Post‐glacial history and introgression in Abies (Pinaceae) species of the Russian Far East inferred from both nuclear and cytoplasmic markers

Aim The main aim of the present study is to infer the post‐glacial history of Abies species from north‐east Asia and to test the hypotheses that coastal Abies populations suffered less from climatic fluctuations during Pleistocene glacial periods than their more continental counterparts, and that Sakhalin was a major area of introgression. Location Natural ranges of the fir species Abies nephrolepis, Abies sachalinensis and Abies holophylla in the Russian Far East, and of Abies gracilis, which is endemic to the Kamchatka Peninsula. Methods Nineteen populations were sampled for allozyme analysis. Seventeen of these populations were also screened for variation at two paternally inherited chloroplast DNA microsatellite loci (cpSSR) and variation at one maternally inherited mitochondrial marker (nad4‐3/4). Finally a subset of 11 populations was analysed with amplified fragment length polymorphism (AFLP). Comparisons were made with already available Abies sibirica data. For all sets of markers, we estimated genetic diversity and differentiation using an analysis of molecular variance (AMOVA). Population clustering was assessed with a Bayesian approach implemented in structure v.2.3. Results Among the three major species, A. sibirica, A. nephrolepis and A. sachalinensis, A. sachalinensis demonstrated the highest cytoplasmic and nuclear diversity and the most continental species, A. sibirica, the lowest. Both nuclear and mitochondrial DNA markers revealed the presence of a transitional zone on Sakhalin Island between A. nephrolepis and A. sachalinensis of south Sakhalin. The structure analysis delivered very clear results confirming the admixed origin of A. sachalinensis, with a genetic contribution from A. nephrolepis. No variation in cytoplasmic markers was found in A. gracilis, suggesting the occurrence of a recent bottleneck. Main conclusions There is a clear reduction of genetic diversity in Abies species from the Pacific coast into the continent. The higher diversity in A. sachalinensis could have two causes: a larger effective population size in the islands due to relatively stable climatic conditions and consequently less pronounced demographic fluctuations in population size and/or hybridization with continental and Japanese populations. Sakhalin Island is a major transitional zone for conifer species. Finally, the fir from Kamchatka, A. gracilis, should be regarded as a separate species closely related to the A. nephrolepis–A. sachalinensis complex.     

1988 Gordon Conference on Theoretical Biology and Biomathematics Tilton Academy, Tilton, New Hampshire, U.S.A., 13–17 June 1988

Announcement

1988 Gordon Conference on Theoretical Biology and Biomathematics Tilton Academy, Tilton, New Hampshire, U.S.A., 13–17 June 1988     

Spectral analysis of ventilation in elderly subjects awake and asleep

We studied the periodicities of ventilation in elderly subjects using digital comb filtering. Two groups of subjects were studied, those with and without sleep apnea. Measurements were made in wakefulness, stage 1-2 sleep, and where possible in stage 3-4 sleep. For each of the digital filters we calculated the average power of the oscillatory output. To compare subject groups we first specifically determined the average power in the filter with the maximum output. The mean of this measurement was greater in elderly subjects with apnea compared with those without apnea, both during wakefulness and stage 1-2 sleep. In both groups of subjects the cycle time of the major ventilatory oscillations was on the order of 40-60 s. There was no difference in this cycle time between the two groups of subjects in wakefulness or stage 1-2 sleep. Thus, whereas similar oscillatory processes occur in subjects with and without apnea, it is the magnitude of the oscillation that differs between the two groups. These conclusions are supported by analysis of the output of individual filters of the digital comb filter. In both groups, stage 1-2 sleep produced significantly increased oscillations in ventilation. Both in wakefulness and stage 1-2 sleep, significantly greater periodicities occurred in the apneic compared with the nonapneic group. In the few subjects who had sufficient data in stage 3-4 sleep for spectral analysis, ventilatory oscillations were virtually absent in this state. Our data suggest that subjects who develop apnea during sleep have an increased propensity for periodic breathing even while awake.     

Characterization of a new hybrid mink-mouse clone panel: chromosomal and regional assignments of the GLO,ACY, NP,CKBB, ADH2, and ME1 loci in mink (Mustela vison)

To expand the mink map, we established a new panel consisting of 23 mink-mouse clones. On the basis of statistical criteria (Wijnen et al. 1977; Burgerhout 1978), we developed a computer program for choice of clones of the panel. Assignments of the following mink genes were achieved with the use of the hybrid panel: glyoxalase (GLO), Chromosome (Chr) 1; acetyl acylase (ACY), Chr 5; creatine phosphokinase B (CKBB), Chr 10; alcohol dehydrogenase-2 (subunit B) (ADH2), Chr 8. Using a series of clones carrying rearrangements involving mink Chr 1 and 8, we assigned the gene for ME1 to the short arm of Chr 1 and that for ADH2 to Chr 8, in the region 8p12-p24. Mapping results confirm the ones we previously obtained with a mink-Chinese hamster panel. However, by means of an improved electrophoretic technique, we revised the localization of the gene for purine nucleoside phosphorylase (NP), which has been thought to be on mink Chr 2. It is reassigned to mink Chr 10.     

