Soft tissue landmark for ultrasound identification of the sciatic nerve in the infragluteal region: the tendon of the long head of the biceps femoris muscle.

BACKGROUND AND OBJECTIVES: The sciatic nerve block represents one of the more difficult ultrasound-guided nerve blocks. Easy and reliable internal ultrasound landmarks would be helpful for localization of the sciatic nerve. Earlier, during ultrasound-guided posterior approaches to the infragluteal sciatic nerve, the authors recognized a hyperechoic structure at the medial border of the long head of biceps femoris muscle (BFL). The present study was performed to determine whether this is a potential internal landmark to identify the infragluteal sciatic nerve. METHODS: The depth and the thickness of this hyperechoic structure, its relationship with the sciatic nerve and the ultrasound visibility of both were recorded in the proximal upper leg of 21 adult volunteers using a linear ultrasound probe in the range of 7-13 MHz. The findings were verified by an anatomical study in two cadavers. RESULTS: The hyperechoic structure at the medial border of the BFL extended in a dorsoventral direction between 1.4+/-0.6 cm (mean+/-SD) and 2.8+/-0.8 cm deep from the surface, with a width of 2.2+/-0.9 mm. Between 2.6+/-0.9 and 10.0+/-1.5 cm distal to the subgluteal fold, the sciatic nerve was consistently identified directly at the ventral end of the hyperechoic structure in all volunteers. The anatomical study revealed that this hyperechoic structure corresponds to tendinous fibres inside and at the medial border of the BFL. CONCLUSION: The hyperechoic BFL tendon might be a reliable soft tissue landmark for ultrasound localization of the infragluteal sciatic nerve.

Detection and localization of quantitative trait loci affecting fatness in broilers.

A cross between 2 genetically different outcross broiler dam lines, originating from the White Plymouth Rock breed, was used to produce a large 3-generation broiler population. This population was used to detect and localize QTL affecting fatness in chicken. Twenty full-sib birds in generation 1 and 456 full-sib birds in generation 2 were typed for microsatellite markers, and phenotypic observations were collected for 3 groups of generation 3 birds (approximately 1,800 birds per group). Body weight, abdominal fat weight, and percentage abdominal fat was recorded at the age of 7, 9, and 10 wk. To study the presence of QTL, an across-family weighted regression interval mapping approach was used in a full-sib QTL analysis. Genotypes from 410 markers mapped on 25 chromosomes were available. For the 3 traits, 26 QTL were found for 18 regions on 12 chromosomes. Two genomewise significant QTL (P < 0.05) were detected, one for percentage abdominal fat at the age of 10 wk on chicken chromosome 1 at 241 cM (MCW0058 to MCW0101) with a test statistic of 2.75 and the other for BW at the age of 10 wk on chicken chromosome 13 at 9 cM (MCW0322 to MCW0110) with a test statistic of 2.77. Significance levels were obtained using the permutation test. Multiple suggestive QTL were found on chromosomes 1, 2, 4, 13, 15, and 18, whereas chromosomes 3, 7, 10, 11, 14, and 27 had a single suggestive QTL.

Typing single-nucleotide polymorphisms using a gel-based sequencer: a new data analysis tool and suggestions for improved efficiency.

Single-nucleotide polymorphisms (SNPs) are increasingly used as genetic markers. Although a high number of SNP-genotyping techniques have been described, most techniques still have low throughput or require major investments. For laboratories that have access to an automated sequencer, a single-base extension (SBE) assay can be implemented using the ABI SNaPshot trade mark kit. Here we present a modified protocol comprising multiplex template generation, multiplex SBE reaction, and multiplex sample analysis on a gel-based sequencer such as the ABI 377. These sequencers run on a Macintosh platform, but on this platform the software available for analysis of data from the ABI 377 has limitations. First, analysis of the size standard included with the kit is not facilitated. Therefore a new size standard was designed. Second, using Genotyper (ABI), the analysis of the data is very tedious and time consuming. To enable automated batch analysis of 96 samples, with 10 SNPs each, we developed SNPtyper. This is a spreadsheet-based tool that uses the data from Genotyper and offers the user a convenient interface to set parameters required for correct allele calling. In conclusion, the method described will enable any lab having access to an ABI sequencer to genotype up to 1000 SNPs per day for a single experimenter, without investing in new equipment.

