Since its identification in April 2009 an A(H1N1) virus containing a unique combination of gene segments from both North American and Eurasian swine lineages has continued to circulate in humans. The 2009 A(H1N1) virus is distantly related to its nearest relatives, indicating that its gene segments have been circulating undetected for an extended period. Low genetic diversity among the viruses suggests the introduction into humans was a single event or multiple events of similar viruses. Molecular markers predicted for adaptation to humans are not currently present in 2009 A(H1N1) viruses, suggesting previously unrecognized molecular determinants could be responsible for the transmission among humans. Antigenically the viruses are homogeneous and similar to North American swine A(H1N1) viruses but distinct from seasonal human A(H1N1).
It has been considered that the yeast Saccharomyces cerevisiae, like many other microorganisms, synthesizes glutamate through the action of NADP ؉ -glutamate dehydrogenase (NADP ؉ -GDH), encoded by GDH1, or through the combined action of glutamine synthetase and glutamate synthase (GOGAT), encoded by GLN1 and GLT1, respectively. A double mutant of S. cerevisiae lacking NADP ؉ -GDH and GOGAT activities was constructed. This strain was able to grow on ammonium as the sole nitrogen source and thus to synthesize glutamate through an alternative pathway. A computer search for similarities between the GDH1 nucleotide sequence and the complete yeast genome was carried out. In addition to identifying its cognate sequence at chromosome XIV, the search found that GDH1 showed high identity with a previously recognized open reading frame (GDH3) of chromosome I. Triple mutants impaired in GDH1, GLT1, and GDH3 were obtained. These were strict glutamate auxotrophs. Our results indicate that GDH3 plays a significant physiological role, providing glutamate when GDH1 and GLT1 are impaired. This is the first example of a microorganism possessing three pathways for glutamate biosynthesis.Two pathways for ammonium assimilation and glutamate biosynthesis have been found in a variety of organisms. The first one, described by Holzer and Schneider in 1957 (12), is mediated by NADP ϩ -glutamate dehydrogenase (NADP ϩ -GDH; EC 1.4.1.4), which catalyzes the reductive amination of 2-oxoglutarate to form glutamate. In an alternative pathway demonstrated by Tempest et al. (25), glutamate is aminated to form glutamine by glutamine synthetase (GS; EC 6.3.1.2), the amide group of which is then transferred reductively to 2-oxoglutarate by glutamate synthase (GOGAT; EC 1.4.1.13), resulting in the net conversion of ammonium and 2-oxoglutarate to glutamate. The GS-GOGAT pathway has been found in several microorganisms (2,13,16,23) and in higher plants (18).In Saccharomyces cerevisiae, both pathways for glutamate biosynthesis are present (7,19). Mutants altered in NADP ϩ -GDH have been isolated (6); these show a higher doubling time than that of the wild type when both strains are grown on minimal medium supplemented with ammonia as the sole nitrogen source. Mutants impaired in GOGAT activity were selected from NADP ϩ -GDH-less mutants as glutamate auxotrophs (7,19). Genetic analysis of one of these mutants showed that the lack of GOGAT activity was due to the presence of two mutations (gus1 and gus2), which suggested the existence of two GOGAT enzymes in S. cerevisiae (7). Cloning of the GOGAT structural gene (GLT1) and construction of null GOGAT mutants definitively established that this yeast possesses a single NADH-GOGAT enzyme (4) and that GOGATless mutants (7) which cannot be complemented with GLT1 (unpublished results) are probably impaired in GLT1 regulation. In this paper we report the characterization of strains impaired in either GDH1, GLT1, or both. Our results show that there is a third pathway for glutamate biosynthesis, mediated by an N...
