Kinetic modeling and mathematical analysis indicate that acute phase gene expression in Hep 3B cells is regulated by both transcriptional and posttranscriptional mechanisms.

To evaluate the possible role of posttranscriptional mechanisms in the acute phase response, we determined the kinetics of transcription (by nuclear run-on assay) and mRNA accumulation of five human acute phase genes in Hep 3B cells incubated with conditioned medium from LPS-stimulated monocytes. Increase in mRNA accumulation was comparable to increase in transcription rate for fibrinogen-alpha and alpha-1 protease inhibitor, suggesting largely transcriptional regulation. In contrast, mRNA accumulation was about 10-20-fold greater than transcriptional increase for serum amyloid A, C3, and factor B, suggesting participation of posttranscriptional mechanisms. Since finding a disparity between the magnitudes of increase in mRNA and transcription does not definitively establish involvement of posttranscriptional mechanisms, we subjected our data to modeling studies and dynamic mathematical analysis to evaluate this possibility more rigorously. In modeling studies, accumulation curves resembling those observed for these three mRNAs could be generated from the nuclear run-on results only if posttranscriptional regulation was assumed. Dynamic mathematical analysis of relative transcription rates and relative mRNA abundance also strongly supported participation of posttranscriptional mechanisms. These observations suggest that posttranscriptional regulation plays a substantial role in induction of some, but not all acute phase proteins.

Control of the expression of c-sis mRNA in human glioblastoma cells by phorbol ester and transforming growth factor beta 1.

The regulation of c-sis oncogene expression in human glioblastoma cell line A172 has been investigated using a sensitive RNA-RNA solution hybridization method. Enhanced expression of c-sis mRNA was induced by phorbol ester (PMA) and diacylglycerol, each of which activates protein kinase C. c-sis mRNA was also induced by transforming growth factor beta (TGF-beta). The response to PMA and TGF-beta was transient, and in each case the decrease in c-sis mRNA level following maximum stimulation occurred with a half-life similar to the mRNA half-life previously determined. Cycloheximide had no significant effect on the induction of c-sis mRNA by either PMA or TGF-beta. The increases in c-sis mRNA following addition of either PMA or TGF-beta correlated well with increases in c-sis transcription as observed by the nuclear run-on technique. In cells in which protein kinase C had been down-regulated, there was no inhibition of the c-sis mRNA response to TGF-beta. Furthermore in cells pretreated with TGF-beta, induction by PMA was unaffected. Thus the TGF-beta signal pathway does not involve activation of protein kinase C, and at least two initially distinct intracellular signaling routes lead to activation of c-sis gene expression in this glioblastoma cell line. The protein kinase inhibitor H7 abolished the ability of not only PMA but also of TGF-beta to induce c-sis mRNA. The ability of H7 to inhibit the TGF-beta stimulation suggests that a protein kinase other than protein kinase C is involved in the signal transduction by TGF-beta.

The genes for 18S, 5.8S and 28S ribosomal RNA of Bombyx mori are organized into tandem repeats of uniform length.

The organization of the multiple genes for 18S, 5.8S and 28S rRNA in the genome of the silkworm, Bombyx mori was determined by restriction endonuclease digestion and Southern blot hybridization. The ribosomal genes (rDNA) are tandemly reiterated, with a uniform repeat length of 6.9 . 10(6) daltons. Each rDNA repeat has a single site for EcoRI, HindIII, HpaI and SmaI and each of these sites has been mapped with respect to the others and to the rRNA genes; each repeat consists of a transcribed region (6 . 10(6)daltons) containing the 18S, 5.8S and 28S rRNA genes (5' leads to 3') and also a small non-transcribed spacer (approximately 10(6) daltons). Complete rDNA repeats were cloned using the vector RSF2124 and grown in Escherichia coli. Characterization of the rDNA plasmids confirmed the conclusions from studies of the total rDNA. The organization of B. mori rDNA is similar to that of other eukaryotes, except for the absence of heterogeneity in the rDNA repeat length; thus, there is neither variation in the length of the non-transcribed spacer nor the presence of inserts in a detectable portion of the rDNA. The utility of this map, and particularly of the rDNA plasmids, for detailed studies of rRNA transcription and processing is discussed.

