A genomewide suppressor and enhancer analysis of cdc13-1 reveals varied cellular processes influencing telomere capping in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Cdc13 binds telomeric DNA to recruit telomerase and to "cap" chromosome ends. In temperature-sensitive cdc13-1 mutants telomeric DNA is degraded and cell-cycle progression is inhibited. To identify novel proteins and pathways that cap telomeres, or that respond to uncapped telomeres, we combined cdc13-1 with the yeast gene deletion collection and used high-throughput spot-test assays to measure growth. We identified 369 gene deletions, in eight different phenotypic classes, that reproducibly demonstrated subtle genetic interactions with the cdc13-1 mutation. As expected, we identified DNA damage checkpoint, nonsense-mediated decay and telomerase components in our screen. However, we also identified genes affecting casein kinase II activity, cell polarity, mRNA degradation, mitochondrial function, phosphate transport, iron transport, protein degradation, and other functions. We also identified a number of genes of previously unknown function that we term RTC, for restriction of telomere capping, or MTC, for maintenance of telomere capping. It seems likely that many of the newly identified pathways/processes that affect growth of budding yeast cdc13-1 mutants will play evolutionarily conserved roles at telomeres. The high-throughput spot-testing approach that we describe is generally applicable and could aid in understanding other aspects of eukaryotic cell biology.

Stable albinism induced without mutagenesis: a model for ribosome-free plastid inheritance.

Maternally inherited chlorophyll deficiency, or albinism, is a standard marker in plant cytoplasmic genetics. Its stability is consistent with mutations in the plastid genome. Nuclear mutations inducing plastid ribosome deficiency (PRD) also lead to maternally inherited chlorophyll deficiency. Here we report that stable chlorophyll deficiency can be efficiently generated in cruciferous plants without mutagenesis by a short exposure to spectinomycin, an inhibitor of plastid protein synthesis. We show that the chlorophyll-deficient phenotype results from a deficiency in plastid ribosomes and plastid translation products. Loss of plastid ribosomes is irreversible. The data suggest that mutations are not essential for generating inheritable PRD. It allows the formulation of a more general model in which stable PRD can be induced by a variety of factors that prevent the formation of functional plastid ribosomes. A non-mutational mechanisms for generating inheritable chlorophyll deficiency has implications for the origin and inheritance of green-white variegation in nature.

Differential regulation of genes transcribed by nucleus-encoded plastid RNA polymerase, and DNA amplification, within ribosome-deficient plastids in stable phenocopies of cereal albino mutants.

We isolated stable albino plants of barley and maize by inhibiting plastid protein synthesis with streptomycin and propagating bleached seedlings in the absence of antibiotics in vitro. Albino plants are deficient in plastid translation products and plastid ribosomal RNAs, and are stable phenocopies of the barley albostrians and maize iojap mutants, which contain ribosome-free plastids. Once plastid ribosomes are lost they cannot be re-synthesized because about one-third of plastid ribosomal proteins are themselves plastid encoded. The group II/subgroup IIA intron in plastid rpl2 transcripts was not processed in albinos, providing strong evidence for the absence of plastid translation. Photosynthesis-related plastid mRNAs and plastid tRNAs were down-regulated in albino leaves. A differential influence of plastid ribosome deficiency on mRNA levels allowed us to divide genes transcribed by nucleus-encoded plastid RNA polymerase into two groups. Northern analysis revealed increases in the levels of clpP, rpl2, rpl23, rps15 and rpoB mRNAs in total RNA from albino leaves relative to those in green leaves. In contrast, albinism did not increase the band intensities of rps2 and rps4 messages. Plastid ribosome-associated factor(s) or plastid-encoded product(s) play a role in the initiation, termination, processing or stability of transcripts containing trnG(UCC) and rps4. Excision and 100-fold amplification of a 5.2-kb region of plastid DNA encompassing the trnG(UCC) and trnE(UUC) genes was observed in one of four albino barley plants. Gene amplification was correlated with the accumulation of abundant novel transcripts derived from regions flanking the trnG(UCC) gene.

Marking cell layers with spectinomycin provides a new tool for monitoring cell fate during leaf development.

