Dynamic expression patterns of the new protocadherin families CNRs and Pcdh-gamma during mouse odontogenesis: comparison with reelin expression.

Protocadherins are transmembrane glycoproteins belonging to the cadherin superfamily of molecules, which are involved in many biological processes such as cell adhesion, cytoskeletal organization and morphogenesis. Protocadherins generally exhibit only moderate adhesive activity and are highly expressed in the nervous system. Here, we report on the expression pattern of two novel families of protocadherins (CNRs and Pcdh-gamma) during rodent teeth development. Furthermore, we compare their expression with that of reelin, which is the potential ligand of CNRs. Throughout odontogenesis, CNRs, Pcdh-gamma and reelin show dynamic spatiotemporal expression patterns, which relate to both morphogenesis and cell differentiation events.

Juxtaposition of CNR protocadherins and reelin expression in the developing spinal cord.

The CNR (cadherin-related neuronal receptors) family of protocadherins is of great interest because of their potential roles as molecular tags in the formation of specific synaptic connections, and as receptors for reelin, during neuronal migration, and cell body positioning. In order to know more about potential functions of CNRs we have mapped their expression during mouse nervous system development and compared their expression with that of reelin and its intracellular effector Dab1 in several tissues. In spinal cord, CNRs and Dab1 are expressed in motoneurons, while reelin is located in adjacent cells. In the hindbrain, there is a differential expression of CNRs and Dab1 in various motor nuclei. In the retina and olfactory system, we observe CNR and reelin expression but not that of Dab1. These results provide new insights into the potential functions of CNRs and their possible integration in the reelin pathway during development.

Death of motoneurons induced by trophic deprivation or by excitotoxicity is not prevented by overexpression of SMN.

The telomeric copy of the survival motor neuron gene (SMN1) is deleted or mutated in all spinal muscular atrophy (SMA) patients and these patients present mainly a loss in spinal motoneurons. Although studies performed in HeLa cells suggest that SMN may be involved in the biogenesis and possibly in recycling of spliceosomal small nuclear ribonucleoproteins (snRNPs), no link has been established between this function and the consequence of the absence of SMN in the specific loss of motoneurons. We attempted to answer the question of whether SMN plays a direct role in motoneuron survival by transducing cultured motoneurons with lentiviral vectors coding either for an antisense Smn mRNA or for full-length or truncated forms of SMN. We studied their effect on survival under different anti- or proapoptotic culture conditions. Our results show that increased levels of SMN are unable to protect motoneurons from death induced by trophic deprivation or by excitotoxicity. These results suggest that SMN is not a survival factor per se for motoneurons. In addition, overexpression of a truncated form of SMN shown to induce a modified subcellular localization and to exert a dominant-negative effect on snRNP biogenesis and RNA splicing in HeLa cells was ineffective in modifying both localization and survival in motoneurons.

Crystallization studies on phase-change optical recording media by use of a two-dimensional periodic mark array.

We present the results of crystallization studies in thin-film samples of amorphous and crystalline Ge(x)Sb(y)Te(z). The experiments, conducted at moderately elevated temperatures, are based on measurements of the first-order diffraction efficiency from a two-dimensional periodic array of recorded marks. When the samples are slowly heated above room temperature, changes in the efficiencies of various diffracted orders give information about the on-going crystallization process within the sample. Two different compositions of the GeSbTe alloy are used in these experiments. Measurements on Ge(2)Sb(2.3)Te(5) films show crystallization dominated by nucleation. For the Sb-rich eutectic composition Ge-(SbTe), crystallization is found to be dominated by growth from crystalline boundaries. We also show that crystalline marks written by relatively high-power laser pulses are different in their optical properties from the regions crystallized by slow heating of the sample to moderate temperatures.

Expression and subcellular localization of two isoforms of the survival motor neuron protein in different cell types.

