An estradiol-dependent protein from chicken liver binds single-stranded DNA and RNA

We have identified two estradiol-dependent single-stranded DNA binding proteins in the nucleus and cytoplasm of chicken hepatocytes that bind the sequence 5'TCACCTTCGCTATG3' in the first exon of the chicken vitellogenin gene. As judged by chromatography on heparin-Sepharose and by proteolytic clipping bandshift assay, the two proteins are different. Furthermore, they only bind to the oligonucleotide corresponding to the upper strand. Depurination and depyrimidination interference experiments with the cytoplasmic protein show that the bases CCTT-G are involved in the protein-DNA interaction. An RNA corresponding to the upper strand of the gene between nucleotide positions -73 and +53 competes for binding to the single-stranded DNA. UV cross-linking experiments performed with bromouridine-substituted single-stranded RNA reveal that an estradiol-dependent hepatocyte cytoplasmic protein with a Mr of 71,000 binds to the mRNA-like single-stranded RNA.     

Cholera toxin as a mucosal adjuvant. Glutaraldehyde treatment dissociates adjuvanticity from toxicity

Cholera toxin (CT), either mixed with or conjugated to unrelated protein Ag, is known to enhance the intestinal IgA response of rodents toward the unrelated Ag. Although relatively low doses of CT exert this gut mucosal adjuvant effect, the inherent toxicity of CT is a hindrance to its use in humans. Our report demonstrates that CT treated with 20 mM glutaraldehyde retains adjuvant properties but exhibits more than 1000-fold lower toxicity than untreated toxin. Glutaraldehyde was also used in a one-stage conjugation procedure to couple CT covalently to Sendai virus. Again, toxicity was reduced more than 1000-fold. This drop in toxicity is consistent with an observed 100-fold loss in binding capacity of the CT B subunit and a 20- to 50-fold reduction in adenylate cyclase activation by the CT A subunit. Oral administration of this virus-toxoid conjugate resulted in increased gut antiviral IgA titers compared with oral administration of either virus alone or of virus mixed with glutaraldehyde-treated toxin. This marked decrease in toxicity may afford a practical approach for the use of CT as a mucosal adjuvant.     

Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

Multiple origins for phenylketonuria in Europe

Phenylketonuria (PKU), a disorder of amino acid metabolism prevalent among Caucasians and other ethnic groups, is caused primarily by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). PKU is a highly heterogeneous disorder, with more than 60 molecular lesions identified in the PAH gene. The haplotype associations, relative frequencies, and distributions of five prevalent PAH mutations (R158Q, R261Q, IVS10nt546, R408W, and IVS12n1) were established in a comprehensive European sample population and subsequently were examined to determine the potential roles of several genetic mechanisms in explaining the present distribution of the major PKU alleles. Each of these five mutations was strongly associated with only one of the more than 70 chromosomal haplotypes defined by eight RFLPs in or near the PAH gene. These findings suggest that each of these mutations arose through a single founding event that occurred within time periods ranging from several hundred to several thousand years ago. From the significant differences observed in the relative frequencies and distributions of these five alleles throughout Europe, four of these putative founding events could be localized to specific ethnic subgroups. Together, these data suggest that there were multiple, geographically and ethnically distinct origins for PKU within the European population.     

Association of a polymorphism of the angiotensin I-converting enzyme gene with essential hypertension.

Angiotensin I-converting enzyme (ACE) is responsible for production of angiotensin II and breakdown of kinins, leading to increased blood pressure (BP). Furthermore, ACE inhibitors are effective antihypertensive agents. A 287 bp insertion/deletion polymorphism in intron 16 of the ACE gene (ACE) was examined by PCR in a cross-sectional study of 80 hypertensive (HT) and 93 normotensive (NT) subjects whose parents had a similar BP status at age greater than or equal to 50. The frequency of the insertion allele was 0.56 in HTs and 0.41 in NTs, and the difference between observed alleles in all subjects in each group was significant (chi 2 = 7.6, P less than 0.01). The data thus provide evidence in favour of an association of HT with a polymorphism at the ACE locus (17q23), so implicating this locus, and possibly a genetic variant of ACE itself, in human essential hypertension.     

Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases.

We have deduced the nucleotide sequence of the genes encoding the three components of 4-chlorobenzoate (4-CBA) dehalogenase from Pseudomonas sp. CBS-3 and examined the origin of these proteins by homology analysis. Open reading frame 1 (ORF1) encodes a 30-kDa 4-CBA-coenzyme A dehalogenase related to enoyl-coenzyme A hydratases functioning in fatty acid beta-oxidation. ORF2 encodes a 57-kDa protein which activates 4-CBA by acyl adenylation/thioesterification. This 4-CBA:coenzyme A ligase shares significant sequence similarity with a large group of proteins, many of which catalyze similar chemistry in beta-oxidation pathways or in siderophore and antibiotic synthetic pathways. These proteins have in common a short stretch of sequence, (T,S)(S,G)G(T,S)(T,E)G(L,X)PK(G,-), which is particularly highly conserved and which may represent an important new class of "signature" sequence. We were unable to find any proteins homologous in sequence to the 16-kDa 4-hydroxybenzoate-coenzyme A thioesterase encoded by ORF3. Analysis of the chemistry and function of the proteins found to be structurally related to the 4-CBA:coenzyme A ligase and the 4-CBA-coenzyme A dehalogenase supports the proposal that they evolved from a beta-oxidation pathway.     

Evidence for an internal pool of nucleoside transporters in mammalian reticulocytes.

Comparisons of the site specific binding of nitrobenzylthioinosine (NBMPR) to intact and lysed red cells from various mammalian and avian species suggest the presence of a cytoplasmic pool of nucleoside transporters. In some species the cytoplasmic pool is about 50% of the total (mouse). On the average, the cytoplasmic pool is approx. 20% of the surface pool of NBMPR-binding sites. In sheep reticulocytes, both pools disappear in an energy-dependent manner during the maturation of the reticulocyte in vitro.     

Effect of genotype and medium on wheat (Triticum aestivum L.) anther culture

Four winter wheat (Triticum aestivum L.) and two spring wheat cultivars were evaluated in anther culture on three to four different media for their ability to initiate callus and green plants. Five media were used in the experiment: stored-potato medium with Ficoll 400, fresh-potato medium with Ficoll 400, fresh-potato medium with agar, fresh-potato liquid medium without agar or Ficoll 400, and a one tep 85D12-3 medium. Greatly different frequencies of calli and/or green plants were obtained from different cultivars and media. The callus initiation frequency varied from 2.7% for Arapahoe to 52% for Pavon, both on the stored potato medium with Ficoll 400. The frequency of green plant regeneration ranged from 0% for Arapahoe and Siouxland on the stored-potato medium with Ficoll 400 and 0% for Redland and Arapahoe in the fresh-potato medium with Ficoll 400 to 12% for Chris in the 85D12-3 medium (one-step procedure). Chris and Centurk 78, previously reported as having high levels of response, had significantly higher (P < 0.05) frequencies of green plant regeneration on the 851312-3 medium than the other cultivars. An unexpected observation is that wet MSC^– medium enhanced callus regeneration more than a drier MSC^– medium.     

大棚黄瓜霜霉病生态防治方法研究——温度控制试验研究初报

黄瓜霜霉病是对黄瓜生长发育危害最大的世界性病害。通过两年共10个处理的田间对比试验发现,大棚黄瓜霜霉病的发生发展与环境温度条件存在着十分密切的关系,在一定温度范围内(15℃—48℃),棚内日最高气温越高,发病期越晚,病越轻,产量越高。控制方法是:在大棚内日最低气温稳定大于10℃后,每隔一天对大棚进行一次40—47℃、并维持2小时左右的高温处理(其中大于42℃持续1.5小时左右,大于45℃持续1小时左右),然后大面积通风换气。方法简便易行、效果很好。