Paul Berg - US grants

The identification of cellular genes whose expression or overexpression can cause cellular transformation and cancer is an important goal. The proposed researches, summarized below, are directed towards that objective. 1. A new method has been developed for cloning cellular mRNAs as cDNAs in E. coli or cultured animal cells. Since the procedure can generate nearly full length cDNA clones (the cDNA segments extend from the nucleotides adjacent to the poly A site up to, or a few nucleotides short of, the cap site) with high efficiency (about 10 5 cDNA recombinants per Mug of cell mRNA), the cloning vector has been constructed in such a way as to permit the expression of the cloned cDNA segment. Thus, it is feasible to attempt to clone cDNAs that could alter the phenotype of selected recipient cells or produce a product which permits the selection or identification of transduced cells. One objective is, therefore, to isolate cDNA copies of known and as yet unrecognized oncogenes by their ability to transduce normal to transformed cells. Similarly, cDNAs prepared from normal cells will be tested to determine if any can suppress the transformed cell phenotype of induced or natural cancer cells. 2. Studies of the structure and function of the SV40 virus early region promoter have shown that a 72 base pair tandem repeat sequence at about position -100 to -25 from the CAP-sites of the early mRNA, has a unique capacity to augment the transcribing activity of weak promoter sequences. The entire SV40 early promoter and its constituent parts (the promoter per se and the 72 bp tandem repeat) have been introduced into bacterial plasmids and will be tested for their ability to transduce normal to transfored cells by integrating at or near cellular genes whose overexpression lead to transformation. If transformed cells arise, the modified and corresponding normal genomic regions will be cloned and an analysis of their structure and oncogenic properties undertaken.

The advent and widespread application of the recombinant DNA methodology during the past twelve years has greatly extended our understanding of the molecular basis of heredity. More significantly, the emergence of a new molecular paradigm - reverse genetics, which relies on the ability to specifically modify the structure of genes in vitro and to test their biological properties, has revolutionized the experimental approaches in molecular genetics. The ability to construct minichromosomes with novel arrangements of genetic elements also provides vectors for the isolation of genes as well as substrates for the analysis of specific genetic reactions. Our proposal, which applies this approach to explore the basic mechanisms of gene regulation in the establishment of immune responsiveness and of genetic recombination, aims (1) identify the factors and mechanisms that limit the epression of antibody genes to only lymphoid cells; (2) isolate and characterize the human gene whose dysfunction is responsible for galactosemia; (3) understand the mechanisms responsible for non- homologous, homologous and non-reciprocal modes of genetic recombination, especially those which permit the rejoining of broken DNA; (4) develop genetic approaches to effect a replacement or repair of chromosomal DNA sequences; (5) developing vectors which can deliver genes to only specific types of cells. It is anticipated that the information we develop will advance our unerstanding of the molecular basis of disease and provide novel approaches to their prevention, treatment and cure.

The advent and widespread application of the recombinant DNA methodology during the past twelve years has greatly extended our understanding of the molecular basis of heredity. More significantly, the emergence of a new molecular paradigm - reverse genetics, which relies on the ability to specifically modify the structure of genes in vitro and to test their biological properties, has revolutionized the experimental approaches in molecular genetics. The ability to construct minichromosomes with novel arrangements of genetic elements also provides vectors for the isolation of genes as well as substrates for the analysis of specific genetic reactions. Our proposal, which applies this approach to explore the basic mechanisms of gene regulation in the establishment of immune responsiveness and of genetic recombination, aims (1) identify the factors and mechanisms that limit the epression of antibody genes to only lymphoid cells; (2) isolate and characterize the human gene whose dysfunction is responsible for galactosemia; (3) understand the mechanisms responsible for non- homologous, homologous and non-reciprocal modes of genetic recombination, especially those which permit the rejoining of broken DNA; (4) develop genetic approaches to effect a replacement or repair of chromosomal DNA sequences; (5) developing vectors which can deliver genes to only specific types of cells. It is anticipated that the information we develop will advance our unerstanding of the molecular basis of disease and provide novel approaches to their prevention, treatment and cure.

This proposal includes a set of six research projects which form a coherent and integrated effort to understand the processes tending to alter genome structure and organization, and the mechanisms cells possess to promote, resist and repair such modifications. Because these processes are undoubtedly fundamental to cell and organismal survival, we presume that they may be important in cellular and organismal senescence. Accordingly, a principal aim of this program project is to characterize the participants and molecular mechanisms of selected DNA transactions, and to explore their status and possible relevance to the aging process. The individual projects focus on: 1. repair of double strand breaks and deletions by recombination in mammalian cells, 2. regulation of chromosomal replication and its influence on the frequency of chromosome breakage, recombinational loss and nondisjunction, 3. an assessment of the role of DNA damage and repair in aging phenomena by studies of the developmental or age-related factors which account for the variation in the repairability of DNA damage in different regions of the genome of terminally differentiated cells, 4. an analysis of the replication and error-correction processes in highly purified replication systems with a view toward establishing assays and criteria for evaluating replication fidelity during aging processes, 5. factors governing genome integrity: the replication fidelity of normal and damaged DNA by Herpes Simplex DNA polymerase, and the mechanism of site-specific intramolecular recombination between homologous sequences catalyzed by a recently detected mammalian enzyme, 6. the mechanism of genome rearrangements leading to the assembly of functional immunoglobin heavy and light chains, particularly the switch recombinations that link a heavy chain variable region to any one of five constant regions.