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Site-directed seamless modification of bacterial artificial chromosomes is enhanced more than tenfold in efficiency by improving the counterselection step. A set of plasmids and oligonucleotide design software also make this E. coli recombineering approach markedly faster and easier. Whereas bacterial artificial chromosomes (BACs) offer many advantages in studies of gene and protein function, generation of seamless, precisely mutated BACs has been difficult. Here we describe a counterselection-based recombineering method and its accompanying reagents. After identifying intramolecular recombination as the major problem in counterselection, we built a strategy to reduce these unwanted events by expressing Redβ alone at the crucial step. We enhanced this method by using phosphothioated oligonucleotides, using a sequence-altered rpsL counterselection gene and developing online software for oligonucleotide design. We illustrated this method by generating transgenic mammalian cell lines carrying small interfering RNA–resistant and point-mutated BAC transgenes. Using this approach, we generated mutated TACC3 transgenes to identify phosphorylation-specific spindle defects after knockdown of endogenous TACC3 expression. Our results highlight the complementary use of precisely mutated BAC transgenes and RNA interference in the study of cell biology at physiological expression levels and regulation.

The human Y chromosome displays an unusual content of repetitive sequences. Y-chromosomal repeats are potential targets for intrachromosomal recombination, which is thought to be involved in a number of Y-associated defects, such as male infertility. Such rearrangements could potentially be investigated by the use of highly polymorphic DNA markers located within the repeat units, such as microsatellites. Here we analyse the two copies of the Y-chromosomal microsatellite DYS385, which we identified and localized to an ∼190 kb duplicated and inverted fragment at Yq11.223. We found a highly significant correlation ( r =0.853, P <0.001) and a nonsignificant difference in a χ 2 -test ( χ 2 =15.45, P >0.05) between the allele frequency distributions at both copies of the Y-STR in a German population sample ( n =70). Such nearly identical allele frequency distribution between two copies of a duplicated highly polymorphic microsatellite cannot be explained by the independent mutational process that creates microsatellite alleles. Instead, this might be interpreted as evidence for a reciprocal intrachromosomal exchange process between the duplicated fragments. However, more detailed analyses using additional human populations as well as additional Y chromosome markers revealed that this phenomenon is highly population-specific and disappears completely when Y-STR diversity is analysed in association with two Y-SNP haplogroups. We found that the diversity of the two DYS385 loci (and other Y-STRs) is highly depending on the haplogroup background, and that equal proportions of both haplogroups in the German sample explains the nearly identical allele frequency distributions at the two DYS385 loci. Thus, we demonstrate here that allele frequency distributions at duplicate loci that are suggestive of intrachromosomal recombination can be explained solely by population history.

Background The Red proteins of lambda phage mediate probably the simplest and most efficient homologous recombination reactions yet described. However the mechanism of dsDNA recombination remains undefined. Results Here we show that the Red proteins can act via full length single stranded intermediates to establish single stranded heteroduplexes at the replication fork. We created asymmetrically digestible dsDNA substrates by exploiting the fact that Redα exonuclease activity requires a 5' phosphorylated end, or is blocked by phosphothioates. Using these substrates, we found that the most efficient configuration for dsDNA recombination occurred when the strand that can prime Okazaki-like synthesis contained both homology regions on the same ssDNA molecule. Furthermore, we show that Red recombination requires replication of the target molecule. Conclusions Hence we propose a new model for dsDNA recombination, termed 'beta' recombination, based on the formation of ssDNA heteroduplexes at the replication fork. Implications of the model were tested using (i) an in situ assay for recombination, which showed that recombination generated mixed wild type and recombinant colonies; and (ii) the predicted asymmetries of the homology arms, which showed that recombination is more sensitive to non-homologies attached to 5' than 3' ends. Whereas beta recombination can generate deletions in target BACs of at least 50 kb at about the same efficiency as small deletions, the converse event of insertion is very sensitive to increasing size. Insertions up to 3 kb are most efficiently achieved using beta recombination, however at greater sizes, an alternative Red-mediated mechanism(s) appears to be equally efficient. These findings define a new intermediate in homologous recombination, which also has practical implications for recombineering with the Red proteins.