The survival motor neuron (SMN) gene is deleted or mutated in over 98% of spinal muscular atrophy patients who show specific motoneuron loss. By performing transfection experiments with rat smn cDNA, we show that two isoforms of SMN with Mr of 32 kDa and 35 kDa are produced by the same cDNA. In cultured motoneurons, both forms colocalize in coiled bodies and not in GEMS bodies as shown for HeLa cells. Subcellular fractionation of cells acutely dissociated from rat embryonic ventral spinal cord shows that the two SMN isoforms have a different subcellular localization, namely, that the 32 kDa isoform is enriched in the cytosol, whereas the 35 kDa isoform is segregating in the microsomal fraction. We show that the 35 kDa isoform of SMN is part of an insoluble complex but is absent from the cytoplasmic membranes and from the mitochondria. Immunostaining studies show that neither SMN isoform colocalizes with Bcl-2, the mitochondrial antiapoptotic protein suggested to bind to SMN in HeLa cells. Our results show that the isoforms of SMN protein have different subcellular localization and may therefore play independent biological roles. Moreover, the absence of colocalization of SMN with Bcl-2 in motoneurons suggests that some of the interactors of SMN may vary depending on the cell type, and this underscores the importance of identifying motoneuron-specific SMN interactors.

The Drosophila modifier of variegation modulo gene product binds specific RNA sequences at the nucleolus and interacts with DNA and chromatin in a phosphorylation-dependent manner.

modulo belongs to the modifier of Position Effect Variegation class of Drosophila genes, suggesting a role for its product in regulating chromatin structure. Genetics assigned a second function to the gene, in protein synthesis capacity. Bifunctionality is consistent with protein localization in two distinct subnuclear compartments, chromatin and nucleolus, and with its organization in modules potentially involved in DNA and RNA binding. In this study, we examine nucleic acid interactions established by Modulo at nucleolus and chromatin and the mechanism that controls the distribution and balances the function of the protein in the two compartments. Structure/function analysis and oligomer selection/amplification experiments indicate that, in vitro, two basic terminal domains independently contact DNA without sequence specificity, whereas a central RNA Recognition Motif (RRM)-containing domain allows recognition of a novel sequence-/motif-specific RNA class. Phosphorylation moreover is shown to down-regulate DNA binding. Evidence is provided that in vivo nucleolar Modulo is highly phosphorylated and belongs to a ribonucleoprotein particle, whereas chromatin-associated protein is not modified. A functional scheme is finally proposed in which modification by phosphorylation modulates Mod subnuclear distribution and balances its function at the nucleolus and chromatin.

Abnormal rearrangements associated with V(D)J recombination in Fanconi anemia.

The hallmark of Fanconi anemia (FA), a rare inherited cancer prone disorder, is a high level of chromosome breakage, spontaneous and induced by cross-linking agents. The increased genomic instability of FA is reflected at the gene level by an overproduction of intragenic deletions. Two of the eight FA genes have been cloned, however, their function remains unknown. We recently demonstrated that the lack of functional FA genes lead to a marked decrease in the fidelity of non-homologous end-joining, a pathway that mammalian cells predominantly use to repair DNA double-strand breaks (DSB). Knowing that specific DSB are generated during V(D)J recombination, here we have examined the molecular features of V(D)J rearrangements in normal and FA lymphoblasts belonging to complementation groups C and D. Using appropriate extrachromosomal recombination substrates, V(D)J coding and signal joint formation have been analysed quantitatively and qualitatively. Our results show that the frequency of coding and signal joint formation was not significantly different in normal and FA cells. However, when the fidelity of the V(D)J reaction was examined, we found that in normal human lymphoblasts V(D)J recombination proceeds with high precision, whereas, in FA cells a several fold increase in the frequency of aberrant rearrangements is associated with V(D)J coding joint formation. The abnormal recombinants that we recovered in FA are consistent with excessive degradation of DNA ends generated during the V(D)J reaction. On the basis of these findings, we propose a working model in which FA genes play a role in the control of the fidelity of rejoining of specific DNA ends. Such a defect may explain several basic features of FA, such as chromosomal instability and deletion proneness.

Dynamics of the sub-nuclear distribution of Modulo and the regulation of position-effect variegation by nucleolus in Drosophila.

modulo belongs to the class of Drosophila genes named 'suppressor of position-effect variegation', suggesting the involvement of the encoded protein in chromatin compaction/relaxation processes. Using complementary procedures of cell fractionation, immunolocalisation on mitotic and polytene chromosomes and cross-linking/immunoprecipitation of genomic DNA targets, we have analysed the sub-nuclear distribution of Modulo. While actually associated to condensed chromatin and heterochromatin sites, the protein is also abundantly found at nucleolus. From a comparison of Modulo pattern on chromosomes of different cell types and mutant lines, we propose a model in which the nucleolus balances the Modulo protein available for chromatin compaction and PEV modification. At a molecular level, repetitive elements instead of rDNA constitute Modulo DNA targets, indicating that the protein directly contacts DNA in heterochromatin but not at the nucleolus. Consistent with a role for Modulo in nucleolus activity and protein synthesis capacity, somatic clones homozygous for a null mutation express a cell-autonomous phenotype consisting of growth alteration and short slender bristles, characteristic traits of Minute mutations, which are known to affect ribosome biogenesis. The results provide evidence suggesting that Modulo participates in distinct molecular networks in the nucleolus and heterochromatin and has distinct functions in the two compartments.

