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At Stanford University from 1971-1977, while working on his Ph.D. with Robert Schimke, Fred Alt developed a mammalian cell subtractive hybridization approach that ultimately allowed him to discover gene amplification and genomic instability in mammalian cancer cells. While working as a postdoctoral fellow at the MIT Center for Cancer Research with David Baltimore from 1977-1982, he helped elucidate basic principles underlying development and function of antibody producing B lineage cells in the immune system. His work with Baltimore included the discovery that in B lineage cells production of membrane-bound (B Cell Receptor or "BCR") versus secreted immunoglobulin (antibodies) is achieved via differential RNA processing. They also discovered that allelic exclusion of Immunoglobulin (Ig) gene rearrangements is controlled by feedback from protein products. With Baltimore, Alt also elucidated major aspects of the V(D)J recombination mechanism that joins variable (V), diversity (D) and joining (J) gene segments to form exons that encode antibody (and T cell receptor) variable regions. Their mechanistic insights included involvement of site-specific DNA double strand breaks (DSBs) that are end joined in the basic recombination mechanism, as well as discovery of “N” regions (non-templated nucleotides) at D to J or V to DJ junctions, which they proposed to be added by the enzyme terminal deoxynucleotidyl transferase (TdT).

As a faculty member at Columbia College of Physicians and Surgeons, Alt and his lab established the role of Ig chains in regulating progression through sequential stages of early B cell development and discovered that all antigen receptor variable region exons are assembled by a common V(D)J recombinase. They also elucidated a role for non-coding gene transcription in mediating "chromatin accessibility" as means to target the lineage, stage, and allele-specific activity of the "V(D)J recombinase". They extended that work to show that, in mature B cells, IgH class switch recombination (CSR) to particular IgH classes is directed by activation of non-coding transcription units that contain the CSR target sequences. At Columbia, the Alt lab also co-discovered the N-myc cellular oncogene, based on its amplification in human neuroblastomas, and went on to characterize the Myc cellular oncogene family.

Early on upon moving to Harvard Medical School based in Boston Children's Hospital (BCH) and the Center for Blood Research (CBR) that became the Program in Cellular and Molecular Medicine (PCMM) at BCH, Dr. Alt's group confirmed the Alt/Baltimore proposal that TdT is a V(D)J recombinase component that adds N regions to V(D)J junctions. As the portion of the antibody IgH and IgL chain variable regions encoded by the V(D)J junctional region serves as the important antigen-contact complementarity-determining region 3 (CDR3), TdT has a major role in diversifying antigen receptor repertoires. During this earlier period at BCH and the CBR, the Alt lab further discovered that the joining activity of the "V(D)J recombinase complex" is carried out by a novel multi-component general cellular non-homologous DNA end joining pathway. This latter discovery also provided the basis for the elucidation of classical non-homologous DNA end-joining (c-NHEJ), one of the two major DSB repair pathways in somatic mammalian cells. Subsequently, the Alt lab discovered several of the first characterized c-NHEJ component factors and also discovered the role of c-NHEJ proteins in maintenance of genomic stability.