Duplications of the X chromosome in males: evidence that most parts of the X chromosome can be active in two copies

Summary We have analysed two duplications of the X chromosome in male patients using chromosome replication and DNA methylation patterns as determinants of the functional status of the duplicated segments. In both cases, the large duplicated regions, Xq12-q22 and Xq26.3-qter, were not inactivated. A review of previously reported male cases revealed that these duplications were also not subject to inactivation. Taken together, the examined duplications cover almost the entire X chromosome except the pericentromeric region and Xq25–26. Thus, most regions of the X chromosome can be present in two functional copies without lethal consequences.     

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene,and expression of inactive mutant enzyme protein in E. coli

Summary A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G⁹⁸⁵. The same assay consistently revealed A⁹⁸⁵ in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G⁹⁸⁵ mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.     

The sulphydryl groups of ox brain and liver glutamate dehydrogenase preparations and the effects of oxidation on their inhibitor sensitivities

Glutamate dehydrogenase preparations from several sources have been shown to have suffered limited proteolysis during purification. This proteolysis has been previously shown to involve removal of the N-terminal tetrapeptide and to result in changes in the regulatory properties of the enzyme. In the present work the previously unidentified N-terminal residue of the unproteolysed enzyme from ox brain and liver is shown to be cysteine. The thiol group of this residue is masked in the native enzyme but it becomes accessible after reduction. Exposure of solutions of the unproteolysed enzyme to air oxidation causes large changes in its sensitivity to inhibition by the antipsychotic drug perphenazine, GTP and by high concentrations of NADH. No such changes occurred in the behaviour of preparations of the enzyme that had suffered proteolysis during purification under these conditions.Special issue dedicated to Dr. Santiago Grisolia.     

Thyroid function and trisomy 21. TSH increase and rT3 deficiency

An excess of thyrotropin (TSH) with normal levels of tetraiodothyronine (T4) and of 3,5,3'-triiodothyronine (T3) was confirmed in the serum of 78 trisomy 21 children. A severe deficiency of 3,3',5'-triiodo-thyronine (rT3 or reverse T3) was observed and the decrease of the rT3/TSH ratio was highly significant. These new facts suggest that the rT3 deficiency plays a peculiar role in trisomy 21 (maybe through the regulation of one or few steps of monocarbons' metabolism). A systematic control of thyroid function (including the patient's rT3 level) is mandatory for the follow-up of every trisomy 21 patient.     

Improved performance of intensive semicontinuous cultures of Scenedesmus by biomass recirculation

The microscopic green alga, Scenedesmus obliquus, was used in a semicontinuous culture system for the tertiary treatment of urban wastewater, with the simultaneous production of usable biomass. Partial biomass recycling was used to increase the productivity of the system by overcoming the limits imposed by the low maximal growth rate of the alga. The biomass to be recycled was collected by simple gravity settling of the removed culture.The culture system was operated at different dilution rates and its productivity measured at each rate. An evaluation of the crude nutrient composition of the algae produced at each dilution rate was also carried out.The system was found to operate stably at dilution rates of up to 0.8 day(-1) which represents a 20% net increase over the maximum dilution rate allowed under the same conditions in a system without recirculation. The composition of the biomass produced varied little over a range of dilution rates, which may be of relevance to its projected end-use.The study indicated that such a system can exploit available light to the full and should be of particular value for the treatment of low-strength wastes such as we employed.     

An M-phase-specific protein kinase of Xenopus oocytes: partial purification and possible mechanism of its periodic activation

