Transformation of Clostridium acetobutylicum Protoplasts with Bacteriophage DNA

Techniques for the transformation of Clostridium acetobutylicum protoplasts with bacteriophage DNA are described. Transformation required regeneration of protoplasts and a 2-h eclipse period.     

Hypoxanthine-guanine phosphoribosyltransferase in human erythroid cells: Properties of the isozymes

We have previously given evidence that the hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8) isozymes in human erythroid cells result from posttranslational modifications of a single gene product [Johnson, G. G., et al. (1982). Biochemistry 21:960]. In the present work we compare the properties of the unmodified and two major modified isozymes, which collectively account for 90% of the HGPRT enzyme activity in cell lysates. The modified isozymes differ from the parent molecule in the pH dependence of activity and in the relative utilization of the two purine base substrates, hypoxanthine and guanine. In contrast to the changes in the catalytic properties of the enzyme, the modifications have no detectable effects on the heat stability or on the equilibrium between enzyme dimers and enzyme tetramers.This work was supported by United States Public Health Service Grant 5 RO1 CA 16754-03 and by the San Diego State University Foundation.     

Synthesis of 2,4-diacetamido-2,4,6-trideoxy-L-altrose, -L-idose, and -L-talose from benzyl 6-deoxy-3,4-O-isopropylidene-beta-L-galactopyranoside

Acetylation of benzyl 6-deoxy-3,4O-isopropylidene-β-L-galactopyranoside gave benzyl 2-O-acetyl-6-deoxy-3,4-O-isopropylidene-β-L-galactopyranoside (1). Removal of the isopropylidene group afforded benzyl 2-O-acetyl-6-deoxy-β-L-galactopyranoside (2), which was converted into benzyl 2-O-acetyl-6-deoxy-3,4-di-O-(methyl-sulfonyl)-β-L-galactopyranoside (3). Benzyl 2,3-anhydro-6-deoxy-4-O-(methyl-sulfonyl)-β-L-gulopyranoside (4) was obtained from 3 by treatment with alkali. Reaction of 4 with sodium azide in N,N-dimethylformamide gave a mixture of two isomeric benzyl 2,4-diazido-2,4,6-trideoxy hexoses, the syrupy diazido derivative 5 and the crystalline benzyl 2,4-diazido-2,4,6-trideoxy-β-L-idopyranoside (6). Acetylation of 6 afforded a compound whose n.m.r. spectrum was completely first order and in agreement with the structure of benzyl 3-O-acetyl-2,4-diazido-2,4,6-trideoxy-β-L-idopyranoside (7). Lithium aluminium hydride reduction of 5, followed by acetylation, afforded a crystalline product (8), shown by n.m.r. spectroscopy to be benzyl 2,4-diacetamido-3-O-acetyl-2,4,6-trideoxy-β-L-altropyranoside. Similar treatment of the diazido derivative 6 afforded benzyl 2,4-diacetamido-3-O-acetyl-2,4,6-trideoxy-β-L-idopyranoside (9). Compounds 8 and 9 could also be obtained from 4 by treatment of the crude diazido mixture with lithium aluminium hydride, with subsequent N-acetylation. The syrupy benzyl 2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranoside (10) and the crystalline benzyl 2,4-diacetamido-2,4,6-trideoxy-β-L-idopyranoside (11) thus obtained were then O-acetylated to give 8 and 9 respectively. Benzyl 2,4-diacetamido-2,4,6-trideoxy-β-L-talopyranoside (15) was obtained from 11 by treatment with methanesulfonyl chloride and subsequent solvolysis. Compound 15 was O-acetylated to yield benzyl 2,4-diacetamido-3-O-acetyl-2,4,6-trideoxy-β-L-talopyranoside (16). the n.m.r. spectrum of which was in full agreement with the assigned structure. The mass spectra of compounds 8–11, 15, and 16 were also in agreement with their proposed structures. Removal of the benzyl groups from 10, 11 and 15 afforded the corresponding 2,4-diacetamido-2,4,6-trideoxyhexoses 12, 13, and 17, having the L-altro, L-ido, and L-talo configurations, respectively.     

