Isolation and identification of a non-specific tandemly repeated DNA sequence in Oryza species

A tandemly repeated DNA sequence (RRS7) was isolated from Oryza alta (CCDD). RRS7-related sequences were also found tandemly arrayed in genomes AA, BB, BBCC, CC, and EE, and a small amount of RRS7-related sequences were detected in genome FF and the Oryza species with unknown genomes. DNA sequence analysis of the 1844-bp insert of RRS7 revealed that it contained six tandemly repeated units, of which five were 155 bp in length and one was 194 bp in length and contained an imperfect internal 39-bp duplication. Southern blot analysis showed that the boundary sequence contained in RRS7 is a single-copy sequence. A 155-bp consensus sequence derived from the six monomeric repeats contained no internal repeat and showed no significant homology to other currently known sequences. The results of Southern blot and sequence analysis revealed that there are at least two subfamilies present in the RRS7 family; these are represented by the DraI site and the MspI site, respectively. Restriction digestion with two pairs of isoschizomers MboI/Sau3A and MspI/HpaII demonstrated that most of the C residues in the GATC sites and the internal C in the CCGG sites of the RRS7 family in O. Alta were methylated. The usefulness of the RRS7 family in determining the evolutionary relationship of the genome DD and other Oryza genomes is discussed.     

Replicon rescue: a novel strategy to clone the genomic DNA flanking insertions of integrating shuttle vector DNA.

A novel cloning strategy, replicon rescue, was developed for cloning genes disrupted by plasmid insertions. After ligation to a tetracycline resistance cassette, fragments containing a bacterial origin of replication from the insertion are recovered in Escherichia coli because they replicate autonomously. Restriction enzymes for cloning are so chosen that the only legitimate two fragment ligation yielding TetR clones involves a fragment spanning the boundary of the insertion. Replicon rescue was used successfully firstly in a test system to clone the chromosomal orl from a Klebsiella aerogenes strain, and secondly to recover a disrupted gene from a phototaxis-deficient mutant of Dictyostelium.     

Synergistic activity of a Bacillus thuringiensis delta-endotoxin and a bacterial endochitinase against Spodoptera littoralis larvae.

In an attempt to increase the insecticidal effect of the delta-endotoxin crystal protein CryIC on the relatively Cry-insensitive larvae of Spodoptera littoralis, a combination of CryIC and endochitinase was used. CryIC comprising the first 756 amino acids from Bacillus thuringiensis K26-21 and endochitinase ChiAII encoded by Serratia marcescens were separately produced in Escherichia coli carrying the genes in overexpression vectors. The endochitinase on its own, even at very low concentrations (0.1 microgram/ml), perforated the larval midgut peritrophic membrane. When applied together with low concentrations of CryIC, a synergistic toxic effect was obtained. In the absence of chitinase, about 20 micrograms of CryIC per ml was required to obtain maximal reduction in larval weight, while only 3.0 micrograms of CryIC per ml caused a similar toxic effect in the presence of endochitinase. Thus, a combination of the Cry protein and an endochitinase could result in effective insect control in transgenic systems in which the Cry protein is not expressed in a crystalline form.     

Myosin VIIA mutation screening in 189 Usher syndrome type 1 patients.

Usher syndrome type 1b (USH1B) is an autosomal recessive disorder characterized by congenital profound hearing loss, vestibular abnormalities, and retinitis pigmentosa. The disorder has recently been shown to be caused by mutations in the myosin VIIa gene (MYO7A) located on 11q14. In the current study, a panel of 189 genetically independent Usher I cases were screened for the presence of mutations in the N-terminal coding portion of the motor domain of MYO7A by heteroduplex analysis of 14 exons. Twenty-three mutations were found segregating with the disease in 20 families. Of the 23 mutations, 13 were unique, and 2 of the 13 unique mutations (Arg212His and Arg212Cys) accounted for the greatest percentage of observed mutant alleles (8/23, 31%). Six of the 13 mutations caused premature stop codons, 6 caused changes in the amino acid sequence of the myosin VIIa protein, and 1 resulted in a splicing defect. Three patients were homozygotes or compound heterozygotes for mutant alleles; these three cases were Tyr333Stop/Tyr333Stop, Arg212His-Arg302His/Arg212His-Arg302His, and IVS13nt-8c-->g/Glu450Gln. All the other USH1B mutations observed were simple heterozygotes, and it is presumed that the mutation on the other allele is present in the unscreened regions of the gene. None of the mutations reported here were observed in 96 unrelated control samples, although several polymorphisms were detected. These results add three patients to single case reported previously where mutations have been found in both alleles and raises the total number of unique mutations in MYO7A to 16.     

Gas exchange in leaves of the root hemiparasitemelampyrum arvense L. before and after attachment to the host plant

Gas exchange characteristics of a hemiparasiteMelampyrum arvense L. before and after attachment to the hostCapsella bursa pastoris (L.) Med. were compared. The net photosynthetic rates (P_N) on a leaf area basis were extremely low and in comparison to the value obtained for the host were about 15 % and 23 % for the unattached and attached hemiparasite, respectively. Also the concentration of photosynthetic pigments was low (as compared with the host the content of chlorophylls was about 33 % and 49 % and of carotenoids about 38 % and 36 % in the unattached and attached hemiparasite, respectively). On the other hand the rates of respiration were high (about 1.8 and 2.6 times higher in the unattached and attached hemiparasite, respectively, than in the host). In darkness stomatal conductance (g_S) of the host and the unattached hemiparasite was rapidly reduced to 10 % of the value obtained in light, g_S of the attached hemiparasite was decreased only by about 30%. A total reduction of g_S occurred at relative water content (RWC) of 85 %, 75% and 45 % for the unattached hemiparasite, the host, and the attached hemiparasite, respectively. The transpiration (E) rate in the preparasitic stage was very low, being 2.6 and 4.5 times smaller than in the host and the attached hemiparasite, respectively. In the attached hemiparasite WUE was 7.5 and 3 times poorer than in the host and in the preparasitic stage, respectively.     

Surface plasmon resonance studies of complex formation between cytochrome c and bovine cytochrome c oxidase incorporated into a supported planar lipid bilayer. I. Binding of cytochrome c to cardiolipin/phosphatidylcholine membranes in the absence of oxidase.

The mechanism of interaction between cytochrome c and a solid-supported planar phosphatidylcholine membrane containing varying amounts of cardiolipin (0-20 mol%) has been studied over a wide range of protein concentrations (0-450 microM) and ionic strength conditions (10-150 mM), by direct measurement of protein binding using surface plasmon resonance (SPR) spectroscopy. The results demonstrate that cytochrome c binds to such phospholipid membranes in two distinct phases characterized by very different (approximately one order of magnitude) affinity constants. The second phase is dependent upon the prior occurrence of the first binding process. Although the binding affinities for both modes of binding are highly sensitive to both the cardiolipin concentration and the ionic strength of the buffer solution, indicating that electrostatic forces are involved in these processes, binding cannot be reversed by salt addition or by dilution. Furthermore, the final saturation levels of adsorbed protein are independent of ionic strength and cardiolipin concentration. These observations suggest that binding involves more than a simple electrostatic interaction. Invariance in the shapes of the SPR spectra indicates that no major structural transitions occur in the proteolipid membrane due to cytochrome c binding, i.e., the bilayer character of the lipid phase appears to be preserved during these interactions. Based on these results, a model of the lipid membrane-cytochrome c interaction is proposed that involves varying degrees of protein unfolding and subsequent binding to the membrane interior via hydrophobic forces.