Seasonal variation in estrous cycling in the mouse: Implications for artificial insemination

Artificial insemination in the C3HeB/FeJ inbred strain of mice has been shown to be more successful at the middle and end of the calendar year. The reasons are twofold: 1) an increase in the number of normal estrous cycles exhibited by females and 2) an increase in the tightness of the phasing of ovarian and vaginal events. The latter phenomenon was found to be the key to the success of artificial insemination, since it permitted the use of vaginal smears to predict accurately the time females could be expected to ovulate and, therefore, the appropriate time for artificial insemination. Seasonal variations in the frequency of estrous cycling also have been observed in SJL/J and B6D2F1/J females.     

Allele-specific PCR can improve the efficiency of experimental resolution of heterozygotes in resequencing studies

Resolution of the two haplotypes present in an individual that is heterozygous at a locus has been a difficult problem for nucleotide sequence-based population genetic studies. Here, we demonstrate a method in which allele-specific polymerase chain reaction (AS-PCR) and computational phasing are combined for relatively high-throughput, efficient resolution of phase in resequencing studies. Using data from multiple loci that were fully experimentally phased, we demonstrate that the popular computational tool PHASE can accurately phase heterozygous individuals with common SNPs (single nucleotide polymorphisms) and/or common haplotypes. However, we also demonstrate that experimental phasing with AS-PCR can efficiently supplement computational phasing, providing a rapid means to phase individuals with rare SNPs or haplotypes and with heterozygous insertion/deletion polymorphisms. By following simple stepwise procedures, AS-PCR can result in much more efficient and accurate experimental phasing of haplotypes than is possible with traditional methods such as cloning.     

Crystal structures of delta1-pyrroline-5-carboxylate reductase from human pathogens Neisseria meningitides and Streptococcus pyogenes

L-proline is an amino acid that plays an important role in proteins uniquely contributing to protein folding, structure, and stability, and this amino acid serves as a sequence-recognition motif. Proline biosynthesis can occur via two pathways, one from glutamate and the other from arginine. In both pathways, the last step of biosynthesis, the conversion of delta1-pyrroline-5-carboxylate (P5C) to L-proline, is catalyzed by delta1-pyrroline-5-carboxylate reductase (P5CR) using NAD(P)H as a cofactor. We have determined the first crystal structure of P5CR from two human pathogens, Neisseria meningitides and Streptococcus pyogenes, at 2.0 angstroms and 2.15 angstroms resolution, respectively. The catalytic unit of P5CR is a dimer composed of two domains, but the biological unit seems to be species-specific. The N-terminal domain of P5CR is an alpha/beta/alpha sandwich, a Rossmann fold. The C-terminal dimerization domain is rich in alpha-helices and shows domain swapping. Comparison of the native structure of P5CR to structures complexed with L-proline and NADP+ in two quite different primary sequence backgrounds provides unique information about key functional features: the active site and the catalytic mechanism. The inhibitory L-proline has been observed in the crystal structure.     

Phasing statistics for alpha helical membrane protein structures

In this report we highlight the latest trends in phasing methods used to solve alpha helical membrane protein structures and analyze the use of heavy atom metals for the purpose of experimental phasing. Our results reveal that molecular replacement is emerging as the most successful method for phasing alpha helical membrane proteins, with the notable exception of the transporter family, where experimentally derived phase information still remains the most effective method. To facilitate selection of heavy atoms salts for experimental phasing an analysis of these was undertaken and indicates that organic mercury salts are still the most successful heavy atoms reagents. Interestingly the use of seleno‐l ‐methionine incorporated protein has increased since earlier studies into membrane protein phasing, so too the use of SAD and MAD as techniques for phase determination. Taken together this study provides a brief snapshot of phasing methods for alpha helical membrane proteins and suggests possible routes for heavy atom selection and phasing methods based on currently available data.     

