Article Figures & Data
Figures
- Fig. 1.
Typical transcriptional activity of GALMBP1-338 in NIH 3T3 cells. Effector plasmid DNA was cotransfected at the indicated concentrations with 5 μg of the GAL4TK CAT reporter plasmid. The total amount of plasmid DNA (10 μg) was kept constant by addition of an empty vector (CMVGAL4) to each transfection mixture. Cell extracts were prepared 48 h posttransfection and assayed for CAT activity. The result indicates that the GALMBP1-338 fusion protein repressed TK promoter activity in a dose-dependent manner.
- Fig. 2.
MBP-1 represses transcription when it is brought to the promoter through the GAL4 DNA binding domain. A 5-μg sample of a GAL4TK CAT or TK CAT reporter plasmid was cotransfected with MBP-1 or an empty vector used as a control. The results shown are average results from four independent assays. A relative CAT activity of 100% was arbitrarily assigned to the vector control.
- Fig. 3.
(A) Schematic representation of deletion mutant GALMBP-1 used in the analysis for MBP-1 transregulatory domains. The hatched box represents the GAL4 1-147 DNA binding domain, and the open box represents MBP-1 sequences. The numbers following MBP are amino acid positions. (B) Transcriptional activity of deletion mutant GALMBP-1 in NIH 3T3 and HeLa cells. Cells were cotransfected with equal amounts of deletion mutant GAL4TK CAT and GALMBP-1. The CAT assay was performed as described in Materials and Methods. The results suggest that MBP-1 possesses two repressor domains (at the N and C termini) and one activation domain (in the middle).
- Fig. 4.
Expression of deletion mutant MBP-1 by Western blot analysis using a monoclonal antibody to the GAL4 DNA binding domain. NIH 3T3 cells were transfected with 2 μg of each expression plasmid, and Western blot analysis was performed as described in Materials and Methods. Polypeptides detected by transfection of CMVGAL4 (lane 1), GALMBP1-338 (lane 2), GALMBP1-244 (lane 3), GALMBP1-155 (lane 4), and GALMBP1-130 (lane 5); by mock-transfected cell extract (lane 6); and by transfection of GALMBP140-244 (lane 7), GALMBP188-244 (lane 8), GALMBP140-338 (lane 9), GALMBP232-338 (lane 10), and GALMBP188-338 (lane 11) are shown. Molecular masses are shown on the right in kilodaltons.
- Fig. 5.
(A) Schematic representation of leucine-to-alanine point mutations in the LXVXL motif of MBP-1 repressor domains. (B) Transcriptional activities of the repressor domains and respective mutant constructs in NIH 3T3 cells are presented as means of three independent experiments. A relative CAT activity of 100% was arbitrarily assigned to the vector control.
- Fig. 6.
The N-terminal region of MBP-1 (amino acids 1 to 47) acts as a transferable repressor domain. (A) The N-terminal repressor domain of MBP-1 was cloned in frame with a heterologous activation domain (3CGln) of Epstein-Barr virus transcription factor EBNA3C. CAT activities from pM3/3CGln were arbitrarily assigned a value of 100%, and CAT activity is shown at the top. (B) Western blot analysis for expression of pM3/3CGln and 3CGln(MBP-1) fusion proteins.
- Fig. 7.
Transcriptional regulation of the c-mycpromoter by wild-type and mutant MBP-1. (A) Schematic diagram of wild-type and mutant MBP-1 under control of the CMV promoter in a pcDNA3 expression vector. CMVMBP-1(wt) represents wild-type MBP-1, and CMVMBP-1(N) and CMVMBP-1(C) represent MBP-1 constructs with leucine-to-alanine mutations in the amino- and carboxy-terminal repressor domains, respectively. (B) Wild-type and mutant MBP-1 cotransfected with a c-myc CAT reporter construct in NIH 3T3 cells. Results are shown as averages of four independent assays. (C) GALMBP1-338, its mutant forms and repressor domains cotransfected with a c-myc CAT construct in NIH 3T3 cells. Results are shown as averages of three independent assays. In all cases, the relative CAT activity of the vector control was arbitrarily assigned a value of 100%.
- Fig. 8.
Western blot analysis of expression of wild-type and mutant MBP-1 as fusion proteins. The arrowhead corresponds to the calculated molecular weight of MBP-1.
Tables
- Table 1.
Oligonucleotides used to construct mutant forms of GALMBP-1
Primer sequencea Initial amino acid of mutant MBP-1 Sense 5′-AACTGAGGATCCAGATGGA-3′ 1 5′-AAAGCTGGATC CCTGATAAG-3′ 140 5′-CTGTACGGATCCTTCATCAA-3′ 188 5′-CCAAAGAGGAT CCCCAAGGCC-3′ 232 Antisense 5′-ACTTTGATCTAGAGGCAGTTG-3′ 244 5′-CCAGACCTCTAGAACTCGGA-3′ 155 5′-GATGACTCTAGAGTTGCCAG-3′ 47 5′-CAGCAGCTCTAGACCTTCTT-3′ 130 5′-GCCTGCCCTCTAGATTACTTG-3′ 338 ↵a Underlined sequences indicate the first codon of the deletion mutant form of MBP-1. Boldface sequences indicate the restriction enzyme sites used for undirectional cloning.
