ABSTRACT
The hygromycin B phosphotransferase gene (hpt) has been widely used in the process of plant genetic engineering to produce plants that can secrete the HPT protein. As part of a safety assessment, sufficient quantities of the protein were produced in Escherichia coli to conduct in vitro digestibility and animal studies. Western blotting analysis showed that the HPT protein was digested by simulated gastric fluid within 40 s. ELISA demonstrated that the protein did not induce detectable levels of specific IgE antibodies or histamine in test animals. Alignment of the amino acid sequence of HPT with those of known allergens did not produce evidence of sequence similarities between these allergens and the HPT protein. We conclude that HPT has a low probability to induce allergenicity.
Subject(s)
Allergens/immunology , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/immunology , Plants, Genetically Modified , Amino Acid Sequence , Animals , Anti-Bacterial Agents/antagonists & inhibitors , Anti-Bacterial Agents/immunology , Blotting, Western , Digestion , Escherichia coli , Gene Expression , Hygromycin B/antagonists & inhibitors , Hygromycin B/immunology , Male , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/immunology , Rats , Sequence Alignment , Sequence Analysis, Protein , Sequence Homology , Streptomyces/enzymologyABSTRACT
Although tightly linked, the TCR alpha and delta genes are expressed specifically in T lymphocytes, whereas the Dad1 gene is ubiquitously expressed. Between TCR alpha and Dad1 are eight DNase I hypersensitive sites (HS). HS1 colocalizes with the TCR alpha enhancer (Ealpha) and is T cell-specific; HS2, -3, -4, -5, and -6 map downstream of HS1 and are tissue-nonspecific. The region spanning HS2-6 was reported to display chromatin-opening activity and to confer copy number-dependent and integration site-independent transgene expression in transgenic mice. Here, we demonstrate that HS2-6 also displays enhancer-blocking activity, as it can block an enhancer from activating a promoter when located between the two in a chromatin-integrated context, and can do so without repressing either the enhancer or the promoter. Multiple enhancer-blocking elements are arrayed across HS2-6. We show that HS2-6 by itself does not activate transcription in chromatin context, but can synergize with an enhancer when located upstream of an enhancer and promoter. We propose that HS2-6 primarily functions as an insulator or boundary element that may be critical for the autonomous regulation of the TCR alpha and Dad1 genes.