Experimental Phasing of MicroED Data Using Radiation Damage

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Conservation and Periodicity of DNA Bend Sites in Eukaryotic Genomes

DNA bend sites appear every 680 bp on average in the human -and ß-globin gene regions. Although most of theirmolecular nature has not been unraveled, a potential bend coresequence A₂N₈A₂N₈A₂ (A/A/A) and its complementary T₂N₈T₂N₈T₂(T/T/T) appeared preferentially either in or very close to mostof the bend sites, whereas other combinations of A₂ and T₂ dinucleotides,A/T/T + A/A/T, T/T/A + T/A/A and A/T/A + T/A/T, did not. Thedistances between any two of the core sequences in the entireß-globin locus showed a strong bias to a length of701–800 bp and multiples thereof, suggesting that thereis periodicity throughout the locus. This bias was not foundfor other combinations of A₂ and T₂. Again, this periodicitywas identified in many eukaryotic genes, whereas the tendencywas absent in mRNAs and prokaryotic as well as viral genomes.     

OLFinder-a program which disentangles DNA sequences containing heterozygous indels

The presence of heterozygous indels in a DNA sequence usually results in the sequence being discarded. If the sequence trace is of high enough quality, however, it will contain enough information to reconstruct the two constituent sequences with very little ambiguity. Solutions already exist using comparisons with a known reference sequence, but this is often unavailable for nonmodel organisms or novel DNA regions. I present a program which determines the sizes and positions of heterozygous indels in a DNA sequence and reconstructs the two constituent haploid sequences. No external data such as a reference sequence or other prior knowledge are required. Simulation suggests an accuracy of >99% from a single read, with errors being eliminable by the inclusion of a second sequencing read, such as one using a reverse primer. Diploid sequences can be fully reconstructed across any number of heterozygous indels, with two overlapping sequencing reads almost always sufficient to infer the entire DNA sequence. This eliminates the need for costly and laborious cloning, and allows data to be used which would otherwise be discarded. With no more laboratory work than is needed to produce two normal sequencing reads, two aligned haploid sequences can be produced quickly and accurately and with extensive phasing information.     

Conformational flexibility in the apolipoprotein E amino-terminal domain structure determined from three new crystal forms: implications for lipid binding

An amino-terminal fragment of human apolipoprotein E3 (residues 1-165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P2(1)2(1)2(1)) crystal form of the amino-terminal 22 kDa fragment of apolipoprotein E (residues 1-191). A second orthorhombic crystal form (P2(1)2(1)2(1) with cell dimensions differing from the first form) and a trigonal (P3(1)21) crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno-methionine multiwavelength anomalous dispersion, and the structure of the second orthorhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high-quality electron-density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four-helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four-helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association.     

Evaluating the possibility of detecting evidence of positive selection across Asia with sparse genotype data from the HUGO Pan-Asian SNP Consortium

Background

The HUGO Pan-Asian SNP Consortium (PASNP) has generated a genetic resource of almost 55,000 autosomal single nucleotide polymorphisms (SNPs) across more than 1,800 individuals from 73 urban and indigenous populations in Asia. This has offered valuable insights into the correlation between the genetic ancestry of these populations with major linguistic systems and geography. Here, we attempt to understand whether adaptation to local climate, diet and environment partly explains the genetic variation present in these populations by investigating the genomic signatures of positive selection.

Results

To evaluate the impact to the selection analyses due to the considerably lower SNP density as compared to other population genetics resources such as the International HapMap Project (HapMap) or the Singapore Genome Variation Project, we evaluated the extent of haplotype phasing switch errors and the consistency of selection signals from three haplotype-based approaches (iHS, XP-EHH, haploPS) when the HapMap data is thinned to a similar density as PASNP. We subsequently applied haploPS to detect and characterize positive selection in the PASNP populations, identifying 59 genomics regions that were selected in at least one PASNP populations. A cluster analysis on the basis of these 59 signals showed that indigenous populations such as the Negrito from Malaysia and Philippines, the China Hmong, and the Taiwan Ami and Atayal shared more of these signals. We also reported evidence of a positive selection signal encompassing the beta globin gene in the Taiwan Ami and Atayal that was distinct from the signal in the HapMap Africans, suggesting the possibility of convergent evolution at this locus due to malarial selection.

