Physical mapping of the von Recklinghausen neurofibromatosis region on chromosome 17

The von Recklinghausen neurofibromatosis (NF1) locus has been linked to chromosome 17, and recent linkage analyses place the gene on the proximal long arm. NF1 probably resides in 17q11.2, since two unrelated NF1 patients have been identified who possess constitutional reciprocal translocations involving 17q11.2 with chromosomes 1 and 22. We have used a somatic-cell hybrid from the t(17;22) individual, along with other hybrid cell lines, to order probes around the NF1 locus. An additional probe, 17L1, has been isolated from a NotI linking library made from flow-sorted chromosome 17 material and has been mapped to a region immediately proximal to the translocation breakpoint. While neither NF1 translocation breakpoint has yet been identified by pulse-field gel analysis, an overlap between two probes, EW206 and EW207, has been detected. Furthermore, we have identified the breakpoint in a non-NF1 translocation, SP-3, on the proximal side of the NF1 locus. This breakpoint has been helpful in creating a 1,000-kb pulsed-field map, which includes the closely linked NF1 probes HHH202 and TH17.19. The combined somatic-cell hybrid and pulsed-field gel analysis we report here favors the probe order D17Z1-HHH202-TH17.19-CRYB1-17L1-NF1- (EW206, EW207, EW203, L581, L946)-(ERBB2, ERBA1). The agreement in probe ordering between linkage analysis and physical mapping is excellent, and the availability of translocation breakpoints in NF1 should now greatly assist the cloning of this locus.     

Molecular cloning of a gene encoding a Chlamydia psittaci 57-kDa protein that shares antigenic determinants with ca. 60-kDa proteins present in many Gram-negative bacteria

In order to develop reagents to study the immune response of guinea pigs to infection by Chlamydia psittaci guinea pig inclusion conjunctivitis strain (GPIC), we constructed a plasmid clone bank with C. psittaci DNA. One of the recombinant clones isolated produced large amounts of a 57-kilodalton (kDa) protein that was immunoreactive with sera from GPIC infected guinea pigs. While investigating this recombinant protein, we discovered that all the Gram-negative bacteria analyzed so far have immunoreactive proteins of similar size. This protein seems to be a 'common antigen' already described in various Gram-negative bacteria.     

Properties of a cGMP-dependent monomeric protein kinase from bovine aorta

A form of cGMP-dependent protein kinase (cGK) that was different from previously described cGK was purified from bovine aorta smooth muscle. The partial amino-terminal sequencing of this enzyme indicated that it was derived by endogenous proteolysis of the type I beta isozyme of cGK. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this form migrated as a smaller protein (Mr = 70,000) than the parent cGK (Mr = 80,000), and since the calculated nondenatured Mr was approximately 89,000 compared to Mr = 170,000 for the dimeric native enzyme, it represented a monomeric form of cGK. The monomer bound approximately 2 mol of [3H]cGMP per mol of monomer, although it had only one rapid component in [3H]cGMP dissociation assays as compared to one rapid and one slow component for the native cGK. The specific catalytic activity of the kinase was similar to that of the native enzyme, suggesting that the catalytic domain was essentially intact. The monomeric cGK incorporated significant 32P when incubated with Mg2+ and [gamma-32P]ATP in the presence of cGMP, although the phosphorylation proceeded at a slower rate than that obtained with native cGK. In contrast to previous reports of monomeric forms of cGK, this monomer was highly cGMP-dependent, although it had a slightly higher Ka (0.8 microM) for cGMP than that of the native enzyme (0.4 microM) and a low Hill coefficient of 1.0 (1.6 for the native enzyme). The cGMP dependence of the monomer did not decrease with dilution, implying that the cGMP dependence was not due to monomer-monomer interactions in the assay. The results indicated that the catalytic domain, cGMP binding domain(s), and inhibitory domain of cGK interact primarily within the same subunit rather than between subunits of the dimer as previously hypothesized for dimeric cGK.     

Nitrification and nitrate uptake: Leaching balance in a declined forest ecosystem in eastern France

Nitrate uptake and leaching were measured during one year in a declined fir forest on the Vosges highlands (eastern France), in order to investigate whether excess nitrification could be responsible for a deleterious acidification of the ecosystem. Nitrate uptake by the vegetation was active mainly from spring to early fall, and then reached about 66 kg N ha^-1. No significant leaching loss occurred during the growth period of the vegetation. Significant nitrate leaching occurred in winter (about 17 kg N ha^-1). During fall and winter the nitrification rate was of the same magnitude as values reported for other ecosystems, and, thus, was not considered to be abnormaly strong. No abnormal temporal discoupling of nitrate production and nitrate uptake occurred in the ecosystem, and forest decline must therefore have some other cause.     

