The interaction of adenovirus‐2 (Ad2) early region IA (EIA) protein (encoded by the 13S mRNA) with DNA was examined using EIA protein synthesized in Escherichia coli extracts directed by a plasmid containing the cloned EIA gene. Without any purification, this protein when chromatographed over calf thymus DNA immobilized on cellulose, showed at least two types of salt‐sensitive activities after associating with equal efficiency to both single‐ and double‐stranded DNA; however, a putative C‐terminal proteolytic fragment of the EIA protein (identified by immunoprecipitation with anti‐serum specific to the EIA carboxy‐terminus) showed 10‐fold greater affinity to double‐ versus single‐stranded DNA. When examined with Ad2 DNA, the EIA protein had a retention that was at least 2‐fold higher compared to calf thymus DNA, suggesting some substrate specificity. It was also found that a 1.0 M salt concentration was required for the elution of the EIA protein from pBR322 DNA containing cloned regulatory sequences of adenovirus early regions II and III. This suggests that the strength of the protein interaction depends on the target DNA sequence. Finally, addition of uninfected HeLa cell extract to bacterial extracts containing EIA‐like protein potentiated the association of the protein to double‐stranded calf thymus DNA up to 7‐fold. These data support the hypothesis that the EIA protein interacts with target DNA, presumably mediated by co‐factor(s) in an indirect fashion.
The transforming region of human adenovirus 2 is located in the left 11.2% of the viral genome and is comprised of two distinct genetic units termed EIA and E1B. cDNAs containing the entire nucleotide sequence of the mature ElA 13S and E1B 22S mRNAs that are complementary to these genetic units have been introduced into bacterial plasmids a short distance downstream from the Escherichia coli lac promoter. Upon transformation into appropriate E. coli hosts, one of these plasmids, pKHAO, directed the synthesis of a 45-kilodalton (kd) protein, and the other, pKHBO, synthesized a protein of 54.9 kd. Both of these plasmidencoded proteins constituted 0.1 to 0.3% of the total cellular protein and were virtually identical to the authentic adenovirus 2 EIA 42-to 50-kd and E1B 53-to 58-kd tumor antigens (T antigen) as determined by gel electrophoresis, immunoprecipitation, and tryptic fingerprint analysis. With the use of our pKHBO expression plasmid we were also able to demonstrate that the second AUG sequence appearing in the E1B 22S mRNA corresponded to the start of the gene encoding the large adenovirus 2 T antigen. This confirms theoretical deductions based on DNA sequencing analysis that translation of the large T antigen initiates translation at an internal ATG rather than at the 5'-proximal AUG.
The (l chain of the T-cell (MHC) class I or class II molecules expressed on the antigenpresenting cell. This antigen recognition occurs via the T-cell receptor (TCR) that is embedded in the membrane of the T cell. This multichain chain receptor complex has been well studied and shown to be responsible for both antigen and MHC recognition via two chains ofthe TCR complex, termed a and P (1). A functional ( chain is formed by two successive in-frame rearrangement events involving variable (V), diversity (D), and joining (J) region segments. As is the case with immunoglobulins, the VDJjunctional regions often contain N regions, which are additional amino acids thought to originate from a template-independent addition of nucleotides during the rearrangement process. Several clusters of complementarity-determining regions (CDR1, -2, and -3) can be recognized on the TCR. Modeling studies have suggested that the CDR1 and -2 regions mainly interact with the MHC whereas the CDR3 region interacts with the peptide when it is associated with the MHC molecule (2-4).Studies on the relationship between the structural elements of the TCR and antigen specificity in animal models have indicated that (i) T cell responses may be highly degenerate, using a wide variety of V, D, and J elements (5, 6), (ii) responses may be somewhat limited with half the antigenspecific clones using the same combination of Va and VB genes within a given MHC context (7), or (iii) responses can be strongly correlated with V8 usage (8, 9). In humans evidence exists for limited TCR heterogeneity in human autoimmune diseases, such as multiple sclerosis (10, 11). However, no evidence exists to date for limited TCR heterogeneity in the recognition of a small peptide in the context of a single HLA.In this study, the T-cell response in one tuberculoid leprosy patient against Mycobacterium leprae and the immunodominant 65-kDa heat shock polypeptide (65hsp) (12)
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