scholarly journals 81. Self-Inactivating Lentiviral Vectors Exhibit Residual Promoter Activity in the Enhancer- and TATA Box-Deleted Long Terminal Repeat

2002 ◽  
Vol 5 (5) ◽  
pp. S29
Keyword(s):  
Long Terminal Repeat ◽  
Promoter Activity ◽  
Lentiviral Vectors ◽  
Terminal Repeat ◽  
Tata Box

Identification of transcriptional elements within the long terminal repeat of Rous sarcoma virus

1983 ◽  
Vol 3 (10) ◽  
pp. 1834-1845
Author(s):  
G M Gilmartin ◽  
J T Parsons
Keyword(s):  
Long Terminal Repeat ◽  
Virus Replication ◽  
Rous Sarcoma Virus ◽  
Regulatory Elements ◽  
Terminal Repeat ◽  
Tata Box ◽  
Transcriptional Regulatory Elements ◽  
Rous Sarcoma ◽  
Transcriptional Regulatory ◽  
Sarcoma Virus

Transcriptional regulatory elements within the Rous sarcoma virus long terminal repeat were examined by the construction of a series of deletions and small insertions within the U3 region of the long terminal repeat. The analysis of these mutations in chicken embryo cells and COS cells permitted the identification of important transcriptional regulatory elements. Sequences within the region 31 to 18 base pairs upstream of the RNA cap site (-31 to -18), encompassing a TATA box-like sequence, function in the selection of the correct site of transcription initiation and, in addition, augment the efficiency of transcription. These sequences are essential for virus replication. Sequences within the region -79 to -59, overlapping a CAAT box-like sequence, are not required for virus replication and have no obvious effect on viral RNA transcription in the presence of an intact TATA box. However, in mutants lacking a functional TATA sequence, mutations in this region serve to decrease the efficiency of correct transcriptional initiation events.

Generation of infectious Moloney murine leukemia viruses with deletions in the U3 portion of the long terminal repeat

1986 ◽  
Vol 6 (12) ◽  
pp. 4634-4640
Author(s):  
R Hanecak ◽  
S Mittal ◽  
B R Davis ◽  
H Fan
Keyword(s):  
Long Terminal Repeat ◽  
Transient Expression ◽  
Leukemia Virus ◽  
Chloramphenicol Acetyltransferase ◽  
Terminal Repeat ◽  
Tata Box ◽  
Moloney Murine Leukemia Virus ◽  
Chloramphenicol Acetyltransferase Gene ◽  
Leukemia Viruses ◽  
Murine Leukemia

Deletional analysis within the long terminal repeat (LTR) of Moloney murine leukemia virus (M-MuLV) was performed. By molecular cloning, deletions were made in the vicinity of the XbaI site at -150 base pairs (bp) in the U3 region, between the tandemly repeated enhancers and the TATA box. The effects of the deletions on LTR function were measured in two ways. First, deleted LTRs were fused to the bacterial chloramphenicol acetyltransferase gene and used in transient expression assays. Second, infectious M-MuLVs were generated by transfection of M-MuLV proviruses containing the deleted LTRs, and the relative infectivity of the mutant viruses was assessed by XC-syncytial assay. Most of the deleted LTRs examined showed relatively high promoter activity in the transient chloramphenicol acetyltransferase assays, with values ranging from 20 to 50% of the wild-type M-MuLV LTR. Thus, the sequences between the enhancers and the TATA box were not absolutely required for transient expression. However, infectivity of viruses carrying the same deleted LTRs showed more pronounced effects. Deletion of sequences from -195 to -174 bp reduced infectivity 20- to 100-fold. Deletion of sequences within the region from -174 to -122 bp did not affect infectivity, indicating that this region is dispensable. On the other hand, deletion of sequences from -150 to -40 bp reduced infectivity from 5 to 6 logs, although the magnitude of the reduction partly may have reflected threshold envelope protein requirements for positive XC assays. The reduced infectivity did not appear to result from a failure of proviral DNA synthesis or integration by the mutant. Thus, the infectivity measurements identified three functional domains in the region between the enhancers and the TATA box.

The long terminal repeat of an endogenous intracisternal A-particle gene functions as a promoter when introduced into eucaryotic cells by transfection

1984 ◽  
Vol 4 (10) ◽  
pp. 2128-2135
Author(s):  
K K Lueders ◽  
J W Fewell ◽  
E L Kuff ◽  
T Koch
Keyword(s):  
Long Terminal Repeat ◽  
Promoter Activity ◽  
Terminal Repeat ◽  
Flanking Sequence ◽  
Murine Sarcoma Virus ◽  
Sarcoma Virus ◽  
Mouse Cells ◽  
Moloney Murine Sarcoma Virus ◽  
Intracisternal A Particle ◽  
Murine Sarcoma

We describe experiments designed to determine whether an endogenous intracisternal A-particle (IAP) gene randomly selected from a mouse embryo library has the potential to be transcriptionally active. Assays for IAP gene transcription were done with permanently transformed rat cells and transiently transfected monkey and mouse cells. The rat cells, which had integrated IAP gene copies, contained IAP RNA. A start site within the IAP 5' long terminal repeat (LTR) was localized by S1 mapping. The promoter activity of the IAP LTR was also measured in cells 48 h after the introduction of recombinant plasmids in which bacterial chloramphenicol acetyl transferase (CAT) encoding sequences were under the control of the LTR. The IAP LTR promoted CAT activity in mouse and monkey cells. In mouse L-cells, the levels of CAT activity were 10 to 25% of those promoted by an analogous recombinant containing the Moloney murine sarcoma virus LTR as the promoter. In contrast to the Moloney murine sarcoma virus LTR, the IAP LTR was five- to eightfold more active in monkey cells than in mouse cells. The 5' and 3' LTRs were equally active, and promoter activity was dependent on having the orientation of the LTRs with respect to the CAT gene the same as their orientation with respect to the IAP gene. A 5'-flanking sequence containing a member of the highly repetitive R-sequence family increased CAT activity in COS cells 11-fold when present along with the LTR. Our results indicate that the LTR of an endogenous mouse IAP gene can function as an efficient promoter in heterologous as well as homologous cells.

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