Study of the DNA primary structure effect on induction of mutations by alkylating agents

Analysis and comparison of mutation spectra is one of the major tasks of molecular biology, since mutation spectra often reveal important properties of various mutagens and proteins involved in the repair/replication systems. Mutability is known to vary significantly along the nucleotide sequence. Mutations are abundant at certain positions (mutation hotspots). In this work, we applied regression analysis based on the basic logic patterns to understand the role of the nucleotide sequence context in mutation induction. The spectra of mutations induced by various alkylating agents were studied. The nucleotide bases at positions -2, -1, +1 and +2 were shown to have the most significant effect in G:C-->A:T replacements.     

The mRNA Codon Environment at the P and E Sites of Human Ribosomes Deduced from Photocrosslinking with pUUUGUU Derivatives

Three mRNA analogs—derivatives of hexaribonucleotide pUUUGUU comprising phenylalanine and valine codons with a perfluoroarylazido group attached to the C5 atom of the uridine residue at the first, second, or third position—were used for photocrosslinking with 80S ribosomes from human placenta. The mRNA analogs were positioned on the ribosome with tRNA recognizing these codons: UUU was at the P site if tRNA^Phe was used, while tRNA^Val was used to put there the GUU codon (UUU at the E site). Thus, the crosslinking group of mRNA analog might occupy positions –3 to +3 with respect to the first nucleotide of the codon at the P site. Irradiation of the complexes with mild UV light ( > 280 nm) resulted in the crosslinking of pUUUGUU derivatives with 18S RNA and proteins in the ribosome small subunit. The crosslinking with rRNA was observed only in the presence of tRNA. The photoactivatable group in positions –1 to +3 binds to G1207, while that in positions –2 or –3 binds to G961 of 18S RNA. In all cases, we observed crosslinking with S2 and S3 proteins irrespective of the presence of tRNA in the complex. Crosslinking with S23 and S26 proteins was observed mainly in the presence of tRNA when modified nucleotide occupied the +1 position (for both proteins) or the –3 position (for S26 protein). The crosslinking with S5/S7 proteins was substantial when modified nucleotide was in the –3 position, this crosslinking was not observed in the absence of tRNA.     

MucAB but not UmuDC proteins enhance ?2 frameshift mutagenesis induced by N-2-acetylaminofluorene at alternating GC sequences

N-2-acetylaminofluorene has been shown efficiently to induce both –1 and –2 frameshift mutations in Escherichia coli as well as in mammalian cells. In E. coli, the genetic characteristics of –1 and –2 frameshift mutations were found to be distinct. The –1 frameshift mutation pathway occurs at monotonous runs of G residues (i.e. GGGGG). This pathway exhibits the same genetic requirements as UV light-induced base substitution mutagenesis. Indeed, optimal mutagenesis requires the expression of both UmuDC and the activated form of RecA. The –2 frameshift mutation pathway operates at short alternating GpC sequences, such as the NarI sequence (i.e. GGCGCCGGCC). In contrast to the –1 frameshift mutation pathway, optimal induction does not require the UmuDC and RecA proteins. This pathway involves a LexA-repressed function tentatively called Npf (for NarI processing factor). In this paper, we show that MucAB efficiently stimulates the –2 frameshift mutation pathway. However, unlike the Npf pathway, MucAB-mediated stimulation requires expression of the RecA protein.     

