Aberrant Electrophoretic Mobility Research Articles

Secondary structure of DNA released from purified capsids of human parvovirus B19 under moderate denaturing conditions.

Parvovirus B19 (B19V) possesses a linear single-stranded DNA genome of either positive or negative polarity. Due to intramolecular sequence homologies, either strand may theoretically be folded in several alternative ways. Viral DNA, when extracted from virions by several procedures, presents as linear single-stranded and/or linear double-stranded molecules, except when one particular commercial kit is used. This protocol yields DNA with an aberrant electrophoretic mobility in addition to linear double-stranded molecules, but never any single-stranded molecules. This peculiar kind of DNA was found in all plasma or serum samples tested and so we decided to analyse its secondary structure. In line with our results for one- and two-dimensional electrophoresis, mobility shift assays, DNA preparation by an in-house extraction method with moderate denaturing conditions, density gradient ultracentrifugation, DNA digestion experiments and competition hybridization assays, we conclude that (i) the unique internal portions of this distinctive single-stranded molecules are folded into tight tangles and (ii) the two terminal redundant regions are associated with each other, yielding non-covalently closed pseudo-circular molecules stabilized by a short (18 nucleotides) intramolecular stem, whereas the extreme 3'- and 5'-ends are folded back on themselves, forming a structure resembling a twin hairpin. The question arises as to whether this fairly unstable structure represents the encapsidated genome structure. The answer to this question remains quite relevant in terms of comprehending the initiation and end of B19V genome replication.

Human Hepatitis B Virus Production in Avian Cells Is Characterized by Enhanced RNA Splicing and the Presence of Capsids Containing Shortened Genomes

Experimental studies on hepatitis B virus (HBV) replication are commonly done with human hepatoma cells to reflect the natural species and tissue tropism of the virus. However, HBV can also replicate, upon transfection of virus coding plasmids, in cells of other species. In such cross-species transfection experiments with chicken LMH hepatoma cells, we previously observed the formation of HBV genomes with aberrant electrophoretic mobility, in addition to the those DNA species commonly seen in human HepG2 hepatoma cells. Here, we report that these aberrant DNA forms are mainly due to excessive splicing of HBV pregenomic RNA and the abundant synthesis of spliced DNA products, equivalent to those also made in human cells, yet at much lower level. Mutation of the common splice acceptor site abolished splicing and in turn enhanced production of DNA from full-length pgRNA in transfected LMH cells. The absence of splicing made other DNA molecules visible, that were shortened due to the lack of sequences in the core protein coding region. Furthermore, there was nearly full-length DNA in the cytoplasm of LMH cells that was not protected in viral capsids. Remarkably, we have previously observed similar shortened genomes and non-protected viral DNA in human HepG2 cells, yet exclusively in the nucleus where uncoating and final release of viral genomes occurs. Hence, two effects reflecting capsid disassembly in the nucleus in human HepG2 cells are seen in the cytoplasm of chicken LMH cells.

The CTCF insulator protein forms an unusual DNA structure

BackgroundThe CTCF insulator protein is a highly conserved zinc finger protein that has been implicated in many aspects of gene regulation and nuclear organization. The protein has been hypothesized to organize the human genome by forming DNA loops.ResultsIn this paper, we report biochemical evidence to support the role for CTCF in forming DNA loops. We have measured DNA bending by CTCF at the chicken HS4 β-globin FII insulator element in vitro and have observed a unique DNA structure with aberrant electrophoretic mobility which we believe to be a DNA loop. CTCF is able to form this unusual DNA structure at two other binding sites: the c-myc P2 promoter and the chicken F1 lysozyme gene silencer. We also demonstrate that the length though not the sequence of the DNA downstream of the binding site is important for the ability of CTCF to form this unusual DNA structure. We hypothesize that a single CTCF protein molecule is able to act as a "looper" possibly through the use of several of its zinc fingers.ConclusionsCTCF is able to form an unusual DNA structure through the zinc finger domain of the protein. This unusual DNA structure is formed in a directional manner by the CTCF protein. The findings described in this paper suggest mechanisms by which CTCF is able to form DNA loops, organize the mammalian genome and function as an insulator protein.

