Showing papers in "Genes & Development in 1989"

Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers.

Abstract: An activating transcription factor (ATF)-binding site (consensus sequence 5'-GTGACGTACAG-3') is a promoter element present in a wide variety of viral and cellular genes. The two best-characterized classes of genes that contain ATF sites are E1A-inducible adenoviral genes and cAMP-inducible cellular genes. Here, we report the isolation of eight ATF cDNA clones, each of which is derived from a separate gene. All ATF cDNA clones examined contain a leucine zipper motif and are significantly similar to one another only within this region. The leucine zipper region of ATF proteins is also similar to that of the AP-1/c-jun family of transcription factors, whose DNA-binding site differs from the ATF-binding site at a single position. DNA binding studies reveal two mechanisms for generating further diversity from the ATF proteins. First, some, but not all, combinations of ATF proteins form heterodimers that efficiently bind to DNA. Second, although all ATF proteins bind to the ATF site, their precise interactions with DNA differ from one another, as evidenced by methylation interference analysis. Our results help to explain how a single promoter element, an ATF site, can be present in a wide variety of promoters.

Novel phytochrome sequences in Arabidopsis thaliana: structure, evolution, and differential expression of a plant regulatory photoreceptor family

Abstract: Phytochrome is a plant regulatory photoreceptor that mediates red light effects on a wide variety of physiological and molecular responses. DNA blot analysis indicates that the Arabidopsis thaliana genome contains four to five phytochrome-related gene sequences. We have isolated and sequenced cDNA clones corresponding to three of these genes and have deduced the amino acid sequence of the full-length polypeptide encoded in each case. One of these proteins (phyA) shows 65-80% amino acid sequence identity with the major, etiolated-tissue phytochrome apoproteins described previously in other plant species. The other two polypeptides (phyB and phyC) are unique in that they have low sequence identity (approximately 50%) with each other, with phyA, and with all previously described phytochromes. The phyA, phyB, and phyC proteins are of similar molecular mass, have related hydropathic profiles, and contain a conserved chromophore attachment region. However, the sequence comparison data indicate that the three phy genes diverged early in plant evolution, well before the divergence of the two major groups of angiosperms, the monocots and dicots. The steady-state level of the phyA transcript is high in dark-grown A. thaliana seedlings and is down-regulated by light. In contrast, the phyB and phyC transcripts are present at lower levels and are not strongly light-regulated. These findings indicate that the red/far light-responsive phytochrome photoreceptor system in A. thaliana, and perhaps in all higher plants, consists of a family of chromoproteins that are heterogeneous in structure and regulation.

A gene with homology to the myc similarity region of MyoD1 is expressed during myogenesis and is sufficient to activate the muscle differentiation program.

Abstract: MyoD1 is a nuclear phosphoprotein that is expressed in skeletal muscle in vivo and in certain muscle cell lines in vitro; it has been shown to convert fibroblasts to myoblasts through a mechanism requiring a domain with homology to the myc family of proteins. The BC3H1 muscle cell line expresses skeletal muscle-specific genes upon exposure to mitogen-deficient medium, but does not express MyoD1 at detectable levels. To determine whether BC3H1 cells may express regulatory genes functionally related to MyoD1, a cDNA library prepared from differentiated BC3H1 myocytes, was screened at reduced stringency with the region of the MyoD1 cDNA that shares homology with c-myc. From this screen, a cDNA was identified that encodes a major open reading frame with 72% homology to the myc domain and basic region of MyoD1. The mRNA encoded by this MyoD1-related gene is expressed in skeletal muscle in vivo and in differentiated skeletal myocytes in vitro and is undetectable in cardiac or smooth muscle, nonmuscle tissues, or nonmyogenic cell types. During myogenesis, the MyoD1-related mRNA accumulates several hours prior to other muscle-specific mRNAs and therefore represents an early molecular marker for entry of myoblasts into the differentiation pathway. Transient transfection of 10T1/2 or 3T3 cells with the MyoD1-related cDNA is sufficient to induce myosin heavy-chain expression and to activate a reporter gene under transcriptional control of the muscle creatine kinase 5' enhancer, which functions only in differentiated myocytes. Expression of this cDNA in stably transfected 10T1/2 cells also leads to fusion and muscle-specific gene expression upon exposure to mitogen-deficient medium. Thus, the product of this MyoD1-related gene is sufficient to activate the muscle differentiation program and may substitute for MyoD1 in certain developmental situations. Together, these results suggest the existence of a family of myogenic regulatory genes that share a conserved motif with c-myc.

