A chicken red cell inhibitor of transcription associated with the terminally differentiated state.

When a red cell nuclear extract (RCE) from adult chickens was injected into Xenopus oocytes along with the chicken beta globin gene, transcript levels were dramatically reduced compared to injection of DNA alone. The inhibitory action of the RCE was not specific to the beta globin gene since the Herpes thymidine kinase and Xenopus 5S RNA gene transcript levels were similarly reduced. Transcriptional repression was observed even after passage of the RCE through oocyte cytoplasm to the nucleus. The inhibitory activity binds to DNA cellulose, which suggests that the inhibitor either binds to DNA or associates with DNA-binding proteins. Nuclease digestion of the chromatin assembled on injected beta globin DNA revealed that inhibition was not associated with local changes in chromatin structure. Extracts from 9-d chicken embryonic erythroid cells, in which the endogenous beta globin gene is actively expressed, did not inhibit transcription. The inhibitory activity is, therefore, restricted to transcriptionally quiescent, adult erythrocytes. Since the inhibitory effects were seen with both polymerase II and III directed genes, we speculate that the activity may be part of the extreme transcriptional repression which occurs in the terminally differentiated erythrocyte.

[Hepatitis B virus morphogenesis]. / Morphogenèse du virus de l'hépatite B.

The human hepatitis B virus (HBV) is a small hepatotropic enveloped virus associated with chronic infection that can lead to cirrhosis and hepatocellular carcinoma. The HBV genome is a DNA molecule contained in an icosahedral capsid. Although HBV is not a retrovirus, the replication of its genome involves reverse transcription. Another distinctive feature of HBV is the production, in great excess over virions, of non-infectious subviral particles (SVP) consisting of membrane phospholipids and the three envelope proteins (small [S], medium [M] and large [L]). These empty non-infectious particles are highly immunogenic, and their in vitro production is at the basis of the current vaccine against hepatitis B. Despite numerous studies that lead to a better understanding of the HBV replication, little is known about the morphogenesis of the virion and its associated SVP. Recent approaches suggest that the mechanisms responsable for assembly of the virions and the SVP could be distinct.

Evidence for torsional stress in transcriptionally activated chromatin.

The existence of torsional stress in eukaryotic chromatin has been controversial. To determine whether it could be detected, we probed the structure of an alternating AT tract. These sequences adopt cruciform geometry when the DNA helix is torsionally strained by negative supercoiling. The single-strand-specific nuclease P1 was used to determine the structure of an alternating AT sequence upstream of the Xenopus beta-globin gene when assembled into chromatin in microinjected Xenopus oocytes. The pattern of cleavage by P1 nuclease strongly suggests that the DNA in this chromatin template is under torsional stress. The cruciform was detected specifically in the most fully reconstituted templates at later stages of chromatin assembly, suggesting that negative supercoiling is associated with chromatin maturation. Furthermore, the number of torsionally strained templates increased dramatically at the time when transcription of assembled chromatin templates began. Transcription itself has been shown to induce supercoiling, but the requisite negative supercoiling for cruciform extrusion by (AT)n in oocytes was not generated in this way since the characteristic P1 cutting pattern was retained even when RNA polymerase elongation was blocked with alpha-amanitin. Thus, torsional stress is associated with transcriptional activation of chromatin templates in the absence of ongoing transcription.

Role for DNA replication in beta-globin gene activation.

Transcriptional activation of the Xenopus laevis beta-globin gene requires the synergistic action of the simian virus 40 enhancer and DNA replication in DEAE-dextran-mediated HeLa cell transfections. Replication does not act through covalent modification of the template, since its requirement was not obviated by the prior replication of the transfected DNA in eucaryotic cells. Transfection of DNA over a 100-fold range demonstrates that replication does not contribute to gene activation simply increasing template copy number. Furthermore, in cotransfections of replicating and nonreplicating constructs, only replicating templates were transcribed. Replication is not simply a requirement of chromatin assembly, since even unreplicated templates generated nucleosomal ladders. Stimulation of beta-globin transcription by DNA replication, though less marked, was also observed in calcium phosphate transfections. We interpret these results as revealing a dynamic role for replication in gene activation.

The maternal CCAAT box transcription factor which controls GATA-2 expression is novel and developmentally regulated and contains a double-stranded-RNA-binding subunit.

