Sex-related growth rate differences in mouse preimplantation embryos in vivo and in vitro

Sex-related growth rate differences in preimplantation mouse embryos were investigated. In experiment I, Day 3 embryos were recovered from reproductive tracts, classified according to developmental stage, and cultured for 24 hr in CZB medium containing glucose. Each embryo was then reclassified and stained for measurement of number of nuclei and finally sexed using the polymerase chain reaction. In experiment II, Day 4 embryos were recovered, classified, stained, and sexed as in experiment I immediately after recovery. Morphologically, there were no differences between the sexes in either of the experiments on Day 4. However, based on number of nuclei, the data showed that in vitro conditions support the development of male embryos to the blastocyst stage compared to female embryos. Furthermore, growth rate differences were observed in vivo on Day 3, as females compacted earlier than males. These results suggest that the increased cell proliferation in cultured male embryos is an artifact caused by the in vitro environment. The variation may be due to sex differences in embryonal energy metabolism during the preimplantation stage. The growth difference implies different in vitro requirements of male and female embryos. © 1995 Wiley-Liss, Inc.     

Expression and purification strategies for the production of single-chain antibody and T-cell receptor fragments inE. coli

This work describes protocols for the production of single-chain antibody and T-cell receptor fragments inE. coli. A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity or metal chelate affinity chromatography. The TCR fragments may have to be denatured and refolded before the fragments attain their proper conformation.     

Evaluation of mass spectrometric techniques for charaterization of engineered proteins

A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described. The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation. The various aspects of the procedure and its application is described in detail.     

Localization of Reactive Cysteine Residues by Maleidoyl Undecagold in the Mitochondrial Creatine Kinase Octamer

Octamers of mitochondrial creatine kinase (Mi-CK) wore modified with the thiol-specific reagents N-ethylmaleimide or the gold-coupled derivative, maleidoyl undecagold. The kinetics of inhibition of the Mi-CK catalysis was shown to be comparable for both reagents, suggesting that the large gold cluster complex is accessible to the reactive cysteines. SDS-PAGE analysis revealed that two of eight cysteines per Mi-CK monomer were labeled with maleidoyl undecagold with a similar affinity for the functional maleimide group. Gel exclusion chromatography of labeled molecules showed that the octameric structure of Mi-CK was preserved after thiol modification. Freeze-dried gold-labeled octamers visualized by electron microscopy under cryoconditions were enhanced in contrast and showed a well-preserved fourfold symmetry of the end-on view, Image analysis of gold-labeled Mi-CK exhibited an averaged end-on view with four strong contrast signals located at the periphery of the notamer, whereas the center of the molecule remained electron translucent. We conclude that the two cysteine residues per monomer labeled with maleidoyl undecagold are located at the octamer's perimeter and we discuss the possible role of these reactive cysteines in enzyme catalysis.     

The spectra of base substitutions induced by the impCAB,mucAB and umuDC error-prone DNA repair operons differ following exposure to methyl methanesulfonate

We have used the lacZ reversion assay to study the mutation spectra induced by the Escherichia coli chromosomal umuDC operon and of its two plasmid-borne analogues impCAB and mucAB following exposure of cells to UV light and methyl methane-sulfonate (MMS). We have shown that the impCAB, mucAB and umuDC operons all produce a similar response to UV light which results almost exclusively in AT → GC transitions. However, we found that the three operons produced different responses to alkylating agents. We found that with MMS the chromosomal umuDC operon produced almost exclusively AT → GC transitions, whilst both mucAB and impCAB produced predominantly transversions. In the case of the impCAB operon the mutation spectrum contained more AT → TA than GC → TA transversions; this balance was reversed with mucAB. The effect of the copy number of the error-prone DNA repair operons upon the mutagenic spectra was also studied. The results obtained suggest that the copy number of the imp operon does not greatly affect the specificity of base substitutions observed. However, an increase in the copy number of the umuDC operon greatly affected the specificity of base substitution, such that virtually no transitions were produced and the spectrum was dominated by GC/AT → TA transversions. It appears that the three error-prone DNA repair operons impCAB, mucAB and umuDC, despite showing strong structural and functional homologies, can display major differences in the spectrum of base changes induced during mutagenesis. We propose that the type of misincorporation/chain extension which DNA polymerase III is allowed to synthesize on a damaged DNA template is extremely sensitive to both the amount and type of error-prone repair proteins present. The modulation of these events by the different proteins can result in widely different mutagenic changes in the repaired DNA.     

Comparative studies on the dynamics of crosslinked insulin

Molecular dynamics simulations were carried out on an insulin crosslinked between the N-terminal A chain and the C-terminal B chain to form a so-called mini-proinsulin: N -^A1-N -^B29-diaminosuberoyl insulin (DASI). To investigate the influence of crosslinking on the dynamics of the insulin moiety, the bridge was removed from a transient DASI structure and simulation was carried on independently with the then unlinked (ULKI) as well as with the crosslinked species. The effects of crystal packing and quaternary interactions were checked by simulating both types of monomers and dimers known from the hexamer structure. All simulations were compared to previous ones of native insulin. DASI shows general similarity to the native simulations in most parts of the structure. Deviations are visible in the segments to which the bridge is directly connected, i.e. their flexibility is reduced. Upon removal of the bridge the ULKI simulations reapproach those of native insulin. The influence of the bridge spreads over the whole molecule, but all of its main structural features remain intact. The simulations suggest that the displacement of the C-terminal B chain of native insulin, considered important for receptor interaction, is prevented by the bridge, which also partially shields some binding residues. This is in accordance with the poor biological potency of A1-B29-crosslinked insulins.Abbreviations DASI-insulin(DASI) bovineN -^A1-N -^B29-di-aminosuberoyl insulin - ULK-insulin (ULKI) Native beef insulin with the bridge of DASI removed     

Statistics of RNA melting kinetics

We present and study the behavior of a simple kinetic model for the melting of RNA secondary structures, given that those structures are known. The model is then used as a map that. assigns structure dependent overall rate constants of melting (or refolding) to a sequence. This induces a landscape of reaction rates, or activation energies, over the space of sequences with fixed length. We study the distribution and the correlation structure of these activation energies. Correspondence to: P. Schuster