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In Southern blot analysis of Paramecium genomic DNA using β-adrenergic specific oligonucleotides labelled with Digoxigenin-ddUTP, the DNA species of the same molecular size were detected by different molecular probes. The 6.5 kb DNA species hybridized with the probes directed to the adjacent β2-receptor transmembrane domains: to TM3 - including Asp 113 involved in β-agonists and antagonists binding, and to TM4 - the oligonucleotide being one of the "gene-specific universal mammalian sequence-tagged site”. Since a profound physiological effect of beta-agonists and antagonists has been previously observed in Paramecium, such a hybridization pattern may suggest an existence of Paramecium DNA sequences homologous to those of metazoan eukaryotes encoding for a membrane beta-adrenergic receptor.
In this study we test three hypotheses. (1) Secretory hairs in the arms and the distal part of the neck of the carnivorous plant Genlisea (Lentibulariaceae) have a different principal function than the digestive hairs in the digestive chamber, that is, prey attraction. (2) Only bacteria and other organisms inside the trap and on the external trap surface lure prey. (3) Substances produced by the plant have a minor influence on prey attraction; more important is trap shape and morphology, because protozoa and microfauna may move to the small interspaces (traps or capillaries) by accidental, nonspecific wandering. We studied the structure of secretory hairs (glands) in the arms and the distal and proximal parts of the trap neck using light, fluorescence and electron microscopy. We tested the hypotheses with several experiments using sterile Genlisea traps as well as glass tubes acting as a Genlisea trap model, and various organisms as prey (Blepharisma sp., Paramecium bursaria, Euglena sp.). Hairs in the arms and the distal part of the Genlisea trap neck represent polysaccharide-protein-secreting hairs. Prey still moved to cleaned traps without chemical attractants. In the proximal part of the neck the secretory hairs have the same ultrastructure as digestive hairs in the digestive chamber of Genlisea. Sterile traps do not need commensals for catching prey. The results of the behavioral experiments reported here support the hypothesis that prey can move to the traps or capillaries by accidental, nonspecific wandering to small objects filled with water. Thus, the complex structure of the Genlisea trap with long arms may help catch prey simply by providing a large surface with many small openings which mimic the interspaces between soil particles, and the plant does not need special mediators for prey attraction.
RT-PCR and Northern blot analysis were performed in order to search for a putative beta-adrenergic receptor (β-AR) in Paramecium using several β2-adrenergic-specific molecular probes.Under strictly defined RT-PCR conditions DNA species of expected molecular size about 360 bp were generated with the primers corresponding to the universal mammalian β2-AR sequence tagged sites (located within the 4th and the 6th transmembrane regions of the receptor). This RT-PCR product hybridized in Southern blot analysis with the oligonucleotide probe designed to the highly conservative β2-AR region involved in G-proteins interaction and located within the amplified region. Northern hybridization was performed on Paramecium total RNA and mRNA with human β2-AR cDNA and two oligonucleotide probes: the first included Phe 290 involved in agonist binding (Strader et al., 1995) and the second was the backward RT-PCR primer. All these probes revealed the presence of about 2 kb mRNA which is consistent with the size of β2-AR transcripts found in higher eukaryotes.
Rab7 is a small GTPase that plays a crucial role in the regulation of transport from early to late endosomes and lysosomes, phagosome maturation and in lysosomal biogenesis in mammalian cells. It contains conserved and unique sequence elements that mediate its function. Two Rab7 genes, Rab7a (703 bp) and Rab7b (707 bp) were identified in the unicellular eukaryote Paramecium by PCR amplification. They contain three short introns of different lengths (28-32 bp) and sequence located at identical positions in both genes. The presence of two Rab7 genes in the Paramecium genome was confirmed by Southern hybridization analysis performed with six different restriction enzymes. Expression of both genes was assessed by Northern blot and RT-PCR. Two transcripts of 1.8 and 2.2 kb were identified by hybridization analysis. The cloned complementary DNAs, both of 618 nucleotides in length, encode polypeptides of 206 amino acids that are 97.6% identical and differ in their C-termini. The predicted protein sequences of Rab7a and Rab7b contain all characteristic domains essential for Rab function: the effector domain (YRATVGADF) and four GTP-binding consensus sequences (GDSGVGKT, WDTAGQ, NKLD, SAK) as well as the prenylation motif (-CC) at the C-terminus indispensable for Rab binding to the membrane. Similarity searches revealed 81.6-82.1% homology of Paramecium Rab7 isoforms to human Rab7 and a lack of an insert typical for the Kinetoplastida - the species that appeared earlier in evolution. Paramecium is the first free-living lower eukaryote in which homologues of Rab7 have been identified that exhibit features similar to those of mammalian Rab7.
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