Severe hydrocephalus in L1-deficient mice.

The neural adhesion molecule L1, a member of the immunoglobulin superfamily of cell recognition molecules, performs important functions in the developing and adult nervous system. This view is confirmed by the fact that mutations in the human L1 gene cause a severe neurological disease, termed CRASH (acronym for: corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraplegia, and hydrocephalus). X-linked hydrocephalus is certainly the most prominent symptom of CRASH syndrome. Mouse mutants deficient in L1 also develop enlarged ventricles. Here, we report that ventricular dilation in L1-deficient mice is not correlated with stenosis of the aqueduct of Sylvius nor with ultrastructural abnormalities of ependymal cells lining the lateral ventricles or the aqueduct. However, a few L1 mutant mice displayed severe hydrocephalus, characterized by a significant enlargement of the skull and an almost complete atrophy of the cerebral cortex. The aqueduct of these severely affected animals was completely closed. Since mutant animals from two independently generated L1-deficient mouse lines displayed a similar phenotype, we consider severe hydrocephalus as a specific consequence of L1-deficiency. However, results of the present study also indicate that severe hydrocephalus represents a secondary rather than a primary defect of the L1 mutation; our combined data suggest that deformations of the brain as a result of massively enlarged ventricles secondarily cause stenosis of the aqueduct and subsequently high pressure hydrocephalus.

The neural recognition molecule L1 is a sialic acid-binding lectin for CD24, which induces promotion and inhibition of neurite outgrowth.

Among the recognition molecules that determine a neuron's interaction with other cells, L1 and CD24 have been suggested to cooperate with each other in neurite outgrowth and signal transduction. Here we report that binding of CD24 to L1 depends on alpha2,3-sialic acid on CD24, which determines the CD24 induced and cell type-specific promotion or inhibition of neurite outgrowth. Using knockout mutants, we could show that the CD24-induced effects on neurite outgrowth are mediated via L1, and not GPI-linked CD24, by trans-interaction of L1 with sialylated CD24. This glycoform is excluded together with L1 from raft microdomains, suggesting that molecular compartmentation in the surface membrane could play a role in signal transduction.

Promoters controlling expression of the alternative nitrogenase and the molybdenum uptake system in Rhodobacter capsulatus are activated by NtrC, independent of sigma54, and repressed by molybdenum.

The alternative nitrogenase of Rhodobacter capsulatus is expressed only under conditions of nitrogen and molybdenum depletion. The analysis of anfA-lacZ fusions demonstrated that this dual control occurred at the level of transcription of anfA, which encodes a transcriptional activator specific for the alternative nitrogenase. The anfA promoter was found to be activated under nitrogen-limiting conditions by NtrC in a sigma54-independent manner. In addition, anfA transcription was repressed by traces of molybdenum. This molybdenum-dependent repression of anfA was released in R. capsulatus mutants carrying either lesions in the high-affinity molybdenum uptake system (modABCD) or a double deletion of mopA and mopB, two genes encoding molybdenum-pterin-binding proteins. The expression of the molybdenum transport system itself was shown to be negatively regulated by molybdenum and, unexpectedly, to be also regulated by NtrC. This finding is in line with the presence of two tandemly arranged DNA motifs located in front of the R. capsulatus mopA-modABCD operon, which are homologous to R. capsulatus NtrC binding sites. Mapping of the transcriptional initiation sites of mopA and anfA revealed promoter sequences exhibiting significant homology to each other but no homology to known prokaryotic promoters. In addition, a conserved DNA sequence of dyad symmetry overlapping the transcriptional initiation sites of mopA and anfA was found. Deletions within this element resulted in molybdenum-independent expression of anfA, indicating that this DNA sequence may be the target of MopA/MopB-mediated repression.

Characterization of anf genes specific for the alternative nitrogenase and identification of nif genes required for both nitrogenases in Rhodobacter capsulatus.