Identification of MRF4: a new member of the muscle regulatory factor gene family.

Abstract: We have identified a rat cDNA encoding MRF4, a new member of the muscle regulatory factor gene family that includes MyoD1, myogenin, and Myf-5. MRF4 encodes a predicted 27-kD protein that contains a conserved helix-loop-helix motif, which is a common feature of this gene family. Northern analyses indicate that MRF4 is expressed solely in skeletal muscle tissue but is not detected in most embryonic muscle cell lines. Transfection of MRF4 into C3H10T1/2 fibroblasts produces stable myogenic lineages at frequencies that are equal to or greater than those obtained when MyoD1 or myogenin are introduced into these cells. Expression of the MRF4 cDNA leads to expression of the endogenous MyoD1 and myogenin genes, although C3H10T1/2 cells expressing MyoD1 or myogenin cDNAs do not express MRF4. Interestingly, the endogenous MyoD1 and myogenin genes are negatively regulated by serum and by purified growth factors since MRF4-transfected C3H10T1/2 cells activate MyoD1 and myogenin expression only in mitogen-depleted, differentiation-induced muscle cultures. The myofiber-specific expression pattern of MyoD1 and myogenin in these cells suggests that the primary role for this muscle regulatory factor gene family may be in regulating specific terminal differentiation events that are crucial for normal skeletal muscle development.

Searching for pattern and mutation in the Drosophila genome with a P-lacZ vector.

Abstract: A P-element vector has been constructed and used to generate lines of flies with single autosomal P-element insertions. The lines were analyzed in two ways: (1) the identification of cis-acting patterning information within the Drosophila genome, as revealed by a lacZ reporter gene within the P element, and (2) the isolation of lethal mutations. We examined 3768 independent lines for the expression of lacZ in embryos and looked among these lines for lethal mutations affecting embryonic neurogenesis. This type of screen appears to be an effective way to find new loci that may play a role in the development of the Drosophila nervous system.

P-element-mediated enhancer detection: a versatile method to study development in Drosophila.

Abstract: We generated and characterized greater than 500 Drosophila strains that carry single copies of a novel P-element enhancer detector. In the majority of the strains, the beta-galactosidase reporter gene in the P-transposon responds to nearby transcriptional regulatory sequences in the genome. A remarkable diversity of spatially and temporally regulated staining patterns is observed in embryos carrying different insertions. We selected numerous strains as markers for different embryonic organs, tissues, and cells. Many of these strains should allow the study of complex developmental processes, such as nervous system development, which have not been convenient to analyze previously. Also, we present genetic evidence that some of the detected regulatory elements control nearby Drosophila genes. In light of our results, we discuss the diversity and complexity of cis-acting regulatory elements in the genome and the general applications of the enhancer detector method for the study of Drosophila development.

RNA-binding proteins as developmental regulators.

Abstract: Many RNA-binding proteins of the nucleus and cytoplasm, including pre-mRNA-, mRNA-, snRNA-, and pre-rRNA-binding proteins, contain a putative RNAbinding domain of approximately 90 amino acids whose amino acid sequence is conserved from yeast to man. The most highly conserved motif within this RNAbinding domain is an octapeptide, termed the ribonucleoprotein consensus sequence {RNP-CSI, which is an identifying feature of this group of proteins. Frequently, these proteins contain several similar, but nonidentical, RNP-CS-type RNA-binding domains. All of these proteins also contain at least one auxiliary domain that is unique to each type of protein and most likely functions in protein-protein interactions. Many, if not all, of the RNP-CS-type proteins display binding preferences for specific RNA sequences, and several have been shown to interact with pre-mRNA sequences important for premRNA processing. Recent work has shown that the proteins encoded by several developmental loci in Drosophila and maize contain RNP-CS and, therefore, are most likely RNA-binding proteins. Here we provide an overview of the structural characteristics of these proteins and speculate on how the modular structure of RNP-CS-type RNA-binding proteins may facilitate their participation in pathways that regulate development at the post-transcriptional level.

Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein.

