PMC8203323_01
Sanger sequencing of exon 4 of USP16 clones. (A) Structure of the human USP16 gene and protein. Blue - ZnF UBP domain; yellow - ubiquitin specific protease (USP) domains; red bars - cysteine (left) and histidine (right) boxes of the C19 family peptidase domain. Asparagine at position 200 and cysteine at position 205 are key residues of the cysteine box; histidine at position 758 and aspartate at position 798 are key residues of the histidine box. The peptidase unit extends from residue 141 to the C terminus at residue 823. The USP domains are members of the C19 family of cysteine peptidases. Drawn from data in and the MEROPS database at https://www.ebi.ac.uk/merops/index.shtml. (B) Sequence of USP16 in edited clones. Upper panel shows the wild type, middle panel shows a heterozygote and lower panel shows a homozygous edited clone. The site of the insertion is indicated by the arrow and the amino acid codons of the wild type allele are shown above the sequence traces. All three founder heterozygous clones had the same insertion of adenine (A; green trace) in one allele. The four homozygous USP16-/- clones had the same insertion of one additional adenine, on both alleles. The nucleotide sequence before and after the insert was identical to wild type. (C) Translation of edited USP16 in homozygous clones by the ExPaSy tool (single letter code). Insertion of the single nucleotide causes a frame shift, which introduces multiple stop codons in the translated sequence. The insert is present in the middle of the zinc finger domain, in codon 91 for the amino acid alanine (pictured here framed by yellow box). Ten codons later, translation would be terminated by an in frame stop codon. Met indicates potential start codons (amino acid methionine), and highlighted sequences show open reading frames. The first gray sequence up to the yellow box is the correct amino acid sequence.
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F1
TIB-202TM), around 50% of cells become adherent in response to PMA, indicating that there is considerable phenotypic heterogeneity (see Supplementary Figure 1 in). To enable a detailed study of the transcriptomic response to PMA, the FANTOM consortium isolated a clonal line that was highly responsive to PMA and uniformly differentiated into a macrophage like state. The karyotype of the clonal line was established by microarray-based comparative genomic hybridization to be 46XY, with minimal chromosomal aberrations (deletions at 6p, 12p, 17p, and on the X chromosome). The THP-1 cells are also predisposed to mutations in tumor suppressor genes (for example TP53 and PTEN) and MLL fusions.