Geminiviruses: Genome structure and gene function

The plant pathogenic single‐strand DNA‐containing geminiviruses have been the recent focus of intense investigation, owing both to their agronomic importance and to their potential as vectors for the expression of foreign genes in plants. Molecular genetic studies have provided detailed information on the genomic organization of many of these viruses. A greater genetic complexity has been demonstrated among the members of this viral family than had previously been suspected, as well as an apparently rapid rate of evolution of genetic diversity. We now recognize fundamental differences in the genome structure and organization of the whitefly‐ and leafhopper‐transmitted viruses, as well as among those geminiviruses infecting dicotyledonous or monocotyledonous hosts. This knowledge has provided new insights into the evolution of these viruses. The viral genes involved in replication and in systemic movement in the plant have been defined, and viral origins for single‐strand (ss) and double‐strand (ds) DNA replication have been mapped to small nucleotide regions. With the structural features of the viral genomes now well defined, current efforts are focused on elucidating the molecular aspects of viral gene regulation and interactions with host‐cell components that lead to the production of disease. Recent progress in determining the mechanism of replication and systemic movement and the contributions of these to symptom and disease development are discussed in the context of the potential for genetically engineering disease‐resistant plants.     

Inhibition of Growth of Escherichia coli and of Homoserine O-Transsuccinylase by α-Methylmethionine

The methionine analogue, alpha-methylmethionine, inhibits bacterial growth, but its action is overcome by methionine, homocysteine, and cystathionine. The effect of the analogue on growth is attributed to its ability to mimic methionine as a feed-back inhibitor of the first enzyme specific to methionine biosynthesis. This conclusion is based on the findings that (i) alpha-methylmethionine inhibits excretion of O-succinylhomoserine, the product of the first enzyme, by a methionine auxotroph unable to convert succinylhomoserine to cystahionine, and that (ii) the enzyme homoserine O-transsuccinylase is inhibited by alpha-methylmethionine in extracts of Escherichia coli. alpha-Methylmethionine also inhibits methionyl-ribonucleic acid synthetase in extracts, but this inhibition probably does not affect growth.     

Molecular Basis of Differential Sensitivity of Myeloma Cells to Clinically Relevant Bolus Treatment with Bortezomib

The proteasome inhibitor bortezomib (Velcade) is prescribed for the treatment of multiple myeloma. Clinically achievable concentrations of bortezomib cause less than 85% inhibition of the chymotrypsin-like activity of the proteasome, but little attention has been paid as to whether in vitro studies are representative of this level of inhibition. Patients receive bortezomib as an intravenous or subcutaneous bolus injection, resulting in maximum proteasome inhibition within one hour followed by a gradual recovery of activity. In contrast, most in vitro studies use continuous treatment so that activity never recovers. Replacing continuous treatment with 1 h-pulse treatment increases differences in sensitivity in a panel of 7 multiple myeloma cell lines from 5.3-fold to 18-fold, and reveals that the more sensitive cell lines undergo apoptosis at faster rates. Clinically achievable inhibition of active sites was sufficient to induce cytotoxicity only in one cell line. At concentrations of bortezomib that produced similar inhibition of peptidase activities a different extent of inhibition of protein degradation was observed, providing an explanation for the differential sensitivity. The amount of protein degraded per number of active proteasomes correlated with sensitivity to bortezomib. Thus, (i) in vitro studies of proteasome inhibitors should be conducted at pharmacologically achievable concentrations and duration of treatment; (ii) a similar level of inhibition of active sites results in a different extent of inhibition of protein breakdown in different cell lines, and hence a difference in sensitivity.     

Analysis of cellular water content in T cells reveals a switch from slow metabolic water gain to rapid water influx prior to cell division

Cell growth is driven by the acquisition and synthesis of both dry biomass and water mass. In this study, we examine the increase of water mass in T cell during cell growth. We found that T-cell growth is characterized by an initial phase of slow increase in cellular water, followed by a second phase of rapid increase in water content. To study the origin of the water gain, we developed a novel methodology we call cold aqua trap-isotope ratio mass spectrometry, which allows analysis of the isotope composition of intracellular water. Applying cold aqua trap-isotope ratio mass spectrometry, we discovered that glycolysis-coupled metabolism of water accounts on average for 11 fl out of the 20 fl of water gained per cell during the initial slow phase. In addition, we show that at the end of the rapid phase before initiation of cell division, a water influx occurs, increasing the cellular water mass by threefold. Thus, we conclude that activated T cells switch from metabolizing water to rapidly taking up water from the extracellular medium prior to cell division. Our work provides a method to analyze cell water content as well as insights into the ways cells regulate their water mass.     

Polymorphism and evolution in the constant region of the T-cell receptor beta chain in an advanced teleost fish

Sequence, PCR and Southern data are presented as evidence that, as in mammals, two gene loci encode C regions of the TCR beta chain in the bicolor damselfish, Stegastes partitus. The loci are distinguished by an insertion of ten amino acids in the c-d loop at one locus and by a high interlocus divergence of the third intron and fourth exon sequences. Unlike their mammalian counterparts, the damselfish TCRBC genes encode highly polymorphic regions. None of the eight complete cDNA or four partial genomic DNA sequences presented from a single animal are identical; and three of the four animals examined are heterozygous at both loci, suggesting high heterozygosity in the damselfish population. Coding regions of the eight cDNA clones differ by up to 12% at the DNA level and 23% at the amino acid level. Polymorphism is concentrated primarily in the less evolutionarily conserved regions, suggesting that this variation may be selectively neutral. However, a comparison of the variation between synonymous and non-synonymous sites suggests that at least a portion of the observed variation results from selection. As in mammals, a gradient of sequence homogenization between the two loci is observed. Data presented here suggest that both interlocus homogenization and the sharing of polymorphic segments are likely achieved by partial gene conversion.     

Systematic exploration of essential yeast gene function with temperature-sensitive mutants

Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (～45%) of the 1,101 essential yeast genes, with ～30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)-based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.     

Regulated Expression of a Sindbis Virus Replicon by Herpesvirus Promoters

We describe the use of herpesvirus promoters to regulate the expression of a Sindbis virus replicon (SINrep/LacZ). We isolated cell lines that contain the cDNA of SINrep/LacZ under the control of a promoter from a herpesvirus early gene which requires regulatory proteins encoded by immediate-early genes for expression. Wild-type Sindbis virus and replicons derived from this virus cause death of most vertebrate cells, but the cells discussed here grew normally and expressed the replicon and β-galactosidase only after infection with a herpesvirus. Vero cell lines in which the expression of SINrep/LacZ was regulated by the herpes simplex virus type 1 (HSV-1) infected-cell protein 8 promoter were generated. One Vero cell line (V3-45N) contained, in addition to the SINrep/LacZ cDNA, a Sindbis virus-defective helper cDNA which provides the structural proteins for packaging the replicon. Infection of V3-45N cells with HSV-1 resulted in the production of packaged SINrep/LacZ replicons. HSV-1 induction of the Sindbis virus replicon and packaging and spread of the replicon led to enhanced expression of the reporter gene, suggesting that this type of cell could be used to develop sensitive assays to detect herpesviruses. We also isolated a mink lung cell line that was transformed with SINrep/LacZ cDNA under the control of the promoter from the human cytomegalovirus (HCMV) early gene UL45. HCMV carries out an abortive infection in mink lung cells, but it was able to induce the SINrep/LacZ replicon. These results, and those obtained with an HSV-1 mutant, demonstrate that this type of signal amplification system could be valuable for detecting herpesviruses for which a permissive cell culture system is not available.