Stacking geometry between two sheared Watson-Crick basepairs: Computational chemistry and bioinformatics based prediction

BackgroundMolecular modeling of RNA double helices is possible using most probable values of basepair parameters obtained from crystal structure database. The A:A w:wC non-canonical basepair, involving Watson-Crick edges of two Adenines in cis orientation, appears quite frequently in database. Bimodal distribution of its Shear, due to two different H-bonding schemes, introduces the confusion in assigning most the probable value. Its effect is pronounced when the A:A w:wC basepair stacks on Sheared wobble G:U W:WC basepairs.MethodsWe employed molecular dynamics simulations of three possible double helices with GAG, UAG and GAU sequence motifs at their centers and quantum chemical calculation for non-canonical A:A w:wC basepair stacked on G:U W:WC basepair.ResultsWe noticed stable structures of GAG motif with specifically negative Shear of the A:A basepair but stabilities of the other motifs were not found with A:A w:wC basepairing. Hybrid DFT-D and MP2 stacking energy analyses on dinucleotide step sequences, A:A w:wC::G:U W:WC and A:A w:wC::U:G W:WC reveal that viable orientation of A:A::G:U prefers one of the H-bonding modes with negative Shear, supported by crystal structure database. The A:A::U:G dinucleotide, however, prefers structure with only positive Shear.ConclusionsThe quantum chemical calculations explain why MD simulations of GAG sequence motif only appear stable. In the cases of the GAU and UAG motifs “tug of war” situation between positive and negative Shears of A:A w:wC basepair induces conformational plasticity.General significanceWe have projected comprehensive reason behind the promiscuous nature of A:A w:wC basepair which brings occasional structural plasticity.     

Role of cysteine amino acid residues on the RNA binding activity of human thymidylate synthase

The role of cysteine sulfhydryl residues on the RNA binding activity of human thymidylate synthase (TS) was investigated by mutating each cysteine residue on human TS to a corresponding alanine residue. Enzymatic activities of TS:C43A and TS:C210A mutant proteins were nearly identical to wild-type TS, while TS:C180A and TS:C199A mutants expressed >80% of wild-type enzyme activity. In contrast, TS:C195A was completely inactive. Mutant proteins, TS:C195A, TS:C199A and TS:C210A, retained RNA binding activity to nearly the same degree as wild-type human TS. RNA binding activity of TS:C43A was reduced by 30% when compared to wild-type TS, while TS:C180A was completely devoid of RNA binding activity. In vitro translation studies confirmed that mutant proteins TS:C43A, TS:C195A, TS:C199A and TS:C210A, significantly repressed human TS mRNA translation, while TS:C180A was unable to do so. To confirm the in vivo significance of the cysteine sulfhydryl residue, mutant proteins TS:C180A and TS:C195A were each expressed in human colon cancer HCT-C18:TS(–) cells that expressed a functionally inactive TS. A recombinant luciferase reporter gene under the control of a TS-response element was co-transfected into these same cells, and luciferase activity increased in the presence of the TS:C195A mutant TS protein to a level similar to that observed upon expression of wild-type TS protein. In contrast, luciferase activity remained unchanged in cells expressing the TS:C180A mutant protein. Taken together, these findings identify Cys-180 as a critical residue for the in vitro and in vivo translational regulatory effects of human TS.     

Interaction of the high density lipoprotein conversion factor with recombinant discoidal complexes of egg phosphatidylcholine, free cholesterol, and apolipoprotein A-I

