However, there may be some situations and some promoters where the use of the lac operator system is advantageous. Our specific plans are to investigate this issue by inserting the lac operator between the glucocorticoid-responsive element (GRE, an inducible enhancer) and the promoter region of the human metallo-. Let us return to a more detailed discussion of the use of the lac operator-repressor system to control eukaryotic gene expression after gene transfer.
The lac repressor can specifically bind to a cognate lac operator sequence downstream of the lac promoter. As described in Chapter 3, a high level of repression is achieved with the lac repressor with a single lac operator sequence inserted into a control region of the test plasmid injected into Xenopus oocytes.
13 Laboratory, Cold Spring Harbor, N.Y
CHAPTER 2
The Inducible lac Operator-Repressor System Is Functional in Mammalian Cells
CHAPTER 3
3, the lac repressor (at approximately 70 ng protein per oocyte) was first injected into the oocyte cytoplasm, and 2 h later the plasmids pSMEO I and pTK-5 were injected into the nucleus of the previously injected oocytes. IPTG does not have non-specific effects on the expression of the HSV-1 tk gene (Fig. As an additional control, it was important to determine whether insertion of the lac operator sequence op-.
Effects of lac operator insertions on gene expression in the absence of the repressor in oocytes. Kathleen Matthews (Rice University) and Sheldon York (University of Denver) for the gifts of the Rev. E.
CHAPTER 4
During muscle tissue differentiation, the P- and 1-cytoskeletal actins are downregulated, whereas the sarcomeric α-cardiac and α-skeletal actins are upregulated, synthesized in large amounts, but with distinct tissue-specific and developmental. expression patterns (Devlin and Emerson, 1979; Minty et al., 1982; Gunning et al., 1983; Bains et al., 1984; . Mohun et al., 1986; Nudel et al., 1986). Coinciding with this transition, the relative level of skeletal α-actin mRNA is induced at least 25-fold above its level in perfused myoblasts both in vivo and in vitro (Hayward and Schwartz, 1986). It is therefore of particular interest to investigate the molecular mechanisms underlying the selective induction of the skeletal α-actin family member during muscle cell development.
What may be the molecular mechanism(s) governing skeletal α-actin gene regulation in myogenic and nonmyogenic cells. One approach to answering this question is the molecular cloning and characterization of the genes that code for the skeletal α-actin proteins. Therefore, we isolated and characterized the genomic clone of the mouse skeletal actin gene from BALB/c mouse sperm DNA.
As described in Chapter 5, the structure and the complete nucleotide sequence of the single genomic copy of the mouse skeletal a-actin gene has been determined. A comparison of the nucleotide sequences of several vertebrate skeletal α-actin genes reveals several highly conserved sequences in the 5'-flanking region and in both 5'- and 3'-untranslated regions. In addition, two potential inverted repeat sequences, partially within the conserved regions, were identified within the large first intron of rodent skeletal α-actin genes.
Furthermore, a tissue-specific transcriptional enhancer has very recently been identified within the first large intron of the human P-actin gene (Kawamoto et al., 1988). Therefore, in the future it will be interesting to investigate whether these partially conserved inverted repeat sequences in the first intron of the mammalian skeletal α-actin genes serve as a regulator or not. elements, such as enhancers, in differentiated muscle cells. We have successfully used this approach to map the transcription start site of the mouse skeletal a-actin gene. eds.).
CHAPTER 5
The complete sequence of the mouse skeletal α-actin gene reveals several conserved and inverted repeated sequences beyond it. Here we present the complete nucleotide sequence of the single genomic copy of the mouse skeletal α-actin gene. RNA from a differentiated culture of the myogenic mouse cell line BC3H-1 (42) was isolated by guanidine thiocyanate extraction (9) and two cycles of oligo(dT)-cellulose chromatography (1).
The structure of the mouse skeletal α-actin gene is shown between the two homology plots. Comparison of the nucleotide sequence of the 5'-flanking and the 5'-untranslated regions of vertebrate skeletal α-actin genes. Predicted inverted repeat structures in the 5'-flanking region and the 5'- and 3'-untranslated regions of the mouse, rat, and chicken skeletal α-actin genes.
Three potential configurations are shown in the 5'-flanking and 5'-untranslated regions of the chick skeletal a-actin gene. A potential stem-loop is demonstrated in the 3'-untranslated region of the mouse skeletal α-actin gene. A potential stem loop is shown in the 3'-untranslated region of the chicken skeletal α-actin gene.
Thus, the 3' untranslated region of the mouse skeletal α-actin mRNA is ca. 245 nucleotides long [excluding the poly(A) tail]. We have aligned the nucleotide sequence of the mouse skeletal α-actin gene with those of rat and chicken using percent homology profiles (Fig. 5). A similar comparison of the 5′-untranslated regions of nonmuscle J3 actins (human eDNA and rat genomic sequence) also shows a high degree of sequence conservation ( 38 ).
CHAPTER 6
Since T4 DNA polymerase will not displace the mRNA:DNA hybrid, synthesis is blocked at the first nucleotide of the mRNA molecule. We used this approach to map the tsp ex-actin gene of the mouse skeleton. We successfully used this approach to map the tsp gene of murine skeletal a-actin.