To identify Rhodobacter capsulatus nif genes necessary for the alternative nitrogenase, strains carrying defined mutations in 32 genes and open reading frames of nif region A, B or C were constructed. The ability of these mutants to grow on nitrogen-free medium with molybdenum (Nif phenotype) or in a nifHDK deletion background on medium without molybdenum (Anf phenotype) was tested. Nine nif genes and nif-associated coding regions are absolutely essential for the alternative nitrogenase. These genes comprise nifV and nifB, the nif-specific ntr system (nifR1, R2, R4) and four open reading frames, which exhibit no homology to known genes. In addition, a significantly reduced activity of both the alternative nitrogenase and the molybdenum-dependent nitrogenase was found for fdxN mutants. By random Tn5 mutagenesis of a nifHDK deletion strain 42 Anf- mutants were isolated. Southern hybridization experiments demonstrated that 17 of these Tn5 mutants were localized in at least 13 different restriction fragments outside of known nif regions. Ten different Anf- Tn5 mutations are clustered on a 6 kb DNA fragment of the chromosome designated anf region A. DNA sequence analysis revealed that this region contained the structural genes of the alternative nitrogenase (anfHDGK). The identification of several Tn5 insertions mapping outside of anf region A indicated that at least 10 genes specific for the alternative nitrogenase are present in R. capsulatus.

Functional analysis of the cysteine motifs in the ferredoxin-like protein FdxN of Rhizobium meliloti involved in symbiotic nitrogen fixation.

The Rhizobium meliloti fdxN gene, which is part of the nifA-nifB-fdxN operon, is absolutely required for symbiotic nitrogen fixation. The deduced sequence of the FdxN protein is characterized by two cysteine motifs typical of bacterial-type ferredoxins. The Fix-phenotype of an R. meliloti fdxN::[Tc] mutant could be rescued by the R. leguminosarum fdxN gene, whereas no complementation was observed with nif-associated genes encoding ferredoxins from Bradyrhizobium japonicum, Azotobacter vinelandii, A. chroococcum and Rhodobacter capsulatus. In addition to these heterologous genes, several R. meliloti fdxN mutant genes constructed by site-directed mutagenesis were analyzed. Not only a cysteine residue within the second cysteine motif (position 42), which is known to coordinate the Fe-S cluster in homologous proteins, but also a cysteine located down-stream of this motif (position 61), was found to be essential for the activity of the R. meliloti FdxN protein. Changing the amino acid residue proline in position 56 into methionine resulted in a FdxN mutant protein with decreased activity, whereas changes in positions 35 (Asp35Glu) and 45 (Gly45Glu) had no significant effect on the function of the FdxN mutant proteins. In contrast to bacterial-type ferredoxins, which contain two identical cysteine motifs of the form C-X2-C-X2-C-X3-C, nif-associated ferredoxins, including R. meliloti FdxN, are characterized by two different cysteine motifs. Six "additional" amino acids separate the second (Cys42) and the third cysteine (Cys51) in the C-terminal motif (C-X2-C-X8-C-X3-C).(ABSTRACT TRUNCATED AT 250 WORDS)

Selective malformation of the splenic white pulp border in L1-deficient mice.

Lymphocytes enter the splenic white pulp by crossing the poorly characterized boundary of the marginal sinus. In this study, we describe the importance of L1, an adhesion molecule of the Ig superfamily, for marginal sinus integrity. We find that germline insertional mutation of L1 is associated with a selective malformation of the splenic marginal sinus. Other splenic structures remain intact. Immunofluorescence analysis of the extracellular framework of the spleen, using an Ab to laminin, reveals that L1 knockout mice have an irregularly shaped, discontinuous white pulp margin. Electron microscopic analysis shows that it is associated with bizarrely shaped marginal sinus lining cells at the periphery of the white pulp. These abnormalities correlate with the localization of L1 in normal mice in that L1 is normally expressed on marginal sinus lining cells at the white pulp border. These L1-immunopositive lining cells coexpress high levels of mucosal addressin cell adhesion molecule-1 and vimentin, indicating that they are of fibroblastic lineage and express a well-characterized addressin. Our findings are the first to implicate L1 in splenic lymphoid architectural development. Moreover, these findings help define the poorly characterized sinusoidal boundary across which mononuclear cells cross to enter the splenic white pulp.