The transcription factor GATA-2 is expressed at high levels in the nonneural ectoderm of the Xenopus embryo at neurula stages, with lower amounts of RNA present in the ventral mesoderm and endoderm. The promoter of the GATA-2 gene contains an inverted CCAAT box conserved among Xenopus laevis, humans, chickens, and mice. We have shown that this sequence is essential for GATA-2 transcription during early development and that the factor binding it is maternal. The DNA-binding activity of this factor is detectable in nuclei and chromatin bound only when zygotic GATA-2 transcription starts. Here we report the characterization of this factor, which we call CBTF (CCAAT box transcription factor). CBTF activity mainly appears late in oogenesis, when it is nuclear, and the complex has multiple subunits. We have identified one subunit of the factor as p122, a Xenopus double-stranded-RNA-binding protein. The p122 protein is perinuclear during early embryonic development but moves from the cytoplasm into the nuclei of embryonic cells at stage 9, prior to the detection of CBTF activity in the nucleus. Thus, the accumulation of CBTF activity in the nucleus is a multistep process. We show that the p122 protein is expressed mainly in the ectoderm. Expression of p122 mRNA is more restricted, mainly to the anterior ectoderm and mesoderm and to the neural tube. Two properties of CBTF, its dual role and its cytoplasm-to-nucleus translocation, are shared with other vertebrate maternal transcription factors and may be general properties of these proteins.

Facile cruciform formation by an (A-T)34 sequence from a Xenopus globin gene.

We have studied the structure adopted by an (A-T)34 sequence from a Xenopus globin gene when present in a negatively supercoiled plasmid. A variety of enzyme and chemical probing experiments and electrophoretic migration shift methods reveal that the sequence adopts cruciform geometry at moderate levels of supercoiling. The structure has the lowest free energy of formation yet observed for a cruciform, and no detectable kinetic barrier preventing rapid interconversion between extruded and unextruded conformations. Analysis of band-shift experiments reveals a twist change on cruciform formation of -5.8, slightly smaller than the -6.5 we would predict on the basis of a transition from B DNA. An attractive explanation consistent with this discrepancy is that the (A-T)34 stretch is locally underwound to about 11.7 base-pairs/helical turn at low levels of supercoiling. This calculation is made on the assumption that the cruciform junction is structurally similar to those examined previously, which is supported by the nuclease digestion results. This perturbed helical structure could be of considerable biological significance.

5' structural motifs and Xenopus beta globin gene activation.

We have analysed the structure of the Xenopus beta globin gene 5' flanking region in erythroid and non-erythroid chromatin, in supercoiled plasmids and in minichromosomes assembled in HeLa cell transfections. We have identified two erythroid chromatin-specific, nuclease-hypersensitive sites (HSs), one centred on the cap site, the other located 1000 base-pairs further upstream. An (AT)n tract is located 200 base-pairs upstream from each of these sites. In supercoiled plasmids, the (AT)n tracts, and not the chromatin HSs, are preferentially cleaved by single strand and double strand-specific nucleases. Using restriction enzymes, we have looked at the structure of the cap site HS in minichromosomes assembled in HeLa cell transfections. We find that the structure is indistinguishable from that found in erythroid chromatin, thus reinforcing our previous suggestion, based only on DNase I studies, that the formation of this HS is not dependent on erythroid-specific factors. In view of this close structural mimicry of the situation in vivo, we have used the HeLa cell model system to study the sequences required for cap site HS formation. We find that deletion of the (AT)n tract immediately upstream influenced neither the formation of the HS nor transcription of the globin gene. Indeed, these features remained unaffected by further deletion of upstream sequences, including 50 base-pairs of the HS itself. In this construct, the dimensions of the HS remained the same as in the undeleted construct, with the plasmid sequences that replaced the deleted Xenopus sequences becoming hypersensitive. Thus, HS formation is directed by sequences downstream from --116 acting over a distance of at least 50 base-pairs.

Structure and expression of a newt cardio-skeletal myosin gene. Implications for the C value paradox.

As part of our studies on the fate of the muscle lineage during amphibian limb regeneration, we have isolated genomic and cDNA sequences from a myosin heavy chain in the newt (Notophthalmus viridescens). Notwithstanding the technical problems inherent in analysing the large newt genome, genomic and cDNA sequences have been isolated and subjected to analysis by restriction mapping. Northern hybridization, Southern hybridization and DNA sequencing. We believe these to be the first single copy newt gene sequences to have been subjected to this type of analysis. The newt gene sequences showed a striking difference from mammalian myosins in both the estimated sizes of the gene and its intervening sequences; these being much larger than in the mammalian models, it is speculated that this could contribute to the exceptional size of the newt genome. By contrast, the coding sequences displayed very high levels of sequence homology to mammalian myosins. In particular, the amino acid sequence of the newt myosin was found to have greatest homology with rat and human myosin isotypes having a similar cardio-skeletal muscle expression pattern. Despite a long evolutionary separation, newt and mammalian cardio-skeletal myosins have remained more similar to each other than have the human or rat cardiac forms to skeletal myosins within their own respective species.

A graded response to BMP-4 spatially coordinates patterning of the mesoderm and ectoderm in the zebrafish.

