Measurement of 60Co-λ ray-induced DNA damage by capillary electrophoresis

Capillary electrophoresis was employed in this study to monitor ⁶⁰Co-γ-ray-induced damage to a 1 kb DNA ladder which consists of restriction fragments ranging from 75 to 12 000 bp. DNA samples (0.5 mg/ml) were exposed to 0–60 Gy of γ-radiation in the presence and absence of 110 μmol/l ethidium bromide (EB). The analysis showed peak broadening without significant changes in the size distribution of irradiated fragments. Radiation-induced conformational changes may account for this peak broadening. EB addition caused small increases in the retention times of DNA fragments without affecting the overall DNA damage. This indicates that the presence of intercalated EB during radiation will not stabilize the DNA against ⁶⁰Co-γ ray-induced damage.     

利用多重PCR反应同时筛选番茄Cf-9和Tm-1基因

利用同一PCR反应体系,对分别与番茄抗叶霉病的Cf-9基因和抗番茄烟草花叶病毒病的Tm-1基因紧密连锁的PCR标记进行了同时扩增筛选,扩增的特异性片段与单引物扩增片段吻合。其中与Cf-9基因紧密连锁的CAPs标记在抗感试材均可扩增出560bp的特异片段,且都存在TaqⅠ酶切位点,抗病基因型酶切后分别产生了450bp、330bp和290bp的不同特异性片段,而感病基因型试材酶切后产生450bp和290bp的特异性片段;与Tm-1基因紧密连锁的SCAR标记为显性标记,只有抗病试材产生750bp的特异片段,不能被TaqⅠ酶切。经反复验证,结果稳定准确,可用于在同一PCR反应体系中对两个抗病基因进行同时筛选鉴定。该体系的建立不仅省时、省工、节省费用,而且可用于苗期辅助选育,加快番茄抗病育种进程。     

Near ultraviolet circular dichroism spectroscopy of plasma fibronectin and fibronectin fragments

Comparison of the near uv CD spectrum of human plasma fibronectin with the spectra of the three major leukocyte elastase fragments 25 Kd, 60 Kd and $\text{140}\text{160}$ shows that the aromatic residues of these fragments are in different environments. In particular, the ellipticity bands associated with tryptophan in the 290–300 nm region differ for each fragment. The results also show that intrachain disulfide bridges are important in stabilizing regions of the fibronectin molecule against the structure-disrupting effects of 8M urea.     

A method for the generation of small pre-determined deletions in plasmid DNA: deletion analysis of the tetR region of vector pBR322

A general method is described that allows precise deletion of a chosen restriction fragment(s) from a plasmid having many cleavage sites for that restriction enzyme. The DNA to be deleted is first separated from the rest of the plasmid on a larger DNA fragment contained between two different unique restriction sites. This fragment is then subdigested by the restriction endonuclease of interest, which recognises two or more tetranucleotide (cohesive end or blunt end) sequences on the fragment, and is recloned between the two original unique restriction sites. The method is rapid, efficient, and the results are predictable. Examples are given in which predetermined HpaII (9 bp, 147 bp), TaqI (141 bp) and AluI (15 bp, 403 bp) fragments have been selectively removed from the tetR region of plasmid pBR322.     

Restriction endonucleases in Azospirillum

Azospirillum brasilense, A. amazonense, and A. lipoferum strains were screened for restriction endonucleases using phage lambda DNA. The extract of A. brasilense 29711 cleaved lambda DNA into specific fragments. It was concluded that this strain possesses a class II restriction endonuclease which was named AbrI. AbrI has a single recognition site on lambda DNA at position of approx. 33 500 bp. AbrI was characterized as an isoschizomer of XhoI, which cuts lambda DNA at 33 498 bp and cleaves double-stranded DNA at the sequence 5'-C TCGAG-3'. From other Azospirilla strains only A. amazonense QRZ42 extracts (AamI activity) cleaved DNA into specific fragments under certain conditions.     

