Two genetically separable steps in the differentiation of thymic epithelium.

The development of the thymus depends initially on epithelial-mesenchymal and subsequently on reciprocal lympho-stromal interactions. The genetic steps governing development and differentiation of the thymic microenvironment are unknown. With the use of a targeted disruption of the whn gene, which recapitulates the phenotype of the athymic nude mouse, the WHN transcription factor was shown to be the product of the nude locus. Formation of the thymic epithelial primordium before the entry of lymphocyte progenitors did not require the activity of WHN. However, subsequent differentiation of primitive precursor cells into subcapsular, cortical, and medullary epithelial cells of the postnatal thymus did depend on activity of the whn gene. These results define the first genetically separable steps during thymic epithelial differentiation.

The whn transcription factor encoded by the nude locus contains an evolutionarily conserved and functionally indispensable activation domain.

Mutations in the whn gene are associated with the phenotype of congenital athymia and hairlessness in mouse and rat. The whn gene encodes a presumptive transcription factor with a DNA binding domain of the forkhead/ winged-helix class. Two previously described null alleles encode truncated whn proteins lacking the characteristic DNA binding domain. In the rat rnu allele described here, a nonsense mutation in exon 8 of the whn gene was identified. The truncated whnrnu protein contains the DNA binding domain but lacks the 175 C-terminal amino acids of the wild-type protein. To facilitate the identification of functionally important regions in this region, a whn homolog from the pufferfish Fugu rubripes was isolated. Comparison of derived protein sequences with the mouse whn gene revealed the presence of a conserved acidic protein domain in the C terminus, in addition to the highly conserved DNA binding domain. Using fusions with a heterologous DNA binding domain, a strong transcriptional activation domain was localized to the C-terminal cluster of acidic amino acids. As the whnrnu mutant protein lacks this domain, our results indicate that a transactivation function is essential for the activity of the whn transcription factor.

Expression of peptides encoded by exons in cloned mammalian DNA.

New synthetic approaches, such as combinatorial chemistry, provide a rich source of potential drug candidates. At the same time, the human genome initiative and other large-scale sequencing projects provide a large number of novel drug targets. However, the functional analysis of thousands of new genes remains a major challenge for the future. A systematic strategy for genome-wide functional analysis of genes could employ the fact that at least some modules in multi-domain proteins are encoded in individual exons. Exon amplification provides information about coding regions of most genes that is independent of their transcriptional status; exon amplification from entire mammalian genomes has been demonstrated. Here, we describe the development of an exon-trap system, lambdaGEE (for genomic exon expression), that couples exon amplification with the expression of exon-encoded peptides.

Detection of the in vivo incorporation of a metal cluster into a protein. The FeMo cofactor is inserted into the FeFe protein of the alternative nitrogenase of Rhodobacter capsulatus.

The photosynthetic bacterium Rhodobacter capsulatus has, in addition to the Mo nitrogenase, a second Mo-independent nitrogen-fixing system, an 'iron-only' nitrogenase which is strongly repressed by molybdate. The MoO4(2-) concentration causing 50% repression of the alternative nitrogenase in nifHDK- cells was 6 nM. If MoO4(2-) was added to a growing nifHDK- culture which had already expressed the alternative nitrogenase, the production of ethane from acetylene, by whole cells, was stimulated dramatically. In spite of the fact that C2H4 formation decreased continuously during the duration of the experiment (3 days), the total C2H6 production increased about twofold within the first 24 h, whereas the relative yield of C2H6 increased from 2% (C2H6/C2H4 x 100) in the absence of MoO4(2-), to a maximal value of 69% in the presence of MoO4(2-) (1 mM) after 72 h incubation. This 'Mo effect' appeared to be stronger the higher the MoO4(2-) concentration in the medium and the longer the incubation time. In the presence of ReO4-, WO4(2-) or VO4(3-), a similar effect did not occur. The 'Mo effect' was not observed in a nifHDK- nifE- double mutant which is unable to synthesize the FeMo cofactor and was diminished in a nifHDK- nifQ- mutant. Crude extracts from nifHDK- cells cultivated in the presence of MoO4(2-), also showed enhanced production of ethane. Component 1, purified from those extracts, displayed an S = 3/2 EPR signal which was relatively weak but characteristic for the FeMoco. These results strongly support the suggestion that the 'Mo effect' is a consequence of the formation of a hybrid enzyme consisting of the apoprotein of the alternative nitrogenase and the FeMo cofactor of the conventional nitrogenase. The 'Mo effect' was not influenced by the addition of chloramphenicol to the cultures. The occurrence of the 'Mo effect' appeared, therefore, to be independent of de-novo protein synthesis. The analysis of nifE-lacZ and nifN-lacZ fusions proved that both genes necessary for the FeMo cofactor synthesis are also expressed under conditions of MoO4(2-) deficiency. The possible explanations for incorporation of the FeMoco into component 1 of the alternative nitrogenase are discussed.

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.