Showing papers in "Genes & Development in 1988"

Isolation of a recombinant copy of the gene encoding C/EBP.

Abstract: In two previous studies we described the properties of a heat-stable DNA-binding protein present in rat liver nuclei. This protein, hereafter termed C/EBP, is capable of selective binding to the CCAAT homology of several viral promoters (Graves et al. 1986), as well as the core homology common to many viral enhancers (Johnson et al. 1987). We now report the isolation of a recombinant clone of the gene that encodes C/EBP. Expression of the clone in bacterial cells yields a protein that binds in vitro to both the CCAAT homology and the enhancer core homology, providing conclusive evidence that a single gene product accounts for both binding activities. By examining the properties of protease-derived fragments of C/EBP, we have localized its DNA-binding domain to a 14-kD fragment. A 60-amino-acid segment located within the DNA-binding domain of C/EBP bears sequence similarity to the products of the myc and fos oncogenes.

Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression.

Abstract: Mature virions of herpes simplex virus type 1 contain an activating factor that primes transcription from the five virally encoded immediate early (IE) genes. This activator is specified by a 65-kD polypeptide termed VP16. The action of VP16 is mediated through cis-regulatory elements located in regions adjacent to each IE gene. Although VP16 is normally introduced into cells by infecting virions, its trans-activating function can also be observed by cotransfecting cells with a plasmid that encodes VP16 along with a reporter gene driven by IE cis-regulatory sequences. We have used such an assay to examine the function of mutant forms of VP16. Our results provide tentative identification of two domains of VP16 that are crucial to its role in the induction of IE gene expression. One domain is located within the carboxy-terminal 78 amino acids of VP16 and is characterized by its acidity. Another domain, located in a more amino-terminal region of the protein, appears to tailor the specificity of VP16 for IE genes. According to the results presented in this and the accompanying paper, we predict that VP16 achieves IE gene specificity via protein: protein, rather than protein: DNA, interaction.

The POU domain: a large conserved region in the mammalian pit-1, oct-1, oct-2, and Caenorhabditis elegans unc-86 gene products

Abstract: The POU domain1 (pronounced 'pow') is a highly charged 155–162-amino-acid (aa) region of sequence similarity contained within three mammalian transcription factors. Pit-1 (ref. 2), Oct-1 (ref. 3) and Oct-2 (ref. 4), and the product of the nematode gene unc-86 (ref. 5) which is involved in determining neural cell lineage. This domain consists of two subdomains, a C-terminal homoeo domain and an N-terminal POL -specific region separated by a short nonconserved linker; the sequence relationship shows that the POU homoeo domains form a distinct POU-related family. In the ubiquitous and lymphoid-specific octamer-motif binding proteins Oct-1 and Oct-2, the POU domain is sufficient for sequence-specific DNA binding3,4. Homoeobox domains contain a helix-turn-helix DNA-binding motif6,7, first identified in bacterial repressers8. The helix-turn-helix region of the POU domain is important for DNA binding3,9 and, in other classes of homoeo-containing proteins, the entire homoeo domain is sufficient for DNA binding10–12; thus the new POU-specific region could be involved in other functions such as protein–protein interactions. Nevertheless, we show here that in fact the POU domain is a novel bipartite DNA-binding structure in which the POU homoeo and POU-specific regions form two subdomains that are both required for DNA binding but are held together by a flexible linker.

Trans-acting protein factors and the regulation of eukaryotic transcription: lessons from studies on DNA tumor viruses.

Abstract: Our knowledge of the mechanisms that regulate transcription in higher eukaryotic cells has increased enormously during the past 2 years. Earlier studies, using a combination of in vitro mutagenesis and DNA-mediated gene transfer, identified two distinct types of cis-acting regulatory sequences: promoters, which are located close to the initiation site and act in a position-dependent fashion, and enhancers, which can be located far from the initiation site and act in a position- and orientation-independent fashion. Promoters can be subdivided into proximal elements, including the cap site itself and the TATA box, which is involved in fixing the site of initiation, and distal elements, which can be spread over several hundred base pairs. It is now clear that many promoters, particularly those of 'housekeeping' genes, lack TATA boxes and are instead composed of GC-rich elements that are often located within methylation-free islands (Bird 1986). Transcription controlled by this latter class of promoters often initiates at multiple sites. Many enhancer elements, for example, those of the immunoglobulin, insulin, and elastase genes, impose tissue-specific expression on adjacent promoters. These cis-acting elements operate by interacting with protein factors, many of which have now been identified by gel retardation and footprinting assays and some of which have been purified to homogeneity. In a few cases the corresponding genes have been cloned. In many of these studies well-characterized cis-acting elements of viruses, particularly of the DNA tumor viruses, have played a major role. At the recent ICRF-sponsored

Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter

Abstract: Cytosine methylation within CpG dinucleotides has been implicated in the regulation of gene expression in vertebrates and, in some cases, has been shown to be causative in repression of transcription . We have examined whether methylation of CpG dinucleotides located within the binding site for a specific transcription factor, MLTF or USF, affects its binding to DNA. This HeLa cell factor binds to the adenovirus major late promoter (AdMLP), as well as endogenous cellular genes, and stimulates transcription in an in vitro assay. Synthetic oligonucleotides in which 5-methylcytosine replaces cytosine at specific sites were used to generate duplex DNAs, and the formation of complexes of these oligomers with MLTF was studied using a gel retardation assay. Methylation at a CpG site centrally located within the binding site strongly inhibited complex formation, whereas methylation at a site 6 bases away had no demonstrable effect. Methylation at the central site was also shown to inhibit specific transcription in vitro from the AdMLP. Methylation at the central site on only one strand caused a partial inhibition of binding, the effect being greater when the noncoding strand was methylated. The results indicate that in some cases, site-specific methylation may inhibit gene expression directly by blocking binding to DNA of factors required for optimal transcription. Along with other recent findings, they suggest an interplay between DNA methylation and transcription factors in the regulation of gene expression.

The ubiquitous octamer-binding protein Oct-1 contains a POU domain with a homeo box subdomain.

Abstract: The octamer motif ATGCAAAT is recognized indistinguishably by two mammalian transcription factors: one that is expressed ubiquitously and referred to here as Oct-1, and another, Oct-2, that is expressed in lymphoid cells. We report the cDNA cloning of the human oct-1 gene, which encodes Oct-1, by screening lambda gt11 recombinant phage in situ for octamer motif-specific DNA binding. One lambda gt11 recombinant expressed a beta-galactosidase-octamer-binding fusion protein with a DNA-binding specificity indistinguishable from human HeLa cell Oct-1 protein. As expected for a ubiquitously expressed protein, Oct-1 mRNA is expressed in all five human and two mouse cell lines tested. Polyclonal rabbit antiserum raised against the beta-galactosidase fusion protein shows that the DNA-binding domains of Oct-1 and Oct-2 proteins are related antigenically. Deletion analysis of the 743-amino-acid-long oct-1 open reading frame shows that the DNA-binding activity lies within a central highly charged domain of 160 amino acids. Comparison of the Oct-1 and Oct-2 sequences reveals that this domain is nearly identical between the two proteins. Highly similar domains are also present in the pituitary-specific transcription factor Pit-1 and the Caenorhabditis elegans unc-86 cell lineage gene product (see Herr et al. 1988). Within this shared POU (Pit-1, Oct-1 and Oct-2, unc-86) domain (pronounced 'pow') lie two subdomains: a POU-related homeo box and a POU-specific box. The Oct-1 protein is unique among the POU-related proteins and other homeo box proteins because it is expressed ubiquitously.

Cloning and expression of AP-2, a cell-type-specific transcription factor that activates inducible enhancer elements.

Abstract: Human AP-2 is a sequence-specific DNA-binding protein that interacts with inducible viral and cellular enhancer elements to stimulate transcription of selected genes. Here, we report the isolation and characterization of a human cDNA clone containing the entire protein-coding region of AP-2. The deduced primary amino acid sequence of AP-2 does not contain a domain resembling any previously identified DNA binding motif. However, an interesting feature of the AP-2 protein is a clustered arrangement of proline and glutamine residues that have been found recently within the activation domains of other transcription factors. Expression of the AP-2 clone in bacteria yields a protein that binds to DNA and activates transcription in vitro in a comparable manner to native human AP-2. Transfection of cDNA clones into Drosophila cells indicates that the AP-2 gene product can also activate gene expression in vivo in a DNA template-dependent manner. Expression of endogenous AP-2 is repressed in a hepatoma cell line and stimulated following retinoic-acid-induced differentiation of a human teratocarcinoma cell line. This indicates that AP-2 may be a transcription factor involved in the control of developmentally regulated gene expression.