Abstract: This paper presents the results of experiments that determine the chromosomal location of the mouse gene encoding CCAAT/enhancer binding protein (C/EBP) and measure its expression as a function of tissue type and temporal period of development in mice and rats Three alleles of the C/EBP gene were identified according to restriction fragment length polymorphisms The strain distribution pattern of the three alleles was determined in recombinant inbred mouse strains and compared to that of other mouse genes These results mapped the gene to a position within 25 centimorgans (cM) of the structural gene encoding glucose phosphate isomerase on chromosome 7 of the mouse The expression pattern of the C/EBP gene was studied by a combination of nucleic acid hybridization and antibody staining assays High levels of C/EBP mRNA were observed in tissues known to metabolize lipid and cholesterol-related compounds at uncommonly high rates These included liver, fat, intestine, lung, adrenal gland, and placenta More detailed analysis of two of these tissues, liver and fat, showed that C/EBP expression was limited to fully differentiated cells Moreover, analysis of the temporal pattern of expression of C/EBP mRNA in two tissues, liver and intestine, revealed a coordinated induction just prior to birth These observations raise the possibility that the synthesis of C/EBP may be responsive to humoral factors and that modulation in C/EBP expression might mediate coordinated changes in gene expression that facilitate adaptive challenges met during development or during the fluctuating physiological states of adult life

CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation.

Abstract: In mammals and other vertebrates, cytosine methylation in CpG sites is often negatively correlated with gene activity. Because methylation of the promoter region is most crucial for this effect, the simplest hypothesis is that CpG methylation interferes with the binding of specific transcription factors. We have examined this hypothesis with two different transcription factor-binding sites that contain a CpG dinucleotide, namely the cAMP-responsive element (CRE; 5'-TGACGTCA) and the Sp1-binding site (5'-GTGAGGCGGTGAGACT). We have reported previously that CpG methylation of the Sp1-binding site affected neither factor binding nor transcription in HeLa cells, which may be related to the fact that Sp1 is typically associated with promoters of housekeeping genes. In contrast, CREs are often associated with promoters of cell type-specific genes. A synthetic oligonucleotide containing two tandem CREs derived from the gene encoding the human glycoprotein hormone alpha-subunit was cloned upstream of a reporter gene. Transcription of this gene was dependent on the CRE sequences in both PC12 and HeLa cells. Bandshift and methylation interference assays show that similar, if not the same, factor(s) bind to the CRE in both cell lines, even though induction by cAMP was only observed in PC12 cells. CpG methylation of the CRE consensus sequences (TGACGTCA) resulted in loss of specific factor binding, as well as loss of transcriptional activity in vitro and in vivo, in both cell types. This suggests that the inactivity of methylated promoters can, at least in some cases, be explained by their inability to bind specific transcription factors.

The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways.

Abstract: Rapid degradation of c-fos proto-oncogene mRNA is crucial for transient c-fos gene expression. Experiments were performed to investigate the cellular mechanisms responsible for the extremely short half-life of human c-fos mRNA in growth-factor-stimulated fibroblasts. These experiments demonstrate the existence of two distinct cellular pathways for rapid c-fos mRNA degradation. Each of these pathways recognizes a different, functionally independent instability determinant within the c-fos transcript. One instability determinant, which is located within the c-fos 3'-untranslated region, is a 75-nucleotide AU-rich segment. Insertion of this element into beta-globin mRNA markedly reduces the half-life of that normally long-lived message. Nevertheless, specific deletion of the AU-rich element from c-fos mRNA has little effect on the transcript's cytoplasmic half-life due to the presence of the other c-fos instability determinant, which is located in the protein-coding segment of the c-fos message. Examination of mRNA decay in cells treated with transcription inhibitors indicates that one c-fos mRNA degradation pathway is dependent on RNA synthesis, whereas the other is not.

Differentiation-induced gene expression in 3T3-L1 preadipocytes: CCAAT/enhancer binding protein interacts with and activates the promoters of two adipocyte-specific genes.

