Multiple base-pair mutations in yeast

The nucleotide changes associated with both forward and reverse mutations at the CYC1 locus in the yeast Saccharomyces cerevisiae have been investigated by sequencing the mutated gene product, iso-1-cytochrome c and, more directly, by sequencing appropriate DNA segments. Although the majority of these mutations are the result of single base-pair changes, approximately 10% are the result of multiple mutations and these occur predominantly at certain sites and with certain patterns. Most multiple base-pair changes occur within 20 nucleotides of each other and are generally within six nucleotides. On the basis of the frequencies and patterns of mutations, these nucleotide changes are considered to have occurred as single, concerted events, rather than as multiple independent mutations. Analysis of these mutations indicates that multiple base-pair changes can arise by widely differing mechanisms. We have recognized the following classes of mutations: multiple base-pair changes that yield (1) direct repeats or (2) inverted repeats of local DNA sequences; (3) substitutions of two tandem base-pairs; (4) frameshift and contiguous single base-pair substitutions; and (5) recombination of the CYC1 gene with a non-allelic gene, resulting in alterations within contiguous segments that can be over 150 nucleotides in length. Some of the multiple base-pair changes do not fall into any of these categories. We suggest mechanisms to account for each of these five classes.     

Strong doublet preferences in nucleotide sequences and DNA geometry

Summary Analysis of the sequence data available today, comprising more than 500,000 bases, confirms the previously observed phenomenon that there are distinct dinucleotide preferences in DNA sequences. Consistent behaviour is observed in the major sequence groups analysed here in prokaryotes, eukaryotes and mitochondria. Some doublet preferences are common to all groups and are found in most sequences of the Los Alamos Library. The patterns seen in such large data sets are very significant statistically and biologically. Since they are present in numerous and diverse nucleotide sequences, one may conclude that they confer evolutionary advantages on the organism.In eukaryotes RR and YY dinucleotides are preferred over YR and RY (where R is a purine and Y a pyrimidine). Since opposite-chain nearest-neighbour purine clashes are major determinants of DNA structure, it appears that the tight packaging of DNA in nucleosomes disfavors, in general, such (YR and RY) steric repulsion.     

The ordering of nucleotides in the DNA: strong pyrimidine-purine patterns near homooligomer tracts

Here, we study the frequencies of occurrence of homooligomers flanked by one base, XnU or UXn, where X = A, C, G, T and U not equal to X. Specifically, we search for preferences (or discriminations) in their nearest neighbor doublet, VV. Extensive analysis of the data base reveals striking patterns in such VVUXn or UXn VV oligomers (V = A, C, G, T). With very few exceptions, if the VV and Xn are composed of complementary nucleotides, those oligomers having a pyrimidine (Y)-purine (R) junction are preferred over those with an RY one. If the VV and Xn nucleotides are not complementary, the RY junction oligomers are preferred over their YR counterparts. These trends are observed consistently in eukaryotic and prokaryotic sequences. They are particularly striking in the YR greater than RY oligomers containing complementary nucleotides. The general preferences and discriminations described here are in the same direction as our previous results for homooligomer tracts. These recurrences, along with some additional universal "rules", aid in our understanding of the ordering of nucleotides in the DNA.     

Purification of DNA complementary to nucleotide sequences required for neoplastic transformation of fibroblasts by avian sarcoma viruses.

We have prepared radioactive DNA (cDNA_sarc) complementary to nucleotide sequences which represent at least a portion of the viral gene(s) required for neoplastic transformation of fibroblasts by an avian sarcoma virus. The genetic complexity of cDNA_sarc (~1600 nucleotides) is sufficient to represent an entire cistron. The genomes of three independent isolates of avian sarcoma viruses share nucleotide sequences closely related to cDNA_sarc, whereas the sequences are absent from transformation-defective mutants of avian sarcoma viruses, several avian leukosis viruses, a non-pathogenic endogenous virus of chickens (Rous-associated virus-O), sarcoma-leukosis viruses of mice and cats, and mouse mammary tumor virus. We conclude that the transforming gene(s) of all avian sarcoma viruses have closely related or common genetic lineages distinct from the transforming genes in sarcoma viruses of other species. Our results conform to previous reports that transformation-defective variants of avian sarcoma viruses are mutants with identical regions deleted from each subunit of a polyploid genome.     

BK virus DNA sequence coding for the t and T antigens and evaluation of methods for determining sequence homology.