Interferon-induced nuclear factors that bind a shared promoter element correlate with positive and negative transcriptional control.

Abstract: Human alpha- and beta-interferons (IFNs) stimulate rapid but transient increases in transcription from a set of previously quiescent genes. Protein synthesis is not required for initial stimulation, but duration of the response is limited to a few hours by a process requiring synthesis of new proteins. An IFN-stimulated response element (ISRE) was identified 5' to an inducible gene by deletion analysis and point mutagenesis, and sequence comparisons with other promoters defined the consensus element YAGTTTC(A/T)YTTTYCC. Two classes of IFN-inducible nuclear factors were found that bind to the ISRE. The most rapidly induced factor appeared without new protein synthesis, whereas a second factor required active protein synthesis for its appearance and maintenance. The kinetics of appearance and loss of these binding activities correlate with the activation and repression of IFN-stimulated genes. These different IFN-activated or induced factors may bind sequentially to the same essential promoter element to first increase and then repress transcription.

Splice site selection, rate of splicing, and alternative splicing on nascent transcripts.

Abstract: Based on ultrastructural analysis of actively transcribing genes seen in electron micrographs, we present evidence that pre-mRNA splicing occurs with a reasonable frequency on the nascent transcripts of early Drosophila embryo genes and that splice site selection may generally precede polyadenylation. The details of the process observed are in agreement with results from in vitro splicing systems but differ in the more rapid completion of in vivo splicing. For those introns that are removed cotranscriptionally, a series of events is initiated following 3' splice site synthesis, beginning with ribonucleoprotein (RNP) particle formation at the 3' splice site within 48 sec, intron loop formation within 2 min, and splicing within 3 min. The initiation of the process is correlated with 3' splice site synthesis but is independent of 5' splice site synthesis, the position of the intron within the transcript, and the age or length of the transcript. In some cases, introns are removed from the 5' end of a transcript before introns are synthesized at the 3' end, supporting a possible role for the order of transcription in splice site pairing. In general, our observations are consistent with the 'first-come-first-served' principle of splice site selection, although an observed example of exon skipping indicates that alternative splicing possibilities can be accommodated within this general framework.

Hormone-mediated repression: a negative glucocorticoid response element from the bovine prolactin gene.

Abstract: We have defined and characterized a region upstream of the bovine prolactin gene that confers repression by glucocorticoids. This 'negative glucocorticoid response element' (nGRE) contains multiple footprinting sites for purified glucocorticoid receptor protein between -51 and -562 bp. A strong consensus sequence for receptor binding within the nGRE has not yet been defined, but it is apparent that nGRE sequences differ from the GRE consensus elements that confer positive glucocorticoid regulation. Unlike 'positive' GREs, the nGRE enhances promoter activity in the absence of glucocorticoids or receptor, presumably through the action of a protein that binds in the same region and activates transcription. The hormone-receptor complex appears to negate this enhancement by competing or inactivating the second factor. As with positive GREs, nGRE sequences confer hormonal regulation upon linked heterologous promoters within various cell types; a 34-bp subfragment containing a single receptor binding site is sufficient for nGRE activity. We speculate that nGRE sequences might alter the structure of bound receptor, thereby preventing it from functioning as a positive regulator when bound at those sites.

Fos and Jun bind cooperatively to the AP-1 site: reconstitution in vitro.

Abstract: The protein products of the fos (Fos) and jun (Jun) proto-oncogenes have been shown to associate with a DNA element known as the transcription factor activator protein-1 (AP-1) binding site. Jun (previously known as the Fos-binding protein p39) and Fos form a protein complex in the nucleus. To investigate the nature of the association of Fos and Jun with the AP-1 site, and to determine the role of protein complex formation in DNA-binding, we have reconstituted the protein-protein and protein-DNA interactions in vitro using Fos and Jun synthesized in reticulocyte lysates. The Fos-Jun complex formed extremely rapidly in vitro and possessed similar, though not identical, chromatographic and sedimentation properties to the complex isolated from cell extracts. Jun exhibited a low level of AP-1 binding activity; however, this was evident only at high concentrations of DNA. Fos did not bind to the AP-1 site on its own; however, it acted cooperatively with Jun to give enhanced DNA-binding activity. The increased affinity of the Fos-Jun complex for DNA resulted from a stabilization of the protein-DNA complex. These data demonstrate a cooperative interaction between the protein products of two proto-oncogenes with a DNA element involved in transcriptional regulation.