Primary structure of the 5 S subunit of transcarboxylase as deduced from the genomic DNA sequence.

Transcarboxylase from Propionibacterium shermanii is a complex biotin-containing enzyme composed of 30 polypeptides of three different types. It is composed of six dimeric outer subunits associated with a central cylindrical hexameric subunit through 12 biotinyl subunits; three outer subunits on each face of the central hexamer. Each outer dimer is termed a 5 S subunit which associates with two biotinyl subunits. The enzyme catalyzes a two-step reaction in which methylmalonyl-CoA and pyruvate form propionyl-CoA and oxalacetate, the 5 S subunit specifically catalyzing one of these reactions. We report here the cloning, sequencing and expression of the monomer of the 5 S subunit. The gene was identified by matching amino acid sequences derived from isolated authentic 5 S peptides with the deduced sequence of an open reading frame present on a cloned P. shermanii genomic fragment known to contain the gene encoding the 1.3 S biotinyl subunit. The cloned 5 S gene encodes a protein of 519 amino acids, M(r) 57,793. The deduced sequence shows regions of extensive homology with that of pyruvate carboxylase and oxalacetate decarboxylase, two enzymes which catalyze the same or reverse reaction. A fragment was subcloned into pUC19 in an orientation such that the 5 S open reading frame could be expressed from the lac promoter of the vector. Crude extracts prepared from these cells contained an immunoreactive band on Western blots which co-migrated with authentic 5 S and were fully active in catalyzing the 5 S partial reaction. We conclude that we have cloned, sequenced and expressed the monomer of the 5 S subunit and that the expressed product is catalytically active.

Antigenic and differentiative heterogeneity among human glioblastomas.

Increasing evidence suggests that in mammals, astrocytes are a heterogenous family of cells all of which share certain properties, but differ in lineage, biochemical and functional aspects. It seems likely that glioblastomas, arising from glial precursors, may also represent a family of related but distinct cell types. We have examined the antigenic characteristics and differentiative potential of 7 different human glioblastoma cell lines in vitro. All the cell lines were labeled with a monoclonal antibody 7B11 which labels all classes of astrocytes and their precursors in the rat CNS. U138MG and Tm3 cells expressed antigens on their surfaces recognized by the monoclonal antibodies A2B5 and HNK-1. When grown in serum-free medium in the presence of cAMP and theophylline, U138MG cells assumed a process-bearing morphology and some cells expressed the Gal-C antigen specific for oligodendrocytes. Under identical conditions, Tm3 cells converted to process-bearing cells, some of which expressed glial fibrillary acidic protein (GFAP) specific for astrocytes. Other cell lines with similar antigenic characteristics did not respond similarly to cAMP and theophylline. Finally, A2781 cells were GFAP immunoreactive and unlabeled by either A2B5 or HNK-1 antibodies. These observations suggest that individual glioblastoma cell lines may be derived from distinct glial precursor cells in the vertebrate CNS.

Transcriptional patterns of growth factors and proto-oncogenes in human glioblastomas and normal glial cells.

To document over-expression of proto-oncogenes in tumors, it is necessary to determine the level of expression in the progenitor normal tissue. These studies compare the levels of nuclear transcription of a series of growth-factor related genes and proto-oncogenes in human glioblastoma cell lines with those in three normal glial cell populations. The unusual finding was that levels in the three normal glial cell populations varied considerably for several genes and thus overexpression of a specific gene in a tumor cell when compared to just one normal glial cell population would not necessarily represent overexpression. In this study, we compared the level of 17 genes in 7 tumors to the highest level of each gene found in any of three normal glial cell populations. Over-expression of PDGF-B in 4/7 glioblastoma cell lines, EGFR in 1/7, neu in 1/7 IGF-2 in 1/7 and ros in 2/7 was observed. The variation observed in the normal glial cell populations emphasizes the possibility that the normal glial cell populations represent different glial cell lineages and/or stages of differentiation and that the tumors could have arisen from different normal glial cells. Matching lineages of normal and tumor cells, probably by monoclonal antibody reactions, may be required to accurately define over-expression.

