scholarly journals DNA sequence requirements for transcriptional initiator activity in mammalian cells.

1994 ◽  
Vol 14 (1) ◽  
pp. 116-127 ◽  
Author(s):  
R Javahery ◽  
A Khachi ◽  
K Lo ◽  
B Zenzie-Gregory ◽  
S T Smale
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Tata Box ◽  
Start Site ◽  
Sequence Element ◽  
Synthetic Promoters ◽  
Sequence Requirements

A transcriptional initiator (Inr) for mammalian RNA polymerase II can be defined as a DNA sequence element that overlaps a transcription start site and is sufficient for (i) determining the start site location in a promoter that lacks a TATA box and (ii) enhancing the strength of a promoter that contains a TATA box. We have prepared synthetic promoters containing random nucleotides downstream of Sp1 binding sites to determine the range of DNA sequences that convey Inr activity. Numerous sequences behaved as functional Inrs in an in vitro transcription assay, but the Inr activities varied dramatically. An examination of the functional elements revealed loose but consistent sequence requirements, with the approximate consensus sequence Py Py A+1 N T/A Py Py. Most importantly, almost every functional Inr that has been described fits into the consensus sequence that we have defined. Although several proteins have been reported to bind to specific Inrs, manipulation of those elements failed to correlate protein binding with Inr activity. The simplest model to explain these results is that all or most Inrs are recognized by a universal binding protein, similar to the functional recognition of all TATA sequences by the same TATA-binding protein. The previously reported proteins that bind near specific Inr elements may augment the strength of an Inr or may impart transcriptional regulation through an Inr.

DNA sequence requirements for transcriptional initiator activity in mammalian cells

1994 ◽  
Vol 14 (1) ◽  
pp. 116-127
Author(s):  
R Javahery ◽  
A Khachi ◽  
K Lo ◽  
B Zenzie-Gregory ◽  
S T Smale
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Tata Box ◽  
Start Site ◽  
Sequence Element ◽  
Synthetic Promoters ◽  
Sequence Requirements

A transcriptional initiator (Inr) for mammalian RNA polymerase II can be defined as a DNA sequence element that overlaps a transcription start site and is sufficient for (i) determining the start site location in a promoter that lacks a TATA box and (ii) enhancing the strength of a promoter that contains a TATA box. We have prepared synthetic promoters containing random nucleotides downstream of Sp1 binding sites to determine the range of DNA sequences that convey Inr activity. Numerous sequences behaved as functional Inrs in an in vitro transcription assay, but the Inr activities varied dramatically. An examination of the functional elements revealed loose but consistent sequence requirements, with the approximate consensus sequence Py Py A+1 N T/A Py Py. Most importantly, almost every functional Inr that has been described fits into the consensus sequence that we have defined. Although several proteins have been reported to bind to specific Inrs, manipulation of those elements failed to correlate protein binding with Inr activity. The simplest model to explain these results is that all or most Inrs are recognized by a universal binding protein, similar to the functional recognition of all TATA sequences by the same TATA-binding protein. The previously reported proteins that bind near specific Inr elements may augment the strength of an Inr or may impart transcriptional regulation through an Inr.

Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells

1985 ◽  
Vol 5 (4) ◽  
pp. 619-627
Author(s):  
M Montoya-Zavala ◽  
J L Hamlin
Keyword(s):  
Dna Sequence ◽  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Chinese Hamster Cells ◽  
Reductase Gene ◽  
Reductase Domain

We have isolated overlapping recombinant cosmids that represent 150 kilobases of contiguous DNA sequence from the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). This sequence includes the 25-kilobase dihydrofolate reductase gene and an origin of DNA synthesis. Eight cosmids that span this domain have been utilized as radioactive hybridization probes to analyze the similarities among the dihydrofolate reductase amplicons in four independently derived methotrexate-resistant Chinese hamster cell lines. We have observed no significant differences among the four cell lines within the 150-kilobase DNA sequence that we have examined, except for polymorphisms that result from the amplification of one or the other of two possible alleles of the dihydrofolate reductase domain. We also show that the restriction patterns of the amplicons in these four resistant cell lines are virtually identical to that of the corresponding, unamplified sequence in drug-susceptible parental cells. Furthermore, measurements of the relative copy numbers of fragments from widely separated regions of the amplicon suggest that all fragments in this 150-kilobase region may be amplified in unison. Our data show that in methotrexate-resistant Chinese hamster cells, the amplified unit is large relative to the dihydrofolate reductase gene itself. Furthermore, within the 150-kilobase amplified consensus sequence that we have examined, significant rearrangements do not seem to occur during the amplification process.

scholarly journals Striking similarities between the regulatory mechanisms governing yeast mating-type genes and mammalian major histocompatibility complex genes.

1991 ◽  
Vol 11 (8) ◽  
pp. 4228-4234 ◽  
Author(s):  
J D Weissman ◽  
D S Singer
Keyword(s):  
Major Histocompatibility Complex ◽  
Mhc Class I ◽  
Dna Sequence ◽  
Mating Type ◽  
Mammalian Cells ◽  
Regulatory System ◽  
Class I ◽  
Major Histocompatibility ◽  
Sequence Element ◽  
Histocompatibility Complex

Expression of a mammalian major histocompatibility complex (MHC) class I gene is in part regulated by a silencer DNA sequence element which binds a complex of silencer factors. This negative regulatory system is shown to be strikingly similar to the yeast alpha 2 mating-type repression system. A moderate DNA sequence homology exists between the MHC class I silencer DNA element and the yeast alpha 2 operator. Mammalian silencer factors specifically bind to the yeast alpha 2 operator DNA and also specifically interact with a yeast alpha 2-binding protein. Furthermore, the alpha 2 operator functions as a silencer element in mammalian cells when placed upstream of a MHC class I promoter.