In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage.

Abstract: We have used a recombinant bacteriophage that expresses the DNA-binding domain of C/EBP to optimize conditions for a screening technique that may facilitate the cloning of genes that encode sequence-specific DNA-binding proteins. The method relies on the expression of cDNA inserts in bacteriophage lambda gt11. Fusion protein adsorbed onto nitrocellulose filters is probed with radioactive, double-stranded DNA as a ligand. Two procedures greatly increase the level of binding between ligand and recombinant fusion protein. First, nitrocellulose filters are processed through a denaturation/renaturation regimen using 6 M guanidine hydrochloride. Second, synthetic DNA corresponding to the specific binding site is catenated extensively using DNA ligase. The combination of these procedures leads to remarkably strong detection signals. Specific DNA-binding signals can be detected on duplicate filters, and filters can be washed and reused by repeating the cycle of denaturation/renaturation.

Expression patterns of the homeo box-containing genes En-1 and En-2 and the proto-oncogene int-1 diverge during mouse development.

Abstract: We have compared the expression of the murine genes En-1,En-2, and in-1 during development by in situ hybridization. Expression of all three genes was first detected at 8.0 days in overlapping bands of the anterior neural folds. By 12.0 days the expression patterns diverged. En-1 and En-2 were expressed in a similar ring of cells in the central nervous system (CNS) at the midbrain/hindbrain junction. En-1 was also expressed de novo in two lateral stripes extending the length of the hindbrain and spinal cord, in the developing vertebral column, in two lateral stripes of dermatome-derived cells, and in the tail and limb buds. By 12.0 days int-1 expression showed little overlap with the En genes and could not be detected at later stages. At 15.5 days En gene expression was primarily limited to the midbrain/hindbrain in overlapping but nonidentical sets of differentiated cells. In the adult, En-1 and En-2 marked the same sets of cells in the pons, but En-2 alone was detected in the granular layer of the cerebellum. The results are consistent with int-1 and the En genes playing a role early in development in defining spatial domains in the CNS. Later in development the En genes may have an additional function in neurogenesis. En-1 expression in the developing pericordal tube suggests that it may also be involved in vertebral assembly.

Induction of proto-oncogene fos transcription through the adenylate cyclase pathway: characterization of a cAMP-responsive element.

Abstract: Transcription of proto-oncogene fos is induced by elevated levels of intracellular cAMP. We report that human c-fos promoter recombinants transfected into rat pheochromocytoma cells (PC12) and human choriocarcinoma cells (JEG-3) are induced by stimulation of adenylate cyclase and that this induction is diminished considerably in the mutant PC12 cell line A126-1B2, which is deficient in cAMP-dependent protein kinase II. An element centered at position -60 of the c-fos promoter, which encompasses a consensus cAMP response element (CRE), is sufficient to confer cAMP responsiveness to a herpes thymidine kinase/CAT fusion gene. The specific binding of a nuclear protein to the c-fos CRE can be competed by the somatostatin and alpha-chorionic gonadotropin (alpha-CG) promoter regions that contain CREs. Gel mobility shift assays with double-stranded oligonucleotides containing either the wild-type or mutated c-fos CRE sequence have demonstrated that binding occurs only to the wild-type CRE. The nuclear factor binding to the c-fos CRE is likely to be transcription factor CREB (CRE nuclear binding protein), because an affinity-purified 43-kD CREB isolated from PC12 cells binds efficiently in a DNA footprinting assay. Thus, regulation of the c-fos gene transcription appears to involve a mechanism common to many genes that respond to cAMP as a second message leading to cell growth and differentiation.

Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins.

Abstract: Heterogeneous nuclear RNA-ribonucleoprotein (hnRNP) particles can be efficiently purified by a specific, rapid, and mild procedure using monoclonal antibodies to hnRNP proteins. We report here on the detailed analysis of the protein composition of immunopurified hnRNP particles from human HeLa cells. By two-dimensional gel electrophoresis, immunopurified hnRNP particles contain at least 24 polypeptides in the range of 34,000-120,000 daltons. The abundant 30,000-40,000 dalton proteins, A, B, and C, described previously, are a subset of these polypeptides. The protein compositions of hnRNP particles found in the nucleoplasm fraction and in the chromatin-nucleolar fraction are very similar. Upon addition of the polyanion heparin, most of the major proteins remain associated in heparin-resistant particles, and only several, mostly minor, proteins dissociate. This provides an aid in the classification of the proteins and an additional criterion for the definition of hnRNP particle components. Chromatography on single-stranded DNA (ssDNA)-agarose in a heparin- and moderate or high salt (higher than 300 mM NaCl)-resistant manner suggests that most, if not all, of these proteins are single-stranded nucleic acid-binding proteins. We describe a general method for the large-scale purification of hnRNP proteins by affinity chromatography on ssDNA columns and its use for the production of new monoclonal antibodies to hnRNP proteins.

