We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was > or = 50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/%28SICI%291097-0061%28199807%2914%3A10%3C943%3A%3AAID-YEA292%3E3.0.CO%3B2-Y

Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2–5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site.

LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actindependent and the other actin-independent, underlie polarity development. The actin-dependent pathway localizes secretory vesicles and a putative vesicle docking complex to sites of cell surface growth, providing an explanation for the dependence of polarized cell surface growth on actin function. Unexpectedly, several proteins that function with actin during cell polarity development, including an unconventional myosin (Myo2p), calmodulin, and an actin-interacting protein (Bud6/Aip3p), achieved polarized localization by an actin-independent pathway, revealing interdependence among cell polarity pathways. Finally, transient actin depolymerization caused many cells to abandon one bud site or mating projection and to initiate growth at a second site. Thus, actin filaments are also required for maintenance of an axis of cell polarity.

/pdf/high-rates-of-actin-filament-turnover-in-budding-yeast-and-2z8mv7ssrf.pdf

High Rates of Actin Filament Turnover in Budding Yeast and Roles for Actin in Establishment and Maintenance of Cell Polarity Revealed Using the Actin Inhibitor Latrunculin-A

Plants in their natural habitats adapt to drought stress in the environment through a variety of mechanisms, ranging from transient responses to low soil moisture to major survival mechanisms of escape by early flowering in absence of seasonal rainfall. However, crop plants selected by humans to yield products such as grain, vegetable, or fruit in favorable environments with high inputs of water and fertilizer are expected to yield an economic product in response to inputs. Crop plants selected for their economic yield need to survive drought stress through mechanisms that maintain crop yield. Studies on model plants for their survival under stress do not, therefore, always translate to yield of crop plants under stress, and different aspects of drought stress response need to be emphasized. The crop plant model rice ( Oryza sativa) is used here as an example to highlight mechanisms and genes for adaptation of crop plants to drought stress.

Plant adaptation to drought stress.

An E3-like Factor that Promotes SUMO Conjugation to the Yeast Septins

Colorectal cancer (CRC) is a significant cause of morbidity and mortality worldwide. However, colon cancer incidence and mortality is declining over the past decade owing to adoption of effective screening programs. Nevertheless, in some parts of the world, CRC incidence and mortality remain on the rise, likely due to factors including "westernized" diet, lifestyle, and lack of health-care infrastructure and resources. Participation and adherence to different national screening programs remain obstacles limiting the achievement of screening goals. Different modalities are available ranging from stool based tests to radiology and endoscopy with varying sensitivity and specificity. However, the availability of these tests is limited to areas with high economic resources. Recently, FDA approved a blood-based test (Epi procolon®) for CRC screening. This blood based test may serve to increase the participation and adherence rates. Hence, leading to increase in colon cancer detection and prevention. This article will discuss various CRC screening tests with a particular focus on the data regarding the new approved blood test. Finally, we will propose an algorithm for a simple cost-effective CRC screening program.

Colorectal cancer screening: An updated review of the available options

The septins: Roles in cytokinesis and other processes

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The septins: Roles in cytokinesis and other processes