Insertion Of DNA Sequences Research Articles

Sumoylation of CCAAT/Enhancer-binding Protein α and Its Functional Roles in Hepatocyte Differentiation

The sumoylation of CCAAT/enhancer-binding proteins (C/EBPs) by small ubiquitin-related modifier-1 (SUMO-1) has been reported recently. In this study, we investigated the functional role of the sumoylation of C/EBPalpha in the differentiation of hepatocytes. The amount of sumoylated C/EBPalpha gradually decreased during the differentiation, which suggests that the sumoylation is important for the control of growth/differentiation especially in the fetal liver. To analyze the function of the sumoylation of C/EBPalpha in liver-specific gene expression, we studied its effects on the expression of the albumin gene. The C/EBPalpha-mediated transactivation of the albumin gene was reduced by sumoylation of C/EBPalpha in primary fetal hepatocytes. The enhancement of C/EBPalpha-mediated transactivation by BRG1, a core subunit of the SWI/SNF chromatin remodeling complex, was hampered by sumoylation in a luciferase reporter assay. In addition, we discovered that sumoylation of C/EBPalpha blocked its inhibitory effect on cell proliferation by leading to the disruption of a proliferation-inhibitory complex because of a failure of the sumoylated C/EBPalpha to interact with BRG1. BRG1 was recruited to the dihydrofolate reductase promoter in nonproliferating C33a cells but was not detected in proliferating cells where C/EBPalpha, BRG1, and SUMO-1 were overexpressed. This result suggests that BRG1 down-regulates the expression of the dihydrofolate reductase gene. These findings provide the insight that SUMO acts as a space regulator, which affects protein-protein interactions.

Recombinational Cloning

AbstractThe study of protein en masse, or functional proteomics, depends on the availability of full‐length cDNAs in appropriate expression‐ready plasmid vectors for protein expression and functional analysis. Recombinational cloning is a universal cloning technique based on site‐specific recombination that is independent of the insert DNA sequence to be cloned, which differentiates this method from the classical restriction enzyme‐based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression‐ready clones using two of the most popular recombinational cloning technologies, now commercially available from Invitrogen (Gateway) and BD Clontech (Creator).

193. Direct Comparison of Integration Efficiency and Stable Gene Expression Mediated by the Sleeping Beauty Transposon System and by the PhiC31 Phage Integrase System in Cultured Human Fibroblasts and in Various Stem Cell Targets

Top of pageAbstract Significant research efforts are directed toward achieving non-viral, DNA-mediated transfer and stable expression of genes in somatic cells for the treatment of diseases. Plasmid DNA has been successfully used to deliver genes into cells, but its usefulness is restricted by the inability to provide sustained expression. One alternative is to include components of an integrating system in the vector design. The Sleeping Beauty (SB) transposase and phiC31 integrase both bring about insertion of DNA sequences into the genomes of human cells, suggesting an alternative to viral transduction. Because each approach requires its own unique genetic elements and catalytic enzyme to mediate gene insertion into cellular chromosomes, we chose to directly compare the effectiveness of integration and stable expression using a two-component system that is internally controlled for the integrating sequence. The integrating vector (pKT2/NAG) consists of (i) a phiC31 integrase recognition site (attB), (ii) eF1-alpha regulated GFP expression unit, and (iii) neomycin resistance gene regulated by the PGK promoter, all flanked by (iv) T2 transposase binding sites (IR/DRs) separated by (v) a colE1 bacterial origin and kanR gene. Overexpression inhibition of the SB transposase suggests a potential limitation with respect to integration efficiency that can be achieved by this vector. To address this issue, we first studied the integrating efficiency of pKT2/NAG by evaluating the effect of the dose of the co-delivered enzyme-encoding plasmid on integration and stable gene expression as determined by G418-resistant colony formation and GFP fluorescence. In each case, pKT2/NAG (500 ng) was co-transfected with 150 ng, 500 ng, or 1500 ng of either CMV-regulated luciferase (pCMV-Luc), transposase (pCMV-SB), or integrase (pCMV-Int) expression plasmids into cultured human HT1080 cells. G418-resistant colony formation exceeded levels achieved by random integration (+ pCMV-Luc) at all tested concentrations of the SB transposase-encoding plasmid, with diminished efficiency at higher concentrations. The inverse relationship was observed for phiC31 integrase, where gene transfer improved as the concentration of pCMV-Int increased relative to pKT2/NAG. Drug-resistant clones have been isolated for studies into: (i) gene silencing by flow cytometric analysis of GFP fluorescence, (ii) insertional preference by cloning and sequencing vector::chromosome junctions, and (iii) insertion frequency by Southern analysis. We have also co-delivered both components into rat multipotent adult progenitor cells and found both systems to effectively mediate G418-resistant colony formation above background. Studies underway in mouse ES cells and in human CD34+ cells using this bifunctional approach will provide a broad assessment of the relative usefulness of these two major non-viral integrating systems for the purpose of ex vivo gene therapy applications in a range of human diseases.

