Ultrastructural localization of silver-staining nuclear proteins at the onset of transcription in early bovine embryos.

Argyrophilic nuclear proteins, known to be functionally associated with ribosomal genes, were localized, in four-, eight-, and 16-cell bovine embryo blastomere nuclei using two different silver-staining procedures. Within the eight-cell cleavage stage by the process of embryonal nucleologenesis in the cow embryo the full-capacity ribosome-producing machinery is established. In the four-cell embryo, many patches and islands of argyrophilic (Ag+) material were detected in the nucleoplasm. The nucleolus-precursor bodies (NPBs), composed uniformly of a homogeneous compact mass, were completely devoid of any silver staining. On the other hand, clear-cut localization of argyrophilic proteins was detected during the eight-cell stage either inside the transforming NPBs or in the close vicinity, or in the already differentiated nucleolus. In compact, nonvacuolated NPB, an intensive Ag+ area was detected, in the form of a lenticle, at the periphery of the NPB. During and following vacuolation of the NPB, no Ag+ was detected inside these vacuoles. It was seen, however, in the dense fibrillar nucleolar component surrounding the smaller vacuoles formed at the time of the establishment of nucleolar structure. Ag+ areas were seen repeatedly in the vicinity of NPBs, probably a part of the nucleolus-associated chromatin or, alternatively, representing the extranucleolar bodies. In blastomere nuclei of 16-cell embryos, already possessing reticulated nucleoli known from intensively synthesizing somatic cells, the silver-staining pattern corresponded to the usual situation in differentiated cells: slight staining of fibrillar centers, heavy labelling in the dense fibrillar component, and absence of silver deposits in the granular component.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K _mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na⁺ (l-methionine inhibition constant K_iA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (K_mB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na⁺ (K_iB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b ^0,+ and y⁺, respectively. Received: 14 August 1995/Revised: 2 April 1996     

Replication banding in the chromosomes of the European eel (Anguilla anguilla)

The chromosomes of the European eel Anguilla anguilla have been analyzed with a replication banding technique from lymphocyte cultures treated with 5-BrdU. This technique allows us to identify with high resolution the individual chromosome pairs and to differentiate classes of chromatin by the order of replication. The replication banding obtained on the chromosomes of European eel can be related with the structural bands described in this species.     

Xp-duplications with and without sex reversal

Duplications in Xp including the DSS (dosage sensitive sex reversal) region cause male to female sex reversal. We investigated two patients from families with Xp duplications. The first case was one of two sisters with karyotype 46,XY, der(22), t(X;22)(p11.3;p11)mat and unambiguous female genitalia. The living sister was developmentally retarded, and showed multiple dysmorphic features and an acrocallosal syndrome. The second case was a boy with a maternally inherited direct duplication of Xp21.3-pter with the breakpoint close to the DSS locus. He had multiple abnormalities and micropenis, but otherwise unambiguous male genitalia. We performed quantitative Southern blot analysis with probes from Xp22.13 to p21.2 to define the duplicated region. Clinical, cytogenetic, and molecular data from both patients were compared with those of previously reported related cases. A comparison of the extragenital symptoms revealed no differences between patients with or without sex reversal. In both cases, the symptoms were non-specific. Among 22 patients with a duplication in Xp, nine had unambiguous female genitalia and a well-documented duplication of the DSS region. Two patients with duplication of DSS showed ambiguous external genitalia. From these data, we conclude that induction of testicular tissue may start in these patients, but that the type of genitalia depends on the degree of subsequent degeneration by a gene in DSS.     

Mutants blocked in penicillin biosynthesis show a deletion of the entire penicillin gene cluster at a specific site within a conserved hexanucleotide sequence

The organization of the genes of the penicillin cluster has been studied in three different mutants of P. chrysogenum impaired in penicillin biosynthesis. The three blocked mutants (derived from the parental strain P. chrysogenum Bb-1) lacked the genes pcbAB, pcbC and penDE of the penicillin biosynthetic pathway and were unable to form isopenicillin N synthase and isopenicillin N acyltransferase. All strains were identified as P. chrysogenum derivatives by fingerprinting analysis with (GTG)_n as a probe. The borders of the deleted region were cloned and sequenced, showing the same junction point in the three mutants. The deleted DNA region was found to be identical to that described in P. chrysogenum npe10. The frequent deletion of the pen gene cluster at this point may indicate that this cluster is located in an unstable genetic region, flanked by hot spots of recombination, that is easily lost by mutagen-induced recombination.     

Neural control of the expression of a Ca2+-activated K+ channel involved in the induction of myotonic-like characteristics

Summary 1. Expression of the apamin-sensitive K⁺ channel (SK⁺) in rat skeletal muscle is neurally regulated. The regulatory effect of the nerve over the expression of some muscle ion channels has been attributed to the electrical activity triggered by the nerve and/or to a trophic effect of some molecules transported from the soma to the axonal endings. 2. SK⁺ channels apparently are involved in myotonic dystrophy (MD), therefore understanding the factors that regulate their expression may ultimately have important clinical relevance. 3. To establish if axoplasmic transport is involved in this process, we used two experimental approaches in adult rats: (a) Both sciatic nerves were severed, leaving a short or a long nerve stump attached to the anterior tibialis (AT). (b) Colchicine or vinblastine (VBL), two axonal transport blockers of different potencies, was applied on one leg to the sciatic nerve. To determine whether electrical activity affects the expression of SK⁺ channels, denervated AT were directly stimulated. The corresponding contralateral muscles were used as controls. 4. With these experimental conditions we measured (a) apamin binding to muscle membranes, (b) muscle contractile characteristics, and (c) electromyographic activity. 5. In the short- and long-nerve stump experiments, 5 days after denervation¹²⁵I-apamin binding to AT membranes was 2.0 times higher in the short-stump side. This difference disappeared at longer times. The delayed expression of SK⁺ channels in the muscle left with a longer nerve stump can be attributed to the extra axoplasm contained in the longer stump, which maintains a normally repressive signal for a longer period of time. Ten to 15 days after application of axonal transport blockers we found that the muscle half-relaxation time increased in the drug-treated side and apamin partially reverted the prolonged relaxation. Myotonic-like discharges specifically blockable by apamin were always present in the drug-treated leg.¹²⁵I-Apamin binding, which is undetectable in a microsomal preparation from hind leg control muscles, was increased in the drug-treated preparations. Apamin binding to denervated and stimulated AT muscles was lower than in the contralateral unstimulated muscles [3.3±1.0 vs 6.8±0.8 (n=4) fmol/mg protein]. 6. Our results demonstrate that electrical activity and axoplasmic transport are involved in the control of expression of SK⁺ in rat skeletal muscle. However, the increased expression of this channel induces myotonic-like characteristics that are reversed by apamin. This myotonic activity could be a model for MD.     

Evaluation of the use of phase-specific gene promoters for the expression of enological enzymes in an industrial wine yeast strain

Summary Genes as POT1, HSP104 and SSA3, which are late expressed in laboratory culture conditions are expressed only during the first few days in microvinifications in wine yeast cells. This effect is probably due to the different growth conditions and leads to useless levels of enzyme activity for a reporter gene. However the ACT1 promoter, which is constitutively expressed in laboratory conditions, produces sufficient amounts of enzyme activity in late fermentation phases.