Structural characterization of supercoiled DNA containing a minisatellite repeat that has polypurine/polypyrimidine stretch

The pUC19 derivative pAL79 contains six tandem repeats of a 30-bp unit sequence. Supercoiled pAL79 molecules show aberrant electrophoretic mobility under acidic conditions, and microscopic analysis suggests pAL79 molecules conform to tight plectonemes when deposited on a substrate under acidic conditions. This may mean that a short inserted DNA sequence affects the global topology of plasmid DNA.

Molecular Cloning and Characterization of a Transcription Regulator with Homology to GC-binding Factor

GC-binding factor (GCF) represses transcription of certain genes and is encoded by a 3.0-kilobase mRNA (Kageyama, R., and Pastan, I. (1989) Cell 59, 815-825). The GCF cDNA hybridizes to two additional mRNA species, 4.2 and 1.2 kilobases. We have used differential hybridization to identify a cDNA clone (termed GCF2) for the 4. 2-kilobase mRNA and find that it is highly expressed in HUT-102 cells. The open reading frame consists of 2256 nucleotides and encodes a protein of 752 amino acids with a calculated molecular mass of 83 kilodaltons. GCF2 expressed in vitro using reticulocyte lysates and Escherichia coli migrates as a 160-kilodalton protein in SDS-polyacrylamide gel electrophoresis but has a molecular mass of 83 kilodaltons as determined by mass spectrum analysis. GCF2 binds to epidermal growth factor receptor promoter fragments, and the major binding site is located between nucleotides -249 and -233. Cotransfection assays show that GCF2 acts to repress transcription from the epidermal growth factor receptor promoter in constructs containing the major GCF2 binding site and not when the site had been mutated. Thus, GCF2 is a newly identified transcriptional repressor with aberrant electrophoretic mobility.

Characterization of N93S, I312T, and A333P missense mutations in two Japanese families with mitochondrial acetoacetyl-CoA thiolase deficiency.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of ketone body and isoleucine catabolisms. Japanese patients, GK01 and GK19, were found to be compound heterozygotes of 149delC and A333P, and N93S and I312T, respectively. The latter three missense mutations were individually characterized by analyses of transient expression of the cDNAs and heat stability. A333P and I312T subunits showed aberrant electrophoretic mobility on SDS-PAGE. T2 protein was destabilized by A333P and existed as an insoluble form in the mitochondria. I312T mutation also destabilized T2 protein; however, some T2 protein was retained in soluble form and reduced residual activity was apparent. N93S mutation did not change the heat stability of T2 activity and the reduced residual activity was retained, however a considerable amount was observed in an insoluble form. The effects of mutations were interpreted based on a tertiary structural model of a subunit of the human T2. This model was constructed from the X-ray crystal structure of the homologous peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae. On the basis of this model, the positions of Ala333 and Ile312 were far from the active site and the mutations would be expected to destabilize the tertiary structure of T2 subunit. By contrast, Asn93 is located near the active site and may function to maintain a local loop structure. The mutation of Asn93 could directly disrupt disposition of the active site.

Dna insertional mutagenesis for the elucidation of a Photosystem II repair process in the green alga Chlamydomonas reinhardtii

The work outlines the isolation of transformant Chlamydomonas reinhardtii cells that appear to be unable to repair Photosystem II from photoinhibitory damage. A physiological and biochemical characterization of three mutants is presented. The results show differential stability for the D1 reaction center protein in the three mutants compared to the wild type and suggest lesions that affect different aspects of the Photosystem II repair mechanism. In the ag16.2 mutant, significantly greater amounts of D1 accumulate in the thylakoid membrane than in the wild type under steady-state growth conditions, and D1 loss is significantly retarded in the presence of the protein biosynthesis inhibitor chloramphenicol. Moreover, aberrant electrophoretic mobility of D1 in the ag16.2 suggests that this protein is modified to an as yet unknown configuration. These results indicate that the biosynthesis and/or degradation of D1 is altered in this strain. A different type of mutation occurred in the kn66.7 and kn27.4 mutants of C. reinhardtii. The stability of D1 declined much faster as a function of light intensity in these mutants than in the wild type. Thereby, the threshold of photoinhibition in these mutants was significantly lower than that in the wild type. It appears that kn66.7 and kn27.4 are similar conditional mutants, with the only difference between them being the amplitude of the chloroplast response to the mutation and the differential sensitivity they display to the level of irradiance.