Meta‐Analysis of gross insertions causing human genetic disease: Novel mutational mechanisms and the role of replication slippage

Although gross insertions (>20 bp) comprise <1% of disease-causing mutations, they nevertheless represent an important category of pathological lesion. In an attempt to study these insertions in a systematic way, 158 gross insertions ranging in size between 21 bp and approximately 10 kb were identified using the Human Gene Mutation Database (www.hgmd.org). A careful meta-analytical study revealed extensive diversity in terms of the nature of the inserted DNA sequence and has provided new insights into the underlying mutational mechanisms. Some 70% of gross insertions were found to represent sequence duplications of different types (tandem, partial tandem, or complex). Although most of the tandem duplications were explicable by simple replication slippage, the three complex duplications appear to result from multiple slippage events. Some 11% of gross insertions were attributable to nonpolyglutamine repeat expansions (including octapeptide repeat expansions in the prion protein gene [PRNP] and polyalanine tract expansions) and evidence is presented to support the contention that these mutations are also caused by replication slippage rather than by unequal crossing over. Some 17% of gross insertions, all >or=276 bp in length, were found to be due to LINE-1 (L1) retrotransposition involving different types of element (L1 trans-driven Alu, L1 direct, and L1 trans-driven SVA). A second example of pathological mitochondrial-nuclear sequence transfer was identified in the USH1C gene but appears to arise via a novel mechanism, trans-replication slippage. Finally, evidence for another novel mechanism of human genetic disease, involving the possible capture of DNA oligonucleotides, is presented in the context of a 26-bp insertion into the ERCC6 gene.

811. The Hotspots of Integration Targeted by the Site-Specific Integrase PhiC31 May Be Cell Line Dependent

Recent reports on insertional mutagenesis due to integration of gene therapy vectors into the host genome have raised concerns about the genetic manipulation of somatic cells. Previously, it was demonstrated that the phage integrase phiC31 derived from a Streptomyces phage mediates site-specific integration into the host genome of mammalian cells in vitro and in vivo by recombining the attB recognition site in an episomal plasmid and one or more pseudo attP sites in the host chromosome. However, a study which carefully analyzes the consequences of phage integrase mediated integration for each independent integration event has yet to be performed. In the present study we characterized the chromosomal integration sites after phiC31 mediated integration from extrachromosomal plasmids in the two liver derived cell lines Huh7 (human hepatoma cell line) and Hep1A (mouse hepatoma cell line) and the human embryonic kidney cell line 293. The number of cell clones selected in a colony forming assay from all three cell lines revealed a 6.0 fold, 17.2 fold and 4.5 fold increase in the number of colonies in Huh7 cells, Hep1A cells and 293 cells compared to the mutant integrase control group, respectively. Single cell clones from all three cell lines were amplified and the insertion sites were identified and characterized based on a plasmid rescue protocol. We found frequent smaller deletions of up to 26 bp in the chromosomal target sites (13 of 32 integrations) and deletions of up to 10 bp in the phage integrase attachment site attB (8 of 32 integrations) in both, Huh-7 and 293 cells. Surprisingly, we also detected insertions of up to 177 bp (7 of 32 integrants); most of the insertions were < 4 bp. The origin of these inserted DNA sequences remains to be determined. Out of the 32 independent integration events analyzed so far, three integrated into human chromosome 19 (19q13.31), a site not previously reported. Because these initial results do not yet have a statistically significant number of independent observations, further analyses of additional clones from all three cell lines are ongoing. In summary, these results suggest that hotspots in the host genome targeted by the site-specific integrase phiC31 may depend on the cell line and perhaps on the tissue from which the cell line is derived. These findings will have important implications on improving the safety of ex vivo and in vivo gene therapy approaches in which stable and long-term transgene expression levels are required.

