Search

International audience; Figure 1. Le trouble bipolaire est une mala-die psychiatrique relevant des troubles de l'humeur. Les troubles de l'humeur se dis-tribuent selon un spectre, en fonction de la durée et l'intensité des épisodes dépressifs et maniaques. Le trouble unipolaire, ou dépression majeure, présente seulement des épisodes dépressifs ; le trouble bipo-laire de type II présente en plus des épi-sodes hypomaniaques ; le trouble bipolaire de type I comporte des phases dépressives et maniaques sévères. La cyclothymie est caractéri-sée par une succession de phases dépressives et euphoriques de faible intensité. La dysthymie correspond à un état dépressif chronique, moins intense que la dépression majeure.

Endoneurial fibroblast-like cells (EFLCs) have been described for more than 60 years, but the embryology, functions, and pathology of these cells are not well defined. Several hypotheses of their origin have been proposed. A previous study suggesting that they were of neural crest origin is supported by our data in humans. This lineage might account for EFLCs having multiple biologic functions and involvement in pathological processes. Here, we review what is known about the origin; functions in collagen synthesis, phagocytosis, inflammatory responses, and immune surveillance; and the pathological alterations of EFLCs based on the literature and on our personal observations.

The transcription factor EGR2 is expressed in Schwann cells, where it controls peripheral nerve myelination. Mutations of EGR2 have been found in patients with congenital hypomyelinating neuropathy or Charcot-Marie-Tooth disease type 1D. In a patient with congenital amyelinating neuropathy, we observed pathological abnormalities recapitulating the peripheral nervous system phenotype of homozygous Egr2-null mice. This patient, born from consanguineous parents, showed no EGR2 immunoreactivity in Schwann cells and harbored a homozygous 10.7-kilobase-long deletion encompassing a myelin-specific enhancer of EGR2. This regulatory mutation is the first genetic abnormality associated with congenital amyelinating neuropathy in humans.

The transcription factor Sox10 is expressed throughout Schwann cell development and has already been shown to be essential for specification and for the identity and further development of immature Schwann cells. Here, we show that Sox10 is also required in Schwann cells for establishing the myelinating state. This is concluded from the fact that a peripheral neuropathy develops in mice in which Sox10 is deleted by a Cre recombinase whose expression is under control of Krox20 regulatory elements. This neuropathy is characterized by altered marker gene expression along the peripheral nerve, decreased conductivity, and severe persistent hypomyelination. As the Cre recombinase is additionally active in boundary cap cells, we also analyzed the role of Sox10 during embryogenesis in establishment and maintenance of the boundary between central and peripheral nervous systems. Sox10 deletion did not affect establishment or survival of boundary cap cells but appeared to compromise barrier function as cells expressing oligodendrocyte and astrocyte markers were no longer restricted to the central nervous system, and instead found in peripheral nerves. We infer that in addition to its many roles in Schwann cells, Sox10 is also important for the integrity of the boundary between central and peripheral nervous systems. © 2012 Wiley Periodicals, Inc.

Boundary cap cells (BC), which express the transcription factor Krox20, participate in the formation of the boundary between the central nervous system and the peripheral nervous system. To study BC stemness, we developed a method to purify and amplify BC in vitro from Krox20 Cre/+ , R26R YFP/+ mouse embryos. We show that BC progeny are EGF/FGF2-responsive, form spheres, and express neural crest markers. Upon growth factor withdrawal, BC progeny gave rise to multiple neural crest and CNS lineages. Transplanted into the developing murine forebrain, they successfully survived, migrated, and integrated within the host environment. Surprisingly, BC progeny generated exclusively CNS cells, including neurons, astrocytes, and myelin-forming oligodendrocytes. In vitro experiments indicated that a sequential combination of ventralizing morphogens and glial growth factors was necessary to reprogram BC into oligodendrocytes. Thus, BC progeny are endowed with differentiation plasticity beyond the peripheral nervous system. The demonstration that CNS developmental cues can reprogram neural crest-derived stem cells into CNS derivatives suggests that BC could serve as a source of cell type-specific lineages, including oligodendrocytes, for cell-based therapies to treat CNS disorders.