Expression of rabbit C-reactive protein in transgenic mice inhibits development of antigen-induced arthritis.

OBJECTIVE: C-reactive protein (CRP) is a plasma protein of hepatic origin thought to play an important role in host defences. We used transgenic mice, capable of expressing high levels of rabbit CRP (serum concentration>50 microg/mL) in response to dietary manipulation, to determine whether high levels of this acute-phase reactant can alter the course of experimentally induced monoarticular arthritis. METHOD: Arthritis was induced by a single injection of methylated bovine serum albumin (mBSA) on day 0 followed by injections of interleukin (IL)-1beta. RESULTS: In transgenic animals in which CRP expression had been suppressed (serum concentration<10 microg/mL), inflammatory arthritis began to develop by day 4 and was fully developed by 7 days after the mBSA challenge. This arthritis was characterized by marked inflammatory cell infiltrates in soft tissues, synovitis, pannus, cartilage loss, and bone erosion. By contrast, when CRP expression was induced, resulting in serum concentrations>50 microg/mL on the day of mBSA and IL-1beta injections, the inflammatory response was dramatically reduced at day 7. These mice manifested little to no evidence of joint inflammation. This anti-inflammatory effect of CRP was seen in animals with high CRP levels on days 0-1 following immunization and did not require elevated CRP levels during the period of rapid inflammatory progression, 4-7 days after challenge. CONCLUSION: CRP, expressed at the time of antigenic stimulation, effectively blocked the subsequent development of inflammatory arthritis in this model by altering the immune or inflammatory responses.

Transgenic mice expressing rabbit C-reactive protein are resistant to endotoxemia.

C-reactive protein (CRP), the prototypic acute-phase reactant in humans, is synthesized in liver in response to a wide variety of inflammatory stimuli. We have generated a line of transgenic mice that express rabbit CRP from the rat phosphoenolpyruvate carboxykinase (PEPCK) promoter in response to gluconeogenic signals. Here we show that transgenic mice expressing high levels of CRP were partially protected from a lethal challenge of bacterial lipopolysaccharide compared with littermates in which CRP expression had been suppressed. Similar protection was observed with challenges from platelet-activating factor (PAF) and the combination of tumor necrosis factor alpha (TNF-alpha) plus interleukin 1beta, but not with TNF-alpha alone. We further demonstrate that although PAF was able to bind CRP, the mechanism by which CRP provides protection probably does not involve sequestration of PAF. The biologically inactive precursor of PAF, lyso-PAF, also bound CRP but did not render the transgenic mice sensitive to PAF when CRP-expressing animals were simultaneously challenged with PAF and an excess of lyso-PAF. These results suggest that CRP functions in vivo by modulating host defense systems.

Genomic organization in the flesh fly Sarcophaga bullata.

The genome of the flesh fly Sarcophaga bullata has been characterized both cytologically and biochemically. S. bullata has a haploid DNA level of 0.61 picograms which is five times larger than the haploid genome size of Drosophila melanogaster. Reassociation kinetics of Sarcophaga DNA shows that its sequence organization is very similar to that of D. melanogaster in having a very large proportion of single copy DNA (81%) and only small amounts of highly and moderately repetitive DNA (9% and 6%, respectively). cRNAs from all three sequence classes were prepared and their cytological distributions on biploid and polytene cells determined by in situ hybridization. The cytological distribution of the highly repetitive probe was found to be restricted to the centromeric heterochromatin of two of the five autosomes and this sequence class was also found to be markedly underreplicated in polytene foot-pad cells. No highly repetitive DNA was localized on either of the sex chromosomes, but only on the two large centromeric regions of chromosomes C and E. Moderately repetitive DNA was found uniformly distributed on all of the autosomes in both testis and polytene foot-pad squashes. As in the case of the highly repetitive sequence probe, no moderately repetitive DNA was detected on either the X or Y chromosomes. Moderately repetitive DNA in Sarcophaga was also shown to have the "Drosophila type" pattern of sequence interspersion with a moderately repetitive element of congruent to 5,000 nucleotides adjacent to a unique element of greater than 10,000 nucleotides. The Sarcophaga genome is the largest for which this type of interspersion has so far been demonstrated.

