The yeast homologue of U3 snRNA.

snR17, one of the most abundant capped small nuclear RNAs of Saccharomyces cerevisiae, is equivalent to U3 snRNA of other eukaryotes. It is 328 nucleotides in length, 1.5 times as long as other U3 RNAs, but shares significant homology both in nucleotide sequence and in predicted secondary structure. Human scleroderma antiserum specific to nucleolar U3 RNP can enrich snR17 from sonicated yeast nuclear extracts. Unlike other yeast snRNAs which are encoded by single copy genes, snR17 is encoded by two genetically unlinked genes: SNR17A and SNR17B. The RNA snR17A is more abundant than snR17B. Deleting one or other of the genes has no obvious phenotypic effect, except that the steady-state level of snR17B is increased in snr17a- strains. Haploid strains with both genes deleted are inviable, therefore yeast U3 is essential.     

Polymorphisms distinguishing different mouse species and t haplotypes.

Three anonymous chromosome 17 DNA markers, D17Tu36, D17Tu43, and D17Le66B, differentiate between house mouse species and/or between t chromosomes. The D17Tu36 probe, which maps near the Fu locus and to the In(17)4 on t chromosomes, identifies at least 15 haplotypes, each haplotype characterized by a particular combination of DNA fragments obtained after digestion with the Taq I restriction endonuclease. Ten of these haplotypes occur in Mus domesticus, while the remaining five occur in M. musculus. In each of these two species, one haplotype is borne by t chromosomes while the other haplotypes are present on non-t chromosomes. The D17Tu43 probe, which maps near the D17Leh122 locus and to the In(17)3 on t chromosomes, also identifies at least 15 haplotypes in Taq I DNA digests, of which nine occur in M. domesticus and six in M. musculus. One of the nine M. domesticus haplotypes is borne by t chromosomes, the other haplotypes are borne by non-t chromosomes; two of the six M. musculus haplotypes are borne by t chromosomes and the remaining four by non-t chromosomes. Some of the D17Tu43 haplotypes are widely distributed in a given species, while others appear to be population-specific. Exceptions to species-specificity are found only in a few mice captured near the M. domesticus-M. musculus hybrid zone or in t chromosomes that appear to be of hybrid origin. The D17Leh66B probe, which maps to the In(17)2, distinguishes three haplotypes of M. domesticus-derived t chromosomes and one haplotype of M. musculus-derived t chromosomes. Because of these characteristics, the three markers are well suited for the study of mouse population genetics in general and of t chromosome population genetics in particular. A preliminary survey of wild M. domesticus and M. musculus populations has not uncovered any evidence of widespread introgression of genes from one species to the other; possible minor introgressions were found only in the vicinity of the hybrid zone. Typing of inbred strains has revealed the contribution of only M. domesticus DNA to the chromosome 17 of the laboratory mouse.     

Ring chromosome 17. Case report and review of the literature.

A 46,XX,r(17) karyotype was observed in a 9-year-old infant with short stature, moderate mental retardation but without other physical abnormality. Eight cases with an r(17) have since been reported: 4 can be compared with our patient, one was detected by amniocentesis, and 3 have Miller-Dieker syndrome. Submicroscopic deletions in the subband p13.3 are probably the cause of Miller-Dieker syndrome. They are present in some cases of r(17) but, in others, this short arm region is entirely preserved.     

Properties of the multiple forms of the soluble 17alpha-hydroxy steroid dehydrogenase of rabbit liver.

The six forms of the 17alpha-hydroxy steroid dehydrogenase purified from rabbit liver cytosol have very similar physical properties. The molecular weights of all the enzymes were within 3% of the average mol.wt of 39 600. Only one of the six enzymes showed a significant difference in amino acid composition. All but one form of the 17alpha-hydroxy steroid dehydrogenases exhibited greater activities towards the androgen, epitestosterone, than towards oestrogen substrates. With oestrogen substrates one enzyme displayed a high specificity towards the substrate oestradiol-17alpha 3-glucuronide. This high activity was lost if the glucuronic acid moiety was removed or replaced by glucose or galacturonic acid. The other enzyme forms had approximately equal activity toward oestradiol-17alpha and its glucuronide or glucoside derivative. However, substitution of galacturonic acid at C-3 of oestradiol-17alpha substantially decreased the activity of all but one enzyme form.     