International audience; The hindbrain transcription factors Phox2b and Egr2 (also known as Krox20) are linked to the development of the autonomic nervous system and rhombomere-related regulation of breathing, respectively. Mutations in these proteins can lead to abnormal breathing behavior as a result of an alteration in an unidentified neuronal system. We characterized a bilateral embryonic parafacial (e-pF) population of rhythmically bursting neurons at embryonic day (E) 14.5 in mice. These cells expressed Phox2b, were derived from Egr2-expressing precursors and their development was dependent on the integrity of the Egr2 gene. Silencing or eliminating the e-pF oscillator, but not the putative inspiratory oscillator (preBötzinger complex, preBötC), led to an abnormally slow rhythm, demonstrating that the e-pF controls the respiratory rhythm. The e-pF oscillator, the only one active at E14.5, entrained and then coupled with the preBötC, which emerged independently at E15.5. These data establish the dual organization of the respiratory rhythm generator at the time of its inception, when it begins to drive fetal breathing.

The zinc finger transcription factor Krox20 plays an essential role in the vertebrate hindbrain segmentation process. It positively or negatively controls a large variety of other regulatory genes, coordinating delimitation of segmental territories, specification of their identity, and maintenance of their integrity. We have investigated the molecular mechanisms of Krox20 transcriptional control by performing a detailed structure-function analysis of the protein in the developing chick hindbrain. This revealed an unsuspected diversity in the modes of action of a transcription factor in a single tissue, since regulation of each of the five tested target genes requires different parts of the protein and/or presumably different co-factors. The multiplicity of Krox20 functions might rely on this diversity. Investigation of known Krox20 co-factors was initiated in relation to this analysis. Nab was shown to act as a negative feedback modulator of the different Krox20 activating functions in the hindbrain. HCF-1 was found to bind to a Krox20 N-terminal region, which was shown to rely on multiple elements, including acidic domains, to convey Nab activation and Krox20 autoregulation.

The inner ear, the sensory organ responsible for hearing and balance, contains specialized sensory and non-sensory epithelia arranged in a highly complex three-dimensional structure. To achieve this complexity, a tight coordination between morphogenesis and cell fate specification is essential during otic development. Tissues surrounding the otic primordium, and more particularly the adjacent segmented hindbrain, have been implicated in specifying structures along the anteroposterior and dorsoventral axes of the inner ear. In this work we have first characterized the generation and axial specification of the otic neurogenic domain, and second, we have investigated the effects of the mutation of kreisler/MafB - a gene transiently expressed in rhombomeres 5 and 6 of the developing hindbrain - in early otic patterning and cell specification. We show that kr/kr embryos display an expansion of the otic neurogenic domain, due to defects in otic patterning. Although many reports have pointed to the role of FGF3 in otic regionalisation, we provide evidence that FGF3 is not sufficient to govern this process. Neither Krox20 nor Fgf3 mutant embryos, characterized by a downregulation or absence of Fgf3 in r5 and r6, display ectopic neuroblasts in the otic primordium. However, Fgf3-/-Fgf10-/- double mutants show a phenotype very similar to kr/kr embryos: they present ectopic neuroblasts along the AP and DV otic axes. Finally, partial rescue of the kr/kr phenotype is obtained when Fgf3 or Fgf10 are ectopically expressed in the hindbrain of kr/kr embryos. These results highlight the importance of hindbrain-derived signals in the regulation of otic neurogenesis. © 2008 Elsevier Inc. All rights reserved., This work was supported by the grant BFU2006-05604 from the Spanish Ministry of Education and Science to C.P. and BFU2007-61030, Tercel, Ciberned and Junta de Castilla y León to T.S. C.V.E. was supported by a FI fellowship from AGAUR (Generalitat de Catalunya), and by CONACYT (Mexico); E.D.F. was supported by a FPI fellowship from the MEC, Spain.