Positional effect of gene insertion on genetic stability of a clover yellow vein virus-based expression vector

The stability of the inserted genes in the viral expression vector varied depending on the sequence introduced and the position of insertion. Infectious cDNA to Clover yellow vein virus (pClYVV) was modified to insert a foreign gene at two independent sites: one, along with a polylinker, between the NIb and CP genes (pClYVV/CP/W) and the other between P1 and HC-Pro (pClYVV-Pst/CP). The green fluorescent protein (GFP) gene was inserted into either pClYVV/CP/W or pClYVV-Pst/CP. GFP gene was stably maintained and expressed in both vectors following serial passages in plants. Progeny viruses from both constructs accumulated in similar amounts and at rates of 70%–80% of that of the wild-type virus. On the other hand, progeny viruses carrying the human interferon-Α (hIFN) gene cloned in pClYVV-Pst/CP were genetically unstable owing to frequent deletions of the cloned gene during passage through plants. In contrast, the hIFN sequence cloned in pClYVV/CP/W was stably maintained in viruses after several passages in broad bean plants, and the progeny virus accumulated at the rate of about 50%–100% of that of the wild-type virus. The nucleotide sequence analyses indicated that the genetic instability of the inserted sequence results from homologous recombination of viral vector and inserted DNA sequences; it is not due to the inserted sequence alone.

Regeneration of transgenic citrus plants under non selective conditions results in high-frequency recovery of plants with silenced transgenes

Insertion of foreign DNA into plant genomes frequently results in the recovery of transgenic plants with silenced transgenes. To investigate to what extent regeneration under selective conditions limits the recovery of transgenic plants showing gene silencing in woody species, Mexican lime [ Citrus aurantifolia (Christm.) Swing.] plants were transformed with the p25 coat protein gene of Citrus tristeza virus (CTV) with or without selection for nptII and uidA. Strikingly, more than 30% of the transgenic limes regenerated under non-selective conditions had silenced transgenes, and in all cases silencing affected all the three transgenes incorporated. These results indicate that the frequency of transgene silencing may be greatly underestimated when the rate of silencing is estimated from the number of regenerants obtained under selective conditions. To our knowledge, this is the first report in which the frequency of gene silencing after transformation has been quantified. When the integration pattern of T-DNA was analyzed in silenced and non-silenced lines, it was observed that inverted repeats as well as direct repeats and even single integrations were able to trigger gene silencing. Gene silencing has often been associated with the insertion of DNA sequences as inverted repeats. Interestingly, here, direct repeats and single-copy insertions were found in both silenced and non-silenced lines, suggesting that the presence of inverted-repeat T-DNAs and the subsequent formation of dsRNAs triggering gene silencing cannot account for all silencing events.

