ITSxpress: Software to rapidly trim internally transcribed spacer sequences with quality scores for marker gene analysis.

The internally transcribed spacer (ITS) region between the small subunit ribosomal RNA gene and large subunit ribosomal RNA gene is a widely used phylogenetic marker for fungi and other taxa. The eukaryotic ITS contains the conserved 5.8S rRNA and is divided into the ITS1 and ITS2 hypervariable regions. These regions are variable in length and are amplified using primers complementary to the conserved regions of their flanking genes. Previous work has shown that removing the conserved regions results in more accurate taxonomic classification. An existing software program, ITSx, is capable of trimming FASTA sequences by matching hidden Markov model profiles to the ends of the conserved genes using the software suite HMMER. ITSxpress was developed to extend this technique from marker gene studies using Operational Taxonomic Units (OTU's) to studies using exact sequence variants; a method used by the software packages Dada2, Deblur, QIIME 2, and Unoise. The sequence variant approach uses the quality scores of each read to identify sequences that are statistically likely to represent real sequences. ITSxpress enables this by processing FASTQ rather than FASTA files. The software also speeds up the trimming of reads by a factor of 14-23 times on a 4-core computer by temporarily clustering highly similar sequences that are common in amplicon data and utilizing optimized parameters for Hmmsearch. ITSxpress is available as a QIIME 2 plugin and a stand-alone application installable from the Python package index, Bioconda, and Github.

Mutational analysis of conserved amino acids in the T cell receptor alpha-chain transmembrane region: a critical role of leucine 112 and phenylalanine 127 for assembly and surface expression.

Correct assembly of all TCR complex polypeptides is essential for its cell surface expression and function. The transmembrane region of the TCRalpha chain is highly conserved and to gain insight into the structural and functional role of these residues, single amino acid substitutions were introduced and surface expression and signaling ability studied in T hybridoma cells. Introduction of acid residues within the TCRalpha chain transmembrane region were mostly tolerated, indicating that the net charge within this region of the TCR complex is not crucial to either assembly or signaling. However, mutations of leucine 112 or phenylalanine 127 to aspartic acids (L112D or F127D, respectively) resulted in dramatic loss of surface expression and, therefore, their signaling ability. Intracellular flow cytometry showed that the mutant TCRalpha polypeptides were present at levels comparable to wild-type, indicating that the reduced surface expression was not a consequence of impaired protein survival. The defect was characterized by immunoprecipitation and showed that residues L112 and F127 were involved in early interactions with the CD3 complex. A large proportion of the TCRalpha chain mutants L112D and F127D consisted of immature protein, indicative of a problem during early assembly of the TCR. Our findings provide evidence for the involvement of the conserved L112 and F127 residues of the TCRalpha chain transmembrane region in the assembly process of the TCR complex.