Showing papers in "Genes & Development in 1990"

Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily.

Abstract: HNF-4 (hepatocyte nuclear factor 4) is a protein enriched in liver extracts that binds to sites required for the transcription of the genes for transthyretin (TTR), the carrier protein in the serum for vitamin A and thyroid hormone, and for apolipoprotein CIII (apoCIII), a major constituent of chylomicrons and very low-density lipoproteins (VLDL) Synthetic oligonucleotides derived from amino acid sequence of affinity-purified HNF-4 protein (54 kD) were used in the polymerase chain reaction (PCR) to isolate a cDNA clone encoding the protein HNF-4 is a member of the steroid hormone receptor superfamily with an unusual amino acid in the conserved "knuckle" of the first zinc finger (DGCKG) Studies with in vitro-translated HNF-4 protein show that it binds to its recognition site as a dimer, and cotransfection assays indicate that it activates transcription in a sequence-specific fashion in nonhepatic (HeLa) cells Northern blot analysis reveals that HNF-4 mRNA is present in kidney and intestine, as well as liver, but is absent in other tissues DNA-binding and antisera reactivity data suggest that HNF-4 could be identical to liver factor A1 (LF-A1), a DNA-binding activity implicated in the regulation of transcription of the alpha 1-antitrypsin, apolipoprotein A1, and pyruvate kinase genes The similarity between HNF-4 and other ligand-dependent transcription factors raises the possibility that HNF-4 and the genes it regulates respond to an as yet unidentified ligand

Pituitary cell phenotypes involve cell-specific Pit-1 mRNA translation and synergistic interactions with other classes of transcription factors.

Abstract: Development of the anterior pituitary gland involves proliferation and differentiation of ectodermal cells in Rathke's pouch to generate five distinct cell types that are defined by the trophic hormones they produce. A detailed ontogenetic analysis of specific gene expression has revealed novel aspects of organogenesis in this model system. The expression of transcripts encoding the c~-subunit common to three pituitary glycoprotein hormones in the single layer of somatic ectoderm on embryonic day 11 establishes that primordial pituitary cell commitment occurs prior to formation of a definitive Rathke's pouch. Activation of Pit-1 gene expression occurs as an organ-specific event, with Pit-1 transcripts initially detected in anterior pituitary cells on embryonic day 15. Levels of Pit-1 protein closely parallel those of Pit-1 transcripts without a significant lag. Unexpectedly, Pit-1 transcripts remain highly expressed in all five cell types of the mature pituitary gland, but the Pit-I protein is detected in only three cell types--lactotrophs, somatotrophs, and thyrotrophs and not in gonadotrophs or corticotrophs. The presence of Pit-1 protein in thyrotrophs suggests that combinatorial actions of specific activating and restricting factors act to confine prolactin and growth hormone gene expression to lactotrophs and somatotrophs, respectively. A linkage between the initial appearance of Pit-1 protein and the surprising coactivation of prolactin and growth hormone gene expression is consistent with the model that Pit-1 is responsible for the initial transcriptional activation of both genes. The estrogen receptor, which has been reported to be activated in a stereotypic fashion subsequent to the appearance of Pit-l, appears to be capable, in part, of mediating the progressive increase in prolactin gene expression characteristic of the mature lactotroph phenotype. This is a consequence of synergistic transcriptional effects with Pit-l, on the basis of binding of the estrogen receptor to a response element in the prolactin gene distal enhancer. These data imply that both transcriptional and post-transcriptional regulation of Pit-1 gene expression and combinatorial actions with other classes of transcription factors activated in distinct temporal patterns, are required for the mature physiological patterns of gene expression that define distinct cell types within the anterior pituitary gland.

Activity and tissue-specific expression of the transcription factor NF-E1 multigene family.

