DNA markers tightly linked to a gall midge resistance gene (Gm2) are potentially useful for marker-aided selection in rice breeding

We have developed a polymerase chain reaction (PCR)-based assay that could effectively reduce the time period required to screen and select for Gall Midgeresistant rice lines under field conditions. The primers for the assay were designed on the basis of sequence information of two phenotype specific random amplified polymorphic DNA fragments which were found to be tightly linked to Gall Midge biotype-1 resistance gene (Gm2). The two RAPD fragments, F8₁₇₀₀ in the susceptible parent ARC6650 and F10₆₀₀ in the resistant parent Phalguna, were identified after screening 5450 loci using 520 random primers on genomic DNAs of ARC6650 and Phalguna. These primers, when used in a multiplexed PCR, amplified specifically a 1.7-kb and 0.6-kb fragment in the susceptible and resistant parents, respectively. When this assay was performed on genomic DNAs of 44 recombinant inbred lines derived from ARC6650 x Phalguna and 5 lines derived from other crosses where one of the parents was Phalguna, ARC6650 or their derivatives, the primers amplified a 1.7-kb fragment in all of the susceptible lines or a 0.6-kb fragment in all of the resistant ones. These markers can be of potential use in the marker-aided selection of Gall Midge biotype-1 resistant phenotypes. As screening for resistance can now be conducted independent of the availability of insects, the breeding of resistant varieties can be hastened.     

Adoptions expérimentales de larves entre des fourmis de genres différents (II):Myrmica laevinodis Nylander etAnergates atratulus Schenck

Résumé Nous avons fait élever des larves d'Anergates atratulus par des ouvrières deMyrmica laevinodis à 22°C. Pour y parvenir, il n'est pas utile de faire hivernerensemble les larves d'Anergates et les ouvrières deMyrmica. La présence de larves autochtones n'empêche pas lesMyrmica d'élever des larves d'Anergates. Dans toutes les expériences lesMyrmica ont été soumises au fridavant de recevoir des larves d'Anergates. Aucune reine deMyrmica n'a été utilisée dans ces expériences.Sur les 64 larves d'Anergates que nous avons utilisées, 38 se sont transformées en imagos. C'est au début de l'adoption et au moment des métamorphoses que périrent la plupart des 26Anergates perdus. Les femelles vécurent en général 2 ou 3 jours et cherchèrent très tôt à quitter le nid natal. Les mâles vécurent 2 à 3 semaines.

---

Summary Larvae ofAnergates atratulus were experimentally reared by workers ofMyrmica laevinodis, at 22°C. An overwintering of both larvae ofAnergates and workers ofMyrmica is not necessary for the success of that experiment. The presence of larvae ofMyrmica does not keep theMyrmica from rearing larvae ofAnergates. The workers ofMyrmica have been cooled, in all the experiments, before receiving larvae ofAnergates. No queen ofMyrmica have been used in that experiments.38 of the 64 larvae ofAnergates used became imagos. Most of the 26 lostAnergates died at the beginning of the adoption and during the metamorphosis. The females lived generally 2 or 3 days and tried, very early, to leave their native nest. The males lived 2 or 3 weeks.

---

Anergates atratulus Myrmica laevinodis, 22 . bmecme Anergates Myrmica. Myrmica Anergates. Myrmica Anergates. Myrmica . 64 Anergates , 38 . 26 Anergates 2 3 . 2 3 .

