Carsten Petersen

Similar Splice-Site Mutations of the ATP7A Gene Lead to Different Phenotypes: Classical Menkes Disease or Occipital Horn Syndrome

More than 150 point mutations have now been identified in the ATP7A gene. Most of these mutations lead to the classic form of Menkes disease (MD), and a few lead to the milder occipital horn syndrome (OHS). To get a better understanding of molecular changes leading to classic MD and OHS, we took advantage of the unique finding of three patients with similar mutations but different phenotypes. Although all three patients had mutations located in the splice-donor site of intron 6, only two of the patients had the MD phenotype; the third had the OHS phenotype. Fibroblast cultures from the three patients were analyzed by reverse transcriptase (RT)-PCR to try to find an explanation of the different phenotypes. In all three patients, exon 6 was deleted in the majority of the ATP7A transcripts. However, by RT-PCR amplification with an exon 6-specific primer, we were able to amplify exon 6-containing mRNA products from all three patients, even though they were in low abundance. Sequencing of these products indicated that only the patient with OHS had correctly spliced exon 6-containing transcripts. We used two different methods of quantitative RT-PCR analysis and found that the level of correctly spliced mRNA in this patient was 2%-5% of the level found in unaffected individuals. These findings indicate that the presence of barely detectable amounts of correctly spliced ATP7A transcript is sufficient to permit the development of the milder OHS phenotype, as opposed to classic MD.

Control of copper homeostasis in Escherichia coli by a P-type ATPase, CopA, and a MerR-like transcriptional activator, CopR.

We have isolated and characterized a copper sensitive Escherichia coli mutant that is deficient in the copper transporting P-type ATPase encoded by the copA gene (previously ybaR). Measurements of uptake and efflux of 64Cu by wild-type and mutant cells implicated the CopA protein in copper efflux from the cytoplasm, and further demonstrated that cell-associated copper in intact E. coli cells is distributed between two kinetically distinguishable pools, the ratio of which was dramatically disturbed by the copA mutation. Using a copA-lacZ gene fusion the copA promoter was found to be specifically induced by copper, and this induction was shown to be dependent on a MerR-like transcriptional activator encoded by a previously uncharacterized gene, copR (previously ybbI). In the copA deficient background the copA-lacZ fusion was super induced to very high levels even in the absence of copper addition to the medium, and this induction was dependent on CopR. These results indicated that the cytoplasmic copper concentration was dramatically increased in the copA mutant, in agreement with the 64Cu uptake experiments. Moreover, they implied, that the copper concentration in wild type cells is determined primarily by the CopA efflux pump, while copper is taken up by an essentially constitutive mechanism.

Invariance of the Nucleoside Triphosphate Pools ofEscherichia coli with Growth Rate

The ATP and GTP pools of Escherichia coli have recently been reported to increase approximately 10-fold with increasing growth rates in the range from 0.4 to 1.4 generations/hour (Gaal, T., Bartlett, M. S., Ross, W., Turnbough, C. L., and Gourse, R. L. (1997) Science 278, 2092–2097). Moreover, it was proposed that this variation of the nucleotide pools, particularly the ATP pool, might be responsible for the well known growth rate-dependent regulation of rRNA synthesis in E. coli. To test this hypothesis we have measured the nucleoside triphosphate pools as a function of growth rate for several E. coli strains. We found that the size of all four RNA precursor pools are essentially invariant with growth rate, in the range from 0.5 to 2.3 generations/hour. Nevertheless we observed the expected growth rate-dependent increase of RNA accumulation in these strains. In light of these results, it seems unlikely that nucleotide pool variations should be responsible for the growth rate-dependent regulation of rRNA synthesis.