Abstract: NF-E1, a DNA-binding protein that recognizes the general consensus motif WGATAR, is the first tissue-specific factor to be identified in erythroid cells. Using a probe from the murine GF-1 (NF-E1) cDNA clone, we isolated three homologous chicken cDNAs: One of these corresponds to an mRNA (NF-E1a) that is abundantly and exclusively expressed in erythroid cells; a second mRNA (NF-E1b) is also expressed in all developmental stages of erythroid cells but is additionally found in a limited subset of other chicken tissues; mRNA representative of a third gene (NF-E1c) is expressed only in definitive (adult) erythrocytes within the red cell lineage but is also abundantly expressed in T lymphocytes and brain. All NF-E1 proteins are highly conserved within the DNA-binding domain and bind to the consensus motif with similar affinities in vitro; they are also all stimulatory trans-acting factors in vivo. The factors differ quantitatively in their ability to trans-activate reporter genes in which the number and position of cognate binding sites is varied relative to the transcriptional initiation site. These data suggest that the NF-E1 consensus motif directs a broader and more complicated array of developmental transcriptional regulatory processes than has been assumed and that NF-E1c may play a unique regulatory role in the developing chicken brain and in T lymphocytes.

Clonal and systemic analysis of long-term hematopoiesis in the mouse

Abstract: We have analyzed the temporal in vivo fate of 142 individual stem cell clones in 63 reconstituted mice. Long-term sequential analyses of the four major peripheral blood lineages, obtained from animals engrafted with genetically marked stem cells, indicate that developmental behavior is primarily a function of time. As such, the first 4-6 months post-engraftment is characterized by frequent fluctuations in stem cell proliferation and differentiation behavior. Gradually, a stable hematopoietic system emerges, dominated by a small number of totipotent clones. We demonstrate that single stem cell clones are sufficient to maintain hematopoiesis over the lifetime of an animal and suggest that mono- or oligoclonality may be a hallmark of long-term reconstituted systems. A model is proposed, wherein lineage-restricted differentiation and dramatic clonal flux are consequences of mechanisms acting on an expanding pool of totipotent cells and are not indicative of intrinsically distinct stem cell classes.

LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein.

Abstract: A gene, encoding a liver-enriched transcriptional activator protein (LAP) has been isolated. LAP is a 32-kD protein that stimulates the transcription of chimeric genes containing albumin D-promoter elements both in vivo and in vitro. LAP shares extensive sequence homology (71%) in its DNA-binding and leucine zipper domains with C/EBP. As a consequence, these two proteins show an indistinguishable DNA-binding specificity and readily heterodimerize. In addition, both genes, lap and cebp, are devoid of intervening sequences. Although correctly initiated transcripts from the LAP gene accumulate in the six examined tissues--liver, lung, spleen, kidney, brain, and testis--LAP protein is highly enriched in liver nuclei. Thus, the preferential accumulation of LAP protein in liver appears to be regulated post-transcriptionally.

slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains.

Abstract: The Drosophila slit locus encodes a protein with four regions containing tandem arrays of a 24-amino-acid leucine-rich repeat (LRR) with conserved flanking sequences (flank-LRR-flank surrounding these arrays), followed by two regions with epidermal growth factor (EGF)-like repeats. Each of these motifs has been implicated in protein-protein interactions as part of an extracellular domain in a variety of other proteins. Analysis of slit cDNA clones reveals that as a consequence of alternative splicing, the locus can code for two distinct protein species differing by 11 amino acids at the carboxyl terminus of the last EGF repeat. The existence of a putative signal sequence and the absence of a transmembrane domain suggest that slit is secreted, an observation supported by an analysis of its expression in tissue culture. Examining the expression pattern of slit in the embryo by antibody staining, enhancer trap detection, and in situ hybridization, we demonstrate that the protein is expressed by a subset of glial cells along the midline of the developing central nervous system. Through immunoelectron microscopy, slit can be seen on the commissural axons traversing the glial cells although it is absent from the cell bodies of these neurons, implying that slit is exported by the glia and distributed along the axons. Finally, we demonstrate that a reduction in slit expression results in a disruption of the developing midline cells and the commissural axon pathways. The embryonic localization, mutant phenotype, and homology of slit to both receptor-binding EGF-like ligands and adhesive glycoproteins suggest that it may be involved in interactions between the midline glial cells, their extracellular environment, and the commissural axons that cross the midline.

The adenovirus major late transcription factor USF is a member of the helix-loop-helix group of regulatory proteins and binds to DNA as a dimer.