The activity of a Ca2+- and cyclic nucleotide-independent protein kinase(s) which catalyzes hyperphosphorylation of a set of endogenous proteins, including a 95-kDa soluble phosphoprotein, is found to fluctuate in both the meiotic and mitotic cell cycles of Xenopus oocytes and activated eggs. The activity is high in M-phase and hardly detectable in interphase. The activity copurifies with a major histone kinase(s) throughout four purification steps: ammonium sulfate precipitation, DEAE-cellulose chromatography, high-performance liquid chromatography on TSK G3000, and CM-Sepharose chromatography. This suggests that a single enzyme shares activity against endogenous proteins and added histones. Changes in the activity of the M-phase-specific protein kinase(s) as assayed in vitro correlate with changes in the extent of protein phosphorylation in oocytes pulse-labeled with 32P-phosphate by microinjection during meiotic maturation and the early embryonic cell cycle. This suggests that the kinase(s) has a broad specificity and plays a key role in the increased protein phosphorylation which occurs at the transition to M-phase. Microinjection of the maturation-promoting factor (MPF) into immature oocytes triggers, after a 10-min lag period, the activation of the M-phase specific kinase(s), even in the absence of protein synthesis. In contrast MPF microinjection does not induce kinase activation in cycloheximide-treated oocytes arrested after completion of the first meiotic cell cycle or in activated eggs arrested in S-phase by incubation in cycloheximide. This suggests that immature oocytes contain an inactive kinase precursor (prokinase) which is synthesized at each of the following cell cycles. In the absence of MPF addition, the prokinase to kinase transition occurs "spontaneously" after a 2-hr lag period in high-speed supernatants prepared from prophase-arrested oocytes if low-molecular-weight metabolites are eliminated by gel filtration. Addition of ATP, but not of AMP-PNP (adenylyl-imidodiphosphate), prevents spontaneous kinase activation in gel-filtered extracts. We propose that MPF activates the M-phase-specific protein kinase in the intact cell by inactivating a factor which requires phosphorylation conditions to inhibit the prokinase to kinase transition.     

Transplant experiments. Growth-limiting factors for diatoms and bluegreen algae in Norwegian lakes

Population densities and total phosphorus concentrations in samples from different lakes of south-eastern Norway were determined. In addition some transplant experiments with dilute phytoplankton populations were carried out. A laboratory batch culture method was used.The diatoms studied may be divided into three ecological groups based on their cell densities and total phosphorus concentrations in the samples. This classification was supported by the experimental results. Cyclotella spp., Asterionella formosa and Tabellaria fenestrata did not grow or had low growth rates above pH 9. Synedra cf. acus and Fragilaria crontonensis had often high growth rates within the pH 9–10 range, but were not able to grow at pH values above 10. High pH-values had no effect on the growth rate of Oscillatoria. Oscillatoria, Synedra and Stephanodiscus were severely growth-limited in filtered water from oligotrophic lakes. Maximum growth rates of all the populations studied were often obtained after addition of phosphate and chelated iron (FeEDTA) in combination to filtered water samples from oligotrophic/mesotrophic lakes.     

Differences in ovarian activity between booroola X merino ewes which were homozygous, heterozygous and non-carriers of a major gene influencing their ovulation rate

Differences in the function and composition of individual ovarian follicles were noted in Booroola Merino ewes which had previously been segregated on at least one ovulation rate record of greater than 5 (FF ewes, N = 15), 3-4 (F+ ewes, N = 18) or less than 3 (++ ewes, N = 18). Follicles in FF and F+ ewes produced oestradiol and reached maturity at a smaller diameter than in ++ ewes. In FF (N = 3), F+ (N = 3) and ++ (N = 3) ewes, the respective mean +/- s.e.m. diameters for the presumptive preovulatory follicles were 3.4 +/- 0.3, 4.1 +/- 0.2 and 6.8 +/- 0.3 mm and in each of these follicles the respective mean +/- s.e.m. numbers of granulosa cells (X 10(6)) were 1.8 +/- 0.3, 2.2 +/- 0.3 and 6.6 +/- 0.3. During a cloprostenol-induced follicular phase, the oestradiol secretion rates from FF ewes with 4.8 +/- 0.4 'oestrogenic' follicles, F+ ewes with 3.2 +/- 0.2 'oestrogenic' follicles and ++ ewes with 1.5 +/- 0.02 'oestrogenic' follicles were not significantly different from one another. Moreover, the mean total numbers of granulosa cells from the 'oestrogenic' follicles from each genotype were identical, namely 5.4 X 10(6) cells. Irrespective of genotype the mean weight of each corpus luteum was inversely correlated to the ovulation rate (R = 0.91, P less than 0.001). Collectively, these findings support the notion that the maturation of greater than or equal to 5 follicles in FF ewes and 3-4 follicles in F+ ewes may each be necessary to provide a follicular-cell mass capable of producing the same quantity of oestradiol as that from 1-2 preovulatory follicles in ++ ewes.