Structural wrinkles and the genomic regulatory sites of eukaryotes

Summary Calculations of DNA angular parameters in 50 eukaryotic sequences reveal regions of large conformational deviations from ideal DNA around regulatory sites. Frequently, discrete peaks of structural variation are present upstream of genes. Known regulatory regions often include variants of consensus sequences. Thus, imprecise sequences and structures are recognized within large genomic stretches. The existence of structurally wrinkled regions in the vicinity of regulatory sequences is likely to facilitate greatly their recognition by proteins and enzymes.     

A histochemical study of the changes occurring in the protein-carbohydrate composition of the cumulus-oocyte complex and zona pellucida in immature mice in response to gonadotrophin stimulation

Summary A histochemical account is presented of the changes that occur in the protein—carbohydrate composition of the cumulus—oocyte complex in immature mice after gonadotrophin treatment. The distribution and nature of the glycosaminoglycans (GAG) present was established by enzymic digestion of tissue sections with testicular orStreptomyces hyaluronidase prior to staining with periodic—acid Schiff (PAS) or Alcian Blue. Treatment with exogenous gonadotrophins [pregnant mare's serum and human chorionic gonadotrophin (hCG)] induced gross changes in the appearance of the zona pellucida (and in the histochemical staining of the cumulus—oocyte complex). A reduction was observed in the amount of PAS-positive material present within the zona pellucida of oocytes located in large Graafian follicles examined 40 h after stimulation with pregnant mare's serum. After the injection of hCG, the zona pellucida was further depleted of PAS-positive naterial. Most of the PAS-positive material became confined to the plasma membrane of the oocyte, while the oocyte itself also became increasingly PAS-positive. All the GAGs disappeared from zona pellucida within 4 h of hCG stimulation. The changes observed in the protein—carbohydrate composition of the zona pellucida in preovulatory oocytes immediately prior to ovulation may be a prerequisite for successful sperm-egg interactions.     

Effect of light intensity on ammonia assimilation in maize leaves

The effect of light on the metabolism of ammonia was studied by subjecting detached maize leaves to 150 or 1350 mol m^–2 s^–1 PAR during incubation with the leaf base in 2 mM ¹⁵NH₄Cl. After up to 60 min, leaves were extracted. Ammonia, glutamine, glycine, serine, alanine, and aspartate were separated by isothermal distillation and ion exchange chromatography. ¹⁵N enrichments were analyzed by emission spectroscopy. The uptake of ammonium chloride did not influence CO₂ assimilation (8.3 and 17.4 mol m^–1 s^–1 at 150 and 1350 mol m^–2 s^–1 PAR, respectively). Leaves kept at high light intensity contained more serine and less alanine than leaves from low light treatments. Within 1 h of incubation the enrichment of ammonia extracted from leaves rose to approximately 20% ¹⁵N. In the high light regime the amino acids contained up to 15% ¹⁵N, whereas in low light ¹⁵N enrichments were small (up to 6%). The kinetics of ¹⁵N incorporation indicated that NH₃ was firstly assimilated into glutamine and then into glutamate. After 15 min ¹⁵N was also found in glycine, serine and alanine. At high light intensity nearly half of the ¹⁵N was incorporated in glycine. On the other hand, at low light intensity alanine was the predominant ¹⁵N sink. It is concluded that light influences ammonia assimilation at the glutamine synthetase reaction.     

Structural features are as important as sequence homologies inDrosophila heat shock gene upstream regions

Summary Pelham has shown that theDrosophila hsp 70 gene is not transcribed under heat shock conditions unless a given upstream region is present. Davidson et al. have recently compiled a list of sequences homologous to this region in otherDrosophila heat shock genes. They proposed that a set of unlinked genes, such as the heat shock genes, could be coordinately induced through an interaction in cis with a common regulatory molecule. That this interaction involves structural elements is suggested by the fact that these upstream regions share inverted repeats as well as areas of Z-DNA potential. Furthermore, using the Calladine-Dickerson rules for local helical parameters, we show that these regions share structural homology. This is significant because the presence of regions homologous to a derived consensus sequence does not necessarily imply structural similarity. Therefore, we suggest that these structural features are at least as important as the sequence homologies in enabling the heat shock response.     

Qa alloantigen expression on functional T lymphocytes from spleen and thymus

The cell-surface expression of the class I alloantigen Qa-2 was analyzed on resting and activated spleen and thymus cells using cytotoxic elimination and immunofluorescence and flow cytometry. Spleen cells activated by mitogens or alloantigen were homogeneously positive for cell surface Qa-2, but activated splenic T cells expressed only about one-third as much Qa-2 per cell as did nonstimulated T cells. These data correlated with the ability to perform cytotoxic elimination with Qa-2-specific monoclonal antibodies (mAbs) in that cytotoxic T lymphocyte (CTL) activity was completely abrogated by pretreatment of spleen cells prior to in vitro culture but was only partially eliminated by treatment of CTL effectors. Qa-2-positive cells constituted only a small subpopulation of fresh normal thymocytes, but were enriched (>40% positive) among cortisone-resistant thymocytes (CRT). These Qa-2-positive CRT contained mature thymocytes as defined by Ly phenotype Ly-2^–, Ly-1^hi. When normal thymocytes were treated with Qa-2-specific mAb and complement prior to in vitro sensitization for generation of allogeneic CTL, CTL activity was completely abrogated despite the fact that the fraction of cells eliminated were undetectable as assessed by cell recovery. CTL effectors from alloantigen-stimulated thymocytes were also susceptible to cytotoxic elimination with Qa-2-specific mAb. These data suggest that the Qa-2 molecule may serve not only as a marker on resting and activated peripheral T cells, but also as a unique marker for functionally mature T cells in the thymus.     

DNA-dependent in vitro synthesis of enzymes of the galactose operon of Escherichia coli

Summary Two active enzymes of the galactose operon of Escherichia coli, uridyl transferase and galactokinase have been synthesized with high yields in a DNA dependent system for protein synthesis. The unspecific blank values amount to less than two percent of the rate obtained under optimal conditions and permit the accurate determination of even a small fraction of the maximum synthesis rate. Therefore this system provides a sensitive assay for the biological activity of DNA that contains the intact galactose operon of Escherichia coli.The synthesis of these galactose enzymes is to a high extent dependent on the presence of cyclic adenosine-3:5-monophosphate.D-fucose, known as an inducer of the galactose operon in vivo, stimulates the synthesis of galactokinase, indicating that the repressor of the galactose operon in active under these conditions. This stimulation is not observed, if the bacterial extract is prepared from a strain defective for the galactose repressor or if the DNA carries an operator constitutive mutation in the galactose operon. Therefore the stimulation by D-fucose is true derepression.     

The reaction of alkylating agents with bacteriophage R17. Biological effects of phosphotriester formation

The extent of biological inactivation and of the degradation of the RNA after reaction of bacteriophage R17 with ethyl methanesulphonate, isopropyl methanesulphonate and N-ethyl-N-nitrosourea was studied. Formation of breaks in the RNA chain probably results from hydrolysis of phosphotriesters formed in the alkylation reactions. Near neutral pH the ethyl and isopropyl phosphotriesters are sufficiently stable for the kinetics of the hydrolysis reaction to be followed. Results indicate that the rate of hydrolysis increases rapidly as the pH is raised. The evidence shows that a phosphotriester group does not itself constitute a lethal lesion. The extent of phosphotriester formation by the different agents is discussed in terms of reaction mechanism.