Abstract: Previous studies have shown that differentiation of 3T3-L1 preadipocytes leads to the transcriptional activation of a group of adipose-specific genes. As an approach to defining the mechanism responsible for activating the expression of these genes, we investigated the binding of nuclear factors to the promoters of two differentiation-induced genes, the 422(aP2) and stearoyl-CoA desaturase 1 (SCD1) genes. DNase I footprinting and gel retardation analysis identified two binding regions within the promoters of each gene that interact with nuclear factors present in differentiated 3T3-L1 adipocytes. One differentiation-induced nuclear factor interacts specifically with a single binding site in the promoter of each gene. Competition experiments showed that the interaction of this nuclear factor with the SCD1 promoter was prevented specifically by a synthetic oligonucleotide corresponding to the site footprinted in the 422(aP2) promoter. Several lines of evidence indicate that the differentiation-induced nuclear factor is CCAAT/enhancer binding protein (C/EBP), a DNA-binding protein first isolated from rat liver. Bacterially expressed recombinant C/EBP binds to the same site at which the differentiation-specific nuclear factor interacts within the promoter of each gene. Northern analysis with RNA from 3T3-L1 cells shows that C/EBP mRNA abundance increases markedly during differentiation. Transient cotransfection studies using a C/EBP expression vector demonstrate that C/EBP can function as a trans-activator of both the 422(aP2) and SCD1 gene promoters.

Expression of c-kit gene products in known cellular targets of W mutations in normal and W mutant mice--evidence for an impaired c-kit kinase in mutant mice.

Abstract: The proto-oncogene c-kit, a transmembrane tyrosine protein kinase receptor for an unknown ligand, was shown recently to map to the dominant white spotting locus (W) of the mouse. Mutations at the W locus affect various aspects of hematopoiesis, as well as the proliferation and/or migration of primordial germ cells and melanoblasts during development. Here, we show that c-kit is expressed in tissues known to be affected by W mutations in fetal and adult erythropoietic tissues, mast cells, and neural-crest-derived melanocytes. We demonstrate that the c-kit associated tyrosine-specific protein kinase is functionally impaired in W/WV mast cells, thus providing a molecular basis for understanding the developmental defects that result from these mutations.

Growth factors and membrane depolarization activate distinct programs of early response gene expression: dissociation of fos and jun induction.

Abstract: A set of early response genes has been identified whose transcription in fibroblasts is rapidly induced in response to growth factors. Prototype members of this group, c-fos and c-jun, encode products that form a heterodimer and have been implicated in the regulation of gene expression and cell growth. It is thought that other early response genes also encode critical mediators of the cell's response to external stimuli. We have used PC12 pheochromocytoma cells as a model system to test the hypothesis that different extracellular signals induce distinct patterns of expression of early response genes. Our results indicate that membrane depolarization, induced either by potassium chloride or by the neurotransmitter analog nicotine, activates a program of gene expression distinct from that activated by nerve growth factor or epidermal growth factor. Notably, c-fos and c-jun activation can be dissociated; whereas c-jun is coinduced with c-fos and jun-B after growth factor stimulation, membrane depolarization activates c-fos and jun-B without stimulating c-jun. Fos may therefore form transcription complexes with alternative cofactors under different stimulation conditions. nur/77 and zif/268, which encode putative transcription factors, also show markedly different responses to growth factors and depolarization. We conclude that multiple nonconvergent signal transduction pathways control early response gene expression. Our findings also indicate that the diversity and specificity of cellular response to environmental change can be accounted for by the differential combinatorial induction of a relatively small number of early response genes.

Notch is required for successive cell decisions in the developing Drosophila retina.

Abstract: Mutations in the Notch locus affect a variety of developmental decisions in Drosophila. In this paper, we examine the role of Notch in the developing retina. We reduced Notch activity at successive intervals during development of the retina, and then examined the effect on individual cells. When Notch activity was reduced, cells responded by selecting inappropriate developmental pathways. We found that all cell types appear to require Notch when establishing their fate. To examine further Notch's role in eye development, we examined two alleles of Notch—split and facet-glossy, split flies show defects in the initial clustering of photoreceptors, whereas the defects in facet-glossy flies are due to the misrouting of presumptive primary pigment cells into the secondary pigment cell pathway. Our results suggest that Notch plays a permissive role in the cell-cell interactions used to assemble the eye.

Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-beta-like genes coordinately regulate aspects of embryonic development.