The DNA sequence of the early region of the human papovavirus BK (MM strain) was determined. A potential initiation signal for translation is located at nucleotides 3,047 to 3,045 or map position 0.614. Extending counterclockwise from this AUG signal there is only one open reading frame, which can code for a putative t antigen of 100 amino acids in length. If the early mRNA of BKV is spliced, then the regions between nucleotides 3,047 to 2,808 and 2,725 to 884 can code for a T antigen 694 amino acids in length. The sequences of the deduced T antigens in BK virus share 71% amino acid homology with those in simian virus 40, whereas the coding sequences of the two viruses share 70% DNA homology. Comparison of DNA sequences and evaluation of homology measurements between these two viruses are discussed.     

DNA sequence of the viral and cellular src gene of chickens. II. Comparison of the src genes of two strains of avian sarcoma virus and of the cellular homolog

The nucleotide sequence of the src gene and flanking regions of the Schmidt-Ruppin strain of Rous sarcoma virus (SR-A) was determined. The src region of SR-A was very homologous to that of recovered avian sarcoma virus (rASV1441), with only 17 differences among 1,578 nucleotides. The size of the predicted protein was 526 amino acids in both viruses, of which 6 amino acids were different. The differences in nucleotides and amino acids between the two viruses localized within the 5' two-thirds of the src coding region. There were also viruses localized within the 5' two-thirds of the src coding region. There were also some differences in the region flanking the 5' end of src. Since rASVs are considered to be recombinatns between deletion mutants of SR-A and cellular-src (c-src) sequences, several segments of c-src DNA were also sequenced to understand the molecular basis for the recombination. At 14 of 17 bases where SR-A and rASV1441 differed, rASV1441 had the same sequence as c-src. Three of these sequences corresponded to sequences of oligonucleotides which were previously identified in RNAs of nearly all isolates of rASV but which were absent in SR-A RNA. In the 5'-flanking sequences of the src gene, c-src was more similar to rASV1441 than to SR-A. These results confirm the cellular origin of the src sequences of rASVs and provide information about the possible sites of the recombination.     

Top-heavy trophic structure within benthic viral dark matter

A better understanding of system-specific viral ecology in diverse environments is needed to predict patterns of virus–host trophic structure in the Anthropocene. This study characterised viral-host trophic structure within coral reef benthic cyanobacterial mats—a globally proliferating cause and consequence of coral reef degradation. We employed deep longitudinal multi-omic sequencing to characterise the viral assemblage (ssDNA, dsDNA, and dsRNA viruses) and profile lineage-specific host–virus interactions within benthic cyanobacterial mats sampled from Bonaire, Caribbean Netherlands. We recovered 11,012 unique viral populations spanning at least 10 viral families across the orders Caudovirales, Petitvirales, and Mindivirales. Gene-sharing network analyses provided evidence for extensive genomic novelty of mat viruses from reference and environmental viral sequences. Analysis of coverage ratios of viral sequences and computationally predicted hosts spanning 15 phyla and 21 classes revealed virus–host abundance (from DNA) and activity (from RNA) ratios consistently exceeding 1:1, suggesting a top-heavy intra-mat trophic structure with respect to virus–host interactions. Overall, our article contributes a curated database of viral sequences found in Caribbean coral reef benthic cyanobacterial mats (vMAT database) and provides multiple lines of field-based evidence demonstrating that viruses are active members of mat communities, with broader implications for mat functional ecology and demography.     

Micrococcal nuclease as a DNA structural probe: its recognition sequences, their genomic distribution and correlation with DNA structure determinants

We have analyzed micrococcal nuclease (MNase) DNA cleavage patterns at the sequence level by examining 2.3 X 10(3) base-pairs of data derived from the Drosophila melanogaster 44D larval cuticle locus. Within this region, MNase preferentially cleaved 140 sites. Clusters of these sites appear to generate the preferential MNase eukaryotic DNA cleavage sites seen on agarose gels at roughly 100 to 300 base-pair intervals. These clusters of preferential cleavage sites rarely occur within gene coding regions. The analysis revealed that duplex DNA sequences preferentially cleaved by MNase are generally determined by a single strand sequence: d(A-T)n, where n greater than or equal to 1, flanked by a 5' dC or dG. Cleavage of the other strand is generally staggered 5' by several nucleotides and occurs even if such sequences are absent on that strand. An empirical predictive DNA cleavage model derived from a statistical analysis of the sequence level data was applied to seven eukaryotic gene loci of known sequence. The predicted patterns were in good general agreement with the previously observed eukaryotic gene/spacer cleavage pattern. Statistical analysis also revealed that sites of predicted preferential DNA cleavage occur less frequently in protein coding regions than for randomized sequences of the same length and nucleotide content. Comparison of the MNase cleavage patterns to the sequence-dependent pattern of binding energies between duplex DNA strands indicates that MNase preferentially cleaves sequences with low helix stability.     