High-speed autoradiography of 3H-thymidine-labelled nuclei

Summary High-speed autoradiography with stripping film of ³H-thymidine-labelled cells was tested. The tests involved: (a) various times of immersion of emulsion-covered cell preparations in the mixture of dioxane-PPO-POPOP, at 20°C, (b) exposure of cell preparations and blanks for various times at either –70°C or +20°C, with different humidity levels. Autoradiographs of good quality could be produced by 2-min immersion in the scintillator, exposure time 1 h at either temperature and relative humidity 20–30%. A linear relationship between autoradiographic efficiency and exposure time of 1–7 h was found at either temperature, although the efficiency of autoradiographs exposed at –70°C was by approximately 30% higher than that of autoradiographs exposed at +20°C. Background values of autoradiographs dried with a fan and exposed for 1/4–7h at either –70°C or +20°C were 0.6–0.8 grain/100 m². Theoretical calculations and experimental data showed that high-speed autoradiographs are 30–50 times more efficient as compared with conventional stripping film autoradiographs, thus allowing a shortening of the respective exposure time. Theoretical aspects of efficiency and resolution of high-speed autoradiography are considered.This investigation was supported in part by MR II.1 grant. The technical assistance of Mrs. S. Bie is gratefully acknowledged     

Covalent Structure of Botulinum Neurotoxin Type E: Location of Sulfhydryl Groups, and Disulfide Bridges and Identification of C-Termini of Light and Heavy Chains

Botulinum neurotoxin (NT) serotype E is synthesized by Clostridium botulinum as an 150-kDa single-chain polypeptide of 1252 amino acid residues of which 8 are Cys residues [Puolet et al. (1992), Biochem. Biophys. Res. Commun. 183, 107–113]. The posttranslational processing of the gene product removes only the initiating methionine. A very narrow segment of this 1251-residue-long mature protein—at one-third the distance from the N-terminus (between residues Lys 418 and Arg 421)—is highly sensitive to proteases, such as trypsin. The single-chain NT easily undergoes an exogenous posttranslational modification by trypsin; residues 419–421 (Gly–Ile–Arg) are excised. The proteolytically processed NT is a dichain protein in which Pro 1–Lys 418 constitute the 50–kDa light chain, Lys 422–Lys 1251 constitute the 100–kDa heavy chain; Cys 411–Cys 425 and Cys 1196–Cys 1237 form the interchain and intrachain disulfide bonds, respectively; the other four Cys residues at positions 25, 346, 941, and 1035 remain as free sulfhydryl groups. The 150–kDa dichain NT, and separated light and heavy chains, were fragmented with CNBr and endoproteases (pepsin and clostripain); some of these fragments were carboxymethylated with iodoacetamide (with or without ^I4C label) before and after fragmentation. The fragments were separated and analyzed for amino acid compositions and sequences by Edman degradation to determine the complete covalent structure of the dichain type E NT. A total of 208 amino acid residues, i.e., 16.5% of the entire protein's sequence deduced from nucleotide sequence, was identified. Direct chemical identification of these amino acids was in complete agreement with that deduced from nucleotide sequence.     

Nature and frequency of mutations in the argininosuccinate synthetase gene that cause classical citrullinemia

Citrullinemia is an autosomal recessive disorder caused by a genetic deficiency of argininosuccinate synthetase (ASS). So far 20 mutations in ASS mRNA have been identified in human classical citrullinemia, including 14 single base changes causing missense mutations in the coding sequence of the enzyme, 4 mutations associated with an absence of exons 5, 6, 7, or 13 in mRNA, 1 mutation with a deletion of the first 7 bases in exon 16 (which is caused by abnormal splicing), and 1 mutation with an insertion of 37 bases between the exon 15 and 16 regions in mRNA. In order to identify the abnormality in the ASS gene causing the exon 7 and 13 deletion mutations and the 37-base insertion mutation between exons 15 and 16 in mRNA, and to establish a DNA diagnostic test, we isolated and sequenced the genomic DNA surrounding each exon. The absence of exon 7 or 13 in ASS mRNA resulted from abnormal splicing caused by a single base change in the intron region: IVS-6^–2 (a transition of A to G at the second nucleotide position within the 3 splice cleavage site of intron 6) and IVS-13⁺⁵ (a transition of G to A at the fifth nucleotide position within the 5 splice cleavage site of intron 13), respectively. The IVS-6^–2 mutation resulted in the creation of an MspI restriction site. DNA diagnostic analysis of 33 Japanese alleles with classical citrullinemia showed that 19 alleles had the IVS-6^–2 mutation (over 50% of the mutated alleles in Japanese patients). It was thus confirmed that one mutation is predominant in Japan. This differs from the situation in the USA where there is far greater heterogeneity. The insertion mutation in mRNA on the other hand resulted from abnormal splicing caused by a 13-bp deletion at the splice-junction between exon 15 and intron 15. The deletion had a short direct repeat (CTCAGG) at the breakpoint junction and presumably resulted from slipped mispairing.     