Abstract: The murine Vgr-1 (Vg-related) and BMP-2a (bone morphogenetic protein 2a) genes are members of the decapentaplegic subgroup of the transforming growth factor-beta (TGF beta) superfamily. Although genetic and biochemical studies suggest that the members of this subgroup play important roles in development, little is known about their function in mammals. Therefore, we investigated the expression of Vgr-1 and BMP-2a RNAs in embryonic, newborn, and adult tissues by in situ hybridization. Vgr-1 RNA is maternally encoded in ovarian oocytes but declines in fertilized eggs and is undectable by the two- to four-cell stage. Only low levels of transcripts are seen in blastocysts and early postimplantation stages. From mid-gestation on, Vgr-1 RNA is expressed at high levels in developing skin, especially in the suprabasal cells of the proliferating epidermis but not in the dermis or hair follicles, both of which contain TGF beta 1 and/or TGF beta 2 RNAs. In contrast, BMP-2a transcripts are seen only in the hair follicles in the cells of the hair bulb cortex. Temporally and spatially distinct patterns of BMP-2a, Vgr-1, TGF beta 1, and TGF beta 2 expression are also seen in different populations of mesenchymal cells in the developing skeletal system (cartilage and bone). Our results suggest that the coordinated expression of several members of the TGF beta superfamily is required to control the progression of specific cell types through their differentiation pathways.

A 65-kappaD subunit of active NF-kappaB is required for inhibition of NF-kappaB by I kappaB.

Abstract: The NF-kappaB transcription factor was affinity-purified from deoxycholate (DOC)-treated cytosol of HeLa cells and shown to contain both a 50-kappaD polypeptide (p50) with a DNA-binding specificity identical to that of nuclear NF-kappaB and a 65-kappaD protein (p65) lacking DNA binding activity. Electrophoretically purified p50, after renaturation, gave rise to a protein-DNA complex that migrated faster than that made by native NF-kappaB. Reconstitution of p50 and p65 together produced a protein that combined with DNA to form a complex with electrophoretic mobility indistinguishable from that of the complex formed by nuclear extracts and DOC-treated cytosolic fractions. Sedimentation and gel filtration analyses indicate that alone, the p50 protein exists as a dimer; two molecules of p65 bind to it to form a heterotetramer. Unlike I kappaB, the specific inhibitor of NF-kappaB, p65 displayed no inhibitor activity and was not released from NF-kappaB by DOC. p65 did not change the DNA binding specificity or the stimulatory effect of GTP on the p50 homodimer. Surprisingly, NF-kappaB could only be inactivated by I kappaB when p65 was bound. It would appear that one function of p65 is to make NF-kappaB susceptible to inhibition by I kappaB.

Cytoplasmic activation of ISGF3, the positive regulator of interferon-alpha-stimulated transcription, reconstituted in vitro.

Abstract: The signal transduction pathway through which interferon-alpha (IFN alpha) stimulates transcription of a defined set of genes involves activation of DNA-binding factors specific for the IFN alpha-stimulated response element (ISRE). IFN-stimulated gene factor-3 (ISGF3), the positive regulator of transcription, was derived in response to IFN alpha treatment from preexisting protein components that were activated first in the cell cytoplasm prior to appearance in the nucleus. Nuclear translocation of ISGF3 required several minutes and could be inhibited by NaF. Formation of active ISGF3 was mimicked in vitro by mixing cytoplasmic extracts from IFN alpha-stimulated cells with extracts of cells treated to contain high amounts of the unactivated factor. Active ISGF3 was found to be formed from association of two latent polypeptide precursors that were distinguished biochemically by differential sensitivity to N-ethyl maleimide. One precursor was modified in response to IFN alpha occupation of its cell-surface receptor, thus enabling association with the second subunit. The resulting complex then was competent for nuclear translocation and binding to ISRE. Cytoplasmically localized transcription factor precursors thus serve as second messengers to translate directly an extracellular signal into specific transcriptional activity in the nucleus.

CCAAT/enhancer binding protein activates the promoter of the serum albumin gene in cultured hepatoma cells.

Abstract: An expression vector capable of encoding full-length CCAAT/enhancer-binding protein (C/EBP) has been constructed and tested in transient transfection assays for its capacity to activate transcription from the promoter of the serum albumin gene When tested in cultured hepatoma cells, the C/EBP expression vector achieved potent trans-activation of the albumin promoter Less substantial activation was observed when the same experiment was conducted using cultured mouse fibroblasts Expression vectors that encoded defective forms of C/EBP failed to activate the albumin promoter Moreover, mutated variants of the albumin promoter that lack the C/EBP-binding site failed to be trans-activated The data are consistent with the interpretation that C/EBP is a bona fide transcription factor During the course of these experiments it was noted also that C/EBP is more than an order of magnitude less concentrated in cultured hepatoma cells than it is in adult liver cells Given these findings, we speculate that C/EBP may play a general role in establishing and maintaining the differentiated, nonproliferative state

V(D)J recombination: a functional definition of the joining signals.

