In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene

1985 ◽  
Vol 5 (8) ◽  
pp. 1887-1893
Author(s):  
D Wolf ◽  
Z Laver-Rudich ◽  
V Rotter
Keyword(s):  
Cdna Clone ◽  
P53 Protein ◽  
Cdna Probe ◽  
P53 Gene ◽  
Heteroduplex Analysis ◽  
Cdna Clones ◽  
Free System ◽  
Human Cdna ◽  
Mrna Molecule

The human p53 gene was cloned and characterized by using a battery of p53 DNA clones. A series of human cDNA clones of various sizes and relative localizations to the mRNA molecule were isolated by using the human p53-H14 (2.35-kilobase) cDNA probe which we previously cloned. One such isolate, clone p53-H7 (2.65 kilobases), spans the entire human mature p53 mRNA molecule. Construction of the human cDNA clones in the pSP65 RNA transcription vector facilitated the generation of p53 transcripts by the SP6 bacteriophage RNA polymerase. The p53-specific RNA transcripts obtained without further processing were translated into p53 proteins in a cell-free system. By using this rapid in vitro transcription-translation assay, we found that whereas clone p53-H7 (2.65 kilobases) coded for a mature-sized p53 protein, a shorter cDNA clone, p53-H13 (1.8 kilobases), dictated the synthesis of a smaller-sized p53 protein (45 kilodaltons). The p53 proteins synthesized in vitro immunoprecipitated efficiently with human-specific anti-p53 antibodies. Genomic analysis of human DNA revealed the presence of a single p53 gene residing within two EcoRI fragments. Heteroduplex analysis between the full-length cDNA clone p53-H7 and the cloned p53 gene indicated the presence of seven major exons.

scholarly journals In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene.

1985 ◽  
Vol 5 (8) ◽  
pp. 1887-1893 ◽  
Author(s):  
D Wolf ◽  
Z Laver-Rudich ◽  
V Rotter
Keyword(s):  
Cdna Clone ◽  
P53 Protein ◽  
Cdna Probe ◽  
P53 Gene ◽  
Heteroduplex Analysis ◽  
Cdna Clones ◽  
Free System ◽  
Human Cdna ◽  
Mrna Molecule

The human p53 gene was cloned and characterized by using a battery of p53 DNA clones. A series of human cDNA clones of various sizes and relative localizations to the mRNA molecule were isolated by using the human p53-H14 (2.35-kilobase) cDNA probe which we previously cloned. One such isolate, clone p53-H7 (2.65 kilobases), spans the entire human mature p53 mRNA molecule. Construction of the human cDNA clones in the pSP65 RNA transcription vector facilitated the generation of p53 transcripts by the SP6 bacteriophage RNA polymerase. The p53-specific RNA transcripts obtained without further processing were translated into p53 proteins in a cell-free system. By using this rapid in vitro transcription-translation assay, we found that whereas clone p53-H7 (2.65 kilobases) coded for a mature-sized p53 protein, a shorter cDNA clone, p53-H13 (1.8 kilobases), dictated the synthesis of a smaller-sized p53 protein (45 kilodaltons). The p53 proteins synthesized in vitro immunoprecipitated efficiently with human-specific anti-p53 antibodies. Genomic analysis of human DNA revealed the presence of a single p53 gene residing within two EcoRI fragments. Heteroduplex analysis between the full-length cDNA clone p53-H7 and the cloned p53 gene indicated the presence of seven major exons.

Molecular basis for heterogeneity of the human p53 protein

1986 ◽  
Vol 6 (12) ◽  
pp. 4650-4656
Author(s):  
N Harris ◽  
E Brill ◽  
O Shohat ◽  
M Prokocimer ◽  
D Wolf ◽  
...  
Keyword(s):  
Electrophoretic Mobility ◽  
P53 Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Antigenic Determinants ◽  
Sequence Difference ◽  
Cdna Clones ◽  
Protein Species ◽  
Free System ◽  
The Individual