The homeodomain transcription factor vHNF1 plays an essential role in the patterning of the caudal segmented hindbrain, where it participates in the definition of the boundary between rhombomeres (r) 4 and 5 and in the specification of the identity of r5 and r6. Understanding the molecular basis of vHnf1 own expression therefore constitutes an important issue to decipher the regulatory network governing hindbrain patterning. We have identified a highly conserved 800-bp enhancer element located in the fourth intron of vHnf1 and whose activity recapitulates vHnf1 neural expression in transgenic mice. Functional analysis of this enhancer revealed that it contains two types of essential motifs, a retinoic acid response element and two half T-MARE sites, indicating that it integrates direct inputs from the retinoic acid signaling cascade and MAF-related factors. Our data suggest that MAFB, which is itself regulated by vHNF1, acts as a positive modulator of vHnf1 in r5 and r6, whereas another MAF-related factor is absolutely required for the expression of vHnf1 in both the hindbrain and the spinal cord. We propose a model accounting for the initiation and maintenance phases of vHnf1 expression and for the establishment of the r4/r5 boundary, based on cooperative contributions of Maf factors and retinoic acid signaling.

The secreted frizzled-related proteins (Sfrp) are a family of soluble proteins with diverse biological functions having the capacity to bind Wnt ligands, to modulate Wnt signalling, and to signal directly via the Wnt receptor, Frizzled. In an enhancer trap screen for embryonic expression in zebrafish we identified an sfrp1 gene. Previous studies suggest an important role for sfrp1 in eye development, however, no data have been reported using the zebrafish model. In this paper, we describe duplicate sfrp1 genes in zebrafish and present a detailed analysis of the expression profile of both genes. Whole mount in situ hybridisation analyses of sfrp1a during embryonic and larval development revealed a dynamic expression profile, including: the central nervous system, where sfrp1a was regionally expressed throughout the brain and developing eye; the posterior gut, from the time of endodermal cell condensation; the lateral line, where sfrp1a was expressed in the migrating primordia and interneuromast cells that give rise to the sensory organs. Other sites included the blastoderm, segmenting mesoderm, olfactory placode, developing ear, pronephros and fin-bud. We have also analysed sfrp1b expression during embryonic development. Surprisingly this gene exhibited a divergent expression profile being limited to the yolk syncytium under the elongating tail-bud, which later covered the distal yolk extension, and transiently in the tail-bud mesenchyme. Overall, our studies provide a basis for future analyses of these developmentally important factors using the zebrafish model.

The zinc-finger transcription factor Krox20 constitutes a key regulator of hindbrain development, essential for the formation and specification of rhombomeres (r) 3 and 5. It is in particular responsible for the respective activation and repression of odd- and even-numbered rhombomere-specific genes, which include Hox genes. In this study, we have identified PIASxbeta as a novel direct interactor of Krox20. In addition, we found that PIASxbeta is able to activate the r4-specific gene Hoxb1. Binding of Krox20 prevents this activation, providing a molecular basis for the repression of Hoxb1 by Krox20. The same domain in the Krox20 protein, the zinc-fingers, is involved in DNA binding for transcriptional activation and in interaction with PIASxbeta for transcriptional repression, although the actual precise contacts are different. Our findings add an additional level in the complexity of Hox gene regulation and provide an example of how a single regulator can coordinate the activation and repression of a set of genes by very different mechanisms, acting as a molecular switch to specify cell identity and fate.

PMID:16615895, Neuronal migration and axon guidance constitute fundamental processes in brain development that are generally studied independently. Although both share common mechanisms of cell biology and biochemistry, little is known about their coordinated integration in the formation of neural circuits. Here we show that the development of the thalamocortical projection, one of the most prominent tracts in the mammalian brain, depends on the early tangential migration of a population of neurons derived from the ventral telencephalon. This tangential migration contributes to the establishment of a permissive corridor that is essential for thalamocortical axon pathfinding. Our results also demonstrate that in this process two different products of the Neuregulin-1 gene, CRD-NRG1 and Ig-NRG1, mediate the guidance of thalamocortical axons. These results show that neuronal tangential migration constitutes a novel mechanism to control the timely arrangement of guidance cues required for axonal tract formation in the mammalian brain., We have been supported by grants from Spanish Government BMC2002-03337, GVA GRUPOS03/053, NARSAD, the European Commission through STREP contract number 005139 (INTERDEVO), and the EURYI program to O.M.; by grants from INSERM, MENRT, ARC, and AFM to P.C.; by NIH grant NS29071 to L.R. and D.T.; and by the Picasso PAI/Programa de Acciones Integradas to O.M. and S.G. G.L.-B. is a “Ramón y Cajal” Investigator from the CSIC. F.B. was supported by a fellowship from the Académie Nationale de Médecine. S.G. is a recipient of the Human Frontier Science Program Organization CDA. O.M. is an EMBO Young Investigator, a NARSAD Young Investigator, and an EURYI Awardee.