Characterization of extrachromosomal DNA in the flesh fly Sarcophaga bullata.

The polytene pupal foot pad cells of the flesh fly Sarcophaga bullata contain numerous extrachromosomal DNA containing granules. We have determined both the origin and the nature of the DNA sequences present in these granules. Studies done with quinacrine staining of seven day old pupal foot-pad polytene nuclei showed that the granules fluoresced very brightly while the chromosomal bands to which the granules were attached did not. The only other highly fluorescent regions of the polytene karyotype were the centromeric heterochromatin of chromosomes C and E and several bands associated with the nucleolus of Chromsome A. When polytene nuclei were hybridized in situ with cRNA made from highly repetitive DNA, many of the granules positively labeled. Most of the label on these slides was concentrated on the centromeric heterochromatin of chromosomes C and E. Quinacrine staining of the foot-pad cells at very early stages of pupal development showed that when granules were present, they were always closely associated with the same two centromeric regions, those of chromosomes C and E. Since the highly repetitive DNA located in these centromeric regions is underreplicated, we conclude that the granules result from an extrusion process which takes place early during the polytenization of these cells. The chromosomal integrity of the centromeric heterochromatin of chromosomes C and E is apparently disrupted and repetitive sequences are dissociated from the chromosomes as DNA granules which then secondarily become associated with chromosomal bands throughout the nucleus.

Mutagenesis affecting the carboxyl terminus of the biotinyl subunit of transcarboxylase. Effects on biotination.

Biotin is added to biotin-containing enzymes as a post-translational modification catalyzed by holoenzyme synthetase. This reaction is fairly general in that synthetase from one organism will modify enzymes from heterologous sources. This suggests that the polypeptides share some structural characteristic(s) that define(s) them as biotin enzymes. We have reported previously that when the gene coding for the 1.3 S biotinyl subunit of transcarboxylase is expressed in Escherichia coli, the polypeptide produced is biotinated by the cellular synthetase. Using in vitro mutagenesis of this gene, we have begun to define the primary structure involved in the enzymatic addition of biotin to a lysine residue. We show here that the carboxyl terminus of the 1.3 S subunit is critical in biotination. Mutations affecting the COOH-terminal residue do not influence the modification, but elimination of the hydrophobic side chain of the penultimate residue abolishes biotin addition.

Two carboxylesterases bind C-reactive protein within the endoplasmic reticulum and regulate its secretion during the acute phase response.

We have previously reported that C-reactive protein (CRP) is normally synthesized by rabbit hepatocytes at relatively low rates and is retained in the endoplasmic reticulum (ER), apparently by specific interaction with a 60-kDa lumenal ER protein. During the acute phase response to tissue injury, a marked increase in CRP synthesis is associated with a decrease in the CRP binding capacity of the 60-kDa protein, with accompanying rapid secretion of CRP. In the present studies, we purified two 60-kDa ER lumenal glycoproteins (referred to as gp60a and gp60b) capable of binding CRP. gp60b, though present at only 5% the level of gp60a, was found to account for 80% of the total CRP binding capacity. Amino-terminal amino acid sequence analysis and biochemical characterization identified gp60a and gp60b as two microsomal carboxylesterases previously reported by others to contain COOH-terminal ER retention signals (HIEL and HTEL). The CRP binding activities of gp60a and gp60b were found to be independent of their esterase activities. In animals undergoing the acute phase response, the levels of gp60a and gp60b were diminished by about 50%, but the CRP binding capacities were reduced by 4-6-fold for gp60a and 25-30-fold for gp60b. These findings indicate that CRP is normally retained within the ER via interaction with gp60a and gp60b, while during the acute phase response a decrease in the CRP binding affinity of these proteins, particularly gp60b, results in efficient secretion of CRP.

