An RNA-binding protein gene (rbpl) from Drosophila melanogaster, encoding an RNA recognition motif and an Arg-Ser rich (RS) domain, has been characterized. The predicted amino acid sequence of rbpl is similar to those of the human splicing factor ASF/SF2, the Drosophila nuclear phosphoprotein SRp55, and the Drosophila puff-associated protein B52. Northern and immunohistochemical analyses showed that rbpl is expressed at all stages in all tissues and that the RBP1 protein is localized to the nucleus. Consistent with a role in mRNA metabolism, indirect immunofluorescence reveals that the RBP1 protein colocalizes with RNA polymerase II on larval salivary gland polytene chromosomes. RBP1 protein made in Escherichia coli was tested for splicing activity using human cell extracts in which ASF has been shown previously both to activate splicing and to affect the choice of splice sites in alternatively spliced pre-mRNAs. In these assays, RBP1 protein, like ASF, is capable of both activating splicing and switching splice site selection. However, in each case, clear differences in the behavior of the two proteins were detected, suggesting that they have related but not identical functions. The general nuclear expression pattern, colocalization on chromosomes with RNA polymerase II, the similarity to ASF/SF2, SRp55, and B52, along with the effect on alternative splicing shown in vitro, suggest that rbpl is involved in the processing of precursor mRNAs.
The alpha-like and beta-like subunits of human hemoglobin are encoded by a small family of genes that are differentially expressed during development. Through the use of molecular cloning procedures, each member of this gene family has been isolated and extensively characterized. Although the alpha-like and beta-like globin genes are located on different chromosomes, both sets of genes are arranged in closely linked clusters. In both clusters, each of the genes is transcribed from the same DNA strand, and the genes are arranged in the order of their expressions during development. Structural comparisons of immediately adjacent genes within each cluster have provided evidence for the occurrence of gene duplication and correction during evolution and have led to the discovery of pseudogenes, genes that have acquired numerous mutations that prevent their normal expression. Recently, in vivo and in vitro systems for studying the expression of cloned eukaryotic genes have been developed as a means of identifying DNA sequences that are necessary for normal gene function. This article describes the application of an in vitro transcription procedure to the study of human globin gene expression.
We investigated the nucleotide sequence requirements of the adenovirus 2 late promoter when activated by either a trans-acting regulatory protein or a cis-acting enhancer element. Using deletion mutants in transient expression assays, we determined that the 5' limit of the region required for activation by a trans-acting regulatory protein, the adenovirus early region la gene product, and the simian virus 40 enhancer is the same in both 293 and HeLa cells. Surprisingly, the 3' limit of required sequences varied, depending on the mechanism of activation. Activation mediated by the early region la protein endogenous in 293 cells or produced after cotransfection of HeLa cells requires the region around the transcriptional start site, whereas activation brought about by an enhancer element in HeLa cells has no requirement for these sequences. Under no conditions tested did the simian virus 40 enhancer activate the late promoter in 293 cells, even when sequences sufficient for enhancer-mediated activation in HeLa cells, but not for early region la activation, were present. These results suggest the existence of at least two different mechanisms for positive regulation of promoter activity.A major factor in gene regulation is differential promoter activation. Gene expression can be controlled at the level of transcription initiation by a number of mechanisms, all of which require either cis-or trans-acting factors, or both. Transcriptional enhancers are an example of cis-acting regulatory squences. (see references 18 and 35, for reviews.) The prototype for this class of controlling element, the simian virus 40 (SV40) 72-base-pair (bp) repeated sequence, was first identified as a far upstream component of the early promoter of this virus (4, 23). However, the so-called 72-bp repeats were soon shown to have unprecedented properties. When DNA fragments containing these sequences were excised from viral DNA and joined to recombinant molecules containing various heterologous genes, it was found that expression of the linked gene was dramatically enhanced, in a manner essentially independent of the position and orientation of the viral DNA fragment (3, 49).Enhancers have since been identified in a number of viral genomes, including polyomavirus (13), retroviral long terminal repeats (20,37), adenovirus (26, 31) and bovine papillomavirus (41). More recently, controlling elements that behave like enhancers have been found in or near certain cellular genes, including immunoglobulins (2, 17, 51) and insulin and chymotrypsin (62). The enhancers described to date show various degrees of specificity, ranging from a slight species specificity in the case of the papovavirus and retroviral enhancers (37) to an almost absolute cell-type specificity in the case of the immunoglobulin enhancers, which are functional only in lymphoid cells.Enhancers in turn interact with factors in the cell nucleus (55). Such trans-acting factors constitute a second category of transcriptional regulators. One of the best studied is the SV40 T antigen, whi...