The B-cell-specific Oct-2 protein contains POU box- and homeo box-type domains.

Abstract: Transcription of promoters of immunoglobulin genes is controlled by an octanucleotide sequence element. The sequence of a cDNA encoding a B-cell-specific protein, Oct-2, has been determined. This protein specifically recognizes the octanucleotide element and is part of the previously identified NF-A2 family of proteins. The DNA-binding domain of Oct-2 is structurally related to the homeo box consensus and thus contains a potential helix-turn-helix sequence. Oct-2 also possesses a potential 'leucine zipper' domain, where four leucines are each separated by exactly seven residues. Comparisons of Oct-2 with protein Oct-1, which also recognizes the octanucleotide element but is constitutively expressed in all cell types, show high sequence conservation through the 60-residue DNA-binding domain, as well as an adjacent tract of 75 residues. The latter conserved region is also found in regulatory genes expressed in pituitary cells and nematodes and has been termed a POU box. Because two different cDNAs were isolated, it is proposed that the oct-2 gene is expressed as multiple mRNAs that vary in splicing patterns. Most interestingly, the oct-2 cDNA contains a second overlapping open reading frame, 278 residues in length, which might also specify a protein important for B-cell development.

A functional mRNA polyadenylation signal is required for transcription termination by RNA polymerase II.

Abstract: Polyadenylation of pre-mRNAs requires the conserved hexanucleotide AAUAAA, as well as sequences located downstream from the poly(A) addition site. The role of these sequences in the production of functional mRNAs was studied by analyzing a series of mutants containing deletions or substitutions in the SV40 early region poly(A) site. As expected, both a previously defined GU-rich downstream element and an AAUAAA sequence were required for efficient usage of the wild-type poly(A) addition site. However, when either of these elements was deleted, greatly increased levels of SV40-specific RNA were detected in the nuclei of transfected cells. Evidence is presented that this accumulation of RNA resulted from a failure of transcription termination, leading to multiple rounds of transcription of the circular templates. We conclude that the sequences required for efficient cleavage/polyadenylation of the SV40 early pre-mRNA also constitute an important element of an RNA polymerase II termination signal. A model proposing a mechanism by which the act of pre-mRNA 3' end formation is signaled to the elongating RNA polymerase, resulting in termination, is presented.

The human beta-globin gene 3' enhancer contains multiple binding sites for an erythroid-specific protein.

Abstract: We have shown that the minimal enhancer fragment present in the 3'-flanking region of the human beta-globin gene contains four regions that bind nuclear proteins in vitro. By using gel mobility shift and DNase I footprinting assays, we were able to show that each of these regions binds an erythroid-cell-specific nuclear factor which we name NF-E1. This factor is present in erythroid cells at different developmental stages of globin gene expression. The recognition sequence of this protein (A/C Py T/A ATC A/T Py) is also present in the intragenic enhancer and the promoter of the beta-globin gene as well as in the promoter of other erythroid-cell specific genes. In addition to NF-E1, each of the four binding regions interacts with at least one other protein factor.

Dwarf mice produced by genetic ablation of growth hormone-expressing cells.

Abstract: Fusion of the 310 bp located 5' of the rat growth hormone (GH) gene to the human GH structural gene resulted in somatotrope-specific expression in transgenic mice. Human GH transcripts were detected only in pituitaries of these mice, and immunocytochemical analyses revealed that this expression was limited to GH-expressing cell types. The rat GH 5' sequences were then used to direct the expression of diphtheria toxin to the GH-expressing cells of transgenic mice. A line of mice was established which lacks detectable levels of circulating GH. This deficiency resulted in dwarfism; transgenic animals grew only to half the size of nontransgenic littermates. Nearly all somatotropes were absent, as shown by GH immunostaining in the transgenic pituitaries. Prolactin (PRL)-producing lactotropes, thought to share a common cellular origin with somatotropes, were also reduced in numbers. A model for the lineal relationships between GH- and PRL-synthesizing cells is proposed.

