cDNA structure of murine C4b-binding protein, a regulatory component of the serum complement system

A cDNA library representing total poly(A+) RNA from the livers of male B10.WR mice was screened with a 1097 base pair (bp) probe obtained from a partial human C4b-binding protein (C4BP) cDNA clone. Two cDNA clones were isolated, the largest of which was sequenced and found to be 1889 bp in length exclusive of the poly(A) tail. The predicted mouse C4BP polypeptide chain encoded by 1239 bp is 413 amino acid residues in length and has a calculated molecular weight of 45,281. The 370-nucleotide sequence upstream from the codon for the predicted amino terminus contains two possible in-phase translational start signals which yield leader sequences of 56 and 13 amino acid residues, respectively. The 3'-untranslated region is 277 bp long, and there are two potential overlapping poly(A) recognition signals, AATTAA and ATTAAAA, located 26 and 25 bp, respectively, upstream from the poly(A) tail; these are preceded by five other potential polyadenylation signals. Beginning at the amino terminus and continuing through to residue 358, there are six contiguous regions of internal homology, each about 60 amino acids in length. The carboxy-terminal 55 amino acid sequence shares no homology with the repeating units. Extensive homology was found with human C4BP at the amino acid level (61%) as well as at the nucleotide level for both the coding and 3'-untranslated regions. Significant differences, however, were observed between mouse and human C4BP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue-specific and developmental expression of human transthyretin gene in transgenic mice

To analyze the regulation of transthyretin gene expression we have produced transgenic mice by microinjecting cloned human transthyretin genes into fertilized eggs of C57BL/6 mice. The 7.6-kilobase (kb) human transthyretin gene containing about 500 base pairs (bp) in the upstream region was used for microinjection. Seven out of nine transgenic mice had detectable amounts of human transthyretin in serum when analyzed by enzyme-linked immunosorbent assay. Transthyretin mRNA was detected in liver and yolk sac but not in other tissues including brain. The amount of mRNA was variable among transgenic mice and was about one-tenth of mouse endogenous transthyretin mRNA. Human and mouse transthyretin mRNAs were detected in liver of fetus and yolk sac at 13 days of gestation and unlike yolk sac the level of mRNA in liver increased gradually during development and reached the maximum at around 17 days of gestation. Human transthyretin was associated with mouse transthyretin to form tetramers as judged from the dilution curve of enzyme-linked immunosorbent assay and the spur formation in Ouchterlony assay.     

Identifying functional miRNA-mRNA regulatory modules with correspondence latent dirichlet allocation

MOTIVATION: MicroRNAs (miRNAs) are small non-coding RNAs that cause mRNA degradation and translational inhibition. They are important regulators of development and cellular homeostasis through their control of diverse processes. Recently, great efforts have been made to elucidate their regulatory mechanism, but the functions of most miRNAs and their precise regulatory mechanisms remain elusive. With more and more matched expression profiles of miRNAs and mRNAs having been made available, it is of great interest to utilize both expression profiles to discover the functional regulatory networks of miRNAs and their target mRNAs for potential biological processes that they may participate in. RESULTS: We present a probabilistic graphical model to discover functional miRNA regulatory modules at potential biological levels by integrating heterogeneous datasets, including expression profiles of miRNAs and mRNAs, with or without the prior target binding information. We applied this model to a mouse mammary dataset. It effectively captured several biological process specific modules involving miRNAs and their target mRNAs. Furthermore, without using prior target binding information, the identified miRNAs and mRNAs in each module show a large proportion of overlap with predicted miRNA target relationships, suggesting that expression profiles are crucial for both target identification and discovery of regulatory modules.     

Positive and negative regulation of the human heme oxygenase-1 gene expression in cultured cells.

To elucidate the regulation of the human heme oxygenase-1 (hHO-1) gene expression, we assessed approximately 4 kb of the 5'-flanking region of the hHO-1 gene for basal promoter activity and sequenced approximately 2 kb of the 5'-flanking region. A series of deletion mutants of the 5'-flanking region linked to the luciferase gene was constructed. Basal level expression of these constructs was tested in HepG2 human hepatoma cells and HeLa cervical cancer cells. By measuring luciferase activity, which was transiently expressed in the transfected cells, we found a positive regulatory region at position -1976 to -1655 bp. This region functions in HepG2 cells but not in HeLa cells. A negative regulatory region was also found at position -981 to -412 bp that functions in both HepG2 cells and HeLa cells.