The human p53 tumor antigen comprises several physically distinct proteins. Two p53 proteins, separable by polyacrylamide gel electrophoresis, are expressed by the human transformed cell line SV-80. The individual cDNAs which code for these proteins were isolated and constructed into the SP6 transcription vector. The proteins encoded by these clones were identified by in vitro transcription with the SP6 vector and translation in a cell-free system. p53-H-1 and p53-H-19 cDNA clones code for the faster- and slower-migrating p53 protein species, respectively, of SV-80. The in vitro-expressed proteins of p53-H-1 and p53-H-19 had the same antigenic determinants and were structurally indistinguishable from their in vivo counterparts. By expressing defined restricted cDNA fragments in vitro, the region of heterogeneity between the respective cDNAs was located at the 5' end of the cDNAs. Exchanging the 5' fragments of interest and expressing the chimeric clones in vitro confirmed that the DNA heterogeneity was responsible for the difference in the electrophoretic mobility of these proteins. The sequences of the two cDNAs revealed a single base pair difference (G versus C) in the coding region of the clones. This sequence difference resulted in an arginine being coded for in clone p53-H-1 and a proline being coded for at the equivalent position in clone p53-H-19. This variation accounted for the change in the electrophoretic mobility of the individual p53 protein species.

Immunologically distinct p53 molecules generated by alternative splicing

1986 ◽  
Vol 6 (9) ◽  
pp. 3232-3239
Author(s):  
N Arai ◽  
D Nomura ◽  
K Yokota ◽  
D Wolf ◽  
E Brill ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cdna Clone ◽  
P53 Protein ◽  
Lymphoid Cells ◽  
Antigenic Determinants ◽  
Cdna Clones ◽  
P53 Expression ◽  
Mrna Species

Transfection of a functional cloned p53 gene into an L12 p53 nonproducer cell line efficiently reconstituted p53 expression. The p53 protein synthesized in these clones was indistinguishable from that occurring naturally in tumor cells. When a p53 cDNA clone was used instead, we observed that the L12-derived clones exhibited a distinct immunological profile. In the present experiments we compared the immunological epitopes of p53 proteins encoded by several full-length cDNA clones. Immunoprecipitation of p53 proteins generated by in vitro transcription and translation of the various cDNA clones indicated variations in the content of immunological epitopes. Basically, two p53 protein species were detected. Both species contained the same antigenic determinants except the PAb421-PAb122 site, which was present in proteins encoded by p53-M11 and pcD-p53, but not in the p53 protein encoded by the p53-M8 cDNA clone. Sequence analysis of the various cDNA clones indicated the existence of a 96-base-pair (bp) insert in clone p53-M8 as compared with clone p53-M11 or pCD-p53. The 96-bp insert contained a termination signal which caused the premature termination of the protein, leading to the generation of a p53 product 9 amino acids shorter than usual. The existence of this insert also accounted for the lack of the PAb421-PAb122 epitope which was mapped to the 3' end of the cDNA clone, following the 96-bp insert. This insert shared complete homology with the p53 intron 10 sequences mapping 96 bp upstream of the 5' acceptor splicing site of p53 exon 11. It was therefore concluded that the different cDNA clones represented p53 mRNA species which were generated by an alternative splicing mechanism. Differential hybridization of the mRNA population of transformed fibroblastic or lymphoid cells with either the 96-bp synthetic oligonucleotide or the p53-M11 cDNA indicated that the various mRNA species are expressed in vivo.

scholarly journals Immunologically distinct p53 molecules generated by alternative splicing.

1986 ◽  
Vol 6 (9) ◽  
pp. 3232-3239 ◽  
Author(s):  
N Arai ◽  
D Nomura ◽  
K Yokota ◽  
D Wolf ◽  
E Brill ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cdna Clone ◽  
P53 Protein ◽  
Lymphoid Cells ◽  
Antigenic Determinants ◽  
Cdna Clones ◽  
P53 Expression ◽  
Mrna Species

