An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5 + or Escherichia coli kan r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C-or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N-or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in

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.

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Patients infected with the SARS-CoV-2 virus can present with a wide variety of symptoms including being entirely asymptomatic. Despite having no or minimal symptoms, some patients may have markedly reduced pulse oximetry readings. This has been referred to as "silent" or "apathetic" hypoxia (Ottestad et al., 2020 [1]). We present a case of a 72-year-old male with COVID-19 syndrome who presented to the emergency department with minimal symptoms but low peripheral oxygen saturation readings. The patient deteriorated over the following days and eventually died as a result of overwhelming multi-organ system failure. This case highlights the utility of peripheral oxygen measurements in the evaluation of patients with SARS-CoV-2 infection. Selfmonitoring of pulse oximetry by patients discharged from the emergency department is a potential way to identify patients needing to return for further evaluation.