     

Inhibition of protein synthesis enhances the lytic effects of tumor necrosis factor α and interferon γ in cell lines derived from gynecological malignancies

Summary Few clinical responses have occurred in preliminary studies using the cytokines tumor necrosis factor (TNF) or interferon (IFN) in cancer patients. This may be related to the observation that many malignant cell lines are resistant to lysis by these cytokinesin vitro. Resistance to lysis by TNF or IFN in many cells is controlled by a protein-synthesis-dependent mechanism, such that when protein synthesis is inhibited cells become sensitive to lysis by these cytokines. Because there is some evidence that TNF and IFN act through different lytic mechanisms and are opposed by different resistance mechanisms, we treated a panel of eight cell lines, five derived from human cervical carcinomas (ME-180, MS751, SiHa, HT-3, and C-33A) and three derived from ovarian carcinomas (Caov-3, SK-OV-3, and NIH: OVCAR-3) with both TNF and IFN to determine whether such combination treatment might maximizein vitro cell lysis. Our results showed that pretreatment with IFN followed by exposure to TNF in the presence of protein synthesis inhibitors increased lysis of seven of the eight cell lines above that seen with either TNF or IFN and inhibitors of protein synthesis. Only the cell line C-33A was resistant to lysis by TNF and IFN, when exposed to these agents both alone and in combination with protein synthesis inhibitors. Clinically, combining the cytokines TNF and IFN with protein synthesis inhibitors may maximize thein vivo lytic effects of these cytokines.Supported by American Cancer Society Career Development Award 90-221     

Purification and characterization of β‐methylaspartase from Fusobacterium varium

Methylaspartase (EC 4.3.1.2) was purified 20fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAESepharose. The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg²⁺ (K_m = 0.27 ± 0.01 mM) and K⁺ (K_m = 3.3 ± 0.8 mM) for maximum activity. Methylaspartasecatalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)3methylaspartate (major product) and (2S,3R)3methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)3methylaspartate (K_m = 0.51 ± 0.04 mM) was observed at pH 9.7. The Nterminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.     

Cytological aspects of in vitro androgenesis in wheat (Triticum aestivum L.) using fluorescent microscopy

Summary Two winter wheat genotypes (Diószegi 200 and Mv 15) were compared for their in vitro androgenic capacity. On average, the induction frequency of embryogenic structures was 71.7% in Diószegi 200 and only 4.3% in Mv 15. The haploid induction ability of the two genotypes differed considerably, with Diószegi 200 being much higher. The difference in the in vitro inductability of the microspores may result from genetic differences which are manifested in the survival rate of the microspores during the culture period and their adaptability to in vitro conditions. Special DNA fluorochrornes were suitable for studying the different pathways of in vitro androgenesis. Our data indicate that the repeated equal divisions of the microspore nucleus might lead to pollen embryo formation, and subsequent divisions of the vegetative portion of the pollen grain after the first asymmetric microspore mitosis can result in pollen callus formation.     

Preparation of a series of sulfated tetrasaccharides from shark cartilage chondroitin sulfate D using testicular hyaluronidase and structure determination by 500 MHz1H NMR spectroscopy

Six tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz¹H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the tetrasaccharides. One fraction was found to contain two tetrasaccharide components. All the seven tetrasaccharides shared the common core structure GlcA1-3GalNAc1-4GlcA1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcA1-3GalNAc(4-sulfate) and/or GlcA1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcA1-3GalNAc(4-or 6-sulfate). These sulfated tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum -N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.Abbreviations NFU National formulary unit - COSY correlation spectroscopy - HOHAHA homonuclear Hartmann-Hahn - 1D or 2D one- or two-dimensional - IdoA l-iduronic acid - GlcA d-gluco-4-enepyranosyluronic acid - Di-0S GlcA1-3GalNAc - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-diS _d GlcA(2-sulfate)1-3GalNAc(6-sulfate) - Di-diS_E GlcA1-3GalNAc(4, 6-disulfate) - U G, U, 2S, 4S, and 6S represent GlcA, GalNAc, GlcA, 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively     

Full 1H NMR assignment of a 24-nucleotide RNA hairpin: Application of the 1H 3D-NOE/2QC experiment

The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the ¹H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5/H5 for RNA plus H2/H2 for DNA), as well as for H5/H6, for H3/H4 in sugars with substantial populations of the N-pucker, for H1/H2 for S-puckered sugars, and usually for H2/H3. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignment of all of the non-exchangeable protons, eliminating the need for stable-isotope labeling.     

