Tagging of a maize gene involved in kernel development by an activated Uq transposable element

Summary A quiescent Uq transposable element has been activated in a maize plant treated with 5-aza-2-deoxycyti-dine. This activated Uq cosegregates with a heritable dominant miniature (Mn) kernel phenotype, indicating its physical association with a maize miniature locus (Mn:: Uq). The Mn:: Uq mutant is dominant in producing a miniature seed phenotype of variable size and in reducing seedling vigor in the early growth stage. Genetic experiments indicate that the Mn:: Uq mutant also affects the activity of the male gametophyte, whereby pollen germination is inhibited, thus lacking pollen tube growth resulting in the male nontransmissibility of this mutant. Proof for the Uq element in this mutant is derived by its ability to transactivate the standard a-ruq reporter allele to yield spotted aleurone tissue. However, the Mn:: Uq mutant does not transactivate a normally Uq-responsive c-ruq allele, suggesting a structural difference between the two ruq receptors at the A1 and C1 loci. It is anticipated that cloning of the Uq transposable element would facilitate the molecular cloning and characterization of the maize miniature gene.Journal Paper No. J-13425 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa 50011, USA, Project No. 2850     

Sequence analysis of alpha 1(VI) and alpha 2(VI) chains of human type VI collagen reveals internal triplication of globular domains similar to the A domains of von Willebrand factor and two alpha 2(VI) chain variants that differ in the carboxy terminus.

Amino acid sequences of human collagen alpha 1(VI) and alpha 2(VI) chains were completed by cDNA sequencing and Edman degradation demonstrating that the mature polypeptides contain 1009 and 998 amino acid residues respectively. In addition, they contain small signal peptide sequences. Both chains show 31% identity in the N-terminal (approximately 235 residues) and C-terminal (approximately 430 residues) globular domains which are connected by a triple helical segment (335-336 residues). Internal alignment of the globular sequences indicates a repetitive 200-residue structure (15-23% identity) occurring three times (N1, C1, C2) in each chain. These repeating subdomains are connected to each other and to the triple helix by short (15-30 residues) cysteine-rich segments. The globular domains possess several N-glycosylation sites but no cell-binding RGD sequences, which are exclusively found in the triple helical segment. Sequencing of alpha 2(VI) cDNA clones revealed two variant chains with a distinct C2 subdomain and 3' non-coding region. The repetitive segments C1, C2 and, to a lesser extent, N1 show significant identity (15-18%) to the collagen-binding A domains of von Willebrand factor (vWF) and they are also similar to some integrin receptors, complement components and a cartilage matrix protein. Since the globular domains of collagen VI come into close contact with triple helical segments during the formation of tissue microfibrils it suggests that the globular domains bind to collagenous structures in a manner similar to the binding of vWF to collagen I.     

Differences in growth of transplantable ascites hepatoma among various lines of Donryu rat

A total of 48 Donryu rats from 8 colonies of 5 lines were inoculated intravenously with 10(7) cells of the ascites hepatoma strain AH 66. All the conventional rats of the lines D-1 and D-2 died between 9 and 15 days after inoculation with a good growth of implanted tumor cells. On the other hand, SPF rats of the lines A-1, B-1, C and E survived for 60 days showing complete rejection of the implanted tumor cells. A 50% of conventionalized ex-SPF rats of the lines A-2 and B-2, which had been once established as SPF colonies, and thereafter had been "re-conventionalized", rejected the tumor cells. The present observations indicate that the microbial conditions of the animal, e.g. whether the animal is SPF or not, might play an important role in the growth of the implanted ascites hepatoma.     

Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells

A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11. DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail. Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase. Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase. The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed. Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase. These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase. Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated. Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.     

1H NMR studies of T4 gene 32 protein: effects of zinc removal and reconstitution

Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol bound in a tetrahedral ligand field. 113Cd NMR studies of Cd-substituted wild-type and mutant (Cys166----Ser166) g32Ps show Cys77, Cys87, and Cys90 to provide three sulfur donor atoms as ligands to the metal ion [Giedroc, D. P., Johnson, B. A., Armitage, I. M., & Coleman, J. E. (1989) Biochemistry 28, 2410]. Proton NMR signals from the His and Trp side chains of the protein have been followed as a function of pH and metal ion removal by biosynthesizing the protein with amino acids carrying protons at specific positions in a background of perdeuteriated aromatic amino acids. Only one of the two pairs of His resonances (from His64 and His81) titrates over the pH range 8.0-5.9. The nontitrating His side chain is most likely ligated to the metal ion. Upon Zn(II) removal, 1H NMR spectra of the fully protonated g32P-(A + B) exhibit substantial signal broadening in several regions of the spectrum, while the His 2,4-1H resonances are broadened beyond detection. The 1H NMR spectral characteristics of the original protein are restored by reconstitution with stoichiometric Zn(II). The broadening of the 1H NMR signals is not due to oligomerization of the protein, since small-angle X-ray scattering experiments show that the average radius of gyration of the apo-g32P-(A + B) is 25.0 A and that of the reconstituted Zn(II)-g32P-(A + B) is 31.2 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Australine, a pyrrolizidine alkaloid that inhibits amyloglucosidase and glycoprotein processing

