Structure and myofiber-specific expression of the rat muscle regulatory gene MRF4.

We have cloned an 11.3-kb rat genomic DNA fragment encompassing the muscle regulatory factor 4 (MRF4) protein-coding sequence, 8.5 kb of 5'-flanking sequence, and 1.0 kb of 3'-flanking sequence. In order to study MRF4 gene expression, the rat myogenic cell line, L6J1-C, which expresses the endogenous MRF4 gene only in differentiated myofibers, was transfected stably with the full-length genomic clone and various 5' deletions. RNase protection assays demonstrated that MRF4 genes containing as little as 430 bp of 5'-flanking sequence exhibited an increase in expression as the cells differentiated into myofibers, indicating that elements responsible for fiber-specific expression are contained within this cloned DNA fragment. Similar up-regulation was observed with genes containing 1.5 kb of 5'-flanking sequence. Interestingly, MRF4 genes containing 5.0 kb and 8.5 kb of 5'-flanking sequence were up-regulated to even higher levels, suggesting that additional myofiber-specific regulatory elements located between 1.5 and 5.0 kb upstream from the coding region play a role in regulating the expression of this muscle-specific gene.     

Molecular Characterization of Equine APRIL and its Expression Analysis During the Adipogenic Differentiation of Equine Adipose-Derived Stem Cell In Vitro

A proliferation inducing ligand (APRIL) is a member of the TNF superfamily. It shares two receptors with B-cell activating factor (BAFF), B-cell maturation antigen (BCMA), and transmembrane activator and CAML interactor (TACI). Herein, the equine APRIL was identified from equine adipose-derived stem cell (ASC), and the protein expression of APRIL and its related molecules were detected during the adipogenic differentiation of equine ASC in vitro. The equine APRIL gene was located on chromosome 11, spans 1852 base pairs (bp). Its open reading frame covers 753 bp, encoding a 250-amino acid protein with the typical TNF structure domain. During the two weeks’ adipogenic differentiation of equine ASC, although the protein expression of APRIL and TACI had an insignificant change, that of BCMA increased significantly. Moreover, with the addition of recombinant protein His₆-sAPRIL, a reduced differentiation of equine ASC toward adipocyte was detected. These results may provide the basis for investigating the role of APRIL in ASC adipogenic differentiation.     

B-cell maturation protein, which binds the tumor necrosis factor family members BAFF and APRIL, is dispensable for humoral immune responses

B-cell maturation protein (BCMA) is a member of the tumor necrosis factor (TNF) receptor family and is expressed in B lymphocytes. BCMA binds two TNF family members, BAFF and APRIL, that stimulate cellular proliferation. BAFF in particular has been shown to influence B-cell survival and activation, and transgenic mice overexpressing BAFF have a lupus-like autoimmune disorder. We have inactivated BCMA in the mouse germ line. BCMA(-/-) mice have normal B-cell development, and the life span of mutant B lymphocytes is comparable to that of wild-type B cells. The humoral immune responses of BCMA(-/-) mice to T-cell-independent antigens as well as high and low doses of T-cell-dependent antigens are also intact. In addition, mutant mice have normal splenic architecture, and germinal centers are formed during an ongoing immune response. These data suggest a functional redundancy of BCMA in B-cell physiology that is probably due to the presence of TACI, another TNF receptor family member that is expressed on B cells and that can also bind BAFF and APRIL.     

Testis-Specific Expression of a Novel Human H3 Histone Gene

We have investigated the expression of a recently described, solitary human H3 histone gene. Using RNase protection assays, the corresponding mRNA could only be detected in RNA preparations from human testis, whereas several human cell lines and somatic tissues did not exhibit expression of this gene.In situhybridization of sections from human testis revealed expression to be confined to primary spermatocytes. In addition to H1t, this novel H3 gene, which is located on chromosome 1, is the second tissue-specific human histone gene that has been found to be expressed solely in the testis.     

TNF receptor family member BCMA (B cell maturation) associates with TNF receptor-associated factor (TRAF) 1, TRAF2, and TRAF3 and activates NF-kappa B, elk-1, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase

BCMA (B cell maturation) is a nonglycosylated integral membrane type I protein that is preferentially expressed in mature B lymphocytes. Previously, we reported in a human malignant myeloma cell line that BCMA is not primarily present on the cell surface but lies in a perinuclear structure that partially overlaps the Golgi apparatus. We now show that in transiently or stably transfected cells, BCMA is located on the cell surface, as well as in a perinulear Golgi-like structure. We also show that overexpression of BCMA in 293 cells activates NF-kappa B, Elk-1, the c-Jun N-terminal kinase, and the p38 mitogen-activated protein kinase. Coimmunoprecipitation experiments performed in transfected cells showed that BCMA associates with TNFR-associated factor (TRAF) 1, TRAF2, and TRAF3 adaptor proteins. Analysis of deletion mutants of the intracytoplasmic tail of BCMA showed that the 25-aa protein segment, from position 119 to 143, conserved between mouse and human BCMA, is essential for its association with the TRAFs and the activation of NF-kappa B, Elk-1, and c-Jun N-terminal kinase. BCMA belongs structurally to the TNFR family. Its unique TNFR motif corresponds to a variant motif present in the fourth repeat of the TNFRI molecule. This study confirms that BCMA is a functional member of the TNFR superfamily. Furthermore, as BCMA is lacking a "death domain" and its overexpression activates NF-kappa B and c-Jun N-terminal kinase, we can reasonably hypothesize that upon binding of its corresponding ligand BCMA transduces signals for cell survival and proliferation.     

Cell cycle regulation of human interleukin-8 gene expression by the human immunodeficiency virus type 1 Tat protein

The human immunodeficiency virus type 1 (HIV-1) Tat protein has been reported to transactivate several cellular genes, including the potent chemotactic factor interleukin-8 (IL-8). Consistent with these in vitro assays, elevated levels of IL-8 protein are found in the serum of HIV-infected individuals. We now extend these observations by demonstrating that Tat induction of IL-8 is linked to the cell cycle. Cells that constitutively express the Tat(1-86) protein (eTat) and control cells (pCEP) were reversibly blocked at the G(1)/S border with hydroxyurea or thymidine. The cells were subsequently released, and IL-8 expression was monitored by RNase protection assays and enzyme-linked immunosorbent assay (ELISA). RNase protection assays demonstrated that IL-8 mRNA expression is transiently induced, approximately fourfold, as the Tat-expressing cells enter S phase. Consistent with the RNase protection assay, an increase in IL-8 protein was observed in the cell supernatant using an IL-8 ELISA. Similar experiments were performed following a reversible block at the G(2)/M border with nocodazole and release into G(1). Using the RNase protection assay and ELISA, little or no increase in IL-8 expression was observed during G(1). Using gel shift as well as an immobilized DNA binding assay, we demonstrate that the increase in IL-8 gene expression correlates with a specific increase in p65 NF-kappa B binding activity only in the nucleus of the Tat-expressing cells. Moreover, the CREB-binding protein coactivator is present in the complex in the Tat cell line. Finally, we demonstrate that the presence of the proteasome inhibitor MG-132 inhibits the induction of NF-kappa B binding, as well as IL-8 expression, supporting the role of NF-kappa B.     

A solution hybridization/RNase protection assay with riboprobes to determine absolute levels of apoB, A-I, and E mRNA in human hepatoma cell lines

A solution hybridization/RNase protection assay with riboprobes was developed to quantitate apolipoprotein mRNA concentrations. Previously, radiolabeled DNA probes have been used in solution hybridization/S1 nuclease protection assays for this purpose. The new assay requires less time for probe preparation and hybridization compared to previous assays. In addition, the vector used for riboprobe preparation can also be used to conveniently produce cRNA required to generate the standard curve to quantitate absolute apolipoprotein mRNA levels. The solution hybridization RNase protection assay was used to quantitate apoB, A-I, and E mRNA levels in four human hepatoma cell lines, HepG2, Hep3B, WRL-68, SK-Hep2. HepG2 and Hep3B, but not WRL-68 and SK-Hep2 cells had concentrations of all three apolipoprotein mRNAs comparable to liver in vivo. These data suggest that HepG2 and Hep3B are suitable models to study liver specific apolipoprotein gene expression.     

RNase9, an androgen-dependent member of the RNase A family, is specifically expressed in the rat epididymis

Members of the RNase superfamily participate in a diverse array of biological processes, including RNA degradation, antipathogen activities, angiogenesis, and digestion. In the present study, we cloned the rat RNase9 gene by in silico methods and genome walking based on homology to the Macaca mulatta (rhesus monkey) epididymal RNase9. The gene is located on chromosome 15p14, spanning two exons, and is clustered with other members of the RNase A superfamily. It contains 1279 bp and encodes 182 amino acids, including a 24-amino acid signal peptide, and it has unique features known from other RNases. Unlike those other members, the rat RNase9 mRNA was specifically expressed in the epididymis, especially in the caput and corpus, and exhibited an androgen-dependent expression pattern but was downregulated in an epididymitis animal model. The RNASE9 was expressed in a principal cell-specific pattern. Interestingly, most of the principal cells in the caput expressed the RNASE9; however, in the distal caput, the principal cells showed a checkerboard-like pattern of immunoreactivity. We also observed that the RNASE9 was bound on the acrosomal domain of sperm. Its potential roles in sperm maturation are discussed.     

