scholarly journals Sequences involved in initiation of simian virus 40 late transcription in the absence of T antigen.

1986 ◽  
Vol 6 (6) ◽  
pp. 1875-1885 ◽  
Author(s):  
F Omilli ◽  
M Ernoult-Lange ◽  
J Borde ◽  
E May
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Late Promoter ◽  
Late Gene Expression ◽  
Rich Region ◽  
Enhancer Element ◽  
Late Initiation ◽  
Late Transcription

We analyzed the sequences involved in vivo in the initiation of simian virus 40 (SV40) late transcription occurring in the absence of both SV40 origin sequences and T antigen. The constituent elements of the SV40 late promoters have already been the subject of extensive studies. In vitro studies have resulted in the description of two putative domains of the late promoters. The first domain consists of an 11-base-pair (bp) sequence, 5'-GGTACCTAACC-3', located 25 nucleotides (nt) upstream of the SV40 major late initiation site (MLIS) (J. Brady, M. Radonovich, M. Vodkin, V. Natarajan, M. Thoren, G. Das, J. Janik, and N. P. Salzman, Cell 31:624-633, 1982). The second domain is located within the G-C-rich region (J. Brady, M. Radonovich, M. Thoren, G. Das, and N. P. Salzman, Mol. Cell. Biol. 4:133-141; U. Hansen and P. A. Sharp, EMBO J. 2:2293-2303). Our previous in vivo studies permitted us to define a domain of the late promoter which extends from nt 332 to nt 113 and includes the 72-bp enhancer sequences. Here, by using transfection of the appropriate chimeric plasmids into HeLa cells in conjunction with quantitative S1 nuclease analysis, we analyzed in more detail the sequences required for the control of SV40 late-gene expression occurring before the onset of viral DNA replication. We showed that the major late promoter element is in fact the 72-bp repeat enhancer element. This element was able to drive efficient late transcription in the absence of T antigen. Under our experimental conditions, removal of the G-C-rich region (21-bp repeats) entailed a significant increase in the level of late-gene expression. Moreover, translocation of this element closer to the MLIS (53 nt upstream of the MLIS) enhanced the level of transcripts initiated at natural late initiation sites. Our results suggest that the G-C-rich regions have to be positioned between the enhancer element and the initiation sites to stimulate transcription from downstream sites. Thus, the relative arrangement of the various promoter elements is a critical factor contributing to the situation in which the early promoter is stronger than late promoters before viral DNA replication.

Sequences involved in initiation of simian virus 40 late transcription in the absence of T antigen

1986 ◽  
Vol 6 (6) ◽  
pp. 1875-1885
Author(s):  
F Omilli ◽  
M Ernoult-Lange ◽  
J Borde ◽  
E May
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Late Promoter ◽  
Late Gene Expression ◽  
Rich Region ◽  
Enhancer Element ◽  
Late Initiation ◽  
Late Transcription

We analyzed the sequences involved in vivo in the initiation of simian virus 40 (SV40) late transcription occurring in the absence of both SV40 origin sequences and T antigen. The constituent elements of the SV40 late promoters have already been the subject of extensive studies. In vitro studies have resulted in the description of two putative domains of the late promoters. The first domain consists of an 11-base-pair (bp) sequence, 5'-GGTACCTAACC-3', located 25 nucleotides (nt) upstream of the SV40 major late initiation site (MLIS) (J. Brady, M. Radonovich, M. Vodkin, V. Natarajan, M. Thoren, G. Das, J. Janik, and N. P. Salzman, Cell 31:624-633, 1982). The second domain is located within the G-C-rich region (J. Brady, M. Radonovich, M. Thoren, G. Das, and N. P. Salzman, Mol. Cell. Biol. 4:133-141; U. Hansen and P. A. Sharp, EMBO J. 2:2293-2303). Our previous in vivo studies permitted us to define a domain of the late promoter which extends from nt 332 to nt 113 and includes the 72-bp enhancer sequences. Here, by using transfection of the appropriate chimeric plasmids into HeLa cells in conjunction with quantitative S1 nuclease analysis, we analyzed in more detail the sequences required for the control of SV40 late-gene expression occurring before the onset of viral DNA replication. We showed that the major late promoter element is in fact the 72-bp repeat enhancer element. This element was able to drive efficient late transcription in the absence of T antigen. Under our experimental conditions, removal of the G-C-rich region (21-bp repeats) entailed a significant increase in the level of late-gene expression. Moreover, translocation of this element closer to the MLIS (53 nt upstream of the MLIS) enhanced the level of transcripts initiated at natural late initiation sites. Our results suggest that the G-C-rich regions have to be positioned between the enhancer element and the initiation sites to stimulate transcription from downstream sites. Thus, the relative arrangement of the various promoter elements is a critical factor contributing to the situation in which the early promoter is stronger than late promoters before viral DNA replication.