Minimal requirements for in vitro reconstitution of the structural subunit of light-harvesting complexes of photosynthetic bacteria

Unlike the and polypeptides of the core light-harvesting complex (LH1) of Rhodobacter (Rb.) sphaeroides, the and polypeptides of the peripheral light-harvesting complex (LH2) of this organism will not form a subunit complex by in vitro reconstitution with bacteriochlorophyll. Guided by prior experiments with the LH1 polypeptides of Rb. sphaeroides and Rhodospirillum rubrum, which defined a set of interactions required to stabilize the subunit complex, a series of mutations to the Rb. sphaeroides LH2 polypeptide was prepared and studied to determine the minimal changes necessary to enable it to form a subunit-type complex. Three mutants were prepared: Arg at position –10 was changed to Asn (numbering is from the conserved His residue which is known to be coordinated to bacteriochlorophyll); Arg at position –10 and Thr at position +7 were changed to Asn and Arg, respectively; and Arg at position –10 was changed to Trp and the C-terminus from +4 to +10 was replaced with the amino acids found at the corresponding positions in the LH1 polypeptide of Rb. sphaeroides. Only this last multiple mutant polypeptide formed subunit-type complexes in vitro. Thus, the importance of the C-terminal region, which encompasses conserved residues at positions +4, +6 and +7, is confirmed. Two mutants of the LH1 polypeptide of Rb. sphaeroides were also constructed to further evaluate the interactions stabilizing the subunit complex and those necessary for oligomerization of subunits to form LH1 complexes. In one of these mutants, Trp at position –10 was changed to Arg, as found in LH2 at this position, and in the other His at position –18 was changed to Val. The results from these mutants allow us to conclude that the residue at the –10 position is unimportant in subunit formation or oligomerization, while the strictly conserved His at –18 is not required for subunit formation but is very important in oligomerization of subunits to form LH1.     

Genetic Mechanisms of Resistance and Susceptibility to Leukemia in Ayrshire and Black Pied Cattle Breeds Determined by Allelic Distribution of Gene BoLA-DRB3

In the herds of Ayrshire and Black Pied cattle breeds of Russian selection, comparative analysis of allelic distribution of BoLA-DRB3 was performed in animal groups with different status of persistent lymphocytosis (PL) caused by the bovine leukemia virus (BLV). Alleles were typed by PCR–RFLP. Different spectra of BoLA-DRB3 alleles mediating susceptibility and resistance to leukemia were detected in the studied breeds. The role of amino acid motives in 1 domain of BoLA-DRB3 antigens was confirmed: ER (in positions 70–71), in resistance to leukemia and VDTY and VDTV (75–78), in susceptibility to leukemia. The nucleotide sequence of allele BoLA-DRB3.2*7with deletion of codon 65, which resulted in the changed conformation of the corresponding antigen molecule, was associated with resistance to PL. Cows of Black Pied and Ayrshire breeds with genotypes coding VDTY/VDTV (RR = 11.67, P = 0,014) and VDTY/VDTY (RR = 4.71, P = 0.022), respectively, were shown to be susceptible to PL. The role of heterozygosity level was demonstrated (estimated by BoLA-DRB3 alleles and by amino acid motives in positions 75–78 of the antigen) as an unspecific factor of resistance to PL. The lowest heterozygosity level by amino acid motives (75–78) was revealed in PL animals, for which sample inbreeding coefficients were detected: F = 0.324 and 0.084 in Ayrshire and Black Pied breeds, respectively.     