Abstract: We isolated full-length cDNAs encoding the 43-kD form of human upstream stimulatory factor (USF), a cellular factor required for efficient transcription of the adenovirus major late (AdML) promoter in vitro. Sequence analysis showed USF to be a member of the c-myc-related family of DNA-binding proteins. Using proteins translated in vitro, we identified a DNA-binding domain near the carboxyl terminus, which includes both a helix-loop-helix motif and a leucine repeat. We show that USF interacts with its target DNA as a dimer. The leucine repeat is required for efficient DNA binding of the intact protein and for interactions between full-length and truncated USF proteins. Interestingly, it is not required for DNA binding of the isolated helix-loop-helix domain. The structure of different cDNA clones indicates that USF RNA is differentially spliced, and alternative exon usage may regulate the levels of functional USF protein.

A cloned human cDNA determines expression of a mouse stage-specific embryonic antigen and the Lewis blood group alpha(1,3/1,4)fucosyltransferase.

Abstract: The stage-specific embryonic antigen SSEA-1 is a cell-surface oligosaccharide molecule expressed with temporal precision during the murine preimplantation period and implicated in adhesive events involving the process of compaction. We used a mammalian transient expression system to isolate a cloned human cDNA that determines expression of the SSEA-1 molecule. The cDNA sequence predicts a type II transmembrane protein with a domain structure similar to mammalian glycosyltransferases, but without primary sequence similarity to these enzymes. The carboxy-terminal domain of this protein was shown to be catalytically active as a fucosyltransferase when expressed in COS-1 cells as a portion of a secreted protein A fusion peptide. The enzyme is an exceptional glycosyltransferase in that it can use both type I and type II oligosaccharides as acceptor substrates to generate subterminal Fuc alpha(1,4)- and Fuc alpha(1,3)-linkages, respectively, in a manner analogous to the human Lewis blood group fucosyltransferase. Southern blot analysis shows that the cDNA corresponds to sequences syntenic to the Lewis locus on chromosome 19. These results indicate that this cDNA is the product of the human Lewis blood group locus, provide genetic confirmation of the hypothesis that this enzyme can catalyze two distinct transglycosylation reactions, and outline an approach to the isolation of other sequences that determine expression of developmentally regulated oligosaccharide antigens.

The E75 ecdysone-inducible gene responsible for the 75B early puff in Drosophila encodes two new members of the steroid receptor superfamily.

Abstract: A pulse of the steroid hormone ecdysone at the end of Drosophila larval development triggers coordinate changes in both larval and imaginal tissues that result in metamorphosis to the adult fly. In larval salivary glands, this pulse activates a genetic regulatory hierarchy manifested by the induction of two kinds of transcription puffs in the polytene chromosomes: a small set of "early" puffs representing a primary response to the hormone, and a complex set of "late" puffs whose delayed appearance is dependent on proteins synthesized during the primary response. We isolated a 50-kb ecdysone-inducible gene, E75, that occupies the early puff locus at 75B. E75 contains two overlapping transcription units. The E75 A unit is a coextensive with the E75 gene and contains six exons: two 5'-proximal exons, A0 and A1, which are specific to this unit, and exons 2-5, which are common to both units. The E75 B unit is 20 kb long and contains five exons, a 5'-terminal exon, B1, located within the second intron of E75 A, and the common exons 2-5. Large open reading frames start within the first exon of each unit and continue into the last exon and therefore encode two different proteins. Both proteins exhibit sequence similarity to the conserved DNA-binding and hormone-binding domains of proteins in the steroid receptor superfamily. The two putative zinc fingers that characterize the DNA-binding domain are encoded by exon A1 and exon 2, so that the E75 A protein contains both fingers, whereas the E75 B protein contains only the second. Both proteins contain the same putative hormone-binding domain encoded by exon 4.

Expression of multiple novel Wnt-1/int-1-related genes during fetal and adult mouse development.