The effects of signal perturbation on expression domains of molecular markers for the mesoderm and ectoderm have been analysed across the dorso-ventral axis in zebrafish embryos. Injection of RNA encoding bone morphogenetic protein-4 (BMP-4) ventralised the embryo, expanding the intermediate mesoderm and non-neural ectoderm at the expense of the dorso-anterior mesoderm and neural plate. A dose-dependent response was observed both morphologically and in expression of gta3, MyoD and pax2. Conversely, increases in dorso-anterior mesoderm and neurectoderm were generated by injection of RNA encoding either a dominant-negative BMP receptor (delta BMPR) or noggin, as demonstrated by goosecoid and pax2 expression. Ventral BMP-4 expression was also inhibited. Thus, patterning of both the mesoderm and the ectoderm during gastrulation appears to depend, directly or indirectly, on the level of BMP activity. Consistent with their locations prior to formation of the neural tube, elevated BMP-4 increased the number of dorsal spinal cord neurons whilst sonic hedgehog and islet1 expression in the ventral spinal cord were reduced. However, the ectopic neurons were not positioned more ventrally, implicating a prepattern in the dorsal neural tube that is independent of the ventral central nervous system.

Expression of zebrafish GATA 3 (gta3) during gastrulation and neurulation suggests a role in the specification of cell fate.

In order to understand the role of the transcription factor GATA 3 in vertebrate development, we have examined its expression and some aspects of its regulation during gastrulation and neurulation in the zebrafish. The complete coding sequence of the cDNA encoding the zebrafish GATA 3 homologue, termed gta3, is described. Analysis of expression patterns by in situ hybridisation shows the gene to be expressed during gastrulation in the ventral region of the embryo which includes tissue fated to form the non-neural ectoderm. By the end of gastrulation, there is a clear border to the gta3 expression domain that is close to the edge of the neural plate. Subsequently, gta3 expresses in the pronephric duct and in defined regions of the central nervous system which include specific cells in each segment of the spinal cord and nuclei in the brain. Double labelling embryos with a probe for gta3 and antibodies which identify differentiated neurons suggest that gta3 is dynamically expressed during the early differentiation phase of a subset of neurons but not in the terminal phase. Analysis of gta3 expression in dorsalised embryos and in cyc and spt mutant embryos indicates that the neural expression of the gene is subject to control by signals from the mesoderm, including both the notochord and the somites, which influence the segmental organisation of expression in the spinal cord.

GATA factors and the origins of adult and embryonic blood in Xenopus: responses to retinoic acid.

The transcription factors, GATA-1, -2 and -3 play essential roles in the differentiation of haematopoietic cells. To study the process of blood formation during vertebrate development we have used the expression of these GATA factors to locate haematopoietic cells in Xenopus embryos and to act as sensors for the effects of all-trans retinoic acid (RA), a signalling molecule which influences both anteroposterior patterning and haematopoietic differentiation. GATA factor expression was detected in the leading edge of the gastrulating mesoderm, in the ventral blood island (VBI) and dorsolateral plate (DLP) mesoderms and in a population of cells between the VBI and DLP. The VBI contributes to both embryonic and adult blood, whereas the DLP contains precursors of adult blood only, which have not been identified previously with molecular markers. The possibility that the GATA-2-expressing cells between the VBI and DLP were haematopoietic progenitors migrating from the VBI to the DLP was ruled out by transplantation analysis. Differential effects of RA on the expression of GATA-1 and GATA-2 suggest that RA has a direct action on haematopoietic differentiation, rather than on the formation of haematopoietic mesoderm.

Insights into early vasculogenesis revealed by expression of the ETS-domain transcription factor Fli-1 in wild-type and mutant zebrafish embryos.

Fli-1 is an ETS-domain transcription factor whose locus is disrupted in Ewing's Sarcoma and F-MuLV induced erythroleukaemia. To gain a better understanding of its normal function, we have isolated the zebrafish homologue. Similarities with other vertebrates, in the amino acid sequence and DNA binding properties of Fli-1 from zebrafish, suggest that its function has been conserved during vertebrate evolution. The initial expression of zebrafish fli-1 in the posterior lateral mesoderm overlaps with that of gata2 in a potential haemangioblast population which likely contains precursors of blood and endothelium. Subsequently, fli-1 and gata2 expression patterns diverge, with separate fli-1 and gata2 expression domains arising in the developing vasculature and in sites of blood formation respectively. Elsewhere in the embryo, fli-1 is expressed in sites of vasculogenesis. The expression of fli-1 was investigated in a number of zebrafish mutants, which affect the circulatory system. In cloche, endothelium is absent and blood is drastically reduced. In contrast to the blood and endothelial markers that have been studied previously, fli-1 expression was initiated normally in cloche embryos, indicating that induction of fli-1 is one of the earliest indicators of haemangioblast formation. Furthermore, although fli-1 expression in the trunk was not maintained, the normal expression pattern in the anterior half of the embryo was retained. These anterior cells did not, however, condense to form blood vessels. These data indicate that cloche has previously unsuspected roles at multiple stages in the formation of the vasculature. Analysis of fli-1 expression in midline patterning mutants floating head and squint, confirms a requirement for the notochord in the formation of the dorsal-aorta. The formation of endothelium in one-eyed pinhead, cyclops and squint embryos indicates a novel role for the endoderm in the formation of the axial vein. The phenotype of sonic-you mutants implies a likely role for Sonic Hedgehog in mediating these processes.

