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Characterization of a unique embedded gene
Abstract
The lysis gene region of bacteriophage [] consists of a cluster of three gene: S, R, and Rz. While the S and R genes are essential for [] lysis under all conditions, the Rz gene is required for [] lysis only under certain conditions. Transposon insertions in the [] Rz gene block lysis if the medium of host cell culture contains Mg²⁺ ions or other divalent cations at 5 mM or higher concentrations, but otherwise cause no phenotypes. The Rz gene product is thought to encode an endopeptidase activity previously reported in [] lysates and which might be involved in the cleavages of oligopeptide crosslinks between glycan strands and the lipoprotein (LPP) from the outer membrane. Recently, a small lipoprotein (41 amino acids in length) has been reported as the product of a short reading frame that is, designated Rz1, totally embedded within the distal half of Rz gene in the +1 register. This protein has been detected in membranes of induced [] lysogens. To determine whether Rz1 has a function in [] vegetative life cycle, amber nonsense alleles of Rz and Rz1 have been constructed by site-directed mutagenesis and used for complementation and suppression analysis. It is demonstrated that, Rzam and Rz1am alleles have phenotypes identical to the original Rz insertion mutant alleles, complement in a non-suppressing host and are fully suppressed in a supE host, indicating that Rz and Rz1 are two distinct, trans-acting genes encoding diffusible proteins required for lysis in the presence of divalent cations. Moreover, it has been observed that, supF host suppresses Rzam but not Rz1am. And the defective Rz1am product (Rz1W38Y) in the supF host shows partial dominant character and significantly retards lysis even in the absence of additional cations in the medium. Rz and Rz1 are unique in biology: two genes occupying the same nucleotide sequence in different reading frames, encoding different proteins, but required in the same physiological pathway. To further investigate the possible interactions between Rz and Rz1 proteins, and also to localize Rz1 protein onto the cell, epitome tag sequences were inserted onto C-terminal domains of Rz or Rz1 by standard PCR and DNA cloning techniques. The tagged alleles Rz_AU1, Rz1-AU1, and Rz1_IRS were constructed and shown to be functional.
Description
Due to the character of the original source materials and the nature of batch digitization, quality control issues may be present in this document. Please report any quality issues you encounter to digital@library.tamu.edu, referencing the URI of the item.Includes bibliographical references (leaves 86-91).
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Citation
Zhang, Ning (1999). Characterization of a unique embedded gene. Master's thesis, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /ETD -TAMU -1999 -THESIS -Z431.
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