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R G Schoner

Publications and source records attributed to R G Schoner.

11 recordsLinked to original sources
PubMed1992

Production and functional analysis of normal and variant recombinant human transthyretin proteins.

The most common form of hereditary systemic amyloidosis is familial amyloidotic polyneuropathy associated with single amino acid changes in the plasma protein, transthyretin. In addition, there are two variants of transthyretin (Ser6 and Thr109) not associated with familial amyloidotic polyneuropathy but with familial euthyroid hyperthyroxinemia, also an autosomal dominant disorder. In these autosomal dominant diseases, most affected individuals are heterozygous and therefore have hybrid forms of the tetrameric plasma transthyretin. In order to study the structure/function relationships of homozygous variant transthyretins, normal human transthyretin and five variant transthyretins (Gly6----Ser, Leu58----His, Thr60----Ala, Ile84----Ser, and Ala109----Thr) were produced in Escherichia coli using the expression vector, pCZ11, and site-directed mutagenesis. These recombinant transthyretin (r-TTR) proteins showed the correct size (14 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis and self-associated into tetramers as determined by size exclusion chromatography. Recombinant normal, Ser6, and Ala60 r-TTRs had an affinity for thyroxine indistinguishable from normal human TTR purified from plasma, whereas His58 and Ser84 r-TTRs had significantly reduced affinity. On the other hand, Thr109 r-TTR had a much higher affinity, probably due to its position within the thyroxine-binding pocket. Expression of mutant transthyretins in E. coli provides the opportunity to study structure/function relationships and amyloid-forming capabilities induced by single amino acid substitutions in the transthyretin molecule.

Amino Acid Sequence↗
PubMed1986

Translation of a synthetic two-cistron mRNA in Escherichia coli.

A synthetic two-cistron expression system was constructed for the high-level expression of eukaryotic genes in Escherichia coli. This system was designed to overcome translational inhibition of mRNAs containing eukaryotic sequences. The first cistron in this system is a 31-base A + T-rich synthetic sequence that provides for efficient translation initiation. The second cistron contains the protein coding sequence for the eukaryotic gene. Insertion of the first cistron between the 5' untranslated region of the mRNA and the protein coding region separates the two and thereby potentially minimizes the formation of local secondary structures that might prevent ribosomes from binding and initiating translation. The 31-base cistron contains three nonsense codons (TAA), one in each of the three translational reading frames, and an 8-base Shine-Dalgarno sequence that is complementary to the 3' end of the 16S rRNA. The effects of translation of the first cistron in all three reading frames on the expression of the second cistron was examined. The most efficient expression of the second cistron seemed to occur when the stop codon that terminates translation of the first cistron is located 3' to the Shine-Dalgarno sequence and close to the AUG start codon for the second cistron. When the Shine-Dalgarno sequence was deleted from the first cistron, no detectable expression of the second cistron was observed. This two-cistron system has been used to express the gene encoding methionylalanyl bovine growth hormone with its native codons and the gene encoding methionyl human growth hormone at a level greater than 20% of total cell protein. In the case of human growth hormone, we show that the amount of gene product is not significantly diminished by placing a "functional" first cistron in front of a gene that can be expressed without a cistron.

Animals↗
PubMed1984

Role of mRNA translational efficiency in bovine growth hormone expression in Escherichia coli.

The conditions necessary for high-level expression of methionyl bovine growth hormone (Met-bGH) in Escherichia coli were investigated. Plasmids were constructed that contain a thermoinducible runaway replicon and either the E. coli tryptophan or lipoprotein promoter and ribosome binding sites, which served as transcriptional and translational initiation sites for the expression of the bGH gene. The expression of Met-bGH was low with either system. However, expression levels of up to 30% of total cell protein were obtained after the introduction of additional codons 3' to the initiating AUG codon, thus altering the NH2-terminal amino acid sequence of bGH. To obtain high-level expression of Met-bGH a two-cistron system was constructed in which the codons that enhanced the expression of bGH were incorporated into the first cistron, and the coding region for Met-bGH was incorporated into the second cistron. This approach may be generally applicable to achieving high-level expression of a gene that contains NH2-terminal sequences that do not allow for its efficient expression. Analyses of the stabilities of the bGH derivatives and their transcripts in vivo suggested that the variations in the level of expression were due to variations in the efficiency of mRNA translation.

