Genetic Analysis of the Heterochromatin of Chromosome 3 in Drosophila Melanogaster. II. Vital Loci Identified through Ems Mutagenesis

Chromosome 3 of Drosophila melanogaster contains the last major blocks of heterochromatin in this species to be genetically analyzed. Deficiencies of heterochromatin generated through the detachment of compound-3 chromosomes revealed the presence of vital loci in the heterochromatin of chromosome 3, but an extensive complementation analysis with various combinations of lethal and nonlethal detachment products gave no evidence of tandemly repeated vital genes in this region. These findings indicate that the heterochromatin of chromosome 3 is genetically similar to that of chromosome 2. A more thorough genetic analysis of the heterochromatic regions has been carried out using the chemical mutagen ethyl methanesulfonate (EMS). Seventy-five EMS-induced lethals allelic to loci uncovered by detachment-product deficiencies were recovered and tested for complementation. In total, 12 complementation groups were identified, ten in the heterochromatin to the left of the centromere and two to the right. All but two complementation groups in the left heterochromatic block could be identified as separate loci through deficiency mapping. The interallelic complementation observed between some EMS-induced lethals, as well as the recovery of a temperature-sensitive allele for each of the two loci, provided further evidence that single-copy, transcribed vital genes reside in the heterochromatin of chromosome 3. Cytological analysis of three detachment-product deficiencies provided evidence that at least some of the genes uncovered in this study are located in the most distal segments of the heterochromatin in both arms. This study provides a detailed genetic analysis of chromosome 3 heterochromatin and offers further information on the genetic nature and heterogeneity of Drosophila heterochromatin.     

Dual-Label Radioisotope Method for Simultaneously Measuring Bacterial Production and Metabolism in Natural Waters

Bacterial production and amino acid metabolism in aquatic systems can be estimated by simultaneous incubation of water samples with both tritiated methyl-thymidine and ¹⁴C-labeled amino acids. This dual-label method not only saves time, labor, and materials, but also allows determination of these two parameters in the same microbial subcommunity. Both organic carbon incorporation and respiration can be estimated. The results obtained with the dual-label technique are not significantly different from single-radiolabel methods over a wide range of bacterial activity. The method is particularly suitable for large-scale field programs and has been used successfully with eutrophic estuarine samples as well as with oligotrophic oceanic water. In the mesohaline portion of Chesapeake Bay, thymidine incorporation ranged seasonally from 2 to 635 pmol liter⁻¹ h⁻¹ and amino acid turnover rates ranged from 0.01 to 28.4% h⁻¹. Comparison of thymidine incorporation with amino acid turnover measurements made at a deep, midbay station in 1985 suggested a close coupling between bacterial production and amino acid metabolism during most of the year. However, production-specific amino acid turnover rates increased dramatically in deep bay waters during the spring phytoplankton bloom, indicating transient decoupling of bacterial production from metabolism. Ecological features such as this are readily detectable with the dual-label method.     

Anion effects on the reaction of lecithin-cholesterol acyltransferase with discoidal complexes of phosphatidylcholines . apolipoprotein A-I . cholesterol

Discoidal complexes of phosphatidylcholine (PC) . apolipoprotein A-I . cholesterol were prepared with egg PC, palmitoyloleoylPC, dipalmitoylPC, or dimyristoylPC, and were used as substrates of purified lecithin-cholesterol acyltransferase to investigate the effects of neutral salts on the enzymatic reaction. Sodium fluoride, chloride and bromide concentrations up to 1 M, did not affect the properties of the substrate particles, but caused marked and distinct changes in the activity of the enzyme with the various PC particles. The effects of salts were largely due to the anions, which followed the order of the lyotropic series in their inactivating capacity: F- less than Cl- less than Br- less than NO3- less than I- less than SCN-. Sodium salts (F-, Cl-, and Br-) produced a very large increase in the pH optimum of the enzymatic reaction (7.4 to at least 8.5) essentially obliterating the ionization of a functional group with pK of 8.1. The kinetics of the enzymatic reaction revealed major differences among the PC particles, and different responses of their kinetic parameters with increasing salt concentrations. The conclusions reached in this work are the following: (1) The relative reactivity of PC substrates, in discoidal particles, with lecithin-cholesterol acyltransferase depends strongly on the concentration and type of salts in the medium. (2) Anions (in lyotropic series) rather than cations affect the enzymatic reaction. (3) There are functional groups with pK of 8.1 which are affected markedly in their ionization behavior by anion binding. (4) The active site of lecithin-cholesterol acyltransferase and its interaction with anions are affected by the exact nature of the PC-apolipoprotein interface.     

