A low molecular weight DNA polymerase from wheat embryos

The study of plant DNA polymerases lags far behind that concerning their animal or yeast counterpart. In this work we describe the first extensive purification to apparent homogeneity, as well as a detailed biochemical and immunological characterization, of a low molecular weight DNA polymerase (DNA polymerase C_I) purified from wheat embryos. The monomeric enzyme is a basic protein having a molecular weight of 52 kDa. Polyclonal antibodies raised in rabbits against DNA polymerase C_I did not inhibit animal DNA polymerases and or wheat DNA polymerase A, whereas wheat DNA polymerases C_II and B were much less affected than the C_I enzyme. Several properties of enzyme C_I were studied. Some known inhibitors of DNA polymerase activity including aphidicolin, phosphonoacetic acid and heparin, did not affect DNA polymerase C_I while the activity of this enzyme was strongly inhibited by ddTTP and N-ethylmaleimide. The polyamine spermine decreased markedly the enzyme activity, while spermidine produced a strong stimulation at the same concentrations that spermine inhibited the enzyme. The best template for this enzyme is poly dA-oligo dT, although polymerase C_I can recognize significantly some synthetic polyribonucleotide templates (poly rC-oligo dG, poly rA-oligo dT) but only at a given protein/template primer ratio. The enzyme is blocked at the amino terminus, thus preventing the automatic sequencing of the protein. The amino acid analysis showed a striking similarity with the animal low molecular weight DNA polymerase . The latter observation, as well as the effect of inhibitors (except N-ethylmaleimide which does not inhibit the animal polymerase) indicate that the DNA polymerase described in this work is a plant DNA polymerase very similar to the low molecular weight animal DNA polymerase , an enzyme believed to be involved in nuclear DNA repair.     

Isolation of DNA from plants high in polyphenolics

DNA suitable for digestion with restriction endonucleases has been isolated fromTheobroma cacao, a plant high in polyphenolic compounds, and several other plant taxa using the method presented in this paper. The method relies on concentrating nuclei away from most of the cytoplasmic components and inhibiting the formation of oxidized polyphenolic compounds and their interaction with the DNA fraction in the remaining steps.     

High recovery of large molecular weight DNA from sorted maize chromosomes

High recovery of large molecular weight DNA from single types of sorted chromosomes is a requirement for the construction of partially digested chromosome-specific libraries. We have developed a simple procedure, based on salting out, for rapidly obtaining large quantities of chromosomal DNA from flow-sorted chromosomes in maize. DNA isolated from sorted frozen chromosomes was of good quality, over 120 kb in size, and suitable for restriction enzyme analysis and construction of phage libraries.     

The isolation of high molecular weight DNA from wheat,barley and rye for analysis by pulse-field gel electrophoresis

A method is presented for the preparation of large DNA molecules from protoplasts embedded in agarose blocks of three different cereals-hexaploid bread wheat (Triticum aestivum), barley (Hordeum vulgare) and rye (Secale cereale). Pulse-field gel electrophoresis (PFGE) analysis of these DNA preparations using a contour-clamped homogeneous field (CHEF) apparatus indicated that the size of the DNA molecules was greater than 6 Mb. DNA samples prepared by this method were shown to be useful for restriction analysis using both frequent and rare cutting enzymes.     

Isolation of genomic DNA from the earthworm speciesEisenia fetida

Our interest in detecting genotoxic exposure in earthworms led us to isolate high quality DNA from theEisenia fetida species. For that, we compared a modification of the conventional phenol-chloroform extraction procedure, usually refered to as the Maniatis procedure, to two commercially available kits reportedly eliminating multiple partitions in phenol and chloroform, namely the Qiagen and Nucleon protocols. From the 260 nm optical density values, the commercial kits extracts hinted toward higher DNA recovery with those procedures. However, the 260/280 nm ratios indicated that the quality of the DNA isolated with the modified Maniatis procedure was purer than that isolated with the commercial kits, the latter being most probably contaminated by proteins and/or RNA. The Maniatis procedure was slightly modified by the introduction of a potassium acetate step for protein precipitation and by shortening the proteinase K treatment from 12–18 h to only 2 h. The higher quality of the DNA isolated by phenol-chloroform extraction was confirmed by quantification with the fluorescent 3,5-diaminobenzoic acid assay. Preliminary results suggest that the modified Maniatis procedure herein described is not only applicable for DNA adducts studies using³²P-postlabelling techniques but is also suitable for DNA extraction from other earthworm species such asLumbricus terrestris.     