The addition of Dasypyrum villosum (L.) Candargy chromosomes to durum wheat (Triticum durum Desf.)

Summary Six monosomic addition lines were produced in which different Dasypyrum villosum (L.) Candargy chromosomes were added to the chromosome complement of Triticum durum Desf. cv. Creso. Each added alien chromosome was found to have a specific effect on plant morphology and fertility. Transmission rate varied widely (from 7.5 to 27.7%) among the six univalent chromosomes. Different monotelosomic addition plants derived by a relatively high frequency of chromosome misdivision were isolated. The addition lines should be useful for studying Dasypyrum chromosome homoeology and the introduction of alien variation into durum and common wheats.Research supported by a grant from the Italian Research Council for Finalized Project IPRA. Sub-project Plant Breeding, Paper No. 1095     

Isospora lutrae n. sp. (Apicomplexa: Eimeriidae), a new coccidium from the European otter Lutra lutra (L.) (Carnivora: Mustelidae) from Spain

Parasitological examination of European otter originating from Extremadura, Spain revealed the presence of a new isosporan species. Oöcysts of Isospora lutrae n. sp. are spherical to subspherical, 31.2 (27.5–32) × 29.6 (28–31) m and have a smooth wall c. 1 m thick. Sporocysts are ellipsoidal, 18.2 (17–19) × 14.4 (14–16) m and lack Stieda and substieda bodies. A spherical sporocyst residuum is present, consisting of granules scattered among the sporozoites. Sporozoites are spindle-shaped, 12.4 × 2.5 m and have anterior and posterior refractile bodies. Based on its unique morphologic structure and host, I. lutrae is considered to be new.     

Interaction of alpha-lactalbumin with mini-alphaA-crystallin

A-Crystallin can function like a molecular chaperone. We have recently shown that residues 71-88 in A-crystallin represent the chaperone active site of the protein. A peptide containing the sequence of A-crystallin sequence DFVIFLDVKHFSPEDLTVK (mini A-crystallin) by itself displays the antiaggregation property of A-crystallin. We have prepared a complex of reduced -lactalbumin and mini-A-crystallin and investigated the nature, conformation, and properties of the complex by dynamic light scattering, HPLC analysis, CD spectroscopy, and fluorescence studies. Although mini-A was able to prevent the precipitation of reduced -lactalbumin, large aggregates (50-500 nm) of the complex were formed during the assay. Amino acid composition estimation revealed that -lactalbumin and mini-A-crystallin were present in 1:2 ratio in the aggregates. During our study significant red shift in the Trp fluorescence emission maximum and an increase in Bis-ANS binding to the mini A-crystallin-bound -lacatalbumin were observed. The CD spectra of the complex showed a significant loss of -helical content but the -sheet content appeared to be less affected, indicating the molten-globule state of the reduced lactalbumin in the complex. These data show that the active site of A-crystallin by itself can maintain a significantly denatured and unfolded protein in soluble form.     

Forskolin Modulates Acetylcholine Receptor Gating by Interacting with the Small Extracellular Loop Between the M2 and M3 Transmembrane Domains

1. Forskolin acts as an allosteric modulator of muscle-type nicotinic acetylcholine receptors. Receptors from mouse muscle and Torpedo electroplax demonstrate differential sensitivity to inhibition by forskolin. Previous work from this laboratory suggested that the subunit is responsible for this differential sensitivity.2. We have used a series of mouse/Torpedo species-chimeric subunits to further define the site of forskolin interaction with the subunit. Analysis of the patterns of forskolin inhibition of receptors containing mouse/Torpedo chimeric subunits along with the mouse , , and subunits suggests that forskolin interacts with the small extracellular domain that links the M2 and M3 transmembrane domains (the M2–M3 linker).3. We suggest that the M2–M3 linker domain plays an important role in the transduction of ligand binding to the conformational changes that result in channel opening.     

