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R C Mierendorf

Publications and source records attributed to R C Mierendorf.

17 recordsLinked to original sources
PubMed1990

Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning.

We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the lambda EXLX(+) and lambda EXLX(-) vectors that can be used for the expression in Escherichia coli of proteins encoded by cDNA inserts. This is achieved by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the lambda SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage lambda to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage lambda arms, lambda LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of a specialized plasmid between these sites will convert it into a phage lambda cDNA cloning vector with automatic plasmid subcloning capability.

Bacteriophage lambda↗
PubMed1986

Initial events involved in the synthesis of the lysosomal enzyme alpha-mannosidase in Dictyostelium discoideum.

In Dictyostelium discoideum the lysosomal enzyme alpha-mannosidase is initially synthesized in vivo as a 140,000 Mr protein which is subsequently processed into two mature acidic glycoproteins of 60,000 and 58,000 Mr. To investigate the initial events involved in the synthesis of this protein, mRNA isolated from growing cells was translated in vitro and the resulting protein products were immunoprecipitated with antibodies prepared against the purified enzyme. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of an immunoprecipitable 120K protein that was identified as the alpha-mannosidase primary translation product by a variety of criteria. Translation in vitro in the presence of dog pancreas microsomes resulted in the conversion of the 120K primary translation product to a 140K form. This 140K species was not accessible to added trypsin under conditions preserving membrane integrity, suggesting it is sequestered in the lumen of the endoplasmic reticulum following synthesis. Treatment of either the in vitro modified or cellular 140K alpha-mannosidase precursors with endoglycosidase H resulted in the appearance of proteins 2K larger than the primary translation product. The pulse-labeled cellular precursor and the in vitro processed form have similar isoelectric points as revealed by two-dimensional gel electrophoresis. These results imply that the precursor is N-glycosylated in the endoplasmic reticulum possibly without removal of the signal sequence and that the majority of acidic modifications are added late in the post-translational pathway.

Animals↗
PubMed1985

A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure.

The in vitro synthesis of extraneous RNA sequences by SP6 and T7 RNA polymerases from specific DNA templates is described. Transcription of templates prepared by digestion with restriction enzymes that leave 3' protruding ends resulted in the production of significant amounts of long, template-sized RNA transcripts which hybridized to vector DNA. Sequences copied from the noncoding template strand were among the extraneous transcripts. The presence of these sequences in probe preparations were detected in Southern and RNase protection hybridization assays. In contrast, transcription of DNA templates with blunt or 5' protruding ends yielded few RNA products as extraneous sequences.

Base Sequence↗
PubMed1985

Regulation of lysosomal alpha-mannosidase-1 synthesis during development in Dictyostelium discoideum.

The cellular specific activity of lysosomal alpha-mannosidase-1 increases dramatically during development in Dictyostelium discoideum. alpha-Mannosidase-1 is composed of two subunits (Mr = 58,000 and 60,000) which are derived from a common precursor polypeptide (Mr = 140,000). Using enzyme-specific monoclonal antibodies we have determined that throughout development (a) the relative rate of precursor biosynthesis closely parallels the rate of accumulation of cellular enzyme activity and (b) the newly synthesized precursor is efficiently processed to mature enzyme (t1/2 less than 10 min). This indicates that the developmental accumulation of alpha-mannosidase-1 activity is primarily controlled by de novo enzyme synthesis. Furthermore, the change in the relative rate of enzyme precursor synthesis can be accounted for by an increase in the cellular level of functional alpha-mannosidase-1 mRNA during development.

Dictyostelium↗
PubMed1985

Pathways involved in targeting and secretion of a lysosomal enzyme in Dictyostelium discoideum.

In Dictyostelium discoideum, the lysosomal enzyme alpha-mannosidase is first synthesized as an N-glycosylated precursor of Mr 140,000. After a 20-30-min lag period, up to 30% of the precursor molecules are rapidly secreted, whereas the rest remain cellular and are proteolytically processed (t 1/2 = 8 min) to mature subunits of Mr 58,000 and 60,000. The secreted precursor is modified more extensively than the cellular form, as is revealed by differences in size, charge, and sensitivity to endoglycosidase H. Subcellular fractionation has shown that, following synthesis in the rough endoplasmic reticulum, the precursor is transported to a low density membrane fraction that contains Golgi membranes. Proteolytic processing takes place in these vesicles, since newly cleaved mature enzyme, but no precursor, co-fractionates with lysosomes. Under conditions that disrupt vesicular membranes, the precursor remains associated with the membrane fraction, whereas the newly processed mature enzyme is soluble. Proteolytic cleavage of the precursor thus coincides with the release of the mature enzyme into the lumen of a lysosomal compartment. These findings suggest a possible mechanism for lysosomal targeting that involves the specific association of enzyme precursors with Golgi membranes.

Biological Transport↗
PubMed1984

Sulfated oligosaccharides block antibodies to many Dictyostelium discoideum acid hydrolases.

The lysosomal hydrolases of the cellular slime mold, Dictyostelium discoideum, possess a common posttranslational modification which is extremely antigenic in rabbits and mice. Rabbit antisera and mouse monoclonal antibodies that recognize this determinant cross-react with a group of at least 40-50 highly negatively charged proteins which include most or all of the lysosomal enzymes. (Knecht, D. A., Dimond, R. L., Wheeler, S., and Loomis, W. F. (1984) J. Biol. Chem. 259, 10633-10640). The present study demonstrates that the determinant is found on certain N-linked oligosaccharides derived from one of these proteins. An esterified sulfate is absolutely required for antigenicity.