Extraction of high molecular weight DNA from microbial mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes. The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 μg of HMW DNA (35-50 kb) per gram of mat sample, with an A(260/280) ratio of 1.6. Furthermore, amplification of 16S rRNA genes suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.     

In vitro phosphorylation of high molecular weight glutenin subunits from wheat endosperm

We have investigated the in vitro phosphorylation of high molecular weight glutenin subunits (HMW-GS), a group of non-soluble proteins present in wheat endosperm. Computer aided searches of potential biological sites in the known sequences of these proteins have evidenced the presence of sequence motifs specific for protein kinase C (PKC), calcium-dependent protein kinase from wheat, casein kinase II, tyrosine protein kinase and glycosylation. We have demonstrated that subunit 1Bx7 is a substrate of a partially purified PKC from rat brain. Further experiments have shown that this subunit is phosphorylated by an endogenous protein kinase activity found in wheat flour. These preliminary results are important for the possible implications on the structure-function relationships of these proteins and could probably suggest, for the first time, a potential physiological role in particular situations for some HMW-GS.     

Isolation and characterization of satellite DNA from mustard seedlings

The DNA from mustard (Sinapis alba L.) seedlings was examined by neutral CsCl and Ag⁺/Cs₂SO₄ density gradient centrifugation. Different satellite fractions were revealed by these two methods. The satellite fractions obtained from the Ag⁺/Cs₂SO₄ density gradient could not be generally correlated with satellite DNA fractions observed in CsCl. In CsCl density gradient centrifugation, a main band at density 1,695 g/cm³ and a heavy shoulder at density 1,703 g/cm³ are found. By preparative CsCl gradient centrifugation the heavy shoulder can be enriched but not completely separated from the main band DNA.—Gradient centrifugation by complexing the DNA with Ag⁺ rf. 0.25 to DNA phosphate reveals three distinct fractions which are further characterized: The heavy satelite DNA fraction revealed by Ag⁺/Cs₂SO₄ gradient centrifugation has the same density in a CsCl gradient and the same Tm value as the main band, but differs from main band DNA in the details of its melting profile and in its renaturation kinetics. The light Ag⁺/Cs₂SO₄ satellite DNA fraction had a higher melting temperature corresponding to a GC-rich base composition. Differences between these 3 fractions are observed in thermal denaturation and renaturation profiles, hybridization in situ with ribosomal RNA, and their response to restriction endonuclease digestion. The light satellite fraction from the Ag⁺/Cs₂SO₄ gradient, rich in ribosomal cistrons corresponds to the heavy shoulder DNA of neutral CsCl gradients which also is rich in ribosomal cistrons. The heavy satellite fraction from Ag⁺/Cs₂SO₄ gradient which contains highly repetitive short nucleotide sequences could not be revealed by the classical CsCl gradient centrifugation technique.     

Rapid extraction and purification of environmental DNA for molecular cloning applications and molecular diversity studies

A rapid method for the extraction and purification of DNA from environmental samples for molecular cloning applications was developed. The indigenous cells from plant debris, organic materials, sediments, and soils were lysed directly by using DAS-I^Z solution and the nucleic acids were precipitated with isopropanol. A simple purification step using DAS-II^Z solution without binding matrix produced highly pure, colorless and undegraded DNA with molecular weight of more than 20 kb. The superiority of this method was tested for wide applications in molecular cloning, i.e., construction of genomic library by using Lambda DASH^(R)II Vector and Gigapack^(R)III XL, plasmid library, cloning of gene encoding protease, and molecular microbial diversity analysis. An additional advantage of this method is that only 0.1 g of sample is required, if analysis of many samples in short time should be done. To extract large amounts of environmental DNA for molecular cloning lasts only 30 min and to purify it less than 1 h.     