Clinical and biochemical investigations and molecular analysis of subjects with mutations in the androgen receptor gene.

Androgen insensitivity syndromes (AIS) in subjects with 46, XY karyotype and normal or even elevated androgen blood levels are characterized by various aberrations in male differentiation and virilization. AIS is often accompanied by a broad spectrum of abnormal binding characteristics of the androgen receptor (AR). In order to investigate the correlation between the degree of virilization defect and the type of androgen binding abnormalities and/or the nature of the mutation in the AR gene, we determined androgen binding characteristics of the AR protein and the sequence of the AR gene in clinically and biochemically well characterized patients with various degrees of androgen resistance. The activity of 5 alpha-reductase and the binding of androgen to its receptor (KD-values, Bmax, thermolability) were determined in genital skin fibroblasts from 20 patients with various degrees of defects in virilization (2 CAIS, complete AIS; 18 PAIS, partial AIS patients). The AR gene of these 20 subjects was characterized by PCR-SSCP analysis. In case of aberrant electrophoretic mobility the corresponding exon was sequenced. The 2 patients with CAIS and 7 with PAIS showed a mutation in the AR gene. In two, the mutation was in the DNA binding domain, and in all others in the ligand binding domain. In 11 patients with severe virilization defects no abnormal behaviour was detected in the PCR-SSCP. Transcriptional activation studies of two mutant ARs revealed that an approximately tenfold higher androgen concentration (methyltrienolone) is necessary to achieve maximal response as compared to the wild type AR. There is no obvious relation between the degree of androgen resistance and the binding parameters of the AR and/or the nature of mutation in the AR gene. Androgen insensitivity syndrome can occur despite normal androgen binding and presumably non-mutated AR genes. Even if there is abnormal binding of androgen and/or a mutation in the AR gene there is no clear-cut relationship between these parameters and the degree of virilization defects. Thus, in a proportion of patients, neither the determination of binding parameters of the AR nor the detection of mutations in the AR gene are sufficient to understand the mechanisms underlying the androgen insensitivity syndrome.

Imaging the Asymmetrical DNA Bend Induced by Repressor Activator Protein 1 with Scanning Tunneling Microscopy

The yeast Repressor Activator Protein 1 (RAP1) binds a 13-bp consensus found in many transcriptional regulatory regions, in silencer elements, and in telomeric repeat DNA of Saccharomyces cerevisiae. Gel retardation assays suggest that RAP1 bends DNA as it binds, with the vertex of the angle located 5′ of the consensus. We show that removal of 230 aa in the N-terminus of RAP1 reduces the aberrant electrophoretic mobility of the protein-DNA complex, while removal of a C-terminal domain of RAP1 causes even greater distortion. To demonstrate that the aberrant electrophoretic mobility is really due to a bend in the double helix, the RAP1-DNA complex was analyzed by Scanning Tunnelling Microscopy (STM). The efficiency and accuracy of binding is checked in parallel by standard Transmission Electron Microscopy (TEM). Due to the use of high-angle shadowing of freeze-dried samples at low temperatures, the STM images allow us to confirm that RAP1 binding induces a DNA bend >50°, while the binding of the minimal DNA-binding domain shows significantly less distortion of the DNA helix.