Boundary cap (BC) cells are neural crest derivatives that form clusters at the surface of the neural tube, at entry and exit points of peripheral nerve roots. Using various knock-in alleles of the mouse gene Egr2 (also known as Krox20), the expression of which, in trunk regions, is initially restricted to BC cells, we were able to trace BC cell progeny during development and analyze their fate. Trunk BC-derived cells migrated along peripheral axons and colonized spinal nerve roots and dorsal root ganglia (DRG). All Schwann cell precursors occupying the dorsal roots were derived from BC cells. In the DRG, BC-derived cells were the progenitors of both neurons, mainly nociceptive afferents, and satellite cells. These data indicate that BC cells constitute a source of peripheral nervous system (PNS) components that, after the major neural crest ventrolateral migratory stream, feeds a secondary wave of migration to the PNS.

Helix-loop-helix transcription factors of the Ebf/Olf1 family have previously been implicated in the control of neurogenesis in the central nervous system in both Xenopus laevis and the mouse, but their precise roles have remained unclear. We have characterised two family members in the chick, and have performed a functional analysis by gain- and loss-of-function experiments. This study revealed several specific roles for Ebf genes in the spinal cord and hindbrain regions of higher vertebrates, and enabled their precise positioning along the neurogenic cascade.During neurogenesis, cell cycle exit appears to be tightly coupled to migration to the mantle layer and to neuronal differentiation. We show that antagonizing Ebf gene activity allows the uncoupling of these processes. Ebf gene function is necessary to initiate neuronal differentiation and migration toward the mantle layer in neuroepithelial progenitors, but it is not required for cell cycle exit. Ebf genes therefore appear to be master controllers of neuronal differentiation and migration, coupling them to cell cycle exit and earlier steps of neurogenesis.Mutual activation between proneural and Ebf genes suggests that besides their involvement in the engagement of differentiation, Ebf genes may also participate in the stabilisation of the committed state. Finally,gain-of-function data raise the possibility that, in addition to these general roles, Ebf genes may be involved in neuronal subtype specification in particular regions of the CNS.

E-cadherin is thought to mediate intercellular adhesion in the mammalian epidermis and in hair follicles as the adhesive component of adherens junctions. We have tested this role of E-cadherin directly by conditional gene ablation in the mouse. We show that postnatal loss of E-cadherin in keratinocytes leads to a loss of adherens junctions and altered epidermal differentiation without accompanying signs of inflammation. Overall tissue integrity and desmosomal structures were maintained, but skin hair follicles were progressively lost. Tumors were not observed and beta-catenin levels were not strongly altered in the mutant skin. We conclude that E-cadherin is required for maintaining the adhesive properties of adherens junctions in keratinocytes and proper skin differentiation. Furthermore, continuous hair follicle cycling is dependent on E-cadherin.

Neuroepithelial b2T cells were derived from the hindbrain and the spinal cord of mouse transgenic embryos, which expressed SV40 T antigen under the control of a Hoxb2 enhancer. Strikingly, b2T cell lines of either origin exhibit a very similar gene expression pattern, including markers of the hindbrain and the spinal cord, such as Hox genes, but not of more anterior cephalic regions. In addition, the broad expression pattern of b2T cells, probably linked to culture conditions, appeared to be appropriately modulated when the cells were reimplanted at different longitudinal levels into chick host embryos, suggesting that these cells are responsive to exogenous signalling mechanisms. Further support for these allegations was obtained by culturing b2T cells in defined medium and by assessing the expression of Krox20, an odd-numbered rhombomere marker, which appeared to be modulated by a complex interplay between FGF, retinoic acid (RA), and noggin. With respect to these as yet unique properties, b2T cells constitute an original alternative tool to in vivo models for the analysis of molecular pathways involved in the patterning of the neural tube.

