Search

3T3-L1 cells have been used as a model to study the differentiation and physiology of adipocytes. Exogenous expression of proteins in these cells offers the prospect of understanding the protein's function(s) in adipose tissue. Viral vectors, in particular, adenovirus, have proven to be a powerful means for introduction of genes into many cell types. However, we have previously shown that 3T3-L1 cells are inefficiently transduced by adenovirus (Orlicky, D. J., and J. Schaack. 2001. J. Lipid Res. 42: 460-466). To overcome the inefficient transduction, we have stably introduced the gene-encoding coxsackie and adenovirus receptor (CAR), which was modified by deletion of the region encoding the cytoplasmic tail, into 3T3-L1 cells. 3T3-L1 CARDelta1 cells are transduced approximately 100-fold more efficiently than parental 3T3-L1 cells. 3T3-L1 CARDelta1 cells should prove to be a useful tool for examination of exogenous protein expression in fat cells.

3T3-L1 cells offer an excellent model system for studies of differentiation and biochemistry of fat cells. However, these cells are limited in their utility by the low efficiency with which DNA can be introduced by transfection. Gene delivery by viral vectors, particularly adenovirus, has proven a powerful means for introduction of genes into certain cell types. Furthermore, adenovirus transduction has been used to study mechanisms involved in the differentiation of 3T3-L1 cells into mature fat cells. We show in this study that 3T3-L1 cells are inefficiently transduced by adenovirus. The potential advantages offered by adenovirus transduction led us to examine methods designed to enhance transduction of 3T3-L1 cells by adenovirus. Of these methods, polylysine-mediated enhancement demonstrates considerable promise because it permits up to 100% of cells to be transduced and because it does not inhibit differentiation of 3T3-L1 cells. -- Orlicky D. J., and J. Schaack. Adenovirus transduction of 3T3-L1 cells. J. Lipid Res. 2001. 42: 460--466.

Previous studies have established that Ca2+-sensitive adenylyl cyclases, whether endogenously or heterologously expressed, are preferentially regulated by capacitative Ca2+ entry, compared with other means of elevating cytosolic Ca2+ (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F. (1995) J. Biol. Chem. 270, 1149-1155; Fagan, K. A., Mahey, R., and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438-12444; Fagan, K. A., Mons, N., and Cooper, D. M. F. (1998) J. Biol. Chem. 273, 9297-9305). These findings led to the suggestion that adenylyl cyclases and capacitative Ca2+ entry channels were localized in the same functional domain of the plasma membrane. In the present study, we have asked whether a heterologously expressed Ca2+-permeable channel could regulate the Ca2+-inhibitable adenylyl cyclase of C6-2B glioma cells. The cDNA coding for the rat olfactory cyclic nucleotide-gated channel was inserted into an adenovirus construct to achieve high levels of expression. Electrophysiological measurements confirmed the preservation of the properties of the expressed olfactory channel. Stimulation of the channel with cGMP analogs yielded a robust elevation in cytosolic Ca2+, which was associated with an inhibition of cAMP accumulation, comparable with that elicited by capacitative Ca2+ entry. These findings not only extend the means whereby Ca2+-sensitive adenylyl cyclases may be regulated, they also suggest that in tissues where they co-exist, cyclic nucleotide-gated channels and Ca2+-sensitive adenylyl cyclases may reciprocally modulate each other's activity.

We determined the sequence of a Drosophila tRNA gene cluster containing a tRNAHis gene and a tRNAHis pseudogene in close proximity on the same DNA strand. The pseudogene contains eight consecutive base pairs different from the region of the bona fide gene which codes for the 3' portion of the anticodon stem of tRNAHis. The tRNAHis gene is transcribed efficiently in Drosophila Kc cell extract, whereas the pseudogene is not. The pseudogene is also a much poorer competitor than the real gene in a stable transcription complex formation assay, even though the sequence alteration in the pseudogene does not affect the sequence or spacing of the putative internal transcription control regions. Recombinant clones were constructed in which the 5'-flanking regions are exchanged. The transcription efficiencies and competitive abilities of the recombinant clones resemble those of the genes from which the 5' flank was derived; for example, the tRNAHis pseudogene with the 5'-flanking sequence of the tRNAHis gene is now efficiently transcribed. Deletion analysis of the pseudogene 5' flank failed to uncover an inhibitory element. Deletion analysis of the real gene showed very high dependence on the presence of the wild-type 5'-flanking sequence for factor binding to the internal control regions and stable complex formation. The 5'-flanking sequence of a Drosophila tRNAArg gene active in the Drosophila Kc cell extract does not restore transcriptional activity or stable complex formation. The tRNAHis gene and pseudogene behave atypically in HeLa cell extract. Both genes compete for HeLa transcription factors, but neither of them is efficiently transcribed. Removal of the 5'-flanking sequences of each gene and replacement with various sequences, including the tRNAArg gene 5' flank, does not allow increased transcription in HeLa cell extract.