An HDL conversion factor which promotes the conversion of HDL3 to populations of larger and smaller particles has recently been identified in human plasma. In the present report a partially purified preparation of this factor has been used to examine the conversion of 79:0:1, 77:5:1, and 74:10:1 (mol:mol:mol) egg phosphatidylcholine-free cholesterol-apolipoprotein A-I (apoA-I) recombinant discoidal complexes. The study was carried out in order to ascertain whether the conversion process is regulated by the concentration of free cholesterol in the complexes. The complexes comprised one major and two minor populations of particles with respective Stokes' diameters of 96 A, 84 A, and 78 A. The 74:10:1 complexes also contained a population of particles 112 A in diameter. The 79:0:1 and 77:5:1 complexes contained two molecules of apoA-I per particle. The 74:10:1 complexes comprised two classes of particles with two or three molecules of apoA-I. When the 74:10:1 complexes were incubated with the conversion factor, the 96 A and 84 A particles were converted to a population of particles 78 A in diameter that contained two apoA-I molecules. In the case of the 79:0:1 and 77:5:1 complexes, the 96 A particles were converted to 78 A particles but the concentration of 84 A particles did not change. The rate of conversion of 96 A particles to 78 A particles was dependent on the concentration of free cholesterol in the complexes. When the 74:10:1 complexes were incubated for 24 hr with the conversion factor, the 96 A particles were completely converted to particles 78 A in diameter. In the case of the 77:5:1 complexes, complete conversion was achieved by 48 hr. Conversion of the 79:0:1 complexes did not proceed to completion, even when the incubation was extended beyond 48 hr. The rate of conversion of 96 A particles to 78 A particles was also dependent on the concentration of the conversion factor in the incubation mixtures. The previous incubations contained equivalent concentrations of apoA-I and conversion factor. When the concentration of the conversion factor relative to apoA-I was reduced, there was a concomitant decrease in the rate of conversion of 96 A particles to 78 A particles. Conversion was not evident when the concentration of the conversion factor was reduced to one-tenth that of apoA-I.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of heavy exercise on spectral baroreflex sensitivity, heart rate, and blood pressure variability in well-trained humans

The aim of the study was to assess the instantaneous spectral components of heart rate variability (HRV) and systolic blood pressure variability (SBPV) and determine the low-frequency (LF) and high-frequency baroreflex sensitivity (HF-BRS) during a graded maximal exercise test. The first hypothesis was that the hyperpnea elicited by heavy exercise could entail a significant increase in HF-SBPV by mechanical effect once the first and second ventilatory thresholds (VTs) were exceeded. It was secondly hypothesized that vagal tone progressively withdrawing with increasing load, HF-BRS could decrease during the exercise test. Fifteen well-trained subjects participated in this study. Electrocardiogram (ECG), blood pressure, and gas exchanges were recorded during a cycloergometer test. Ventilatory equivalents were computed from gas exchange parameters to assess VTs. Spectral analysis was applied on cardiovascular series to compute RR and systolic blood pressure power spectral densities, cross-spectral coherence, gain, and alpha index of BRS. Three exercise intensity stages were compared: below (A1), between (A2), and above (A3) VTs. From A1 to A3, both HF-SBPV (A1: 45 +/- 6, A2: 65 +/- 10, and A3: 120 +/- 23 mm2Hg, P < 0.001) and HF-HRV increased (A1: 20 +/- 5, A2: 23 +/- 8, and A3:40 +/- 11 ms2, P < 0.02), maintaining HF-BRS (gain, A1: 0.68 +/- 0.12, A2: 0.63 +/- 0.08, and A3: 0.57 +/- 0.09; alpha index, A1: 0.58 +/- 0.08, A2: 0.48 +/- 0.06, and A3: 0.50 +/- 0.09 ms/mmHg, not significant). However, LF-BRS decreased (gain, A1: 0.39 +/- 0.06, A2: 0.17 +/- 0.02, and A3: 0.11 +/- 0.01, P < 0.001; alpha index, A1: 0.46 +/- 0.07, A2: 0.20 +/- 0.02, and A3: 0.14 +/- 0.01 ms/mmHg, P < 0.001). As expected, once VTs were exceeded, hyperpnea induced a marked increase in both HF-HRV and HF-SBPV. However, this concomitant increase allowed the maintenance of HF-BRS, presumably by a mechanoelectric feedback mechanism.     

Analysis for linkage between F13A and three chromosome 6 marker loci: evidence for 6pter: F13A:HLA:GL01:cen gene order

Summary The results of the present study provide independent support for F13A:HLA linkage and refine the F13A: HLA and F13A: GLO1 linkage relationships. Analysis of the corresponding recombination fractions for the total paternal F13A:HLA and F13A:GLO1 peak lod scores() indicates a locus order of 6pter: F13A:HLA:GLO1:cen. Lod scores between F13A and PLG, a locus recently assigned to chromosome 6, exclude close linkage between these loci.     