Spectinomycin, an inhibitor of plastid protein synthesis, can be used to mark specific cell layers in the shoot meristem of Brassica napus. Pale yellow-green (YG) plants resulting from spectinomycin-treatment can be propagated indefinitely in vitro. Microscopic examination showed that YG-plants result from inactivation of plastids in the L2 and L3 layers and are composed of a pale green epidermis covering a white mesophyll layer. Epidermal cells of YG and normal green plants are similar and contain 10-20 small pale green plastids. YG plants are equivalent to periclinal chimeras with the important distinction that there is no genotypic difference between the white and green cell layers. Periclinal divisions of epidermal cells take place at all stages of leaf development to produce invaginations of green mesophyll located in sectors of widely varying sizes. A periclinal division rate of 1 in 3000-4000 anticlinal divisions for the adaxial epidermis, was 2-3-fold higher than that estimated for the abaxial epidermis. Analysis of white and green mesophyll showed that chloroplasts are essential for palisade cell differentiation and this requirement is cell-autonomous. Stable marking of cell lineages with spectinomycin is simple, rapid and reveals the requirement for functional plastids in cellular differentiation.

Selection of anthocyanin-accumulating potato (Solanum tuberosum L.) cell lines from calli derived from seedlings produced by gamma-irradiated seeds.

Callus cell lines of potato (Solanum tuberosum L. cv. Zarevo) were obtained from seedlings germinated from gamma-irradiated seeds (200 Gy). Some of these cell lines produce red-violet pigments which were identified as acylated anthocyanins. The major anthocyanin was determined to be peonidin 3-O-[6-O-(4-O-E-p-coumaroyl-rhamnosyl)-glucoside]-5-O-glucoside ("peonanin"). Single cell-derived protoclones from non-pigmented protoplasts sometimes also gave rise to pigmented cell clusters thus indicating that the changes in the expression of the anthocyanin pathway can also occur after the stage of initial callus induction.

Somatic hybridization in potato: use of γ-irradiated protoplasts of Solanum pinnatisectum in genetic reconstruction.

Leaf mesophyll protoplasts of Solanum pinnatisectum (2n=24) γ-irradiated at doses of 200 Gy and consequently unable to divide were fused with untreated protoplasts of genomic chlorophyll deficient mutant IvP 841-1 (2n=24) containing the germplasms of S. tuberosum and S. phureja. Two types of plants differing in their pigmentation characteristics were selected. The regenerants of one group were identified as true somatic hybrids by using isozyme analyses of esterase and aspartate aminotransferase. The anthocyanin marker of S. pinnatisectum was phenotypically expressed in these regenerants and could be used as an additional selection trait for hybrid screening in this species combination. The regenerants of the second group were corrected for the gene controlling chlorophyll deficiency but contained species-specific isozymes of the potato cultivar only. Restriction analysis of chloroplast DNA revealed chloroplasts of the S. pinnatisectum type in all but one of the plants tested. The fusion experiments involving γ-irradiated protoplasts show that this approach in potato reconstruction has the advantage of producing a wide range of genetically novel plants.

Extensive developmental and metabolic alterations in cybrids Nicotiana tabacum (+ Hyoscyamus niger) are caused by complex nucleo-cytoplasmic incompatibility.

The genetic basis of multiple phenotypic alterations was studied in cell-engineered cybrids Nicotiana tabacum (+ Hyoscyamus niger) combining the nuclear genome of N. tabacum, plastome of H. niger and recombinant mitochondria. The plants possess a complex, maternally inheritable syndrome of nucleo-cytoplasmic incompatibility, severely affecting growth, metabolism and development. In vivo, the syndrome was manifested as: late germination of seeds; dramatic decrease of chlorophyll and carotenoids in cotyledons and leaves; altered morphology of cotyledons, leaves and flowers; and dwarfism. The leaf phenotype depended on light intensity. In 'green flowers' (an extreme phenotype), homeotic function B was downregulated. In vitro, the incompatibility syndrome was restricted to the pigment deficiency of cotyledons. Electron microscopy revealed perturbations in the differentiation of chloroplasts and palisade parenchyma cells in bleached leaves. The pigment deficiency accompanied by retarded growth is discussed as a result of plastome-genome incompatibility, whereas other features are likely to be due to nucleo-mitochondrial incompatibilities.