Preferential flow in sandy loam soils as affected by irrigation intensity

Abstract Dye-tracer studies in the field using Brilliant Blue FCF as tracer were performed to investigate the effect of irrigation intensity and soil heterogeneity on preferential flow. In two fields, both level and newly tilled in terms of seed bed preparation, to plots of 1.6 × 1.6 m were applied 50 mm of dye solution at rates of 10 and 50 mm h−1. In the second year level, plots of grass of similar size were applied with 25 mm dye solution at a rate of 3.1, 6.2, 12.5, and 25 mm h−1. For all plots the stained patterns were examined one or two days after application of dye solution by the excavation of 11 vertical cross sections of 100 × 100 cm and 10 cm apart from each other. Flow patterns were digitized and depth functions for the degree of dye coverage and the number of activated flow channels were calculated. Furthermore, the structural features of each cross section were examined visually. The results show that deep penetration of water into the soil profile took place as preferential flow through macropores, mainly earthworm channels, with much of the water thus bypassing the soil matrix. In the top 0–25 cm layer, the degree of dye coverage tended to be larger for the lower irrigation intensities indicating that water flow in the top soil took place through a relatively great proportion of the pores in the soil matrix. In the 35–100 cm subsoil layer the number of stained macropores tended to be larger for the higher irrigation intensities. Thus, at higher irrigation intensity a positive pressure potential apparently developed more extensively in the topsoil initiating preferential flow through a greater number of macropores in the subsoil. In the newly tilled soil, water flow took place through a relatively great part of the topsoil matrix. Deeply penetrating stained earthworm channels originated, predominantly, in the well defined transition zone between topsoil and subsoil. In the soil left untilled and grass covered for about one year the continuity of macropores was more pronounced, and stained channels could frequently be traced from the subsoil all the way to the soil surface, in particular at low irrigation intensity.

Characterization of the hCTR1 gene: Genomic organization, functional expression, and identification of a highly homologous processed gene

The human hCTR1 gene was originally identified by its ability to complement a yeast mutant deficient in high-affinity copper uptake (Zhou, B., Gitschier, J., 1997. A human gene for copper uptake identified by complementation in yeast. Proc. Natl. Acad. Sci. USA 94, 7481-7486). Here, we have determined the DNA sequence of the exon-intron borders of the hCTR1 structural gene and report that the coding sequence is disrupted by three introns, all of which comply with the GT/AG rule. Furthermore, human fibroblasts, transfected with hCTR1 cDNA, were shown to have a dramatically increased capacity for (64)Cu uptake, indicating that the hCtr1 protein is functional in copper uptake in human cells. In contrast, no evidence was found for involvement of the hCTR2 gene product in copper uptake. Finally, we have identified a highly homologous processed pseudogene, hCTR1psi, which was localized to chromosome 3q25/26. The processed gene was found to be transcribed, but due to a frame shift mutation, it only had the potential to encode a truncated protein of 95 amino acid residues, and cells transfected with hCTR1psi DNA showed no increase of (64)Cu uptake.

The effect of lupins as compared with peas and oats on the yield of the subsequent winter barley crop

Abstract New high yielding early maturing cultivars of lupins have been introduced in north-west Europe as grain protein crops in crop rotations. This paper reports on a comparative study of lupins with peas and oats, and of their effect on yield of subsequent winter barley crops. These crops were given five levels of N under irrigated and non-irrigated conditions on sand and loam. Under rain fed conditions the grain yield of pea, oat and lupin varied between 24–36, 34–53 and 18–37 hkg DM ha−1, respectively. Supplemental irrigation raised grain yield of oat to 50–60 hkg DM ha−1, while grain yield in pea was not affected and grain yield in lupin in most cases decreased due to gray mould attack and excessive vegetative growth in the indeterminate lupin variety. Under rain fed conditions, the grain nitrogen content of pea, oat and lupin varied between 137–172, 61–80 and 189–226 kg N ha−1, respectively, and was significantly higher in lupin as compared with pea. On sandy soil, similar low-root densities were found for pea, oat and lupin below 30 cm depth. On sand, at final harvest the residual soil-N of lupin and pea, as measured in a subsequent winter barley crop not supplied with N fertilizer, was 15 and 8–10 kg N ha−1 higher than in winter barley following oat, respectively. The nature of the probably more N-root residues of lupin is discussed. On loam, the residual N of lupin and pea was similar, 18–27 kg N ha−1. On sand, under rain fed conditions preceding lupin and pea as compared with oat, increased the barley grain yield at zero N-application 77 and 49%, respectively; the effect of lupin was significantly higher than that of pea until the highest N-level 120 kg N-application ha−1. On loam under rain fed conditions preceding lupin and pea increased the barley grain yield at zero N-application by 36 and 62%, respectively, as compared with oat; at N-application >60 kg N ha−1 the grain yield was similar after all three crops. For both soil types the same level of effect was found under irrigated conditions. Conclusions: Supplemental irrigation might result in lower grain yield in lupin due to gray mould attack and excessive growth if indeterminate lupin varieties are used. Grain nitrogen yield of lupin is significantly higher than that of pea. On sand, the effect of lupin on the subsequent winter barley grain yield is significantly higher than that of pea, probably due to greater N-root nitrogen residues. On loam, lupin and pea have similar effects on the subsequent winter barley crop.

