Isolation of high-molecular-weight plant DNA for DNA damage quantitation: relative effects of solar 297 nm UVB and 365 nm radiation

Quantitation of UV-induced DNA damages in nanogram quantities of non-radiactive DNA from irradiated plants by gel electrophoresis requires a prompt, efficient, high-yield method of isolating DNA yielding high-molecular-weight, enzymatically digestible DNA. To meet these criteria we devised a high-yield method for isolating from plant tissue, DNA whose single-strand molecular length is greater than about 170 kb. Leaf tissue is embedded in agarose plugs, digested with Proteinase K in the presence of detergent, and treated with phenylmethylsulfonyl fluoride (PMSF). The agarose plugs are then soaked with buffer appropriate to the desired enzyme treatment. Evaluation of the DNA on neutral and alkaline gels indicates its high molecular length and low frequency of single-strand breaks. The DNA can be digested with damage-specific and other endonucleases. The method is especially suitable for DNA damage quantitation, as tissue processing is carried out immediately after harvesting (allowing DNA lesion measurement at precisely known times after irradiation), and many samples can be easily handled at once. It should also be useful for molecular analysis of large numbers of plant samples available only in small quantities. We here use this method to quantitate DNA damage induced by 297 and 365 nm radiation, and calculate the relative damaging effects of these wavebands in today's solar spectrum.     

The legumin gene family: a reconstructed Vicia faba legumin gene encoding a high-molecular-weight subunit is related to type B genes

Nucleotide sequence information from a partial genomic clone, a cDNA clone, a RACE clone and a PCR fragment was combined to reconstruct the first reported complete gene sequence encoding a large legumin subunit, designated LelB3. The length difference to the well-characterized major legumin subunits is caused by an extended glutamin/glutamic acid-rich region encoded by the C-terminal part of the chain. Amino acid sequence comparisons reveal that gene LelB3 is more closely related to B-type than to A-type legumin genes of Vicia faba. Gene LelB3 is a member of a small gene family as indicated by published (Pich and Schubert, Biol Zbl 112 (1993); 342–350) and limited own data.     

Contact System. New Concepts on Activation Mechanisms and Bioregulatory Functions

This review considers the data of recent years concerning the contact system initiating the activation of blood plasma proteolytic systems, such as hemocoagulation, fibrinolysis, kininogenesis, and also complement and angiotensinogenesis. The main proteins of the contact system are the factors XII and XI, prekallikrein, and high-molecular-weight kininogen. The data on the structure, functions, and biosynthesis of these proteins and on their genes are presented. Studies in detail on the protein–protein interactions during formation of the ensemble of the contact system components on the anionic surface resulted in the postulation of the mechanism of activation of this system associated with generation of the XIIa factor and of kallikrein. This mechanism is traditionally considered a trigger of processes for the internal pathway of the hemocoagulating cascade. However, the absence of direct confirmation of such activation in vivo and the absence of hemorrhagia in the deficiency of these components stimulated the studies designed to find another mechanism of their activation and physiological role outside of the hemostasis system. As a result, a new concept on the contact system activation on the endothelial cell membrane was proposed. This concept is based on the isolation of a complex of proteins, which in addition to the above-mentioned proteins includes cytokeratin 1 and the receptors of the urokinase-like plasminogen activator and of the complement q-component. The ideas on the role of this system in the biology of vessels are developed. Some of our findings on the effect of leukocytic elastase on the key components of the contact system are also presented.     

A Simple and Rapid Extraction of High Molecular Weight Chromosomal DNA from Bacillus subtilis Protoplasts for Cosmid Cloning and Interspecific Transformation

After conversion of Bacillus subtilis vegetative cells to protoplasts, a simple and rapid method for extracting high-molecular-weight chromosomal DNA was devised with the inclusion of bovine serum albumin and phenol-chloroform treatments. The DNA sample thus prepared was the size of 100-450 kb and could be used for cosmid cloning and interspecific transformation.     

一株高效降解芘的细菌分离、鉴定及其降解效果

摘要：【目的】获得高效降解高分子量多环芳烃的细菌,并研究其对多环芳烃的降解能力。【方法】利用富集培养和芘升华平板方法,从焦化厂污染土壤中分离多环芳烃降解细菌,对分离菌株通过形态特征、16S rRNA基因和gyrb基因序列相似性分析进行鉴定,并研究该菌对高分子量多环芳烃（HMW-PAHs）的降解效果。【结果】筛选到一株能以芘、苯并蒽、屈、苯并芘、茚并芘、苯并苝、荧恩为碳源和能源生长并降解这些底物的菌株HBS1,该菌株的16S rRNA基因和gyrb基因序列与Gordonia amicalis的相应基因的相似     