Characterization of a secreted cholesterol oxidase from Rhodococcus sp.GKI (CIP 105335)

Extracellular cholesterol oxidase (COX) (EC 1.1.3.6) was produced by Rhodococcus sp. GK1 cells grown in a defined mineral salt medium containing a mixture of phytosterols (sitosterol, campesterol, stigmasterol) as the sole source of carbon and energy. In the same time, the sterols acted as enzyme inducers. The medium was enriched with yeast extract in order to stimulate enzyme secretion. COX was purified from the culture supernatants by affinity-like chromatography on a column packed with kieselguhr and cholesterol. Enzyme bound onto the column was eluted with 0.05 M phosphate buffer pH 7.0 containing Triton X-100 at 0.1% (w/v). Some properties of the purified COX were determined. Its specific activity at pH 7.0 and 30 °C, was around 5.5 units mg^–1. The molecular mass of the enzyme, as estimated by SDS-PAGE, was 59 kDa. Its isoelectrofocusing point was around pH 8.9. The C-5 double bond and the alkyl chain moiety in sterol molecules were necessary for an adequate oxidation of the sterol 3-ol. Enzyme inhibition by the ions (0.1 mM): AsO₂ ^–, Ba²⁺, Co²⁺, Cd²⁺, Cu²⁺, N₃ ^–, Ni²⁺, and Pb²⁺ was negligible (around 10%). However, COX inhibition by 0.1 mM of either Zn²⁺, 2-[(ethylmercurio)-thio]benzoic acid, or Hg²⁺ was 18%, 22% and 93% respectively. Inhibition of activity by Hg²⁺ was significant, even at 1 M. The purified COX (0.1–0.15 mg ml^–1 in 0.05 M phosphate pH 7.0) was relatively heat-stable at temperatures up to 50 °C. At this temperature, the half-life of its activity was around 70 min. However, 90% of the enzyme initial activity was lost by 20 min incubation at 60 °C. The aminoacid sequence of the COX N-terminal segment was: H2N–Ala–Pro–Pro–Val–Ala–Ser–X–Arg–Tyr–X–(Phe)– (X might be 2 Cys residues).     

Co-regulation with genes of phospholipid biosynthesis of the CTR/HNM1-encoded choline/nitrogen mustard permease in Saccbaromyces cerevisiae

An 815 by region of the promoter of the Saccharomyces cerevisiae gene CTR/HNM1, encoding choline permease was sequenced and its regulatory function analysed by deletion studies in an in-frame promoter-lacZ construct. In addition to the TATA box, a 10 by motif (consensus 5-CATGTGAAAT-3) was found to be mandatory for CTR/HNM1 expression. This decamer motif is located between nucleotides –262 and –271 and is identical in 9 of 10 by with the regulatory motif found in the S. cerevisiae INO1 and CHO1 genes. Constructs with the 10 by sequence show high constitutive expression, while elimination or alterations at three nucleotide positions, of the decamer motif in the context of an otherwise unchanged promoter leads to total loss of -galactosidase production. Expression of the CTR/HNM1 gene in wild-type cells is regulated by the phospholipid precursors inositol and choline; no such influence is seen in cells bearing mutations in the phospholipid regulatory genes INO2, INO4, and OPI1. There is no regulation by INO2 and OPI1 in the absence of the decamer motif. However constructs not containing this sequence (promoter intact to positions –213 or –152) are still controlled by INO4. Other substrates of the choline permease, i.e. ethanolamine, nitrogen mustard and nitrogen half mustard do not regulate expression of CTR/HNM1.     