Functional analysis of the role of the A + T-rich region and upstream flanking sequences in simian virus 40 DNA replication

1986 ◽  
Vol 6 (12) ◽  
pp. 4570-4577
Author(s):  
R Gerard ◽  
Y Gluzman
Keyword(s):  
Dna Replication ◽  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Rich Region ◽  
Viral Dnas ◽  
Origin Function

One boundary of the minimal origin of replication of simian virus 40 DNA lies within the A + T-rich region. Deletion of only a few bases into the adenine-thymine (AT) stretch results in a DNA template which is defective for replication both in vivo and in vitro (B. Stillman, R. D. Gerard, R. A. Guggenheimer, and Y. Gluzman, EMBO J. 4:2933-2939, 1985). In the present study, such deletion mutations have been reconstructed into a simian virus 40 genome containing an intact early promoter-enhancer region. The resulting mutants synthesized wild-type levels of T antigen, but were defective for replication and would not form plaques on CV-1 monkey cells. Replication-competent phenotypic revertants were selected after transfection of large quantities of the replication-defective viral DNAs into CV-1 cells. DNA sequence analysis showed that most of these revertants contained insertions or point mutations which partially regenerate the length of the AT stretch. These genotypic alterations were shown to be responsible for the revertant phenotype by replication analysis in vivo of subcloned revertant origin fragments. In general, our results emphasize the importance of the AT region to simian virus 40 origin function. However, one revertant retained the altered AT region but deleted six nucleotides upstream. Experiments using this mutant indicate that the 21-base-pair repeats identified as part of the early transcriptional promoter may compensate for defects in simian virus 40 DNA replication in vivo caused by mutations in the A + T-rich region when positioned at an appropriate distance from the core origin.

trans Activation of the simian virus 40 late transcription unit by T-antigen

1985 ◽  
Vol 5 (6) ◽  
pp. 1391-1399
Author(s):  
J Brady ◽  
G Khoury
Keyword(s):  
Gene Expression ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Transcription Unit ◽  
T Antigen ◽  
Late Gene ◽  
Antigen Binding ◽  
Late Gene Expression ◽  
Antigen Binding Site

We have investigated the role of simian virus 40 (SV40) T-antigen in the induction of late gene expression independent of its function in amplifying templates through DNA replication. Northern blot and S1 nuclease analyses showed that stimulation occurred at the transcriptional level. At least two template elements, the T-antigen-binding sites and the 72-base-pair repeats, appeared to be important for this induction. Using template mutants, we demonstrated that deletions within T-antigen-binding site II decreased T-antigen-mediated late gene expression approximately 10- to 20-fold. In addition, multiple point mutations within a single retained copy of the SV40 72-base-pair repeat decreased T-antigen-mediated late gene expression. Using in vivo competition studies, we demonstrated that competitor DNA fragments containing the SV40 control region (nucleotides 5171 through 272) quantitatively decreased SV40 late gene expression in COS-1 cells. In contrast, competition with a plasmid containing SV40 nucleotides 1 through 294 (which removes all of T-antigen-binding site I and half of site II) was much less efficient. Finally, we demonstrated that in vivo competition experiments employing competitor fragments distal to the T-antigen-binding sites within the late template region (SV40 nucleotides 180 through 2533) resulted in superinduction of late gene expression in COS-1 cells. This finding suggests that negative factors such as repressors or attenuators may modulate late SV40 gene expression before induction. Our results are consistent with a model in which induction of late gene expression involves an interaction of the SV40 origin region with DNA-binding proteins, one of which may be T-antigen. Activation of the SV40 late transcription unit may involve induction of the SV40 enhancer or removal of a repressor-like protein or both.

scholarly journals trans Activation of the simian virus 40 late transcription unit by T-antigen.