Australine [(1R,2R,3R,7S,7aR)-3-(hydroxymethyl)-1,2,7-trihydroxypyrrolizid ine] is a polyhydroxylated pyrrolizidine alkaloid that was isolated from the seeds of the Australian tree Castanospermum australe and characterized by NMR and X-ray diffraction analysis [Molyneux et al. (1988) J. Nat. Prod. (in press)]. Since swainsonine and catanospermine are polyhydroxylated indolizidine alkaloids that inhibit specific glycosidases, we tested australine against a variety of exoglycosidases to determine whether it would inhibit any of these enzymes. This alkaloid proved to be a good inhibitor of the alpha-glucosidase amyloglucosidase (50% inhibition at 5.8 microM), but it did not inhibit beta-glucosidase, alpha- or beta-mannosidase, or alpha- or beta-galactosidase. The inhibition of amyloglucosidase was of a competitive nature. Australine also inhibited the glycoprotein processing enzyme glucosidase I, but had only slight activity toward glucosidase II. When incubated with cultured cells, this alkaloid inhibited glycoprotein processing at the glucosidase I step and caused the accumulation of glycoproteins with Glc3Man7-9(GlcNAc)2-oligosaccharides.     

Thyroxine effects on parameters of glucose turnover in BHE rats fed menhaden oil

The effect of hyperthyroidism on glucose turnover in BHE rats fed menhaden oil was studied. Thyroxine-treated rats had a greater glucose mass, a greater absolute glucose synthesis rate, less hepatic and muscle glycogen levels, and greater hepatic and peripheral fat cell lipogenic rates than nontreated rats. No differences in body weight gain were observed, nor were there differences in blood glucose levels, glucose space, or fractional reversible or irreversible glucose use. These observations suggest that thyroxine and menhaden oil were additive in their effects on glucose metabolism in BHE rats, which are genetically programmed to develop non-insulin-dependent diabetes mellitus.     

Purification and properties of arylmannosidases from mung bean seedlings and soybean cells

Two arylmannosidases (signified as A and B) were purified tohomogeneity from soluble and microsomal fractions of mung beanseedlings. Arylmannosidase A from the microsomes appeared thesame on native gels and on SDS gels as soluble arylmannosidaseA, the same was true for arylmannosidase B. Sedimentation velocitystudies indicated that both enzymes were homogeneous, and thatarylmannosidase A had a molecular mass of 237 kd while B hada molecular mass of 243 kd. Arylmannosidase A showed two majorprotein bands on SDS gels with molecular masses of 60 and 55kd, and minor bands of 79, 39 and 35 kd. All of these bandswere N-linked since they were susceptible to digestion by endo-glucosaminidaseH. In addition, at least the major bands could be detected byWestern blots with antibody raised against the xylose moietyof N-linked plant oligosaccharides, and they could also be labeledin soybean suspension cells with [2–3H]mannose. ArylmannosidaseB showed three major bands with molecular masses of 72, 55 and45 kd, and minor bands of 42 and 39 kd. With the possible exceptionof the 45 and 42 kd bands, all of these bands are glycoproteins.Arylmannosidases A and B showed somewhat different kineticsin terms of mannose release from high-mannose oligosaccharides,but they were equally susceptible to inhibition by swainsonineand mannostatin A. Polyclonal antibody raised against the arylmannosidaseB cross-reacted equally well with arylmannosidase A from mungbean seedlings and with arylmannosidase from soybean cells.However, monoclonal antibody against mung bean arylmannosidaseA was much less effective against arylmannosidase B. Antibodywas used to examine the biosynthesis and structure of the carbohydratechains of arylmannosidase in soybean cells grown in [2–³H]mannose.Treatment of the purified enzyme with Endo H released 50% ofthe radioactivity, and these labeled oligosaccharides were ofthe high-mannose type, i.e. mostly Man9GlcNAc. The precipitatedprotein isolated from the Endo H treatment still contained 50%of the radioactivity, and this was present in modified structuresthat probably contain xylose residues. Mung beans mannosidases glycoproteins -soybean--mannosidases xylose-containing N-linked glycoproteins     

Anti-GM3-lactam monoclonal antibodies of the IgG type recognize natural GM3-ganglioside lactone but not GM3-ganglioside

Immunization of mice with a synthetic GM₃-lactam-BSA (bovine serum albumin) conjugate (designed to emulate the corresponding natural GM₃-lactone conjugate), followed by fusion of splenocytes with myeloma cells, gave rise to more than 300 monoclonal hybridomas producing antibodies to GM₃-lactam-BSA, which did not react with Glc-BSA and BSA. Eight antibody clones were randomly chosen from the positive 300 hybridomas. The eight clones, all belonging to the IgG class, were unreactive against GM₃-ganglioside, whereas two antibodies (P5-1 and P5-3, both IgG₁, ) reacted with GM₃-ganglioside lactone. Binding of these two antibodies to the GM₃-lactam-BSA conjugate was inhibited by soluble glycosides of GM₂-, GM₃-, and GM₄-lactam and by GM₃- and GM₄-lactam, respectively, but not by Gb₃ or asialo-GM₁ and GM₂-saccharides. A third antibody (P3; IgG_2b, ) was inhibited by GM₂-, GM₃-, and GM₄-lactam, but did not recognize GM₃-ganglioside lactone.