Pattern of selective constraint in C. elegans and C. briggsae genomes.

Similarity between related genomes may carry information on selective constraint in each of them. We analysed patterns of similarity between several homologous regions of Caenorhabditis elegans and C. briggsae genomes. All homologous exons are quite similar. Alignments of introns and of intergenic sequences contain long gaps, segments where similarity is low and close to that between random sequences aligned using the same parameters, and segments of high similarity. Conservative estimates of the fractions of selectively constrained nucleotides are 72%, 17% and 18% for exons, introns and intergenic sequences, respectively. This implies that the total number of constrained nucleotides within non-coding sequences is comparable to that within coding sequences, so that at least one-third of nucleotides in C. elegans and C. briggsae genomes are under strong stabilizing selection.     

Previously unknown and highly divergent ssDNA viruses populate the oceans

Single-stranded DNA (ssDNA) viruses are economically important pathogens of plants and animals, and are widespread in oceans; yet, the diversity and evolutionary relationships among marine ssDNA viruses remain largely unknown. Here we present the results from a metagenomic study of composite samples from temperate (Saanich Inlet, 11 samples; Strait of Georgia, 85 samples) and subtropical (46 samples, Gulf of Mexico) seawater. Most sequences (84%) had no evident similarity to sequenced viruses. In total, 608 putative complete genomes of ssDNA viruses were assembled, almost doubling the number of ssDNA viral genomes in databases. These comprised 129 genetically distinct groups, each represented by at least one complete genome that had no recognizable similarity to each other or to other virus sequences. Given that the seven recognized families of ssDNA viruses have considerable sequence homology within them, this suggests that many of these genetic groups may represent new viral families. Moreover, nearly 70% of the sequences were similar to one of these genomes, indicating that most of the sequences could be assigned to a genetically distinct group. Most sequences fell within 11 well-defined gene groups, each sharing a common gene. Some of these encoded putative replication and coat proteins that had similarity to sequences from viruses infecting eukaryotes, suggesting that these were likely from viruses infecting eukaryotic phytoplankton and zooplankton.     

Polymerase errors accumulating during natural evolution of the glycoprotein gene of vesicular stomatitis virus Indiana serotype isolates

We report the entire glycoprotein (G) gene nucleotide sequences of 26 vesicular stomatitis virus Indiana serotype (VSV IND) type 1 isolates from North and Central America. These sequences are also compared with partial G gene sequences of VSV IND type 2 (Cocal) and type 3 (Alagoas) viruses and the complete G gene sequences of the more distantly related VSV New Jersey (NJ) and Chandipura viruses. Phylogenetic analysis of the G gene sequences by maximum parsimony revealed four major lineages or subtypes within the classical VSV IND (type 1) viruses, each with a distinct geographic distribution. A high degree of VSV genetic diversity was found in Central America, with several virus subtypes of both VSV IND and NJ serotypes existing in this mainly enzootic disease region. Nineteen percent sequence variation but no deletions or insertions were evident within the 5' noncoding and the coding regions of the VSV IND type 1 G genes. In addition to numerous base substitutions, the 3' noncoding regions of these viruses also contained numerous base insertions and deletions. This resulted in striking variation in G gene sizes, with gene lengths ranging from 1,652 to 1,868 nucleotides. As the VSV IND type 1 subtypes have diverged from the common ancestor with the NJ subtypes, their G mRNAs have accumulated more 3' noncoding sequence inserts, ranging up to 303 nucleotides in length. These primarily consist of an imprecise reiteration of the sequence UUUUUAA, apparently generated by a unique polymerase stuttering error. Analysis of the deduced amino acid sequence differences among VSV IND type 1 viruses revealed numerous substitutions within defined antigenic epitopes, suggesting that immune selection may play a role in the evolution of these viruses.     

Profound flanking sequence preference of Dnmt3a and Dnmt3b mammalian DNA methyltransferases shape the human epigenome

Mammalian DNA methyltransferases methylate cytosine residues within CG dinucleotides. By statistical analysis of published data of the Human Epigenome Project we have determined flanking sequences of up to +/-four base-pairs surrounding the central CG site that are characteristic of high (5'-CTTGCGCAAG-3') and low (5'-TGTTCGGTGG-3') levels of methylation in human genomic DNA. We have investigated the influence of flanking sequence on the catalytic activity of the Dnmt3a and Dnmt3b de novo DNA methyltransferases using a set of synthetic oligonucleotide substrates that covers all possible +/-1 flanks in quantitative terms. Methylation kinetics experiments revealed a >13-fold difference between the preferred (RCGY) and disfavored +/-1 flanking base-pairs (YCGR). In addition, AT-rich flanks are preferred over GC-rich ones. These experimental preferences coincide with the genomic methylation patterns. Therefore, we have expanded our experimental analysis and found a >500-fold difference in the methylation rates of the consensus sequences for high and low levels of methylation in the genome. This result demonstrates a very pronounced flanking sequence preference of Dnmt3a and Dnmt3b. It suggests that the methylation pattern of human DNA is due, in part, to the flanking sequence preferences of the de novo DNA MTases and that flanking sequence preferences could be involved in the origin of CG islands. Furthermore, similar flanking sequence preferences have been found for the stimulation of the immune system by unmethylated CGs, suggesting a co-evolution of DNA MTases and the immune system.     