Daisy: Model Use, Calibration, and Validation

Daisy is a soil-plant-atmosphere system model focusing on agro-ecosystems. It simulates water, heat, carbon, and nitrogen balances as well as crop production and pesticide fate in agro-ecosystems subjected to various management strategies. The basic scale of application is the field (management unit), which may be simulated in one or two dimensions. Daisy allows several different process descriptions for water flow, evapotranspiration, crop growth, and solute transport. Furthermore, it can operate in a distributed mode (several fields) and link up with distributed hydrological models. In this case, statistical and remote sensing data are relevant. Considerations concerning the objective of a given study and available data determine the choice of process descriptions. All applications require information concerning weather (at minimum, daily values of solar radiation, air temperature, and precipitation), soil (texture, organic matter, hydraulic parameters, etc.), location of groundwater, and vegetation cover. Applications that focus on nitrogen dynamics require a description of crop rotation, tillage, use of fertilizer and manure, irrigation, sowing, harvesting, and organic matter turnover in the soil. In carbon and nitrogen balance simulations, the uncertainty associated with crop growth is particularly important because Daisy only considers water and nitrogen stress. Dry matter and nitrogen yield may require calibration. Uncertainty associated with initialization of the organic matter pools and the parameterization of organic fertilizers is considered to be of major importance. For pesticide transport calculations, descriptions of macroporosity and hydraulic conditions close to the surface are critical. Daisy has been validated in several international comparative validation studies.

Parameter assessment for simulation of biomass production and nitrogen uptake in winter rape

Field experiments were conducted with winter rape at two locations in 1991/1992. The effects of three different amounts of nitrogen were compared. Plants were protected against weeds, diseases and pests and irrigated to minimize detrimental effects on yield. The results are used to assess a set of crop parameters for use in a dynamic simulation model for the soil-plant system, DAISY. The model is briefly described. The time course of biomass production, nitrogen uptake and green crop area index development is simulated for potential and for nitrogen-limited crop production. Results are compared with observations. The agreement was quite satisfactory for both locations. Shortcomings of the present model are demonstrated, however, in its ability to simulate nitrogen dynamics in plants late in the growth period and also in its ability to simulate green area index (and growth) during the autumn period.

Long-range translational coupling in the rplJL-rpoBC operon of Escherichia coli☆

In Escherichia coli the genes encoding ribosomal proteins L10 and L7/L12, rplJ and rplL, are cotranscribed, and translation of both cistrons is regulated by binding of L10 or a complex of L10 and L7/L12 to a single target in the mRNA leader region. Co-ordinated regulation is assured by some kind of translational coupling, the mechanism of which was investigated here by deletion analysis of plasmids carrying either the intact rplL gene or rplL-lacZ gene fusions. Unless the rplL ribosome binding site was modified by deletion, efficient initiation of translation required translation of a region located more than 500 nucleotides upstream on the transcript within the rplJ cistron. It is proposed that the wild-type rplL ribosome binding site is blocked by long-range RNA base-pairing to this region, when translation of the rplJ sequence is inhibited.

The functional stability of the lacZ transcript is sensitive towards sequence alterations immediately downstream of the ribosome binding site

SummaryVarious synthetic DNA sequences were inserted downstream of the fourth codon of the Escherichia coli lacZ gene on plasmids containing a hybrid lacZ-galK operon. Several different sequences, one as short as 10 bp, reduced the functional stability of the lacZ message three- to fourfold, whereas others had little or no effect. Introduction of synthetic sequences into a plasmid containing the intact lac operon resulted in similar reductions of mRNA stability. The sequence alterations also reduced the translational efficiency and transcription through lacZ as monitored by measurements of galactokinase synthesis from the downstream galK gene. There was no correlation between the average translational frequency and the stability of the lacZ message indicating that some of the inserted sequences reduced mRNA stability directly and not as a consequence of their effect on translation. The reduction of transcription through the lacZ gene correlated with the reduction of translation in agreement with current models of transcriptional polarity.