Biomedical subjects

R J Rickles

Publications and source records attributed to R J Rickles.

23 records · Page 2Linked to original sources

PubMed1988

Tissue plasminogen activator mRNA in murine tissues.

The urokinase-type and tissue-type plasminogen activators are the two enzymes found in mammals, which specifically convert the zymogen plasminogen to plasmin. Using cDNA probes, we have assayed for the presence of the two types of plasminogen activator mRNAs in murine tissues. We demonstrate that tissue-type plasminogen activator mRNA can be detected in a wide variety of tissues. In contrast, the accumulation of urokinase-type plasminogen activator mRNA is observed in only a few of the tissues analyzed. Using an S1 nuclease assay, we demonstrate that the tPA mRNA detected contains the complete sequences encoding the non-protease finger, growth-factor and kringle domains.

Animals↗

PubMed1988

Molecular cloning of complementary DNA to mouse tissue plasminogen activator mRNA and its expression during F9 teratocarcinoma cell differentiation.

Two tissue plasminogen activator (tPA) cDNA clones were isolated from mouse cDNA libraries and characterized. The sequence coding for mature mouse tPA protein including its 29-residue signal peptide, as well as that for the 3' and part of the 5'-nontranslated regions of the mRNA was determined. The mature protein consists of 530 amino acids and is encoded by a transcript of approximately 2800 nucleotides. The mouse tPA protein has 81% homology with its human counterpart, and amino acid sequence conservations suggest that the multidomain-like structure found for human tPA is maintained in the mouse enzyme. The tPA cDNA has been used as a probe to analyze tPA gene expression during F9 cell differentiation. tPA mRNA is not detected in F9 stem cells. When F9 cells are induced to differentiate with retinoic acid and dibutyryl cyclic AMP, tPA mRNA accumulates. We demonstrate that this induction occurs at least in part because of an increase in tPA gene transcription.

Animals↗

PubMed1988

Anti-sense inhibition of tissue plasminogen activator production in differentiated F9 teratocarcinoma cells.

F9 teratocarcinoma cells secrete the serine protease, tissue plasminogen activator (t-PA), upon differentiation induced in vitro by retinoic acid (RA) or RA and dibutyryl cAMP (RA/dbcAMP). A recombinant plasmid capable of directing the production of t-PA anti-sense RNA was constructed and transfected into F9 stem cells in an attempt to create a hypomorphic phenotype for t-PA synthesis. Several colonies were isolated which contained anti-sense RNA and which showed greater than a 50% reduction in t-PA activity upon differentiation. One such colony, 3b4, exhibited a 75% reduction in t-PA activity and was analyzed further. Large quantities of t-PA anti-sense transcript were expressed in the stem cells which are characterized by the absence of t-PA gene expression. In the induced cells, which normally express t-PA, the amount of detectable anti-sense transcript was significantly decreased. The amount of t-PA mRNA in differentiated cells containing t-PA anti-sense RNA was comparable to that in differentiated control cells. Subcellular localization of the mRNA in induced 3b4 cells appeared to be the same as induced control cells. Expression of collagen type IV, another marker of differentiation, was also monitored and was unaffected by the presence of t-PA anti-sense RNA in RA/dbcAMP-treated cells. The inhibition of differentiation-specific gene expression by anti-sense RNA may be useful for further studies of developmentally regulated genes.

Animals↗

PubMed1987

Chromosomal assignments of genes for tissue plasminogen activator and urokinase in mouse.

The genes encoding the two plasminogen activators, tissue plasminogen activator and urokinase, were mapped to mouse chromosomes using probes derived from the respective mouse cDNAs. DNA from mouse-Chinese hamster and mouse-rat somatic cell hybrids was digested with BamHI and EcoRI, respectively, and analyzed by Southern blot hybridization for the segregation of the two genes. Tissue plasminogen activator and urokinase cosegregated with mouse chromosomes 8 and 14, respectively. The plasminogen activator genes thus fall into two syntenic groups that are conserved in human and mouse.

Animals↗

PubMed1982

Genome-length copies of poliovirion RNA are synthesized in vitro by the poliovirus RNA-dependent RNA polymerase.

A soluble RNA-dependent RNA polymerase was purified from the cytoplasm of poliovirus-infected HeLa cells. A single virus-specific protein designated as p63 (or NCVP4) copurified with this activity. The purified polymerase was free of ribonuclease activity and was shown to copy poliovirion RNA when oligo(U) was added to the in vitro reaction mixture. Characterization of the product RNA by electrophoresis in methylmercury (II) hydroxide-agarose gels showed that genome-sized copies of poliovirion RNA were synthesized in vitro by the purified polymerase. The product RNA was shown to be heteropolymeric, complementary to virion RNA, and covalently linked to oligo(U). The product RNA contained the expected distribution of UMP and GMP containing dinucleotide pairs which included a very low frequency of CpG pairs. The amount, size distribution, and rate of synthesis of product RNA was very dependent on the in vitro reaction conditions. Full sized product RNA was synthesized in about 6 min when reaction conditions were used that yielded maximum elongation rates (pH 8.0, 7 mM Mg2+, 37 degrees C). Under these conditions, most of the product RNA recovered from a 1-h reaction was full sized. Thus, the polymerase was found to specifically initiate synthesis at the 3'-end of the template using an oligo(U) primer and to carry out an elongation reaction at about 1250 nucleotides/min that resulted in the synthesis of full sized product RNA.

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