1985 ◽  
Vol 5 (6) ◽  
pp. 1391-1399 ◽  
Author(s):  
J Brady ◽  
G Khoury
Keyword(s):  
Gene Expression ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Transcription Unit ◽  
T Antigen ◽  
Late Gene ◽  
Antigen Binding ◽  
Late Gene Expression ◽  
Antigen Binding Site

We have investigated the role of simian virus 40 (SV40) T-antigen in the induction of late gene expression independent of its function in amplifying templates through DNA replication. Northern blot and S1 nuclease analyses showed that stimulation occurred at the transcriptional level. At least two template elements, the T-antigen-binding sites and the 72-base-pair repeats, appeared to be important for this induction. Using template mutants, we demonstrated that deletions within T-antigen-binding site II decreased T-antigen-mediated late gene expression approximately 10- to 20-fold. In addition, multiple point mutations within a single retained copy of the SV40 72-base-pair repeat decreased T-antigen-mediated late gene expression. Using in vivo competition studies, we demonstrated that competitor DNA fragments containing the SV40 control region (nucleotides 5171 through 272) quantitatively decreased SV40 late gene expression in COS-1 cells. In contrast, competition with a plasmid containing SV40 nucleotides 1 through 294 (which removes all of T-antigen-binding site I and half of site II) was much less efficient. Finally, we demonstrated that in vivo competition experiments employing competitor fragments distal to the T-antigen-binding sites within the late template region (SV40 nucleotides 180 through 2533) resulted in superinduction of late gene expression in COS-1 cells. This finding suggests that negative factors such as repressors or attenuators may modulate late SV40 gene expression before induction. Our results are consistent with a model in which induction of late gene expression involves an interaction of the SV40 origin region with DNA-binding proteins, one of which may be T-antigen. Activation of the SV40 late transcription unit may involve induction of the SV40 enhancer or removal of a repressor-like protein or both.

scholarly journals Functional analysis of the role of the A + T-rich region and upstream flanking sequences in simian virus 40 DNA replication.

1986 ◽  
Vol 6 (12) ◽  
pp. 4570-4577 ◽  
Author(s):  
R Gerard ◽  
Y Gluzman
Keyword(s):  
Dna Replication ◽  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Rich Region ◽  
Viral Dnas ◽  
Origin Function

One boundary of the minimal origin of replication of simian virus 40 DNA lies within the A + T-rich region. Deletion of only a few bases into the adenine-thymine (AT) stretch results in a DNA template which is defective for replication both in vivo and in vitro (B. Stillman, R. D. Gerard, R. A. Guggenheimer, and Y. Gluzman, EMBO J. 4:2933-2939, 1985). In the present study, such deletion mutations have been reconstructed into a simian virus 40 genome containing an intact early promoter-enhancer region. The resulting mutants synthesized wild-type levels of T antigen, but were defective for replication and would not form plaques on CV-1 monkey cells. Replication-competent phenotypic revertants were selected after transfection of large quantities of the replication-defective viral DNAs into CV-1 cells. DNA sequence analysis showed that most of these revertants contained insertions or point mutations which partially regenerate the length of the AT stretch. These genotypic alterations were shown to be responsible for the revertant phenotype by replication analysis in vivo of subcloned revertant origin fragments. In general, our results emphasize the importance of the AT region to simian virus 40 origin function. However, one revertant retained the altered AT region but deleted six nucleotides upstream. Experiments using this mutant indicate that the 21-base-pair repeats identified as part of the early transcriptional promoter may compensate for defects in simian virus 40 DNA replication in vivo caused by mutations in the A + T-rich region when positioned at an appropriate distance from the core origin.

BMP9 Can Induce Schwann Cells Expressing Simian Virus 40 T Antigen to Differentiate into Fat and Bone In Vivo and In Vitro

2021 ◽  
Vol 23 (2) ◽  
pp. 108-116
Author(s):  
Rui-Fang Li ◽  
Guo-Xin Nan ◽  
Dan Wang ◽  
Chang Gao ◽  
Juan Yang ◽  
...  
Keyword(s):  
Schwann Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen

Sequential transcription-translation of simian virus 40 by using mammalian cell extracts

1981 ◽  
Vol 1 (10) ◽  
pp. 919-931
Author(s):  
C L Cepko ◽  
U Hansen ◽  
H Handa ◽  
P A Sharp
Keyword(s):  
Transcription Initiation ◽  
Simian Virus 40 ◽  
Restriction Enzymes ◽  
Simian Virus ◽  
T Antigen ◽  
Coding Regions ◽  
Cell Extracts ◽  
Initiation Of Translation

Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980) were tested for their efficiency and fidelity in directing protein synthesis in reticulocyte lysates. Simian virus 40 deoxyribonucleic acid (DNA), cleaved by various restriction endonucleases, was used as the template. Successful translation of the small tumor antigen t, as well as the capsid proteins VP1, VP2, and VP3, was detected by immunoprecipitation analysis. Although no synthesis of large T antigen was detected, use of this technology allows detection of large T synthesis resulting from the correct splicing of as little as 0.2% of the in vitro RNA transcripts, making it ideal for use as an in vitro splicing assay. Transcripts synthesized in vitro were used as messages at least as efficiently as were viral messenger RNA's (mRNA's) synthesized in vivo; and in the case of small t, there was more efficient translation of small t mRNA synthesized in vitro than of small t mRNA synthesized in vivo. The transcripts that served as mRNA's for the various polypeptides were identified by using the following two criteria. (i) The sensitivity of synthesis of a given protein to digestion of the template DNA with restriction enzymes allowed the localization of the promoter and coding regions. (ii) Translation of size-fractionated RNA allowed confirmation of the transcript-mRNA assignments. With these techniques we found that VP2, VP3 and, in some cases, VP1 synthesis resulted from the initiation of translation at internal AUG codons. In fact, families of polypeptides were produced by initiation of translation at AUG codons within sequences coding for VP1 and T, presumably as a result of transcription initiation events that generated 5' ends immediately upstream from these AUGs. Application of this technology for the identification of coding regions within cloned DNA fragments is discussed.

scholarly journals Long-term testosterone treatment prevents gonadal and adrenal tumorigenesis of mice transgenic for the mouse inhibin-alpha subunit promoter/simian virus 40 T-antigen fusion gene

2000 ◽  
Vol 166 (1) ◽  
pp. 77-85 ◽  
Author(s):  
◽  
J Kero ◽  
T Paukku ◽  
I Huhtaniemi
Keyword(s):  
Fusion Gene ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Alpha Subunit ◽  
Adrenal Tumors ◽  
Microscopical Examination ◽  
Cell Hyperplasia ◽  
Testosterone Treatment

We have developed a transgenic (TG) mouse model for tumorigenesis of gonadal somatic cells using a 6 kb fragment of the mouse inhibin-alpha subunit promoter (Inh-alpha) fused with the simian virus 40 T-antigen (Tag) coding sequence. Gonadal tumors, of Leydig or granulosa cell origin, develop in the TG mice with 100% penetrance by the age of 5-8 months. Conspicuously, if the mice are gonadectomized, they develop adrenal tumors. Gonadal and adrenal tumorigenesis in these mice seem to be gonadotropin dependent. On the other hand, testosterone stimulates the proliferation of a cell line (C alpha 1) established from one of the adrenal tumors. The purpose of the present study was therefore to investigate further whether testosterone affects the growth of these gonadal and adrenal tumors in vivo. Two experimental models were used: (1) Tag TG/hypogonadotropic (hpg) double mutant mice and (2) castrated Tag TG mice. Both were treated between 1-2 and 7-8 months of age with Silastic rods (length 2 cm) containing testosterone. None of the control or testosterone-treated Tag/hpg mice developed gonadal or adrenal tumors. The castrated Tag TG mice displayed, upon microscopical examination, early stages of adrenal tumors, whereas those receiving testosterone did not show such changes. Testosterone increased the weights of gonads in the Tag/hpg mice, and those of uteri and seminal vesicles in both groups. In contrast, the adrenal weights were significantly reduced in both groups by testosterone treatment. Gonadal histology of the testosterone-treated mice showed hyperplasia of testicular Leydig cells and ovarian stroma. Spermatogenesis was induced by testosterone in the Tag/hpg mice. Adrenal histology of the testosterone-treated animals demonstrated the disappearance of the X-zone. Serum levels of FSH in testosterone-treated Tag/hpg mice were significantly increased, while those of serum LH were decreased. In conclusion, the present result indicate that the suppression of gonadotropins by testosterone implants in castrated Inh-alpha/Tag TG mice prevents the tumorigenesis of their adrenals. In intact Tag/hpg mice, testosterone implants were not able to induce gonadal or adrenal tumorigenesis. Although testosterone treatment was able to induce interstitial cell hyperplasia in gonads of the Inh-alpha/Tag mice, direct gonadotropin action is responsible for gonadal and adrenal tumorigenesis.

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