Construction of Replication‐Defective Herpes Simplex Virus Vectors

Advances in identification and characterization of gene products responsible for specific diseases of the nervous system have opened opportunities for novel therapies using gene transfer vectors for gene replacement. Herpes simplex virus (HSV)-based vectors are particularly well suited for gene delivery to neurons of the central and peripheral nervous systems. The authors have developed methods to delete HSV-1 IE gene functions and to subsequently introduce foreign genes into the HSV-1 genome using homologous recombination. This unit describes methods for generating cell lines that complement multiple essential gene deletion mutants as well for generating such replication-defective virus recombinants and inserting foreign DNA sequences into replication-defective viral genomes, the last step in preparing a vector. Three support protocols describe methods for preparing virus stocks, titering virus, and preparing viral DNA.

ShcA tyrosine phosphorylation sites can replace ShcA binding in signalling by middle T-antigen.

ShcA and Grb2 are crucial components in signalling by most tyrosine kinase-associated receptors. How ever, it is not clear whether Grb2 bound directly to the receptor is equivalent to Grb2 associated via ShcA. We have used signalling stimulated by the middle T-antigen (MT) of polyoma virus to address this question. The two known Grb2-binding sites from murine ShcA, 313Y and 239/240YY, could functionally replace the MT ShcA-interacting region in transformation assays using Rat2 fibroblasts. This demonstrates that signal output from membrane-bound ShcA requires only these two sequences and the ShcA-binding site in MT does not recruit other signalling molecules. Two standard Grb2-interacting sequences, either from the EGF receptor or the ShcA 313Y region, could not replace the requirement for ShcA binding to MT, indicating an enhanced role for the ShcA 239/240YY motif. Sos1 and the docking protein Gab1 are brought into the MT complex through Grb2 association and this may be more effective using the 239/240YY sequence.

Experimental models of protein-RNA interaction: isolation and analyses of tRNA(Phe) and U1 snRNA-binding peptides from bacteriophage display libraries.

Peptides that bind either U1 small nuclear RNA (U1 snRNA) or the anticodon stem and loop of yeast tRNA(Phe) (tRNA(ACPhe)) were selected from a random-sequence, 15-amino acid bacteriophage display library. An experimental system, including an affinity selection method, was designed to identify primary RNA-binding peptide sequences without bias to known amino acid sequences and without incorporating nonspecific binding of the anionic RNA backbone. Nitrocellulose binding assays were used to evaluate the binding of RNA by peptide-displaying bacteriophage. Amino acid sequences of RNA-binding bacteriophage were determined from the foreign insert DNA sequences, and peptides corresponding to the RNA-binding bacteriophage inserts were chemically synthesized. Peptide affinities for the RNAs (Kd approximately 0.1-5.0 microM) were analyzed successfully using fluorescence and circular dichroism spectroscopies. These methodologies demonstrate the feasibility of rapidly identifying, isolating, and initiating the analyses of small peptides that bind to RNAs in an effort to define better the chemistry, structure, and function of protein-RNA complexes.

A rapid technique for the determination of unknown plasmid library insert DNA sequence directly from intact yeast cells.

A polymerase chain reaction (PCR)-based technique is described which allows for the determination of library plasmid insert DNA sequence directly and rapidly from intact yeast cells. Yeast spheroplasts are used to template a PCR reaction to amplify the insert sequence. This PCR product is then purified and its sequence directly determined using thermal cycle sequencing. Readable sequence can reproducibly be obtained from multiple yeast colonies in just two days. Uses of this technique in yeast two-hybrid screening as well as other types of yeast library screens are discussed.