Sp1 transcription factor binds DNA and activates transcription even when the binding site is CpG methylated.

Abstract: In vertebrates, a negative correlation between gene activity and CpG methylation of DNA, notably in the promoter region, is well established. Therefore, it is conceivable that differential binding of transcription factors to methylated versus unmethylated binding sites is crucial for gene activity. Since the consensus binding site of transcription factor Sp1 contains a central CpG, we have investigated the binding of Sp1 factor to unmethylated and synthetically CpG-methylated DNA. A strong Sp1 binding site was methylated on both strands at two CpG positions, located in the center and at the periphery of the recognition sequence. Our studies show that neither binding in vitro, nor transcription in vivo and in vitro are affected by methylation of the Sp1 binding site. We discuss the possibility that binding of Sp1 factor, which is often associated with promoters of housekeeping genes, prevents CpG methylation.

Expression of homeo box genes during mouse development: a review.

Abstract: The aim of this review is to summarize briefly w^hat is presently known about the spatial and temporal patterns of homeo box gene expression during mouse development. Since information about mouse homeo box genes is accumulating very rapidly, this review does not offer a complete compendium of facts about topics such as gene sequence and organization. Instead, we have tried to assemble rather fragmentary published and unpublished data about expression into a tentative framework which may provide some clues as to the function of these genes. To simplify our analysis, we have concentrated upon three different stages of mouse development; early presomite gastrula (7-7.75 days post coitum*), early somite neurula (8-8.5 days p.c), and a stage about twothirds through gestation (12.5 days p.c.) when most of the organ systems have been established but are still undergoing morphogenesis. Detailed accounts of the anatomy of these embryos and the overall process of mouse development can be found in Snell and Stevens (1966), Theiler (1972), Rugh (1968), and Hogan et al. (1985, 1986). The homeo box is a DNA sequence of about 180 bp, originally identified within the coding region of several Drosophila genes controlling embryonic development (for review, see Gehring 1987). It is generally accepted that the protein products of Drosophila homeo box genes act as sequence-specific DNA binding proteins, regulating gene expression. This suggestion is consistent with the nuclear localization of several Drosophila homeo box gene products (White and Wilcox 1984; Beachy et al. 1985; Carroll and Scott 1985; Di Nardo et al. 1985), with their reported in vitro DNA binding properties (Desplan et al. 1985), and with their predicted protein structure (Shepherd et al. 1984; Laughon et al. 1985). Drosophila homeo box genes constitute a rather variable gene family. DNA sequence comparisons, primarily of the homeo box region itself, indicate that classes of genes exist within which the genes share a more recent evolutionary origin than that shared by the homeo box gene family as a whole. The most extensive class, for which Antennapedia (Antp) is the prototype, contains several of the Drosophila homeotic genes (including Antp, Sex combs reduced, Deformed, and infraabdo-

Evidence of DNA: protein interactions that mediate HSV-1 immediate early gene activation by VP16.

Abstract: The viral genes first expressed upon lytic infection by herpes simplex virus type 1 (HSV-1) encode the five immediate early (IE) proteins. IE gene expression is potently and specifically induced by a virion protein termed VP16. Previous studies have shown that the activating properties of VP16 are IE gene specific and mediated by upstream regulatory elements common to each IE gene. Paradoxically, however, VP16 does not appear to be a sequence-specific DNA-binding protein. To understand the specificity of VP16 activation, we identified the cis-regulatory sequences of an IE gene that mediate VP16 response. Two distinct DNA sequence motifs enable the ICP4 gene to respond to VP16. Biochemical fractionation of nuclear proteins from uninfected cells revealed the existence of cellular proteins that bind directly to each of these VP16 cis-response elements. These observations, in concert with the identification of functional domains of the VP16 protein, lead to the hypothesis that VP16 achieves activation specificity via protein: protein, rather than protein: DNA, interactions.

5' splice site selection in yeast: genetic alterations in base-pairing with U1 reveal additional requirements.

