The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new sequence-tagged site marker−based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished by the use of a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new ~936 cM linkage map comprising 2469 marker loci. In silico anchoring approaches used genetic and physical maps from the diploid potato genotype RH89-039-16 (RH) and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (~93%) of the 723 Mb genome assembly and 37,482 (~96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules are closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal “pseudomolecules”.
The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the a subunit of translation initiation factor 2 (eIF-2a) in amino acid-starved cells. Phosphorylation of eIF-2a reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these GCN27 alleles map in the protein kinase moiety, and two in this group alter the presumed ATP-binding domain, suggesting that ATP binding is a regulated aspect of GCN2 function. Six GCN2' alleles map in a region related to histidyl-tRNA synthetases, and two in this group alter a sequence motif conserved among class H aminoacyl-tRNA synthetases that directly interacts with the acceptor stem of tRNA. These results support the idea that GCN2 kinase function is activated under starvation conditions by binding uncharged tRNA to the domain related to histidyl-tRNA synthetase. The remaining GCN2C alleles map at the extreme C terminus, a domain required for ribosome association of the protein. Representative mutations in each domain were shown to depend on the phosphorylation site in eIF-2c for their effects on GCN4 expression and to increase the level of eIF-2a phosphorylation in the absence of amino acid starvation. Synthetic GCN2" double mutations show greater derepression ofGCN4 expression than the parental single mutations, and they have a slow-growth phenotype that we attribute to inhibition of general translation initiation. The phenotpes of the GCN2C alleles are dependent on GCNI and GCN3, indicating that these two positive regulators of GCN4 expression mediate the inhibitory effects on translation initiation associated with activation of the yeast eIF-2am kinase GCN2.Phosphorylation of the a subunit of translation initiation factor 2 (eIF-2a) in mammalian cells leads to inhibition of protein synthesis at the initiation step. Two different mammalian (eIF-2a) kinases have been identified: the doublestranded-RNA-activated inhibitor of translation (DAI) that is activated in response to viral infections and the hemecontrolled repressor (HCR) that is activated in reticulocytes by heme deficiency (for a review, see reference 26). Both kinases phosphorylate eIF-2a on the serine residue at position 51 (Ser-51) (9, 47). The phosphorylation of mammalian eIF-2a inhibits translation initiation by impairing the conversion of eIF-2-GDP to eIF-2-GTP at the completion of each initiation cycle, a reaction carried out by the guanine nucleotide exchange factor eIF-2B. Only the GTP-bound form of eIF-2 is able to form a ternary complex with the initiator tRNAMet and catalyze new rounds of translation initiation (41).GCN2 to increased synthesis of GCN4, a transcriptional activator of numerous genes encoding enzymes involved in amino acid biosynthesis. The kinase activity of GCN2 is required f...
The GCN4 gene of the yeast Saccharomyces cerevisiae encodes a transcriptional activator of amino acid biosynthetic genes that is regulated at the translational level according to the availability of amino acids. GCN2 is a protein kinase required for increased translation of GCN4 mRNA in amino acid-starved cells. Centrifugation of cell extracts in sucrose gradients indicated that GCN2 comigrates with ribosomal subunits and polysomes. The fraction of GCN2 cosedimenting with polysomes was reduced under conditions in which polysomes were dissociated, suggesting that GCN2 is physically bound to these structures. When the association of 40S and 60S subunits was prevented by omitting Mg2e from the gradient, almost all of the GCN2 comigrated with 60S ribosomal subunits, and it remained bound to these particles during gel electrophoresis under nondenaturing conditions. GCN2 could be dissociated from 60S subunits by 0.5 M KCI, suggesting that it is loosely associated with ribosomes rather than being an integral ribosomal protein. Accumulation of GCN2 on free 43S-48S particles and 60S subunits occurred during polysome runoff in vitro and under conditions of reduced growth rate in vivo. These observations, plus the fact that GCN2 shows preferential association with free ribosomal subunits during exponential growth, suggest that GCN2 interacts with ribosomes during the translation initiation cycle. The extreme carboxyl-terminal segment of GCN2 is essential for its interaction with ribosomes. These sequences are also required for the ability of GCN2 to stimulate GCN4 translation in vivo, leading us to propose that ribosome association by GCN2 is important for its access to substrates in the translational machinery or for detecting uncharged tRNA in amino acid-starved cells.
Wine Saccharomyces cerevisiae strains producing a new killer toxin (Klus) were isolated. They killed all the previously known S. cerevisiae killer strains, in addition to other yeast species, including Kluyveromyces lactis and Candida albicans. The Klus phenotype is conferred by a medium-size double-stranded RNA (dsRNA) virus, Saccharomyces cerevisiae virus Mlus (ScV-Mlus), whose genome size ranged from 2.1 to 2.3 kb. ScV-Mlus depends on ScV-L-A for stable maintenance and replication. We cloned and sequenced Mlus. Its genome structure is similar to that of M1, M2, or M28 dsRNA, with a 5-terminal coding region followed by two internal A-rich sequences and a 3-terminal region without coding capacity. Saccharomyces cerevisiae killer strains produce and secrete protein toxins that are lethal to sensitive strains of the same or related yeast species. These toxins have been grouped into three types, K1, K2, or K28, based on their killing profiles and lack of cross-immunity. Members of each group can kill nonkiller yeasts as well as killer yeasts belonging to the other types. They are immune, however, to their own toxin or to toxins produced by strains of the same killer type (for reviews, see references 21, 32, 33, and 47).K1, K2, and K28 killer toxins are genetically encoded by medium-size double-stranded RNA (dsRNA) viruses grouped into three types, M1, M2, and M28, of 1.6, 1.5, and 1.8 kb, respectively. Only one strand (the positive strand) has coding capacity. In each case, the 5Ј-end region contains an open reading frame (ORF) that codes for the toxin precursor, or preprotoxin (pptox), which also provides immunity. The three toxin-coding M dsRNAs show no sequence homology to each other (35). M viruses depend on a second large (4.6-kb) dsRNA helper virus, L-A, for maintenance and replication. L-A provides the capsids in which both L-A and M dsRNAs are separately encapsidated (reviewed by Schmitt and Breinig [33]). L-BC virus is an L-A-related virus, with a similar 4.6-kb genome size, which coexists with L-A in most killer and nonkiller S. cerevisiae strains (1, 37). L-BC shows no sequence homology with L-A, and it has no known helper activity. L-A and L-BC, however, share the same genomic organization. They code for two proteins, the major coat protein Gag and a minor Gag-Pol fusion protein translated by a Ϫ1 ribosomal frameshifting mechanism (7,10,17,26). These viruses, called Saccharomyces cerevisiae viruses (ScVs), belong to the Totiviridae family and are cytoplasmically inherited, spreading horizontally by cell-cell mating or by heterokaryon formation (47). In addition to the M dsRNA-encoded killer toxins, other S. cerevisiae killer toxins, named KHR and KHS, showing weak killer activity, are encoded on chromosomal DNA (13,14).The positive strands of both L-A and M viruses contain cis signals in their 3Ј-terminal regions essential for packaging and replication (46). The signal for transcription initiation has been proposed to be present in the first 25 nucleotides (nt) of L-A, probably in the 5Ј-terminal sequ...
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