As part of the process-characterization campaign of a candidate vaccine product, a recently developed class of three-level designs—definitive-screening designs—was employed to select a quadratic model that describes the effect of six input process parameters, including protein concentration, formaldehyde-to-protein ratio, lysine concentration, reaction duration, pH, and reaction temperature, on a formylation protein-crosslinking reaction. This design requires only 17 experimental runs. The resulting model was then used to simulate 10,000 runs that account for the variability in the inputs expected on manufacturing scale. The extent of protein polymerization was predicted to be within specifications for all simulated runs, demonstrating the robustness of the unit operation for subsequent process validation and future commercial manufacturing.

Single strand annealing proteins (SSAPs) like Redβ initiate homologous recombination by annealing complementary DNA strands. We show that C-terminally truncated Redβ, whilst still able to promote annealing and nucleoprotein filament formation, is unable to mediate homologous recombination. Mutations of the C-terminal domain were evaluated using both single- and double stranded (ss and ds) substrates in recombination assays. Mutations of critical amino acids affected either dsDNA recombination or both ssDNA and dsDNA recombination indicating two separable functions, one of which is critical for dsDNA recombination and the second for recombination per se . As evaluated by co-immunoprecipitation experiments, the dsDNA recombination function relates to the Redα-Redβ protein-protein interaction, which requires not only contacts in the C-terminal domain but also a region near the N-terminus. Because the nucleoprotein filament formed with C-terminally truncated Redβ has altered properties, the second C-terminal function could be due to an interaction required for functional filaments. Alternatively the second C-terminal function could indicate a requirement for a Redβ-host factor interaction. These data further advance the model for Red recombination and the proposition that Redβ and RAD52 SSAPs share ancestral and mechanistic roots.

Single strand annealing proteins (SSAPs) like Redβ initiate homologous recombination by annealing complementary DNA strands. We show that C-terminally truncated Redβ, whilst still able to promote annealing and nucleoprotein filament formation, is unable to mediate homologous recombination. Mutations of the C-terminal domain were evaluated using both single- and double stranded (ss and ds) substrates in recombination assays. Mutations of critical amino acids affected either dsDNA recombination or both ssDNA and dsDNA recombination indicating two separable functions, one of which is critical for dsDNA recombination and the second for recombination per se. As evaluated by co-immunoprecipitation experiments, the dsDNA recombination function relates to the Redα-Redβ protein-protein interaction, which requires not only contacts in the C-terminal domain but also a region near the N-terminus. Because the nucleoprotein filament formed with C-terminally truncated Redβ has altered properties, the second C-terminal function could be due to an interaction required for functional filaments. Alternatively the second C-terminal function could indicate a requirement for a Redβ-host factor interaction. These data further advance the model for Red recombination and the proposition that Redβ and RAD52 SSAPs share ancestral and mechanistic roots.

The Red proteins of lambda phage mediate probably the simplest and most efficient homologous recombination reactions yet described. However the mechanism of dsDNA recombination remains undefined. Here we show that the Red proteins can act via full length single stranded intermediates to establish single stranded heteroduplexes at the replication fork. We created asymmetrically digestible dsDNA substrates by exploiting the fact that Red[alpha] exonuclease activity requires a 5' phosphorylated end, or is blocked by phosphothioates. Using these substrates, we found that the most efficient configuration for dsDNA recombination occurred when the strand that can prime Okazaki-like synthesis contained both homology regions on the same ssDNA molecule. Furthermore, we show that Red recombination requires replication of the target molecule. Hence we propose a new model for dsDNA recombination, termed 'beta' recombination, based on the formation of ssDNA heteroduplexes at the replication fork. Implications of the model were tested using (i) an in situ assay for recombination, which showed that recombination generated mixed wild type and recombinant colonies; and (ii) the predicted asymmetries of the homology arms, which showed that recombination is more sensitive to non-homologies attached to 5' than 3' ends. Whereas beta recombination can generate deletions in target BACs of at least 50 kb at about the same efficiency as small deletions, the converse event of insertion is very sensitive to increasing size. Insertions up to 3 kb are most efficiently achieved using beta recombination, however at greater sizes, an alternative Red-mediated mechanism(s) appears to be equally efficient. These findings define a new intermediate in homologous recombination, which also has practical implications for recombineering with the Red proteins.

