Previously, we reported the isolation of cDNA clones representing four alternative splice forms of TCF-1, a T-cell-specific transcription factor. In the present study, Western blotting (immunoblotting) yielded a multitude of TCF-1 proteins ranging from 25 to 55 kDa, a pattern not simply explained from the known splice alternatives. Subsequent cDNA cloning, PCR amplification, and analysis by rapid amplification of 5 cDNA ends revealed (i) the presence of an alternative upstream promoter, which extended the known N terminus by 116 amino acids, (ii) the presence of four alternative exons, and (iii) the existence of a second reading frame in the last exon encoding an extended C terminus. Inclusion of the extended N terminus into the originally reported protein resulted in a striking similarity to the lymphoid factor Lef-1. Several of the TCF-1 isoforms, although less potent, mimicked Lef-1 in transactivating transcription through the T-cell receptor ␣-chain (TCR-␣) enhancer. These data provide a molecular basis for the complexity of the expressed TCF-1 proteins and establish the existence of functional differences between these isoforms. Furthermore, the functional redundancy between Tcf-1 and Lef-1 explains the apparently normal TCR-␣ expression in single Tcf-1 or Lef-1 knockout mice despite the firm in vitro evidence for the importance of the Tcf/Lef site in the TCR-␣ enhancer.

Small nuclear ribonucleoprotein (snRNP) particles are a class of RNA-containing particles in the nucleus of eukaryotic cells. Sera from patients with connective tissue diseases often contain antibodies against the proteins present in these snRNPs. Antibodies against the RNA components of snRNPs, the U snRNAs, are thought to be rare.We tested 118 anti-snRNP sera for the presence of antisnRNA antibodies and found them in 45 sera (38%). In all sera the antibodies (IgG and F(ab)2 fragments thereof) were exclusively directed against Ul snRNA.The anti-(Ul)RNA antibodies were always accompanied by anti-(Ul)RNP antibodies but were not found in sera which contain antibodies of the Sm serotype directed against all nucleoplasmic U snRNP particles. Like anti-RNP antibodies, anti-Ul RNA activity is confined to sera from patients with SLE or SLE overlap syndromes and is rarely found in patients with other connective tissue diseases. By analyzing binding to subfragments of Ul snRNA made in vitro, it was demonstrated that anti-(Ul)RNA antibodies recognize epitopes distributed throughout the Ul RNA molecule. In most sera, however, either the second or the fourth hairpin loop is the main target of the antibody.The possible mechanisms that could lead to the production of this new type of autoantibody are discussed. (J. Clin. Invest.

Detection of biologically active compounds is one of the most important topics in molecular biology and biochemistry. One of the most promising detection methods is based on the application of surface plasmon resonance for label-free detection of biologically active compounds. This method allows one to monitor binding events in real time without labeling. The system can therefore be used to determine both affinity and rate constants for interactions between various types of molecules. Here, we describe the application of a surface plasmon resonance biosensor for label-free investigation of the interaction between an immobilized antigen bovine serum albumin (BSA) and antibody rabbit anti-cow albumin IgG1 (anti-BSA). The formation of a self-assembled monolayer (SAM) over a gold surface is introduced into this laboratory training protocol as an effective immobilization method, which is very promising in biosensing systems based on detection of affinity interactions. In the next step, covalent attachment via artificially formed amide bonds is applied for the immobilization of proteins on the formed SAM surface. These experiments provide suitable experience for postgraduate students to help them understand immobilization of biologically active materials via SAMs, fundamentals of surface plasmon resonance biosensor applications, and determination of non-covalent biomolecular interactions. The experiment is designed for master and/or Ph.D. students. In some particular cases, this protocol might be adoptable for bachelor students that already have completed an extended biochemistry program that included a background in immunology.

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Physical interaction between the lymphoid high mobility group (HMG)-box architectural transcription factors TCF/ LEF and b-catenin is associated with translocation of the heteromeric complex to the nucleus and regulation of target gene expression. Since formation of molecular complexes among b-catenin, E-cadherin, p300 apc and TCF/LEF depends on balanced expression of these constituents, we investigated the biosynthesis of TCF-1 in colorectal cancer. Here we report detailed analyses of activation and overexpression of lymphoid transcription factor TCF-1 in human colorectal cancer-derived cell lines. Northern blot analyses revealed considerable steady-state expression levels of TCF-1 mRNA of normal size. Genomic rearrangement of the 58 flanking region of the TCF-1 gene was excluded as a cause of ectopic expression. By contrast, CAT-reporter constructs depending on a 515-bp T-cell-regulated TCF-1 genomic upstream region were significantly activated in epithelial tumor cells. RT-PCR analyses revealed a heterogeneic population of mRNA isoforms due to alternative splicing in the TCF-1 gene. On Western blots of colorectal cancer cells, the TCF-1-specific monoclonal antibody 7H3 detected a similar heterogeneous spectrum of TCF-1 specific polypeptide chains. Interestingly, overexpression of TCF-1-specific splice forms correlated with the metastatic behavior of the analyzed cells and with overproduction of lymphoid tyrosine protein kinase p56 lck . We conclude that ectopic expression of the HMG-box factor TCF-1 is associated with late events in tumor progression. Int.