Expression and stability of c-sis mRNA in human glioblastoma cells.

The production of platelet-derived growth factor like (PDGF-like) material by glioblastomas may be involved in the conversion of normal cells to tumor cells. In an investigation of this problem, we have examined some of the properties of the platelet-derived growth factor B-chain mRNA (c-sis mRNA) by a sensitive and quantitative RNA-RNA solution hybridization method. In 5 out of 8 human glioblastoma cell lines, c-sis mRNA was present, and in the line with the highest level, there were approximately 4-10 molecules per cell. The half-lives of the c-sis mRNA in two glioblastoma cell lines were 2.6 and 3.4 h, while in human umbilical vein endothelial (HUVE) and bladder carcinoma (T24) cells they were 1.6 and 2.5 h, respectively. Inhibiting protein synthesis produced no significant alteration of the c-sis mRNA half-lives in the glioblastoma or HUVE cells. The A-U-rich sequence at the 3' end of the c-sis mRNA therefore does not appear to affect the mRNA stability in the presence of cycloheximide as it does in other transcripts. The similarity of the c-sis mRNA half-lives in normal and tumor cells suggests that regulation of stability of c-sis mRNA is not a major factor in tumorigenesis in the glioblastoma cell lines examined.

Identification of Alu transposition in human lung carcinoma cells.

We have demonstrated genetic transposition in human cells. An experimental system was established in which the Ecogpt (gpt) gene was employed as a target for inactivation. The human lung carcinoma cell line A549 containing this target was fused to UV-irradiated A549 cells that did not contain the target. From the fusion products, sublines carrying an inactivated gpt gene were analyzed. UV irradiation increased the frequency of inactivated gpt genes in the fusion cells by 100-fold. One subline was found to contain a complete Alu sequence in the coding region of the gpt gene. The inserted element differed from the Blur8 sequence by only 7 out of the 270 nucleotides. The insertion of this Alu element created a 5 bp insertion site duplication.

A rapid procedure to identify newborn transgenic mice.

We have developed a rapid procedure to identify newborn transgenic mice containing foreign genetic material in their genome. The protocol involves collagenase digestion of a small amount of tail tissue which can be taken very early after birth, phenol and chloroform extraction, polymerase chain reaction, and polyacrylamide gel electrophoresis. The entire procedure, from tissue biopsy to final results, can be completed in 1 day.

Induction of transcription from the long terminal repeat of Moloney murine sarcoma provirus by UV-irradiation, x-irradiation, and phorbol ester.

The long terminal repeat (LTR) of Moloney murine sarcoma virus (Mo-MuSV) was used as a model system to study the stress response of mammalian cells to physical carcinogens. The chloramphenicol acetyltransferase (CAT) gene was inserted between two Mo-MuSV LTRs, and the LTR-CAT-LTR construct was used for virus production and was integrated into the genome of NIH 3T3 cells in the proviral form. This construct was used to assure that the integrated CAT gene was driven by the promoter of the LTR. Expression of the CAT gene was stimulated 4-fold by UV irradiation, and the peak of activity was observed at 18 hr. In contrast, stimulation of the CAT expression after x-irradiation was 2-fold and occurred at 6 hr. Phorbol myristate acetate also stimulated CAT activity 4-fold with a peak at 6 hr. Down-regulation of protein kinase C blocked totally the response to x-irradiation but only partially the response to UV. The protein kinase inhibitor H7 blocked the response to treatment by UV, x-ray, and phorbol ester.