Biomedical subjects
S J Eccles
Publications and source records attributed to S J Eccles.
Rapid detection of ultraviolet-induced reversion of an amber mutation in mouse L cells.
An amber codon (TAG) was introduced into the N-terminal coding region of the murine H-2Kb gene. The mutant gene was transfected into mouse L cells, and a clone containing a single unrearranged chromosomally integrated copy of the mutant gene was mutagenized with 254-nm UV radiation. Surviving cells were scored for surface expression of H-2Kb protein with in situ immunoperoxidase staining. Revertants were detected at a frequency of 3 X 10(-6) at a dose of 40 J/m2 (3-5% survival). Revertant genes, cloned by plasmid rescue, contained the expected thymine-to-cytosine transitions at the amber codon. These data show that revertants can be rapidly detected in mammalian cells without selection and provide a basis for the development of mammalian cell lines that could be used to study mutational phenomena. During this study the steady-state level of mRNA was reduced in L cells carrying the amber mutant H-2Kb gene compared with L cells containing a wild-type or revertant H-2Kb gene. This reduction was shown not to be due to transcriptional differences, suggesting that the amber mutation decreases stability of the H-2Kb mRNA.
Differential expression of RT1 class II genes in fibroblast cell lines: recognition by allogeneic and xenogeneic T lymphocytes.
A cosmid (cos a 13.1) containing RT1 class II B alpha and B beta genes was introduced into mouse and rat fibroblast cell lines by transformation. Mouse L cells transformed with cos a 13.1-synthesized cell surface class II molecules that were similar, with respect to apparent molecular weight and binding of xenogeneic- and allogeneic-specific antibodies, to class II molecules on rat B cells. RT1 class II molecules on the surface of mouse L cells were recognized by both allogeneic and xenogeneic T cells. In contrast, rat-2 cells transformed with cos a 13.1 did not synthesize any detectable RT1 class II molecules at the cell surface, and the levels of B alpha and B beta mRNA were generally very low or undetectable. This differential expression of exogenous class II genes was not exclusively due to a trans-acting positive regulatory factor in L cells. Other possible explanations for this difference are discussed.
DNA sequence analysis of a rat RT1 class II A beta gene.
The rat major histocompatibility complex (RT1) encodes twin sets of class II molecules, each consisting of two polypeptide chains referred to as A alpha and A beta, and E alpha and E beta. A gene encoding the RT1.A beta chain was isolated from a rat genomic library using an HLA-DQ beta chain cDNA as a probe. The nucleotide sequence of the coding regions of this gene was determined. Comparison of this sequence with those of the corresponding genes of mouse (H-2A beta) and human (HLA-DQ beta) revealed that this gene has been highly conserved during evolution, and that some parts of the molecule are more conserved than others. Analysis of the nucleotide sequence encoding the two external domains suggests that the membrane proximal domain has been subject to conservative selection, whereas replacement substitutions have been selected positively at certain residues within the amino terminal domain. The overall organization of the RT1.A beta gene is similar to that of the H-2A beta gene.
Biochemical and genetic characterization of the tet determinant of Bacillus plasmid pAB124.
A fragment of Bacillus plasmid pAB124 carrying the genes encoding tetracycline resistance was previously cloned into Escherichia coli plasmid pSF2124 (S.J. Eccles, A. Docherty, I. Chopra, S. Shales, and P. Ball, J. Bacteriol. 145:1417-1420, 1981). The cloned pAB124 tet fragment conferred low-level resistance in E. coli, but exposure of this strain to a subinhibitory level of tetracycline led to selection of a mutant plasmid in which high-level resistance, associated with decreased drug accumulation, was expressed constitutively. In this plasmid, the Bacillus tet determinant appeared to be transcribed from a promoter on the vector. Construction of tetracycline-sensitive derivatives of this plasmid by transposon insertion mutagenesis allowed identification of a 32,000-dalton membrane-located protein, which apparently promoted decreased accumulation of tetracycline. This protein was also synthesized as a 32,000-dalton polypeptide in a coupled, in vitro transcription-translation system directed by plasmid DNA. The pAB124 tet determinant differed from the tetA through tetD determinants found in gram-negative bacteria in DNA-DNA hybridization and in the ability to prevent accumulation of different tetracycline derivatives, but was closely related to the tet determinant of another plasmid isolated from Bacillus species, pBC16.
Diffusion of tetracycline across the outer membrane of Escherichia coli K-12: involvement of protein Ia.
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Accumulation of tetracyclines by Escherichia coli K-12.
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Tetracycline resistance in Escherichia coli K12 is not associated with a decrease in cyclopropane fatty acid content.
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