The presence of direct repeats does not influence coding joint formation during V(D)J recombination.

During the recombination process that assembles immunoglobulin and T-cell receptor gene segments, the coding ends to be joined are extensively processed. Contradictory reports have been made in the past about the existence of homology directed mechanisms in V(D)J recombination. In this study we analyse coding end processing and the influence of the presence of homology stretches on coding joint formation using artificial substrates in which short sequence changes creating direct repeats have been introduced. These changes were monitored 3 bp away from the termini in order to avoid any differences due to the initiation steps of V(D)J recombination. Our results show that the sequence of the coding ends influences joint formation, but no evidence was found for a mechanistic bias due to the presence of direct repeats.

Methylation status of immunoglobulin kappa gene segments correlates with their recombination potential.

We have previously shown that unlike endogenous chi genes, unrearranged chi transgenes undergo V chi-J chi recombination in Tas well as B cells of transgenic mice. To determine whether the difference in recombination specificity of the transgenic and endogenous chi genes is associated with differences in DNA structure, the methylation status of the endogenous genes and three unrearranged chi transgenes was compared. The J chi-C chi locus of the transgenes was found to be hypomethylated in all tissues of the transgenic mice. In contrast, methylation of the endogenous chi genes was tissue and developmentally regulated. Hypomethylation of the endogenous J chi-C chi region occurs only in cells of the B lineage undergoing, or having completed chi gene recombination. Transfection of fibroblasts from transgenic and control mice with the recombination activating genes, Rag1 and Rag2, led to a high level of rearrangement of the hypomethylated transgenic, but not the endogenous chi genes. These results suggest that hypomethylation defines an accessible state of the chi locus and that methylation/demethylation could be involved in the control of chi gene rearrangement during lymphocyte differentiation.

The intronic immunoglobulin kappa gene enhancer acts independently on rearrangement and on transcription.

It is not known which cis-acting elements regulate kappa gene rearrangement. However, the onset of rearrangement coincides with the onset of transcription suggesting that common regulatory elements are used. We have investigated the role of the intronic enhancer on rearrangement using mice transgenic for kappa minigenes. The rabbit kappa enhancer, which is defective in activation of transcription in mouse cells, allows a high level of rearrangement. However, transgenes which possess no enhancer region at all are rearranged a hundred times less compared to transgenes which possess the rabbit enhancer. Our results suggest that while the enhancer region can activate rearrangement as well as transcription, its action on both phenomena can be uncoupled.

Rag-1: a topoisomerase?

Recombination activating genes Rag-1 and Rag-2 were isolated on the basis of their ability to confer V(D)J recombination activity when co-expressed in fibroblasts. The mode of action of the confer V(D)J recombination activity when co-expressed in fibroblasts. The mode of action of the products of these genes is not known. Based on sequence comparison data, it was suggested that Rag-1 protein could act like a topoisomerase and that tyrosine in position 998 could be the active site tyrosine. We tested this hypothesis by introducing a point mutation on the Rag-1 cDNA, transforming the tyrosine codon into a phenylalanine codon. We show that the mutation has no effect on site specific recombination implying that Tyr-998 is not essential for the recombination reaction.

A V(D)J site-specific recombination model involving no compulsory double-stranded break formation at the coding segments.

Complete immunoglobulin and T-cell-receptor genes are assembled by site-specific recombination of separately encoded gene segments. We present a novel recombination model which accounts for all the characteristics of V(D)J recombination that have been described. The sequence of events proposed implies no formation of double-stranded breaks at the coding ends, ensuring continuity between the recombination partners during the reaction, and solves the problem of the ligation of extremities which have no complementarity. According to this recombination model, the formation of covalently sealed coding ends does not constitute a compulsory step in the recombination process.

Three lymphoid-specific factors account for all junctional diversity characteristic of somatic assembly of T-cell receptor and immunoglobulin genes.