Abstract: The mammary tumor-associated proto-oncogene Wnt-1/int-1 encodes a secreted protein implicated in the regulation of neural development in vertebrates and segmental pattern in Drosophila. Using a PCR-based strategy, we isolated cDNAs encoding six novel, related proteins that are expressed in fetal mice. Predicted proteins are of similar molecular masses (38-42 kD) and share between 50% and 85% of amino acids. All contain a putative hydrophobic signal sequence, and comparative analysis reveals 83 absolutely conserved amino acid residues, including 21 cysteines. Transcripts were detected throughout fetal development by Northern blot analysis. Detailed examination of the expression of two of these genes by in situ hybridization revealed complex temporal and spatial patterns of transcription. All new Wnt family members are expressed in adult tissues, particularly in brain and lung. These data support the view that the Wnt-1/int-1 family constitutes a large family of signaling peptides with diverse roles in mouse development.

Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein.

Abstract: Translation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal internal entry into the 5'-nontranslated region (5' NTR) rather than by ribosomal scanning. The internal ribosomal entry site (IRES) in the EMCV 5' NTR was determined by in vitro translation with RNAs that were generated by in vitro transcription of EMCV cDNAs containing serial deletions from either the 5' or 3' end of the EMCV 5' NTR. Regions downstream of nucleotide 403 and upstream of nucleotide 811 of EMCV were required for efficient translation. Site-directed mutagenesis revealed that a stem-loop structure (400 nucleotides upstream of the initiation codon) was essential for IRES function. We discovered a 57-kD cellular protein whose specific interaction with this stem-loop appears to be prerequisite for IRES function. A A pyrimidine-rich stretch proximal to the initiation codon was also crucial for efficient translation of EMCV mRNA. We propose that ribosomes bind directly to the initiating AUG without scanning.

HNF-3A, a hepatocyte-enriched transcription factor of novel structure is regulated transcriptionally.

Abstract: Hepatocyte-specific gene expression requires the interaction of many proteins with multiple binding sites in the regulatory regions. HNF-3 is a site found to be important in the maximal hepatocyte-specific expression of several genes. We find that liver nuclear extracts contain three major binding activities for this site, which we call HNF-3A, HNF-3B, and HNF-3C. Purification from rat liver nuclear extracts of HNF-3A and HNF-3C reveals that each activity corresponds to a distinct polypeptide, as determined by SDS-PAGE. Peptide sequence derived from the most abundant species, HNF-3A, was used for synthesizing probes with which to isolate a cDNA clone of this protein. The encoded protein contains 466 amino acids (48.7 kD) and has binding properties identical to those of the purified protein. A 160-amino-acid region that does not resemble the binding domain of any known transcription factor is essential for DNA binding. The mRNA for HNF-3A is present in the rat liver but not in brain, kidney, intestine, or spleen, and the basis for this difference is cell-specific regulation of HNF-3A gene transcription.

A deletion of the human beta-globin locus activation region causes a major alteration in chromatin structure and replication across the entire beta-globin locus.

Abstract: Naturally occurring deletions that remove sequences located approximately 60 kb upstream of the human adult beta-globin gene result in the failure to transcriptionally activate the cis-linked globin genes in erythroid cells. In addition, transfection, transgenic, and somatic cell hybrid studies have revealed that sequences within this region are essential for the developmentally regulated high-level expression of cis-linked globin genes. This regulatory region located at the 5' end of the beta-globin locus has been termed the locus activation region (LAR). Using somatic cell hybrids, we have studied the chromatin structure and timing of DNA replication of the normal human beta-globin locus and a locus containing a de novo 25-kb deletion that removes elements of the LAR. As a result of this deletion, the entire beta-globin locus and sequences approximately 100 kb 5' and 3' of the adult beta-globin gene are DNase I-resistant and do not form characteristic distant hypersensitive sites. These sequences also replicate late in S phase in an erythroid cell background. In contrast, the sequences of the normal locus are DNase I sensitive and early replicating. These results suggest that the LAR is required for both the erythroid-specific chromatin structure and timing of DNA replication over a large physical distance.

New light on Myc and Myb. Part II. Myb.

