Targeted deep-intronic sequencing in a cohort of unexplained cases of suspected Lynch syndrome.

Lynch syndrome (LS) is caused by germline defects in DNA mismatch repair (MMR) pathway, resulting in microsatellite instability (MSI-H) and loss of immunohistochemical staining (IHC) of the respective protein in tumor tissue. However, not in all clinically suspected LS patients with MSI-H tumors and IHC-loss, causative germline alterations in the MMR genes can be detected. Here, we investigated 128 of these patients to possibly define new pathomechanisms. A search for large genomic rearrangements and deep-intronic regulatory variants was performed via targeted next-generation sequencing (NGS) of exonic, intronic, and chromosomal regions upstream and downstream of MLH1, MSH2, MSH6, PMS2, MLH3, MSH3, PMS1, and EPCAM. Within this cohort, two different large rearrangements causative for LS were detected in three cases, belonging to two families (2.3%). The sensitivity to detect large rearrangements or copy number variations (CNV) was evaluated to be 50%. In 9 of the 128 patients (7%), previously overlooked pathogenic single-nucleotide variants (SNV) and two variants of uncertain significance (VUS) were identified in MLH1, MSH2, and MSH6. Pathogenic aberrations were not found in MLH3, MSH3, and PMS1. A potential effect on regulation was exerted for 19% of deep-intronic SNVs, predominantly located in chromosomal regions where the modification of histone proteins suggests an enhancer function. In conclusion, conventional variation analysis of coding regions is missing rare genomic rearrangements, nevertheless they should be analyzed. Assessment of deep-intronic SNVs is so far non-conclusive for medical questioning.

Protein function microarrays based on self-immobilizing and self-labeling fusion proteins.

Protein microarrays are an attractive approach for the high-throughput analysis of protein function, but their impact on proteomics has been limited by the technical difficulties associated with their generation. Here we demonstrate that fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT) can be used for the simple and reliable generation of protein microarrays for the analysis of protein function. Important features of the approach are the selectivity of the covalent immobilization; this allows for direct immobilization of proteins out of cell extracts, and the option both to label and to immobilize AGT fusion proteins, which allows for direct screening for protein-protein interactions between different AGT fusion proteins. In addition to the identification of protein-protein interactions, AGT-based protein microarrays can be used for the characterization of small molecule-protein interactions or post-translational modifications. The potential of the approach was demonstrated by investigating the post-translational modification of acyl carrier protein (ACP) from E. coli by different phosphopantetheine transferases (PPTases), yielding insights into the role of selected ACP amino acids in the ACP-PPTase interaction.

Protein-functionalized polymer brushes.

A new strategy for the preparation of protein-functionalized polymer brushes is reported, which is based on a combination of surface-initiated atom transfer radical polymerization (ATRP), p-nitrophenyl chloroformate activation of the surface hydroxyl groups, and subsequent O(6)-benzylguanine (BG) functionalization. The BG-functionalized brushes are used to chemoselectively immobilize O(6)-alkylguanine-DNA-alkyltransferase (AGT) fusion proteins with a defined orientation and surface density. These protein-modified polymer brushes are attractive candidates for the development of protein microarrays.

Sequence and transcriptional analysis of a gene cluster of Pseudomonas putida 86 involved in quinoline degradation.

Although quinoline 2-oxidoreductase (Qor) and 1H-2-oxoquinoline 8-monooxygenase (OxoOR), which catalyse the first two steps of quinoline degradation by Pseudomonas putida 86, and their genes have been investigated in some detail, the genetic organization and regulation of the catabolic pathway are not known yet. A gene cluster involved in quinoline degradation was characterized. Upstream of oxoO encoding the oxygenase component of OxoOR, the gene oxoS coding for a XylS-type protein is located. The DNA region downstream of oxoO comprises potential open reading frames (ORFs) that may code for further catabolic enzymes (an alpha/beta-hydrolase fold protein, and an amidase), and for accessory proteins presumably required for the assembly of metal cofactor containing holoenzymes (XdhC-like protein, MoeC- and MobA-like protein(s), IscS and IscU). The potential iscU gene is followed by the genes qorMSL that encode the structural subunits of Qor. Three potential ORFs (ORFs7-9) are located between qorMSL and oxoR, which codes for the reductase component of OxoOR. ORFs7-9 have counterparts in the cox (CO oxidizing system) and nic (nicotine degradation) gene clusters. Transcription of all these genes and ORFs located downstream of oxoS is induced by quinoline or 1H-2-oxoquinoline. Insertional inactivation of oxoS abolished quinoline-induced transcription. However, weak transcription of ORFs7-9 also occurred independent of quinoline and OxoS. The typical tandem recognition site for a XylS-type transcriptional activator was identified in the putative promoter region of qorM, and archetypal XylS indeed was found to activate synthesis of Qor. Motifs corresponding to single half-sites of a XylS-type binding site are located upstream of oxoO, the xdhC-like gene, and oxoR. Putative quinoline-specific transcriptional start sites were identified for these genes, and for qorM. The gene cluster probably is transcribed from several promoters, resulting in multiple overlapping polycistronic mRNAs.