Characterization of mammalian orthologues of the Drosophila osa gene: cDNA cloning, expression, chromosomal localization, and direct physical interaction with Brahma chromatin-remodeling complex

The osa gene of Drosophila melanogaster encodes a nuclear protein that is a component of the Brahma chromatin-remodeling complex. Osa is required for embryonic segmentation, development of the notum and wing margin, and photoreceptor differentiation. In these tissues, osa mutations have effects opposite to those caused by wingless (wg) mutations, suggesting that osa functions as an antagonist of wg signaling. Here we describe the cloning and characterization of mammalian orthologues of osa. Three evolutionarily conserved domains were identified in Osa family members: the N-terminal Bright domain and C-terminally located Osa homology domains 1 and 2. RNase protection analysis indicates a widespread expression of the Osa1 gene during mouse development, in adult tissues, and in cultured cell lines. The Osa1 gene was localized to mouse chromosome 4, within the region syntenic to chromosomal position 1p35-p36 of its human counterpart. We present evidence that the OSA1 product is localized in the nucleus and associates with human Brahma complex, which suggests evolutionarily conserved function for Osa in gene regulation between mammals and Drosophila.     

R‐loop induced stress response by second (p)ppGpp synthetase in Mycobacterium smegmatis: functional and domain interdependence

Persistent R‐loops lead to replicative stress due to RNA polymerase stalling and DNA damage. RNase H enzymes facilitate the organisms to survive in the hostile condition by removing these R‐loops. MS_RHII‐RSD was previously identified to be the second (p)ppGpp synthetase in Mycobacterium smegmatis. The unique presence of an additional RNase HII domain raises an important question regarding the significance of this bifunctional protein. In this report, we demonstrate its ability to hydrolyze R‐loops in Escherichia coli exposed to UV stress. MS_RHII‐RSD gene expression was upregulated under UV stress, and this gene deleted strain showed increased R‐loop accumulation as compared to the wild type. The domains in isolation are known to be inactive, and the full length protein is required for its function. Domain interdependence studies using active site mutants reveal the necessity of a hexamer form with high alpha helical content. In previous studies, bacterial RNase type HI has been mainly implicated in R‐loop hydrolysis, but in this study, the RNase HII domain containing protein showed the activity. The prospective of this differential RNase HII activity is discussed. This is the first report to implicate a (p)ppGpp synthetase protein in R‐loop‐induced stress response.     

A novel RNase G mutant that is defective in degradation of adhE mRNA but proficient in the processing of 16S rRNA precursor.

Escherichia coli RNase G, encoded by the rng gene, is involved in both the processing of 16S rRNA precursor and the degradation of adhE mRNA. Consequently, defects in RNase G result in elevation of AdhE levels. Furthermore, the adhR430 mutant strain, DC430, is reported to overproduce the AdhE protein in a manner dependent on the adhC81 mutation. We found that overproduction of AdhE by DC430 was reversed to wild-type levels by introduction of a plasmid carrying the wild-type allele of rng. Mapping by P1-phage-mediated transduction also indicated that a mutation involved in AdhE overproduction was located around the rng region in DC430. DNA sequencing of the rng region revealed that DC430 indeed had a mutation in the rng gene: a G1022 to A transition that caused substitution of Gly341 with Ser and which was named rng430. This lies in the highly conserved region of the RNase E/RNase G family, called high similarity region 2 (HSR2). However, very interestingly, rng430 mutant strains did not accumulate the 16.3S precursor of 16S rRNA unlike rng::cat mutants. We also found that the Rng1 mutant protein, which is truncated in its C-terminal domain encompassing HSR2, exhibited a residual processing activity against the 16S rRNA precursor, when overproduced. These results indicate that the HSR2 of RNase G plays an important role in substrate recognition and/or ribonucleolytic action.     

Quail Pax-6 (Pax-QNR) mRNAs are expressed from two promoters used differentially during retina development and neuronal differentiation.

During investigations on the regulation of the Pax-6 gene, we characterized a cDNA from quail neuroretina showing a 5' untranslated region distinct from that previously described and initiated from an internal promoter. Using RNase protection and primer extension mapping, we localized this second quail Pax-6 promoter, termed P1. As reported for the already described P0 promoter, P1 was also transactivated in vitro by the p46Pax-QNR protein. RNase protection assays performed with quail neuroretina RNA showed that P1-initiated mRNAs were detected before the P0-initiated mRNAs, remained constant up to embryonic day 8, and decreased slowly thereafter whereas, P0-initiated mRNAs accumulated up to embryonic day 8. In contrast, quail retinal pigmented epithelium expressed only the P1-initiated mRNAs. Transformation of these cells by the v-myc oncogene induced neuronal traits in the culture, which thereafter, in addition to the P1-initiated mRNAs, expressed Pax-QNR from the P0 promoter. These results suggest that expression of the quail Pax-6 gene is under the control of different regulators through alternate promoters, P0 being activated at the onset of neuronal differentiation.