A DNA sequence of Drosophila melanogaster with a differential telomeric distribution

A DNA sequence (8–19T) of 2.3 kilobase pairs (kb) of Drosophila melanogaster was localized by in situ hybridization to the extreme ends of polytene chromosomes and to the chromocenter. The relative abundance of this sequence at the ends of polytene chromosomes X2L2R3L3R is 13.41.902.7. This differential distribution is probably due to different copy numbers at the individual telomeric regions. Restriction enzyme analysis of genomic DNA shows that 8–19T sequences are interspersed with other sequences. The clone 8–19T, which contains most of this interspersed repetitive sequence, is itself not internally repetitive but has a complex sequence composition. Some of these sequences are transcribed into poly(A)⁺RNA. We suggest that the ends of Drosophila chromosomes are of a complex arrangement with some sequences common to all ends.     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo     

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.     

Production of β-mannosidase and β-mannanase by an alkalophilic Bacillus sp.

Summary An alkalophilic bacterium producing high amounts of the cell-associated -mannosidase and extracellular -mannanase was isolated from soil. The isolate (AM-001) that grew well in alkaline pH media was identified as a strain of Bacillus sp. The optimal cultivation temperature for enzyme production was 31° C for -mannosidase and 37° C for -mannanase with the optimum production medium composed of 1% konjac powder, 0.2% yeast extract, 2% Polypepton, 0.1% K₂HPO₄, 0.02% MgSO₄ · 7H₂O and 0.5% Na₂CO₃. Optimum pH and temperature for -mannosidase were 7.0 and 55° C, and for -mannanase were 9.0 and 65° C.     

Export and activity of hybrid FhuA''-''Iut receptor proteins and of truncated FhuA'' proteins of the outer membrane of Escherichia coli

Summary The FhuA protein in the outer membrane of Escherichia coli serves as a multifunctional receptor for the phages T5, T1, 80, for colicin M, for ferrichrome (Fe³⁺-siderophore) and for the structurally related antibiotic, albomycin. To determine structural domains required for these receptor functions and for export, a fusion protein between FhuA and Iut (receptor for Fe³⁺-aerobactin and cloacin DF13) was constructed. In the FhuA-Iut hybrid protein, 24 amino acids of FhuA were replaced by 19 amino acids, 18 of which were from Iut. The number of plaque forming units of phage T5 and T1 on cells expressing FhuA-Iut was nearly as high as on cells expressing plasmid-encoded wild-type FhuA. However, 10⁷-fold higher concentrations of phage 80 and 10³ times more colicin M were required to obtain a zone of growth inhibition. Truncated FhuA proteins in which the last 24 amino acids at the carboxy-terminus were replaced by 16 (FhuA2) or 3 (FhuAT) amino acids could hardly be detected on polyacrylamide electrophoretograms of outer membrane proteins, due to proteolytic degradation. Sensitivity of cells expressing FhuA2 to phage T5 and T1 was reduced by several orders of magnitude and sensitivity to phage 80 and colicin M was totally abolished. In contrast, cells expressing FhuAT were nearly as sensitive to phage T5, T1, and 80 and to colicin M as cells containing FhuA-Iut. None of the constructs could grow on ferrichrome as sole iron source and none was sensitive to albomycin. Ferrichrome did not inhibit binding of T5 to TonB^– cells expressing FhuA-Iut (as it did in FhuA⁺ cells) due to the lack of ferrichrome binding. It is concluded that very small deletions (relative to the size of FhuA with 714 amino acids) at the C-terminal end render FhuA susceptible to proteolytic cleavage. The C-terminal alterations affect sensitivity to FhuA-specific agents very differently. Apparently, the C-terminus is a very important part of FhuA regarding stability and activity. Expression of active FhuA and partially inactive FhuA derivatives in the same cell revealed no negative complementation, suggesting a FhuA monomer as functional unit.     