Nitric oxide reductase from Pseudomonas stutzeri, a novel cytochrome bc complex

The nitric oxide reductase (NOR) from Pseudomonas stutzeri is a cytochrome bc complex which shows on SDS/PAGE two subunits with apparent molecular masses of 17 kDa and 38 kDa. Two other species of approximately 45 kDa and 74-78 kDa represent the undissociated enzyme complex and an aggregate of the cytochrome b subunit, respectively. The cytochrome b subunit is highly hydrophobic and results in aberrant electrophoretic mobility. The stability of the enzyme in various detergents and at different pH was investigated. The highest specific activity of 60 mumol NO min-1 mg-1 protein was obtained after electrophoresis in the presence of laurylpropanediol-3-phosphorylcholine ether. Purified NOR contained cardiolipin, phosphatidylglycerol, and phosphatidylethanolamine, the latter as the major component. A phospholipid was required for high catalytic activity with either cardiolipin or phosphatidylglycerol increasing the activity of the enzyme as isolated by a factor of up to 5. Free fatty acids inhibited NOR, with cis-9-octadecenoic acid (oleic acid) showing the most pronounced effect. Certain detergents substituted for the phospholipid requirement of NOR. The enzyme, as isolated, in 0.1% Triton X-100, 20 mM Tris/HCl pH 8.5, exhibited a complex set of EPR resonances at low magnetic field, with a prominent peak at g 6.34 resulting from Fe(III) high-spin cytochrome b. The second prominent feature arose from a low-spin Fe(III) heme center with strong lines at apparent g values of 3.02 and 2.29, and a broad resonance at g approximately 1.5 which we assigned to the cytochrome c component of the enzyme. From spin quantitation and computer simulations of the various EPR signals a ratio close to 1:1 for the low-spin/high-spin heme centers in NOR was estimated. Shifting the pH from 8.5 to 5.0, replacing Triton X-100 by other detergents, or adding soybean phospholipids to the protein, led to pronounced changes of the EPR signals in the g = 6 region. In contrast, the strong inhibitor oleic acid did not cause significant spectral changes. NOR which had been reduced by L-ascorbate/phenazine methosulfate prior to incubation with its substrate NO gave the characteristic Fe(II) nitrosyl triplet centered at g approximately 2.01, with a hyperfine splitting of 1.70 mT. In the absence of dioxygen, NOR was quantitatively reduced by either sodium dithionite, or photochemically with deazaflavin and oxalate; the enzyme was reoxidizable by ferricyanide in a fully reversible reaction. Spectroelectrochemical oxidoreductive titrations gave E'o (versus standard hydrogen electrode) = +322 mV for the cytochrome b and +280 mV for the cytochrome c component.

Albumin Hawkes Bay; a low level variant caused by loss of a sulphydryl group at position 177

A slow migrating minor albumin component, representing 5% of total circulating albumin, was detected by routine serum protein electrophoresis and immunofixation. After treatment with 5 mM dithiothreitol the abnormal component was found to migrate normally suggesting the attachment of some component to the free thiol at position 34. However, purification and analysis by SDS-PAGE showed that the abnormal component had a slightly lower apparent molecular weight than normal albumin. Limited tryptic cleavage indicated the abnormal site to be in the N-terminal third of the molecule. HPLC analysis of tryptic peptides from this domain showed the presence of a new peptide of sequence Ala-Ala-Phe-Leu-Leu-Pro-Lys, indicating either a point mutation of 177 Cys → Phe or the deletion of residues 166–177. DNA sequencing of PCR-amplified DNA confirmed the former Cys → Phe substitution by indicating a point mutation of C to A at nucleotide position 5185. It appears that the aberrant electrophoretic mobility of the variant might be due to a gross conformational change associated with the formation of a new disulphide bond between Cys-168 and Cys-124.

