for transformed cells which have lost specific growth factor requirements. We have used this system to show that several cloned onc genes generate different transformed phenotypes with respect to growth factor requirements. BPV-1 is active in relieving contact inhibition, yet these transformed NIH/3T3 cells retain their stringent requirement for FGF. In contrast, sis and H-ras were equally proficient at relieving contact inhibition and the requirement for FGF. Sis induced equal numbers of colonies regardless of the presence or absence of insulin, however, H-ras-mediated colony formation decreased four-fold when insulin was removed. This suggests that H-ras is less efficient in relieving the insulin requirement than is sis. To determine if colony formation by H-ras is a function of dosage, we are conducting experiments to measure the level of expression of p21 in transformants selected with and without insulin in the media. We have also presented data to show that loss of contact inhibition and loss of growth factor requirements are dissociable phenotypes under separate control in some cells. Thus, it should be possible to use this selective system to identify transforming genes in tumor DNA. Since some of these genes may be undetectable by the standard focus forming assay, selection in MSF medium may prove to be a useful tool for identifying and elucidating the action of activated cellular onc genes.

Cancer is a malfunction of cellular growth control. The discovery of oncogenes, first in transforming retroviruses, and later in human and animal tumors, may have uncovered the key to understanding one of the most elusive subjects of basic cell biology, namely, the controlling mechanisms of cell growth. The ras gene family encodes a group of closely related 21,000 dalton (p21) proteins with special affinity for guanine nucleotides. Other cellular proteins with similar biochemical properties, collectively known as G-proteins, include the regulatory G proteins of adenylate cyclase, the alpha subunit of transducin of retina rod outer segments, the recently identified rho gene proteins, and perhaps also the elongation factors, EF-Tu and EF-G, of the protein synthesis system. These G-proteins have roles in cellular signal transduction; by analogy p21 may have a similar cellular function in mediating the flow of growth control signals. Recent progress in the cloning and sequencing of these genes, overproduction of gene products in E. coli, protein engineering, detailed biochemical characterization, and the molecular structure determined by high resolution X-ray crystallography, have helped to elucidate in great detail the structure and function of p21 ras proteins. p21 appears to have a small membrane binding domain at the C-terminus, which contains a palmitylation site at cysteine-186, four amino acid residues from the end. Separated by a variable "hinge" region, most of the rest of ras amino acid sequences are highly conserved in nature. Four regions of extensive sequence homology among G-proteins constitute the GTP/GDP binding domain. In the crystal structure of EF-Tu, four peptide loops connecting beta sheets and alpha helices form the pocket for binding GDP. Studies using site-directed mutagenesis and immnochemical probes, indicate that the basic structure of the GDP binding site is conserved between p21 and EF-Tu. Furthermore, these studies also conclude that GTP binding is crucial for p21 ras cellular function. Although the precise target molecules for p21 are still unknown, the finding of the on/off switch function for ras genes have provided a better understanding of the mechanism of proto-oncogene activation, and may also provide further impetus to explore means of cancer intervention by interfering with the switch function.

The mammalian homologues of the ets-region from the transforming gene of avian erythroblastosis virus, E26, consists of two distinct domains located on different chromosomes. Using somatic cell hybrid panels, the mammalian homolog of the 5' v-ets-domain (ets-1) was mapped to chromosome 11 in man, to chromosome 9 in mouse, and to chromosome D1 in cat. The mammalian homolog of the 3' v-ets domain (ets-2) was similarly mapped to human chromosome 21, to mouse chromosome 16, and to feline chromosome C2. To better define the human proto-ets domains, the genomic DNA was molecularly cloned and sequences analyzed. The ets-related sequences of human DNA on chromosomes 11 and 21 were found to be discontiguous, unlike that of the chicken and avian E26 virus genome, except for a small overlap region. We conclude that the ets sequence shared by the virus, the chicken and man is likely to contain at least two dissociable functional domains, identifiable as ets-1 and ets-2. The human ets-1 locus is transcriptionally active and encodes a single mRNA of 6.8 kb, while the second locus, human ets-2 encodes three mRNAs of 4.7, 3.2 and 2.7 kb. By contrast, the chicken homolog, having a contiguous ets-1 and ets-2 sequence, is primarily expressed in normal chicken cells as a single 7.5 kb mRNA. Because chromosome translocations have been associated with different human disorders, we have used our human probes with two panels of rodent-human cell hybrids to study specific translocations occurring in acute myeloid leukemias (AML). The human ets-1 gene was found to translocate from chromosome 11 to 4 in t(4;11)(q21;q23) and the human ets-2 gene was found to translocate from chromosome 21 to 8 in t(8;21)(q22;q22). Both translocations were found associated with the altered expression of ets.