Transfection of a functional cloned p53 gene into an L12 p53 nonproducer cell line efficiently reconstituted p53 expression. The p53 protein synthesized in these clones was indistinguishable from that occurring naturally in tumor cells. When a p53 cDNA clone was used instead, we observed that the L12-derived clones exhibited a distinct immunological profile. In the present experiments we compared the immunological epitopes of p53 proteins encoded by several full-length cDNA clones. Immunoprecipitation of p53 proteins generated by in vitro transcription and translation of the various cDNA clones indicated variations in the content of immunological epitopes. Basically, two p53 protein species were detected. Both species contained the same antigenic determinants except the PAb421-PAb122 site, which was present in proteins encoded by p53-M11 and pcD-p53, but not in the p53 protein encoded by the p53-M8 cDNA clone. Sequence analysis of the various cDNA clones indicated the existence of a 96-base-pair (bp) insert in clone p53-M8 as compared with clone p53-M11 or pCD-p53. The 96-bp insert contained a termination signal which caused the premature termination of the protein, leading to the generation of a p53 product 9 amino acids shorter than usual. The existence of this insert also accounted for the lack of the PAb421-PAb122 epitope which was mapped to the 3' end of the cDNA clone, following the 96-bp insert. This insert shared complete homology with the p53 intron 10 sequences mapping 96 bp upstream of the 5' acceptor splicing site of p53 exon 11. It was therefore concluded that the different cDNA clones represented p53 mRNA species which were generated by an alternative splicing mechanism. Differential hybridization of the mRNA population of transformed fibroblastic or lymphoid cells with either the 96-bp synthetic oligonucleotide or the p53-M11 cDNA indicated that the various mRNA species are expressed in vivo.

scholarly journals Molecular basis for heterogeneity of the human p53 protein.

1986 ◽  
Vol 6 (12) ◽  
pp. 4650-4656 ◽  
Author(s):  
N Harris ◽  
E Brill ◽  
O Shohat ◽  
M Prokocimer ◽  
D Wolf ◽  
...  
Keyword(s):  
Electrophoretic Mobility ◽  
P53 Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Antigenic Determinants ◽  
Sequence Difference ◽  
Cdna Clones ◽  
Protein Species ◽  
Free System ◽  
The Individual

The human p53 tumor antigen comprises several physically distinct proteins. Two p53 proteins, separable by polyacrylamide gel electrophoresis, are expressed by the human transformed cell line SV-80. The individual cDNAs which code for these proteins were isolated and constructed into the SP6 transcription vector. The proteins encoded by these clones were identified by in vitro transcription with the SP6 vector and translation in a cell-free system. p53-H-1 and p53-H-19 cDNA clones code for the faster- and slower-migrating p53 protein species, respectively, of SV-80. The in vitro-expressed proteins of p53-H-1 and p53-H-19 had the same antigenic determinants and were structurally indistinguishable from their in vivo counterparts. By expressing defined restricted cDNA fragments in vitro, the region of heterogeneity between the respective cDNAs was located at the 5' end of the cDNAs. Exchanging the 5' fragments of interest and expressing the chimeric clones in vitro confirmed that the DNA heterogeneity was responsible for the difference in the electrophoretic mobility of these proteins. The sequences of the two cDNAs revealed a single base pair difference (G versus C) in the coding region of the clones. This sequence difference resulted in an arginine being coded for in clone p53-H-1 and a proline being coded for at the equivalent position in clone p53-H-19. This variation accounted for the change in the electrophoretic mobility of the individual p53 protein species.

scholarly journals Alterations of p53 and c-myc in the clonal evolution of malignant lymphoma

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 452-459 ◽  
Author(s):  
H Chang ◽  
S Benchimol ◽  
MD Minden ◽  
HA Messner
Keyword(s):  
Growth Factors ◽  
T Cell ◽  
Cell Lines ◽  
P53 Protein ◽  
Clonal Evolution ◽  
Cdna Probe ◽  
Cell Phenotype ◽  
Coding Region ◽  
P53 Genes

Abstract We derived the lymphoma cell lines OCI-Ly 13.1 and OCI-Ly 13.2 from a patient with non-Hodgkin's lymphoma at the time of presentation and during chemotherapy-resistant relapse. These lines were of T-cell phenotype and contained the identical T-cell receptor beta-chain rearrangement, indicating that both lines were members of the same malignant clone. The lines differed in their growth characteristics; OCI-Ly 13.1 grew slowly and required growth factors for colony formation, whereas OCI-Ly 13.2 grew rapidly and formed colonies without addition of growth factors. To test whether or not these biologic differences were associated with specific genetic changes, we evaluated the status of the c-myc and p53 genes of both cell lines. The p53 and c- myc genes of OCI-Ly 13.1 were in germline configuration and produced normal-sized transcripts. The p53 protein expressed in OCI-Ly 13.1 was recognized by the anti-p53 monoclonal antibody, PAb240, indicating a conformation typical of p53 proteins expressed by p53 alleles containing a missense mutation. However, sequencing studies of the entire p53 coding region did not reveal any point mutations. In contrast, the cell line OCI-Ly 13.2 contained structural abnormalities of both the c-myc and p53 genes. In addition, one of the p53 alleles was lost as determined by a cDNA probe for the p53 gene (17p 13.1) and the YNZ22.1 probe (17p 13.3). These changes resulted in the absence of p53 protein and mRNA in OCI-Ly 13.2 as detected by immunoprecipitation and Northern blot analysis, respectively. They may be a reflection of disease progression and may be associated with the altered behavior of the malignant cell population within the patient and in vitro.

