Transformation of Saccharomyces cerevisiae and Schizosaccharomyces pombe with linear plasmids containing 2 micron sequences.

Linear plasmids were constructed by adding telomeres prepared from Tetrahymena pyriformis rDNA to a circular hybrid Escherichia coli-yeast vector and transforming Saccharomyces cerevisiae. The parental vector contained the entire 2 mu yeast circle and the LEU gene from S. cerevisiae. Three transformed clones were shown to contain linear plasmids which were characterized by restriction analysis and shown to be rearranged versions of the desired linear plasmids. The plasmids obtained were imperfect palindromes: part of the parental vector was present in duplicated form, part as unique sequences and part was absent. The sequences that had been lost included a large portion of the 2 mu circle. The telomeres were approximately 450 bp longer than those of T. pyriformis. DNA prepared from transformed S. cerevisiae clones was used to transform Schizosaccharomyces pombe. The transformed S. pombe clones contained linear plasmids identical in structure to their linear parents in S. cerevisiae. No structural re-arrangements or integration into S. pombe was observed. Little or no telomere growth had occurred after transfer from S. cerevisiae to S. pombe. A model is proposed to explain the genesis of the plasmids.     

Yeast replicative DNA polymerases and their role at the replication fork.

The budding yeast, Saccharomyces cerevisiae, is an excellent model system for the study of DNA polymerases and their roles in DNA replication, repair, and recombination. Presently ten DNA polymerases have been purified and characterized from S. cerevisiae. Rapid advances in genome sequencing projects for yeast and other organisms have greatly facilitated and accelerated the identification of yeast enzymes and their homologues in other eukaryotic species. This article reviews current available research on yeast DNA polymerases and their functional roles in DNA metabolism. Relevant information about eukaryotic homologues of these enzymes will also be discussed.     

ER quality control: towards an understanding at the molecular level.

The process of 'quality control' in the endoplasmic reticulum (ER) involves a variety of mechanisms that collectively ensure that only correctly folded, assembled and modified proteins are transported along the secretory pathway. In contrast, non-native proteins are retained and eventually targeted for degradation. Recent work provides the first structural insights into the process of glycoprotein folding in the ER involving the lectin chaperones calnexin and calreticulin. Underlying principles governing the choice of chaperone system engaged by different proteins have also been discovered.     

Yeast plasmids have fewer superhelical turns than predicted by the nucleosome repeat of yeast DNA

The superhelical density of three Saccharomyces cerevisiae plasmids was determined with respect to a defined reference state during vegetative growth and stationary phase. The levels of supercoiling determined were approximately 20% lower than predicted by comparisons with SV40 DNA and reconstituted minichromosomes using histones from higher eukaryotes. In two different plasmids with the ARS1 origin of replication, the level of supercoiling changed substantially as the host cells entered stationary phase. Supercoiling of the endogenous 2-microns plasmid during vegetative growth was lower than in the ARS1-containing plasmids but did not change significantly upon entry of the cells into stationary phase.     

Exaptation at the molecular genetic level

The realization that body parts of animals and plants can be recruited or coopted for novel functions dates back to, or even predates the observations of Darwin. S.J. Gould and E.S. Vrba recognized a mode of evolution of characters that differs from adaptation. The umbrella term aptation was supplemented with the concept of exaptation. Unlike adaptations, which are restricted to features built by selection for their current role, exaptations are features that currently enhance fitness, even though their present role was not a result of natural selection. Exaptations can also arise from nonaptations; these are characters which had previously been evolving neutrally. All nonaptations are potential exaptations. The concept of exaptation was expanded to the molecular genetic level which aided greatly in understanding the enormous potential of neutrally evolving repetitive DNA—including transposed elements, formerly considered junk DNA—for the evolution of genes and genomes. The distinction between adaptations and exaptations is outlined in this review and examples are given. Also elaborated on is the fact that such distinctions are sometimes more difficult to determine; this is a widespread phenomenon in biology, where continua abound and clear borders between states and definitions are rare.     

The yeast 2 micron plasmid: strategies for the survival of a selfish DNA

Summary The designation of the yeast 2 circle as a selfish DNA molecule has been confirmed by demonstrating that the plasmid is lost with exponential kinetics from haploid yeast populations grown in continuous culture. We show that plasmid-free yeast cells have a growth rate advantage of some 1.5%–3% over their plasmid-containing counterparts. This finding makes the ubiquity of this selfish DNA in yeast strains puzzling. Two other factors probably account for its survival. First, the rate of plasmid loss was reduced by allowing haploid populations to enter stationary phase periodically. Second, it was not possible to isolate a plasmid-free segregant from a diploid yeast strain. Competition experiments demonstrated that stability in a diploid is conferred at the level of segregation and that plasmid-free diploid cells are at a selective advantage compared with their plasmid-containing counterparts. Yeast cells in nature are usually homothallic and must frequently pass through both diploid and stationary phases. The 2 plasmid appears to have evolved a survival strategy which exploits these two features of its host's life cycle.     