Fractional Isolation and Study of the Structure of Galactomannan from Sophora (Styphnolobium japonicum) Seeds

Two fractions (1 and 2) of the galactomannan from seeds of sophora (Styphnolobium japonicum) were isolated using cold and hot aqueous extraction with a total yield of 12.88%. The two fractions differed by the ratio between mannose (Man) and galactose (Gal) residues (4.8 : 1 and 5.3 : 1, respectively) and molecular weight (1190 and 1400 kDa, respectively). Aqueous solutions of these fractions were optically active ([]_D = +4.80° and –3.36°, respectively) and highly viscous ([] 1028.8 and 1211.2 ml/g). ¹³C NMR spectra of both fractions were identical with respect to the number and positions of signals, which indicates that their primary structures were identical. Using chemical and spectroscopic (IR and NMR) methods, it was shown that the galactomannan has a main chain consisting of 1,4--D-mannopyranose, some residues of which (16 and 17% in fractions 1 and 2, respectively) are -galactosylated at the C-6 position. Frequencies of differently substituted mannobiose blocks in the chain, calculated for fraction 1 using NMR spectroscopic data, were 0.13 for the disubstitited blocks Gal(Man–Man)Gal, 0.37 for the sum of monosubstituted blocks Gal(Man–Man) and (Man–Man)Gal, and 0.50 for the unsubstituted block Man–Man.     

Action of a cell-envelope proteinase (CEPIII-type) from Lactococcus lactis subsp. cremoris AM1 on bovine κ-casein

The specificity of the cell-envelope proteinase (CEP_III-type) from Lactococcus lactis subsp. cremoris AM₁ in its action on bovine -casein was studied. A 4-h digest (pH 6.2, 15°C) of -casein was made with the purified proteinase. The pH-4.6 soluble fraction, representing more than 95% of the whole hydrolysate, was ultrafiltered to obtain a high-molecular-mass (HMM) and a low-molecular-mass (LMM) fraction, which were separately further purified by electrophoretic and chromatographic techniques. Isolated HMM and LMM products were identified by amino acid analysis, end-group determination and mass spectrometry. On-line HPLC/mass spectrometry was also used for the separation of an LMM peptide mixture and the identification of its components. The HMM products formed were the fragments 1–160, 1–151, 1–95 and 1–79 of -casein, whereas the main LMM products found were the 161–169 and 152–160 fragments. The enzyme specificity was concluded to be primarily directed towards the C-terminal region of the substrate molecule by cleavage of the 160–161 and 151–152 peptide bonds. Two minor LMM products were identified as the fragments 96–104 and 103–106, indicating additional cleavage at positions 102–103, 104–105 and 106–107 of the sequence. Also several peptide bonds within the 161–169 sequence were found to be subject to secondary cleavage by the proteinase. From electrophoretic and identification data it is concluded that the lactococaal CEP_I, CEP_III and several mixed-type proteinases all act on the peptide bonds at positions 79–80 and 95–96. However, the C-terminal region of the -casein sequence is the exclusive target of the CEP_III-aand, to variable extents, of the mixed-type enzymes.     

HMG1 Domains: The Victims of the Circumstances

The method of circular dichroism (CD) was used to compare DNA behavior during its interaction with linker histone H1 and with nonhistone chromosomal protein HMG1 at different ionic strength and at different protein content in the system. The role of the negatively charged C-terminal segment of HMG1 was analyzed using recombinant protein HMG1-(A+B), which lacks the C-terminal amino acid sequence. The -type CD spectra were common for DNA interaction with histone H1, but no spectra of this type were observed in HMG1–DNA systems even at high ionic strength. The CD spectrum of the truncated recombinant protein at high salt concentration somewhat resembled the ⁺-type spectrum. Two very intense positive bands were located near 215 nm and near 272 nm, and the whole CD spectrum was positive. The role of the C-terminal part of HMG1 in the formation of ordered DNA–protein complexes is discussed.     