The restriction endonuclease Alu I induces chromosomal aberrations in human peripheral lymphocytes in vitro

The restriction endonuclease Alu I (recognition site AG/CT) produces chromosomal aberrations in isolated human peripheral lymphocytes in vitro. The aberrations are of the chromosome-type when the cells are treated in G1 and of the chromatid-type when the cells are treated in late S, early G2. Additional treatment with ammonium sulphate leads to higher aberration frequencies than treatment with Alu I alone.     

Isolation by ion-exchange high performance liquid chromatography of rabbit liver cytochrome P-450 with regioselectivity for omega-hydroxylation of prostaglandins

Previous studies suggested that rabbit liver microsomes contain cytochrome P-450 monooxygenase(s) with low affinity for (omega-1)-hydroxylation and high affinity for omega-hydroxylation of prostaglandins (Theoharides, A. D., and Kupfer, D. (1981) J. Biol. Chem. 256, 2168-2175). The current investigation describes the isolation from livers of untreated rabbits of a cytochrome P-450 catalyzing, with regioselectivity, the omega-hydroxylation of prostaglandins E1 and E2. The isolation of the enzyme involved enrichment of the omega-hydroxylase activity by polyethylene glycol 8000 fractionation, followed by ion-exchange high performance liquid chromatography. Based on Mr of 59,000-60,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the isolated enzyme is referred to as P-450 form 7. This P-450 exhibits a low spin spectrum (lambda max = 417 nm) and a difference spectrum of the CO-reduced complex versus reduced (lambda max = 451 nm). For catalytic activity, the P-450 form 7 was reconstituted with NADPH-P-450 reductase, cytochrome b5, and lipid. There was no activity in the absence of the reductase, and deletion of cytochrome b5 yielded a minimal amount of product (heme could not substitute for cytochrome b5), demonstrating an absolute requirement for these components.     

The value of provoked expectoration in obtaining sputum samples for cytologic investigation. A prospective, consecutive and controlled investigation of 134 patients

A prospective controlled investigation in 134 consecutive outpatients compared the cytologic adequacy of sputum samples obtained by spontaneous and provoked expectoration. Inhalation of nebulized 10% sodium chloride was used for provoked expectoration. A significantly higher number of adequate samples was produced after provocation, as judged by the presence of alveolar macrophages (X2 = 5.63; p less than 0.02). The improvement in sample adequacy was limited to the nonsmokers and ex-smokers in the study. This result, together with the relatively high cost of cytologic sputum examinations, indicates that provoked expectoration should at least be applied to the collection of sputum samples from nonsmokers and ex-smokers.     

Regioselectivity of hydroxylation of prostaglandins by liver microsomes supported by NADPH versus H2O2 in methylcholanthrene-treated and control rats: formation of novel prostaglandin metabolites

The effects of methylcholanthrene (MC) treatment of male rats on the regioselectivity of hydroxylation of prostaglandins E1 and E2 (PGE1 and PGE2) by liver microsomes, supplemented with NADPH or H2O2, was examined. In the presence of NADPH, control microsomes catalyzed the hydroxylation at omega-1 (C19) and at omega-(C20) sites with minimal formation of novel monohydroxy metabolites of PGE1 and PGE2, referred to as compounds X1 and X2, respectively. Similarly, H2O2 supported the 19-hydroxylation and the formation of compounds X1 and X2, but yielded only minimal amounts of 20-hydroxy products. With NADPH, MC-treated microsomal incubations demonstrated only minor quantitative change in the 19- and 20-hydroxylation as compared with controls, but showed a 7- to 11-fold increase in formation of compound X1 and a 10-fold increase in formation of X2. By contrast with H2O2, MC-treatment increased by about 3-fold the 19- and 20-hydroxylation of PGE1 and by 35- to 46-fold the formation of X1; similarly, there was an approximate 2-fold increase in 19- and 20-hydroxylation of PGE2 and a 10-fold increase in formation of X2. These findings suggest that several monooxygenases are involved in catalyzing the hydroxylation at the various sites of the PGE molecule. Inhibitors of monooxygenases (SKF 525A, alpha-naphthoflavone, and imidazole derivatives) provided further evidence that the hydroxylation at the three sites of PGEs is catalyzed by different P-450 monooxygenases. It is striking that the inhibitors had a much lesser effect on the 20-hydroxylation of PGE1 as compared with other sites of hydroxylation. Structural identification of compounds X1 and X2 was elucidated as follows. Resistance of the PGB derivative of X1 to periodate oxidation and mass fragmentation analysis of the t-butyldimethylsilyl ether methyl ester, placed the hydroxylation at C17 or C18. Finally, mass fragmentation of trimethylsilyl ether methyl ester PGB derivatives of X1 and X2 provided conclusive evidence that X1 and X2 are 18-hydroxy-PGE1 and 18-hydroxy-PGE2, respectively. The above findings indicate that the high regioselectivity of hydroxylation of PGE1 and PGE2, resulting in the formation of 18-hydroxy-PGE1 and 18-hydroxy-PGE2, respectively, is catalyzed by P-450 isozyme(s) which are induced by MC, possibly by P-450c.     