Abstract: Two conserved DNA sequences serve as joining signals in the assembly of immunoglobulins and T-cell receptors from V-, (D)-, and J-coding segments during lymphoid differentiation. We have examined V(D)J recombination as a function of joining signal sequence. Plasmid substrates with mutations in one or both of the heptamer-spacer-nonamer sequences were tested for recombination in a pre-B-cell line active in V(D)J recombination. No signal variant recombines more efficiently than the consensus forms of the joining signals. We find the heptamer sequence to be the most important; specifically, the three bases closest to the recombination crossover site are critical. The nonamer is not as rigidly defined, and it is not important to maintain the five consecutive As that distinguish the consensus nonamer sequence. Both types of signals display very similar sequence requirements and have in common an intolerance for changes in spacer length greater than 1 bp. Although the two signal types share sequence motifs, we find no evidence of a role in recombination for homology between the signals, suggesting that they serve primarily as protein recognition and binding sites.

Identification of the sigma E subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression.

Abstract: The rpoH gene of Escherichia coli encodes sigma 32, the 32-kD sigma-factor responsible for the heat-inducible transcription of the heat shock genes. rpoH is transcribed from at least three promoters. Two of these promoters are recognized by RNA polymerase containing sigma 70, the predominant sigma-factor. We purified the factor responsible for recognizing the third rpoH promoter (rpoH P3) and identified it as RNA polymerase containing a novel sigma-factor with an apparent Mr of 24,000. This new sigma, which we call sigma E, is distinct from the known sigma factors in molecular weight and promoter specificity. sigma E holoenzyme will not recognize the sigma 70- or sigma 32-controlled promoters we tested, but it does transcribe the htrA gene, which is required for viability at temperatures greater than 42 degrees C. The in vivo role of sigma E is not known. The transcripts from the sigma E-controlled rpoH P3 and htrA promoters are most abundant at very high temperature, suggesting the sigma E holoenzyme may transcribe a second set of heat-inducible genes that are involved in growth at high temperature or in thermotolerance.

The growth hormone-binding protein in rat serum is an alternatively spliced form of the rat growth hormone receptor.

Abstract: A cDNA clone isolated from rat liver was demonstrated to encode a soluble, secreted growth hormone (GH)binding protein consistent with the properties of the newly discovered serum GH-binding protein. The protein coding region of this cDNA was identical in sequence to the extracellular domain of the rat liver GH receptor up to three amino acids before the putative transmembrane domain. At this point, an additional 17 amino acids were encoded in the GH-binding protein before a stop codon was encountered. This cDNA clone was shown to be representative of the structure of the mRNA present in rat liver. These results suggest that the mechanism for production of the rat serum GH-binding protein is by alternative splicing of the gene for the rat GH receptor.

Identification and characterization of HAP4: a third component of the CCAAT-bound HAP2/HAP3 heteromer.

Abstract: The CYCl gene of Saccharomyces cerevisiae is positively regulated by the HAP2 and HAPS proteins, which form a heteromeric complex that binds to a CCAAT box in the upstream activation site, UAS2, and which activate transcription in a nonfermentable carbon source. We carried out a genetic analysis to identify additional trans-acting regulatory factors exerting their effects through UAS2. We present the identification and characterization of a new locus, HAP4, which is shown to encode a subunit of the DNA-binding complex at UAS2. In the bap4 mutant, the binding of HAP2 and HAP3 (HAP2/3) is not observed in vitro. The HAP4 gene is regulated transcriptionally by a carbon source, suggesting that it encodes a regulatory subunit of the bound complex. The sequence of HAP4 shows a highly acidic region, which innactivated the protein when deleted. Replacement of this region with the activation domain of GAL4 restored activity, suggesting that it provides the principal activation domain to the bound HAP2/3/4 complex.

P-element-mediated enhancer detection: an efficient method for isolating and characterizing developmentally regulated genes in Drosophila.