Antibodies, Monoclonal↗
PubMed1983

Synthesis of related forms of the lysosomal enzyme alpha-mannosidase in Dictyostelium discoideum.

Purified lysosomal alpha-mannosidase from the cellular slime mold, Dictyostelium discoideum, is composed of two subunits of Mr = 58,000 and 60,000 as revealed by polyacrylamide gel electrophoresis under denaturing conditions. The pattern of peptide fragments produced when these two species are digested with proteases indicates that they are related but not identical. Using monoclonal antibodies prepared against purified alpha-mannosidase, we have analyzed the different forms of the enzyme synthesized in vivo. In addition to two bands that co-migrate with the pure enzyme, a large Mr (140,000) species is found in immunoprecipitated [35S] methionine-labeled extracts of cellular and secreted proteins. The precipitation of all three bands is inhibited by preadsorption of the antibodies with pure enzyme and all three proteins are absent in extracts of an alpha-mannosidase structural gene mutant. One-dimensional peptide maps indicate that all sequences present in the smaller species are found in the Mr = 140,000 form. In addition, the large form accounts for about 20% of the extracellular alpha-mannosidase activity secreted by amoebae during growth in axenic culture. These results lead to the conclusion that alpha-mannosidase is first synthesized as a large enzymatically active precursor which is modified and proteolytically cleaved to form the smaller subunits.

Dictyostelium↗
PubMed1983

Functional heterogeneity of monoclonal antibodies obtained using different screening assays.

Two alternate screening methods have enabled the detection of monoclonal antibodies with different specificities toward the lysosomal enzyme alpha-mannosidase of Dictyostelium discoideum. Spleen/myeloma hybrid cell cultures were screened for antibody production by separate assays: an indirect enzyme-linked immunoadsorbent assay (ELISA) based on the antibody binding to enzyme adsorbed on plastic, and a direct assay of the antibodies' ability to precipitate enzyme activity with fixed Staphylococcus aureus cells (Pansorbin). Fourteen stable antibody-producing cell lines resulted from a single fusion; these fell into three distinct classes based on their screening characteristics. A group of eight were positive in both assays, and these immunoprecipitated a 140,000 Mr precursor form of alpha-mannosidase in addition to the 58,000 and 60,000 Mr mature enzyme subunits from [35S]methionine-labeled total secreted protein preparations. Two of the antibodies were positive only in the immunoprecipitation assay; these failed to precipitate the 140,000 Mr precursor. The third class consisted of four antibodies that were positive only in the ELISA method. These exclusively recognized an altered conformation of the enzyme (precursor and mature forms) that was immobilized either on plastic or on nitrocellulose paper. In addition, only members of this class were able to bind to immobilized fragments of protease-treated enzyme. The implications of these findings for the general design of monoclonal antibody screenings and for the alternative structures of this enzyme are discussed.

Animals↗
PubMed1982

The effect of dexamethasone on the initiation of beta-globin gene transcription in differentiating Friend cells.

Plasmid subclones of the 5' and 3' regions of the beta major-globin gene have been used as probes to assay the level and character of the transcripts of this gene in differentiating Friend erythroleukemia cells treated with dexamethasone. Using an S1 nuclease protection protocol, it is shown that dexamethasone reduces the amount of precursor-specific sequences in differentiating Friend cells within 30 min without significantly affecting the nuclear turnover of these molecules. The sites of initiation and termination of transcription remained the same during the shutdown of transcription by the hormone. In contrast to the situation in Friend cells, dexamethasone failed to inhibit globin gene transcription in three normal erythroid tissues. The manner in which the Friend virus complex might render the globin genes sensitive to glucocorticoids is discussed.

Animals↗
PubMed1981

Role of dexamethasone in globin gene expression in differentiating Friend cells.

The expression of the globin genes which accompanies the chemically induced differentiation of Friend erythroleukemia cells is subject to inhibition by glucocorticoid hormones. The present study inquires into the possible mechanisms for this suppression. It is shown that the synthetic glucocorticoid, dexamethasone, can both prevent the initial appearance of beta-globin mRNA during the induction of differentiation and inhibit the ongoing production of this RNA in induced cells. Isolated nuclei from dexamethasone-treated cells also exhibited a depressed ability to synthesize beta-globin mRNA. These effects were achieved without altering the turnover rate of the mature messenger RNA. Electrophoretic analysis of pulse-labeled transcripts before and after a chase interval indicated that large Mr beta-globin precursor molecules were processed normally to mature nuclear beta-globin mRNA in cells treated with dexamethasone. S1 nuclease protection experiments showed further that dexamethasone treatment of induced cells uniquely depressed the amount of precursor-specific beta-globin sequences contained in unlabeled nuclear RNA preparations. The data support the view that dexamethasone regulates globin gene expression at or very close to the transcriptional level.

Animals↗
PubMed1979

Possible role of glucocorticoid receptors in globin gene expression in differentiating Friend cells.

Previous studies from this laboratory have shown that the expression of globin genes during the dimethyl sulfoxide-induced differentiation of T3C12 Friend erythroleukemia cells in inhibited by dexamethasone and related glucocorticoids. The present report shows that dexamethasone exerts a similar effect on the accumulation of globin mRNA and hemoglobin in differentiating GM86 Friend cells, but that these cells are less sensitive to this hormone. The higher sensitivity of T3C12 cells has been correlated with the presence of 6 times as many cytoplasmic glucocorticoid receptors as in the GM86 cells. The receptors of T3C12 cells have been shown to have properties in common with glucocorticoid receptors found in other tissues. It is proposed that these receptors may be responsible for the steroid control of the globin genes in Friend cells.

Animals↗