Isolation, sequencing, and functional analysis of the TATA-less murine ATPase II promoter and structural analysis of the ATPase II gene

Abstract

The P-type Mg 2+ -ATPase, termed ATPase II (Atp8a1), is a putative aminophospholipid transporting enzyme, which helps to maintain phospholipid asymmetry in cell membranes. In this project we have elucidated the organization of the mouse ATPase II gene and identified its promoter. Located within chromosome 5, this gene spans about 224 kb and consists of 38 exons, three of which are alternatively spliced (exons 7, 8 and 16), giving rise to two transcript variants. Translation of these transcripts results in two ATPase II isoforms (1 and 2) composed of 1164 and 1149 amino acids, respectively. Using RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) we identified multiple transcription start sites (TSS) in messages obtained from heart, lung, liver, and spleen. The mouse ATPase II promoter is TATA-less and lacks a consensus initiator sequence. Luciferase reporter analysis of full and core promoters revealed strong activity and little cell type specificity, possibly because more flanking, regulatory sequences are required to cause such tissue specificity. In the neuronal HN2, N18, SN48 cells and the NIH3T3 fibroblast cells, but not in the B16F10 melanoma cells, the core promoter (-318/+193 with respect to the most common TSS) displayed significantly higher activity than the full promoter (-1026/+193). Serial 5′ deletion of the core promoter revealed significant cell type-specific activity of the fragments, suggesting differential expression and use of transcription factors in the five cell lines tested. Additionally distribution of the TSS was organ specific. Such observations suggest tissue-specific differences in transcription initiation complex assembly and regulation of ATPase II gene expression. Information presented here form the groundwork for further studies on the expression of this gene in apoptotic cells.

Comparison of Sessile Drop and Captive Bubble Methods on Rough Homogeneous Surfaces: A Numerical Study

“…Wetting is ubiquitous in many emerging disciplines such as self-cleaning surfaces, microfluidics, microelectromechanical and nanoelectromechanical systems, and so forth. − The wettability of a solid surface by a probe liquid is usually monitored with contact angle measurements, from which the surface energy of the solid may be evaluated. − The wettability of practical solid surfaces is commonly described by a range of observable contact angles, known as the contact angle hysteresis range. The limits of this range are known as the theoretical advancing contact angle (TACA) and the theoretical receding contact angle (TRCA). , However, in practice, TACA and TRCA are rarely reproduced in experimental studies because any wetting system is frequently affected by environmental perturbations, which reduce the experimentally accessible hysteresis range.…”

Interactive effect between ATPase-related genes and early-life tobacco smoke exposure on bronchial hyper-responsiveness detected in asthma-ascertained families

“…The ATP8A1 gene codes for a putative aminophospholipid transporting enzyme which helps maintain phospholipid asymmetry in cell membranes 38. Recently, in non-small cell lung cancer, ATP8A1 was found to be a novel direct target of miR-140–3p,39 a small non-coding RNA molecule known to regulate gene expression, and that may contribute substantially to airway epithelium abnormalities 40.…”

A genetic strategy involving a glycosyltransferase promoter and a lipid translocating enzyme to eliminate cancer cells

“…Preparing expression vectors for Atp8a1, Atp8a2, and GnT-V Mouse brain RNA was isolated as described earlier [24]. Using reverse transcriptase-PCR (RT-PCR) and flanking primers, in which the downstream primer (the antisense primer) included the coding sequence but not the stop codon, the cDNA sequences for both Atp8a1 and Atp8a2 were amplified and then inserted into pcDNA 6.1/myc-His A in frame with myc-His A sequences to eventually achieve expression of epitopetagged Atp8a1 and Atp8a2.…”