Conclusions

We established that the lower SNP content of the PASNP data conferred weaker ability to detect signatures of positive selection, but the availability of the new approach haploPS retained modest power. Out of all the populations in PASNP, we identified only 59 signals, suggesting a strong need for high-density population-level genotyping data or sequencing data in order to achieve a comprehensive survey of positive selection in Asian populations.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-332) contains supplementary material, which is available to authorized users.     

Production and crystallization of a selenomethionyl variant of UmuD′, an Echerichia coli SOS response protein

Crystals of both native and mutant Escherichia coli UmuD′ protein were obtained using the hanging drop method. Soaking the native crystals in solutions of heavy metal ions failed to produce good isomorphous derivatives, and selenomethionine substituted wild-type protein did not crystallize under conditions that gave native crystals. Site-directed mutagenesis was used to change the penultimate residue, a methionine amino acid, to either a valine or a threonine amino acid. Crystals were subsequently obtained from these mutant proteins with and without selenomethionine Incorporation. Crystals of the native, the mutant, and the selenomethionine Incorporated protein were all similar, crystallizing in the P4₁2₁2 space group. © 1996 Wiley-Liss, Inc.     

Linked‐read sequencing enables haplotype‐resolved resequencing at population scale

The feasibility to sequence entire genomes of virtually any organism provides unprecedented insights into the evolutionary history of populations and species. Nevertheless, many population genomic inferences – including the quantification and dating of admixture, introgression and demographic events, and inference of selective sweeps – are still limited by the lack of high‐quality haplotype information. The newest generation of sequencing technology now promises significant progress. To establish the feasibility of haplotype‐resolved genome resequencing at population scale, we investigated properties of linked‐read sequencing data of songbirds of the genus Oenanthe across a range of sequencing depths. Our results based on the comparison of downsampled (25×, 20×, 15×, 10×, 7×, and 5×) with high‐coverage data (46–68×) of seven bird genomes mapped to a reference suggest that phasing contiguities and accuracies adequate for most population genomic analyses can be reached already with moderate sequencing effort. At 15× coverage, phased haplotypes span about 90% of the genome assembly, with 50% and 90% of phased sequences located in phase blocks longer than 1.25–4.6 Mb (N50) and 0.27–0.72 Mb (N90). Phasing accuracy reaches beyond 99% starting from 15× coverage. Higher coverages yielded higher contiguities (up to about 7 Mb/1 Mb [N50/N90] at 25× coverage), but only marginally improved phasing accuracy. Phase block contiguity improved with input DNA molecule length; thus, higher‐quality DNA may help keeping sequencing costs at bay. In conclusion, even for organisms with gigabase‐sized genomes like birds, linked‐read sequencing at moderate depth opens an affordable avenue towards haplotype‐resolved genome resequencing at population scale.     

Old fold in a new X‐ray diffraction dataset? Low‐resolution molecular replacement using representative structural templates can provide phase information

The advent of structural genomics has led to a dramatic increase in the number of structures deposited in the Protein Data Bank. The number of new folds, however, still remains a very small fraction of the total number of deposited structures. Recent data on the progress of the structural genomics initiative reveals that more than 85% of target proteins that progress to the stage of data collection and structure determination have a known fold. Enzymes, which tend to exploit reaction space while adopting a common stable scaffold, contribute significantly to this observation. Herein, we evaluate a method to examine the "old fold in a new dataset" scenario likely to be encountered in the structural genomics pipeline. We demonstrate that a fold detection strategy based on secondary structure signatures followed by molecular replacement using a minimalist model can be effectively used to solve the phase problem in X-ray crystallography without further recourse to heavy atom derivatives or multiple anomalous dispersion techniques. Three common folds-the triosephosphate isomerase (TIM), adenine nucleotide alpha hydrolase-like (HUP), and RNA recognition motif (RRM)-were examined using this approach. The results presented herein also provide an estimate of the extent of phase information that can be derived from a single domain in a large multidomain structure.     

