Laser cross-linking of nucleic acids to proteins. Methodology and first applications to the phage T4 DNA replication system

Single-pulse (approximately 8 ns) ultraviolet laser excitation of protein-nucleic acid complexes can result in efficient and rapid covalent cross-linking of proteins to nucleic acids. The reaction produces no nucleic acid-nucleic acid or protein-protein cross-links, and no nucleic acid degradation. The efficiency of cross-linking is dependent on the wavelength of the exciting radiation, on the nucleotide composition of the nucleic acid, and on the total photon flux. The yield of cross-links/laser pulse is largest between 245 and 280 nm; cross-links are obtained with far UV photons (200-240 nm) as well, but in this range appreciable protein degradation is also observed. The method has been calibrated using the phage T4-coded gene 32 (single-stranded DNA-binding) protein interaction with oligonucleotides, for which binding constants have been measured previously by standard physical chemical methods (Kowalczykowski, S. C., Lonberg, N., Newport, J. W., and von Hippel, P. H. (1981) J. Mol. Biol. 145, 75-104). Photoactivation occurs primarily through the nucleotide residues of DNA and RNA at excitation wavelengths greater than 245 nm, with reaction through thymidine being greatly favored. The nucleotide residues may be ranked in order of decreasing photoreactivity as: dT much greater than dC greater than rU greater than rC, dA, dG. Cross-linking appears to be a single-photon process and occurs through single nucleotide (dT) residues; pyrimidine dimer formation is not involved. Preliminary studies of the individual proteins of the five-protein T4 DNA replication complex show that gene 43 protein (polymerase), gene 32 protein, and gene 44 and 45 (polymerase accessory) proteins all make contact with DNA, and can be cross-linked to it, whereas gene 62 (polymerase accessory) protein cannot. A survey of other nucleic acid-binding proteins has shown that E. coli RNA polymerase, DNA polymerase I, and rho protein can all be cross-linked to various nucleic acids by the laser technique. The potential uses of this procedure in probing protein-nucleic acid interactions are discussed.     

Regulation of Flavonoid Biosynthetic Genes in Germinating Arabidopsis Seedlings

Many higher plants, including Arabidopsis, transiently display purple anthocyanin pigments just after seed germination. We observed that steady state levels of mRNAs encoded by four flavonoid biosynthetic genes, PAL1 (encoding phenylalanine ammonia-lyase 1), CHS (encoding chalcone synthase), CHI (encoding chalcone isomerase), and DFR (encoding dihydroflavonol reductase), were temporally regulated, peaking in 3-day-old seedlings grown in continuous white light. Except for the case of PAL1 mRNA, mRNA levels for these flavonoid genes were very low in seedlings grown in darkness. Light induction studies using seedlings grown in darkness showed that PAL1 mRNA began to accumulate before CHS and CHI mRNAs, which, in turn, began to accumulate before DFR mRNA. This order of induction is the same as the order of the biosynthetic steps in flavonoid biosynthesis. Our results suggest that the flavonoid biosynthetic pathway is coordinately regulated by a developmental timing mechanism during germination. Blue light and UVB light induction experiments using red light- and dark-grown seedlings showed that the flavonoid biosynthetic genes are induced most effectively by UVB light and that blue light induction is mediated by a specific blue light receptor.     

Raman spectra of the model B-DNA oligomer d(CGCGAATTCGCG)2 and of the DNA in living salmon sperm show that both have very similar B-type conformations

Raman spectra were obtained from aqueous solutions of the deoxyoligonucleotide d(CGCGAATTCGCG)2 (I), which has been suggested as a model for B-type DNA conformation. These spectra were compared with the Raman spectra of the aqueous solutions of several DNAs of natural origin taken under identical solution conditions. Since the model sequence has a high percent GC (66%), the Raman spectrum was compared with the Raman spectrum of the DNA from Micrococcus lysodeikticus (72% GC), and the spectra of the two different DNAs were found to be rather similar in both 50 mM salt and 6 M salt solutions. Computer-aided band-shape analysis of the backbone vibrational region of the Raman spectra shows the existence of several bands corresponding to different furanose ring puckers. This appears to indicate a heterogeneity of furanose ring pucker in both the model dodecamer and the native DNA. Significant differences were found in the intensity of the conformational marker band at 810 cm-1, which indicates corresponding differences in furanose ring pucker heterogeneities in these two high GC content DNAs. The Raman spectrum of the dodecamer (I) was used to analyze the Raman spectrum of the DNA inside the head of living intact salmon sperm. Sperm spectra were taken with both our conventional Raman spectrograph and a newly developed intracavity laser Raman microscope system. Although the DNA in the sperm head is required by packing considerations to be in a highly compact and condensed state, the Raman spectra of the intact sperm are almost identical with that of the model dodecamer (I) if the difference in base composition is taken into account.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis

Eleven loci that play a role in the synthesis of flavonoids in Arabidopsis are described. Mutations at these loci, collectively named transparent testa (tt) , disrupt the synthesis of brown pigments in the seed coat (testa). Several of these loci ( tt3, tt4, tt5 and ttg ) are also required for the accumulation of purple anthocyanins in leaves and stems and one locus ( ttg ) plays additional roles in trichome and root hair development. Specific functions were previously assigned to tt1–7 and ttg . Here, the results of additional genetic, biochemical and molecular analyses of these mutants are described. Genetic map positions were determined for tt8, tt9 and tt10 . Thin-layer chromatography identified tissue- and locus-specific differences in the flavonols and anthocyanidins synthesized by mutant and wild-type plants. It was found that UV light reveals distinct differences in the floral tissues of tt3, tt4, tt5, tt6 and ttg , even though these tissues are indistinguishable under visible light. Evidence was also uncovered that tt8 and ttg specifically affect dihydroflavonol reductase gene expression. A summary of these and previously published results are incorporated into an overview of the genetics of flavonoid biosynthesis in Arabidopsis .     

Plasma lipoproteins of lambs and sheep.

The major plasma lipoproteins of the adult sheep were high density lipoprotein (76%) having alpha-mobility on electrophoresis and low density lipoprotein (20%) having beta-mobility. Chylomicrons and very low density lipoproteins were minor constituents (less than 5%). The postabsorptive hyperlipidaemia in suckling lambs is mainly a result of increased concentration of low density and high density lipoproteins although the relative contribution of very low density lipoproteins was increased to 7-15% of the total lipoproteins. The hyperlipiaemia was markedly greater in an intact male lamb than in female or castrated male lambs. In suckling lambs a new lipoprotein (density 1-090 g/ml) appeared in the high density lipoprotein fraction but disappeared before weaning.     

A light-independent developmental mechanism potentiates flavonoid gene expression in Arabidopsis seedlings

Throughout the plant kingdom expression of the flavonoid biosynthetic pathway is precisely regulated in response to developmental signals, nutrient status, and environmental stimuli such as light, heat and pathogen attack. Previously we showed that, in developing Arabidopsis seedlings, flavonoid genes are transiently expressed during germination in a light-dependent manner, with maximal mRNA levels occurring in 3-day-old seedlings. Here we describe the relationship between developmental and environmental regulation of flavonoid biosynthesis by examining phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) mRNA levels in germinating Arabidopsis seedlings as a function of light, developmental stage and temperature. We show that seedlings exhibit a transient potential for induction of these four genes, which is distinct from that observed for chlorophyll a/b-binding protein (CAB). The potential for flavonoid gene induction was similar in seedlings grown in darkness and red light, indicating that induction potential is not linked to cotyledon expansion or the development of photosynthetic capacity. The evidence for metabolic regulation of flavonoid genes during seedling development is discussed.     

Environmentally induced conformational changes in B-type DNA: comparison of the conformation of the oligonucleotide d(TCGCGAATTCGCG) in solution and in its crystalline complex with the restriction nuclease EcoRI

Raman spectroscopic analysis of the secondary structure of the crystalline restriction endonuclease EcoRI, the oligonucleotide d(TCGCGAATTCGCG) in solution, and the corresponding crystalline EcoRI-oligonucleotide complex reveals structural differences between the complexed and uncomplexed protein and oligonucleotide components that appear to be linked to complex formation. Structural differences that are spectroscopically identified include (1) an increase in the population of furanose rings adopting the C3'-endo conformation and (2) spectroscopically observed changes in base stacking which are probably associated with the crystallographically observed distortion of the phosphate backbone about positions C(3)-G(4) and C(9)-G(10) and unwinding between the symmetry-related segments GAA-TTC which make up the central recognition core (McClarin et al., 1986). Changes in base stacking due to distortions and unwinding along the oligonucleotide result in differences in the base vibrational region between the spectra of the complex and the oligonucleotide in solution. The spectroscopic analysis indicates that the C2'-endo population is similar for the oligonucleotide in solution and in the complex. The additional C3'-endo population in the complex appears to arise from the conversion of rings adopting alternative conformations such as C1'-exo and O1'-endo. Analysis of the vibrational bands derived from guanine indicates that the population of guanine residues associated with furanose rings in a C2'-endo conformation is similar for the oligonucleotide in solution and in the crystalline complex. This implies that the increase in C3'-endo population is not associated with guanine residues. Large conformational distortions such as those observed in the crystal distortions are not observed in either the crystal or the solution of the oligomer d(CGCGAATTCGCG).(ABSTRACT TRUNCATED AT 250 WORDS)