scholarly journals Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule.

1985 ◽  
Vol 5 (1) ◽  
pp. 127-132 ◽  
Author(s):  
D Wolf ◽  
N Harris ◽  
N Goldfinger ◽  
V Rotter
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cells ◽  
Cdna Clone ◽  
P53 Protein ◽  
P53 Gene ◽  
Full Length ◽  
Antigenic Determinants ◽  
Entire Group ◽  
P53 Expression ◽  
P53 Antibodies

Transfection of a cloned p53 gene into a p53 nonproducer Abelson murine leukemia virus-transformed cell line, L12, reconstituted p53 expression. The protein expressed in these cells was indistinguishable from that naturally expressed in p53 producer tumor cells. Conversely, p53 protein expressed in L12-derived clones that were established by transfection with a full-length p53 cDNA clone (pM8) exhibited a discrete immunological form. Immunoprecipitation of p53 with a panel of monoclonal anti-p53 antibodies showed that L12-derived clones that were transfected with the genomic p53 clone contained the same antigenic determinants as those found in the p53 protein expressed in tumor cells. These p53 proteins bound all monoclonal antibody types as well as the polyclonal anti-p53 tested. However, L12-derived clones established by transfection of the p53 cDNA clone (pM8) expressed a p53 protein that bound the RA3-2C2 and PAb200.47 anti-p53 monoclonal antibodies as well as polyclonal anti-p53 serum but totally lacked the antigenic receptor for the PAb122 and PAb421 monoclonal antibodies. The p53 proteins expressed by either genomic or cDNA p53 clones exhibited the same apparent molecular sizes and identical partial peptide maps. We suggest that transfection of the p53 gene induced expression of the entire group of the possible mRNA species, whereas cloned p53 cDNA (pM8) represented a single mRNA molecule that codes for a discrete species of p53 protein.

scholarly journals High-expression of ROCK1 modulates the apoptosis of lens epithelial cells in age-related cataracts by targeting p53 gene

2020 ◽  
Author(s):  
Shanshan Hu ◽  
Dongmei Su ◽  
Lei Sun ◽  
Zhongying Wang ◽  
Lina Guan ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Protein Level ◽  
P53 Protein ◽  
P53 Gene ◽  
Lens Epithelial Cells ◽  
Protein Levels ◽  
P53 Signaling ◽  
Age Related ◽  
Gene And Protein Expression

Abstract Background: Age-related cataract (ARC) is a serious visual impairment disease, and its pathogenesis is unclear. This article aims to investigate the role of ROCK1 in the apoptosis of lens epithelial cells (LECs) in age-related cataracts. Methods: We collect anterior capsule samples from normal people, patients with age-related cataracts, young mice and naturally aging cataract mice. The Oxidative stress-induced apoptosis model was constructed by cultivating HLE-B3 cells with H2O2. MTT, Hoechst 33342, and TUNEL assay were performed to explore proliferation and apoptosis. HE assay was used to observe cell morphology. The gene and protein expression were assessed by quantitative real-time PCR, western blot, immunofluorescence, and immunohistochemical staining.Result: The results from the clinic and mice experiments showed that the numbers of lens epithelial cells from cataract individuals were less than the control individuals. In vitro, the apoptotic cells were increased in lens epithelial cells under H2O2 treatment. The ROCK1 protein level increased in the lens epithelial cells from age-related cataract patients and the old mice, respectively. Meanwhile, the up-regulation of the ROCK1 gene was associated with H2O2-induced HLE-B3 cells apoptosis. MTT and apoptosis assay showed ROCK1 was necessary in mediating H2O2-induced lens epithelial cells apoptosis through ROCK1 over-expression and knockdown experiment, respectively. Further investigation showed that p53 protein levels had been increased during ROCK1-mediated apoptosis in response to H2O2. Besides, ROCK1 phosphorylated p53 at ser15 to up-regulate its protein level. Conclusions: This study established the novel association of ROCK1/p53 signaling with lens epithelial cells apoptosis and age-related cataract genesis.