Spinal motor neurons must extend their axons into the periphery through motor exit points (MEPs), but their cell bodies remain within spinal motor columns. It is not known how this partitioning is established in development. We show here that motor neuron somata are confined to the CNS by interactions with a neural crest subpopulation, boundary cap (BC) cells that prefigure the sites of spinal MEPs. Elimination of BC cells by surgical or targeted genetic ablation does not perturb motor axon outgrowth but results in motor neuron somata migrating out of the spinal cord by translocating along their axons. Heterologous neural crest grafts in crest-ablated embryos stop motor neuron emigration. Thus, before the formation of a mature transitional zone at the MEP, BC cells maintain a cell-tight boundary that allows motor axons to cross but blocks neuron migration.

The bZip transcription factor Mafb is expressed in two segments of the developing vertebrate hindbrain: the rhombomeres 5 and 6. Loss of Mafb expression in the mouse mutant kreisler leads to elimination of r5 and to alterations of r6 regional identity. Here, we further investigated the role of Mafb in hindbrain patterning using gain-of-function experiments in the chick embryo. Our work has revealed novel functions for Mafb, including a positive autoregulatory activity, the capacity to repress Hoxb1 expression, and the capacity to synergise with or antagonise Krox20 activity. These different activities appear to be spatially restricted in the hindbrain, presumably due to interactions with other factors. Reinvestigation of the kreisler mutation indicated that it also results in an ectopic activation of Mafb in rhombomere 3, accounting for the previously described molecular alterations of this rhombomere in the mutant. Together, these data allow us to refine our view of the dual function of Mafb in both segmentation and specification of anteroposterior identity in the hindbrain.

The calcium-dependent adhesion protein E-cadherin is present in noncompacted regions of myelin sheaths in the peripheral nervous system. There, it is localized to electron-dense structures between membranes of the same Schwann cell referred to as autotypic adherens junctions. It has been suggested that the failure of E-cadherin-mediated adhesion might cause demyelination that proceeds in certain pathological states. To test the requirement of E-cadherin in peripheral nerves, we used tissue-specific gene ablation techniques based on the Cre/LoxP system. We show that E-cadherin deficiency does not cause significant demyelination up to the age of 15 months. Immunostainings for nodal sodium channels, the paranodal protein Caspr1, and the juxtaparanodal potassium channels Kv1.1 and Kv1.2 revealed that E-cadherin is not necessary to maintain the general functional architecture of the nodal region. On the ultrastructural level, we detected a widening of the outer mesaxon accompanied by a loss of electron-dense cytoplasmic areas. We conclude that E-cadherin is required for the proper establishment and/or the maintenance of the outer mesaxon in myelinated PNS fibers but is dispensable for proper nerve function.

International audience; The induction of long-term potentiation (LTP) in the dentate gyrus of the hippocampus is associated with a rapid and robust transcription of the immediate early gene Zif268. We used a mutant mouse with a targeted disruption of Zif268 to ask whether this gene, which encodes a zinc finger transcription factor, is required for the maintenance of late LTP and for the expression of long-term memory. We show that whereas mutant mice exhibit early LTP in the dentate gyrus, late LTP is absent when measured 24 and 48 hours after tetanus in the freely moving animal. In both spatial and non-spatial learning tasks, short-term memory remained intact, whereas performance was impaired in tests requiring long-term memory. Thus, Zif268 is essential for the transition from short- to long-term synaptic plasticity and for the expression of long-term memories.