Abstract: The group of proto-oncogen es found to encode nuclear proteins now includes myc, myb, fos, jun, ski, cbl, eibA, members of the ets family^ and possibly several others. Given that so many cytoplasmic and membrane-asso­ ciated oncoproteins are involved in signal transduction pathways, one rather appealing notion has been that some or all of the nuclear oncoproteins encoded by these genes might act to mediate specific transcriptional re­ sponses to signals originally generated in the plasma membrane or cytoplasm (for review, see Weinberg 1989). During the last several years, a number of these onco­ proteins, including ErbA, Fos, Jun and, most recently, Ets, have been demonstrated to be directly involved in transcriptional regulation. It is somewhat ironic that Myc and Myb, two of the first oncoproteins to be shown localized to the nucleus, have appeared to elude func­ tional characterization. However, recent evidence has demonstrated that Myb also functions in transcription and, while Myc has remained a citadel of incomprehen­ sibility, new studies have begun to bring this mysterious protein into sharper focus. At first glance, Myc and Myb would appear to have little in common, aside from the fact that both are predominantly localized in the nu­ cleus. They are quite different structurally and their pat­ terns of expression are also rather distinct, with Myc present in nearly all cell types while Myb is restricted to hematopoietic cells. However, the functions of both of these oncoproteins appear to be linked to proliferation , and these oncoproteins clearly play major roles in cell differentiation. Furthermore, recent work has cast some new light on both oncoproteins, and in what follows we attempt to meld older with more recent evidence re­ lating to possible functions. This review will appear in two segments. In part I we consider studies relating to the structure and potential function of Myc; part II, to appear in the next issue, will discuss recent findings on Myb.

TFE3: a helix-loop-helix protein that activates transcription through the immunoglobulin enhancer muE3 motif.

Abstract: The muE3 motif within the immunoglobulin heavy-chain enhancer is required for full enhancer activity and is known to bind one, or perhaps a family, of related ubiquitous nuclear proteins Here, we present the isolation of a cDNA that encodes an apparently novel microE3-binding protein designated TFE3 The major open reading frame of the cDNA predicts a protein of 59 kD, with a leucine zipper situated adjacent to an myc-related motif that has been proposed to assume a helix-loop-helix structure Both of these motifs have been shown (for other proteins) to facilitate protein-protein interactions and DNA binding Expression of the cDNA in 3T3 cells stimulates transcription from an artificial promoter consisting of four muE3 sites linked to a TATA box and also augments transcription of a reporter gene when it is linked to multiple copies of a particular heavy-chain enhancer subfragment but not when it is linked to the intact enhancer Using GAL4 fusion proteins, we mapped a strong transcription activation domain within TFE3 that is distinct from the leucine zipper and helix-loop-helix motifs and includes a potential negative amphipathic helix Like the other muE3-binding proteins detected in nuclear extracts, in vitro-synthesized TFE3 also binds to the USF/MLTF site found in the adenovirus major late promoter

Transcriptional activation and DNA binding by the erythroid factor GF-1/NF-E1/Eryf 1.

Abstract: The murine, erythroid DNA-binding protein GF-1 (also known as NF-El, Eryf 1), a 413-amino acid polypeptide with two novel finger domains of the C,-C, variety, recognizes a consensus GATA motif present in cis elements of the majority of erythroid-expressed genes. We have performed a structure-function analysis of this protein to evaluate its potential as a transcriptional activator and to examine the role of the finger domains in DNA binding. Using a cotransfection assay, we find that GF-1 is a potent transcriptional activator with several activation domains but that this is revealed only in heterologous cells and with reporters containing minimal promoters onto which either a single or multiple GATA-binding sites are placed. The two fingers of GF-1 are functionally distinct and cooperate to achieve specific, stable DNA binding. The amino finger is necessary only for full specificity and stability of binding, whereas the carboxyl finger is required for binding. The role of each finger is more pronounced with some GATA-binding sites than with others, suggesting a diversity of interactions between GF-1 and different target sites. The complex activation and DNA-binding properties of GF-1 are likely to contribute to the ability of this single protein to participate widely in gene expression throughout erythroid development.

A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated trans-activation.