Redβ anneals DNA to initiate homologous recombination ined recent prominence through the development of the DNA engineering technology known as ‘recombineering' or ‘Red/ET'. It originates from the red-operon of λ phage where it is co-expressed in the early life cycle stage with Redα a processive 5'-3' exonuclease and Redγ, a DNA mimetic and RecBCD inhibitor. Unlike RecA/RAD51, Redβ is not an ATPase and it's mechanism for initiating homologous recombination is poorly understood. To examine the structure and dynamics of Redβ complexes at sub-molecular resolution we performed tapping mode atomic force microscopy (AFM) of Redβ protein alone and in complex with DNA. Without DNA, Redβ forms a ‘split lock washer' structure with a shallow right-handed helicity. Sequentially adding complementary ssDNA generates a stable left-handed helical filament. Importantly, the contour length of the helical filament equated linearly to the lengths of complementary ssDNA, giving the number of nucleotides per Redβ monomer. Additionally, the monomer width along the filament was quantified. These new quantities as well as the observed helical transition reveals new insights into the mechanism of DNA annealing mediated by Red and led us to suggest new mechanistic models.

Background The prevalence of restless legs syndrome (RLS) is lower among children than adults. This could be due to RLS manifestation later in life or false diagnoses in children caused by symptom similarity to other diseases such as attention deficient hyperactivity disorder (ADHD). Objective The aim of this study was to test the reliability and validity of a questionnaire designed to identify RLS-related symptoms in children and adolescents. Materials and methods The questionnaire was designed for two age groups (6–12 and 13–18 years). It was distributed among 11 pediatric RLS patients and 22 healthy children and adolescents (control group). The control group answered the questionnaire twice (M1, M2; 14-day separation). Reliability was analyzed descriptively and with Spearman correlation coefficients, intraclass correlation coefficients (ICC), and test–retest variability (TRV). An index was calculated by transferring the 12 answers concerning RLS symptoms into a coding system, grading each answer between −2 and 2 and adding the grades to an index value. Low index values hint at the absence of RLS-related symptoms. Student’s t‑test was used to test the difference between healthy controls and pediatric patients. Results In the control group, 84% of answer pairs were identical between M1 and M2. Mean TRV was 8% (range: 2–16%), Spearman correlation coefficient 0.762, and ICC 0.754 (confidence interval: 0.473–0.899). Comparison between controls and pediatric RLS patients showed that RLS patients had significantly higher ( p  < 0.001) index values (mean ± SD: 18.8 ± 4) compared to controls (mean ± SD: −3.8 ± 7.6). Conclusion The questionnaire can distinguish between supposedly healthy and RLS-diagnosed children and adolescents in most cases. Nonetheless, the questionnaire cannot be used alone as a diagnostic instrument.

BackgroundThe prevalence of restless legs syndrome (RLS) is lower among children than adults. This could be due to RLS manifestation later in life or false diagnoses in children caused by symptom similarity to other diseases such as attention deficient hyperactivity disorder (ADHD).ObjectiveThe aim of this study was to test the reliability and validity of a questionnaire designed to identify RLS-related symptoms in children and adolescents.Materials and methodsThe questionnaire was designed for two age groups (6–12 and 13–18 years). It was distributed among 11 pediatric RLS patients and 22 healthy children and adolescents (control group). The control group answered the questionnaire twice (M1, M2; 14-day separation). Reliability was analyzed descriptively and with Spearman correlation coefficients, intraclass correlation coefficients (ICC), and test–retest variability (TRV). An index was calculated by transferring the 12 answers concerning RLS symptoms into a coding system, grading each answer between −2 and 2 and adding the grades to an index value. Low index values hint at the absence of RLS-related symptoms. Student’s t‑test was used to test the difference between healthy controls and pediatric patients.ResultsIn the control group, 84% of answer pairs were identical between M1 and M2. Mean TRV was 8% (range: 2–16%), Spearman correlation coefficient 0.762, and ICC 0.754 (confidence interval: 0.473–0.899). Comparison between controls and pediatric RLS patients showed that RLS patients had significantly higher (p < 0.001) index values (mean ± SD: 18.8 ± 4) compared to controls (mean ± SD: −3.8 ± 7.6).ConclusionThe questionnaire can distinguish between supposedly healthy and RLS-diagnosed children and adolescents in most cases. Nonetheless, the questionnaire cannot be used alone as a diagnostic instrument.