Scapharca inæquivalvis Tetrameric Hemoglobin A and B Genes: Evidence for a Minigene

Vertebrate and many invertebrate globin genes have a three-exon/two-intron organization, with introns in highly conserved positions. According to the ``intron early' hypothesis, introns are the vestigial segments which flank previously independent coding sequences, thus providing evidence for the assembly of the ancient proteins by ``exon shuffling.' In this paper, we report the analysis of the genes of the bivalve mollusk Scapharca inaequivalvis tetrameric hemoglobin (HbII), which support this hypothesis, at least for the hemoglobin genes. We show the existence of ``minigenes' in the IIA and IIB globin genes, spanning part of the first and second introns, ``in frame' with the heme-binding domain coded by the second exon. Further support for the exon shuffling hypothesis can be found in the degree of identity of the ``new' translated sequences with those flanking the central protein domain of some invertebrate hemoglobins. Received: 31 July 1997 / Accepted: 12 December 1997     

Characterization of the molecular mechanisms of the coupling between intracellular loops of prostacyclin receptor with the C-terminal domain of the Galphas protein in human coronary artery smooth muscle cells

The C-terminal domain of the Gs protein alpha subunit (Galphas Ct) and the first intracellular loop (iLP1) of prostacyclin receptor (IP) have been predicted to be involved in the receptor signaling mediated through the IP/Gs protein coupling by our previous NMR studies using synthetic peptides. To test whether the results of the peptide studies can be applied to the protein interaction between the IP receptor and the Gs protein in cells, a minigene technique was used to construct cDNAs that encoded either the amino acid residues of the Galphas or that of the individual intracellular loops of the IP receptor. The effects of the minigene-expressed protein fragments on cAMP production mediated by the IP/Gs coupling were evaluated through experiments that co-expressed peptides either through the Galphas Ct or the IP intracellular loops with the IP receptor in HEK293 cells. The first (iLP1) and third (iLP3) IP intracellular loops, as well as the Galphas Ct, which are important to the IP/Gs coupling-mediated signaling, were identified by the significant reduction of cAMP production when the corresponding peptides were expressed in the cells. Furthermore, the cAMP productions were significantly impaired in Galphas-knockout cells co-expressing the IP receptor with the Galphas C-terminal mutants (E392A, L393A and L394A), compared with the Galphas wild type. Blocking of the endogenous IP/Gs coupling by the minigene-expressed peptides of the Galphas CT, iLP1 and iLP3 was further observed in the human coronary artery smooth muscle cells (SMCs). These results indicate that the three residues (E392-L394) of the Galphas protein predicted from NMR peptide studies, and the IP iLP1 and iLP3 play important roles in the Galphas-mediated IP receptor signaling in the cells, which may be a general binding site for the corresponding regions of the other prostanoid receptors that couple to Gs protein.     

Antagonist minigenes identify genes regulated by parathyroid hormone through G protein-selective and G protein co-regulated mechanisms in osteoblastic cells

Parathyroid hormone (PTH) is the major hormone regulating bone remodeling. Binding of PTH to the PTH1 receptor (PTH1R), a heterotrimeric G protein coupled receptor (GPCR), can potentially trigger multiple signal transduction pathways mediated through several different G proteins. In this study, we employed G protein antagonist minigenes inhibiting Gα_s, Gα_q or Gα₁₂ to selectively dissect out which of these G proteins were responsible for effects of PTH(1-34) in targeted signaling and osteogenesis arrays consisting of 159 genes. Among the 32 genes significantly regulated by 24 h PTH treatment in UMR-106 osteoblastic cells, 9 genes were exclusively regulated through G_s, 6 genes were solely mediated through G_q, and 3 genes were only controlled through G₁₂. Such findings support the concept that there is some absolute specificity in downstream responses initiated at the G protein level following binding of PTH to the PTH1R. On the other hand, 6 PTH-regulated genes were regulated by both G_s and G_q, 3 genes were regulated by both G_s and G₁₂, and 3 genes were controlled by G_s, G_q and G₁₂. These findings indicate potential overlapping or sequential interactions among different G protein-mediated pathways. In addition, two PTH-regulated genes were not regulated through any of the G proteins examined, suggesting that additional signaling mechanisms may be involved. Selectivity was largely maintained over a 2-48-hour time period. The minigene effects were mimicked by downstream inhibitors. The dissection of the differential effects of multiple G protein pathways on gene regulation provides a more complete understanding of PTH signaling in osteoblastic cells.     

