ABSTRACT
Globin genes of the bivalve mollusk Scapharca inaequivalvis have the two intron/three exon organization typical of vertebrate and many invertebrate globins, with introns in highly conserved positions. Sequence studies on the A and B globin genes of the mollusk tetrameric hemoglobin gave evidence for the existence of 'minigenes' spanning part of the first and second intron, in-frame with the heme binding domain encoded by the central exon. Putative promoter and regulatory sequences flanking these minigenes were identified in the 3' regions of intron I. Here we report cloning and functional analysis of these regions ( approximately 400bp) and their promoter activity, which was assessed in K562 cells by transient transfection, was established. Moreover, in vitro reverse transcriptase-polymerase chain reaction (RT-PCR) on total cytoplasmatic RNA demonstrated that the A and B minigenes are transcriptionally active in the erythrocytes of S. inaequivalvis. Thus, the present results lead to the conclusion that the present-day organization of the globin genes of S. inaequivalvis tetrameric hemoglobin is still reminiscent of an ancestral globin gene before exon shuffling.
Subject(s)
Bivalvia/genetics , Hemoglobins/genetics , Animals , Base Sequence , DNA/genetics , Humans , K562 Cells , Molecular Sequence Data , Promoter Regions, Genetic/genetics , RNA/genetics , RNA/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Regulatory Sequences, Nucleic Acid , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Transfection , beta-Galactosidase/genetics , beta-Galactosidase/metabolismABSTRACT
In this study, we have investigated the positions of introns in the globin gene of Scapharca inaequivalvis homodimeric hemoglobin. We found the three exon/two intron organization typical of vertebrate globin genes, with the two introns in highly conserved positions, as it occurs in the A and B globin genes of the tetrameric hemoglobin from the same organism, confirming the absence of the so-called 'central intron' found in the globin genes of plants and of some invertebrates. We identified two homodimeric globin genes (3207 and 2723 bp) that differ only with respect to the size of the first intron. Sequence analysis of the two first introns (1668 and 1364 bp) has revealed that they are highly homologous, except for a 569- and 296-bp insertion in each intron I. Interestingly, the two first introns contain regions with an unusually high identity (approximately 80%) with regions of the first intron of the congeneric clam Anadara trapezia and the related clam Barbatia reveana globin genes, suggesting that these uncoding regions may have played a regulatory role that has subsequently been lost during the course of the evolution.
Subject(s)
Bivalvia/genetics , Exons , Hemoglobins/genetics , Introns , Animals , Base Sequence , Bivalvia/chemistry , DNA/chemistry , DNA/genetics , DNA Transposable Elements , Globins/genetics , Molecular Sequence Data , Multigene Family , Sequence Analysis, DNA , Sequence Deletion , Sequence Homology, Nucleic AcidABSTRACT
A new water-soluble benzodiazepine, midazolam maleate, was used at a dosage of 0.15 mg X kg-1 to induce general anaesthesia for eye surgery in 30 patients. The following parameters were measured at times C (control), t1, t2 and t3 (2, 5 and 10 min after induction): systemic arterial blood pressure, heart rate, respiratory rate, arterial blood gases and eye tone. Results showed very significant changes in acid-base balance, with an increase in PaCO2 and a significant fall in eye tone; changes in haemodynamic parameters were not significant. Midazolam therefore appeared to be a good drug to use for inducing anaesthesia in eye surgery, particularly in the elderly.
