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At least 901 records · Page 50Linked to original sources
PubMed1990

Pseudorabies virus immediate-early gene overlaps with an oppositely oriented open reading frame: characterization of their promoter and enhancer regions.

The immediate-early (IE) gene of pseudorabies virus (PRV) has recently been sequenced for two virus strains. To investigate IE gene regulation and to examine the genome segment reported to encode latency-related transcripts in opposite polarity to the IE gene, sequence analysis has been extended by 5 kb from each end of the IE gene. The IE promoter (P1) was found to be more complex than previously recognized: it consisted of nine imperfect repeats, each containing five to six different consensus elements for transcription factor binding. A second promoter (P2) was discovered downstream of the IE gene. It contained numerous octamer consensus sequences (ATGCAAAT) and recognition sites for transcription factor Sp1; specific binding of nuclear proteins to four Sp1 sites was detected. An open reading frame (ORF3) bordering on P2 was identified, oriented antiparallel to the IE gene. Potential enhancer elements (E3 and E4) were isolated by the enhancer trap technique. Linked to P1 and a CAT indicator gene, E3 acted as an enhancer and E4 as a silencer. The PRV IE gene product repressed transcription from its own promoter and activated the SV40 early promoter. The transactivating virion protein Vmw65 of HSV1 had an opposite effect on these promoters.

Base Sequence↗
PubMed1989

Enzymatic approaches to probing of RNA secondary and tertiary structure.

To make strong statements about possible tertiary structure or the relative stability of regions of secondary structure, the structure-probing experiments must go further than single-hit reactions. Some elements of the environment of the RNA molecule must be altered systematically. Knowledge of the effects of ions or other interacting factors on the activity or physical parameters (e.g., NMR and melting cooperativity) of the RNA help in experimental design. For example, the copious work on tRNA(Phe) compared the crystal and solution structures and allowed the direct correlation of Mg2+ stabilization of the tertiary structure of that molecule. Figure 3 demonstrates that pre-tRNA(Leu-3) responds to Mg2+ depletion in the same manner as detected by the appearance of highly sensitive RNase cleavage sites in the D and T psi C loops. Similar experiments titrating polyamine concentrations suggested that secondary structure was more efficiently stabilized by polyamines than by Mg2+. The variation of Mg2+ concentrations has been used to gain additional information about other RNA structures. Others have used protein-RNA interactions to approach the question of the functional structure of a RNA (for examples, see Ref. 3). Thus, the ideal parameters to choose would be those known to affect the function of the RNA. The variation of Mg2+ and polyamine concentrations would minimally suggest regions of greater or lesser secondary or tertiary structure stability.

Base Sequence↗
PubMed1987

Lac repressor is a transient gene-activating protein.

We show, using a combination of methods, that contrary to the usual view, lac repressor increases, by more than 100-fold, the initial binding of RNA polymerase to E. coli lac UV5 promoter DNA. Kinetic studies revealed that the repressor acts to block the isomerization step in transcription initiation. When IPTG, a gratuitous inducer, is added, formation of open complex and productive transcription proceed. Because of the large increases in the binding constant, at low polymerase concentrations the presence of lac repressor (and then inducer) actually increases the rate of the first round of productive transcription, thus allowing the system to respond rapidly to the release of repression. This dual role of stabilization of a pretranscriptional complex coupled with blockage of transcription initiation may be a more general model for genetic regulation than that provided by the concept of simple repression.

DNA, Bacterial↗
PubMed1987

Identification and purification of a human lymphoid-specific octamer-binding protein (OTF-2) that activates transcription of an immunoglobulin promoter in vitro.

The octamer sequence 5'-ATGCAAAT, in either orientation, serves as an upstream element in a variety of promoters and also occurs as a modular enhancer element. It is of particular interest in immunoglobulin genes since it is found in the upstream regions of all heavy and light chain promoters and in the heavy chain enhancer, both of which are known to be necessary for cell-specific expression. We report here the chromatographic separation of ubiquitous and B cell-specific octamer-binding proteins. The B cell factor was purified to homogeneity using affinity chromatography and consists of three peptides of 62, 61, and 58.5 +/- 1.5 kd. Each of the polypeptides was renatured after SDS-PAGE and shown to bind to the octamer sequence. The specific DNA binding activity of the pure B cell-specific factor was indistinguishable from that of the affinity-purified ubiquitous factor. This B cell-specific octamer-binding factor, in pure form, activated transcription from a kappa light chain promoter in vitro, thus demonstrating that it is indeed a B cell-specific transcription factor for this gene. In addition to the ubiquitous and B cell-specific octamer-binding factors, we identified several additional proteins, one of which is B cell-specific, that interact with the kappa promoter.

B-Lymphocytes↗
PubMed1988

Sequence requirements for nucleolar localization of human T cell leukemia virus type I pX protein, which regulates viral RNA processing.

The posttranscriptional regulator (p27x-III) of human T cell leukemia virus type I (HTLV-I) is located predominantly in the cell nucleolus. A highly basic amino-terminal sequence (NH2-Met-Pro-Lys-Thr-Arg-Arg-Arg-Pro-Arg-Arg-Ser-Gln-Arg-Lys-Arg-Pro-Pro -Thr- Pro) in this protein, when fused to the amino termini of beta-galactosidase and p40x of HTLV-I, acts as an autonomous signal capable of directing the hybrid proteins to the cell nucleolus.

Amino Acid Sequence↗