Identification and sequencing of a cDNA encoding 6-phosphogluconate dehydrogenase from a fungus, Cunninghamella elegans and expression of the gene in Escherichia coli

The fungus, Cunninghamella elegans has been widely used in bioremediation and microbial models of mammalian studies in many laboratories. Using the polymerase chain reaction to randomly amplify the insert directly from the single non-blue plaques of a C. elegans cDNA library, then partly sequencing and comparing with GenBank sequences, we have identified a clone which contains C. elegans 6-phosphogluconate dehydrogenase gene. The polymerase chain reaction product was cloned into a plasmid, pGEM-T Easy vector for full insert DNA sequencing. The 6-phosphogluconate dehydrogenase gene (1458 bases) and the deduced protein sequence were determined from the insert DNA sequence. The gene was found by open reading frame analysis and confirmed by the alignment of the deduced protein sequence with other published 6-phosphogluconate dehydrogenase sequences. Several highly conserved regions were found for the 6-phosphogluconate dehydrogenase sequences. The 6-phosphogluconate dehydrogenase gene was subcloned and over-expressed in a plasmid– E. coli system (pQE30). The cell lysate of this clone has a very high 6-phosphogluconate dehydrogenase enzyme activity. Most of the recombinant protein in this system was formed as insoluble inclusion bodies, but soluble in high concentration of urea-buffer. Ni-NTA resin was used to purify the recombinant protein which showed 6-phosphogluconate dehydrogenase enzyme activity. The recombinant protein has a predicted molecular size correlating with that revealed by sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. The C. elegans 6-phosphogluconate dehydrogenase was in a cluster with yeast' 6-phosphogluconate dehydrogenase in the phylogenetic tree. Bacterial 6-phosphogluconate dehydrogenase and higher organisms' 6-phosphogluconate dehydrogenase were found in different clusters.

A holistic approach for determining the entomopathogenic potential of Bacillus thuringiensis strains.

The cry gene content of Bacillus thuringiensis subsp. aizawai HD-133 was analyzed by a combination of high-pressure liquid chromatography (HPLC) and exclusive PCR. A total of six cry genes were detected in genomic DNA purified from HD-133, four from the cry1 family (cry1Aa, cry1Ab, cry1C, and cry1D) as well as a gene each from the cry2 (cry2B) and the cry1I families. To directly determine which genes were expressed and crystallized in the purified parasporal inclusions, solubilized and trypsinized HD-133 crystals were subjected to chromatographic separation by HPLC. Only three proteins, Cry1Ab, Cry1C, and Cry1D, were found, in a 60/37/3 ratio. Dot blot analysis of total mRNA purified from HD-133 showed that both the cry2B and cry1I genes, but not the cry1Aa gene, were transcribed. Cloning and sequencing of the cry1Aa gene revealed an inserted DNA sequence within the cry coding sequence, resulting in a disrupted reading frame. Taken together, our results show that combining crystal protein analysis with a genetic approach is a highly complementary and powerful way to assess the potential of B. thuringiensis isolates for new insecticidal genes and specificities. Furthermore, based on the number of cryptic genes found in HD-133, the total cry gene content of B. thuringiensis strains may be higher than previously thought.

SINEs of destruction

Fukuyama-type congenital muscular dystrophy (FCMD) is among the most common autosomal recessive disorders in Japan. Kobayashi et al., 1998xAn ancient retrotransposal insertion causes Fukuyama-type congenital muscular dystrophy. Kobayashi, K. et al. Nature. 1998; 394: 388–392Crossref | PubMed | Scopus (0)See all ReferencesKobayashi et al., 1998have now shown that in 87% of cases it is associated with a 3-kb insertion into the 3′ untranslated region of the FCMD gene located on Chromosome 9 of the human genome. FCMD codes for fukutin, which is thought to be a secreted protein but whose function in the pathogenesis of muscular dystrophies is not yet understood. The inserted DNA sequence causes a decrease of transcription and/or an instability of mRNA, resulting in loss of function. It represents a complex retrotransposon-like insertion containing tandemly repeated sequences and a SINE (short interspersed nuclear element). This mutation appears to be the result of an ancient retrotransposition event, indicated by a 17-bp target-site duplication flanking the inserted sequence. SINE sequences included in this retrotranscript infest the human genome and comprise >10% of its DNA. Alu, a SINE, is already known to cause new mutations, such as an insertion in the NF1 gene, which results in neurofibromatosis. The insertion of another mobile element, the human retrotransposon LINE 1 (long interspersed nuclear element 1) into specific genes has been shown to be the cause of human diseases such as haemophilia, Duchenne muscular dystrophy and colon cancer. We should be prepared to hear more in the future about these mobile elements as a cause of genetic disorders.

