Secondary structure of β-hydroxydecanoyl thiol ester dehydrase, a 39-kDa protein, derived from Hα, Cα, Cβ and CO signal assignments and the Chemical Shift Index: Comparison with the crystal structure

Summary Nearly complete backbone ¹H, ¹⁵N and ¹³C signal assignments are reported for -hydroxydecanoyl thiol ester dehydrase, a 39-kDa homodimer containing 342 amino acids. Although ¹⁵N relaxation data show that the protein has a rotational correlation time of 18 ns, assignments were derived from triple-resonance experiments recorded at 500 MHz and pH 6.8, without deuteration. The Chemical Shift Index, CSI, identified two long helices and numerous -strands in dehydrase. The CSI predictions are in close agreement with the secondary structure identified in the recently derived crystal structure, particularly when one takes account of the numerous bulges in the -strands. The assignment of dehydrase and a large deuterated protein [Yamazaki et al. (1994) J. Am. Chem. Soc., 116, 11655–11666] suggest that assignment of 40–60 kDa proteins is feasible. Hence, further progress in understanding the chemical shift/structure relationship could open the way to determine the structures of such large proteins. Supplementary Material is available on request, comprising Table S1 listing the spectral parameters; Table S2 listing the assignments; Fig. S1 showing the 2D ¹H–¹⁵N HSQC spectrum; Fig. S2 showing sequential NOEs, secondary shifts, H-exchange and ³J_HN data; and Fig. S3 showing plots of the H, C, CO and C Chemical Shift Indexes.To whom correspondence should be addressed.     

Molecular modelling of the photoconduction mechanism by charged solitons intrans-polyacetylene: I

It is possible to rationalize the different steps involved in the photoconductivity observed intrans-polyacetylene (t-PA) in terms of a simple chemical model. The interchain electron transfer (IET) and the intrachain polaron capture by neutral solitons proposed by Orensteinat al. may be represented within a simple molecular orbital (MO) picture. An efficiency index for the IET is proposed in terms of the binding energy of the charged polarons and of the electronic chemical potential. The predictive power of the efficiency index is analyzed in the context of the global softness concept. An MO protocol is described to obtain estimates of the IET hopping probabilities.     

Gtl2 lacZ,an insertional mutation on mouse Chromosome 12 with parental origin-dependent phenotype

We have produced a transgenic mouse line, Gtl2 ^lacZ (Gene trap locus 2), that carries an insertional mutation with a dominant modified pattern of inheritance:heterozygous Gtl2 ^lacZ mice that inherited the transgene from the father show a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype is strongly reduced in Gtl2 ^lacZ mice that inherited the transgene from the mother. On a mixed genetic background this pattern of inheritance was reversible upon transmission of the transgene through the germ line of the opposite sex. On a predominantly 129/Sv genetic background, however, transgene passage through the female germ line modified the transgene effect, such that the penetrance of the mutation was drastically reduced and the phenotype was no longer obvious after subsequent male germ line transmission. Expression of the transgene, however, was neither affected by genetic background nor by parental legacy. Gtl2 ^lacZ maps to mouse Chromosome 12 in a region that displays imprinting effects associated with maternal and paternal disomy. Our results suggest that the transgene insertion in Gtl2 ^lacZ mice affects an endogenous gene(s) required for fetal and postnatal growth and that this gene(s) is predominantly paternally expressed. Received: 30 May 1995 / Accepted: 7 August 1995     

Cleavage of double-stranded DNA by manganese tris(methylpyridiniumyl)porphyrin linked to 3′-spermine oligonucleotides

 Cleavage of double-stranded DNA was performed with cationic manganese porphyrin complexes linked via a spermine tether to the 3′- or 5′-side of triple-helix-forming oligonucleotides (cleaver-TFO conjugates). The targeted sequence was a 15-polypurine sequence present in the env gene of HIV-1 (positions 7301–7315). The presently used TFOs contain only thymine and 5-methylcytosine residues and one adenine at the 3′-end in order to be able to easily introduce a 3′-polyamine linker by reductive amination of the corresponding 3′-apurinic polypyrimidine oligonucleotides. With this method we prepared these TFO-cleaver conjugates in 45% yield with only two equivalents of the Mn-TrisMPyP-COOH precursor. These new metalloporphyrin-TFO conjugates were able to cleave a complementary 45-mer duplex at 10 nM concentration with only ten equivalents of TFO-cleaver. Conjugates without spermine, without 5-methylcytosine, with a random sequence or with the managanese porphyrin-spermine entity on the 5′-end of TFOs were synthesized for comparative studies. Received: 6 December 1995 / Accepted: 5 February 1996     

Xanthine accumulation and vacuolization inChlamydomonas reinhardtii cells

Summary Utilization of xanthine as the sole nitrogen source for growth byChlamydomonas reinhardtii cells involved the formation of a transient, intracellular pool of xanthine. Up to 20% of the total xanthine supplied to the medium was not assimilated after uptake but stored in the cells at concentrations that exceeded xanthine solubility in water. At the subcellular level, a massive accumulation of starch grains in the chloroplast and the appearance of many vacuoles in the cytoplasm distinguished xanthine-grown from ammonium-grown cells. Starch accumulation, but not development of vacuoles, was also observed in N-starved cells. Uptake experiments with radio-labelled xanthine showed that this accumulates only in the cytoplasm, most probably inside vacuoles. The electron-dense material observed in vacuoles of xanthine-grown cells suggests that the intracellular xanthine is in part solid xanthine.     

Absence of plasma membrane H+-ATPase in plasmodesmata located in pit-fields of the young reactive pulvinus ofMimosa pudica L.

Summary Immunocytochemical techniques were employed to study the spatial distribution of the plasma membrane H⁺-ATPase within various cell types of the young reactive primary pulvinus ofMimosa pudica L. These cells were interconnected by large numbers of plasmodesmata, being concentrated within pit-fields. Although we could routinely detect evidence of the H⁺-ATPase along the plasma membrane, immunolabelling was rarely, if ever, observed along the plasma membranes of the plasmodesmata. This finding is discussed with respect to the likely specialized supramolecular structure of the plasmodesma.Abbreviations SEL size exclusion limit of plasmodesmata     

uidA gene transfer and expression in maize microspores using the biolistic method

Summary The ability to recover male gametophyte derived plants, which is necessary to get transformed haploid plants, was verified for a hybrid of maize. Using the isolated microspore culture technique, a 9 × 10^–5 plant regeneration frequency was obtained. Maize microspores were bombarded with tungsten particles using a PDS He/1000 apparatus. GUS expression in the microspores was maximum with 1.1 m diameter tungsten microprojectiles for 1100 and 1350 psi helium pressures at a 6 cm distance between the launch point and the target cells. Increasing the amount of DNA coated on the microparticles from 1.66 to 4 g DNA/mg of particles allowed a two-fold and four-fold increase of the GUS-expressing microspore frequency for 1100 and 1350 psi helium pressure bombardment, respectively. Optimal concentration of solidifying agent in the bombardment support culture medium was found to be 1%. Cell density ranging from 25000 microspores/bombardment to 100000 microspores/bombardment did not affect the frequency of GUS-expressing microspores. Using these optimal conditions, the maximum frequency of GUS-expressing microspores was found to be about 9 × 10^–4, while maintaining an embryo formation frequency about 5 × 10^–4.Abbreviations GUS -glucuronidase - PEG polyethylene glycol