Sequence analysis of 22 kDa-like α-coixin genes and their comparison with homologous zein and kafirin genes reveals highly conserved protein structure and regulatory elements

Several genomic and cDNA clones encoding the 22 kDa-like -coixin, the -prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like -coixin genes designated -3A, -3B and -3C were found in the 15 kb -3 genomic clone. The -3A and -3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the -3B gene, suggesting that the three -coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication.Comparison of the deduced amino acid sequences of -coixin clones with the published sequences of 22 kDa -zein and 22 kDa-like -kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15–20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like -prolamins and the 19 kDa -zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins.Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5 and 3 flanking regions of -3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. –300 prolamin box are present at conserved positions in -3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in -3B that occupies approximately the same positions as those identified for the 22 kDa -zein and -kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes.The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like -prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation.     

Cloning and expression in Saccharomyces cerevisiae of a β-amylase gene from the oomycete Saprolegnia ferax

Genomic DNA and cDNA encoding the -amylase from the oomycete, Saprolegnia ferax, were cloned into Saccharomyces cerevisiae and analyzed. The Spl. ferax -amylase gene consisted of a 1350 bp open reading frame, encoding a protein of 450 amino acids with a calculated mass of 49353 Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 42% similarity to the -amylase of Arabidopsis thaliana. The -amylase gene was expressed in Sacc. cerevisiae and its product was secreted into the culture medium.     

A method for the investigation of those transformations under which the visual recognition of a given object is invariant

An experiment is described which shows in operation the program set out in Foster (1972a) for the investigation of the invariance transformations of visual recognition. The concern in the present study is with the Lie group of rotations SO(2), and a certain centrally located foveal Landolt ring. By presenting to the visual system this Landolt ring and a rotated image in rapid succession, one attempted to induce a specified rotation-type phi-motion. Two subjects were employed. Both reported the existence of the required type of phi-motion for rotations ₀ of the Landolt ring about the visual axis with -2/72/7. By appealing to the basic Proposition 2 of Foster (1972 a), the conclusion is reached that the visual system appears capable of effecting upon a certain centrally located foveal annulus the local 1-parameter group of rotations about the visual axis ₀, [–2/7,2/7].     

Structural studies of the glycopeptides of B-chain of cinnamomin – a type II ribosome-inactivating protein by nuclear magnetic resonance

Cinnamomin is a plant type II ribosome-inactivating protein (RIP) isolated from the seeds of Cinnamomum camphora. It consists of two nonidentical polypeptide chains (A- and B-chain) held together through one disulfide linkage. Its A- and B-chain contain 0.3% and 3.9% sugars respectively. The B-chain of cinnamomin was digested by pronase E and then the liberated glycopeptides were separated from non-glycopeptides by gel filtration chromatography on a Bio-Gel P-4 column. Three crude glycopeptides were obtained by continuing chromatography over anion-exchange resin (AG1-X2) in the buffer of 2% pyridine-acetic acid (pH 8.3) with a polygradient elution system. Through further purification by the gel filtration chromatography and HPLC, three major glycopeptides, GP1, GP2 and GP3 were obtained. Mainly by two-dimensional Nuclear Magnetic Resonance (NMR) including TOCSY, DQF-COSY, NOESY, HMQC and HMBC, their primary structures were analyzed as: Man1,3Man1,6(Man1,3)(Xyl1,2)Man1,4GlcNAc1,4GlcNAc1-(Gly-)Asn-Asn-Thr(GP1), Man1,6(Man1,3)(Xyl1,2)Man1,4GlcNAc1,4(Fuc1,3)GlcNAc1-Asn-Ala-Thr(GP2),Man1,6(Man1,3)Man1,6(Man1,2 Man1,3)Man1,4GlcNAc1,4GlcNAc1-(Ala-)Asn-Gly-Thr(GP3).