No abstract

The accuracy of NIPT for ChrX and ChrY can be improved substantially by integrating the results of maternal-plasma sequencing with those for maternal-WBC sequencing. The relatively high frequency of maternal mosaicism warrants mandatory WBC testing in both shotgun sequencing- and single-nucleotide polymorphism-based clinical NIPT after the finding of a potential fetal SCA.

Resistance to intercalating dyes (ethidium, acriflavine) and other organic cations, such as quaternary ammonium-type antiseptic compounds, mediated by the Staphylococcus aureus plasmid pSK1 is specified by an energy-dependent export mechanism encoded by the qacA gene. From nucleotide sequence analysis, qacA is predicted to encode a protein of Mr 55017 containing 514 amino acids. The gene is likely to initiate with a CUG codon, and a 36 bp palindrome immediately preceding qacA, along with an upstream reading frame with homology to the TetR repressors, may be components of a regulatory circuit. The putative polypeptide specified by qacA has properties typical of a cytoplasmic membrane protein, and is indicated to be a member of a transport protein family that includes proteins responsible for export-mediated resistance to tetracycline and methylenomycin, and uptake of sugars and quinate. The analysis suggests that N- and C-terminal regions of these proteins are involved in energy coupling (proton translocation) and substrate transport, respectively. The last common ancestor of the qacA and related tet (tetracycline resistance) lineages is inferred to have been repressor controlled, as occurs for modern tet determinants from Gram-negative, but not those from Gram-positive, bacteria.

Extracellular factors that regulate the growth and differentiation of cell lineages in the pancreatic primordia are poorly understood. Identification of these factors for pancreatic islet beta-cells could open new avenues for the treatment of insulin-dependent diabetes. We developed a low cell density serum-free culture system for dissociated pancreatic cells from the 13.5-day mouse fetus and investigated the effects of extracellular matrix proteins on differentiation of islet cells. After 4 days in culture, total cell number decreased by two-thirds, but insulin-positive beta-cell number increased 10-fold. Both of collagens I and IV inhibited cell survival (by >50%), whereas fibronectin had no effect. In the presence of soluble laminin-1, however, the number of beta-cells increased linearly by 60-fold without an increase in the total cell number; glucagon-positive cell number was unchanged, and somatostatin and pancreatic polypeptide-positive cells were not detected. The effect of laminin-1 was completely blocked by a monoclonal rat anti-laminin-1 antibody. In the presence of laminin-1, the thymidine analogue, BrdU, was incorporated into only 2.5% of cells, which were mainly insulin-negative at days 1-3. Laminin-1 appeared, therefore, to induce differentiation of beta-cells from precursor cells in day-13.5 fetal pancreas. Laminin-1 was shown to be expressed in the epithelial basement membrane of the 13.5- to 17.5-day fetal pancreas. These findings provide the first evidence of a role for laminin-1 to promote differentiation of pancreatic beta-cells.

Detection of chromosome copy number variation (CNV) plays an important role in the diagnosis of patients with unexplained clinical symptoms and for the identification of chromosome disease syndromes in the established fetus. In current clinical practice, karyotyping, in conjunction with array-based methods, is the gold standard for detection of CNV. To increase accessibility and reduce patient costs for diagnostic CNV tests, we speculated that next-generation sequencing methods could provide a similar degree of sensitivity and specificity as commercial arrays. CNV in patient samples was assessed on a medium-density single nucleotide polymorphism array and by low-coverage massively parallel CNV sequencing (CNV-seq), with mate pair sequencing used to confirm selected CNV deletion breakpoints. A total of 10 ng of input DNA was sufficient for accurate CNV-seq diagnosis, although 50 ng was optimal. Validation studies of samples with small CNVs showed that CNV-seq was specific and reproducible, suggesting that CNV-seq may have a potential genome resolution of approximately 0.1 Mb. In a blinded study of 72 samples with known gross and submicroscopic CNVs originally detected by single nucleotide polymorphism array, there was high diagnostic concordance with CNV-seq. We conclude that CNV-seq is a viable alternative to arrays for the diagnosis of chromosome disease syndromes.

No abstract

This pilot study suggests that trophectoderm biopsy and blastocyst transfer may be more advantageous than cleavage stage biopsy with respect to outcome of PGD for monogenic diseases.

The genes mvhDGA, which encode the subunit polypeptides of the methyl viologen-reducing hydrogenase in Methanobacterium thermoautotrophicum strain AH, have been cloned and sequenced. These genes, together with a fourth open reading frame designated mvhB, are tightly linked and appear to form an operon that is transcribed starting 42 base pairs upstream of mvhD. The organization and sequences ofthe mvhG and mvhA genes indicate a common evolutionary ancestry with genes encoding the small and large subunits of hydrogenases in eubacterial species. The product of the mvhB gene is predicted to contain six tandomly repeated bacterial-ferredoxinlike domains and, therefore, is predicted to be a polyferredoxin that could contain as many as 48 iron atoms in 12 Fe4S4 clusters.Methanobacterium thermoautotrophicum reduces CO2 to CH4 using H2 as the reductant. Therefore, hydrogenase activity is essential for methanogenesis in this species, and two hydrogenases have been purified and characterized from extracts of M. thermoautotrophicum (1-3). In this report we describe the organization and structure of the clustered genes (mvhDGA) that encode subunits ofthe hydrogenase that does not reduce cofactor F420, the enzyme conventionally designated as the methyl viologen-reducing hydrogenase (MV hydrogenase desired recombinant clones by their ability to direct the synthesis of antigens in Escherichia coli that bound rabbit antibodies raised against the a subunit of the F420-reducing hydrogenase, purified as previously described from M. thermoautotrophicum AH (2). DNA prepared from positive clones was subcloned into pUC8 (11) and sequenced (Fig. 1). § § Determination of Amino Acid Sequences. The aminoterminal sequences of the subunit polypeptides of MVhydrogenase purified from M. thermoautotrophicum AH and separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis were determined by using an Applied Biosystems 470A gas-phase protein sequencer. The subunits did not have N-terminal methionyl residues. The N-terminal amino acid sequence of the a subunit was found to be '40% identical to the N-terminal sequence of the a subunit of the F420-reducing hydrogenase (2). This conservation of amino acid residues presumably accounts for the immunological cross-reactivity of the two polypeptides.RNA Preparation, Primer Extension, and RNA Sequencing. Total cellular RNA was prepared from lysates of M. thermoautotrophicum strain AH. 32P-labeled oligonucleotide primers were synthesized and hybridized to the M. thermoautotrophicum AH RNA, and the hybrid molecules were used in primer extension procedures to determine the 5' end of the mvh transcript (9) and to sequence the transcript of the mvhG gene (12) in the region of the cloned TGA codon (see Results).Primer-Directed Amplification and Sequencing of M. thermoautotrophicum AH Genomic DNA. Oligonucleotide primers (24 mers) were synthesized complementary to the sequences located 42 bp 5' and 42 bp 3' from the position at which the TGA codon had been detected in the cloned mvhG gen...