Mutational specificity of N-nitrosodimethylamine: Comparison between in vivo and in vitro assays

We have determined the DNA alterations recovered after treatment with Aroclor 1254-treated rat S9-activated N-nitrosodimethylamine (NDMA) in the N-terminal region of the LacI gene of E. coli. A total of 125 independent Lacl^-d mutants of E. Coli were characterized by DNA sequencing. Consistent with the known methylating ability of this compound, the predominant mutation was the G:C → A:T transition, which accounted for 90% of all the mutations recovered. Non G:C → A:T events include 2 G:C → T:A 2 G:C → C:G, 2 A:T → G:C, 3 A:T → T:A and 3 frameshifts. Contingency analysis reveals that NDMA-induced mutation recovered after in vitro activation (S9) have a spectrum very similar to that previously obtained after in vivo activation employing a mouse host-mediated assay. In both systems, G:C → A:T events clearly dominate and their distribution reveals similar site-specificity Moreover, the proportion and kind of non-G:C → A::T events are also similar     

NMR studies of G:A mismatches in oligodeoxyribonucleotide duplexes modelled after ribozymes.

The structures of two oligodeoxyribonucleotide duplexes, the base sequences of which were modelled after both a hammerhead ribozyme and a small metalloribozyme, were studied by NMR. Both duplexes contain adjacent G:A mismatches; one has a PyGAPu:PyGAPu sequence and the other a PyGAPy:PuGAPu sequence. It is concluded on the basis of many characteristic NOEs that in both duplexes G:A base pairs are formed in the unique 'sheared' form, where an amino proton instead of an imino proton of G is involved in the hydrogen bonding, and G and A bases are arranged 'side by side' instead of 'head to head'. A photo-CIDNP experiment, which gives unique and independent information on the solvent accessibility of nucleotide bases, also supports G:A base pairing rather than a bulged-out structure of G and A residues. This is the first demonstration that not only the PyGAPu:PyGAPu sequence but also the PyGAPy:PuGAPu sequence can form the unique sheared G:A base pairs. Taking the previous studies on G:A mismatches into account, the idea is suggested that a PyGA:GAPu sequence is a minimum and essential element for the formation of the sheared G:A base pairs. The sheared G:A base pairs in the PyGAPu:PyGAPu sequence are suggested to be more stable than those in the PyGAPy:PuGAPu sequence. This is explained rationally by the idea proposed above.     

中国湖北地区汉族家系补体第四成分（C4）单倍型的检测

用我室仿国际标准C4定型程序改进后建立的方法及羧肽酶B处理后C4分型方法对湖北地区93个无血缘关系的汉族家系进行C4单倍型的检测,对310个C4单倍型分析可见,我国汉族以A3B1频率最高(0.4194),A3B2次之(0.1161),A2B1与A4B2均为0.0903。以下依次是AQOB1(0.0645)、A3BQO(0.0548)、AQOBQO(0.0322)、A2B2(0.0256)、A4B1(0.0161)、A2B92(0.0129)等。从连锁不平衡参数(Δ)的卡方数值可见,A4B2、AQOBQO及A2B92具有极显著意义的阳性△值;而A4B1与AQOB2则具有极显著意义的阴性△值。将我们的结果与日本人、美国及德国白人,南非黑人的资料进行了对比,并进行了一些讨论。     

Book Reviews

Book reviewed in this article: Palaeobiology of ammonoids: A review by M. J Benton: Ammonoid Paleobiology. Topics in Geobiology. Vol. 13 . By N. H. Landman, K. Tanabe and R. A. Davis. Reconstructing the tree of life: A review by M. J. Benton: Multicellular Animals: A new approach to the phylogenetic order in nature. Volume I . By P. Ax. Springer The reverse of the medal: A review by A. Minelli: Homoplasy. The Recurrence of Similarity in Evolution . By Michael J. Sanderson and Larry Hufford (Eds.).     

Thermodynamic studies of base pairing involving 2,6-diaminopurine.

The thermal stabilities of oligodeoxyribonucleotide duplexes containing 2,6-diaminopurine (D) matched with each of the four normal DNA bases were determined by optical melting techniques. Comparison of optical melting curves yielded relative stabilities for the D-containing standard base pairs in an otherwise identical base-pair sequence. The D:T pair was found to be more stable than the A:T pair in dC3DG3:dC3TG3, as stable as the A:T in dCT3DT3G:dCA3TA3G, and less stable than the A:T in dCA3DA3G:dCT7G. The order of stabilities for X:Y in the DNA duplex dCA3XA3G:dCT3YT3G is: (A:T) greater than (T:D) congruent to (D:T) greater than or equal to (T:A) greater than (C:D) congruent to (D:A) congruent to (D:G) greater than or equal to (D:C) congruent to (G:D) congruent to (D:D) greater than or equal to (A:D). Implications of these results for design of DNA oligonucleotide probes are discussed.     