Large structural variations in the haplotype-resolved African cassava genome

Cassava (Manihot esculenta Crantz, 2n = 36) is a global food security crop. It has a highly heterozygous genome, high genetic load, and genotype-dependent asynchronous flowering. It is typically propagated by stem cuttings and any genetic variation between haplotypes, including large structural variations, is preserved by such clonal propagation. Traditional genome assembly approaches generate a collapsed haplotype representation of the genome. In highly heterozygous plants, this results in artifacts and an oversimplification of heterozygous regions. We used a combination of Pacific Biosciences (PacBio), Illumina, and Hi-C to resolve each haplotype of the genome of a farmer-preferred cassava line, TME7 (Oko-iyawo). PacBio reads were assembled using the FALCON suite. Phase switch errors were corrected using FALCON-Phase and Hi-C read data. The ultralong-range information from Hi-C sequencing was also used for scaffolding. Comparison of the two phases revealed >5000 large haplotype-specific structural variants affecting over 8 Mb, including insertions and deletions spanning thousands of base pairs. The potential of these variants to affect allele-specific expression was further explored. RNA-sequencing data from 11 different tissue types were mapped against the scaffolded haploid assembly and gene expression data are incorporated into our existing easy-to-use web-based interface to facilitate use by the broader plant science community. These two assemblies provide an excellent means to study the effects of heterozygosity, haplotype-specific structural variation, gene hemizygosity, and allele-specific gene expression contributing to important agricultural traits and further our understanding of the genetics and domestication of cassava.     

Comparative analysis of the nucleosomal structure of rye,wheat and their relatives

Analysis of the structure of chromatin in cereal species using micrococcal nuclease (MNase) cleavage showed nucleosomal organization and a ladder with typical nucleosomal spacing of 175–185 bp. Probing with a set of DNA probes localized in the authentic telomeres, subtelomeric regions and bulk chromatin revealed that these chromosomal regions have nucleosomal organization but differ in size of nucleosomes and rate of cleavage between both species and regions. Chromatin from Secale and Dasypyrum cleaved more quickly than that from wheat and barley, perhaps because of their higher content of repetitive sequences with hairpin structures accessible to MNase cleavage. In all species, the telomeric chromatin showed more rapid cleavage kinetics and a shorter nucleosome length (160 bp spacing) than bulk chromatin. Rye telomeric repeat arrays were shortest, ranging from 8 kb to 50 kb while those of wheat ranged from 15 kb up to 175 kb. A gradient of sensitivity to MNase was detected along rye chromosomes. The rye-specific subtelomeric sequences pSc200 and pSc250 have nucleosomes of two lengths, those of the telomeric and of bulk nucleosomes, indicating that the telomeric structure may extended into the chromosomes. More proximal sequences common to rye and wheat, the short tandem-repeat pSc119.2 and rDNA sequence pTa71, showed longer nucleosomal sizes characteristic of bulk chromatin in both species. A strictly defined spacing arrangement (phasing) of nucleosomes was demonstrated along arrays of tandem repeats with different monomer lengths (118, 350 and 550 bp) by combining MNase and restriction enzyme digestion.     

Production of selenomethionyl-derivatized proteins in baculovirus-infected insect cells

A protocol is described for the production of both intracellularly expressed and secreted selenomethionyl-derivatized recombinant proteins in baculovirus-infected insect cells. The method results in the production of recombinant soluble proteins with an SeMet occupancy of approximately 75% and with a recovery of approximately 20% that of native protein expression. The method is independent of the percentage methionine content of the protein and is reliable and consistent. Similar results are obtained using either Spodoptera frugiperda Sf9 or Trichoplusia ni High Five insect cells as the expression host, and when cultures are grown in either shake flasks or in Wave BioReactors.