The thermodynamic stability of nanoconstructions based on the double-stranded DNA molecules

The temperature stability of nanoconstructions formed by double-stranded DNA molecules fixed in the structure of their liquid crystalline dispersions and cross-linked by nanobridges was determined. It was shown that the heating of nanoconstructions is accompanied by a decrease in the amplitude of the negative bands in the CD spectrum both at lambda approximately 310 and lambda approximately 505 nm. Temperature "melting curves" were derived and characterzed by T(M) values. The T(M) values at lambda approximately 310 and lambda approximately 505 nm coincided with each other but differed from the T(M) value characteristic of the DNA cholesteric liquid crystalline dispersion.     

Linear dichroism characteristics of ethidium-and proflavine-supercoiled DNA complexes

A flow linear dichroism technique is utilized to study the unwinding of supercoiled DNA induced by the binding of ethidium bromide (EB) and proflavine (PF) at different ratios r (drug added/DNA base). In the case of either EB or PF bound to linear calf thymus DNA, the reduced linear dichroism signals LD/A (LD: linear dichroism; A: absorbance, both measured at the same wavelength), determined at 258, and 520 or 462 nm (corresponding to contributions predominantly from the partially oriented DNA bases, intercalated EB, or PF, respectively) are nearly independent of drug concentration. In the case of supercoiled DNA, the magnitude of LD/A at 258 nm first increases to a maximum value near r = 0.04-0.05, and then decreases as r is increased further, mimicking the behavior of the sedimentation coefficients, viscosities, and gel electrophoresis patterns measured by other workers at similar values of r. However, LD/A at 520 nm, which is due to DNA-bound EB molecules, is constant within the range of r values of 0.02-0.06 in which the magnitude of LD/A determined at 258 nm due to the DNA bases exhibits a pronounced maximum. In contrast, in the case of PF, the magnitudes of LD/A determined at 258 or 462 nm are characterized by similar dependencies on r, both exhibiting pronounced maxima at r = 0.05; this parallel behavior is expected according to a simple intercalation model in which the DNA bases and drug molecules are stacked on top of one another, and in which both are oriented to similar extents in the flow gradient. The unexpected differences in the dependencies of (LD/A)258 and (LD/A)520 on r in the case of EB bound to supercoiled DNA, are attributed to differences in the net overall alignment of the EB molecules and DNA bases in the flow gradient. The magnitude of the LD signal at 258 nm reflects the overall degree of orientation of the supercoiled DNA molecules that, in turn, depends on their hydrodynamic shapes and sizes; the LD signals characterizing the bound EB molecules may reflect this orientation also, as well as the partial alignment of individual DNA segments containing bound EB molecules.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular cloning and sequencing of lysozyme gene of bacteriophage SF6 ofBacillus subtilis

Digestion of bacteriophage SF6 (ofBacillus subtilis) DNA with restriction endonuclease Pst I generates seven fragments (A-G). These fragments were cloned in pBR322 DNA, and the recombinant clones carrying the lysozyme gene were identified by measuring lysozyme activity. The complete nucleotide sequence of 996 bp fragment D, containing lysozymze gene, was determined. One open reading frame was present at 13 bp, and the termination codons were present at 961 bp. The deduced amino acid sequence of the lysozyme gene was also determined.     

Sequence variation in class 1 outer membrane protein in Neisseria meningitidis isolated from patients with meningococcal infection and close household contacts

Abstract Aspergillus oryzae , which is widely used for Japanese traditional fermentation, produced at least two lipolytic enzymes (L1 and L2). Southern hybridization analysis of restriction enzyme-digested genomic DNA fragments of Aspergillus oryzae with 23-mer oligonucleotides synthesized according to the amino acid sequence of the enzyme L1 as probes suggested that there is single copy of the L1 gene in the genome. DNA fragments containing the L1 gene were cloned in Escherichia coli . Nucleotide sequencing of the DNA fragments revealed an open reading frame consisting of 213 amino acid residues. It had three putative introns whose sizes were 52 bp, 48 bp and 53 bp, respectively. Putative CAAT and TATA boxes were found at positions −147 and −100 from A (+1) of the translational initiation codon, and a polyadenylation site at 158 bp downstream of the stop codon. The deduced amino acid sequence of the L1 gene was highly similar to those of cutinases from phytopathogenic fungi. Thus, it is interesting to note that the non-phytopathogenic fungus, A. oryzae , produces cutinase, which seems to play an important role in flavor formation.     