Active Ras-induced effects on skeletal myoblast differentiation and apoptosis are independent of constitutive PI3-kinase activity

23A2 myoblasts expressing GAP-resistant, constitutively active G12V:H-Ras (A2:G12V:H-Ras myoblasts) display a transformed morphology and do not undergo mitogen-deprivation-induced differentiation or the associated apoptosis. To determine the phenotype induced by F156L:H-Ras, a constitutively active mutant with enhanced nucleotide exchange activity rather than impaired GAP-stimulated GTPase activity, myoblast cell lines were established that stably express F156L:H-Ras at levels of H-Ras comparable to the A2:G12V:H-Ras myoblasts. These A2:F156L:H-Ras myoblast cell lines do not possess a transformed morphology, and while differentiation and apoptosis are impaired, these processes are not abrogated as in the A2:G12V:H-Ras myoblasts. Surprisingly, while expression of either G12V:H-Ras or F156L:H-Ras results in constitutive signaling through PI3-kinase, only cells expressing G12V:H-Ras additionally possess constitutive signaling through MAPK, and NFkappaB. Pharmacological abrogation of the Ras-induced constitutive PI3-kinase signal, however, is not responsible for the impaired differentiation or apoptosis in either A2:G12V:H-Ras myoblasts or A2:F156L:H-Ras myoblasts. Thus, our data suggest that a pathway distinct from those that signals through MAPK, NFkappaB or PI3-kinase is responsible for the impaired differentiation and apoptosis in 23A2 skeletal myoblasts expressing constitutively active Ras.     

HLA genetic profile of Mapuche (Araucanian) Amerindians from Chile

Amerindian Mapuche (Araucanians) are now living in Chile and Argentina at both sides of Andean Mountains. They are anthropologically and genetically different from southernmost South America Patagonian Amerindians. Most of the HLA alleles found in our Mapuche sample are frequent or very frequent in North and South America Amerindians: (1) Class I: A*02:01, A*03:01, A*68:01, B*39:09, B*51:01, (2) Class II: DRB1*03:01, DRB1*04:03, DRB1*07:01, DRB1*08:02, DRB1*14:02, DRB1*16:02. One of the nine most frequent extended haplotypes seems to be from European origin, suggesting the existence of a degree of admixture with Europeans in our Mapuche sample. It has been calculated of about 11 % admixture. Three of the extended haplotypes are also found in other Amerindians and five of them are newly found in Mapuche Amerindians: A*68:01-B*39:09-DRB1*08:02-DQB1*04:02; A*68:01-B*51:01-DRB1*04:03-DQB1*03:02; A*29:01-B*08:01-DRB1*03:01-DQB1*02:01; A*02:01-B*15:01-DRB1*04:03-DQB1*03:02; A*33:01-B*14:02-DRB1*07:01-DQB1*03:03. The medical importance of calculating HLA profile is discussed on the diagnostic (HLA and disease) and therapeutical bases of HLA pharmacogenomics and on the construction of a virtual transplantation HLA list profile. Also, anthropological conclusions are drawn.     

Fatty acid composition of lipid A in Pseudomonas fluorescens

The fatty acid composition of lipid A was studied using gas-liquid chromatography (GLC) and GLC-mass spectrometry in Pseudomonas fluorescens strains of biovars A, B, C, i, F and G, the type strain ATCC 13525 (biovar A) inclusive. The following fatty acids were identified as predominant in the composition of lipid A in the strains representing biovars A, B, C, i, F and G: 3-hydroxydecanoic (3-OH C10:0), 2-hydroxydodecanoic (2-OH C12:0), 3-hydroxydodecanoic (3-OH C12:0), dodecanoic (C12:0), hexadecanoic (C16:0), octadecanoic (C18:0), hexadecenoic (C16:1) and octadecenoic (C18:1) acids. Lipid A of a biovar G strain differed noticeably from other strains in its fatty acid composition. Its main components were as follows: 3-hydroxytetradecanoic (3-OH C14:0), 3-hydroxypentadecanoic (3-OH C15:0) and dodecanoic (C12:0) fatty acids. The coefficients of similarity were determined for lipid A specimens isolated from the studied strains of P. fluorescens by calculating their fatty acid composition with a computer.     