Abstract: Using a strategy of compensatory nucleotide changes between yeast U1 and a 5' splice site, we have analyzed the contribution of base-pairing to the efficiency and fidelity of pre-mRNA splicing in vivo. Watson-Crick base-pairing interactions with U1 can be demonstrated at intron positions 1 and 5 but not at position 4. Moreover, restoration of the ability to pair with U1 is not sufficient to restore activity in the second step of splicing to intron position 1 mutants. Finally, in contrast to recent observations in mammalian systems, we find that the precise position of 5' splice site cleavage is not determined solely by the base-pairing interaction with U1. Rather, the presence of a G residue at position 5 is required for the correct localization of the nucleolytic event. Taken together, these results indicate that the demands for 5' splice site selection and utilization are more complex than a simple maximization of Watson-Crick interactions with U1.

Delta, a Drosophila neurogenic gene, is transcriptionally complex and encodes a protein related to blood coagulation factors and epidermal growth factor of vertebrates.

Abstract: Delta (D1) is required for normal segregation of the embryonic ectoderm into neural and epidermal cell lineages in Drosophila melanogaster. Loss-of-function mutations in D1 and other zygotic neurogenic loci lead to expansion of the neuroblast population at the expense of the dermoblast population within the ectoderm. Characterization of the transcriptional organization and maternal/embryonic expression within the chromosomal interval corresponding to D1 reveals that the locus encodes multiple transcripts: a minimum of two maternal transcripts, approximately 4.5 and 3.6 kb in length, and four zygotic transcripts, approximately 5.4 (two distinct species), 3.5, and 2.8 kb in length. These transcripts differ on the bases of differential splicing and differential polyadenylation site choice. The DNA sequence of a cDNA clone representing the predominant transcripts of the locus indicates that D1 encodes a transmembrane protein homologous to blood coagulation factors and epidermal growth factor. The relationship between coding sequences and transcript-specific exons within the locus suggests that D1 encodes multiple translational products.

A liver-specific factor essential for albumin transcription differs between differentiated and dedifferentiated rat hepatoma cells.

Abstract: We have identified and characterized two mutually exclusive nuclear proteins that interact with a single crucial element of the albumin promoter. One, albumin proximal factor (APF), is found only in liver or differentiated hepatoma cells and is probably identical to the liver-specific factor named HNF1, alpha 1TFB, or HP1-binding protein. The other, variant albumin proximal factor (vAPF), is present in dedifferentiated hepatoma cells as well as in somatic cell hybrids that show extinction of the expression of liver-specific proteins, including albumin. Reversion to the hepatic phenotype of either a dedifferentiated variant or an extinguished somatic hybrid clone is accompanied by loss of vAPF and reappearance of APF. These two proteins differ in their thermostability and in their molecular weight, while displaying identical sequence specificities. Both proteins interact with a homologous motif present in promoter regions of several other liver-specific genes. In vitro transcription assays, using a rat liver nuclear extract, indicate that the binding of APF to its target sequence is required for albumin transcription. These results suggest that a modification in the primary structure of a transcription factor is correlated with the differentiated state of the hepatic cell.

Changes in histone gene dosage alter transcription in yeast.

Abstract: Chromatin structure is believed to be important for a number of cellular processes, including transcription. However, the role of nucleosomes in transcription is not well understood. We have identified the yeast histone locus HTB1-HTB1, encoding histones H2A and H2B, as a suppressor of solo 8 insertion mutations that inhibit adjacent gene expression. The HTA1-HTB1 locus causes suppression either when present on a high-copy-number plasmid or when mutant. These changes in HTA1-HTB1 alter transcription of the genes adjacent to the 8 insertions. On the basis of this result, we have examined the effects of increased and decreased histone gene dosage for all four yeast histone loci. From the types of histone gene dosage changes that cause suppression of insertion mutations, we conclude that altered stoichiometry of histone dimer sets can alter transcription in yeast.

A group of genes required for pattern formation in the ventral ectoderm of the Drosophila embryo.

Abstract: Mutations in the genes spitz (spi), Star (S), single-minded (sim), pointed (pnt), rhomboid (rho) (all zygotic), and sichel (sic) (maternal), collectively called the spitz group, cause similar pattern alterations in ventral ectodermal derivatives of the Drosophila embryo. The cuticle structures lacking in mutant embryos normally derive from longitudinal strips of the ventro-lateral blastoderm. Defects were found in the median part of the central nervous system in whole-mount embryos stained with anti-HRP (horseradish peroxidase) antibodies. In addition, the nerve cells expressing the even-skipped protein appeared abnormally arranged. These results suggest that groups of cells from the same region, including both epidermal and neural precursor cells, require spitz-group gene activity for normal development. The members of the spitz group differ from one another: sim affects a more median strip of the ventral ectoderm than the other zygotic genes and pnt causes separation rather than deletion of pattern elements. As shown by pole cell transplantations, spi and S are also required for normal development of the female germ line, while sim, rho, and pnt appear to be exclusively zygotically expressed, and the maternal gene sic acts in the germ line autonomously. Some embryos produced by sic-homozygous females differentiate the spitz phenotype, others develop normally or die early. Of all the spitz-group genes, sim appears to have the most specific effect on the embryonic pattern. The significance of the spitz-group phenotypes for the dorso-ventral pattern formation is discussed.

