George Khoury

Post-translational modifications (PTMs) broadly contribute to the recent explosion of proteomic data and possess a complexity surpassing that of protein design. PTMs are the chemical modification of a protein after its translation, and have wide effects broadening its range of functionality. Based on previous estimates, it is widely believed that more than half of proteins are glycoproteins. Whereas mutations can only occur once per position, different forms of post-translational modifications may occur in tandem. With the number and abundances of modifications constantly being discovered, there is no method to readily assess their relative levels. Here we report the relative abundances of each PTM found experimentally and putatively, from high-quality, manually curated, proteome-wide data, and show that at best, less than one-fifth of proteins are glycosylated. We make available to the academic community a continuously updated resource (http://selene.princeton.edu/PTMCuration) containing the statistics so scientists can assess "how many" of each PTM exists.

The simian virus (SV40) 72-base pair (bp) tandem repeated sequences have recently been shown to function as activators or enhancers of early viral transcription. A recombinant viral genome was recently constructed by inserting 72-bp tandem repeats from the Moloney murine sarcoma virus (MSV) in place of the 72-bp repeats of SV40. Although this genome replicates in monkey kidney cells, its rate of large tumor antigen expression and replication is considerably slower than that of wild-type SV40. In mouse cells, however, equivalent levels of large tumor antigen appear to be expressed from both wild-type and recombinant genomes, suggesting a relationship between the level of enhancer activity and the host cell. To confirm this observation, we have applied a sensitive quantitative assay for gene expression based on the conversion of chloramphenicol to its acetylated forms. The gene encoding the enzymatic function chloramphenicol acetyltransferase was inserted into two vectors in which the enhancer sequences from SV40 or MSV were placed adjacent to the early SV40 promoter. The SV40 tandem repeats appear to activate gene expression to significantly higher levels in monkey kidney cells, but the MSV repeats are more active in two lines of mouse cells. These findings suggest that the tandem repeat elements may interact with host-specific molecules and, furthermore, may constitute one of the elements determining the host range of these eukaryotic viruses.

Human T-lymphotropic virus type 1 (HTLV-1) is a suspected causative agent of adult T-cell leukemia. One of the viral genes encodes a protein (tat) that not only results in transactivation of viral gene expression but may also regulate the expression of certain cellular genes that are important for cell growth. Transgenic mice that expressed the authentic tat protein under the control of the HTLV-1 long terminal repeat were generated, and cell types that are permissive for the viral promoter and the effects of the tat gene on these cells were studied. Three of eight founder mice with high levels of expression of the transgene in muscle were bred and then analyzed. All developed soft tissue tumors at multiple sites between 13 to 17 weeks of age. This phenotype was transmitted to nine of nine offspring that inherited the tat gene and were available for analysis. The remaining five founders expressed the transgene in the thymus, as well as in muscle. This second group of mice all exhibited extensive thymic depletion and growth retardation; in all of these mice, death occurred between 3 to 6 weeks of age before tumors became macroscopically visible. The tat gene under the control of the HTLV-1 regulatory region showed tissue-specific expression and the tat protein efficiently induced mesenchymal tumors. The data establish tat as an oncogenic protein and HTLV-1 as a transforming virus.

On the late side of the simian virus 40 (SV40) DNA replication origin are several sets of tandem repeated sequences, the largest of which is 72 base pairs long. The role of these sequences was examined through construction of deletion mutants of SV40. A mutant from which one of the 72-base-pair repeated units was removed is viable upon transfection of monkey kidney cells with viral DNA. Extension of this deletion into the second repeated unit, however, leads to nonviability, as recognized by the absence of early transcription and of tumor antigen production. These observations indicate that the 72-base-pair repeated sequences form an essential element in the early viral transcriptional promoter and explain the inability of such a deleted genome to complement an early temperature-sensitive mutant of SV40, tsA, as well as the failure to replicate its DNA. In a parallel experiment it was found that the extended deletion mutant was also unable to complement a late temperature-sensitive mutant of SV40, tsB. This suggests that the extended mutant is also defective in DNA replication or late transcription (or both).