Purification and characterization of a high molecular weight endoxylanase from the solid-state culture of an alkali-tolerant Aspergillus fumigatus MKU1

Summary A high molecular weight endoxylanase (XylF2) from the solid state culture of Aspergillus fumigatus MKU1 was purified to homogeneity by a combination of tube gel electrophoresis and electroelution methods. The purity was demonstrated by SDS-PAGE and the molecular mass of the XylF2 was found to be 66 kDa. The optimal pH and temperature for activity were 5.0 and 90 °C, respectively. The apparent K _m and V _max values of XylF2 with oat spelt xylan as substrate were 1.6 mg/ml and 3.25 mmol/min/mg protein respectively. The enzyme showed high activity towards oat spelt xylan while negligible activity was observed on carboxymethylcellulose. The activity of XylF2 was strongly inhibited by Hg²⁺, Ni²⁺, Zn²⁺, SDS and N-bromosuccinimide and stimulated by l-cysteine and iodoacetamide. The hydrolysis of oat spelt xylan by XylF2 released only xylo-oligosaccharides.     

Improved methods for the preparation of high molecular weight DNA from large and small scale cultures of filamentous fungi

Improved methods are described for the isolation of pure, high molecular weight DNA from small and large scale cultures of filamentous fungi. The methods depend on the extraction of DNA under conditions which prevent nuclease activity and contamination by carbohydrate. The small scale method depends on enzymatic digestion of the wall whereas the large scale method uses partial damage followed by autolysis. High yields of DNA are obtained by both methods and the DNA is suitable for restriction analysis. Southern Blotting, RFLP analysis, dot blotting and the production of gene libraries. The small scale method can be used for the simultaneous analysis of multiple cultures.     

带芒草属物种新型高分子量谷蛋白亚基的鉴定

采用SDSPAGE方法对牧草带芒草属3个种8份材料的高分子量谷蛋白进行了检测和鉴定。结果显示,带芒草物种具有的高分子量谷蛋白亚基与普通小麦中发现的不一样,其迁移率存在较大差异。其中,x型亚基均比Dx2亚基迁移率小或接近,y型亚基均比Dx12亚基迁移率大。8份材料中共发现了4种x型亚基新类型(Tax1,Tax2,Tax3和Tax4),5种y型亚基新类型(Tay1,Tay2,Tay3,Tay4和Tay5)和6种亚基组合类型(Tax1+Tay3,Tax3+Tay2,Tax4+Tay1,Tax1+Tay1,Tax2+Tay5,Tax4+Tay2),该项研究结果揭示了带芒草属植物可能具有与普通小麦类似的高分子量谷蛋白亚基,这些亚基在小麦品质遗传改良中具有潜在的利用价值。     

The effect of whey protein hydrolyzate average molecular weight on the lactic acid fermentation

Summary The batch fermentation of whey permeate to lactic acid was improved by supplementing the broth with enzyme-hydrolyzed whey protein. Hydrolyzates prepared with endoprotease were more stimulatory to acid production rates than were those prepared with exo/endo protease. The effect of hydrolyzate average molecular weight on acid production is presented. Results show that the hydrolyzate having an average molecular weight of 700 is the most stimulatory to acid production rates.     

Low molecular weight subunits associated with the cytochrome b 6 f complexes from spinach and Chlamydomonas reinhardtii

Cytochrome b ₆ f complexes, prepared from spinach and Chlamydomonas thylakoids, have been examined for their content of low molecular weight subunits. The spinach complex contains two prominent low molecular weight subunits of 3.7 and 4.1 kD while a single prominent component of 4.5 kD was present in the Chlamydomonas complex. An estimation of the relative stoichiometry of these subunits suggests several are present at levels approximating one copy per cytochrome complex. The low molecular weight subunits were purified by reversed phase HPLC and N-terminal sequences obtained. Both the spinach and Chlamydomonas cytochrome complexes contain a subunit that is identified as the previously characterized petG gene product (4.8 kD in spinach and 4.1 kD in Chlamydomonas). A second subunit (3.8 kD in spinach and 3.7 kD in Chlamydomonas) appears to be homologous in the two complexes and is likely to be a nuclear gene product. The possible presence of other low molecular weight subunits in these complexes is also considered.     