Structural arrangements of transcription control domains within the 5'-untranslated leader regions of the HIV-1 and HIV-2 promoters.

Abstract: Promoter-proximal downstream regions of the human immunodeficiency viruses (HIV-1 and HIV-2) mediate the action of the viral transcription activator protein, Tat. We demonstrate here that the downstream domain of each virus interacts with two RNA polymerase II transcription factors. One of these, CTF/NF I, is a multifunctional protein associated previously with activation of transcription and DNA replication. The other cellular protein, designated LBP-1 (leader-binding protein-1), recognizes repeated elements within an extended region of DNA corresponding to part of the 5'-untranslated leader. Analysis of clustered point mutants in the HIV-1 leader for DNA-binding and transcription activity in vitro and in vivo suggests a role for LBP-1 as part of the basal promoter. A complex overlapping arrangement is observed between sequences required for the interaction of LBP-1 and CTF/NF I proteins and those defined previously for regulation by the HIV-1 Tat protein.

In vitro transcription of the Drosophila engrailed gene.

Abstract: An enzyme system that accurately initiates transcription of the engrailed gene has been prepared from Drosophila embryos. The system has been separated chromatographically into two fractions, both of which are required for specific engrailed transcription. DNase footprint and competition analysis detected at least two sequence-specific DNA-binding proteins in one of these two fractions. Together, these proteins bind to eight regions within 400 bp of the transcription initiation sites. Most of the regions containing these binding sites are required for manimal engrailed transcription in vitro. In addition, a region downstream from the initiation sites and within the first 40 residues of the transcription unit is essential for transcription. Transient in vivo expression assays indicated that these same upstream and downstream sequences are required for transcription in Drosophila tissue culture cells.

From DNA to form: the achaete-scute complex.

Abstract: The large bristles of flies (macrochaetes) are sense organs that form at precise locations on the thorax and the head, so much so that each has been given a name. The first scute mutant, a fly that lacked a few bristles, was discovered in 1916. As more mutations were found, it was observed that different alleles removed different subsets of bristles. A few of the new mutations affected a subset of bristles totally different from the subset elimi­ nated by the original scute mutation, sc^. Such muta­ tions fully complement sc^ and therefore were assumed to define a new gene named achaete. Early investigators were struck by the fact that each scute allele removed a specific subset of bristles, and they set out to understand whether and how the struc­ ture of the gene could explain such specificity. One theory, originating with a group of Russian genetists (who isolated many scute mutations in the late 1920s, after Miiller discovered that X-rays are mutagenic) held that scute contained distinct subgenes, each responsible for the development of one or a few bristles (Serebrowsky and Dubinin 1930; Agol 1931; Dubinin 1932, 1933). However, western geneticists asserted that the specificity was not in scute, but resulted from regional differences in the epidermis and/or involved the genetic system as a whole (Goldschmidt 1931, 1938; Sturtevant and Schultz 1931; Child 1935). At the heart of the disagreement lay the problem of the divisibility of the gene. This was understood clearly by H. Miiller, who had worked with both sides. He dem­ onstrated that the chromosome segment affected by scute mutations contains at least three adjacent genes, two of which {achaete and scute) are essential for the formation of bristles, while the third [lethal of scute) is essential for viability (Miiller and Prokofyeva 1935). Yet, in conclusion to an outstanding piece of work that in­ volved the feat of planning and bringing to fruition 29 consecutive crosses, he admitted that there is no way to decide whether this region may be further subdivided into elements that specify individual bristles, or whether the origin of the specificity must be found else­ where (Raffel and Miiller 1940). Thus, a deadlock had been reached in 1940, with no prospect of a solution. Yet, as we shall see, it now appears that both theories contain an element of truth and reflect two different aspects of a finely engineered piece of genetic ma­ chinery.