The variable codons of H5N1 avian influenza A virus haemagglutinin genes

We investigated the selection pressures on the haemagglutinin genes of H5N1 avian influenza viruses using fixed effects likelihood models. We found evidence of positive selection in the sequences from isolates from 1997 to 2007, except viruses from 2000. The haemagglutinin sequences of viruses from southeast Asia, Hong Kong and mainland China were the most polymorphic and had similar nonsynonymous profiles. Some sites were positively selected in viruses from most regions and a few of these sites displayed different amino acid patterns. Selection appeared to produce different outcomes in viruses from Europe, Africa and Russia and from different host types. One position was found to be positively selected for human isolates only. Although the functions of some positively selected positions are unknown, our analysis provided evidence of different temporal, spatial and host adaptations for H5N1 avian influenza viruses.     

env Gene of Chicken RNA Tumor Viruses: Extent of Conservation in Cellular and Viral Genomes

The env gene of avian sarcoma-leukosis viruses codes for envelope glycoproteins that determine viral host range, antigenic specificity, and interference patterns. We used molecular hybridization to analyze the natural distribution and possible origins of the nucleotide sequences that encode env; our work exploited the availability of radioactive DNA (cDNA(gp)) complementary to most or all of env. env sequences were detectable in the DNAs of chickens which synthesized an env gene product (chick helper factor positive) encoded by an endogenous viral gene and also in the DNAs of chickens which synthesized little or no env gene product (chick helper factor negative). env sequences were not detectable in DNAs from Japanese quail, ring-necked pheasant, golden pheasant, duck, squab, salmon sperm, or calf thymus. The detection of sequences closely related to viral env only in chicken DNA contrasts sharply with the demonstration that the transforming gene (src) of avian sarcoma viruses has readily detectable homologues in the DNAs of all avian species tested [D. Stehelin, H. E. Varmus, J. M. Bishop, and P. K. Vogt, Nature (London) 260: 170-173, 1976] and in the DNAs of other vertebrates (D. Spector, personal communication). Thermal denaturation studies on duplexes formed between cDNA(gp) and chicken DNA and also between cDNA(gp) and RNAs of subgroup A to E viruses derived from chickens indicated that these duplexes were well matched. In contrast, cDNA(gp) did not form stable hybrids with RNAs of viruses which were isolated from ring-necked and golden pheasants. We conclude that substantial portions of nucleotide sequences within the env genes of viruses of subgroups A to E are closely related and that these genes probably have a common, perhaps cellular, evolutionary origin.     

Probing the DNA sequence specificity of Escherichia coli RECA protein

Escherichia coli RecA protein catalyzes the central DNA strand-exchange step of homologous recombination, which is essential for the repair of double-stranded DNA breaks. In this reaction, RecA first polymerizes on single-stranded DNA (ssDNA) to form a right-handed helical filament with one monomer per 3 nt of ssDNA. RecA generally binds to any sequence of ssDNA but has a preference for GT-rich sequences, as found in the recombination hot spot Chi (5′-GCTGGTGG-3′). When this sequence is located within an oligonucleotide, binding of RecA is phased relative to it, with a periodicity of three nucleotides. This implies that there are three separate nucleotide-binding sites within a RecA monomer that may exhibit preferences for the four different nucleotides. Here we have used a RecA coprotease assay to further probe the ssDNA sequence specificity of E.coli RecA protein. The extent of self-cleavage of a λ repressor fragment in the presence of RecA, ADP-AlF₄ and 64 different trinucleotide-repeating 15mer oligonucleotides was determined. The coprotease activity of RecA is strongly dependent on the ssDNA sequence, with TGG-repeating sequences giving by far the highest coprotease activity, and GC and AT-rich sequences the lowest. For selected trinucleotide-repeating sequences, the DNA-dependent ATPase and DNA-binding activities of RecA were also determined. The DNA-binding and coprotease activities of RecA have the same sequence dependence, which is essentially opposite to that of the ATPase activity of RecA. The implications with regard to the biological mechanism of RecA are discussed.