Identification of mycoplasmal promoters in Escherichia coli using a promoter probe vector with Green Fluorescent Protein as reporter system.

A promoter probe vector, pGFPUV2, with Green Fluorescent Protein (GFP) as the reporter system was constructed to identify potential mycoplasmal promoter-containing regulatory sequences in E. coli. Libraries of M. pneumoniae and M. genitalium DNA constructed in pGFPUV2 and transformed into E. coli resulted in GFP-expressing clones. Primer extension analysis with E. coli RNA from five M. pneumoniae clones and two M. genitalium clones indicated that transcription originated from the insert DNA fragments of these promoter constructs. Primers based on the insert DNA sequences were used in primer extension reactions with total RNA isolated from M. pneumoniae or M. genitalium. Of the seven primers used, three generated products by primer extension with mycoplasmal RNA. However, only one of the DNAs had a 5' end similar to that obtained in a comparable reaction with E. coli RNA, and the start site of this transcript appeared to originate one base prior to the predicted open reading frame. These results indicate that E. coli can identify mycoplasmal promoters which have transcriptional elements resembling E. coli promoters.

Deriving Probes From Large‐Insert Clones by PCR Methods

This unit describes several polymerase chain reaction (PCR)-based methods to obtain DNA fragments from clones with large inserts without prior knowledge of the insert DNA sequence. The protocols can be categorized into three groups: (1) methods to generate DNA fragments at random representing the entire length of the cloned insert, (2) methods to generate DNA fragments representing the extremities of an insert, and (3) methods to generate complex probes suitable for fluorescence in situ hybridization. Support protocols describe direct cloning of these PCR products and the isolation of total yeast DNA from yeast artificial chromosome (YAC) clones.

Functional analysis of the ver gene using antisense transgenic wheat.

The function of ver203, a gene related to vernalization in winter wheat, was investigated by expression of a complementary DNA as an antisense RNA in transgenic plants. A verc203:gus fusion-expression plasmid was constructed in pBI221, which contains a CaMV (cauliflower mosaic virus) 35S-promoter, a gus gene and a nos terminator. The construct was then introduced into the plant by the pollen-tube pathway. The results showed that heading was strongly inhibited in 6 of 326 vernalized antisense transgenic winter wheat plants, until both the vernalized control winter wheat and sense transgenic plants ripened. The hybridization analysis of DNA, amplification of the insert DNA sequences with PCR, northern blot analysis with double- and single-stranded probes, and detection of GUS activity by X-gluc assay gave strong positive results. This suggests that the VER203 protein plays an important role in controlling heading and flower development in winter wheat.

DNA sequence insertion and evolutionary variation in gene regulation.

Current evidence on the long-term evolutionary effect of insertion of sequence elements into gene regions is reviewed, restricted to cases where a sequence derived from a past insertion participates in the regulation of expression of a useful gene. Ten such examples in eukaryotes demonstrate that segments of repetitive DNA or mobile elements have been inserted in the past in gene regions, have been preserved, sometimes modified by selection, and now affect control of transcription of the adjacent gene. Included are only examples in which transcription control was modified by the insert. Several cases in which merely transcription initiation occurred in the insert were set aside. Two of the examples involved the long terminal repeats of mammalian endogenous retroviruses. Another two examples were control of transcription by repeated sequence inserts in sea urchin genomes. There are now six published examples in which Alu sequences were inserted long ago into human gene regions, were modified, and now are central in control/enhancement of transcription. The number of published examples of Alu sequences affecting gene control has grown threefold in the last year and is likely to continue growing. Taken together, all of these examples show that the insertion of sequence elements in the genome has been a significant source of regulatory variation in evolution.