Genetic Diversity of Gli-1, Gli-2 and Glu-1 Alleles in Sichuan Wheat Landraces

Genetic diversity at Gli-1, Gli-2 and Glu-1 loci was investigated in 89 Sichuan wheat ( Triticum aestivum L.) landraces by using acid polyacrylamide gel electrophoresis (APAGE) and SDS-PAGE. In these landraces, a total of 32 gliadin and 3 high-molecular-weight (HMW) glutenin patterns were observed. In total, 14, 15 and 5 alleles were identified at Gli-1, Gli-2 and Glu-1, respectively. At each locus, the alleles in higher frequency were Gli-A1a (89%), Gli-B1 h (46%), Gli-D1a (65%), Gli-A2a (64%), Gli-B2j (45%), Gli-D2 a (48%), Glu-A1c (99%), Glu-B1b (99%) and Glu-D1a (100%). The Nei's genetic variation index (H) of Sichuan wheat landraces was 0.3706, varying from 0 to 0.7087. The highest genetic diversity was found at Gli-B2 locus, while the lowest was found at Glu-D1 . The genetic diversity at Gli loci was higher than that of Glu-1 loci among these landraces, but it was much lower than that of modern wheat cultivars. These results indicated a narrow genetic base of Sichuan wheat landraces. In this study, “Chengdu-guangtou” had the identical gliadin and HMW-glutenin patterns with “Chinese Spring”, further supporting the proposal that “Chinese Spring” is a strain of “Chengdu-guangtou”.     

PFGE analysis of the rice genome: estimation of fragment sizes,organization of repetitive sequences and relationships between genetic and physical distances

Pulsed-field gel electrophoresis (PFGE) has been applied to analyze the rice nuclear genome. Probing 56 RFLP probes selected from the 12 rice chromosomes to PFGE blots of nine rare-cutting restriction enzymes revealed that there are relatively high numbers of rare-cutting restriction sites in the rice genome. The average sizes of restriction fragments detected by single-copy probes are smaller than 200 kb for all of the rare-cutting restriction enzymes examined. Sizes of fragments detected by repetitive probes are variable, depending on the probes analyzed. By using PFGE, a tandemly repeated sequence, Os48, was found to be tightly linked to telomeric tandem repeats but not physically linked to r5s genes with which sequence homology had been observed. Relationships between genetic and physical distances have been established for three different chromosomal segments. In these regions 1 cm corresponds to ca. 260 kb on average. Analysis of a cluster of RFLP markers on chromosome 3 revealed that genetically clustered RFLP markers are also physically closely linked, suggesting that clustering of genetic markers may result in part from uneven distribution of single-copy sequences.     

Genetic Diversity of Gli-1，Gli-2 and Glu-1 Alleles Among Chinese Endemic Wheats

Genetic diversity at Gli-1, Gli-2 and Glu-1 loci was investigated in 32 accessions of Chinese endemic wheat by using acid polyacrylamide gel electrophoresis (APAGE) and sodium dodecyl sulfate (SDS)-PAGE. There were 8 gliadin and 3 high-molecular-weight (HMW)-glutenin patterns in 14 Yunnan hulled wheat ( Triticum aestivum ssp. yunnanese King) accessions, 9 gliadin and 4 HMW-glutenin patterns in 9 Tibetan weedrace ( T. aestivum ssp. tibetanum Shao ) accessions, and 9 gliadin and 5 HMW-glutenin patterns in 9 Xinjiang rice wheat ( T. petropavlovskyi Udacz. et Migusch.) accessions. One accession (i.e. Daomai 2) carried new subunits 2.1+10.1 encoded by Glu-D1 . Among the three Chinese endemic wheat groups, a total of 10, 14 and 11 alleles at Gli-1 locus; 11, 14 and 12 alleles at Gli-2 locus; and 5, 6 and 8 alleles at Glu-1 locus were identified, respectively. Among Yunnan hulled wheat, Tibetan weedrace and Xinjiang rice wheat, the Nei's genetic variation indexes were 0.3798, 0.5625 and 0.5693, respectively. These results suggested that Tibetan weedrace and Xinjiang rice wheat had higher genetic diversity than Yunnan hulled wheat.     

Creating Wheat Germplasm for High Quality Breeding by Monosomic Backcrossing

By crossing bread wheat cultlvar GC8901 with the 1D monosonlc line of Xiaoyan No. 6 and backcrosslng the offsprlng with the Xlaoyan No. 6 1D monosonlc llne for 5 years, high-molecular-welght glutenin subunlts 1Dx5＋1Dy10 from GC8901 have been transferred Into wheat cultivar Xiaoyan No. 6. The BC5F1 offspring lines had been detected by using methods of cytology, marker, molecular marker and six elite single plants with high molecular-welght glutenin subunlts： lAx1, 1Bx14＋1 By15, 1Dx5＋1 Dy10 were Identified. Those lines have high-yleld potential with better agronomic characters and have been used In high quality wheat breeding processes as well.