Purification and structure of mutacin B-Ny266: a new lantibiotic produced by Streptococcus mutans

Mutacins are bactericidal substances of proteinaceous nature produced by Streptococcus mutans. Lantibiotics are antibacterial substances containing post-translationally modified amino acids such as lanthionine. Mutacin B-Ny266 was purified from the cell pellet of S. mutans strain Ny266 by ethanol extraction at pH 2.0 followed by reversed-phase chromatography (Sep-Pak® cartridge) and by HPLC on a C₁₈ column. The mean purification factor was 3240±81 and the mean yield was 1.0±0.1%. Molecular mass of mutacin B-Ny266 as determined by mass spectroscopy is 2270.29±0.21 Da. The amino acid sequence of the purified active fraction was obtained by Edman degradation after treatment with alkaline ethanethiol. Twenty-one amino acids were detected in this analysis. Mutacin B-Ny266 belongs to the type A lantibiotics. The proposed sequence is: F–K–

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–W–U–F–

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–

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–P–G–

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–A–K–O–G–

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–F–N–

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–Y–

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. The molecule differs from that of epidermin/staphylococcin 1580 and gallidermin at positions 1, 2, 4, 5 and 6.     

Nucleotide sequence of the 3′-terminal region of chilli vein-banding mottle potyvirus isolated from pepper in Thailand

The sequence of the 2227 nucleotides of the 3-terminus of chilli vein-banding mottle virus-Chiangmai isolate (CVbMV-CM1) genome was determined. This sequence encodes a putative 287 amino acid coat protein. Downstream of coat protein coding region is a 288 nucleotide untranslated sequence terminated by a polyadenylate tract. The amino acid sequence of coat protein contained aspartic acid–alanine–glycine tripeptide, which may correlate with the high frequency of aphid transmissibility. The cleavage site between large nuclear inclusion protein (NIB) and coat protein was glutamine/serine. Comparison of 3 non-coding region nucleotide and coat protein amino acid sequences of CVbMV-CM1 with those of other 18 potyviruses showed 44–56% and 54–65% homology respectively, suggesting CVbMV should be regarded as new potyvirus.     

KATP channels of mouse skeletal muscle: mechanism of channel blockage by AMP-PNP

Single ATP-sensitive potassium channels (K _ATP channels) were studied in inside-out membrane patches excised from mouse skeletal muscle. Channel blockage by the non-hydrolysable ATP analogue AMP-PNP was investigated in the absence or presence of 1 mM MgCl₂ with K⁺-rich solutions bathing the internal membrane surface. Currents through single. K _ATP channels were recorded at –40 and +40 mV AMP-PNP (5 to 500 M; Li salt) reduced the open-probability p_o of K _ATP channels and decreased the single-channel currents at high nucleotide concentrations by approximately 10%. Half maximal reduction of p_o at –40 mV was observed at nucleotide concentrations of 29 M in the absence and of 39 M in the presence of Mg²⁺. The steepness of the AMP-PNP concentration-response curves was strongly affected by Mg²⁺, the Hill coefficients of the curves were 0.6 in the absence and 1.6 in the presence of 1 mM MgCl₂. The efficacies of channel blockage by AMP-PNP at –40 and ⁺40 mV were not significantly different. The results indicate that a K _ATP channel can bind more divalent Mg²⁺-complexes of AMP-PNP than trivalent protonated forms of the nucleotide and that channel blockage is hardly affected by the membrane electric field. To estimate the contribution of lithium ions to the observed results, we studied the effects of LiCl (0.8 to 10 mM) in the Mg²⁺-free solution on the single channel current i. At a Li⁺ concentration of 10 mM, i was hardly affected at –40 mV but reduced by a factor of 0.75 at +40 mV. The results are interpreted by a fast, voltage-dependent blockage of K _ATP channels by internal Li⁺ ions. Correspondence to: B. Neumcke     

The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: Causes and consequences