Plasmid segregation: spatial awareness at the molecular level

In bacteria, low-copy number plasmids ensure their stable inheritance by partition loci (par), which actively distribute plasmid replicates to each side of the cell division plane. Using time-lapse fluorescence microscopic tracking of segregating plasmid molecules, a new study provides novel insight into the workings of the par system from Escherichia coli plasmid R1. Despite its relative simplicity, the plasmid partition spindle shares characteristics with the mitotic machinery of eukaryotic cells.     

The integrase family of site-specific recombinases: regional similarities and global diversity.

A combination of two methods for detecting distant relationships in protein primary sequences was used to compare the site-specific recombination proteins encoded by bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1. This group of proteins exhibits an unexpectedly large diversity of sequences. Despite this diversity, all of the recombinases can be aligned in their C-terminal halves. A 40-residue region near the C terminus is particularly well conserved in all the proteins and is homologous to a region near the C terminus of the yeast 2 mu plasmid Flp protein. This family of recombinases does not appear to be related to any other site-specific recombinases. Three positions are perfectly conserved within this family: histidine, arginine and tyrosine are found at respective alignment positions 396, 399 and 433 within the well-conserved C-terminal region. We speculate that these residues contribute to the active site of this family of recombinases, and suggest that tyrosine-433 forms a transient covalent linkage to DNA during strand cleavage and rejoining.     

Magnetic tweezers: micromanipulation and force measurement at the molecular level

Cantilevers and optical tweezers are widely used for micromanipulating cells or biomolecules for measuring their mechanical properties. However, they do not allow easy rotary motion and can sometimes damage the handled material. We present here a system of magnetic tweezers that overcomes those drawbacks while retaining most of the previous dynamometers properties. Electromagnets are coupled to a microscope-based particle tracking system through a digital feedback loop. Magnetic beads are first trapped in a potential well of stiffness approximately 10(-7) N/m. Thus, they can be manipulated in three dimensions at a speed of approximately 10 microm/s and rotated along the optical axis at a frequency of 10 Hz. In addition, our apparatus can work as a dynamometer relying on either usual calibration against the viscous drag or complete calibration using Brownian fluctuations. By stretching a DNA molecule between a magnetic particle and a glass surface, we applied and measured vertical forces ranging from 50 fN to 20 pN. Similarly, nearly horizontal forces up to 5 pN were obtained. From those experiments, we conclude that magnetic tweezers represent a low-cost and biocompatible setup that could become a suitable alternative to the other available micromanipulators.     

Control of eukaryotic DNA replication at the chromosomal level.

A hypothesis for the control of eukaryotic DNA replication at the chromosomal level is proposed. The specific regulatory problem arises from the subdivision of the genome into thousands of individually replicating units, each of which must be duplicated a single time during S-phase. The hypothesis is based on the finding of direct repeats at replication origins. Such repeats can adopt, beyond the full-length double helical structure, another configuration exposing two single-stranded loops that provide suitable templates for the initiation of DNA replication. Any further initiation at the same origin is excluded as the single strandedness is eliminated by the replication process. Restoration of the initiable loop structure is proposed to occur by DNA-protein rearrangements involved in chromosome condensation and duplication of the chromosomal protein backbone during mitosis. A possible role of the maturation promoting factor (MPF) is suggested.     

Plant-pathogen arms races at the molecular level

Advances in research into the genetics of plant-pathogen interactions include an embracing of evolutionary ideas and methodologies. Recent studies reveal positive selection and selective maintenance of variation in plant resistance and defense-related genes. Coevolution between plants and their enemies involves many interactions at the molecular level.     

Bacterial iron detoxification at the molecular level

Iron is an essential micronutrient, and, in the case of bacteria, its availability is commonly a growth-limiting factor. However, correct functioning of cells requires that the labile pool of chelatable “free” iron be tightly regulated. Correct metalation of proteins requiring iron as a cofactor demands that such a readily accessible source of iron exist, but overaccumulation results in an oxidative burden that, if unchecked, would lead to cell death. The toxicity of iron stems from its potential to catalyze formation of reactive oxygen species that, in addition to causing damage to biological molecules, can also lead to the formation of reactive nitrogen species. To avoid iron-mediated oxidative stress, bacteria utilize iron-dependent global regulators to sense the iron status of the cell and regulate the expression of proteins involved in the acquisition, storage, and efflux of iron accordingly. Here, we survey the current understanding of the structure and mechanism of the important members of each of these classes of protein. Diversity in the details of iron homeostasis mechanisms reflect the differing nutritional stresses resulting from the wide variety of ecological niches that bacteria inhabit. However, in this review, we seek to highlight the similarities of iron homeostasis between different bacteria, while acknowledging important variations. In this way, we hope to illustrate how bacteria have evolved common approaches to overcome the dual problems of the insolubility and potential toxicity of iron.