Predator avoidance behaviour in the buffy-headed marmoset,Callithrix flaviceps

The predator avoidance behaviour of a free-ranging group of buffy-headed marmosets,Callithrix flaviceps, was recorded in detail during the course of a long-term study of behavioural ecology at the Fazenda Montes Claros, southeastern Brazil. Four distinct patterns of predator avoidance behaviour, each with specific vocalisations, were recognised and are described here. The selection and use of sleeping sites by the study group are also described. An analysis of the records indicates that these small monkeys are generally most vulnerable to predation by aerial raptors. Variations in the frequency of alarm calls also indicate that the marmosets tend to be more vigilant at higher levels in the forest and when the leaf cover is less extensive. The implications of group size and social structure for both the evolution and the efficacy of the anti-predator behaviour of marmosets are also discussed.     

Correlation between the release of ovine trophoblast protein-1 by the conceptus and the production of polypeptides by the maternal endometrium of ewes

We studied the biosynthesis of two proteins, p70 (Mr 70,000; pI 4.0) and p15 (Mr 15,000; pI 5.7), by endometrial tissues from ewes between Days 12 and 24 of pregnancy and between Days 12 and 16 of the oestrous cycle to determine whether production of the two was correlated with the period of biosynthesis of ovine trophoblast protein-1 (oTP-1) by the conceptus. We also compared the protein synthetic activities of endometrium from gravid and non-gravid horns of pregnant ewes at Days 14, 16 and 18 in which the conceptus had been confined to one uterine horn. Proteins p70 and p15 were produced maximally between Days 14 and 20 of pregnancy, but synthesis by endometrial cultures from cyclic ewes was low or absent. Furthermore, synthesis of Protein p70 in particular was much greater by the gravid than non-gravid horn of unilaterally pregnant ewes. We conclude that synthesis of Proteins p70 and p15 by the uterus of sheep coincides with the time of oTP-1 production by the conceptus.     

A recombination event that redefines the Huntington disease region

We report both a recombination event that places the Huntington disease gene proximal to the marker D4S98 and an extended linkage-disequilibrium study that uses this marker and confirms the existence of disequilibrium between it and the HD locus. We also report the cloning of other sequences in the region around D4S98, including a new polymorphic marker R10 and conserved sequences that identify a gene in the region of interest.     

Investigation of the selectivity of maltoporin channels using mutant LamB proteins: mutations changing the maltodextrin binding site.

Wild-type and seven mutant maltoporins were purified and their channel-forming activities studied after reconstitution into black lipid membranes. The proteins were assayed for alterations at the maltodextrin binding site by measuring the sugar-dependent blockage of ion flux through these channels. Some substitutions (R8H, W74R) caused reduced channel affinity for all maltodextrins without changing single channel conductivities. The channel with a GlySer insertion after residue 9 was also poorly blocked by sugars but unique to this protein, the channel showed a striking, almost exponential increase of affinity with increasing maltodextrin chain length. In mutants with AspPro insertions after residues 79 and 183, there was an increase in affinity for glucose and maltose but not longer maltodextrins. The additional negative charge in the AspPro insertion mutants increased the cation selectivity of maltoporin channels, as did the decrease in positive charge resulting from the R8H substitution. A mutant with a W120C substitution also showed an increased affinity for glucose and maltose but reduced affinity for longer maltosaccharides. In contrast, a Y118F substitution resulted in an 8-fold increase in maltotriose affinity, but lesser improvements for other sugars. These results are interpreted to reflect changes in subsites contributing to an extended binding site within the channel, which in turn determines the overall sugar affinity of maltoporin.     

The relationship between polyploidy and organogenetic potential in embryo- and root-derived tissue cultures of Vicia faba L.

The cell cycle was examined in embryo and root explants of Vicia faba in culture to test whether or not polyploidy and aneuploidy affected organogenetic potential. Nuclear DNA contents and the mitotic index were measured in the 0–1 mm apical segment of primary roots of 5-day old seedlings and at various times following transfer to modified MS in darkness or Chu's N6 medium in an 8 h light/16h dark cycle (N6-MS programme) at 20°C. Mature embryos were dissected and cut longitudinally. Each half was cultured on the N6-MS programme. Root explants grown on MS in darkness developed into callus but there was no subsequent organogenesis. Only on the N6-MS programme were new roots initiated from root-derived callus. Using the N6-MS programme, embryo-derived callus became green and after 3 to 4 months, produced roots and shoots. Approximately 40% of these cultures regenerated plantlets. Polyploidy occurred within 24 h of culture irrespective of both tissue source and culture protocol. Variations in chromosome number from 2n=2x=12 were also routinely observed. Thus, calluses had the ability to initiate roots and shoots regardless of persistent polyploidy and aneuploidy. Compared with the baseline of cell cycle data for roots in vivo, the proportions of cells in the different cell cycle phases remained constant. Thus, in V. faba induction of organogenesis seems more related to culture protocols than to specific changes to the cell cycle. The mitotic index was significantly lower in vitro compared with meristems of intact roots.