Fluorescence emission spectra of chloroplasts and subchloroplast preparations at low temperature.

A study was made of the chlorophyll fluorescence spectra between 100 and 4.2 K of chloroplasts of various species of higher plants (wild strains and chlorophyll b mutants) and of subchloroplast particles enriched in Photosystem I or II. The chloroplast spectra showed the well known emission bands at about 685, 695 and 715--740 nm; the System I and II particles showed bands at about 675, 695 and 720 nm and near 685 nm, respectively. The effect of temperature lowering was similar for chloroplasts and subchloroplast particles; for the long wave bands an increase in intensity occurred mainly between 100 and 50 K, whereas the bands near 685 nm showed a considerable increase in the region of 50--4.2 K. In addition to this we observed an emission band near 680 nm in chloroplasts, the amplitude of which was less dependent on temperature. The band was missing in barley mutant no. 2, which lacks the light-harvesting chlorophyll a/b-protein complex. At 4.7 K the spectra of the variable fluorescence (Fv) consisted mainly of the emission bands near 685 and 695 nm, and showed only little far-red emission and no contribution of the band at 680 nm. From these and other data it is concluded that the emission at 680 nm is due to the light-harvesting complex, and that the bands at 685 and 695 nm are emitted by the System II pigment-protein complex. At 4.2 K, energy transfer from System II to the light-harvesting complex is blocked, but not from the light-harvesting to the System I and System II complexes. The fluorescence yield of the chlorophyll species emitting at 685 nm appears to be directly modulated by the trapping state of the reaction center.     

毛囊蠕形螨与皮脂蠕形螨基因组DNA的RAPD分析和序列比对

【目的】分析毛囊蠕形螨Demodex folliculorum（D.f.）和皮脂蠕形螨D. brevis（D.b.）基因组DNA的多态性, 对相关条带进行测序分析。【方法】采用改良小昆虫DNA提取法提取两种人体蠕形螨基因组DNA, 选择RAPD技术对其进行多态性分析, 将相关条带分别与pMD18-T载体连接, 克隆、测序后进行酶切鉴定和分析。【结果】毛囊蠕形螨共扩增15条带, 皮脂蠕形螨共扩增12条带;两种蠕形螨既有共有条带, 又有特异性条带;根据条带差异计算得到两种间的遗传距离为0.5556. 毛囊蠕形螨约800 bp处特异性条带测序结果显示, 序列片段长度为855 bp（GenBank登录号为FI277970）;特异性引物扩增和酶切鉴定均为毛囊蠕形螨所特有. 序列比对显示与阿糖胞苷DNA区域结合蛋白有46%的序列相似度。两种人体蠕形螨约300 bp处共有条带序列分析显示, 碱基序列均为341 bp（GenBank登录号分别为D.f. FI520176;D.b. FI520175）, 在第84和第165位点有2个碱基不同, 分别是A/G和C/T互换, 同源性高达99.4%. 但未发现有开放阅读框和相似度高的序列。 【结论】序列片段为855 bp的特异性条带为毛囊蠕形螨所特有;341 bp碱基序列为毛囊蠕形螨和皮脂蠕形螨所共有, 同源性高达99.4%. RAPD技术可用于两种人体蠕形螨基因组DNA的多态性分析和物种鉴定。     

Fluorescence decay measurements for determining the relative content of ethidium bromide to DNA in situ in cell nuclei

