CRISPR/Cas9 Allows Efficient and Complete Knock-In of a Destabilization Domain-Tagged Essential Protein in a Human Cell Line,Allowing Rapid Knockdown of Protein Function

Although modulation of protein levels is an important tool for study of protein function, it is difficult or impossible to knockdown or knockout genes that are critical for cell growth or viability. For such genes, a conditional knockdown approach would be valuable. The FKBP protein-based destabilization domain (DD)-tagging approach, which confers instability to the tagged protein in the absence of the compound Shield-1, has been shown to provide rapid control of protein levels determined by Shield-1 concentration. Although a strategy to knock-in DD-tagged protein at the endogenous loci has been employed in certain parasite studies, partly due to the relative ease of knock-in as a result of their mostly haploid lifecycles, this strategy has not been demonstrated in diploid or hyperploid mammalian cells due to the relative difficulty of achieving complete knock-in in all alleles.The recent advent of CRISPR/Cas9 homing endonuclease-mediated targeted genome cleavage has been shown to allow highly efficient homologous recombination at the targeted locus. We therefore assessed the feasibility of using CRISPR/Cas9 to achieve complete knock-in to DD-tag the essential gene Treacher Collins-Franceschetti syndrome 1 (TCOF1) in human 293T cells. Using a double antibiotic selection strategy to select clones with at least two knock-in alleles, we obtained numerous complete knock-in clones within three weeks of initial transfection. DD-TCOF1 expression in the knock-in cells was Shield-1 concentration-dependent, and removal of Shield-1 resulted in destabilization of DD-TCOF1 over the course of hours. We further confirmed that the tagged TCOF1 retained the nucleolar localization of the wild-type untagged protein, and that destabilization of DD-TCOF1 resulted in impaired cell growth, as expected for a gene implicated in ribosome biogenesis. CRISPR/Cas9-mediated homologous recombination to completely knock-in a DD tag likely represents a generalizable and efficient strategy to achieve rapid modulation of protein levels in mammalian cells.     

Isolation and sequence of cDNA clones coding for a member of the family of high mobility group proteins (HMG-T) in trout and analysis of HMG-T-mRNA''s in trout tissues.

A specific oligonucleotide has been used to isolate a cDNA prepared from the mRNA for a trout High Mobility Group (HMG) protein closely related to trout HMG-T and bovine HMG 1 and 2 proteins. The sequence isolated more closely resembles bovine HMG-1 than the previously sequenced HMG-T protein in regions corresponding to the N terminal half of the protein. Northern blot analysis at low stringency indicated that 2 related sequences are expressed in total trout testis mRNA. Southern blots of total trout DNA indicate that several different forms of the homologous sequence are present in the trout genome and an estimate of copy number by dot-blot shows 4 HMG-T genes per trout sperm DNA equivalent. Analysis of mRNA from several trout tissues, including testis, liver and kidney indicates that expression of genes for histones and the larger HMG proteins in trout is not closely coupled.     

Nucleation of calcite on the sheaths of cyanobacteria using a simple diffusion cell

A method allowing observation of calcium carbonate nucleation on the sheaths of cyanobacteria and other microorganisms is described. Nucleation of calcite was obtained on the sheaths of Lyngbya aestuarii and Scy‐tonema myochrous cultures but not on Entophysalis major and Pseudanabaena minuta. Differences in the rate of nucleation were also noted in media of differing salinity.     

A Note on the Relationship Between Some Saxicolous Lichens and Manganese Ore in North Wales,UK

Manganese oxyhydroxides have been found deposited on the surface of the lichen Catillaria chalybeia on an altered rhodochrosite. It is suggested that the Mn was mobilised from the ore surface via surface-weathering and redeposited in the thallus. The lichen Acarospora smaragdula also grew upon the ore but showed no sign of Mn deposition. Nine further lichens and two bryophytes were also found on the ore.     

Some observations on travertine algae from Stjáni hot spring,Lýsuhóll,Iceland

The photosynthetic microbial flora of an Icelandic hot spring is described. Several cosmopolitan cyanobacteria were common, but diatoms were scarce despite the apparently high silica content of the water. A red alga, Chroothece mobilis occurred among the cyanobacteria and has not been previously recorded from this environment.