The chemistry of 9 alpha-hydroxysteroids. 3. Methods for selective formation and dehydrations of 17 beta-cyano-9 alpha,17 alpha-dihydroxyandrost-4-en-3-one

Two methods to produce the 17-cyanohydrin, using potassium cyanide in acetic acid/methanol or acetone cyanohydrin with aqueous sodium hydroxide, were followed with 9 alpha-hydroxyandrost-4-ene-3,17-dione, both providing 17 beta-cyano-9 alpha,17 alpha-dihydroxyandrost-4-en-3-one. The selectivity of one of these methods, that which uses acetone cyanohydrin, is not in agreement with a comparable reaction with the 9 alpha-unsubstituted androst-4-ene-13,17-dione to give the 17 alpha-cyano-17 beta-hydroxy product, as reported in the literature and confirmed by us. The 9 alpha-hydroxy and 17 alpha-hydroxy groups were used for the regioselective introduction of 9(11)- and 16(17)-double bonds by dehydrating 17 beta-cyano-9 alpha,17 alpha-dihydroxyandrost-4-en-3-one under different conditions.     

A comparison of induced mutation at homologous alleles of the tk locus in human cells. II. Molecular analysis of mutants.

Previously we described the dose-response relationship for X-ray-induced mutation of the two homologous alleles of the thymidine kinase (tk) gene in a human lymphoblastoid cell line (Amundson and Liber, 1991). The two alleles were differentially mutable by X-rays, with one allele 6-10 times more mutable than the other. This difference was shown to be due to the virtual absence of the class of slow growth mutants from one allele. In the present report, restriction fragment length polymorphism (RFLP) analyses of informative markers along chromosome 17 have been used to delineate a region of chromosome 17 in which heterozygosity is lost with relatively high frequency among slow growth TK- mutants from the more mutable allele. However, loss of heterozygosity of this region has never been observed in normal growth mutants obtained from the more mutable allele, or in TK- mutants from the other, less mutable, allele. This may indicate the presence of a heterozygous essential gene on chromosome 17 distal to TK1.     

Oestradiol-17 alpha dehydrogenase from chicken liver.

The NADP+-linked oestradiol-17 alpha dehydrogenase (EC 1.1.1.148) present in cell-free extracts of chicken liver was investigated with the aim of separating it from a closely related oestradiol-17 beta dehydrogenase (EC 1.1.1.62) found in the same subcellular fraction. However, its chromatographic behaviour on CM-cellulose and DEAE-cellulose was almost identical with that previously reported for the latter enzyme, including resolution into two peaks on the anion-exchanger. Both peaks contained oestradiol-17 alpha dehydrogenase and oestradiol-17 beta dehydrogenase activity. Further attempts to separate the putative enzymes by dye-ligand chromatography with the use of the dyes Procion Yellow, Reactive Red and Cibachron Blue linked to Sepharose were unsuccessful, and they behaved identically on affinity columns of adenosine 2',5'-bisphosphate-agarose and 17 beta-oestradiol 3-hemisuccinate bound to Sepharose. A previous report of partial separation on Sephadex G-200 was not confirmed. Slab gel electrophoresis of enzyme preparations after affinity chromatography on adenosine 2',5'-bisphosphate-agarose revealed multiple bands in systems containing sodium dodecyl sulphate, whereas analysis by rod gel electrophoresis gave two major and one minor bands that stained coincidently for oestradiol-17 alpha dehydrogenase, oestradiol-17 beta dehydrogenase, epitestosterone dehydrogenase and testosterone dehydrogenase activities. Isoelectric focusing gave four enzymically active peaks that each oxidized oestradiol-17 alpha and -17 beta. Apparent Km values for the two forms of oestradiol-17 alpha dehydrogenase obtained by DEAE-cellulose chromatography were 17 and 23 microM for oestradiol-17 alpha, and 8.7 and 11.0 microM for NADP+. Limited kinetic studies with oestradiol-17 alpha and -17 beta with the use of the mixed-substrate method showed that the total velocity was equal to the sum of the separate velocities. The active-site inhibitor-alkylating agent 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one did not cause time- or temperature-dependent inhibition, in contrast with the reported case of the oestradiol-17 beta dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities of the human placental oestradiol dehydrogenase. NADP+ appeared to afford some protection against inhibition. Investigation of substrate specificity with a limited range of steroids suggests that the enzyme(s) from chicken liver differs substantially from the oestradiol-17 beta dehydrogenase from human placenta, and although the evidence is not conclusive it suggests the existence of one enzyme.     