Activity domains of the TonB protein

Escherichia coli and related Gram-negative bacteria contain an energy-coupled transport system through the outer membrane which consists of the proteins TonB, ExbB, ExbD anchored in the cytoplasmic membrane and receptors in the outer membrane. Differences in the activities of the Escherichia coli and the Serratia marcescens TonB proteins were used to identify TonB functional domains. In E.coli TonB segments were replaced by equivalent fragments of S. marcescens TonB and the activities of the resulting chimaeric proteins were determined. In addition, E. coli TonB was truncated at the C-terminal end, and point mutants were generated using bisulphite. From the results obtained we draw the following conclusions: an important site of interaction between TonB and ExbB is located in the N-terminal region of TonB within or close to the cytoplasmic membrane since an N-terminal 44-residue fragment of TonB was stabilized by ExbB and interfered with wild-type TonB activity. In addition, the activity of a TonB derivative in which histidine residue 20 was replaced by arginine was strongly reduced, and a double mutant containing arginine-7 to histidine and alanine-22 to threonine substitutions displayed an impaired uptake of ferrichrome. Furthermore, the domain around residue 160 is involved in TonB activity. S. marcescens TonB segments of this region in E. coli TonB conferred S. marcescens TonB activities, and E. coli TonB point mutants displayed strongly impaired activities for the uptake of colicin B and M and ferric siderophores. Plasmid-encoded tonB mutants of this region showed negative complementation of chromosomal wild-type tonB, and certain tonB mutants suppressed colicin B TonB-box mutants. Uptake of colicins required different domains in TonB, for colicin B and M around residue 160 and for colicin Ia, a domain closer to the C-terminal end. Tandem duplication of the E. coli (EP)X(KP) region by insertion of the S. marcescens (EP)X(KP) region (38 residues) and replacement of lysine residue 91 by glutamate did not alter TonB activity so that no evidence was obtained for this region to be implicated in receptor binding. The aberrant electrophoretic mobility of TonB was caused by the proline-rich sequence since its removal resulted in a normal mobility.

Double Strand Conformation Polymorphism (DSCP) Detects Two Point Mutations at Codon 280 (AAC→ATC) and at Codon 431 (TAC→AAC) of the Blood Coagulation Factor VIII Gene

Hemophilia A is a hereditary, X-linked, bleeding disorder that is caused by a defect in the factor VIII gene. Here, we report two novel point mutations in the factor VIII gene that result in an aberrant electrophoretic mobility of double strand PCR fragments (double strand conformation polymorphism, DSCP). In exon 9 a TAC-->AAC mutation at codon 431, replacing Tyr by Asn, was observed in a family (A211) with moderately severe hemophilia A. A family with mild hemophilia A revealed an A-->T mutation in codon 280 (exon 7) that results in the replacement of Asn by Ile. One of these two mutations was not detected in an analysis based on single strand conformation polymorphisms (SSCP). At present we have no explanation for the effect of the nucleotide changes on the electrophoretic mobility of double strand DNA. Although DSCP is not able to detect all mutations the combination of DSCP analysis with SSCP analysis increases the sensitivity in a screening for factor VIII mutations.

Purification, primary structure, bacterial expression and subcellular distribution of an oocyte-specific protein in Xenopus.

This study defines a novel Xenopus laevis protein (P100) that has recently been shown to be recognized by scleroderma patient sera. Using a combination of differential solubility in detergents, hydroxyapatite chromatography and one-dimensional PAGE, P100 was purified to apparent homogeneity and the amino acid sequence was obtained. An oligonucleotide derived from this sequence was used to clone P100 cDNA through a polymerase-chain-reaction cloning strategy. The entire P100 cDNA sequence was determined, identifying a novel 83,000-Da protein. Two alleles for P100 were transcribed in the oocyte, with only one predicted amino acid change between them. Bacterial expression of a clone containing the entire P100 coding region produced a protein that migrated at a mass 15% greater than that predicted from the amino acid sequence, indicating an aberrant electrophoretic mobility. The mRNA transcript for P100 was only expressed during the previtellogenic stages of oogenesis (stages I and II) and was absent from other Xenopus tissues. Similarly, the P100 protein was found only in Xenopus oocytes and was localized to the cytoplasm of these cells. P100 irreversibly bound single-stranded-DNA--cellulose but not double-stranded-DNA--cellulose. These data demonstrate the presence of a novel oocyte-specific protein in Xenopus.