The molecular mechanisms controlling the process of myelination by Schwann cells remain elusive, despite recent progress in the identification and characterization of genes encoding myelin components (reviewed in ref. 1). We have created a null allele in the mouse Krox-20 gene, which encodes a zinc-finger transcription factor, by in-frame insertion of the Escherichia coli lacZ gene, and have shown that hindbrain segmentation is affected in Krox-20-/- embryos. We demonstrate here that Krox-20 is also activated in Schwann cells before the onset of myelination and that its disruption blocks Schwann cells at an early stage in their differentiation, thus preventing myelination in the peripheral nervous system. In Krox-20-/- mice, Schwann cells wrap their cytoplasmic processes only one and a half turns around the axon, and although they express the early myelin marker, myelin-associated glycoprotein, late myelin gene products are absent, including those for protein zero and myelin basic protein. Therefore Krox-20 is likely to control a set of genes required for completion of myelination in the peripheral nervous system.

Neurofibromatosis type 1 (NF1) is one of the most prevalent dominantly inherited genetic diseases of the nervous system. NF1 encodes a tumor suppressor whose functional loss results in the development of benign neurofibromas that can progress to malignancy. Neurofibromas are complex tumors composed of axonal processes, Schwann cells, fibroblasts, perineurial cells, and mast cells. Through use of a conditional (cre/lox) allele, we show that loss of NF1 in the Schwann cell lineage is sufficient to generate tumors. In addition, complete NF1-mediated tumorigenicity requires both a loss of NF1 in cells destined to become neoplastic as well as heterozygosity in non-neoplastic cells. The requirement for a permissive haploinsufficient environment to allow tumorigenesis may have therapeutic implications for NF1 and other familial cancers.

Krox-20, a zinc finger transcription factor with similarity to Sp1, is likely to play an important role in the development of the vertebrate central nervous system. A knowledge of its molecular properties will help to understand its physiological functions. We have therefore performed a structure-function analysis of the protein to identify the regions involved in DNA-binding and transcriptional activation. Our data suggest that only the zinc fingers are required for high affinity, specific DNA-binding. Transcriptional activation was not affected by deletion of the C-terminal tail of the protein. In contrast, deletion of the N-terminal half, upstream of the zinc fingers, completely abolished transactivation without affecting DNA-binding or nuclear localization. Two transcriptional activation domains were identified in this region. They cooperate to establish full activity. They are rich in negatively-charged amino acids and are therefore may constitute acidic activation domains. Comparative analysis of the amino acid sequences of several zinc finger proteins belonging to the Krox-20 subfamily indicates that they contain acidic regions at similar locations within their N-terminal region, suggesting that the functional organization of these proteins has been conserved during evolution.

International audience; Neural crest (NC) cells are a multipotent, highly migratory cell population that generates most of the components of the peripheral nervous system (PNS), including the glial Schwann cells (SC) and boundary cap (BC) cells. These latter cells are located at the interface between the central nervous system and PNS, at the exit/entry points of ventral motor/dorsal sensory axons and give rise to all SC in the nerve roots and to a subset of nociceptive neurons and satellite cells in the dorsal root ganglia. In the present study we have compared BC cells with two closely related cell types, NC and Schwann cell precursors (SCpr), by RNA profiling. This led to the definition of a set of 10 genes that show specific expression in BC cells and/or in their derivatives along the nerve roots. Analysis of the expression of these genes during mouse development revealed novel features, of those most important are: (i) dorsal and ventral nerve root BC cell derivatives express different sets of genes, suggesting that they have distinct properties; (ii) these cells undergo major modifications in their gene expression pattern between embryonic days 14.5 and 17.5, possibly linked to the SCpr-immature Schwann cell transition; (iii) nerve roots SC differ from more distal SC not only in their origins and locations, but also in their gene expression patterns. In conclusion, the identification of these novel makers opens the way for a detailed characterization of BC cells in both mouse and man.

Zinc fingers constitute important eukaryotic DNA-binding domains, being present in many transcription factors. The Cys2/His2 zinc-finger class has conserved motifs of 28-30 amino acids which are usually present as tandem repeats. The structure of a Cys2/His2 zinc finger has been determined by nuclear magnetic resonance, but details of its interaction with DNA were not established. Here we identify amino acids governing DNA-binding specificity using in vitro directed mutagenesis guided by similarities between the zinc fingers of transcription factors Sp1 and Krox-20. Krox-20 is a serum-inducible transcription activator which is possibly involved in the regulation of hindbrain development; it contains three zinc fingers similar to those of Sp1 and binds to a 9-base-pair target sequence which is related to that of Sp1. Our results show that each finger spans three nucleotides and indicate two positions in Krox-20 zinc fingers that are important for base-pair selectivity. Modelling with molecular graphics suggests that these residues could bind directly with the bases and that other amino acid-base contacts are also possible.