Metabolism of 2-3H- and 4-14C-17alpha-ethynylestradiol 3-methyl ether (mestranol) by women.

A mixture of 2-3H and 4-14C-mestranol was administered orally to five women and 2-3H-mestranol alone to one woman. Reactions involving position 2 were extensive as judged by liberation of 3H into body water (14-45% of the dose). 17alpha-Ethynylestradiol, 2-hydroxy-17alpha-ethynylestradiol, 2-methoxy-17alpha-ethynylestradiol, 2-hydroxy-17alpha-ethynylestradiol 3-methyl ether and 16geta-hydroxy-17alpha-ethynylestradiol were measured in the "glucuronide" and pH1 fractions and mestranol, D-homoestrone-17a and D-homoestradiol-17abeta were also measured in the "glucuronide" fraction frum the urine to two of the women by reverse isotope dilution. Radioactive 2-methoxyestradiol accounted for less than 0.011% of the 14C dose in the "glucuronide" fraction of one of the women, consistent with the extent of de-ethynylation previously reported (Steroids, 25, 343 (1975).     

Antigenic determinants of hen egg-white lysozyme in delayed hypersensitivity. II. Antigenicity and immunogenicity of the N- and C-terminal peptide.

Various small fragments of (see article) which is one of the immunodominant groups of hen egg-white lysozyme (HL), were tested for macrophage migration inhibition (MMI) of peritoneal exudate cells (PEC) from guinea pigs immunized with HL. P17 was split in the middle with cyanogen bromide. The terminal portion of (see article) showed positive MMI, whereas the non-terminal half of P17, P17i (sequence 13-27) only showed very weak MMI activity. A fragment derived from the middle portion of P17, P17m (sequence 11-22), was inactive. When P17 were reduced and alkylated, one of the resultant peptides, P17N (sequence 1-[CM-Cys-6]-27) still has MMI activity with PEC taken from guinea pigs immunized with HL, although no antibody reacting with it was detected, but P17C (sequence 123-[CM-Cys-127]-129) was inactive. The peptides P17 and P17N were both immunogenic in guinea pigs in respect to the delayed hypersensitivity response. Again P17t and P17N were immunodominant groups, but the reactivity of P17i in MMI assay of this group of animals was greater than that in guinea pigs immunized with HL. The reactivities of HL with PEC taken from guinea pigs immunized with P17 or P17N were generally weaker than those of the antigens used for immunization.     

Deuterium-labeled steroids for study in humans. II. Preliminary studies on estrogen production rates in pre- and post-menopausal women.

6,7-Dideuterio-3-hydroxy-1,3,5(10)-estratrien-17-one (dideuterio-estrone) and 4-deuterio-1,3,5(10)-estratriene-3,17 beta-diol (monodeuterio-17 beta-estradiol) were used for the estimation of estrogen production rates in pre- and post-menopausal women. The results are concordant with those obtained by radioisotope administration as reported in the literature. This preliminary study suggests that one or more steroids labeled with one or multiple deuterium and/or other stable isotopes may be employed for the measurement of production rates of steroid hormones which are derived from multiple precursors.     

Unfolding of E. coli ribosomes: evidence for pre-existing breaks in the large subunit.

An investigation has been made on structure modifications of E. coli ribosomes following EDTA treatment. When completely deprived of magnesium, the small subunit sediments at 16S while the large one, in the same conditions, shows two components at 17S and 21S. Unfolding causes in both subunits an increase in radius of gyration without substantial change in molecular weight, as shown by light scattering measurements. The occurence of the slower 17S component besides the 21S one has to be connected with a fraction of the large subunit population which presents nucleolytic breaks in its RNA chain. These breaks do not cause fragmentation of the unfolded subunit but lead to a more open configuration sedimenting at lower velocity.     