Characterization of human mammary cell types in primary culture: Immunofluorescent and immunocytochemical indicators of cellular heterogeneity

Summary Parenchymal organoidal structures that were obtained from collagenase digestion of reduction mammoplasty specimens of apparently normal human breasts have been grown in short-term primary cultures, either on plastic or on floating gels of polymerized rat-tail collagen. Three morphologically distinct major cell types are readily observed in both systems: cuboidal cells, which occupy apical positions on collagen gels; larger, epithelioid, or basal cells on gels; and elongated cells which penetrate into the gel. In addition, a fourth cell type, that of a large, flat cell, is observed less readily by phase contrast microscopy on the surface of cultures grown on plastic. Immunofluorescent and immunocytochemical staining of cultures on plastic or histologic sections of cultures on gels have been undertaken with antisera and other histochemical reagents that stain the different parenchymal cell types in vivo. Thus antisera to epithelial membrane antigen(s), monoclonal antibodies (MABs) to the defatted mammary milk fat globule membrane, peanut lectin, and keratin MAB LE61, which preferentially stain the epithelial cells of ducts in vivo, also stain the cuboidal/apical cells in vitro. The large, flat cells are stained intensely by the first three reagents but not by the last one. Antisera to collagen IV, laminin, fibronectin, actin, keratin MAB LP34, MABs to the common acute lymphoblastic leukemia antigen, and MAB LICR-LON-23.10, which showed enhanced staining for the ductal myoepithelial cells in vivo, also stain the epithelioid/elongated cells in vitro. However, the effect of the last four reagents is reduced considerably in most elongated cells, and MAB LP34 stains the large, flat cells intensely. Heterogeneous cells of intermediate morphologies and staining patterns between the cuboidal/flat cells and large epithelioid cells have also been identified. The results suggest that the cuboidal cells and large, flat cells are related to mammary epithelial cells, whereas the large epithelioid/elongated cells have some characteristics of myoepithelial cells, and that intermediate forms may exist in culture between the two parenchymal cell types. This work was supported in part by the Ludwig Institute for Cancer Research and the Cancer and Polio Research Fund. Dr. M. J. Warburton is supported by the Cancer Research Campaign.     

Sister chromatid exchange analysis of the 15q11 region in Prader-Willi syndrome patients

Summary Prader-Willi syndrome (PWS) is a sporadic disorder in which about half of cases have a 15q12 deletion. Although a small number of cases have other rearrangements involving 15q12, the rest of the cases appear to have normal chromosomes. Clinical similarities among all these patients regardless of the karyotype strongly suggests a common etiology. To investigate the nature of this common etiology, we analyzed sister chromatid exchange (SCE) at the 15q11-13 region in 10 PWS patients with the chromosome deletion, 12 PWS patients with normal chromosomes, and 11 normal control individuals. While SCE at the q11-13 region was absent on the 15q12 deleted chromosome, the percentage of SCE on chromosome 15 at q11 was statistically higher for PWS with normal chromosomes (10.1%) compared to that for normal controls (1.9%) and the normal homologue (2.2%) in deleted patients (²=7.7982, df=2, P<0.025). The data suggest relative instability of DNA at the 15q11 region in PWS patients.     

Complotypes in individuals of African origin: frequencies and possible extended MHC haplotypes

We analyzed the frequency distribution of 106 complotypes [four allele sets of the major histocompatibility complex (MHC) genes for the complement proteins factor B, C2, C4A, and C4B] from 32 Black families residing in Boston and Washington, DC. Twenty-five different complotypes were identified, among which there were four complotypes that had not been previously observed in our large database of complotypes compiled from family studies of Boston Caucasians and that are, presumably, unique to individuals of African origin. These four African-derived complotypes areFC(1,90)0, FC63, S1C2,17, andSC(3,2,90)0. The frequencies of two of these four unique Black complotypes,FC(1,90)0 andFC63, were increased significantly when compared to Caucasians (p_corr <0.00042, p_corr=0.00294, respectively). The complotypeFC(1,90)0 was in positive linkage disequilibrium withHLA-DR3 haplotypes containing theB locus antigens Bw42, Bw52, Bw53, and Bw58, whileFC63 was associated withHLA-Bw70,-DR5. These findings demonstrate the extensive polymorphism of complotypes in Blacks, and also suggest that it may be possible to define unique extended haplotypes of African origin.     

The initiation of somatic embryos and adventitious roots from developing zygotic embryo explants of Cercis canadensis L. cultured in vitro

Zygotic embryo explants of Cercis canadensis L. cultured in vitro responded to 1 and 5 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 50 M -naphthaleneacetic acid (NAA) by initiating somatic embryos and adventitious roots. Somatic embryos and adventitous roots were formed from developing zygotic embryos, while fully developed embryos collected from mature seed initiated only adventitious roots. Following 2,4-D application, the number of somatic embryos decreased while adventitious roots increased with increasing developmental age of the explant. The greatest number of somatic embryos were initiated with a 5 to 20 day exposure to 5 M 2,4-D from zygotic embryos collected between 75 and 82 days post-anthesis in 1987. Somatic embryos formed directly from epidermal and subepidermal cells, while adventitious roots developed from interior cortex cells. Normal somatic embryos were recovered after a 20 day exposure to 5 M 2,4-D and acclimated to greenhouse conditions.