Improvement of the comparative map of chicken chromosome 13.

A comparative map was made of chicken chromosome 13 (GGA13) with a part of human chromosome 5 (HSA5). Microsatellite markers specific for GGA13 were used to screen the Wageningen chicken bacterial artificial chromosome (BAC) library. Selected BAC clones were end sequenced and 57 sequence tag site (STS) markers were designed for contig building. In total, 204 BAC clones were identified which resulted in a coverage of about 20% of GGA13. Identification of genes was performed by a bi-directional approach. The first approach starting with sequencing mapped chicken BAC subclones, where sequences were used to identify orthologous genes in human and mouse by a basic local alignment search tool (BLAST) database search. The second approach started with the identification of chicken orthologues of human genes in the HSA5q23-35 region. The chicken orthologous genes were subsequently mapped by fluorescent in situ hybridisation (FISH) and/or single neucleotide polymorphism typing. The total number of genes mapped on GGA13 is increased from 14 to a total of 20 genes. Genes mapped on GGA13 have their orthologues on HSA5q23-5q35 in human and on Mmu11, Mmu13 and Mmu18 in mouse.

A comparative map of chicken chromosome 24 and human chromosome 11.

To improve the physical and comparative map of chicken chromosome 24 (GGA24; former linkage group E49C20W21) bacterial artificial chromosome (BAC) contigs were constructed around loci previously mapped on this chromosome by linkage analysis. The BAC clones were used for both sample sequencing and BAC end sequencing. Sequence tagged site (STS) markers derived from the BAC end sequences were used for chromosome walking. In total 191 BAC clones were isolated, covering almost 30% of GGA24, and 76 STS were developed (65 STS derived from BAC end sequences and 11 STS derived within genes). The partial sequences of the chicken BAC clones were compared with sequences present in the EMBL/GenBank databases, and revealed matches to 19 genes, expressed sequence tags (ESTs) and genomic clones located on human chromosome 11q22-q24 and mouse chromosome 9. Furthermore, 11 chicken orthologues of human genes located on HSA11q22-q24 were directly mapped within BAC contigs of GGA24. These results provide a better alignment of GGA24 with the corresponding regions in human and mouse and identify several intrachromosomal rearrangements between chicken and mammals.

Mapping the sites of interaction between SecY and SecE by cysteine scanning mutagenesis.

In Escherichia coli, the SecYEG complex mediates the translocation and membrane integration of proteins. Both genetic and biochemical data indicate interactions of several transmembrane segments (TMSs) of SecY with SecE. By means of cysteine scanning mutagenesis, we have identified intermolecular sites of contact between TMS7 of SecY and TMS3 of SecE. The cross-linking of SecY to SecE demonstrates that these subunits are present in a one-to-one stoichiometry within the SecYEG complex. Sites in TMS3 of SecE involved in SecE dimerization are confined to a specific alpha-helical interface and occur in an oligomeric SecYEG complex. Although cross-linking reversibly inactivates translocation, the contact between TMS7 of SecY and TMS3 of SecE remains unaltered upon insertion of the preprotein into the translocation channel. These data support a model for an oligomeric translocation channel in which pairs of SecYEG complexes contact each other via SecE.

Whole genome scan in chickens for quantitative trait loci affecting carcass traits.

An experiment was conducted to enable quantitative trait loci (QTL) mapping for carcass traits. The population consisted of 10 full-sib families originating from a cross between male and female founders chosen from two different outcross broiler lines. Founder animals, parents, offspring, and grandoffspring are denoted as generation (G) 0, 1, 2, and 3 animals, respectively. Microsatellite marker genotypes were collected on G1 and G2 animals. Phenotypic observations were collected on G3 animals. Recorded traits were BW at 48 d, carcass weight, carcass percentage, breast meat color, and leg score. Average adjusted progeny trait values were calculated for each G2 animal and for each trait after adjusting phenotypic observations on G3 animals for fixed effects, covariables, the additive genetic contribution of the other parent, and differences between sexes. The average adjusted progeny trait values were used as the dependent variable in the QTL analysis. A QTL analysis was undertaken by modeling the segregation from G1 to G2, using a full-sib across family regression interval mapping approach. In total, 27 autosomal linkage groups covered with 420 markers were analyzed. Genomewise significance thresholds were derived using the permutation test and a Bonferroni correction. Two QTL, affecting two of the five analyzed traits, exceeded suggestive linkage. The most significant QTL was located on Chromosome 1 at 466 cM and showed an effect on carcass percentage. The other QTL, which affected meat color, was located on Chromosome 2 and gave a peak at 345 and 369 cM.