The regions of a Drosophila tRNAArg gene responsible for the "wild type" in vitro transcription level were determined by a transcription-competition assay. Cell-free transcription extracts programmed with 5' and 3' deletion mutants of the tRNAArg gene were used to quantitate the efficiency of transcription and to measure the ability of these DNAs to compete for transcription factors compared to the wild type tRNAArg gene. The results show that those portions of the gene which code for the D-stem/D-loop and T-stem/T-loop of the tRNA product are the regions responsible for competitive ability. These regions were previously shown to contain the intragenic control sequences for eukaryotic tRNA gene transcription and are respectively referred to as the D- and T-control regions. The presence of both the D- and T-control regions is essential for maximum competitive strength. The 5'-flanking and 5' stem regions adjacent to the D-control region have a function in the competitive ability of the D-control region while the 3'-flanking and the 3' stem regions adjacent to the T-control region have a function in the competitive ability of the T-control region. These results are consistent with a model for promotion of tRNA gene transcription that involves recognition by transcription factors of the two control regions. Optimal binding of the transcription factors is dependent upon sequences adjacent to and flanking the intragenic control regions.

Drosophila tRNA genes form stable transcription complexes in vitro, as we have demonstrated by kinetic analyses of transcription experiments in Drosophila Kc cell extracts. tRNA genes added to transcriptionally active cell-free extracts rapidly and stably sequester a transcription factor, inhibiting transcription of a tRNA gene added later. We describe a simplified competition assay dependent on the ability of tRNA genes to form stable complexes. Through the use of this assay with deletion mutations of a Drosophila tRNAArg gene, we demonstrate that stable transcription complex formation is dependent on the DNA region extending from the 5' end of the sequence encoding the T-stem of the tRNA to more than 10 base pairs downstream from the transcription termination sequence. Stable transcription complex formation involves an initial, rapid factor binding followed by rearrangement of the gene-factor complex to a transcriptionally active state. Factor binding to form the stable transcription complex is kinetically dependent on the sequence 5' to the gene region encoding the D-stem, and thermodynamically dependent on the gene region encoding the D-stem and -loop.

Class III transcription components were fractionated from Drosophila Kc cells. By the use of successive DEAE-Sephadex and CM-Sepharose column chromatographies, two fractions were identified to contain distinct positive transcription components. The reconstitution of these two fractions and RNA polymerase III was essential for faithful in vitro transcription of cloned Drosophila tRNA and 5 S RNA genes. Further chromatography of one of these fractions using phosphocellulose and DEAE-cellulose did not separate additional components required for reconstitution of transcription. These fractions may contain generalized class III transcription factors, as suggested by the cofractionation of required tRNA and 5 S RNA transcription components. We present further evidence that tRNA and 5 S RNA genes share at least one common transcription component other than RNA polymerase III by the use of stable transcription complex formation and competition experiments.

Differences in the processing of dimeric tRNASer-tRNAMet precursors derived from the Schizosaccharomyces pombe sup9 wild-type and opal suppressor genes can be attributed to conformational alterations in the tRNASer anticodon/intron domain. A comparison of the patterns obtained upon transcription of the sup9+ (wild-type) and sup9-e (opal suppressor) genes in a coupled transcription/processing extract from Saccharomyces cerevisiae reveals that the latter exhibits a greatly reduced efficiency of 5'-end maturation and is susceptible to specific endonucleolytic cleavage(s) within the intron. Free energy calculations indicate that these effects coincide with a destabilization of the wild-type anticodon/intron stem and suggest that the predominant sup9-e conformer lacks secondary structure in this region. Evidence in support of this hypothesis was obtained by analyzing the processing of sup9+ and sup9-e precursors carrying the intron base substitution, G37:10, which destroys and restores, respectively, the base-pairing potential of the proposed secondary structure and comparing the strength and temperature sensitivity of sup9-e and sup9-e G37:10 suppression in vivo in S. cerevisiae. The data indicate that the anticodon/intron structure of tRNA precursors can influence the rate of RNase P cleavage in vitro and affect tRNA expression in vivo.