The somatic diversity immunoglobulin and T-cell receptor diversity is largely provided by the junctional variation created during site-specific rearrangement of separately encoded gene segments. Using a transient transfection assay, we demonstrate that the recombination activating genes Rag1 and Rag2 direct site-specific rearrangement on an artificial substrate in poorly differentiated as well as in differentiated nonlymphoid cell lines. In addition to a high frequency of precise recombination events, coding joints show deletions and more rarely P-nucleotide insertions, reminiscent of immunoglobulin and T-cell receptor junctions found in fetal tissues. N-region insertions, which are characteristic of adult junctional diversity, are obtained at high frequency upon transfection of a terminal deoxynucleotidyltransferase expression vector together with Rag1 and Rag2. These results show that only three lymphoid-specific factors are needed to generate all types of junctional diversity observed during lymphoid development.

Mutation of immunoglobulin J chi splice sites does not affect the rearrangement frequency of J chi segments.

The rabbit b9 kappa 1 locus contains 5 joining (J chi) gene segments, only two of which, J chi 1 and J chi 2, are utilized in assembly of a complete variable region gene (as shown by Akimenko, M.-A., Mariamé, B. and Rougeon, F. (1986) Proc. natl. Acad. Sci. (Wash.), 83, 5180-5183). J chi 4 and J chi 5 do not rearrange because of deficient recombination signal sequences. The J chi 3 gene segment is also not rearranged and has a non-functional splice site. In view of the proposed relationship between transcription and rearrangement of immunoglobulin genes, we sought to determine whether splicing of the germline transcript is implicated in the rearrangement process. We addressed this question by introducing mutations in the splice sites of the J chi segments of an unrearranged kappa light chain immunoglobulin transgene. Rearrangement was analysed by polymerase chain reaction on transgenic spleen DNA. We observed that mutation of the splice sites had no effect on the utilization of the J chi gene segments, demonstrating that there is no relationship between splicing and rearrangement.

An exogenous albumin promoter can become silent in dedifferentiated hepatoma variants as well as intertypic hybrids.

In order to evaluate the ability of an exogenous tissue-specific promoter to undergo the same dynamic changes in activity as the endogenous one, a 400-base pair fragment of the rat albumin proximal promoter, upstream of the bacterial gpt gene, has been introduced into rat hepatoma cells. Four clones containing a single integrated copy of the construct and producing substantial amounts of albumin and of xanthine phosphoribosyltransferase were isolated. These clones were subjected to two treatments known to result in silencing of the albumin gene: selection for dedifferentiated variants, and fusion with L-cell fibroblasts. In most cases, the albumin-negative progeny obtained no longer expressed the gpt gene: the exogenous promoter of 400 base pairs must contain the sequences required to respond to the mechanisms that block activity of the endogenous gene. However, exceptions were observed: the albumin-deficient variants of one clone remained xanthine phosphoribosyltransferase positive, and some of the albumin-negative hybrids from a different clone continued to produce xanthine phosphoribosyltransferase. These cases of dissociation in expression of the endogenous and the exogenous genes indicate that the site of integration of the alb-gpt construct in one clone renders the sequences insensitive to the mechanisms responsible for albumin gene silencing in dedifferentiated variants, and in the other clone to the mechanism of extinction. Consequently, the mechanisms causing gene silencing in variants and in intertypic hybrids must be different.

Immunoglobulin kappa light chain gene promoter and enhancer are not responsible for B-cell restricted gene rearrangement.

We have produced transgenic mice which synthesize chimeric mouse-rabbit immunoglobulin (Ig) kappa light chains following in vivo recombination of an injected unrearranged kappa gene. The exogenous gene construct contained a mouse germ-line kappa variable (V kappa) gene segment, the mouse germ-line joining (J kappa) locus including the enhancer, and the rabbit b9 constant (C kappa) region. A high level of V-J recombination of the kappa transgene was observed in spleen of the transgenic mice. Surprisingly, a particularly high degree of variability in the exact site of recombination and the presence of non germ-line encoded nucleotides (N-regions) were found at the V-J junction of the rearranged kappa transgene. Furthermore, unlike endogenous kappa genes, rearrangement of the exogenous gene occurred in T-cells of the transgenic mice. These results show that additional sequences, other than the heptamer-nonamer signal sequences and the promoter and enhancer elements, are required to obtain stage- and lineage- specific regulation of Ig kappa light chain gene rearrangement in vivo.