Molecular detection of microscopic and submicroscopic deletions associated with Miller-Dieker syndrome.

Miller-Dieker syndrome (MDS), a disorder manifesting the severe brain malformation lissencephaly ("smooth brain"), is caused, in the majority of cases, by a chromosomal microdeletion of the distal short arm of chromosome 17. Using human chromosome 17-specific DNA probes, we have begun a molecular dissection of the critical region for MDS. To localize cloned DNA sequences to the MDS critical region, a human-rodent somatic cell hybrid panel was constructed which includes hybrids containing the abnormal chromosome 17 from three MDS patients with deletions of various sizes. Three genes (myosin heavy chain 2, tumor antigen p53, and RNA polymerase II) previously mapped to 17p were excluded from the MDS deletion region and therefore are unlikely to play a role in its pathogenesis. In contrast, three highly polymorphic anonymous probes, YNZ22.1 (D17S5), YNH37.3 (D17S28), and 144-D6 (D17S34), were deleted in each of four patients with visible deletions, including one with a ring chromosome 17 that is deleted for a portion of the single telomeric prometaphase subband p13.3. In two MDS patients with normal chromosomes, a combination of somatic cell hybrid, RFLP, and densitometric studies demonstrated deletion for YNZ22.1 and YNH37.3 in the paternally derived 17's of both patients, one of whom is also deleted for 144-D6. The results indicate that MDS can be caused by submicroscopic deletion and raises the possibility that all MDS patients will prove to have deletions at a molecular level. The two probes lie within a critical region of less than 3,000 kb and constitute potential starting points in the isolation of genes implicated in the severe brain maldevelopment in MDS.     

Genetic and structural characterization of the human mitochondrial inner membrane translocase.

Translocation of nuclear-encoded mitochondrial preproteins is mediated by translocases in the outer and inner membranes. In the yeast Saccharomyces cerevisiae, translocation of preproteins into the matrix requires the membrane proteins Tim23, Tim17 and Tim44, which drive translocation in cooperation with mtHsp70 and its co-chaperone Mge1p. We have cloned and functionally analyzed the human homologues of Tim17, Tim23 and Tim44. In contrast to yeast, two TIM17 genes were found to be expressed in humans. TIM44, TIM23 and TIM17a genes were mapped to chromosomes 19p13.2-p13.3, 10q11. 21-q11.23 and 1q32. The TIM17b gene mapped to Xp11.23, near the fusion point where an autosomal region was proposed to have been added to the "ancient" part of the X chromosome about 80-130 MY ago. The primary sequences of the two proteins, hTim17a and hTim17b, are essentially identical, significant differences being restricted to their C termini. They are ubiquitously expressed in fetal and adult tissues, and both show expression levels comparable to that of hTim23. Biochemical characterization of the human Tim components revealed that hTim44 is localized in the matrix and, in contrast to yeast, only loosely associated with the inner membrane. hTim23 is organized into two distinct complexes in the inner membrane, one containing hTim17a and one containing hTim17b. Both TIM complexes display a native molecular mass of 110 kDa. We suggest that the structural organization of TIM23.17 preprotein translocases is conserved from low to high eukaryotes.     

Estrogen-binding proteins of calf uterus. Purification to homogeneity of receptor from cytosol by affinity chromatography.

