Translation Initiation Factor-Dependent Extracts from YeastSaccharomyces cerevisiae

Translation initiation factor-dependent extracts are prepared fromSaccharomyces cerevisiaestrains that have no or reduced activity of a translation initiation factor. Elimination of factor activity can be achieved by deletion of the gene encoding the factor if it is not essential for the survival of the strain. If the gene is essential it is placed under the control of the regulatable GAL1 promoter and its expression is shut offin vivo.Alternatively, a temperature-sensitive mutation can be introduced into the gene and the activity of the gene product eliminatedin vitroby preincubation of the extract at the nonpermissive temperature. Factor-dependent extracts can be complementedin vitrowith purified initiation factor preparations isolated fromS. cerevisiaeor fromEscherichia colicells expressing them from plasmid-encoded constructs. To simplify the purification of the factors they may be expressed as fusion proteins with N- or C-terminal tags. Initiation factor-dependent extracts can be used to study initiation factor structure–function relationships and initiation factor requirements for specific mRNA translation.     

The 5'-leader sequence of tobacco mosaic virus RNA mediates initiation-factor-4E-independent, but still initiation-factor-4A-dependent translation in yeast extracts

Messenger RNAs encoding chloramphenicol acetyltransferase (CAT) with or without the 5'-leader sequence of tobacco mosaic virus (TMV) RNA were synthesized in vitro and translated in Saccharomyces cerevisiae extracts dependent on eukaryotic initiation factors eIF-4E or eIF-4A. The 5'-leader sequence of TMV RNA renders translation of CAT mRNA eIF-4E-independent but still 4A-dependent.     

Identification and characterization of cap-binding proteins from yeast

Photochemical cross-linking of Saccharomyces cerevisiae ribosomal salt wash preparations to cap-labeled mRNA reveals, in addition to the previously characterized 24-kDa cap-binding protein (eIF-4E), the presence of two novel cap-binding proteins (CBPs) of apparent molecular masses of 96 and 150 kDa. Cross-linking of the 96-kDa CBP was found to occur spontaneously without UV light induction. Based on the ATP/Mg2+ requirements, the three CBPs can be subdivided into two classes: 1) ATP/Mg2+ independent (24- and 150 kDa) and 2) Mg2+ dependent (96 kDa). The co-purification of the 24- and 150-kDa CBPs through several different chromatographic steps is consistent with the existence of a yeast CBP complex, possibly analogous to mammalian eIF-4F.     

Combining next‐generation sequencing and progeny testing for rapid identification of induced recessive and dominant mutations in maize M2 individuals

Molecular identification of mutant alleles responsible for certain phenotypic alterations is a central goal of genetic analyses. In this study we describe a rapid procedure suitable for the identification of induced recessive and dominant mutations applied to two Zea mays mutants expressing a dwarf and a pale green phenotype, respectively, which were obtained through pollen ethyl methanesulfonate (EMS) mutagenesis. First, without prior backcrossing, induced mutations (single nucleotide polymorphisms, SNPs) segregating in a (M₂) family derived from a heterozygous (M₁) parent were identified using whole‐genome shotgun (WGS) sequencing of a small number of (M₂) individuals with mutant and wild‐type phenotypes. Second, the state of zygosity of the mutation causing the phenotype was determined for each sequenced individual by phenotypic segregation analysis of the self‐pollinated (M₃) offspring. Finally, we filtered for segregating EMS‐induced SNPs whose state of zygosity matched the determined state of zygosity of the mutant locus in each sequenced (M₂) individuals. Through this procedure, combining sequencing of individuals and Mendelian inheritance, three and four SNPs in linkage passed our zygosity filter for the homozygous dwarf and heterozygous pale green mutation, respectively. The dwarf mutation was found to be allelic to the an1 locus and caused by an insertion in the largest exon of the AN1 gene. The pale green mutation affected the nuclear W2 gene and was caused by a non‐synonymous amino acid exchange in encoded chloroplast DNA polymerase with a predicted deleterious effect. This coincided with lower cpDNA levels in pale green plants.     

Nitrate or ammonium: Influences of nitrogen source on the physiology of a green alga

In freshwaters, algal species are exposed to different inorganic nitrogen (N_i) sources whose incorporation varies in biochemical energy demand. We hypothesized that due to the lesser energy requirement of ammonium ()‐use, in contrast to nitrate ()‐use, more energy remains for other metabolic processes, especially under CO₂‐ and phosphorus (P_i) limiting conditions. Therefore, we tested differences in cell characteristics of the green alga Chlamydomonas acidophila grown on or under covariation of CO₂ and P_i‐supply in order to determine limitations, in a full‐factorial design. As expected, results revealed higher carbon fixation rates for ‐grown cells compared to growth with under low CO₂ conditions. ‐grown cells accumulated more of the nine analyzed amino acids, especially under P_i‐limited conditions, compared to cells provided with . This is probably due to a slower protein synthesis in cells provided with . In contrast to our expectations, compared to ‐grown cells ‐grown cells had higher photosynthetic efficiency under P_i‐limitation. In conclusion, growth on the N_i‐source did not result in a clearly enhanced C_i‐assimilation, as it was highly dependent on P_i and CO₂ conditions (replete or limited). Results are potentially connected to the fact that C. acidophila is able to use only CO₂ as its inorganic carbon (C_i) source.     

Century-scale Methylome Stability in a Recently Diverged Arabidopsis thaliana Lineage

There has been much excitement about the possibility that exposure to specific environments can induce an ecological memory in the form of whole-sale, genome-wide epigenetic changes that are maintained over many generations. In the model plant Arabidopsis thaliana, numerous heritable DNA methylation differences have been identified in greenhouse-grown isogenic lines, but it remains unknown how natural, highly variable environments affect the rate and spectrum of such changes. Here we present detailed methylome analyses in a geographically dispersed A. thaliana population that constitutes a collection of near-isogenic lines, diverged for at least a century from a common ancestor. Methylome variation largely reflected genetic distance, and was in many aspects similar to that of lines raised in uniform conditions. Thus, even when plants are grown in varying and diverse natural sites, genome-wide epigenetic variation accumulates mostly in a clock-like manner, and epigenetic divergence thus parallels the pattern of genome-wide DNA sequence divergence.