scholarly journals DNA Sequence and Functional Analysis of Homologous ARS Elements of Saccharomyces cerevisiae and S. carlsbergensis

Genetics ◽  
1999 ◽  
Vol 152 (3) ◽  
pp. 943-952
Author(s):  
James F Theis ◽  
Chen Yang ◽  
Christopher B Schaefer ◽  
Carol S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Chromosomal Dna ◽  
Functional Elements ◽  
Sequence Comparisons ◽  
Cis Acting ◽  
Homologous Sequences ◽  
Ars Elements

Abstract ARS elements of Saccharomyces cerevisiae are the cis-acting sequences required for the initiation of chromosomal DNA replication. Comparisons of the DNA sequences of unrelated ARS elements from different regions of the genome have revealed no significant DNA sequence conservation. We have compared the sequences of seven pairs of homologous ARS elements from two Saccharomyces species, S. cerevisiae and S. carlsbergensis. In all but one case, the ARS308-ARS308carl pair, significant blocks of homology were detected. In the cases of ARS305, ARS307, and ARS309, previously identified functional elements were found to be conserved in their S. carlsbergensis homologs. Mutation of the conserved sequences in the S. carlsbergensis ARS elements revealed that the homologous sequences are required for function. These observations suggested that the sequences important for ARS function would be conserved in other ARS elements. Sequence comparisons aided in the identification of the essential matches to the ARS consensus sequence (ACS) of ARS304, ARS306, and ARS310carl, though not of ARS310.

scholarly journals Striking similarities between the regulatory mechanisms governing yeast mating-type genes and mammalian major histocompatibility complex genes

1991 ◽  
Vol 11 (8) ◽  
pp. 4228-4234
Author(s):  
J D Weissman ◽  
D S Singer
Keyword(s):  
Major Histocompatibility Complex ◽  
Mhc Class I ◽  
Dna Sequence ◽  
Mating Type ◽  
Mammalian Cells ◽  
Regulatory System ◽  
Class I ◽  
Major Histocompatibility ◽  
Sequence Element ◽  
Histocompatibility Complex

Expression of a mammalian major histocompatibility complex (MHC) class I gene is in part regulated by a silencer DNA sequence element which binds a complex of silencer factors. This negative regulatory system is shown to be strikingly similar to the yeast alpha 2 mating-type repression system. A moderate DNA sequence homology exists between the MHC class I silencer DNA element and the yeast alpha 2 operator. Mammalian silencer factors specifically bind to the yeast alpha 2 operator DNA and also specifically interact with a yeast alpha 2-binding protein. Furthermore, the alpha 2 operator functions as a silencer element in mammalian cells when placed upstream of a MHC class I promoter.

Saturation Mutagenesis of the TATA Box and Upstream Activator Sequence in the Haloarchaeal bop Gene Promoter

1999 ◽  
Vol 181 (8) ◽  
pp. 2513-2518 ◽  
Author(s):  
Nitin S. Baliga ◽  
Shiladitya DasSarma
Keyword(s):  
Transcription Start Site ◽  
Direct Evidence ◽  
Consensus Sequence ◽  
Gene Promoter ◽  
Tata Box ◽  
Saturation Mutagenesis ◽  
Start Site ◽  
Wild Type ◽  
Transcription Start ◽  
Transcriptional Terminator

ABSTRACT Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal bop promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS). The mutagenized bop promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeonHalobacterium sp. strain S9. Active promoters were isolated by screening transformants of an orange (Pum− bop) Halobacterium mutant for purple (Pum+ bop +) colonies on agar plates and analyzed for bop mRNA and/or bacteriorhodopsin content. Sequence analysis yielded the consensus sequence 5′-tyT(T/a)Ta-3′, corresponding to the promoter TATA box element 30 to 25 bp 5′ of the transcription start site. A putative UAS, 5′-ACCcnactagTTnG-3′, located 52 to 39 bp 5′ of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement of the TATA box and UAS for bop promoter activity.

Yeast and human TATA-binding proteins have nearly identical DNA sequence requirements for transcription in vitro

1990 ◽  
Vol 10 (8) ◽  
pp. 3859-3867
Author(s):  
C R Wobbe ◽  
K Struhl
Keyword(s):  
Dna Sequence ◽  
Consensus Sequence ◽  
Yeast Cells ◽  
Tata Element ◽  
Binding Factor ◽  
Transcription In Vitro ◽  
Sequence Requirements ◽  
Element Function

We have analyzed the DNA sequence requirements for TATA element function by assaying the transcriptional activities of 25 promoters, including those representing each of the 18 single-point mutants of the consensus sequence TATAAA, in a reconstituted in vitro system that depends on the TATA element-binding factor TFIID. Interestingly, yeast TFIID and HeLa cell TFIID were virtually identical in terms of their relative activities on this set of promoters. Of the mutated elements, only two had undetectable activity; the rest had activities ranging from 2 to 75% of the activity of the consensus element, which was the most active. In addition, mutations of the nucleotide following the TATAAA core strongly influenced transcriptional activity, although with somewhat different effects on yeast and HeLa TFIID. The activities of all these promoters depended upon TFIID, and the level of TFIID-dependent transcription in vitro correlated strongly with their activities in yeast cells. This suggests that the in vivo activities of these elements reflect their ability to functionally interact with a single TATA-binding factor. However, some elements with similar activities in vitro supported very different levels of transcriptional activation by GAL4 protein in vivo. These results extend the degree of evolutionary conservation between yeast and mammalian TFIID and are useful for predicting the level of TATA element function from the primary sequence.

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