水稻雄性不育新质源的研究──新型水稻细胞质雄性不育系马协A与野败型珍汕97A不育性的遗传学比较

采用套袋自交结实率和自然结实率为主,花粉育性和田间目测整株育性为辅的综合性状,判定新型细胞质雄性不育系马协Ａ以及它与明恢６３的杂种Ｆ_１、Ｆ_２和ＢＦ_１的植株育性,并以野败型珍汕９７Ａ作对照,比较研究了其不育性的遗传规律。结果表明,马协Ａ与珍汕９７Ａ不育性的遗传均由两对基因控制,但新型细胞质雄性不育系马协Ａ两对基因的作用方式与珍汕９７Ａ不同。前者Ｆ_２群体的育性分离符合９：３：３：１的比例,ＢＦ_１符合１：２：１的比例;后者相应群体则符合１２：３：１和２：１：１的比例,两对基因间表现为显性上位。斯米尔诺夫检验也表明马协Ａ／明恢６３和珍汕９７Ａ／明恢６３的Ｆ_２群体的结实率频率分布差异显著（Ｐ＜０．０１）。并讨论了细胞质雄性不育的遗传机理及分子基础。     

DNA alkylation repair limits spontaneous base substitution mutations in Escherichia coli.

The Escherichia coli Ada and Ogt DNA methyltransferases (MTases) are known to transfer simple alkyl groups from O6-alkylguanine and O4-alkylthymine, directly restoring these alkylated DNA lesions to guanine and thymine. In addition to being exquisitely sensitive to the mutagenic effects of methylating agents, E. coli ada ogt null mutants display a higher spontaneous mutation rate than the wild type. Here, we determined which base substitution mutations are elevated in the MTase-deficient cells by monitoring the reversion of six mutated lacZ alleles that revert via each of the six possible base substitution mutations. During exponential growth, the spontaneous rate of G:C to A:T transitions and G:C to C:G transversions was elevated about fourfold in ada ogt double mutant versus wild-type E. coli. Furthermore, compared with the wild type, stationary populations of the MTase-deficient E. coli (under lactose selection) displayed increased G:C to A:T and A:T to G:C transitions (10- and 3-fold, respectively) and increased G:C to C:G, A:T to C:G, and A:T to T:A transversions (10-, 2.5-, and 1.7-fold, respectively). ada and ogt single mutants did not suffer elevated spontaneous mutation rates for any base substitution event, and the cloned ada and ogt genes each restored wild-type spontaneous mutation rates to the ada ogt MTase-deficient strains. We infer that both the Ada MTase and the Ogt MTase can repair the endogenously produced DNA lesions responsible for each of the five base substitution events that are elevated in MTase-deficient cells. Simple methylating and ethylating agents induced G:C to A:T and A:T to G:C transitions in these strains but did not significantly induce G:C to C:G, A:T to C:G, and A:T to T:A transversions. We deduce that S-adenosylmethionine (known to e a weak methylating agent) is not the only metabolite responsible for endogenous DNA alkylation and that at least some of the endogenous metabolites that cause O-alkyl DNA damage in E. coli are not simple methylating or ethylating agents.     

Mutagen specificity and position effects on mutation in T4rII nonsense sites.

In 14 sites in the T4rII region, spontaneous and induced interconversions of ochre (UAA) and opal (UGA) alleles, as well as the reversion of the nonsense sites to r+, were studied. The mutagens employed were 2-aminopurine (2AP), 5-bromouracil (5BU), N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and hydroxylamine (HA). With the test system employed, mutagen specificity (i.e., the preferential induction of A: T leads to G: C or G: C leads to A: T mutation at a given site) can be studied. Simultaneously, the response of similar base pairs at various locations in the same or in different nucleotide triplets throughout the rII region, to a given mutagen, can be compared. 2-Aminopurine can induce transitions of both A: T and G: C base pairs at high rates. This mutagen shows no preference for either direction. Furthermore, there is a correlation between the response to 2AP of an A: T pair occupying a given site, and that of a G: C pair occupying the same site. NTG and HA induce G: C leads to A: T transitions almost exclusively. However, there is a correlation between the low rates of A: T leads to G: C transition induced in each of the various sites by these mutagens and those of G: C leads to A: T. 5-Bromouracil induces transitions from G: C to A: T more readily than from A: T to G: C. With 5BU-induced mutation, there is no correlation between the rates of G: C leads to A: T transitions and those of A: T leads to G: C. In UAA sites, all three adenine:thymidine paris respond to 2AP mutagenesis in a similar pattern, In each position in the triplet, response to 2 AP is correlated with that to 5BU. In UGA sites, there are correlations among the spontaneous as well as the 2AP-, HA- and NTG-induced transition rates. 5BU-induced transition rates are usually not correlated with those induced by other mutagens or with the sponatneous ones.     