Aberrant O-linked oligosaccharide biosynthesis in lymphocytes and platelets from patients with the Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immunodeficiency affecting B lymphocytes, T lymphocytes, and platelets. Previous studies on lymphocytes from WAS patients have revealed that leu-kosialin (CD43), a cell-surface glycoprotein bearing approximately 90 O-linked oligosaccharide chains, shows an aberrant electrophoretic mobility. To determine whether this finding reflects a different pattern of O-linked glycosylation in WAS cells, we have compared healthy individuals and WAS patients with respect to glycosyltransferase activities in T lymphocytes, platelets, and Epstein-Barr virus (EBV)-immortalized B cell lines. Stimulation of peripheral T cells from normal individuals in vitro with anti-CD3 antibodies and interleukin-2 was associated with a 3-fold increase in UDP-GlcNAc:Gal beta 3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T) from 0.8 to 2.2 nmol/mg/h. In contrast, peripheral T lymphocytes from WAS patients showed an inversion of this phenotype with high core 2 GlcNAc-T activity in unstimulated cells (2.3 nmol/mg/h) and a 2-3-fold decrease in activity following stimulation. Core 2 GlcNAc-T activity was also three times higher in platelets from WAS patients than in normal platelets. Glycosyltransferase activities were measured in immortalized B cell lines established from WAS and normal subjects by infection with EBV. Core 2 GlcNAc-T was less than 0.4 nmol/mg/h in WAS EBV-B cell lines compared to 2.4 nmol/mg/h in EBV-B cell lines from healthy individuals, In contrast, CMP-SA:SA alpha 2-3Gal beta 1-3GalNAc-R (where SA represents sialyl (sialic acid to GalNAc) alpha 6-sialyltransferase II activity was 2.0 nmol/mg/h in the WAS EBV-B cell and less than .01 nmol/mg/h in EBV-B cell lines derived from normal subjects. Eleven other glycosyltransferase activities were measured and found to be similar in EBV-B cell lines from WAS and normal individuals. Polylactosamine sequences were much reduced in the O-linked oligosaccharides of CD43 from WAS EBV-B cells consistent with decreased core 2 GlcNAc-T activity and expression of core 1 oligosaccharides in the cells. In conclusion, B cells, T cells, and platelets in WAS patients show abnormal expression of two developmentally regulated glycosyltransferases, consistent with the idea that the WAS immunodeficiency is due to a failure of normal lymphocyte maturation.

A spectroscopic and calorimetric study of the melting behaviors of a "bent" and a "normal" DNA duplex: [d(GA4T4C)]2 versus [d(GT4A4C)]2.

We have calorimetrically detected and energetically characterized a premelting event in the "bent" duplex [d(GA4T4C)]2 that is absent in the "normal" duplex [d(GT4A4C)]2. This premelting may correspond to the thermally induced "unbending" of the duplex. Specifically, we have used a combination of spectroscopic and calorimetric techniques to evaluate whether differences in the electrophoretic and hydrogen exchange properties of the bent duplex, [d(GA4T4C)]2, and the normal duplex, [d(GT4A4C)]2, are paralleled by differences in the equilibrium melting properties of these duplexes. Our results reveal that the bimolecular global meltings of both duplexes exhibit two-state behavior and are characterized by the expected thermodynamic changes, as well as the expected salt-dependencies. Significantly, however, at temperatures below duplex melting and over a similar temperature range in which the aberrant electrophoretic mobility is observed (approximately less than 35 degrees C), the bent duplex exhibits calorimetric premelting behavior absent in the normal duplex. Analysis of the calorimetric data in this preglobal melting domain (approximately less than 35 degrees C) allows us to estimate a lower-limit value of 1.6 kcal/(mol-base pair) for the thermally induced unbending of the decameric duplex.

Isolation and characterization of cDNA encoding a human nuclear antigen predominantly recognized by autoantibodies from patients with primary biliary cirrhosis.