Summary A total of 101 different examples of point mutations, which lie in the vicinity of mRNA splice junctions, and which have been held to be responsible for a human genetic disease by altering the accuracy of efficiency of mRNA splicing, have been collated. These data comprise 62 mutations at 5 splice sites, 26 at 3 splice sites and 13 that result in the creation of novel splice sites. It is estimated that up to 15% of all point mutations causing human genetic disease result in an mRNA splicing defect. Of the 5 splice site mutations, 60% involved the invariant GT dinucleotide; mutations were found to be non-randomly distributed with an excess over expectation at positions +1 and +2, and apparent deficiencies at positions –1 and –2. Of the 3 splice site mutations, 87% involved the invariant AG dinucleotide; an excess of mutations over expectation was noted at position -2. This non-randomness of mutation reflects the evolutionary conservation apparent in splice site consensus sequences drawn up previously from primate genes, and is most probably attributable to detection bias resulting from the differing phenotypic severity of specific lesions. The spectrum of point mutations was also drastically skewed: purines were significantly overrepresented as substituting nucleotides, perhaps because of steric hindrance (e.g. in U1 snRNA binding at 5 splice sites). Furthermore, splice sites affected by point mutations resulting in human genetic disease were markedly different from the splice site consensus sequences. When similarity was quantified by a consensus value, both extremely low and extremely high values were notably absent from the wild-type sequences of the mutated splice sites. Splice sites of intermediate similarity to the consensus sequence may thus be more prone to the deleterious effects of mutation. Regarding the phenotypic effects of mutations on mRNA splicing, exon skipping occurred more frequently than cryptic splice site usage. Evidence is presented that indicates that, at least for 5 splice site mutations, cryptic splice site usage is favoured under conditions where (1) a number of such sites are present in the immediate vicinity and (2) these sites exhibit sufficient homology to the splice site consensus sequence for them to be able to compete successfully with the mutated splice site. The novel concept of a potential for cryptic splice site usage value was introduced in order to quantify these characteristics, and to predict the relative proportion of exon skipping vs cryptic splice site utilization consequent to the introduction of a mutation at a normal splice site.     

Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1.

Summary Previous genetic analyses indicated that translational frameshifting in the –1 direction occurs within the run of six adenines in the sequence 5-TTAAAAAACTC-3 at nucleotide positions 305–315 in IS 1, where the two out-of-phase reading frames insA and B-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84–87. IS 1 mutants with a 1 by insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84–87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-LysLys-Leu. An IS 1 mutant with the DNA segment 5-CTTAAAAACTC-3 at positions 305–315 carrying the termination codon TAA in the B-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting. The -galactosidase activity specified by several IS 1- lacZ fusion plasmids, in which B-insB is in-frame with lacZ, showed that the region 292–377 is sufficient for frameshifting. The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu - Lys - Lys - Leu encoded by the DNA segment 5-TTAAAAAACTC-3, indicating that –1 frameshifting does occur within the run of adenines.     

The nucleotide sequence of the mitochondrial DNA molecule of the grey seal,Halichoerus grypus,and a comparison with mitochondrial sequences of other true seals

The sequence of the mtDNA of the grey seal, Halichoerus grypus, was determined. The length of the molecule was 16,797 base pairs. The organization of the molecule conformed with that of other eutherian mammals but the control region was unusually long due to the presence of two types of repeated motifs. The grey seal and the previously reported harbor seal, Phoca vitulina, belong to different but closely related genera of family Phocidae, true (or earless) seals. In order to determine the degree of differences that may occur between mtDNAs of closely related mammalian genera, the 2 rRNA genes, the 13 peptide coding genes, and the 22 tRNA genes of the 2 species were compared. Total nucleotide difference in the peptide coding genes was 2.0–6.1%. The range of conservative difference was 0.0–1.5%. In the inferred peptide sequences the amino acid difference was 0.0–4.5%, and the difference with respect to chemical properties of amino acids was 0.0–3.0%. A gene that showed a limited degree of difference in one mode of comparison did not necessarily show a corresponding limited difference in another mode. The ratio for differences in codon positions 1, 2, and 3 was 2.7:1:16. The corresponding ratio for conservative differences was 1.8:1. l:1. The evolutionary separation of the two species was calculated to have taken place 2–2.5 million years ago. This dating gives the figure 8 × 10^–9 as the mean rate of substitution per site and year in the entire mtDNA molecule. Comparison with the cytochrome b gene of the Hawaiian monk seal and the Weddell seal suggested that the lineage of these two species and that of the grey and harbor seals separated 8 million years ago. Correspondence to: Ú. Árnason