The estrogen receptor has been purified to homogeneity from calf uterus cytosol by sequential affinity chromatography by using heparin--Sepharose 4B and 17-hemisuccinyl-17beta-estradiol-ovalbumin--Sepharose 4B. The procedure yields about 1.2 mg of receptor protein from 1 kg of calf uteri, with a recovery of 53%. The receptor protein, as a complex with 17beta-[3H]estradiol, is purified more than 99%. A single band is seen on polyacrylamide gel ectrophoresis under nondenaturing conditions. 17beta-[3H]Estradiol comigrates with the protein band. As computed from the specific activity of radioactive hormone, 64,450 g of purified receptor protein binds 1 mol of 17beta-estradiol. 17beta-[3H]Estradiol bound to the protein is displaced by estrogenic steriods but not by progesterone, testosterone, or cortisone. As judged by chromatography on calibrated Sephadex G-200 columns, the purified receptor is identical with native receptor in crude cytosol: both show a Stokes radius of 6.4 nm. On sucrose gradient in low-salt buffer, the purified receptor sediments at 8 S. On electrophoresis in NaDodSO4 gels, the purified receptor migrates as a single protein band with an apparent molecular weight of 70,000. The sedimentation coefficient measured on sucrose gradients in the presence of chaotropic salts [1 M NaBr or NaSCN (0.1 M)] is 4.2 S. We conclude that the estrogen receptor of cytosol consists of a single subunit weighing about 70,000 daltons and endowed with one estrogen binding site. Under native conditions in cytosol, several subunits associate to form a quaternary structure with a Stokes radius of 6.4 nm.     

Studies on epsilon-prototoxin of Clostridium perfringens type D. Physicochemical and chemical properties of epsilon-prototoxin.

epsilon-Prototoxin of clostridium perfringens type D consists of one polypeptide chain of 311 amino acids with the following composition: Asp52 Thr31 Ser25 Glu28 Pro12 Gly17 Ala14 Val28 Met5 Ile15 Leu18 Tyr17 Phe8 Lys31 His2 Arg5 Tyr2. It has no free cysteine but contains one blocked cysteine. The N-terminal as well as the C-terminal residue is lysine. The ultracentrifuge pattern gave one single boundary having S020,w = 2.15 S and Do20,w = 5.56-10(-7) cm2/s. Calculation of the molecular weight from D020,w and S020,w gave a value of 34 250. The molecular weight determined from sedimentation equilibrium using ultraviolet optics gave a value of 33 000 +/- 1000. On the other hand molecular weights calculated from a calibrated Sephadex G-100 column in borate buffer was 25 000 and that in sodium phosphate, ionic strength 0.2, was 27 500. This discrepancy between values obtained in the ultracentrifuge and by gel filtration is attributed to adsorption of epsilon-prototoxin by Sephadex.     

A molecular determinant of human immunodeficiency virus particle assembly located in matrix antigen p17.

We report single-point mutations that are located in the matrix protein domain of the gag gene of human immunodeficiency virus type 1 and that prevent Gag particle formation. We show that mutations of p17 that abolish human immunodeficiency virus particle assembly also prevent the dimerization of p17 protein, as measured directly by a protein-protein binding assay. In the three-dimensional structure of p17, mutations that abolish dimerization are located in a single alpha helix that forms part of a fingerlike projection from one side of the molecule. Peptides derived from this region of p17 also reduce the level of p17 dimer when they are added to p17-expressing cells and compete for p17 self-association when present in protein-protein binding assays. We propose that the dimerization of the Gag precursor that occurs by the interdigitation of alpha helices on adjacent matrix molecules is a key stage in virion assembly and that the prevention of such an interaction is the molecular basis of particle misassembly.     

Evidence of an enzymatic 17alpha leads to 21 hydroxyl transfer in the biosynthesis of cortexolone from 17alpha-hydroperoxyprogesterone by adrenal cortex microsomes.

The concept of a possible biogenetic intramolecular relationship between several pairs of hydroxylated positions of corticosteroids, in particular between positions 17alpha and 21, has been proposed by us some time ago. We now present evidence that to a certain extent such a relationship can indeed exist. 18O-labelled 17alpha-hydroperoxyprogesterone was incubated under ordinary oxygen atmosphere with the microsomal fractions of bovine adrenal cortex. Following extensive purifications by thin-layer chromatography, we have isolated a metabolite with mobility characteristics corresponding to those of authentic 17,21-dihydroxy-4-pregnene-3,20-dione (cortexolone). According to its mass spectrum, this metabolite has a molecular weight of 350, i.e. 4 atomic mass units higher than the molecular weight of non-labelled cortexolone. No conversion of 17alpha-hydroperoxyprogesterone to cortexolone was observed with a previously heat-inactivated preparation. The presence of 4 additional mass units in the cortexolone metabolite means that the latter has preserved two 18O-labels in the molecule, one at position 17alpha and the other one at position 21. This can only be explained by a rearrangement reaction of the hydroperoxide group in such a way that it is accompanied by a C-17alpha to C-21 hydroxyl transfer. In the inverse case, when non-labelled 17alpha-hydroperoxyprogesterone was incubated under 99% 18O2-atmosphere, non-labelled cortexolone of molecular weight 346 was also found.     