The basic principle of the method for mapping a teaspoon to a genomic clone is illustrated in Fig. Synthesis is blocked by the 5' end of the RNA, as T4 DNA polymerase does not catalyze strand displacement synthesis (Masamune and Richardson, 1971). . The overall identification of the logging site is consistent with our lower resolution Exo VII mapping data (Hu et al., 1986).
Figure 3 shows the pattern of elongation products from a serial dilution of poly(A)+ RNA vs. It is clear that the intensity of the mRNA-specific signal (the bands labeled "cap site") increases in accordance with the amount of poly(A) + RNA used. The experimental reasoning was that the primer extension reaction of the T4 DNA polymerase was indeed specifically blocked by the 5' end.
The Mcap" site (tsp) and the T A TA box are shown to the right of the autoradiogram. Expression cloning of cDNAs encoding the p75 subunit of the high-affinity interleukin-2 receptor by a novel selection strategy. The IL peptide molecule -2, supports the idea that both subunits contribute directly to the high-affinity receptor binding site (Robb et al., 1987).
SPECIFIC AIMS
First, I will construct a selected eDNA library using pull-down probes to enrich IL-2-R p75 cDNAs as described (Hedrick et al., 1984). (1) That they should be expressed in YT-clone-2C2 cells, a human leukemic T cell clone that expresses only IL-2-R p75 (Teshigawara et al., 1987), but not in MT-1 cells, a Line human leukemic T cell that does not express the IL-2-R p75 subunit (Tsudo et al., 1987). -poly(A)+-linked polysomal RNA will be prepared from YT-clone-2C2 cells as described (LaPolla et al., 1984); Total cellular poly(A)+ RNA from YT-clone-2C2 and MT-1 cells will be prepared by the procedure as described (Chirgwin et al., 1979).
YT-clone-2C2 eDNA synthesized from total cellular poly(A)+ RNA will be subtracted with MT-1 total poly(A)+ RNA and the subtracted products will be used to synthesize the second strand DNA with DNA polymerase I as described ( Davis et al., 1984). Specifically, in this protocol a spheroplast fusion technique (Sandri-Goldin et al., 1981) will be used to introduce the library into COS cells, where it replicates and expresses. Whether or not the positive cDNAs are derived from IL-2-R p75 mRNA will be determined by transfecting these plasmid DNAs into MT-1 cells, which constitutively express pSS but not p75 subunit (Tsudo et al., 1987) , to reconstitute high - affinity.
Moreover, a 15- to 20-bp sequence within this DNA segment binds a nuclear factor (or factors) from extracts of human Hela cells or mouse ID13 cell nuclei (Hay et al., 1987). The expression of c-myc increases during the response of cells to growth factors (Greenberg and Ziff, 1984; Greenberg et al., 1985; Dean et al., 1986; Levine et al., 1986) and decreases during cell differentiation. into a non-proliferative state (Grosso and Pitot, 1985; Bentley and Groudine, 1986;.
Second, the c-myc gene has been proposed to be under negative transcriptional control via a putative repressor that binds to the 5' flanking region or within the large first untranslated exon (Dunnick et al., 1983; . Leder et al., 1983 ), i.e. Third, modulation of RNA stability has been suggested to play an important role in the regulation of c-myc RNA level (Blanchard et al., 1985; Dani et al. Strikingly, a negative regulator for the human c- myc gene was also identified very recently (Hay et al., 1987).
103 REFERENCES
Similarly, the tissue specificity of the immunoglobulin heavy chain (IgH) enhancer has been shown to be negatively controlled (Imler et al., 1987). Alternatively, IFN-induced transcriptional desensitization (or repression) in transfected cells can be analyzed by nuclear leakage assays (Larner et al., 1986) using plasmid ElB DNA to monitor the transcriptional signal. Molecular cloning of &ene encodin and re&ulatory ne&ative protein(s).
Constitutive transcription of the ISG-54 gene has been found to occur with extracts from uninduced cells (Levy et al., 1986). This should contribute to a better understanding of the mechanisms governing the control of gene expression. This design also prevents the self-ligation between the two free ends of the composite enhancer/streptavidin/SV-CAT molecule in the.
Fortunately, because of the extremely high affinity interaction between biotin and streptavidin [Kd-lo-1 5M, (Green, 1975)], it seems reasonable to assume that the biotin-streptavidin bond will be stable in cells of culture and in vitro transcription system (see below). During the development of the vertebrate nervous system, nerve growth factor (NGF) plays an essential role in the survival and maintenance of sympathetic and sensory neurons (Levi-Montalcini and Angeletti, 1968). I propose the use of the proteolytic dissection technique to identify the functional domains of the human NGF receptor.
The size of the digested peptides will be analyzed by electrophoresis in 15% sodium dodecyl sulfate (SDS) polyacrylamide gels (Laemmli, 1970) in the presence of dithiothreitol. Alternatively, the time course of Pronase E degradation of NGF/NGF-receptor complexes can be performed and monitored by the same gel electrophoresis. The origin of eluted fragments (ie, NGF-binding domains) on the NGF receptor will be determined by automated Edman digestion and amino acid analysis as described (Hunkapiller et al., 1984).
As a control, nonspecific binding of the NGF receptor to Sepharose-4B will be determined by parallel incubation in the column containing no NGF. The flow rate through the gel can be regulated by the angle of the plate to the horizontal.
A model for binding of NGF to NGF receptor to form low and high affinity complexes.