Evidence for non-axial A/P patterning in the nonneural ectoderm of Xenopus and zebrafish pregastrula embryos.

Recent studies in early Xenopus and zebrafish embryos have demonstrated that posteriorizing, non-axial signals arising from outside the organizer (or shield) contribute to A/P patterning of the neural axis, in contradiction to the classical Spemann model in which such signals were proposed to be solely organizer derived. Our studies on the early expression of the transcription factors GATA-2 and 3 in both Xenopus and zebrafish nonneural ectoderm lend support to the existence of such non-axial signaling in the A/P axis. Thus we find that the earliest expression of GATA-2 and 3 is located in nonneural ectoderm and is strongly patterned in a graded manner along the A/P axis, being high anteriorly and absent from the most posterior regions. This results by early neurula stages in three broad zones: an anterior region which is positive for both GATA-2 and 3, a middle region which is positive for GATA-2 alone and a posterior region in which neither gene is expressed. These regions correspond to head, trunk and tail ectoderm and may represent the beginnings of functional segmentation of nonneural ectoderm, as suggested in the concept of the 'ectomere'. We find that A/P patterning of GATA expression in nonneural ectoderm may occur as early as late blastula/early gastrula stages. We investigate which posteriorizing signals might contribute to such distinct non axial ectodermal patterning in the A/P axis and provide evidence that both FGF and a Wnt family member contribute towards the final A/P pattern of GATA expression in nonneural ectoderm.

Quantitative and qualitative analysis of exogenous gene expression by the S1 nuclease protection assay.

A method for determining the beginning and end of gene transcripts along with the position of intron/exon boundaries by S1 nuclease protection is described. Probe strategies and hybridization conditions are considered in detail. Under conditions of probe excess the method is quantitative.

Quantitative and Qualitative Analysis of Exogenous Gene Expression by the S1 Nuclease ProtectionAssay.

The Sinuclease protection procedure allows the precise definition of the beginning and end of gene transcripts as well as the position of intron/ exon boundaries in a gene. Alternatively, when these parameters are already known, the technique can be used to quantify transcript levels in a variety of expression systems.

Primer extension analysis of mRNA isolated from transfected cell lines.

Primer extension is a relatively quick and convenient means by which transcription from a gene transfected into tissue-culture cells can be monitored. The technique can be used to accurately determine the site of transcription initiation or to quantify the amount of cap-site-specific message produced.

Evaluation of extrachromosomal gene copy number of transiently transfected cell lines.

The short-term expression of DNA introduced into eukaryotic cells is now widely used to investigate the biological activities of cellular and viral genes or their products. A number of different transfection methods are in common use and can be broadly divided into two categories, based on the method by which DNA is introduced (either as a complex with a carrier substance that facilitates uptake by the cell [e.g., DEAE-dextran transfection (1,2), calcium phosphate coprecipitation (3), or lipofection (4) or by direct exposure to the cytoplasm [e.g., electroporation (5), microinjection (6) or scrapefection (7). In each case, transfected cells can maintain a considerable number of plasmids in their nuclei for several cell cycles. Plasmids lacking functional replication origins are lost progressively in subsequent mitoses. It appears that, for at least some transfection protocols, the majority of DNA that is taken up, remains circular and extrachromosomal, and is present as chromatin (8,9), and Wilson and Patient, unpublished).

Use of dpn I restriction enzyme to assess newly replicated gene copies in amplifiable vector systems.

Bacterially propagated plasmid DNA can be transfected into established eukaryotic cell lines or primary cell cultures by a variety of techniques, such as electroporation (see Chapter 5 , this vol) (1), scrape-loading (2), and DEAE dextran (see Chapter 3 ) or calcium phosphate mediated gene transfer (see Chapter 2 ) (3-5). At least some of the DNA introduced into the cells enters into the nucleus, where it is thought to be assembled into chromatin (6), and is maintained extrachromosomally for at least 48 h. During this time, the cellular chromosomal DNA may have undergone one or more rounds of DNA replication. However, the extrachromosomal transfected DNA will not replicate unless the DNA sequences contained in the plasmid include a DNA origin of replication recognized by the host cell. Origin sequences have so far proved difficult to identify in eukaryotic chromosomes. In contrast, viral genomes, such as SV40 and polyoma, have well-characterized origins of replication (7), which, when included on DNA.