Complete Genomic Sequence of the Human Retinoblastoma Susceptibility Gene

A 180,388-bp contig encompassing the human retinoblastoma gene was sequenced in its entirety. Partial analysis of the sequence revealed (1) a high (A + T)/(G + C) ratio and a high density of Line-1 (L1) repeat sequences, suggesting that the locus maps to G-bands 13q14.12 or 13q14.2; (2) Alu repeats that are asymmetrically oriented over a region extending 87 kb; (3) an overabundance of non-Alu-associated poly(A) tracts 10 bp or larger oriented in the antisense rather than the sense direction (36 vs 6); (4) an Alu sequence nested within an L1 repeat, indicating that the expansion of L1 repeats predates at least some of the Alu expansions; (5) at least three newly discovered microsatellite polymorphisms, one of which was subsequently found to be identical to a polymorphism in a microsatellite-based linkage map of the human genome published by another group; and (6) the basis of previously discovered intragenic RFLPs. This sequence should enhance studies of this locus and of the organization of the human genome.     

Nuclear Magnetic Resonance Spectroscopy for the Study of B-Cell Epitopes

High-resolution nuclear magnetic resonance (NMR) spectroscopy is a structural technique that is finding increasing use in the study of antibody–antigen interactions. In this review we describe how the dynamic structural parameters obtained from NMR spectroscopy can further our understanding of B-cell epitopes and their function. Specific applications of NMR spectroscopy to examine the residues on peptides and proteins that contact the antibody combining site are also described. These include “footprinting” techniques using H–D exchange–COSY NMR spectroscopy, which are particularly useful for epitope mapping of protein antigens. For smaller systems, such as Fab–or Fv–peptide complexes, nuclear magnetization transfer difference NMR spectroscopy, transferred nuclear Overhauser effect spectroscopy, double-quantum-filtered NOE spectroscopy, and isotope editing techniques have been applied. The interpretation and limitations of the data obtained from these procedures and anticipated improvements in these applications in the future are discussed.     

The distribution of Gossypium hirsutum chromatin in G. barbadense germ plasm: molecular analysis of introgressive plant breeding

Cotton is unusual among major crop plants in that two cross-fertile species are widely cultivated for a common economic product, fiber. Both historical evidence and classical genetic studies suggest that many improved forms of Gossypium barbadense (Sea Island, Egyptian, and Pima cottons) may include chromatin derived from G. hirsutum. Using 106 restriction fragment length polymorphism (RFLP) loci well distributed across the cotton genome, we revealed the amount and genomic distribution of G. hirsutum chromatin in 54 G. barbadense collections from around the world. The average G. barbadense collection was comprised of 8.9% alleles apparently derived from G. hirsutum. Pima cultivars (7.3 %) had fewer G. hirsutum alleles than Sea Island (9.0%) or Egyptian (9.6%) cultivars. G. hirsutum alleles were not randomly distributed, as 57.5% of the total introgression observed was accounted for by five specific chromosomal regions that span less than 10% of the genome. The average length of an introgressed chromosome segment was 12.9 cM. Overlap of introgressed chromatin in different breeding programs hints that retention of these G. hirsutum chromosomal segments may impart a selective advantage to G. barbadense genotypes. Although cluster analysis generally grouped germ plasm from common classes and/or breeding programs together, no 2 genotypes were identical — thus differences in the length and repertoire of introgressed chromosome segments also permit DNA fingerprinting of G. barbadense cultivars.     

Modulation of Intracellular Cyclic AMP Levels by Different Human Dopamine D4 Receptor Variants

Abstract: To investigate whether polymorphic forms of the human dopamine D4 receptor have different functional characteristics, we have stably expressed cDNAs of the D4.2, D4.4, and D4.7 isoforms in several cell lines. Chinese hamster ovary CHO-K1 cell lines expressing D4 receptor variants displayed pharmacological profiles that were in close agreement with previous data from transiently expressed D4 receptors in COS-7 cells. Dopamine stimulation of the D4 receptors resulted in a concentration-dependent inhibition of the forskolin-stimulated cyclic AMP (cAMP) levels. The potency of dopamine to inhibit cAMP formation was about twofold reduced for D4.7 (EC₅₀ of ∼37 n M ) compared with the D4.2 and D4.4 variants (EC₅₀ of ∼16 n M ). Antagonists block the dopamine-mediated inhibition of cAMP formation with a rank order of potency of emonapride > haloperidol = clozapine ≫ raclopride. There was no obvious correlation between the efficacy of inhibition of forskolin-stimulated cAMP levels and the D4 subtypes. Dopamine could completely reverse prostaglandin E₂-stimulated cAMP levels for all three D4 receptor variants. Deletion of the repeat sequence does not affect functional activity of the receptor. The data presented indicate that the polymorphic repeat sequence causes only small changes in the ability of the D4 receptor to block cAMP production in CHO cells.     

Correlation between serum ionised calcium and serum albumin concentrations in two hospital populations.

One quarter of 172 patients from two hospitals with no obvious disturbances of calcium homeostasis and with total serum calcium concentrations that were normal after adjustment for albumin concentration had low serum ionised calcium concentrations. The low values were not due to changes in pH but were associated with hypoalbuminaemia. Significant positive regressions of ionised calcium on albumin concentration were observed in patients from both hospitals and also in 48 healthy laboratory staff. Because the regressions did not differ between patients and healthy subjects the low ionised calcium values associated with hypoalbuminaemia are unlikely to have been of pathological importance. These findings indicate that interpreting serum ionised calcium concentrations in patients with a reduced serum albumin concentration on the basis of a reference range determined in subjects with a normal serum albumin concentration may be clinically misleading.     

Inhibition of thymidine uptake in asynchronous HeLa cells by nitrobenzylthioinosine

Nitrobenzylthioinosine (NBMPR), an inhibitor of nucleoside transport by human erythrocytes, was found to be a potent inhibitor of thymidine uptake by asynchronous monolayer cultures of HeLa cells. Rates of thymidine uptake by the cultures at 20 °C were constant between 10 and 40 sec after thymidine addition and were assayed during this interval; TTP was the principal metabolite of thymidine and the thymidine phosphates accumulated at constant rates which extrapolated through time zero. The lack of an effect of NBMPR on thymidine kinase activity, or on the relative proportions of thymidine metabolites in cell extracts, indicated that NBMPR inhibited thymidine transport. When mediated entry (transport) was eliminated by 2 μM NBMPR, a significant diffusional component of thymidine entry was apparent. The mediated component of thymidine uptake exhibited Michaelis-Menten kinetics and apparent K_m and V_max values of 0.5 μM and 10–21 pmoles/min/10⁶ cells were obtained. When NBMPR-treated cells were transferred to NBMPR-free medium, inhibition of thymidine uptake persisted, suggesting that NBMPR was firmly bound to the transport inhibitory sites.     

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.