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There are many new species to be discovered, including eukaryotic species.
- Recognize Eukaryotic diversity
- We know only a fraction of the number of species out there, and most of the attention has been given to large macroscopic species, a true understanding of microscopic eukaryotic life is not known.
- Even though mammals make up a small percentage of the species that are eukaryotes, several new mammalian species have been identified in the last decade.
- As extinction is increasing, we may never know all the eukaryotes that share the world with us.
- extant: Still alive; not extinct.
- metagenomics: The study of genomes recovered from environmental samples; especially the differentiation of genomes from multiple organisms or individuals, either in a symbiotic relationship, or at a crime scene.
According to the Global Taxonomy Initiative and the European Distributed Institute of Taxonomy, the total number of species for some phyla may be much higher than what was known in 2010:10–30 million insects; (of some 0.9 million we know today) 5–10 million bacteria; 1.5 million fungi; of some 0.075 million we know today. The number of microbial species is not reliably known, but the Global Ocean Sampling Expedition dramatically increased the estimates of genetic diversity by identifying an enormous number of new genes from near-surface plankton samples at various marine locations.
From these studies, it is apparent that less than 1% of all species that have been described have been studied beyond simply noting their existence. The vast majority of Earth’s species are microbial. Contemporary biodiversity is “firmly fixated on the visible world”. For example, microbial life is metabolically and environmentally more diverse than multicellular life (e.g., extremophile). On the tree of life, based on analyses of small-subunit ribosomal RNA, visible life consists of barely noticeable twigs. The inverse relationship of size and population recurs higher on the evolutionary ladder.
Due to the advent of mass sequencing tools, thousands of new viral species have been identified in metagenomics studies, while at the same time hundreds of new viral species have been found. On top of that, there have been numerous fungal species identified. However, as suggested above most of the attention is given to large species, which represent a very small portion of the new species identified Even with this in mind, since the beginning of this century, 5 marsupial species, 25 primate, 1 elephant, 1 sloth, 3 rabbit, several rodent species, at least 30 new bat species have been discovered. On top of that several subspecies have been found. Considering how large an elephant is, this should point out how little we know about the numbers of microscopic eukaryotes that are yet to be discovered.
Of course we may never truly identify many eukaryotic species, since the rate of extinction has increased. Many extant species may become extinct before they are described.
Pneumocystis Encodes a Functional S-Adenosylmethionine Synthetase GeneFIG. 1 . Nucleotide and deduced amino acid sequences of P. carinii sam1 and partial sequences of the putative chd1 gene. Initiation and termination codons are shown in bold and are underlined. The transcription start site is indicated by an arrow, the introns are shown in lowercase letters, and the XbaI site is marked by a solid line above the sequence. FIG. 2 . Alignment of the deduced Pneumocystis Sam1 amino acid sequences with those of other organisms. Sam1 sequences from P. carinii, P. murina, P. jirovecii, S. pombe, and S. cerevisiae, MetK sequence from E. coli, and MAT1 sequences from mice (Mus musculus), rats (Rattas norvegicus), and humans (Homo sapiens) were aligned using Clustal W. Identical amino acid residues are boxed. AdoMet synthetase signature motifs are underlined. ATP binding motifs are boxed in bold. FIG. 3 . Northern and Southern blotting analyses of Pneumocystis sam1. (A) Northern blotting analysis of total RNA from P. carinii organisms or P. murina-infected mouse lung. The blots were hybridized with a DIG-labeled PCR product spanning 552 to 2,020 bp of P. carinii sam1 genomic sequence. A strong hybridization signal (∼1.3 kb) indicated by the solid arrow is seen in P. carinii (lane 1) or P. murina (lane 2) RNA preparations, which is consistent with the size of Pneumocystis sam1 cDNA. The probe also recognized a minor band of ∼5.6 kb, which is indicated by an open arrow. When the blot containing P. carinii RNA was reprobed with DIG-labeled oligonucleotides designed from the coding region of the putative chd1 gene, only the 5.6-kb band was seen (lane 3). (B) Southern blotting