Abstract: We describe a new approach for identifying and studying genes involved in Drosophila development. Single copies of an enhancer detector transposon, P[1ArB], have been introduced into flies at many different genomic locations. The beta-galactosidase reporter gene in this construct is influenced by a wide range of genomic transcriptional regulatory elements in its vicinity. Our results suggest that a significant proportion of these regulatory sequences are control elements of nearby Drosophila genes. These genes need not be disrupted for their regulatory elements to be identified by P[1ArB]. The P[1ArB] transposon has been designed to facilitate both rapid cloning and deletion analysis of genomic sequences into which it inserts. Therefore, the enhancer detection system is an efficient method of screening for genes primarily on the basis of their expression pattern and then rapidly analyzing those of particular interest at the molecular and genetic levels.

Poly(A) elongation during Xenopus oocyte maturation is required for translational recruitment and is mediated by a short sequence element.

Abstract: Xenopus oocytes contain several mRNAs that are mobilized into polysomes only at the completion of meiosis (maturation) or at specific times following fertilization. To investigate the mechanisms that control translation during early development, we have focused on an mRNA, termed G10, that is recruited for translation during oocyte maturation. Coincident with its translation, the poly(A) tail of this message is elongated from approximately 90 to 200 adenylate residues. To identify the cis sequence that is required for this cytoplasmic adenylation and recruitment, we have synthesized wild-type and deletion mutant G10 mRNAs with SP6 polymerase. When injected into oocytes that subsequently were induced to mature with progesterone, wild-type G10 mRNA, but not mutant transcripts lacking a 50-base sequence in the 3'-untranslated region, was polyadenylated and recruited for translation. The 50-base sequence was sufficient to confer polyadenylation and translation when fused to globin mRNA, which does not normally undergo these processes during oocyte maturation. Further mutational analysis of this region revealed that a U-rich sequence 5' to the AAUAAA hexanucleotide nuclear polyadenylation signal, as well as the hexanucleotide itself, were both required for polyadenylation and translation. The 50-base cis element directs polyadenylation, but not translation per se, as a transcript that terminates with 3'-deoxyadenosine (cordycepin) is not recruited for translation. The available data suggest that the dynamic process of polyadenylation, and not the length of the poly(A) tail, is required for translational recruitment during oocyte maturation.

A new family of mouse homeo box-containing genes: molecular structure, chromosomal location, and developmental expression of Hox-7.1.

Abstract: Two families of homeo box-containing genes have been identified in mammals to date, the Antennapedia- and engrailed-like homeo boxes, based on the sequence similarity to those from Drosophila. Here, we report the isolation of a homeo box-containing gene that belongs to a new family of which there are at least three related genes in the mouse genome. The homeo box of this new gene shows remarkable similarity to the Drosophila Msh homeo box that we designate as the prototype for this family. The gene maps to the proximal end of mouse chromosome 5 and does not cosegregate with any known homeo box-containing gene. We designate this locus Hox-7.1. In situ hybridizations to mouse embryos at different stages show a unique pattern of expression, as compared to other homeo box-containing genes described thus far. Hox-7.1 transcripts are detected in 9.5-day-old embryos in the neural crest, developing limb bud, and visceral arches. Later, this gene is expressed in regions of the face that are derived from neural crest and in the interdigital mesenchymal tissues in both the fore- and hindlimbs.

A nucleoprotein complex mediates the integration of retroviral DNA.

Abstract: The integration of viral DNA into the host genome is an essential step in the retrovirus life cycle To understand this process better, we have examined the native state of viral DNA in cells acutely infected by murine leukemia virus (MLV), using both a physical assay for viral DNA and a functional assay for integration activity (Brown et al 1987) The viral DNA and integration activity copurify during velocity sedimentation, gel filtration, and density equilibrium centrifugation, indicating that viral DNA is in a large (approximately 160S) nucleoprotein complex that includes all functions required for integration activity in vitro Analysis by immunoprecipitation shows that the viral capsid protein is part of the active nucleoprotein complex, but recognition of the complex by only a subset of anti-capsid sera implies that the protein is constrained conformationally The viral DNA within this structure is accessible to nucleases; the effects of nucleases on the integrity of the complex suggest that the integration-competent particle is derived from and similar to the core of extracellular virions

Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat.

