Induced overexpression of AID in CH12F3-2 B lymphoma cells augmented class switching from IgM to IgA without cytokine stimulation. AID deficiency caused a complete defect in class switching and showed a hyper-IgM phenotype with enlarged germinal centers containing strongly activated B cells before or after immunization. AID-/- spleen cells stimulated in vitro with LPS and cytokines failed to undergo class switch recombination although they expressed germline transcripts. Immunization of AID-/- chimera with 4-hydroxy-3-nitrophenylacetyl (NP) chicken gamma-globulin induced neither accumulation of mutations in the NP-specific variable region gene nor class switching. These results suggest that AID may be involved in regulation or catalysis of the DNA modification step of both class switching and somatic hypermutation.

C ameleons are genetically encoded fluorescent indicators for Ca 2ϩ based on GFP variants and calmodulin (CaM) (1, 2). They are chimeric proteins composed of a short-wavelength variant of GFP, CaM, a glycylglycine linker, the CaM-binding peptide of myosin light-chain kinase (M13), and a longwavelength variant of GFP. Ca 2ϩ binding to CaM initiates an intramolecular interaction between CaM and M13, which changes the chimeric protein from an extended to a more compact conformation, thereby increasing the efficiency of fluorescence resonance energy transfer (FRET) from the shorter-to the longer-wavelength variant of GFP. Yellow cameleons (YCs) have cyan and yellow fluorescent proteins (CFP and YFP) as the FRET donor and acceptor, respectively. YCs are classified into several groups based on the composition of their Ca 2ϩ -sensing domains. For example, YC2 has an intact CaM and thus shows high affinity for Ca 2ϩ . On the other hand, YC3 and YC4 are low-affinity indicators because of mutations in the Ca 2ϩ -binding loops of their CaM domains. These YCs have been made more resistant to acidification by replacing the original YFP with EYFP.1 (3). The improved YCs include YC2.1 and YC3.1. In addition, some YCs have been made to mature more quickly by using especially bright versions of YFP such as citrine (4) or Venus (5). In this way, YCs have been improved mainly by optimizing the YFP component.Despite the above-mentioned improvements, YCs still suffer from poor dynamic range. The best versions available currently, such as YC2.12 or YC3.12, exhibit at most a 120% change in the ratio of YFP͞CFP upon Ca 2ϩ binding in vitro. These YCs do not have good signal-to-noise ratios, particularly when they are targeted to organelles or submicroscopic environments, because of low levels of signal. It has been also suggested that their dynamic range is attenuated in vivo depending on the abundance of endogenous CaM and CaM-binding proteins that may interact with the sensing domains of YCs.In the present study, we have attempted to modify the acceptor to increase the dynamic range of the indicator. To achieve a Ca 2ϩ -dependent large change in the relative orientation and distance between the fluorophores of CFP and YFP, we assumed that optimization of the length and sequence of the linkers used in YCs would yield only moderate improvement. Thus, we took a more rigorous approach that used a circularly permuted GFP (cpGFP), in which the N and C portions were interchanged and reconnected by a short spacer between the original termini (6, 7). By using cpYFPs that are resistant to acidification and that mature efficiently, we attempted to vary the relative orientation of the two chromophores' transition dipoles. Materials and MethodsGene Construction. The cDNAs of the 5Ј portions of the cpVenus variants were amplified by PCR using sense primers containing a BamHI site and reverse primers containing the sequence encoding the linker (GGSGG) between the natural N and C termini. The cDNAs of their 3Ј portions were extended by PCR at the 5Ј ...

CD9 is an integral membrane protein associated with integrins and other membrane proteins. Mice lacking CD9 were produced by homologous recombination. Both male and female CD9-/- mice were born healthy and grew normally. However, the litter size from CD9-/- females was less than 2% of that of the wild type. In vitro fertilization experiments indicated that the cause of this infertility was due to the failure of sperm-egg fusion. When sperm were injected into oocytes with assisted microfertilization techniques, however, the fertilized eggs developed to term. These results indicate that CD9 has a crucial role in sperm-egg fusion.

Genome stability is regulated by the balance between efficiencies of the repair machinery and genetic alterations such as mutations and chromosomal rearrangements. It has been postulated that deregulation of class switch recombination (CSR) and somatic hypermutation (SHM), which modify the immunoglobulin (Ig) genes in activated B cells, may be responsible for aberrant chromosomal translocations and mutations of non-Ig genes that lead to lymphocyte malignancy. However, the molecular basis for these genetic instabilities is not clearly understood. Activation-induced cytidine deaminase (AID) is shown to be essential and sufficient to induce both CSR and SHM in artificial substrates in fibroblasts as well as B cells. Here we show that constitutive and ubiquitous expression of AID in transgenic mice caused both T cell lymphomas and dysgenetic lesions of epithelium of respiratory bronchioles (micro-adenomas) in all individual mice. Point mutations, but not translocations, were massively introduced in expressed T cell receptor (TCR) and c-myc genes in T lymphoma cells. The results indicate that AID can mutate non-Ig genes including oncogenes, implying that aberrant AID expression could be a cause of human malignancy.

The basic helix-loop-helix (bHLH) gene Hes7, a putative Notch effector, encodes a transcriptional repressor. Here, we found that Hes7 expression oscillates in 2-h cycles in the presomitic mesoderm (PSM). In Hes7-null mice, somites are not properly segmented and their anteriorposterior polarity is disrupted. As a result, the somite derivatives such as vertebrae and ribs are severely disorganized. Although expression of Notch and its ligands is not affected significantly, the oscillator and Notch modulator lunatic fringe is expressed continuously throughout the mutant PSM. These results indicate that Hes7 controls the cyclic expression of lunatic fringe and is essential for coordinated somite segmentation.