The transcription efficiencies of four Drosophila tRNAArg genes located in a tRNA gene cluster at region 42A on chromosome 2, and containing identical coding sequences, were studied in Drosophila Kc cell extracts. Transcription is modulated by the 5' flanking sequences; efficient transcription is dependent on the presence of an optimal 5' flanking sequence. One of the genes, p17D Arg, is not transcribed in the homologous extract but does compete with the other genes for transcription factors. Deletion of a specific sequence from the 5' flank of the gene of p17D Arg leads to an increase in transcription efficiency. All tRNAArg genes are efficiently transcribed in extracts from HeLa cells. However, introduction of small amounts of Drosophila extract reduces the efficiency of transcription in HeLa extracts. This is due to incompatibility between transcriptional components of the two extracts.

We have examined the transcriptional role of the DNA region which lies between the two intragenic control sequences (D-control and T-control) of tRNA genes. Deletion templates (3‘ and 5‘) of a Drosophila tRNAArg gene, which contain either the D- or T-control region, were joined together through XhoI linkers such that the mutant tRNA genes formed now contain an internal cloning site. DNA fragments of different lengths were inserted into the newly formed cloning site to create a series of mutant tRNAArg genes which have an increased separation between the two intragenic control regions of 12 to 1530 nucleotides. Increased separation of the two intragenic control regions did not alter the transcription initiation or termination sites from those of the wild type tRNAArg gene. Transcription, while most efficient in the wild type gene, is still efficient when the two regions are further separated by a distance of 12-77 nucleotides. However, any further increase in length of the sequence separating the control regions resulted in a decreased transcription efficiency and in a decreased ability to compete in the binding of transcription factors in Drosophila Kc cell extracts. The reduction in transcription efficiency is directly related to the decreased ability of the insertion mutant tDNAs to form stable transcription complexes. The tRNAArg gene forms detectable stable complexes up to a separation of the two intragenic control regions by approximately 200 to 400 base pairs. These results suggest a model for tRNA gene transcription that involves factor recognition of sequences within each control region and that the control regions interact only via these factors.

We have examined the 5'-and 3'-flanking sequence requirements for the “wild type transcription properties” of a Drosophila tRNA Arg gene through the use of transcription assays in cell-free extracts. Thirty-three base pairs of the 5' flank immediately adjacent to the sequence encoding the mature tRNA are necessary for efficient transcription in Drosophila Kc cell extract. Sequences affecting factor binding to form stable transcription complexes extend more than 60 base pairs into the 5' flank, and approximately 35 base pairs into the 3' flank. HeLa cell extract exhibits dependence, albeit reduced, on the same 5'-flanking sequence; it also has 3'-flanking sequence requirements for maximal stable complex formation. This requirement of in vitro transcription for flanking sequence is not dependent on the use of a homologous system, but is dependent on the cellular source of the extract.

Under conditions in which cytoplasmic accumulation of HeLa cell mRNAs has been blocked by adenovirus infection, hsp70 family mRNAs are transported from the nucleus to the cytoplasm at near normal efficiency subsequent to heat shock. Heat shock does not reverse the general virus-induced block to host cell mRNA transport. The heat shock mRNAs are translated within the cytoplasm of the infected cell but at substantially reduced efficiency compared with that of uninfected cells. Thus, the hsp70 family of mRNAs can escape the transport block but not the translational block instituted late after adenovirus infection. The beta-tubulin gene family is induced by the viral E1A gene after infection, and its mRNAs also accumulate in the cytoplasmic compartment. Given these two examples, it seems likely that the process of transcriptional induction allows the resulting mRNA to escape the viral block of transport.