Autoantibodies to a novel nuclear Ag, Sp100, have recently been described that recognize a nuclear protein with an apparent molecular mass of 95 to 100 kDa and a dot-like distribution within cell nuclei. By immunoscreening of a lambda gt11 cDNA expression library derived from HeLa cells with an anti-Sp100 autoimmune serum a 0.7-kb cDNA (Sp26) coding for a fragment of Sp100 was isolated. Expression of this cDNA and use of the recombinant protein in ELISA revealed that the fragment carries major Sp100 autoepitopes and that anti-Sp100 autoantibodies predominantly occur in patients suffering from primary biliary cirrhosis (50/184). The Sp26 cDNA was used as hybridization probe for isolation of longer cDNA from human liver- and placenta-derived lambda gt10 cDNA libraries. Overlapping fragments were assembled to generate a full length cDNA coding for a protein with a molecular mass of 53 kDa and an isoelectric point of 4.7. The Sp100 autoantigen expressed in vitro from this cDNA and authenticated by a capture immunoblot assay, comigrated in SDS-PAGE with the authentic HeLa autoantigen of 95 to 100 kDa and thus showed an aberrant electrophoretic mobility. Computer based protein sequence analysis of the Sp100 autoantigen revealed regions of striking sequence similarities to the alpha 1 and alpha 2 domains of various human and non-human MHC class I Ag and to several transacting transcriptional regulatory proteins.

Effect of polyadenine-containing curved DNA on promoter utilization in Bacillus subtilis.

The effect of DNA upstream of the -35 region on promoter function was examined using two promoters isolated from the Bacillus subtilis bacteriophage SP82. The affinity of RNA polymerase for the two promoters in vitro differed significantly. For each promoter the nucleotide sequence of the upstream DNA was characterized by the presence of successive runs of adenines with a 10-11-base pair periodicity. DNA fragments with the polyadenine-containing upstream DNA displayed aberrant electrophoretic mobilities when analyzed on polyacrylamide gels indicative of curved DNA. A series of mutant promoters in which the upstream DNA was deleted or altered was constructed. The curved DNA upstream of the -35 region was required for efficient RNA polymerase binding. Decreased in vitro transcription observed when the upstream DNA was deleted could be partially restored if the template was negatively supercoiled. Measurements of chloramphenicol acetyltransferase specific activity from B. subtilis strains carrying transcriptional fusions indicate that the curved upstream DNA stimulated transcription from the promoter with the weaker affinity for RNA polymerase. The curved DNA reduced the in vivo activity of the promoter with the strong affinity for RNA polymerase. One function of the curved upstream DNA may be to provide RNA polymerase-promoter interactions that facilitate open complex formation.

Function and structure of microvirid phage α3 genome. DNA sequence of H gene and properties of missense H mutant

The nucleotide sequence of wild-type α3 H gene and its surrounding region was determined and compared with those of ∅X174 and G4. The corresponding DNA regions in double mutants amJH22, amJH69 and amJH76 were also sequenced and their missense mutation sites located. A phage strain missH22 having a single missense mutation in gene H was constructed by replacing the J region of amJH22 in vitro with the wild-type DNA. Like amJH22, the missense mutant coded for H protein with aberrant electrophoretic mobility, but formed normal plaques on suppressor-deficient Escherichia coli. Heat stability, plating efficiency on certain hosts and rate of eclipse were higher in strain missH22 than in wild-type phage.

Photodynamic intramolecular crosslinking of myoglobin

Exposure of native myoglobin to visible light in the presence of a sensitizer leads to photooxidation, but not to appreciable intermolecular crosslinking. Despite this absence of intermolecular crosslinking a considerable decrease of free NH 2 groups is observed, suggesting the formation of intramolecular crosslinks. During photooxidation the physical properties of the protein were affected, as shown, for example, by aberrant electrophoretic mobility in urea-SDS gels, a changed isoelectric focusing pattern and increased resistance against thermal denaturation. Photooxidized myoglobin is more sensitive to proteolysis by elastase than is native myoglobin. This was shown both by monitoring hydrolysis by pH-stat titration and by measurements of terminal amino groups. Whereas limited proteolysis of native myoglobin is attended with the appearance of proteolytic fragments in urea-SDS electrophoretograms, such degradation products are not observed after similar proteoylysis of photooxidized myoglobin. This phenomenon, for the first time, clearly indicates the existence of intramolecular crosslink bridges in photooxidized myoglobin, preventing the separation of the proteoyltic fragments.