Abstract: The Tat protein of human immunodeficiency virus type 1 (HIV-1) trans-activates viral gene expression and is obligatory for virus replication. Tat function is mediated through a sequence termed TAR that comprises part of the 5'-noncoding region of all HIV-1 mRNAs. This region forms a stable stem-loop structure in vitro. Recent evidence indicates that Tat binds directly to the TAR RNA sequence, and this binding is independent of the nucleotide sequence in the loop but dependent on the integrity of the upper stem. We used the electrophoretic mobility-shift assay to identify the sequence and structure specificity of this interaction and its correlation with Tat trans-activation. We show that a 3-nucleotide bulge structure (positions +23 to +25) in TAR RNA is important for both Tat interaction with TAR RNA and Tat-mediated trans-activation of gene expression. Single base substitutions at position +23 that impair Tat-mediated trans-activation in vivo also reduce binding of Tat to TAR in vitro, suggesting that the first uridine residue in the bulge is the critical base for both functions. In contrast, mutations in the loop (positions +31 to +34) and the stem (positions +9 to +12 and +49 to +52), which reduce Tat-mediated trans-activation, had no effect on Tat binding. We also show that a Tat peptide that includes the basic region required for nucleolar localization binds to TAR RNA with the same specificity as the full-length protein. We conclude that Tat binding to TAR is necessary but not sufficient by itself to account for trans-activation.

rhomboid, a gene required for dorsoventral axis establishment and peripheral nervous system development in Drosophila melanogaster.

Abstract: rhomboid (rho) belongs to a group of four genes involved in the elaboration of positional information at a ventrolateral level in the Drosophila embryo. Mutations at any of these four loci also lead to a variety of other phenotypes, including reduction in the number of stretch receptor organs (chordotonal organs) in the peripheral nervous system (PNS). We have cloned rho with the aid of a lacZ-bearing P-element inserted into the rho gene. In the early blastoderm stage, a putative rho transcript is expressed in ventrolateral strips corresponding to the domain of activity of the rho gene on the embryonic fate map. Later expression of the transcript correlates with regions of the embryo that are disrupted in rho mutants and includes a cell that may be the precursor for the missing stretch receptor organs. We hypothesize that rho acts very early in differentiation pathways to specify the identities of domains and isolated precursor cells. Sequence analysis suggests that this transcript codes for a trans-membrane protein.

Identification of HTLV-I tax trans-activator mutants exhibiting novel transcriptional phenotypes.

Abstract: The type I human T-cell leukemia virus (HTLV-I) encodes a 40-kD nuclear trans-regulatory protein termed Tax that transcriptionally activates the HTLV-I long terminal repeat (LTR), as well as select [corrected] cellular and heterologous viral promoters. Tax does not bind DNA specifically but, rather, acts in a more indirect manner. Tax activation of the HTLV-I LTR is mediated through constitutively expressed cellular factors that bind to cAMP response elements (CREs) present within the 21-bp enhancers of the LTR. In contrast, Tax transactivation of the interleukin-2 receptor-alpha gene (IL-2R alpha) and LTR of the type 1 human immunodeficiency virus (HIV-1) involves the induced nuclear expression of NF-kappa B. We now report the identification of missense mutations within the tax gene that functionally segregate these two pathways of trans-activation. Additionally, we demonstrate that the carboxyl terminus of the Tax protein, despite its acidic and predicted alpha-helical structure, is completely dispensable for trans-activation through either of these transcription factor pathways. Finally, we demonstrate that mutations within a putative zinc finger domain disrupt the nuclear localization of Tax and abolish trans-activation. These results demonstrate that Tax trans-activation of viral and cellular promoters involves at least two mechanisms of host transcription factor activation and suggest that this activation is likely mediated through distinct functional domains.

NRF-1: a trans-activator of nuclear-encoded respiratory genes in animal cells.