Purification of the glutamate synthase (GOGAT) enzyme from Saccharomyces cerevisiae showed that it is an oligomeric enzyme composed of three identical 199-kDa subunits. The GOGAT structural gene was isolated by screening a yeast genomic library with a yeast PCR probe. This probe was obtained by amplification with degenerate oligonucleotides designed from conserved regions of known GOGAT genes. The derived aminoterminal sequence of the GOGAT gene was confirmed by direct amino-terminal sequence analysis of the purified protein of 199 kDa. Northern (RNA) analysis allowed the identification of an mRNA of about 7 or 8 kb. An internal fragment of the GOGAT gene was used to obtain null GOGAT mutants completely devoid of GOGAT activity. The results show that S. cerevisiae has a single NADH-GOGAT enzyme, consisting of three 199-kDa monomers, that differs from the one found in prokaryotic microorganisms but is similar to those found in other eukaryotic organisms such as alfalfa.The existence of two pathways for glutamate biosynthesis has been demonstrated in several microorganisms (4,16,18,31,36,40) and higher plants (1,6,15,23,32,39). One pathway consists of the action of the NADP-dependent glutamate dehydrogenase, and the other involves the concerted action of glutamine synthetase and glutamate synthase (GOGAT).Escherichia coli NADPH-GOGAT and Azospirillum brasilense NADPH-GOGAT have been extensively studied. They are composed of two subunits with molecular masses of 135 and 53 kDa (24, 29). The genes that code for the large and small polypeptides have been cloned and sequenced previously (9, 27, 28).In higher plants, GOGAT occurs in three forms that differ in molecular mass, kinetics, location within the plant, and cofactor specificity (38). The genes that code for NADH-GOGAT from alfalfa (Medicago sativa) (14) and Fd-GOGAT from maize (Zea mays) (32) have also been cloned. Comparative analysis of the amino acid sequences of these two GOGATs with that of E. coli revealed highly conserved regions (14).A protein which bears GOGAT activity has also been purified from baker's yeast. This protein is a dimer composed of a large subunit (169 kDa) and a small subunit (61 kDa) (19).The isolation of Saccharomyces cerevisiae mutants impaired in GOGAT activity has previously been reported (7,25). Genetic analysis of one of these mutants, CN39 (MAT␣ gdh gus1 gus2), showed that its lack of GOGAT activity was due to the presence of two mutations (gus1 and gus2), which suggests the existence of two GOGAT enzymes in S. cerevisiae (7).In order to definitively establish whether there is one GOGAT enzyme or two GOGAT enzymes in S. cerevisiae, we decided to purify the enzyme and construct null GOGAT mutants.We report here the purification of this enzyme and cloning of the yeast GOGAT structural gene. Our results show that the protein is a homotrimeric enzyme composed of 199-kDa polypeptides. We were also able to isolate GOGAT-disrupted yeast mutants completely devoid of NADH-GOGAT activity. Our results show that S. cerevisiae has a single N...
As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.
Different structural changes of the Sym plasmid were found in a Rhizobium phaseoli strain that loses its symbiotic phenotype at a high frequency. These rearrangements affected both nif genes and TnS mob insertions in the plasmid, and in some cases they modified the expression of the bacterium's nodulation ability. One of the rearrangements was more frequent in heat-treated cells, but was also found under standard culture conditions; other structural changes appeared to be related to the conjugal transfer of the plasmid.The genus Rhizobium comprises the gram-negative bacteria that form nodules on legumes. In this association, the bacteria fix atmospheric nitrogen that is then assimilated by the plant.The genetic information controlling symbiotic activity in the fast-growing rhizobia is encoded in plasmids (10,11,13,19). A symbiotic (Sym) plasmid has been defined as one that determines the plant species specificity for nodulation and contains the nitrogenase enzyme structural genes (nifgenes) (12).Plasmids participate very frequently in recombination events (14,20). It has been proposed (27) that this plasticity enables the bacteria that harbor plasmids to adapt to different environmental changes and permits the rapid spread of newly created functions among very diverse bacteria. There is one report (24) of a change in the structure of a plasmid that resulted in modified metabolic activity in the recipient bacteria. This strongly suggests that plasmid plasticity is important in the generation of new functions in bacteria.In Rhizobium phaseoli, the nitrogen fixation gene sequences are reiterated (25). In strain CFN42, there are three regions of the Sym plasmid that contain nitrogenase structural genes (nifregions) (22). These three regions contain the nitrogenase reductase gene (nifH); the nucleotide sequence of the three copies is identical (26). In addition, two of the regions contain also nifD and nifK genes. The identity of the nifH genes suggests that a recombination event could be involved in the generation or maintenance of their reiteration. Reiterated sequences are not common in bacteria, but they have been found in some strains of R. phaseoli, Rhizobium trifolii, and Rhizobium japonicum (2) and also Streptomyces sp. (21), Halobacterium halobium (29), and Pseudomonas syringae p.v. "phaseolicola" (32). The presence of these reiterations may be related to the instability and genetic rearrangements of these organisms (1, 6, 23).We have found that symbiotically unstable isolates are very common among the R. phaseoli strains isolated in different regions of Mexico, and all have reiterated nifH genes (L. Castrejon and G. Soberon, manuscript in preparation). We suppose that the symbiotic instability of these R. phaseoli strains is due to genetic rearrangements caused by the presence of reiterated sequences. We report here that the loss of the symbiotic phenotype of an unstable R. phaseoli strain is due to changes in the structure of its Sym plasmid, * Corresponding author. and that this Sym plasmid can be involve...
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