Further Characterization of Genetic Elements Associated with the Segregation Distorter Phenomenon in DROSOPHILA MELANOGASTER

Segregation Distorter, SD, associated with the second chromosome of Drosophila melanogaster, is known to cause sperm bearing the non-SD homologue to dysfunction in heterozygous males. In earlier studies, using different, independently derived, SD chromosomes, three major loci were identified as contributing to the distortion of segregation ratios in males. In this study the genetic components of the SD-5 chromosome have been the subjects of further investigation, and our findings offer the following information. Crossover analysis confirms the mapping of the Sd locus to a position distal to but closely linked with the genetic marker pr. Spontaneous and radiation-induced recombinational analyses and deficiency studies provide firm support to the notion that the Rsp (Responder) locus lies within the proximal heterochromatin of chromosome 2, between the genetic markers lt and rl and most likely in the heterochromatin of the right arm. The major focus of this study, however, has been on providing a better definition of the genetic properties of the Enhancer of SD [E(SD)]. Our findings place this locus within the region of the two most proximal essential genes in the heterochromatin of the left arm of chromosome 2. Moreover, our analysis reveals a probable association of the E(SD) locus with a meiotic drive independent of that caused by Sd.     

The human placenta contains two distinct binding and immunoreactive species of insulin-like growth factor-I receptors

Two species of insulin-like growth factor-I (IGF-I) receptors in human placenta have been delineated on the basis of their immunoreactivity with an autoantiserum (B-2) to the insulin receptor. When all the IGF-I binding sites in solubilized human placenta were assayed by polyethylene glycol precipitation, a curvilinear Scatchard plot was obtained which could be resolved into two single classes of binding sites: one immunoprecipitable by B-2 IgG and the other, nonimmunoprecipitable. The B-2 reactive sites bound IGF-I with lower affinity (Kd = 7.1 X 10(-10) M) than the B-2 nonreactive sites (Kd = 2.1 X 10(-10) M) and cross-reacted more readily with insulin, the IGF-I/insulin-binding potencies being congruent to 120 and congruent to 1100, respectively. Both receptor subtypes bound IGF-I with congruent to 30-fold higher affinity than multiplication-stimulating activity, and, after affinity cross-linking with 125I-IGF-I, migrated as specific reduced bands of Mr = 138,000 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit sizes of the B-2 reactive IGF-I receptor were similar to those of the insulin receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-labeled receptors immunoprecipitated by autoantiserum B-2 or autoantiserum B-10 (which recognizes only insulin receptors) revealed, in both cases, specific reduced bands of Mr = 130,000 and 90,000; the same bands were also seen after sequential precipitation with B-10 and B-2 antisera to enrich the proportion of IGF-I receptors recovered. The presence of two distinct binding and immunoreactive species of IGF-I receptors in human placenta raises the possibility that cell- or tissue-specific isotypes of the IGF-I receptor could mediate the different biological actions of IGF-I.     

A high-affinity folate binding protein in normal human leukocytes: Ligand binding characteristics,ionic charge and molecular size

High-affinity binding of [³H]folate to supernatant from homogenized human leukocytes containing large amounts of binding protein displayed apparent positive cooperativity. The DEAE-Sepharose^® CL-6B chromatographic profile of the supernatant at pH 6.3 contained a major peak of folate binding (M_r approx. 25 000) in the front effluent and a smaller more acidic peak (M_r approx. 25 000) that emerged after a rise in NaCl from 30 mmol/l to 1 mol/l. Triton X-100 solubilized ceil sediment from the leukocyte homogenate contained some high-affinity folate binding activity (M_r approx 25 000), typically 5–10% of the total binding activity.