Abstract: The human immunodeficiency virus (HIV-1)-encoded trans-activator (tat) increases HIV gene expression and replication. Previously, we demonstrated that tat facilitates elongation of transcription through the HIV-1 long terminal repeat (LTR) and that short transcripts corresponding to prematurely terminated RNA are released and accumulate in the absence of tat. Here, using a transient expression assay, we tested clustered and compensatory mutations, as well as 3' deletions, in the trans-acting responsive region (tar) and observed that the primary sequence in the loop and secondary structure in the stem of the stem-loop in tar are required for trans-activation by tat. Insertions in the 5' region of tar revealed that tar must be near the site of HIV-1 initiation of transcription for trans-activation by tat. Deletions (3') and an insertion in tar demonstrated that an intact stem-loop is required for the recovery of prematurely terminated transcripts. Short and full-length transcripts were observed also with HIV type 2 (HIV-2) in the absence and presence of tat, respectively. We conclude that an intact stem-loop in tar is essential for trans-activation by tat and that initiation of transcription by HIV-1 promoter factors and elongation of transcription by tat are coupled.

A pituitary POU domain protein, Pit-1, activates both growth hormone and prolactin promoters transcriptionally.

Abstract: The anterior pituitary gland provides a model for investigating the molecular basis for the appearance of phenotypically distinct cell types within an organ, a central question in development. The rat prolactin and growth hormone genes are expressed selectively in distinct cell types (lactotrophs and somatotrophs, respectively) of the anterior pituitary gland, reflecting differential mechanisms of gene activation or restriction, as a result of the interactions of multiple factors binding to these genes. We find that when the pituitary-specific 33-kD transcription factor Pit-1, expressed normally in both lactotrophs and somatotrophs, is expressed in either the heterologous HeLa cell line or in bacteria, it binds to and activates transcription from both growth hormone and prolactin promoters in vitro at levels even 10-fold lower than those normally present in pituitary cells. This suggests that a single factor, Pit-1, may be capable of activating the expression of two genes that define different anterior pituitary cell phenotypes. Because a putative lactotroph cell line (235-1) that does not express the growth hormone gene, but only the prolactin gene, appears to contain high levels of functional Pit-1, a mechanism selectively preventing growth hormone gene expression may, in part, account for the lactotroph phenotype.

Poly(A) addition during maturation of frog oocytes: distinct nuclear and cytoplasmic activities and regulation by the sequence UUUUUAU.

Abstract: In frog oocytes, certain maternal mRNAs receive poly(A) in the cytoplasm during progesterone-induced maturation. To analyze this reaction and to compare it to poly(A) addition in the nucleus, we injected short, synthetic RNA substrates into Xenopus oocytes. These RNAs contain only portions of the 3'-untranslated regions of appropriate mRNAs and end at the natural poly(A) site. We demonstrate that the nuclear and maturation-specific polyadenylation activities are distinct in substrate specificity and subcellular location. The sequence AAUAAA, contained in virtually all pre-mRNAs, is necessary for both activities. A second sequence element, UUUUUAU, activates poly(A) addition during maturation. UUUUUAU and AAUAAA are both necessary and virtually sufficient for maturation-specific polyadenylation: Poly(A) tails of between 50 and 300 nucleotides are added during maturation to RNAs containing both sequences but not to RNAs that lack either sequence. Before maturation, RNAs that contain AAUAAA are extended by just 10 nucleotides, presumably adenosines. The maturation-specific activity first appears within 1 hr of the time the nucleus breaks down but apparently does not require a nuclear component, as it is unaffected by enucleation. These observations, combined with those of others, lead us to speculate that polyadenylation may be responsible for the translational activation of a family of mRNAs essential for maturation.

Isolation and characterization of a gene specifically expressed in different metastatic cells and whose deduced gene product has a high degree of homology to a Ca2+-binding protein family.

Abstract: The gene mts1, which is expressed specifically in metastatic cells, was isolated by molecular cloning coupled with differential DNA reassociation. Transcription of mts1 was found not only in tumor cells, but also in normal cells; homologous RNA was detected only in spleen, thymus, bone marrow, and blood lymphocytes. DNA sequencing of mts1 revealed an open reading frame containing information for a peptide of 101 amino acids, and the amino acid sequence suggested that the mts1 protein was identical to the previously isolated Ca2+-binding mouse protein (Jackson-Grusby et al. 1987; Goto et al. 1988). Thus, the mts1 protein is a member of the calcium-modulated protein family, and our data indicate that mts1 is involved in regulating the metastatic behavior of tumor cells.