A Legumain-based minigene vaccine targets the tumor stroma and suppresses breast cancer growth and angiogenesis

Tumor associated macrophages (TAMs) are well known to play a very important role in tumor angiogenesis and metastasis. The suppression of TAMs in the tumor-microenvironment (TME) provides a novel strategy to inhibit tumor growth and dissemination by remodeling the tumor’s stroma. Here, we tested our hypothesis that suppression of TAMs can be achieved in syngeneic BALB/c mice with oral minigene vaccines against murine MHC class I antigen epitopes of Legumain, an asparaginyl endopeptidase and a member of the C13 family of cystine proteases which is overexpressed on TAMs in the tumor stroma. Vaccine vectors were constructed and transformed into attenuated Salmonella typhimurium (Dam ⁻ , AroA ⁻ ) for oral delivery. Groups of mice received either the expression vectors encoding the Legumain H-2D or 2K epitopes or the control empty vector by gavage. The efficacy of the minigene vaccines was determined by their ability to protect mice from lethal tumor cell challenges, the induction of a specific CTL response as well as IFN-γ release, and inhibition of tumor angiogenesis. We demonstrated that the Legumain minigene vaccine provided effective protection against tumor cell challenge by inducing a specific CD8⁺ T-cell response against Legumain⁺ TAMs in our breast tumor model. The protection, induced by this T-cell response, mediated by the Legumain Kd minigene, is also responsible for lysing D2F2 breast carcinoma cells in syngeneic BALB/c mice and for suppressing tumor angiogenesis. Importantly, in a prophylactic setting, the minigene vaccine proved to be of similar anti-tumor efficacy as a vaccine encoding the entire Legumain gene. Together, our findings establish proof of concept that a Legumain minigene vaccine provides a more flexible alternative to the whole gene vaccine, which may facilitate the future design and clinical applications of such a vaccine for cancer prevention.     

Conserved sequences in the final intron of MDM2 are essential for the regulation of alternative splicing of MDM2 in response to stress

Alternative splicing plays a fundamental role in generating proteome diversity and is critical in regulation of eukaryotic gene expression. It is estimated that 50% of disease-causing mutations alter splicing efficiency and/or patterns of splicing. An alternatively spliced form of murine double-minute 2, MDM2-ALT1, is associated with pediatric rhabdomyosarcoma (RMS) at high frequency in primary human tumors and RMS cell lines. We have identified that this isoform can be induced in response to specific types of stress (UV and cisplatin). However, the mechanism of alternative splicing of MDM2 in human cancer is unknown. Using UV and cisplatin to model alternative splicing of the MDM2 gene, we have developed a damage-inducible in vitro splicing system. This system employs an MDM2 minigene that mimics the damage-induced alternative splicing observed in vivo. Using this in vitro splicing system, we have shown that conserved intronic sequences in intron 11 of MDM2 are required for normal splicing. Furthermore, we showed that these intronic elements are also required for the regulated damage-induced alternative splicing of MDM2. The use of this novel damage-inducible system will allow for the systematic identification of regulatory elements and factors involved in the splicing regulation of the MDM2 gene in response to stress. This study has implications for identification of novel intervention points for development of future therapeutics for rhabdomyosarcoma.     

Use of minigene systems to dissect alternative splicing elements

Pre-mRNA splicing is an essential step for gene expression in higher eukaryotes. The splicing efficiency of individual exons is determined by multiple features involving gene architecture, a variety of cis-acting elements within the exons and flanking introns, and interactions with components of the basal splicing machinery (called the spliceosome) and auxiliary regulatory factors which transiently co-assemble with the spliceosome. Both alternative and constitutive exons are recognized by multiple weak protein:RNA interactions and different exons differ in the interactions which are determinative for exon usage. Alternative exons are often regulated according to cell-specific patterns and regulation is mediated by specific sets of cis-acting elements and trans-acting factors. Transient expression of minigenes is a commonly used in vivo assay to identify the intrinsic features of a gene that control exon usage, identify specific cis-acting elements that control usage of constitutive and alternative exons, identify cis-acting elements that control cell-specific usage of alternative exons, and once regulatory elements have been identified, to identify the trans-acting factors that bind to these elements and modulate splicing. This chapter describes approaches and strategies for using minigenes to define the cis-acting elements that determine splice site usage and to identify and characterize the trans-acting factors that bind to these elements and regulate alternative splicing.