A series of recombinants of adenovirus DNA fragments and pBR322 was used to test the transcriptional activity of the nine known adenovirus promoters in a cell-free extract. Specific initiation was seen at all five early promoters as well as at the major late promotor and at the intermediate promoter for polypeptide IX. The system failed to recognize the two other adenovirus promoters, which were prominent in vivo only at intermediate and late stages in infection. Microheterogeneity of 5' termini at several adenovirus promoters, previously shown in vivo, was reproduced in the in vitro reaction and indeed appeared to result from heterogeneous initiation rather than 5' processing. To test for the presence of soluble factors involved in regulation of nRNA synthesis, the activity of extracts prepared from early and late stages of infection was compared on an assortment of viral promoter sites. Although mock and early extracts showed identical transcription patterns, extracts prepared from late stages gave 5- to 10-fold relative enhancement of the late and polypeptide IX promoters as compared with early promoters.
ABSTRACr We have constructed a frameshift mutation in the simian virus 40 early region using a novel method of oligonucleotide-directed mutagenesis. The mutated DNA specifies an 84,000-dalton large tumor antigen that consists of =75,000 daltons encoded by the wild-type reading frame and 9,000 daltons, by the alternative reading frame (wild-type large tumor antigen is =82,000 daltons). The frameshifted carboxyl terminus of the protein bears a strong similarity to the same region of polyoma virus middle-sized tumor antigen. We have found that the mutant DNA is unable to replicate when introduced into permissive monkey cells and incapable of transforming nonpermissive mouse cells.Polyoma virus encodes three tumor (T) antigens-a small, a middle-sized, and a large-whereas simian virus 40 (SV40) apparently encodes only a large and a small T antigen (1). The polyoma middle-sized T antigen appears to be necessary and sufficient to bring about the transformation of cells in tissue culture. Recombinant plasmids that contain the promoter proximal part of the polyoma early region but lack the distal part (i.e., are unable to encode large T antigen) are able to transform rat cells in tissue culture with high efficiency (2, 3). In addition, plasmids containing the information to encode only middle-sized T antigen transform rat cells with nearly the same efficiency, and to the same extent (i.e., ability to grow in soft agar), as does wild-type polyoma (4). However, very little is known about the biochemical function(s) of middle-sized T antigen. It is a 50-kilodalton (kDa) phosphoprotein, contains a tightly associated protein kinase activity (5-7), and is localized predominantly in the plasma membrane (8,9). Deletion mutations that affect the carboxyl terminus of middle-sized T antigen greatly reduce transformation efficiency (10). This region of the protein consists of an unusual string of six consecutive glutamic acid residues followed by an extremely hydrophobic region, which is responsible for the membrane localization of the protein and is also required for the transformation and associated protein kinase activities (11).Because SV40 does not encode a middle-sized T antigen, the ability of this virus to transform cells must reside with the large or the small T antigen. Mark and Berg (12) pointed out the existence of an apparently unused reading frame located near the 3' end of the SV40 early region. The amino acid residues encoded by it would be extremely hydrophobic (nearly 60% of the codons specify hydrophobic amino acids). Also, at the start of the region with two alternative reading frames, but in the large T-antigen reading frame, there is a stretch of six codons that encode consecutive acidic amino acids. This sequence arrangement bears an obvious and striking similarity to the sequence found in the polyoma middle-sized T antigen, suggesting that SV40 may contain the information to produce a "middle-sized T antiger.-"However,-noe exists icnthat second reading frame is used, either in lytic infection or in transforme...
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