Mutations induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lacZ and cII genes of Muta Mouse

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) found in chewing tobacco, snuff, cigarettes, and cigars is a tobacco-specific nitrosamine and classified as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). NNK given intraperitoneally was seen to induce lung and liver adenomas. To evaluate the genotoxicity of NNK in vivo, NNK was intraperitoneally administered to Muta Mouse at two concentrations (125 and 250 mg/kg, once a week for 4 weeks) followed by the measurement of mutant frequencies in the lacZ and cII genes from lung and liver in the same mice. Characterization of the types of the mutation was determined by sequencing the cII genes from mutant plaques. The mutant frequencies in both target genes from both organs dose-dependently increased up to 10 times compared to those of the control group. For the types of mutations, the ratio of the G:C to A:T mutation in the total number of mutants was less than the ratio of A:T to T:A and A:T to C:G transversion, contrary to a previous report. The A:T to T:A transversion was the most highly induced mutation both in the lung and liver cII genes. The increasing rate of mutant frequencies in lung and liver over the vehicle control was 55 and 56 times, respectively, while the increasing rate of G:C to A:T transition was only 1.9 and 2.8 times, respectively. These observations show that NNK predominantly induces DNA adducts leading to A:T to T:A and/or A:T to C:G mutations in the transgene.     

Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: DNA binding specificity based on energetics of DNA kinking.

The catabolite activator protein (CAP) makes no direct contact with the consensus base-pair T:A at position 6 of the DNA half-site 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' but, nevertheless, exhibits strong specificity for T:A at position 6. Binding of CAP results in formation of a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site. The consensus base-pair T:A at position 6 and the consensus base-pair G:C at position 7 form a T:A/G:C step, which is known to be associated with DNA flexibility. It has been proposed that specificity for T:A at position 6 is a consequence of formation of the DNA kink between positions 6 and 7, and of effects of the T:A(6)/G:C(7) step on the geometry of DNA kinking, or the energetics of DNA kinking. In this work, we determine crystallographic structures of CAP-DNA complexes having the consensus base-pair T:A at position 6 or the non-consensus base-pair C:G at position 6. We show that complexes containing T:A or C:G at position 6 exhibit similar overall DNA bend angles and local geometries of DNA kinking. We infer that indirect readout in this system does not involve differences in the geometry of DNA kinking but, rather, solely differences in the energetics of DNA kinking. We further infer that the main determinant of DNA conformation in this system is protein-DNA interaction, and not DNA sequence.     

The importance of distinct metabolites of N-nitrosodiethylamine for its in vivo mutagenic specificity

Although N-nitrosodiethylamine (NDEA) is a potent carcinogen in rodents and a probable human carcinogen, little attempts were made to characterize its mutation spectrum in higher eukaryotes. We have compared forward mutation frequencies at multiple (700) loci with the mutational spectrum induced at the vermilion gene of Drosophila, after exposure of post- and pre-meiotic male germ cells to NDEA. Among 30 vermilion mutants collected from post-meiotic stages were 12 G:C-->A:T transitions (40%), 8 A:T-->T:A transversions (27%), and 4 structural rearrangements (13%). The remainder were three A:T-->G:C transitions, two G:C-->C:G transversions and one G:C-->T:A transversion. The results show that although NDEA induces predominantly transitions (40% G:C-->A:T and 10% A:T-->G:C), the frequencies of transversions (37%, of which 27% of A:T-->T:A transversions) and especially of rearrangements (13%) are remarkably high. This mutation spectrum differs significantly from that produced by the direct-ethylating agent N-ethylnitrosourea (ENU), although the relative distribution of ethylated DNA adducts is similar for both carcinogens. These differences, in particular the occurrence of rearrangements, are most likely the result of the requirement of NDEA for bioactivation. Since all four rearrangements were collected from non-metabolizing spermatozoa (or late spermatids), it is hypothesized that they derived from acetaldehyde, a stable metabolite of NDEA. Due to its cytotoxicity, attempts to isolate vermilion mutants from NDEA-exposed pre-meiotic cells were largely unsuccessful, because only two mutants (one A:T-->G:C transition and one 1bp insertion) were collected from those stages. Our results show that NDEA is capable of generating carcinogenic lesions other than base pair substitutions.