analysis of genomic DNA from P. carinii. Genomic DNA extracted from P. carinii-infected rat lung was digested with different restriction endonucleases, and the blots were probed with a DIG-labeled PCR product spanning 552 to 2,020 bp of P. carinii sam1 genomic sequence. DNA digested with AseI (lane 1) showed a single band XbaI (lane 2) gave two bands due to the presence of a restriction site within the region of the probe. When the same blot was reprobed with the DIG-labeled oligonucleotide designed from the putative chd1 gene, a single band was seen following digestion with AseI (lane 3) or XbaI (lane 4). The hybridization signals obtained correspond to the same size as the band recognized by the sam1 probe, consistent with a tandem genetic arrangement of sam1 and the putative chd1 gene. Molecular size markers (kb) are shown to the right of each gel. FIG. 4 . Expression of P. carinii recombinant protein Sam1. (A) SDS-PAGE analysis of the bacterial cells expressing recombinant protein showed a band of the expected size (∼45 kDa, indicated by the arrows), when stained with Coomassie blue (lane 1). When vector with no insert was analyzed, no band of that size was observed (lane 2). (B) Bacterial cells expressing the Sam1 protein showed immunoreactivity with the expected size band when probed in a Western blot with His tag antibody (lane 1), while there was no immunoreactivity when vector alone was analyzed (lane 2). (C) Refolded protein showed a band of ∼45 kDa when stained with Coomassie blue (lane 1), while no band of that size was seen when vector alone was analyzed (lane 2). (D) A 45-kDa band showed immunoreactivity when Western blotting analysis shown in panel C was performed using His tag antibody (lane 1), but no immunoreactivity was observed when vector alone (negative control) was analyzed (lane 2). (E) Immunoreactivity to a 45-kDa band (lane 1) was lost when the anti-Sam1 antibody was preincubated with the antigen (recombinant protein) (lane 2). Molecular size markers (kDa) are shown to the right of panels B and D. FIG. 5 . AdoMet synthetase activity. AdoMet synthetase activity of recombinant, refolded P. carinii protein was measured at different time intervals. The figure shows the activity measured in two different experiments and performed in duplicate. Values are means ± standard deviations (error bars) (n = 4). The enzyme activity is expressed as nmol of AdoMet formed/mg protein. The protein preparation obtained by using the vector alone showed no activity. FIG. 6 . Western blotting analysis of immunoprecipitated samples from P. murina. Partially purified P. murina extracts were subjected to immunoprecipitation, using anti-Sam1 antibody (lane 1) or preimmune serum as a negative control (lane 2), followed by Western blotting analysis, using the anti-Sam1 antibody. The former (lane 1) showed a reactive band of 45 kDa (arrow), the size expected for Sam1, while the latter (lane 2) showed no immunoreactivity. The bands corresponding to IgG are marked. When anti-Sam1 antibody that was preincubated with recombinant Sam1 was used for the Western blot, immunoreactivity to the 45-kDa band was blocked (lane 3), demonstrating that the immunoreactivity is specific for Sam1. FIG. 7 . Confocal immunofluorescence microscopic detection of Sam1 in P. murina-infected mouse lung. (A) Dual immunofluorescence staining of P. murina-infected mouse lung tissue sections using anti-Sam1 and anti-Pneumocystis (4D7) antibodies. (B) Dual immunofluorescence staining of P. murina-infected mouse lung tissue sections using anti-Sam1 antibody preabsorbed with insoluble recombinant AdoMet protein and anti-Pneumocystis (4D7) antibody. Panel 1, blue indicates the cell nuclei stained with DAPI. Panel 2, staining of Pneumocystis using 4D7 antibody, biotin-conjugated anti-mouse IgG and streptavidin-conjugated Alex Fluor 594. Red indicates immunoreactivity. Panel 3, staining with rabbit anti-P. murina Sam1 antibody, using Alexa Fluor 488-conjugated goat anti-rabbit IgG as the secondary antibody. Green indicates the immunoreactivity with the anti-Sam1 antibody. Panel 4, merged images. The green anti-Sam1 fluorescence colocalizes with the anti-Pneumocystis red fluorescence staining. An arrow indicates a structure that is consistent with a cyst, within which are multiple DAPI-stained nuclei that, however, did not stain with the anti-Sam1 antibody. The anti-Sam1 immunoreactivity was blocked when the antibody was preabsorbed with recombinant protein, showing that the immunoreactivity is specific for Sam1. The thickness of the slides used for the confocal examination was 5 μm, and each confocal picture is estimated to be ∼1 μm.
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