Isolation and identification of a high molecular weight protein in sow milk

A high molecular weight protein (HMWP) was isolated and purified from sow milk, and some of its biochemical characteristics and biological functions were identified. The origin of HMWP was also investigated. The molecular weight of HMWP was determined to be about 115 000 and 114 800 by SDS-PAGE and gel filtration, respectively. The sequence of 10 amino acids in N-terminal of HMWP was Ala-Leu-Val-Gln-Ser-Cys-Leu-Asn-Leu-Val. The sequence was blasted against GenBank. No protein showed significant similarity with this sequence suggesting the HMWP may be novel. The result of liquid chromatography mass spectrometry (LC-MS) also proved HMWP could be a novel protein. By amino acid assay, HMWP was rich in glutamate (including glutamine), cysteine, glycine, aspartic acid (including asparagines) and proline. The content of hydrophobic amino acids (Ala, Val, Leu, Ile, Met, Phe and Pro) was lower at 18.59% of the total amino acids suggesting HMWP has high solubility in water. Western blots of lectins were used to identify the kinds of carbohydrate residues attached to HMWP qualitatively. The result showed that HMWP was a kind of glycoprotein containing N-acetylneuraminic acid (NeuNAc), mannose (Man) and/or N-acetylglucosamine (GlcNAc). By isoelectric focusing, HMWP pI was found to be 5.1. Compared with milk fat globule membrane protein (MFGMP) isolated from the sow milk in SDS-PAGE, MFGMP did not contain HMWP. HMWP was assumed to be a secretory milk protein. HMWP was not found in bovine, goat, rabbit or human milk in SDS-PAGE gel suggesting HMWP may be unique to sow milk. By Western blot, HMWP could be detected in sow milk, not in sow serum, which suggests it is synthesized and secreted by the mammary gland. HMWP concentrations in sows milk were the lowest in the first day of lactation, rose significantly during lactation 1 to 7 days. The HMWP content of sows milk remained relatively constant ((1.95±0.13) g/l) during lactation 7 to 20 days. HMWP significantly inhibited Escherichia coli in a dose related manner in vitro. Overall, HMWP could be a novel sow milk protein with implications for the mammary gland and the piglet.     

Evidence from subunit molecular weight suggests hybridization was the source of the phosphoglucose isomerase gene duplication in Clarkia

Summary The apparent molecular weight of cytosolic phosphoglucose isomerase (PGI) subunits was evaluated in 18 species of Clarkia which have or do not have duplicated genes specifying this glycolytic enzyme. Species that lack the duplication had subunits of 59,000 or 60,400 whereas species with the duplication generally possessed two types of PGI subunits with these or closely similar molecular weights. The additive pattern in the species with the duplication suggests that the molecular weight divergence preceded the origin of the duplication, and that the duplication arose following hybridization between taxa that represented different lineages within Clarkia.     

A simple and rapid method for preparing genomic DNA from gram-positive bacteria

Molecular epidemiologic and other studies may require preparation of genomic DNA from large numbers of bacteria in sufficiently pure form for restriction endonuclease digestion, cloning, RAPD-PCR, Southern hybridization, and so on.Staphylococcus and other Gram-positive bacteria have a rigid cell wall and can be difficult to lyse. Here, a simple and rapid method for the preparation of genomic DNA from multiple samples is reported. This method produces clean DNA for use in most molecular biology methods in <90 min.     

Distribution of low molecular weight carbohydrates in the subgenusCeratotropis of the genusVigna (Leguminosae)

The seeds of 9 members of the subgenusCeratotropis (Piper) Verdc., namelyVigna aconitifolia (Jacq.) Maréchal,V. angularis (Willd.) Ohwi et Ohashi,V. minima (Roxb.) Ohwi et Ohashi,V. nakashimae (Ohwi) Ohwi et Ohashi,V. reflexo-pilosa Hayata,V. umbellata (Thumb.) Ohwi et Ohashi,V. mungo (L.) Hepper,V. radiata (L.) Wilczek andV. sp., have been examined. On their low molecular weight carbohydrate compositions, this subgenus has been divided into 2 subgroups, mungo-radiata group and angularis group. Four other species referred to the subgeneraPlectotropis (Schumach.) Bak.,Lasiospron (Benth. emend Piper) Maréchal, Mascherpa et Stainier andVigna, V. vexillata (L.) A. Rich.,V. lasiocarpa (Benth.) Verdc.,V. marina (Burm.) Merr. andV. unguiculata (L.) Walp., were also analyzed and they had distinctive carbohydrate compositions. 1d-4-O-methyl-myo-inositol and 1d-5-O-(α-d-galactopyranosyl)-4-O-methyl-myo-inositol were detected in all species examined exceptV. vexillata, V. mungo andV. radiata.