Abstract: The assembly of the respiratory apparatus requires the coordinate expression of a large number of genes from both nuclear and mitochondrial genetic systems. In vertebrate organisms, the molecular mechanisms integrating the activities of these distinct genomic compartments in response to tissue demands for respiratory energy remain unknown. A potential inroad to this problem came with the discovery of nuclear respiratory factor 1 (NRF-1), a novel transcriptional activator defined by mutational and DNA binding analysis of the somatic cytochrome c promoter. Functional NRF-1 sites are now observed in several other recently isolated nuclear genes whose products function in the mitochondria. Among these are genes encoding subunits of the cytochrome c oxidase (subunit VIc) and reductase (ubiquinone-binding protein) complexes. In addition, a functional NRF-1 site resides in the MRP RNA gene encoding the RNA moiety of a ribonucleoprotein endonuclease involved in mitochondrial DNA replication. Synthetic oligomers of these sites competitively displace NRF-1 binding to the cytochrome c promoter. NRF-1-binding activities for each site also have the same thermal lability, copurify chromatographically, and make similar guanosine nucleotide contacts within each recognition sequence. Moreover, NRF-1 recognition in vitro correlates with the ability of each site to stimulate expression in vivo from a truncated cytochrome c promoter. The presence of NRF-1-binding sites in nuclear genes encoding structural components of the mammalian electron transport chain, as well as the mitochondrial DNA replication machinery, suggests a mechanism for coordination of nuclear and mitochondrial genetic systems through the concerted modulation of nuclear genes.

A correlation of thyroid hormone receptor gene expression with amphibian metamorphosis.

Abstract: The expression of the thyroid hormone (TH) receptor genes alpha (TR alpha) and beta (TR beta) in Xenopus laevis begins after the embryo hatches. The TR alpha mRNA increases throughout the premetamorphosis stage of tadpole development, is maximal by prometamorphosis, and falls after climax of metamorphosis to a lower level in frogs. The TR beta mRNA is barely detectable during premetamorphosis. In synchrony with the onset of endogenous TH synthesis by the thyroid gland (prometamorphosis), the level of TR beta mRNA rises in parallel with endogenous TH, reaching a peak at the climax of metamorphosis (stage 61) and drops to approximately 10% of its peak level after metamorphosis. As suggested by this correlation, exogenous TH up-regulates TR beta mRNA as much as 20-fold during premetamorphosis, whereas TH up-regulates TR alpha mRNA by approximately 2-fold during the same period. Up-regulation of TR beta mRNA is the earliest response to exogenous TH by competent tadpoles yet detected.

Posterior localization of vasa protein correlates with, but is not sufficient for, pole cell development.

Abstract: The protein product of the Drosophila materual-effect posterior group gene vasa is localized to the posterior pole of the oocyte and is sequestered by the pole cells as they form. It is, however, present at easily detectable levels throughout the oocyte and pre-blastoderm embryo. The protein is present in the pole cells and their germ line derivatives throughout all stages of development. An antiserum against this protein recognizes a pole-ceUspecific antigen in seven other Drosophila species. Of six other materual-effect loci essential for embryonic pole cell development, none affects expression of vasa, mutations in four abolish vasa protein localization, and mutations in two, tudor and valois, have little, if any, effect on vasa expression or localization. This indicates that vasa protein, when properly localized, is not sufficient for induction of pole cell development, and that at least the tudor and valois wild-type functions are also required specifically for this process. These results are discussed with respect to the multiple functions of the vasa gene.

Identification of a retinoic acid-sensitive period during primary axis formation in Xenopus laevis.

Abstract: Retinoic acid (RA) is able to profoundly alter patterning of the primary body axis in embryos of the frog Xenopus laevis. The response to RA is dose-dependent, and leads to progressive truncation of the anteroposterior axis, with anterior structures most sensitive. Both mesodermal and ectodermal tissues are affected, and in vitro assays demonstrate that induced dorsal ectoderm is one direct target of RA. RA represses expression of anterior-specific genes and concomitantly induces expression of at least one posterior-specific gene. Resistance to RA is acquired gradually, during gastrula and early neurula stages, with posterior structures becoming resistant before anterior structures. These data demarcate in the embryo an anterior "domain," which may define the head rudiment and which transcends germ layers. RA can alter the axial pattern after its initial induction; thus, RA sensitivity defines a labile intermediate that occurs during axial patterning. These data suggest a possible role for RA in normal axis formation.

bag-of-marbles: a Drosophila gene required to initiate both male and female gametogenesis.