Repression of the herpes simplex virus 1 alpha 4 gene by its gene product (ICP4) within the context of the viral genome is conditioned by the distance and stereoaxial alignment of the ICP4 DNA binding site relative to the TATA box

Infected cell protein no. 4 (ICP4), the major regulatory protein encoded by the alpha 4 gene of herpes simplex virus 1, binds to a site (alpha 4-2) at the transcription initiation site of the alpha 4 gene. An earlier report described the construction of recombinant viruses that contained chimeric genes (alpha 4-tk) that consisted of the 5' untranscribed and transcribed noncoding domains of the alpha 4 gene fused to the coding sequences of the thymidine kinase gene and showed that disruption of the alpha 4-2 binding site by mutagenesis derepressed transcription of this gene (N. Michael and B. Roizman, Proc. Natl. Acad. Sci. USA 90:2286-2290, 1993). This experimental design was used to determine the effect of displacement of the alpha 4-2 binding site on the repression of alpha 4 gene transcription by ICP4. We report the following findings. (i) In the absence of the alpha 4-2 binding site, at 4 h after infection, alpha 4-tk RNA levels increased 10-fold relative to the corresponding RNA levels of a gene that contained the alpha 4-2 site at its natural location. Displacement of the alpha 4-2 binding site by approximately one, two, and three turns of the DNA helix, i.e., by 10, 21, and 30 nucleotides downstream of the original site, increased the concentration of alpha 4-tk RNA 2.4-, 3.5-, and 5.8-fold, respectively. (ii) Displacement of 16 nucleotides, i.e., approximately 1.5 helical turns, increased the accumulation of alpha 4-tk by 5.3-fold, i.e., more than predicted by displacement alone. (iii) At 8 h after infection in the absence of the binding site, the accumulation of alpha 4-tk RNA increased 13.6-fold. However, in cells infected with recombinants that carried displaced alpha 4-2 binding sites, RNA accumulation decreased relative to the levels seen at 4 h after infection. The insertion of DNA sequences in order to displace the alpha 4-2 binding site had no effect on accumulation of RNA in the presence of cycloheximide, i.e., in the absence of ICP4, or on maximum accumulation of alpha 4-tk RNA in the absence of the alpha 4-2 binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of interleukin (IL)-11 gene expression in IL-1 induced primate bone marrow stromal cells

Interleukin (IL)-1 alpha treatment of a primate bone marrow stromal cell line, PU-34, transiently increased the steady state level of IL-11 mRNA. Nuclear run-on experiments showed that the transcription rate of the IL-11 gene was not affected appreciably by IL-1 alpha induction, but changes in the half-life of the IL-11 mRNA corresponded well with the changes in the steady state level of the IL-11 mRNA during the induction. Although transient transfection of PU-34 cells with IL-11 promoter constructs failed to respond to IL-1 alpha, a 10-base pair promoter region and JunD.AP-1 complex were found to be responsible for the basal level transcription of the IL-11 gene. The tyrosine kinase inhibitor genistein accelerated the degradation of the IL-11 mRNA without affecting the transcription rate of the IL-11 gene in IL-1 alpha stimulated cells. The insertion of DNA sequences corresponding to the 3'-untranslated region of the IL-11 gene into a rabbit beta-globin gene resulted in destabilization of the chimeric mRNA which failed to be induced by IL-1 alpha. Exogenous IL-11 expression generated from transient transfection with plasmid pCMV-IL-11, however, can be stabilized by IL-1 alpha. In contrast to the hypothesis that AUUUA motifs in the 3'-untranslated region are sufficient to regulate cytokine mRNA stability, our results suggest that IL-1 alpha induced stabilization of the IL-11 mRNA requires participation of RNA sequences from different regions of the IL-11 message.