We report here part of the genomic sequence of the mouse homolog of the human gene encoding the transcription factor Sp1. This gene belongs to a subfamily of zinc finger proteins, which includes another transcription factor, Krox-20. The analysis of the mouse Sp1 nucleotide sequence revealed that (i) the C-terminal part of the protein is conserved between man and mouse, consistent with a possible role in transcriptional activation; (ii) while the there Krox-20 zinc fingers are encoded by a unique exon, an intron separates the regions encoding fingers 2 and 3 of Sp1. On the basis of this latter observation, we propose a model for the evolution of the Sp1/Krox-20 gene family which might shed some light on the role of introns in the evolution of modular proteins.

Zinc fingers of the Cys2/His2 class are conserved 28-30 amino acid motifs that constitute an important and widespread family of eukaryotic DNA-binding domains. It is therefore of great interest to understand the rules that govern specific recognition of DNA by zinc fingers. The DNA-binding domain of the transcription factor Krox-20 consists of three zinc fingers, each of them making its primary contacts with a three-base pair subsite. We have performed a data base-guided site-directed mutagenesis analysis of Krox-20: nine derivatives were generated, in which one to three amino acid changes had been introduced within finger 2, at positions which were likely to affect the specificity of DNA recognition. The affinities of the different proteins for a panel of potential DNA binding sites were estimated by gel retardation assay. Six of the derivatives bound specific targets with affinities comparable to that of wild type Krox-20 for its consensus binding site. However, the specificity of recognition was dramatically modified at the expected bases, in a manner that could be explained by examining the newly introduced amino acids within the context of the overall finger/triplet interaction. These data provide new insights into the details of zinc finger-DNA interactions and, combined with the modular nature of zinc fingers, illustrate both the potential and the difficulties of utilising these motifs for designing DNA-binding proteins with novel specificities.

The structural gene coding for both polypeptides I and II which are the two major polypeptides of the Hepatitis B surface antigen, is found to be localized on the viral genome. This gene, referred to as gene S, is located in the partially single stranded region. It maps between positions 73.6 and 95.1% of the genome length. It is composed of 678 nucleotides, which correspond to a theoretical polypeptide of 25,422 molecular weight.

Bacteriophage vectors derived from lambda plac5 have been constructed. Their genomes have one EcoRI restriction site which is located at the very beginning of the lac Z gene. The major part of this gene was deleted by an in vivo intramolecular recombination. These vectors allow the fusion of a gene or an operon with the beginning of the lac Z gene, placing them under the control of the lac promoter, which carries the UV5 mutation. Some of these vectors (lambda Y) also include the lac Y gene and it too is under the control of the lac promoter. The lambda YEQS, which carries the Qam73 and Sam7 mutations, as safety mutations, has been certified as a B2 (EK2) vector by the French control commission "recombinaison génétique in vitro".

The effect of hemin on the rate of synthesis and the level of globin messenger RNA's in the human erythroid cell line K562 was examined by means of cloned hybridization probes specific for each of the human embryonic, fetal, and adult globin genes. Hemin increases both the rate of transcription and the level of accumulation of zeta-, epsilon-, gamma-, and alpha-globin messenger RNA's by a factor of 3 to 5. Thus, hemin induction of globin gene expression in K562 cells is at the level of transcription.

Immunochemical investigations of the viral antigens and molecular characterization of the viral DNA have elucidated the nature of the hepatitis B virus infection underlying acute, chronic, and oncogenic disorders of the liver in man. Cloning and sequencing of viral DNA have made possible studies on the structure of the genome and on certain aspects of the biology of the virus, hitherto constrained for a lack of tissue culture systems and laboratory animal models useful in its propagation.