Cysteine-directed cross-linking demonstrates that helix 3 of SecE is close to helix 2 of SecY and helix 3 of a neighboring SecE.

Preprotein translocation in Escherichia coli is mediated by translocase, a multimeric membrane protein complex with SecA as the peripheral ATPase and SecYEG as the translocation pore. Unique cysteines were introduced into transmembrane segment (TMS) 2 of SecY and TMS 3 of SecE to probe possible sites of interaction between the integral membrane subunits. The SecY and SecE single-Cys mutants were cloned individually and in pairs into a secYEG expression vector and functionally overexpressed. Oxidation of the single-Cys pairs revealed periodic contacts between SecY and SecE that are confined to a specific alpha-helical face of TMS 2 and 3, respectively. A Cys at the opposite alpha-helical face of TMS 3 of SecE was found to interact with a neighboring SecE molecule. Formation of this SecE dimer did not affect the high-affinity binding of SecA to SecYEG and ATP hydrolysis, but blocked preprotein translocation and thus uncouples the SecA ATPase activity from translocation. Conditions that prevent membrane deinsertion of SecA markedly stimulated the interhelical contact between the SecE molecules. The latter demonstrates a SecA-mediated modulation of the protein translocation channel that is sensed by SecE.

Whole genome scan in chickens for quantitative trait loci affecting growth and feed efficiency.

A feed efficiency experiment was conducted in a population consisting of progeny from 10 full sib families of a cross between two broiler lines. Microsatellite genotypes were determined on Generation (G) 1 and 2. In G3, BW at 23 and 48 d and feed intake were measured and were used to calculate growth between 23 and 48 d, feed intake adjusted for BW, and feed efficiency. Average adjusted progeny trait values were calculated for G2 animals after adjusting phenotypic observations on offspring for fixed effects, covariables, maternal genetic effects, the additive genetic contribution of the mate, and heterogeneity between sexes and were used as dependent variable in the quantitative trait loci (QTL) analysis. A full sib interval mapping approach was applied using genotypes from 420 markers on 27 autosomal linkage groups. Four QTL exceeded significance thresholds. The most significant QTL was located on Chromosome 1 at 235 cM and had a 4% genomewise significance for feed intake between 23 and 48 d. Furthermore, this QTL exceeded suggestive linkage for growth between 23 and 48 d and BW at 48 d. A second QTL was located on linkage group WAU26 at 16 cM and showed suggestive linkage for feed intake between 23 and 48 d. On Chromosome 4, at 147 cM a third QTL, which had an effect on both feed intake traits, was found. Finally, a fourth QTL, which affected feed intake adjusted for BW, was located on Chromosome 2 at 41 cM.

A comprehensive microsatellite linkage map of the chicken genome.

A comprehensive linkage map of the chicken genome has been developed by segregation analysis of 430 microsatellite markers within a cross between two extreme broiler lines. The population used to construct the linkage map consists of 10 families with a total of 458 F2 individuals. The number of informative meioses per marker varied from 100 to 900 with an average of 400. The markers were placed into 27 autosomal linkage groups and a Z-chromosome-specific linkage group. In addition, 6 markers were unlinked, 1 of which was Z chromosome specific. The coverage within linkage groups is 3062 cM. Although, as in other species, the genetic map of the heterogametic sex (female) is shorter than the genetic map of the homogametic sex (male), the overall difference in length is small (1.15%). Forty-five of the markers represent identified genes or ESTs. Database homology searches with the anonymous markers resulted in the identification of a further 9 genes, bringing the total number of genes/ESTs on the current map to 54. The mapping of these genes led to the identification of two new regions of conserved synteny between human and chicken and confirmed other previously identified regions of conserved synteny between human and chicken. The linkage map has 210 markers in common with the linkage maps based on the East Lansing and Compton reference populations, and most of the corresponding linkage groups in the different maps can be readily aligned.