SummaryDNA polymerases of the Y family promote survival by their ability to synthesize past lesions in the DNA template. One Escherichia coli member of this family, DNA pol V (UmuC), which is primarily responsible for UV-induced and chemically induced mutagenesis, possesses a canonical β β β β processivity clamp-binding motif. A detailed analysis of this motif in DNA pol V (UmuC) showed that mutation of only two residues in UmuC is sufficient to result in a loss of UV-induced mutagenesis. Increased levels of wild-type β β β β can partially rescue this loss of mutagenesis. Alterations in this motif of UmuC also cause loss of the cold-sensitive and β β β β -dependent synthetic lethal phenotypes associated with increased levels of UmuD and UmuC that are thought to represent an exaggeration of a DNA damage checkpoint. By designing compensatory mutations in the cleft between domains II and III in β β β β , we restored UV-induced mutagenesis by a UmuC β β β β -binding motif variant. A recent co-crystal structure of the 'little finger' domain of E. coli pol IV (DinB) with β β β β suggests that, in addition to the canonical β β β β -binding motif, a second site of pol IV ( 303 VWP 305 ) interacts with β β β β at the outer rim of the dimer interface. Mutational analysis of the corresponding motif in UmuC showed that it is dispensable for induced mutagenesis, but that alterations in this motif result in loss of the coldsensitive phenotype. These two β β β β interaction sites of UmuC affect the dual functions of UmuC differentially and indicate subtle and sophisticated polymerase management by the β β β β clamp.

We present a set of programs and a website designed to facilitate protein structure comparison and protein structure modeling efforts. Our protein structure analysis and comparison services use the LGA (local-global alignment) program to search for regions of local similarity and to evaluate the level of structural similarity between compared protein structures. To facilitate the homology-based protein structure modeling process, our AL2TS service translates given sequence–structure alignment data into the standard Protein Data Bank (PDB) atom records (coordinates). For a given sequence of amino acids, the AS2TS (amino acid sequence to tertiary structure) system calculates (e.g. using PSI-BLAST PDB analysis) a list of the closest proteins from the PDB, and then a set of draft 3D models is automatically created. Web services are available at .

Recently, several attempts have been made to create a generation of transgenic chickens via chimeric intermediates produced by primordial germ cells (PGCs) transfer. This study aimed to compare the influences of different chicken PGCs isolated from circulating blood (bPGCs) or gonads (gPGCs), purification (ACK, Percoll or trypsin) and transfection methods (electroporation or lipofection) on the expression of transgenes in vitro and the migration of modified donor cells to the recipient gonads. The highest average frequency of pEGFP-N1 plasmid-transfected bPGCs (75.8 %) was achieved with Percoll density gradient centrifugation and electroporation. After ammonium chloride-potassium (ACK) treatment and lipofection, in vitro transgene expression was only detected in 35.2 % of bPGCs. Chimeric chickens were produced from these purified, transfected and cultured cells, and the transgene was detected in the gonads of 44 and 42 % of the recipient embryos that had been injected with bPGCs and gPGCs, respectively. These data confirmed that the combination of PGC purification via Percoll centrifugation and electroporation was an effective method for producing transgenic chickens. Subsequently, we used this method with expression vectors for gene hIFNα 2a/hepatitis B virus surface antigen (HBsAg) under the control of the ovalbumin promoter to generate G0 transgenic chickens. Consequently, we observed that 4.9 % of the hens and 3.5 % of the roosters carried the hIFNα 2a gene, whereas 16.7 % of the hens and 2.4 % of the roosters carried the HBsAg gene, thus undisputedly confirming the exceptional effectiveness of the applied methods.

Improvements in genome editing technology in birds using primordial germ cells (PGCs) have made the development of innovative era genome-edited avian models possible, including specific chicken bioreactors, production of knock-in/out chickens, low-allergenicity eggs, and disease-resistance models. New strategies, including CRISPR/Cas9, have made gene editing easy and highly efficient in comparison to the well-known process of homologous recombination. The clustered regularly interspaced short palindromic repeats (CRISPR) technique enables us to understand the function of genes and/or to modify the animal phenotype to fit a specific scientific or production target. To facilitate chicken genome engineering applications, we present a concise description of the method and current application of the CRISPR/Cas9 system in chickens. Different strategies for delivering sgRNAs and the Cas9 protein, we also present extensively. Furthermore, we describe a new gesicle technology as a way to deliver Cas9/sgRNA complexes into target cells, and we discuss the advantages and describe basal applications of the CRISPR/Cas9 system in a chicken model.