A hydrophobic protein, chargerin II, purified from rat liver mitochondria is encoded in the unidentified reading frame A6L of mitochondrial DNA

Previous studies showed that a hydrophobic protein called chargerin II may have a key role in energy transduction of oxidative phosphorylation, since antibody against chargerin II labeled with monoazide ethidium inhibited ATP synthesis, ATP-Pi exchange, and reversed electron flow from succinate to NAD coupled with succinate oxidation by O2. In the present work, unlabeled chargerin II was purified from intact rat liver mitochondria by high performance liquid chromatography. The purified preparation of chargerin II, which was a single protein as judged by polyacrylamide gel electrophoresis and Western blotting, was digested with lysylendopeptidase. The digest was separated on a reverse-phase column into five peptides, which all cross-reacted with the antibody against chargerin II, indicating that they were fragments of chargerin II. The sequences of two of these peptides (a total of 12 amino acids) were determined and found to be highly homologous with the sequence of the carboxyl-terminal peptide of the putative polypeptide encoded by the unidentified reading frame A6L (URFA6L) of mammalian mitochondrial DNA. The amino acid compositions of the purified preparation of chargerin II were in good accord with those of the putative product of the URFA6L. Thus, we concluded that chargerin II is encoded by the URFA6L. This is the first demonstration that the URFA6L product was identified in rat liver mitochondria and purified from the membranes.     

The production and characterization of a new monoclonal antibody to the trefoil peptide human spasmolytic polypeptide

Summary Human spasmolytic polypeptide (hSP) is a member of the growing family of trefoil peptides which are expressed in discrete regions of the body, most notably the gastrointestinal tract. Much of the research into the localization of the spasmolytic polypeptide has relied on hybridization in situ to detect its mRNA, due to the absence of a suitable antibody. The aim of the present study was to develop and characterize a monoclonal antibody against the human spasmolytic polypeptide, using a combination of immunohistochemistry and hybridization in situ.After immunoblotting, the antibody detected a 14 kDa protein in gastrointestinal tissue extracts from the stomach and small intestine only. Using immunohistochemistry, human spasmolytic polypeptide showed a distinctive staining pattern in the duodenum which co-localized with its mRNA. The co-localization of the immunoreactive peptide with its mRNA provides good evidence that the antibody truly recognized human spasmolytic polypeptide.     

Assignment of the human gene for histidine-rich glycoprotein to chromosome 3

Histidine-rich glycoprotein (HRG) is a monomeric plasma glycoprotein involved in the modulation of coagulation and fibrinolysis. Using Southern analysis of human-rodent somatic cell hybrid DNA with a human HRG-specific cDNA probe, the HRG gene was assigned to chromosome 3. One hybrid that was known to contain only a segment of chromosome 3 also reacted positively with the HRG probe. Hybridization analysis with a set of chromosome 3-specific probes showed that the segment of chromosome 3 present in this hybrid is missing the region pter-p14, which indicates that HRG is not located in this region. No restriction fragment length polymorphisms were detected for HRG with 10 commonly used restriction enzymes.     

A new polymorphic restriction site at the human atrial natriuretic peptide (hANP) gene locus

A unique two allele polymorphism for both HpaII and SmaI is described in the second intron of the human atrial natriuretic peptide gene. It should be a useful marker of this candidate gene in familial susceptibility to hypertension.     

Antibodies against synthetic peptides reveal that the unidentified reading frame A6L, overlapping the ATPase 6 gene, is expressed in human mitochondria

Antibodies prepared against chemically synthesized peptides predicted from the DNA sequence have been used to detect human mitochondrial gene products. In particular, antibodies directed against either the NH2-terminal decapeptide or the COOH-terminal undecapeptide of cytochrome c oxidase subunit II (COII) were both very effective in immunoprecipitating the previously identified COII polypeptide from an SDS lysate of mitochondria from HeLa cells. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative polypeptide encoded in the unidentified reading frame A6L, which overlaps the ATPase 6 gene, immunoprecipitated specifically a component (#25) of the HeLa cell mitochondrial translation products; antibodies directed against the NH2-terminal octapeptide also precipitated protein 25, although less efficiently. The size of protein 25, as estimated from its electrophoretic mobility, is compatible with its being the unidentified reading frame A6L product. Furthermore, a fingerprinting analysis of this protein after trypsin digestion has given results consistent with this identification.     

Transgenic zebrafish model to study translational control mediated by upstream open reading frame of human chop gene

Upstream open reading frame (uORF)-mediated translational inhibition is important in controlling key regulatory genes expression. However, understanding the underlying molecular mechanism of such uORF-mediated control system in vivo is challenging in the absence of an animal model. Therefore, we generated a zebrafish transgenic line, termed huORFZ, harboring a construct in which the uORF sequence from human CCAAT/enhancer-binding protein homologous protein gene (huORF(chop)) is added to the leader of GFP and is driven by a cytomegalovirus promoter. The translation of transgenic huORF(chop)-gfp mRNA was absolutely inhibited by the huORF(chop) cassette in huORFZ embryos during normal conditions, but the downstream GFP was only apparent when the huORFZ embryos were treated with endoplasmic reticulum (ER) stresses. Interestingly, the number and location of GFP-responsive embryonic cells were dependent on the developmental stage and type of ER stresses encountered. These results indicate that the translation of the huORF(chop)-tag downstream reporter gene is controlled in the huORFZ line. Moreover, using cell sorting and microarray analysis of huORFZ embryos, we identified such putative factors as Nrg/ErbB, PI3K and hsp90, which are involved in huORF(chop)-mediated translational control under heat-shock stress. Therefore, using the huORFZ embryos allows us to study the regulatory network involved in human uORF(chop)-mediated translational inhibition.     

Assignment of 35 single-copy and 17 repetitive sequence DNA probes to human chromosome 3: high-resolution physical mapping of 7 DNA probes by in situ hybridization

Thirty-five single-copy and 17 repetitive sequence DNA probes specific for human chromosome 3 were isolated from human chromosome 3-derived genomic libraries. Seven DNA clones, including three that are polymorphic for BglII or MspI, were mapped by in situ hybridization. Four probes were mapped to 3p subregions and 3 were mapped to 3q subregions. Three of the DNA sequences map to regions overlapping a segment of chromosome 3 (3p14-23) frequently deleted in small cell lung cancer cells. By Southern blot analysis on a deletion hybrid panel, we previously mapped 6 of these probes to three distinct chromosome 3 subregions. Our in situ data support these assignments and more precisely determine the localization of each clone to the following regions: D3S34 (3p14-21), D3S35 (3p21), D3S39 (3p21), D3S40 (3p12-13), D3S37 (3q21-23), and D3S36 (3q21). Clone pL84c, a low repeat sequence clone (approximately 30 copies), was mapped to the 3q21-29 subregion. These DNA clones mapped by in situ hybridization can provide useful landmarks for the ordering and localization of other clones.