Abstract: In Drosophila, male and female gametes begin development when a stem cell divides to produce a cyst precursor. Subsequently, four special divisions give rise to a cluster of 16 interconnected cystocytes that develop into a single egg or 64 sperm. We identified and characterized a gene, bag-of-marbles (bam), that disrupts cyst formation in both sexes. An apparent null mutation causes abnormal cysts to form containing an excess number of cells that cannot differentiate into gametes. bam function resides within a simple 2.2-kb transcription unit encoding a single 442-amino-acid protein that shows similarity to the product of the ovarian tumor gene. The specific expression of bam RNA within female cystoblasts suggested that it might be involved in the specific cell-cycle alterations that occur during cystocyte divisions.

DnaK, DnaJ, and GrpE heat shock proteins negatively regulate heat shock gene expression by controlling the synthesis and stability of sigma 32.

Abstract: The Escherichia coli DnaK heat shock protein has been identified previously as a negative regulator of E. coli heat shock gene expression. We report that two other heat shock proteins, DnaJ and GrpE, are also involved in the negative regulation of heat shock gene expression. Strains carrying defective dnaK, dnaJ, or grpE alleles have enhanced synthesis of heat shock proteins at low temperature and fail to shut off the heat shock response after shift to high temperature. These regulatory defects are due to the loss of normal control over the synthesis and stability of sigma 32, the alternate RNA polymerase sigma-factor required for heat shock gene expression. We conclude that DnaK, DnaJ, and GrpE regulate the concentration of sigma 32. We suggest that the synthesis of heat shock proteins is controlled by a homeostatic mechanism linking the function of heat shock proteins to the concentration of sigma 32.

Floral homeotic mutations produced by transposon-mutagenesis in Antirrhinum majus.

Abstract: To isolate and study genes controlling floral development, we have carried out a large-scale transposon-mutagenesis experiment in Antirrhinum majus. Ten independent floral homeotic mutations were obtained that could be divided into three classes, depending on whether they affect (1) the identity of organs within the same whorl; (2) the identity and sometimes also the number of whorls; and (3) the fate of the axillary meristem that normally gives rise to the flower. The classes of floral phenotypes suggest a model for the genetic control of primordium fate in which class 2 genes are proposed to act in overlapping pairs of adjacent whorls so that their combinations at different positions along the radius of the flower can specify the fate and number of whorls. These could interact with class 1 genes, which vary in their action along the vertical axis of the flower to generate bilateral symmetry. Both of these classes may be ultimately regulated by class 3 genes required for flower initiation. The similarity between some of the homeotic phenotypes with those of other species suggests that the mechanisms controlling whorl identity and number have been highly conserved in plant evolution. Many of the mutations obtained show somatic and germinal instability characteristic of transposon insertions, allowing the cell-autonomy of floral homeotic genes to be tested for the first time. In addition, we show that the deficiens (def) gene (class 2) acts throughout organ development, but its action may be different at various developmental stages, accounting for the intermediate phenotypes conferred by certain def alleles. Expression of def early in development is not necessary for its later expression, indicating that other genes act throughout the development of specific organs to maintain def expression. Direct evidence that the mutations obtained were caused by transposons came from molecular analysis of leaf or flower pigmentation mutants, indicating that isolation of the homeotic genes should now be possible.

Expression of the trk proto-oncogene is restricted to the sensory cranial and spinal ganglia of neural crest origin in mouse development.

Abstract: We have cloned and characterized the mouse homolog of the human trk proto-oncogene, a member of the protein tyrosine kinase (TK) receptor gene family. Here, we present the first report of a trk-encoded mRNA species in vivo. In situ hybridization analysis in the mouse embryo reveals a striking temporal and spatial regulation of trk transcription, with expression confined to the sensory cranial (trigeminal, superior, jugular) and dorsal root ganglia (DRG) of neural crest origin. Recent reports have shown that TK receptors can play regulatory roles in embryonic development. Thus, the developmental mutations W in mouse and torso and sevenless in Drosophila represent genes that code for defective TK receptors. Our data show that trk, a gene associated with malignancy in humans, is a specific marker for a set of neural crest-derived sensory neurons, and are consistent with the hypothesis that this protoooncogene may have an important role in the development or phenotype of the neurons where it is expressed.