Estefanía Claudio

Molecular cloning of a mouse homologue for the Drosophila splicing regulator Tra2.

We report the identification of a mouse cDNA, SIG41, encoding a protein of 288 amino acids that is 45% identical (58% similar) to the Drosophila splicing regulator Tra2. SIG41 cDNA contains four polyadenylation signals whose alternative use gives rise to four types of transcripts (2.1, 2.0, 1.5, and 1.4 kb) in mouse cells. Northern analysis and RT‐PCR assays showed that SIG41 mRNA is present in virtually all the cell lines and tissues studied, with remarkable levels of expression in uterus and brain tissues. Differential stability of the SIG41 mRNAs was detected in mouse macrophage cells.

Measurement of cytotoxicity by propidium iodide staining of target cell DNA: Application to the quantification of murine TNF-α

A rapid and sensitive method is described for the determination of murine tumor necrosis factor (TNF-alpha), which can be performed in microtiter plates using a fluorescence plate scanner. The method is based on the binding of propidium iodide (PI), a membrane-impermeant dye, to nucleic acids of WEHI 164 cells, whose plasma membrane permeable due to TNF-alpha-induced cell damage. The analytical range for the proposed method is 0.3-200 pg/ml of TNF-alpha after 5 h of incubation. The optimal number of target cells was found to be 4-5X10(4)/well. The variability obtained for the PI assay was 7.6%; lower than that obtained with a commonly employed method in which MTT is used to determine cell viability (11.3%). Thus, the PI assay appears to be a reliable and reproducible method for the determination of biologically active TNF-alpha. The assay can be performed in a few hours and has the advantage over the current MTT and 51Cr-released assays that kinetic studies of TNF-alpha toxicity are possible since it permits multiple, sequenced measurements of cell viability during the incubation of the sample. The method can also be used for the determination of human TNF-alpha.

Identification of an Additional Member of the Cytochrome c Oxidase Subunit VIIa Family of Proteins

We report the cloning, nucleotide sequence, evolutionary analysis, and intracellular localization of SIG81, a silica-induced cDNA from mouse macrophages. The cDNA encodes a 111-amino acid protein with extensive sequence identity with members of the mammalian cytochrome c oxidase subunit VIIa (COX7a) family. A human SIG81 sequence >80% identical with the mouse cDNA was deduced from homologous sequences in the human expressed tags data base. The deduced amino-terminal region shows features common to mitochondrial targeting sequences. A phylogenetic analysis of the carboxyl-terminal domain homologous to COX7a identifies SIG81 as a divergent member of the family with an ancient origin. Southern blot analysis showed that the mouse genome contains two to three copies of the SIG81 gene. Northern blot analysis revealed that the SIG81 transcript is approximately 1 kb and expressed in every tissue tested, with higher levels of expression observed in kidney and liver. Antibodies raised against a glutathione S-transferase SIG81 fusion protein detected a 13.5-kDa protein that co-fractionates with mitochondrial localized enzymatic activity. Taken together, our data suggest that SIG81 is a novel member of the COX7a family that is constitutively expressed in mouse cells.

Differential regulation of the murine ribosomal protein L26 gene in macrophage activation

In mouse RAW 264.7 macrophages, the gene for ribosomal protein L26 is positively regulated by silica. In order to study L26 gene expression a near full-length cDNA for mouse L26 was isolated and characterized. Sequence analysis revealed that mouse L26 is a 145 amino acid protein highly homologous to other vertebrate L26 proteins. The treatment of RAW 264.7 cells with the inflammatory mediators LPS and IFN gamma induced the expression of L26 mRNA, but the patterns of expression obtained differed markedly from silica. On the contrary, TNF alpha acted as a down-regulator of L26 gene. Our results suggest that the synthesis of ribosomal components in response to macrophage activators is inducer-specific. Mouse genomic DNA analysis revealed the presence of multiple (10-12) sequences related to the L26 gene.