Amino Acid Sequence↗
PubMed1983

Enhanced expression of mouse dihydrofolate reductase in Bacillus subtilis.

pPL608-TR1 is a high-copy plasmid that permits phenotypic expression of the mouse dihydrofolate reductase (DHFR) gene in Bacillus subtilis. A plasmid mutation has been identified that increases expression of mouse DHFR more than ten-fold. The mutation is located in a 0.2-kb segment that intervenes between the DHFR gene and the 0.3-kb promoter fragment needed for transcriptional activation of DHFR. Nucleotide sequence analysis suggests that the effect of the mutation is to facilitate translation, initiated within the promoter fragment, through the 0.2-kb segment to the site of insertion of the DHFR-containing fragment. Additional promoter-containing fragments selected by their ability to promote expression of a plasmid gene located downstream from DHFR, the CAT gene, promote either high, intermediate or no phenotypic expression of DHFR. The results indicate that promoter fragments that allow phenotypic expression of the mouse DHFR gene contain two regulatory signals. One signal is essential to transcription of both DHFR and CAT and therefore functions as a promoter. The second signal may be necessary for translation of that portion of the mRNA specifying mouse DHFR.

Animals↗
PubMed1983

Expression plasmid for B. subtilis.

The collective evidence from several laboratories demonstrates that foreign genes from both prokaryotes and eukaryotes, can be expressed in B. subtilis if the heterologous gene is placed under the control of Bacillus promoters and ribosome binding sites. The nucleotide sequences of several Bacillus promoters are very similar to the consensus E. coli promoter sequence at both the -10 and -35 recognition sites. In spite of this apparent similarity, E. coli promoters appear to be poorly recognized in vitro by B. subtilis RNA polymerase35. Thus, a barrier to heterologous (E. coli) gene expression in B. subtilis may reside at the transcriptional level. In addition, there appears to be a barrier at the level of translation. Rabinowitz and co-workers have shown that ribosomes from gram-positive bacteria, including B. subtilis, are relatively inactive in the in vitro translation of mRNA gram-negative species. This appears to be due to an inability of ribosomes from gram-positive bacteria to use the relatively weak ribosome binding sites that commonly occur in RNA from gram-negative bacteria.

Amino Acid Sequence↗
PubMed1981

The organization of the dihydrofolate reductase gene of baby hamster kidney fibroblasts.

Using cloned DNA complementary to mouse dihydrofolate reductase (DHFR) mRNA, the organization of the hamster DHFR gene has been determined in two baby hamster kidney (BHK) cell lines, A5 and B1. A5 cells are highly methotrexate-resistant, containing 200-fold more copies of the DHFR gene than do the parental B1 cells. The DHFR gene has the same organization in A5 and B1 cells, suggesting that it has not been altered by the amplification process. The BHK DHFR gene spans a maximum of 10.7 kb and contains at least three introns. Thus the BHK DHFR gene is much smaller than the mouse DHFR gene, which has a minimum size of 42 kb and at least five introns. This striking size difference is probably due to much smaller introns in the BHK DHFR gene.

Animals↗
PubMed1981

Expression of Escherichia coli trp genes and the mouse dihydrofolate reductase gene cloned in Bacillus subtilis.

The mouse dihydrofolate reductase gene and a segment of the Escherichia coli trp operon are expressed in Bacillus subtilis when cloned in the "expression plasmid" pPL608. The cloned mouse gene confers trimethoprim resistance on B. subtilis and the cloned trp fragment complements mutations in the B subtilis trpD, C and F genes Expression of both cloned fragments is dependent on a promoter present in the vector plasmid. The E. coli trp fragment is cloned in a HindIII site within a chloramphenicol acetyltransferase gene present on pPL608, and as a result, expression of the E. coli trpC gene product is inducible by chloramphenicol. The mouse gene is inserted at a PstI site preceding the chloramphenicol acetyltransferase gene and its expression is not chloramphenicol inducible. The replication functions and neomycin-resistance of pPL608 are derived from pUB110. Accordingly, pPL608 is stably maintained at high copy number in B. subtilis.

Animals↗
PubMed1981

Distribution of IS5 in bacteria.

Four different strains of Escherichia coli and several other bacteria were examined by Southern analysis for the presence of the insertion element IS5 and IS5-like sequences. Variations in the copy number, degree of homology and restriction pattern of the IS5-like sequence were found among the different organisms. The number and distribution of IS5 sequences do not appear to correlate with the evolutionary relationship of the bacteria in which they occur.

Bacillus subtilis↗