Testicular steroidogenesis in the baboon Papio anubis.

We recently reported that the baboon testis converts pregnenolone to testosterone through the delta-4 pathway. The present studies were to determine the metabolism of intermediates of the delta-4 and delta-5 pathway by the baboon testis. Fragments (50 mg) were incubated for 3 hr with 10 muCi of the following tritium-labelled substrates: pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, or testosterone. Pregnenolone was converted to testosterone primarily through the delta-4 pathway, with accumulation of progesterone, 17-hydroxyprogesterone and 20alpha-dihydroprogesterone as predominant intermediates. Similar results were obtained in progesterone incubations. 17-hydroxyprogesterone was not efficiently metabolized by the fragments, while 17-hydroxypregnenolone and dehydroepiandrosterone were efficiently converted into testosterone and androstenedione. Androstenedione was metabolized primarily to testosterone, while testosterone was not a suitable substrate. Some 5alpha-androstanediol was identified in each incubate. These results suggest that although testosterone is formed from pregnenolone through the delta-4 pathway, the delta-5 intermediates are more suitable substrates for testosterone synthesis in the baboon testis.     

17 long arm isochromosome. A common anomaly in malignat blood disorders.

A cytogenetic anomaly consisting in the replacement of a 17 by its long arm isochromosome was identified as the only alteration in the marrow cells of two patients with acute granulocytic leukemia. In one case, the specific nature of the abnormal chromosome was established by newly available techniques. Since its identification in 1965, this structural anomaly, which implies 17 long arm duplication and short arm deletion, has been observed, as a sole or as an associated finding, in the malignant cells of a spectrum of blood disorders, including acute granulocytic leukemias, the blast crisis of chronic myeloid leukemia and lymphoreticular proliferative disorders. Attention is called to this particular rearrangement for its clinical as well as fundamental implications, as its presence in blood forming cells unfailingly hearalds a fast, fatal course of evolution.     

Analysis of insecticidal Azadirachta indica A. Juss. fractions

As a result of chemical investigation on the ethanolic extract of fresh fruit coatings of Azadirachta indica A. Juss. (neem), twenty-seven compounds were identified in non-polar to less polar fractions which showed pesticidal activity determined by WHO method against Anopheles stephensi Liston. These identifications were basically made through GC-EIMS and were further supported by other spectroscopic techniques, including 13C NMR, UV and FTIR as well as retention indices. Thus sixteen n-alkanes, 1-16; three aromatics 2,6-bis-(1,1-dimethylethyl)-4-methyl phenol (17), 2-(phenylmethylene)-octanal (20), 1,2,4-trimethoxy-5-(1Z-propenyl)-benzene (27); three benzopyranoids 3,4-dihydro-4,4,5,8-tetramethylcoumarin (18), 3,4-dihydro-4,4,7,8-tetramethylcoumarin-6-ol (19), 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta[g]-2-benzopyran (22); one sesquiterpene methyl-3,7,11-trimethyl-2E,6E,10-dodecatrienoate (21); three esters of fatty acids methyl 14-methyl-pentadecanoate (23), ethyl hexadecanoate (24), ethyl 9Z-octadecenoate (25) and one monoterpene 3,7-dimethyl-1-octen-7-ol (26) were identified. Except 6, 8, 24 and 25 all these compounds were identified for the first time from the pericarp and fifteen of these, 1-3, 7, 9, 10, 17-23, 26, 27, are hitherto unreported previously from any part of the tree. Although this tree is a rich source of various natural products, it is the first report of identification of mono- and sesquiterpenes 26 and 21 and a potent antioxidant, 17.