A fluorometric method for the determination of the amount of ethidium bromide (EB) bound to DNA in situ in cell nuclei is discussed. Even when the EB content was very small, the molar ratio of DNA-phosphorus (DNA-p) to dye (P/D ratio) could be estimated by measuring the lifetime of the transient fluorescence of the EB-DNA complex as a function of the P/D ratio. To examine the relationship between the fluorescence intensity, lifetime, and P/D ratio, polyacrylamide gel film containing 4.7 mM DNA-p was used as a model DNA tissue, and its fluorescence was measured using a nanosecond microfluorometer. The fluorescence intensity showed a maximum at P/D = 6. The fluorescence lifetime increased with the P/D ratio, and this was accompanied by a proportional increase in the quantum efficiency. Thus, the lifetime value was an effective parameter for the determination of the P/D ratio in situ in tissue. When this approach was applied to tissue sections of mouse liver treated with solutions of EB at concentrations of 10 and 50 micrograms/ml, the fluorescence lifetimes on cell nuclei were 18.9 and 17.4 ns with P/D ratios of 20 and 12, respectively, as based on the model-tissue experiments. When the P/D ratio was 20, the concentration of EB in the nucleus was approximately 1.5 mM, i.e., 60 times higher than that in the staining solution.     

Interaction of ethidium bromide with DNA: effect of LiCl and ethylene glycol.

The interaction of the intercalating dye ethidium bromide with several native and synthetic polydeoxyribonucleic acids has been studied by means of circular dichroic spectra. The CD of DNA-ethidium bromide complexes in the 290-360 nm region is characterized, especially at high salt and at high ethylene glycol content, by positive and negative bands near 308 nm and 295 nm, respectively. These dye associated CD bands are unaffected by the addition of LiCl or ethylene glycol, suggesting that the relative conformation of dye and neighboring base pairs does not change when the conformation of the rest of the DNA changes.     

Dose response, wavelength dependence and rate of excision of ultraviolet radiation-induced pyrimidine dimers in mouse skin DNA

The yield of thymine-containing dimers produced in mouse skin DNA in vivo by 290 nm ultraviolet radiation was shown to increase with dose up to around 2000 J/m2 and subsequently at a much slower rate up to 8000 J/m2. The study of wavelength dependence of dimer formation in skin indicated that 290 nm was the most effective wavelength of those investigated, followed by 300, 280 and 260 nm, with 310 nm being by far the least effective. A reduction in the number of dimers present in skin DNA was shown to occur by 24 h post-irradiation in a dose-dependent manner. A significant percentage of the dimers was, however, found to persist in the skin until at least 72 h post-irradiation.     

The Complex Satellite DNA of Tropaeolum majus L.: Partial Characterization of Isolated and of Cloned Restriction Fragments

Satellite DNA (satDNA) was purified by repeated neutral CsCl,ethidium bromide/CsCl and actinomycin D/CsCl density centrifugationfrom total nuclear DNA of Tropaeolum majus. Digestion patternsof satDNA and main band DNA as generated with 22 different restrictionenzymes were partially characterized and hybridized by the Southernblot technique with Hpa II-generated satDNA fragments and aribosomal DNA fragment. Some restriction fragments were sequencedby the chemical method, either directly, or after cloning, andpossible secondary structures were determined. The results showthat the satellite is of high complexity and evidently composedof different, interspersed repeats, and is also interspersedwith ribosomal sequences. Some sequences of the satellite aredispersed throughout the Tropaeolum genome, and even found inthe chloroplast genome. Most of the restriction enzymes cutthe satDNA into many fragments of lengths between 14 bp and3000 bp. Hpa II cut the satellite DNA into at least 63 differentsized fragments (more than is known from any other plant satDNA).Sal I cut the satDNA into a single fragment of 1040 bp in size,which probably represented the superrepeat unit. Sequence analysisof satDNA restriction fragments and of cloned fragments isolatedat different times, indicated a rapid diversification in vivoand in vitro. Computer modeling of most likely secondary structuressuggest the occurrence of palindromic loops (cruciform structures).These could be the basis of rapid diversification in being sitesof preferred intragenomic recombination and rearrangement. Cloning, palindromic sequences, restriction analysis, satellite DNA complexity, Tropaeolum majus L, nasturtium     

A novel method for the transport and analysis of genetic material from polyps and zooxanthellae of scleractinian corals

