Patterns of non-ARD variation in more than 300 full-length HLA-DPB1 alleles.

Our understanding of sequence variation in the HLA-DPB1 gene is largely restricted to the hypervariable antigen recognition domain (ARD) encoded by exon 2. Here, we employed a redundant sequencing strategy combining long-read and short-read data to accurately phase and characterise in full length the majority of common and well-documented (CWD) DPB1 alleles as well as alleles with an observed frequency of at least 0.0006% in our predominantly European sample set. We generated 664 DPB1 sequences, comprising 279 distinct allelic variants. This allows us to present the, to date, most comprehensive analysis of the nature and extent of DPB1 sequence variation. The full-length sequence analysis revealed the existence of two highly diverged allele clades. These clades correlate with the rs9277534 A → G variant, a known expression marker located in the 3'-UTR. The two clades are fully differentiated by 174 fixed polymorphisms throughout a 3.6 kb stretch at the 3'-end of DPB1. The region upstream of this differentiation zone is characterised by increasingly shared variation between the clades. The low-expression A clade comprises 59% of the distinct allelic sequences including the three by far most frequent DPB1 alleles, DPB1*04:01, DPB1*02:01 and DPB1*04:02. Alleles in the A clade show reduced nucleotide diversity with an excess of rare variants when compared to the high-expression G clade. This pattern is consistent with a scenario of recent proliferation of A-clade alleles. The full-length characterisation of all but the most rare DPB1 alleles will benefit the application of NGS for DPB1 genotyping and provides a helpful framework for a deeper understanding of high- and low-expression alleles and their implications in the context of unrelated haematopoietic stem-cell transplantation.

Expression of the Bradyrhizobium japonicum type III secretion system in legume nodules and analysis of the associated tts box promoter.

In Bradyrhizobium japonicum, as in some other rhizobia, symbiotic efficiency is influenced by a type III secretion system (T3SS). Most genes encoding the transport machinery and secreted proteins are preceded by a conserved 30-bp motif, the type-three secretion (tts) box. In this study, we found that regions downstream of 34 tts boxes are transcribed. For nopB, nopL, and gunA2, the transcriptional start sites were found to be 12, 11, and 10 bp downstream of their tts boxes, respectively. The deletion of this motif or modification of two or more conserved residues strongly reduced expression of nopB. This indicates that the tts box is an essential promoter element. Data obtained with lacZ reporter gene fusions of five genes preceded by a tts box (gunA2, nopB, rhcV, nopL, and blr1806) revealed that they are expressed in 4-week-old nodules of Macroptilium atropurpureum. These data suggest that the T3SS is active in mature nitrogen-fixing nodules. The two-component response regulator TtsI is required for the expression of rhcV, nopL, and blr1806 in bacteroids. Staining of inoculated roots showed that nopB is also expressed in early infection stages.