Characterization of the mutant N-acetylglucosaminylphosphotransferase in I-cell disease and pseudo-Hurler polydystrophy: complementation analysis and kinetic studies

Complementation was examined among various types of I-cell disease and pseudo-Hurler polydystrophy by monitoring N-acetylglucosaminylphosphotransferase activity in multinucleated cells produced by fusing pair combinations of cultured skin fibroblasts. Patients with the classical forms of these disorders (5 I-cell disease and 3 pseudo-Hurler polydystrophy cell lines) comprised one complementation group and 5 cell lines from patients with variant forms of pseudo-Hurler polydystrophy comprised a distinct complementation group. In the first group, total or partial deficiency of the transferase activity was demonstrated with both natural (lysosomal enzymes) and artificial (alpha-methylmannoside) acceptor substrates with low Vmax but apparently normal Km values for the donor (UDP-GlcNAc) and acceptor (alpha-methylmannoside) substrates. The activity toward artificial substrate could be inhibited by adding exogenous lysosomal enzyme preparations to the reaction mixture. In the second group, the cells demonstrated deficiency of the transferase activity toward lysosomal enzyme acceptors but had normal activity toward alpha-methylmannoside acceptor and this activity could not be inhibited by the addition of exogenous lysosomal enzyme preparations. These findings suggest that N-acetylglucosaminylphosphotransferase is composed of at least two distinct subunits, a catalytic subunit which is absent or defective in the first complementation group, and a recognition subunit which is altered or deficient in the second group.     

Regional assignment of the genes for TK1, GALK, ALDC, and ESD on chromosome 8 in the American mink by chromosome-mediated gene transfer

Summary A panel of clones of mink-Chinese hamster somatic cell hybrids was analysed to obtain data for assigning the genes for thymidine kinase-1 (TK1), galactokinase (GALK), subunit C of aldolase (ALDC), and esterase D (ESD) to specific mink chromosomes. The results demonstrate that the genes for TK1, GALK, ALDC and ESD are syntenic and located on mink chromosome 8. Prometaphase analysis of transformed mouse cells obtained by transfer of mink genes by means of metaphase chromosomes demonstrated the presence of mink chromosome 8 fragments of different sizes in some of the independent transformants. Segregation analysis of these fragments and mink TK1, GALK, ALDC and ESD allowed us to assign the genes for TK1 and GALK to 8p24, ALDC to pter-8p25, and ESD to 8q24-8qter.     

Calculations of the spatial distribution of charge density in the environment of DNA

A technique for modeling the structured environmental charge distribution about isolated polyions of arbitrary geometry is presented and applied to B-DNA. It describes the three-dimensional variation of the continuous space charge and allows estimation of local electrostatic potentials and fields that the electrolytic environment induces at nuclei of the polyion. Calculations involve an iterative solution to the set of equations coupling electrostatic potential and average charge density in space. By dividing the region around a DNA segment into finite volume elements, sets of numerically stable atmospheric charge densities have been obtained over a range of concentrations of added monovalent salt. Results are in good agreement with those of Poisson-Boltzmann calculations on comparable systems and are consistent with findings from Monte Carlo simulations of DNA.     

Chronology of chromosome reduplication in Chinese hamster cells incubated at low temperature

Experiments have been carried out on Chinese hamster fibroblasts. Cultures at the log-stage of growth were incubated at 15 or 25° C for 24 hrs. In two groups of experiments the cells were labeled with H³-TdR for 6 hrs at the respective temperature, washed and further incubated at 37° C. In each group of experiments cultures labeled with H³-TdR at 37° C for 20 min were used as a control. It was found: 1. the delay in the onset of cell passage through the mitotic cycle at 37° in cultures exposed to 15 or 25° C was about equal to 1,5-1 hr resp. and cells proceeded through the life cycle without blockages at any phase of the cycle; 2. the patterns of chromosome reproduction during the second half of the S-phase were the same after labeling at 15, 25 and 37° C. — In the third group the cells were labelled with HP-TdR for 10–60 min and 6 hrs resp. at 25° C. The patterns of reproduction of chromosome pairs 1–4 and small metacentrics were found to be the same in cells labeled briefly and those labeled for 6 hrs. After brief labeling asynchronous reduplication of different segments in many chromosomes became evident. It was masked because of heavy labeling after 6 hrs treatment.     

Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli.

We have designed dimeric antibody fragments that assemble in Escherichia coli. They are based on single-chain FV fragments, with a flexible hinge region from mouse IgG3 and an amphiphilic helix fused to the C-terminus of the antibody fragment. The sequence of the helix was taken either from that of a previously reported four-helix bundle design or from a leucine zipper, optionally extended with a short cysteine-containing peptide. The bivalent fragments associate in vivo, either with covalent linkage or with a monomer-dimer equilibrium, and results from ultracentrifugation sedimentation studies and SDS-PAGE are consistent with dimers. All constructs are able to bind to surface-bound antigen under conditions in which only bivalent but not monovalent antibody fragments bind. The covalent bundle helix construct shows binding characteristics nearly identical to those of the much larger whole mouse antibody, resulting in substantially more stable immunoglobulin-antigen complexes than in the case of monovalent fragments. This modular design of natural and engineered protein domains directly leads to a boost of avidity, and it allows the construction of bispecific antibody fragments in functional form in E. coli.