scholarly journals Analysis of von Willebrand factor mRNA from the lung of pigs with severe von Willebrand disease by using a human cDNA probe

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1341-1346 ◽  
Author(s):  
QY Wu ◽  
BR Bahnak ◽  
L Coulombel ◽  
D Kerbiriou-Nabias ◽  
L Drouet ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Cdna Probe ◽  
Von Willebrand Disease ◽  
Northern Analysis ◽  
Cdna Clones ◽  
Total Rna ◽  
Human Cdna ◽  
Actin Mrna ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract To examine the control of porcine von Willebrand factor (vWF) biosynthesis we cloned human vWF complementary DNA (cDNA) and investigated the expression of the vWF gene in lungs from normal pigs and pigs with severe von Willebrand's disease (vWD). Recombinant clones spanning approximately 90% of human vWF cDNA were identified in a lambda gt10 human lung cDNA library by screening with oligonucleotides. One clone spanning nucleotides 960 to 3,240 of human vWF cDNA was used to investigate the steady-state levels of vWF mRNA in lungs from normal pigs and from pigs phenotypically determined to be homozygous for vWD. This clone hybridized with genomic DNA from pig leukocytes when Southern blots were processed under very stringent conditions; therefore, human cDNA clones were considered valid probes to detect porcine mRNA. Northern blot analysis of total RNA from normal pig lung and human umbilical vein endothelial cells identified the vWF mRNA as a molecular species of approximately 9.0 kilobases (kb). A very faint to undetectable band at 9.0 kb in total RNA from lungs of vWD pigs suggested a decreased rate of transcription of the vWF gene. Sucrose density gradient centrifugation of RNA from the vWD pigs confirmed by Northern analysis that the high-molecular weight fractions contained vWF mRNA and at the same size as normal pig mRNA. Dot blot hybridization analysis of vWF and actin mRNA processed under stringent conditions demonstrated that the relative ratio of vWF mRNA to actin mRNA in the vWD pigs varied from 21% to 41% of the ratio observed in normal pigs. Because the amount of vWF mRNA is not correlated to the amount of vWF activity or antigen in plasma of vWD pigs we conclude that posttranscriptional events are also probably involved in abnormal expression of vWF in these animals.

Assembly of the Alu domain of the signal recognition particle (SRP): dimerization of the two protein components is required for efficient binding to SRP RNA

1990 ◽  
Vol 10 (2) ◽  
pp. 777-784
Author(s):  
K Strub ◽  
P Walter
Keyword(s):  
Cdna Clone ◽  
Repetitive Sequences ◽  
Signal Recognition Particle ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Cdna Clones ◽  
Functional Domain ◽  
Signal Recognition ◽  
Srp Rna

The signal recognition particle (SRP), a cytoplasmic ribonucleoprotein, plays an essential role in targeting secretory proteins to the rough endoplasmic reticulum membrane. In addition to the targeting function, SRP contains an elongation arrest or pausing function. This function is carried out by the Alu domain, which consists of two proteins, SRP9 and SRP14, and the portion of SRP (7SL) RNA which is homologous to the Alu family of repetitive sequences. To study the assembly pathway of the components in the Alu domain, we have isolated a cDNA clone of SRP9, in addition to a previously obtained cDNA clone of SRP14. We show that neither SRP9 nor SRP14 alone interacts specifically with SRP RNA. Rather, the presence of both proteins is required for the formation of a stable RNA-protein complex. Furthermore, heterodimerization of SRP9 and SRP14 occurs in the absence of SRP RNA. Since a partially reconstituted SRP lacking SRP9 and SRP14 [SRP(-9/14)] is deficient in the elongation arrest function, it follows from our results that both proteins are required to assemble a functional domain. In addition, SRP9 and SRP14 synthesized in vitro from synthetic mRNAs derived from their cDNA clones restore elongation arrest activity to SRP(-9/14).

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