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DNA mutations of various types often affect the cellular localization and function of gene products. The role of mutant transcripts in the pathogenesis of human disease is increasingly recognized. Among the pathogenic RNA variants are transcripts with single nucleotide substitutions, small insertions or deletions, aberrantly or alternatively spliced transcripts and RNAs derived from fused genes. To discriminate among transcripts, particularly those of low abundance, showing small or large sequence differences, a highly sensitive and specific RNA imaging method is required. The method that fulfills these criteria is single-molecule fluorescence in situ hybridization (smFISH) combined with probes discriminating among RNA variants. With this method, RNA transcripts produced from individual alleles can be imaged, and differences in their transcription, processing, cellular localization and decay can be revealed. In addition to its applications for studying physiological processes involving RNA variants, smFISH offers several advantages for disease related mutation research. Further development of allele-specific microscopic methods may broaden group of RNA variants analyzed, including RNAs with expanded repeat tract, different variants of 3′UTR, RNAs differing in length of polyA tract or transcripts produced from alternative start codons. Moreover, first attempts for allele-specific RNA live imaging were made adding time-lapse analysis. In this review, we discuss important aspects of the variant-specific smFISH methodology and present examples of its applications in deciphering RNA-mediated pathogenic mechanisms in a variety of human diseases, including cancer, neurological, immunological and cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Abstract: I formulated a systematic approach to screen for colicin resistance-associated genes by using an attenuated colicin mutant and demonstrated its utility in a screen of genes related to colicin E5 resistance. Screening of an Escherichia coli genome library revealed rstA as a partial resistance gene to colicin E5. Transcript expression of BtuB and OmpF, proteins responsible for translocation of nuclease E colicins, was clearly inhibited in an rstA-overexpressing strain. In addition, the tatA::recN fusion gene provides resistance to an attenuated colicin E5 mutant. Improving tatA::recN by directed coevolution, I created a novel gene with enhanced resistance to colicin E5 and concluded that attenuated colicin-based screening is useful for the discovery and creation of novel colicin resistance-associated genes. [Copyright &y& Elsevier]

Abstract: The expression vector, pBI121CTBVP1, containing the fusion of the foot and mouth disease virus (FMDV) VP1 gene and the cholera toxin B subunit (CTB) gene was constructed by fused PCR and transferred into potato (Solanum tuberosum L.) by Agrobacterium-mediated transformation. Transformed plants were obtained by selecting on kanamycin-resistant medium strictly and regenerated. The transgenic plantlets were identified by PCR, Southern-blot and the production of fused protein was confirmed and quantified by Western-blot and ELISA assays. The results showed that the fused genes were expressed stablely under the control of specific-tuber patatin promoter. The expressed fused proteins have a certain degree of immunogenicity. [Copyright &y& Elsevier]

The human repetin gene is a member of the“fused” gene family and localized in the epidermal differentiation complex on chromosome 1q21. The“fused” gene family comprises profilaggrin, trichohyalin, repetin, hornerin, the profilaggrin-related protein and a protein encoded by c1orf10. Functionally, these proteins are associated with keratin intermediate filaments and partially crosslinked to the cell envelope (CE). Here, we report the isolation and characterization of the human repetin gene and of its protein product. The repetin protein of 784 amino acids contains EF (a structure resembling the E helix-calcium-binding loop-F helix domain of parvalbumin) hands of the S100 type and internal tandem repeats typical for CE precursor proteins, a combination which is characteristic for“fused” proteins. Repetin expression is scattered in the normal epidermis but strong in the acrosyringium, the inner hair root sheat and in the filiform papilli of the tongue. Ultrastructurally, repetin is a component of cytoplasmic non-membrane“keratohyalin” F-granules in the stratum granulosum of normal epidermis, similar to profilaggrin. Finally, we show that EF hands are functional and reversibly bind Ca2+. Our results indicate that repetin is indeed a member of the fused gene family similar to the prototypical members profilaggrin and trichohyalin. [ABSTRACT FROM AUTHOR]

The protein encoded by theC1orf10gene was described to be esophageal-specific and a marker for cancer development. This protein, however, has the previously unreported structural features of the“fused gene” family combining sequences and structural similarities of both the S100 proteins and precursor proteins of the cornified cell envelope as in profilaggrin, trichohyalin, and repetin. Since all members of this family are expressed in keratinocytes, we suspected a role in epidermal differentiation and named the protein cornulin. Here, we report that human cornulin mRNA is expressed primarily in the upper layers of differentiated squamous tissues including the epidermis. Using polyclonal peptide antibodies, we show that cornulin is expressed in the granular and lower cornified cell layers of scalp epidermis and foreskin, as well as in calcium-induced differentiated cultured keratinocytes. Ca2+-overlay assay indicated that EF-hand domains of cornulin are functional and bind calcium. In HeLa cells, cornulin, co-transfected with transglutaminase 1, was diffusely distributed throughout the cytoplasm in contrast to small proline-rich 4, which localized to the cell periphery. We conclude that cornulin is a new member of the“fused gene” family, does not appear to be a precursor of the cornified cell envelope by itself, and is a marker of late epidermal differentiation. [ABSTRACT FROM AUTHOR]