Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

Phenotypic hiding: the carryover of mutations in RNA viruses as shown by detection of mar mutants in influenza virus.

When influenza virus monoclonal antibody-resistant (mar) mutants are selected by incubation in vitro with excess antibody, 90 to 99% of the mutants are not detectable. This observation may be explained by encapsidation of mar mutant RNAs within phenotypically wild-type envelopes. This phenotypic hiding can be revealed by selection of mar mutants in vivo after virus uncoating. Using experimental procedures appropriate to detect all viable mar mutants in a virus population, we determined precisely the mutation rates to the mar genotype by the fluctuation test for two nonoverlapping monoclonal antibodies.     

Fine needle aspiration of peripheral neuroepithelioma of soft tissues.

Peripheral neuroepithelioma of soft tissue is a malignant primitive neuroectodermal tumor that appears both in children and adults and usually has a poor outcome. Fine needle aspiration on two patients with tumors in the lower limbs showed small round cells with unipolar processes and a tendency to form Homer-Wright rosettes. The cells had a round to oval nucleus with fine chromatin, up to four small, conspicuous nucleoli and vacuolated, periodic acid-Schiff-positive cytoplasm. The diagnosis was supported by electron microscopic study of the aspirate, which showed features of neuroblastic differentiation (i.e., neurosecretory granules), and by histologic, immunohistochemical and cytogenetic study of the resected tumors.     

Purification and characterization of glutathione reductase from Rhodospirillum rubrum.

Glutathione reductase (NAD(P)H:GSSG oxidoreductase EC 1.6.4.2.) was purified 1160-fold to homogeneity from the nonsulfurous purple bacteria Rhodospirillum rubrum (wild type). Specific activity of the pure preparation was 102 U/mg. The enzyme displayed a typical flavoprotein absorption spectrum with maxima at 274,365, and 459 nm and an absorbance ratio A280/A459 of 7.6. The amino acid analysis revealed an unusually high content of glycine and arginine residues. Titration of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) showed a total of two free thiol groups per subunit, one of which is made accessible only under denaturing conditions. An isoelectric point of 5.2 was found for the native enzyme. Km values, determined at pH 7.5, were 6.1 and 90 microM for NADPH and GSSG, respectively. NADH was about 2% as active as NADPH as an electron donor. The enzyme's second choice in disulfide substrate was the mixed disulfide of coenzyme A and glutathione, for which the specific activity and Km values were 5.1 U/mg and 3.4 mM, respectively. A native molecular weight of 118,000 was found, while denaturing electrophoresis gave a value of 54,400 per subunit, thus suggesting that R. rubrum glutathione reductase exists as a dimeric protein. Other physicochemical constants of the enzyme, such as Stokes radius (4.2 nm) and sedimentation coefficient (5.71 S), were also consistent with a particle of 110,000.     

Interfacial pH modulation of membrane protein function in vivo. Effect of anionic phospholipids.

In yeast cells, the magnitude of the membrane surface potential (phi) is determined to a large extent by the relative amount of anionic phospholipids (Cerbón and Calderón (1990) Biochim. Biophys. Acta 1028, 261-267). When a significant surface potential exists, the pH at the membrane surface (interfacial pH) will be different to that in the bulk suspending medium. We now report that: (1) In cells with higher phi (phosphatidylinositol-rich cells (PI-rich) and phosphatidylserine-rich cells (PS-rich) a 10-times lower proton concentration in the bulk was enough to achieve the maximum transport activity of H(+)-linked transport systems when compared to normal cells. (2) When the phi was reduced by increasing the concentration of cations in the medium, more protons were required to achieve maximum transport, that is, the pH activity curves shifted downwards to a more acidic pH. (3) The magnitude of the downward pH shift was around 2.5-times higher for the more charged membranes. (4) Around 10-times more KCl than MgCl2 was necessary to give an equivalent pH shift, in agreement with their capacity to reduce the phi of artificial bilayers. The interfacial pH calculated from the values of phi indicates that it was 0.4 pH units lower in the anionic phospholipid rich cells as compared to normal cells. The results indicate that membrane surface potential may explain the complex relationship between pH, ionic strength and membrane protein function. Maximum transport activities were found for glutamate at interfacial pH of 4.2-4.8 and were inhibited at interfacial pH = 3.2-3.4, suggesting that surface groups of the carrier proteins with pK values in the region 3.8-4.2 (aspartyl and glutamyl) are involved in binding and/or release of charged substrates.     

Transfer of hygromycin resistance into Brassica napus using total DNA of a transgenic B. nigra line

The successful transfer of a marker gene (hpt gene) from Brassica nigra into B. napus via direct gene transfer was demonstrated. Total DNA was isolated from a hygromycin-resistant callus line, which contained three to five copies of the hpt gene. This line had been produced via direct gene transfer with the hygromycin resistance-conferring plasmid pGL2. The treatment of B. napus protoplasts with genomic DNA of B. nigra (Hyg^R) resulted in relative transformation frequencies of 0.1–0.4%. Similar transformation rates were obtained in direct gene transfer experiments using B. napus protoplasts and plasmid pGL2.     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Purification of thymocyte growth peptide (TGP) from sheep thymus. Relationship to FTS/thymulin

Thymocyte growth peptide (TGP) initiates DNA synthesis in immature thymocytes and has previously been characterized as an acidic peptide isolated from calf thymus. We now report the isolation of TGP from sheep thymus and show it to be a nonapeptide with a large N-terminal blocking moiety characterized by high UV absorbance. The amino acid composition is identical to FTS, consisting of 2 Gly, 2 Ser, 2 Glx, 1 Ala, 1 Lys, 1 Asx. In contrast to FTS, TGP is acidic with an apparent isoelectric point of 4.2 and a high UV absorbance at 270–280 nm. Reverse phase chromatography of TGP at an acidic pH results in a change of the molecule and the appearance of two new compounds TGP-A and TGP-B, both with less than 50% of the original TGP activity. Full activity could be restored by the addition of ZnCl₂ to TGP-A. Both TGP-A and B have some amino acid composition and high UV absorbance as native TGP. We propose that TGP consists of a non-peptide moiety bound to the N-terminal of the nonapeptide Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn and that the active molecule is stabilized by Zn²⁺.