We have developed a new simple method for transport, storage, and analysis of genetic material from the corals Agaricia agaricites, Dendrogyra cylindrica, Eusmilia ancora, Meandrina meandrites, Montastrea annularis, Porites astreoides, Porites furcata, Porites porites, and Siderastrea siderea at room temperature. All species yielded sufficient DNA from a single FTA card (19 microg-43 ng) for subsequent PCR amplification of both coral and zooxanthellar DNA. The D1 and D2 variable region of the large subunit rRNA gene (LSUrDNA) was amplified from the DNA of P. furcata and S. siderea by PCR. Electrophoresis yielded two major DNA bands: an 800-base pair (bp) DNA, which represented the coral ribosomal RNA (rRNA) gene, and a 600-bp DNA, which represented the zooxanthellar srRNA gene. Extraction of DNA from the bands yielded between 290 microg total DNA (S. siderea coral DNA) and 9 microg total DNA (P. furcata zooxanthellar DNA). The ability to transport and store genetic material from scleractinian corals without resort to laboratory facilities in the field allows for the molecular study of a far wider range and variety of coral sites than have been studied to date.     

Fluorescence decay measurements for determining the relative content of ethidium bromide to DNA in situ in cell nuclei

Summary A fluorometric method for the determination of the amount of ethidium bromide (EB) bound to DNA in situ in cell nuclei is discussed. Even when the EB content was very small, the molar ratio of DNA-phosphorus (DNA-p) to dye (P/D ratio) could be estimated by measuring the lifetime of the transient fluorescence of the EB-DNA complex as a function of the P/D ratio. To examine the relationship between the fluorescence intensity, lifetime, and P/D ratio, polyacrylamide gel film containing 4.7 mM DNA-P was used as a model DNA tissue, and its fluorescence was measured using a nanosecond microfluorometer. The fluorescence intensity showed a maximum at P/D=6. The fluorescence lifetime increased with the P/D ratio, and this was accompanied by a proportional increase in the quantum efficiency. Thus, the lifetime value was an effective parameter for the determination of the P/D ratio in situ in tissue. When this approach was applied to tissue sections of mouse liver treated with solutions of EB at concentrations of 10 and 50 g/ml, the fluorescence lifetimes on cell nuclei were 18.9 and 17.4 ns with P/D ratios of 20 and 12, respectively, as based on the model-tissue experiments. When the P/D ratio was 20, the concentration of EB in the nucleus was approximately 1.5 mM, i.e., 60 times higher than that in the staining solution.     

Circular dichroism of squid rhodopsin and its intermediates

Circular dichroism (CD) and absorption spectra of squid (Todarodes pacificus) rhodopsin, isorhodopsin and the intermediates were measured at low temperatures. Squid rhodopsin has positive CD bands at wavelengths corresponding the - and β-absorption bands at liquid nitrogen temperature (CD maxima: 485 nm at -band and 348 nm at β-band) as well as at room temperature (CD maxima: 474 nm at -band and 347 nm at β-band). The rotational strength of the -band has a molecular ellipticity about twice that of cattle rhodopsin. The CD spectrum of bathorhodopsin displays a negative peak at 532 nm, the rotational strength of which has an absolute value slightly larger than that of rhodopsin. The reversal in sign at -band of the CD spectrum may indicate that the isomerization of retinal chromophore from twisted 11-cis form to twisted 11-trans form has occurred in the process of conversion from rhodopsin to bathorhodopsin. Lumirhodopsin has a small negative CD band at 490 nm, the maximum of which lies at 25 nm shorter wavelengths than the absorption maximum (515 nm), and a large positive CD band near 290 nm, which is not observed in rhodopsin and the other intermediates. This band may be derived from a conformational change of the opsin. In the process of changing from lumirhodopsin to LM-rhodopsin, the CD bands at visible and near ultraviolet regions disappear. Both alkaline and acid metarhodopsins have no CD bands at visible and near ultraviolet regions.     

Separation of fluorescence-labelled terminal restriction fragment DNA on a two-dimensional gel (T-RFs-2D) - an efficient approach for microbial consortium characterization

Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (±1) bp and 514 (±1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities.