Distinct spectra of somatic mutations accumulated with age in mouse heart and small intestine

Somatic mutation accumulation has been implicated as a major cause of cancer and aging. By using a transgenic mouse model with a chromosomally integrated lacZ reporter gene, mutational spectra were characterized at young and old age in two organs greatly differing in proliferative activity, i.e., the heart and small intestine. At young age the spectra were nearly identical, mainly consisting of G·C to A·T transitions and 1-bp deletions. At old age, however, distinct patterns of mutations had developed. In small intestine, only point mutations were found to accumulate, including G·C to T·A, G·C to C·G, and A·T to C·G transversions and G·C to A·T transitions. In contrast, in heart about half of the accumulated mutations appeared to be large genome rearrangements, involving up to 34 centimorgans of chromosomal DNA. Virtually all other mutations accumulating in the heart appeared to be G·C to A·T transitions at CpG sites. These results suggest that distinct mechanisms lead to organ-specific genome deterioration and dysfunction at old age.

Bidirectional imprinting of a single gene: GNAS1 encodes maternally, paternally, and biallelically derived proteins

The GNAS1 gene encodes the α subunit of the guanine nucleotide-binding protein Gs, which couples signaling through peptide hormone receptors to cAMP generation. GNAS1 mutations underlie the hormone resistance syndrome pseudohypoparathyroidism type Ia (PHP-Ia), so the maternal inheritance displayed by PHP-Ia has raised suspicions that GNAS1 is imprinted. Despite this suggestion, in most tissues Gsα is biallelically encoded. In contrast, the large G protein XLαs, also encoded by GNAS1, is paternally derived. Because the inheritance of PHP-Ia predicts the existence of maternally, rather than paternally, expressed transcripts, we have investigated the allelic origin of other mRNAs derived from GNAS1. We find this gene to be remarkable in the complexity of its allele-specific regulation. Two upstream promoters, each associated with a large coding exon, lie only 11 kb apart, yet show opposite patterns of allele-specific methylation and monoallelic transcription. The more 5′ of these exons encodes the neuroendocrine secretory protein NESP55, which is expressed exclusively from the maternal allele. The NESP55 exon is 11 kb 5′ to the paternally expressed XLαs exon. The transcripts from these two promoters both splice onto GNAS1 exon 2, yet share no coding sequences. Despite their structural unrelatedness, the encoded proteins, of opposite allelic origin, both have been implicated in regulated secretion in neuroendocrine tissues. Remarkably, maternally (NESP55), paternally (XLαs), and biallelically (Gsα) derived proteins all are produced by different patterns of promoter use and alternative splicing of GNAS1, a gene showing simultaneous imprinting in both the paternal and maternal directions.

Methylation of the minimal promoter of an embryonic globin gene silences transcription in primary erythroid cells

Methylation of cytosines in the dinucleotide CpG has been shown to suppress transcription of a number of tissue-specific genes, yet the precise mechanism is not fully understood. The vertebrate globin genes were among the first examples in which an inverse correlation was shown between CpG methylation and transcription. We studied the methylation pattern of the 235-bp ρ-globin gene promoter in genomic DNA from primary chicken erythroid cells using the sodium bisulfite conversion technique and found all CpGs in the promoter to be methylated in erythroid cells from adult chickens in which the ρ-globin gene is silent but unmethylated in 5-day (primitive) embryonic red cells in which the gene is transcribed. To elucidate further the mechanism of methylation-induced silencing, an expression construct consisting of 235 bp of 5′ promoter sequence of the ρ-globin gene along with a strong 5′ erythroid enhancer driving a chloramphenicol acetyltransferase reporter gene, ρ-CAT, was transfected into primary avian erythroid cells derived from 5-day embryos. Methylation of just the 235-bp ρ-globin gene promoter fragment at every CpG resulted in a 20- to 30-fold inhibition of transcription, and this effect was not overridden by the presence of potent erythroid-specific enhancers. The ability of the 235-bp ρ-globin gene promoter to bind to a DNA Methyl Cytosine binding Protein Complex (MeCPC) was tested in electrophoretic mobility shift assays utilizing primary avian erythroid cell nuclear extract. The results were that fully methylated but not unmethylated 235-bp ρ-globin gene promoter fragment could compete efficiently for MeCPC binding. These results are a direct demonstration that site-specific methylation of a globin gene promoter at the exact CpGs that are methylated in vivo can silence transcription in homologous primary erythroid cells. Further, these data implicate binding of MeCPC to the promoter in the mechanism of silencing.

trEST, trGEN and Hits: access to databases of predicted protein sequences

High throughput genome (HTG) and expressed sequence tag (EST) sequences are currently the most abundant nucleotide sequence classes in the public database. The large volume, high degree of fragmentation and lack of gene structure annotations prevent efficient and effective searches of HTG and EST data for protein sequence homologies by standard search methods. Here, we briefly describe three newly developed resources that should make discovery of interesting genes in these sequence classes easier in the future, especially to biologists not having access to a powerful local bioinformatics environment. trEST and trGEN are regularly regenerated databases of hypothetical protein sequences predicted from EST and HTG sequences, respectively. Hits is a web-based data retrieval and analysis system providing access to precomputed matches between protein sequences (including sequences from trEST and trGEN) and patterns and profiles from Prosite and Pfam. The three resources can be accessed via the Hits home page (http://hits.isb-sib.ch).

Relationships of cereal crops and other grasses

The grass family includes some 10,000 species, and it encompasses tremendous morphological, physiological, ecological, and genetic diversity. The phylogeny of the family is becoming increasingly well understood. There were two major radiations of grasses, an early diversification leading to the subfamilies Pooideae, Bambusoideae, and Oryzoideae, and a later one leading to Panicoideae, Chloridoideae, Centothecoideae, and Arundinoideae. The phylogeny can be used to determine the direction of changes in genome arrangement and genome size.

Plant genome values: How much do we know?

Plants are the basis of life on earth. We cannot overemphasize their importance. The value of plant genome initiatives is self-evident. The need is to identify priorities for action. The angiosperm genome is highly variable, but the extent of this variability is unknown. Uncertainties remain about the number of genes and the number of species living. Many plants will become extinct before they are discovered. We risk losing both genes and vital information about plant uses. There are also major gaps in our karyotypic knowledge. No chromosome count exists for >70% of angiosperm species. DNA C values are known for only ≈1% of angiosperms, a sample unrepresentative of the global flora. Researchers reported new relationships between genome size and characters of major interest for plant breeding and the environment and the need for more data. In 1997, a Royal Botanic Gardens Kew workshop identified gaps and planned international collaboration to fill them. An electronic version of the Angiosperm DNA C value database also was published. Another initiative, which will make a very significant contribution to the conservation of plant genetic diversity on a global scale is Kew’s Millennium Seed Bank, partly funded by the U.K. Millennium Commission, celebrating the year 2000. Costing up to £80 million (£1 = $1.62), its main aims are to collect and conserve the seed of almost all of the U.K. spermatophyte flora by the year 2000, to collect and conserve a further 10% of the world spermatophyte flora principally from the drylands by 2009, and to provide a world class building as the focus of this activity by 2000.

Reovirus reverse genetics: Incorporation of the CAT gene into the reovirus genome

We have modified the infectious reovirus RNA system so as to generate a reovirus reverse genetics system. The system consists of (i) the plus strands of nine wild-type reovirus genome segments; (ii) transcripts of the genetically modified cDNA form of the tenth genome segment; and (iii) a cell line transformed so as to express the protein normally encoded by the tenth genome segment. In the work described here, we have generated a serotype 3 reovirus into the S2 double-stranded RNA genome segment of which the CAT gene has been cloned. The virus is stable, replicates in cells that have been transformed (so as to express the S2 gene product, protein σ2), and expresses high levels of CAT activity. This technology can be extended to members of the orbivirus and rotavirus genera. This technology provides a powerful system for basic studies of double-stranded RNA virus replication; a nonpathogenic viral vector that replicates to high titers and could be used for clinical applications; and a system for providing nonselectable viral variants (the result of mutations, insertions, and deletions) that could be valuable for the construction of viral vaccine strains against human and animal pathogens.

Heat effects on DNA repair after ionising radiation: hyperthermia commonly increases the number of non-repaired double-strand breaks and structural rearrangements

After ionising radiation double-strand breaks (dsb) are lethal if not repaired or misrepaired. Cell killing is greatly enhanced by hyperthermia and it is questioned here whether heat not only affects dsb repair capacity but also fidelity in a chromosomal context. dsb repair experiments were designed so as to mainly score non-homologous end joining, while homologous recombination was largely precluded. Human male G0 fibroblasts were either preheated (45°C, 20 min) or not before X-irradiation. dsb induction and repair were measured by conventional gel electrophoresis and an assay combining restriction digestion using a rare cutting enzyme (NotI) and Southern hybridisation, which detects large chromosomal rearrangements (>100 kb). dsb induction rate in an X-chromosomal NotI fragment was 4.8 × 10–3 dsb/Gy/Mb. Similar values were found for the genome overall and also when cells were preheated. After 50 Gy, fibroblasts were competent to largely restore the original restriction fragment size. Five per cent of dsb remained non-rejoined and 14% were misrejoined. Correct restitution of restriction fragments occurred preferably during the first hour but continued at a slow rate for 12–16 h. In addition, dsb appeared to misrejoin throughout the entire repair period. After hyperthermia the fractions of non-rejoined and misrejoined dsb were similarly increased to 13 and 51%, respectively. It is suggested that heat increases the probability of dsb being incorrectly rejoined but it is not likely to interfere with one dsb repair pathway in particular.

A yeast gene, MGS1, encoding a DNA-dependent AAA+ ATPase is required to maintain genome stability

Changes in DNA superhelicity during DNA replication are mediated primarily by the activities of DNA helicases and topoisomerases. If these activities are defective, the progression of the replication fork can be hindered or blocked, which can lead to double-strand breaks, elevated recombination in regions of repeated DNA, and genome instability. Hereditary diseases like Werner's and Bloom's Syndromes are caused by defects in DNA helicases, and these diseases are associated with genome instability and carcinogenesis in humans. Here we report a Saccharomyces cerevisiae gene, MGS1 (Maintenance of Genome Stability 1), which encodes a protein belonging to the AAA+ class of ATPases, and whose central region is similar to Escherichia coli RuvB, a Holliday junction branch migration motor protein. The Mgs1 orthologues are highly conserved in prokaryotes and eukaryotes. The Mgs1 protein possesses DNA-dependent ATPase and single-strand DNA annealing activities. An mgs1 deletion mutant has an elevated rate of mitotic recombination, which causes genome instability. The mgs1 mutation is synergistic with a mutation in top3 (encoding topoisomerase III), and the double mutant exhibits severe growth defects and markedly increased genome instability. In contrast to the mgs1 mutation, a mutation in the sgs1 gene encoding a DNA helicase homologous to the Werner and Bloom helicases suppresses both the growth defect and the increased genome instability of the top3 mutant. Therefore, evolutionarily conserved Mgs1 may play a role together with RecQ family helicases and DNA topoisomerases in maintaining proper DNA topology, which is essential for genome stability.

Use of cDNA subtraction and RNA interference screens in combination reveals genes required for germ-line development in Caenorhabditis elegans

Caenorhabditis elegans is an ideal organism for the study of the molecular basis of fundamental biological processes such as germ-line development, especially because of availability of the whole genome sequence and applicability of the RNA interference (RNAi) technique. To identify genes involved in germ-line development, we produced subtracted cDNA pools either enriched for or deprived of the cDNAs from germ-line tissues. We then performed differential hybridization on the high-density cDNA grid, on which about 7,600 nonoverlapping expressed sequence tag (EST) clones were spotted, to identify a set of genes specifically expressed in the germ line. One hundred and sixty-eight clones were then tested with the RNAi technique. Of these, 15 clones showed sterility with a variety of defects in germ-line development. Seven of them led to the production of unfertilized eggs, because of defects in spermatogenesis (4 clones), or defects in the oocytes (3 clones). The other 8 clones led to failure of oogenesis. These failures were caused by germ-line proliferation defect (Glp phenotype), meiotic arrest, and defects in sperm–oocyte switch (Mog phenotype) among others. These results demonstrate the efficacy of the screening strategy using the EST library combined with the RNAi technique in C. elegans.

Complete replication of an animal virus and maintenance of expression vectors derived from it in Saccharomyces cerevisiae.

Here we describe the first instances to our knowledge of animal virus genome replication, and of de novo synthesis of infectious virions by a nonendogenous virus, in the yeast Saccharomyces cerevisiae, whose versatile genetics offers significant advantages for studying viral replication and virus-host interactions. Flock house virus (FHV) is the most extensively studied member of the Nodaviridae family of (+) strand RNA animal viruses. Transfection of yeast with FHV genomic RNA induced viral RNA replication, transcription, and assembly of infectious virions. Genome replication and virus synthesis were robust: all replicating FHV RNA species were readily detected in yeast by Northern blot analysis and yields of virions per cell were similar to those from Drosophila cells. We also describe in vivo expression and maintenance of a selectable yeast marker gene from an engineered FHV RNA derivative dependent on FHV-directed RNA replication. Use of these approaches with FHV and their possible extension to other viruses should facilitate identification and characterization of host factors required for genomic replication, gene expression, and virion assembly.

The physical and genomic organization of microsatellites in sugar beet.

Microsatellites, tandem arrays of short (2-5 bp) nucleotide motifs, are present in high numbers in most eukaryotic genomes. We have characterized the physical distribution of microsatellites on chromosomes of sugar beet (Beta vulgaris L.). Each microsatellite sequence shows a characteristic genomic distribution and motif-dependent dispersion, with site-specific amplification on one to seven pairs of centromeres or intercalary chromosomal regions and weaker, dispersed hybridization along chromosomes. Exclusion of some microsatellites from 18S-5.8S-25S rRNA gene sites, centromeres, and intercalary sites was observed. In-gel and in situ hybridization patterns are correlated, with highly repeated restriction fragments indicating major centromeric sites of microsatellite arrays. The results have implications for genome evolution and the suitability of particular microsatellite markers for genetic mapping and genome analysis.

Structural variation of the pseudoautosomal region between and within inbred mouse strains.

The pseudoautosomal region (PAR) is a segment of shared homology between the sex chromosomes. Here we report additional probes for this region of the mouse genome. Genetic and fluorescence in situ hybridization analyses indicate that one probe, PAR-4, hybridizes to the pseudoautosomal telomere and a minor locus at the telomere of chromosome 9 and that a PCR assay based on the PAR-4 sequence amplifies only the pseudoautosomal locus (DXYHgu1). The region detected by PAR-4 is structurally unstable; it shows polymorphism both between mouse strains and between animals of the same inbred strain, which implies an unusually high mutation rate. Variation occurs in the region adjacent to a (TTAGGG)n array. Two pseudoautosomal probes can also hybridize to the distal telomeres of chromosomes 9 and 13, and all three telomeres contain DXYMov15. The similarity between these telomeres may reflect ancestral telomere-telomere exchange.

5-Methyl-2'-deoxycytidine in single-stranded DNA can act in cis to signal de novo DNA methylation.

Methylation of cytosine residues in DNA plays an important role in regulating gene expression during vertebrate embryonic development. Conversely, disruption of normal patterns of methylation is common in tumors and occurs early in progression of some human cancers. In vertebrates, it appears that the same DNA methyltransferase maintains preexisting patterns of methylation during DNA replication and carries out de novo methylation to create new methylation patterns. There are several indications that inherent signals in DNA structure can act in vivo to initiate or block de novo methylation in adjacent DNA regions. To identify sequences capable of enhancing de novo methylation of DNA in vitro, we designed a series of oligodeoxyribonucleotide substrates with substrate cytosine residues in different sequence contexts. We obtained evidence that some 5-methylcytosine residues in these single-stranded DNAs can stimulate de novo methylation of adjacent sites by murine DNA 5-cytosine methyltransferase as effectively as 5-methylcytosine residues in double-stranded DNA stimulate maintenance methylation. This suggests that double-stranded DNA may not be the primary natural substrate for de novo methylation and that looped single-stranded structures formed during the normal course of DNA replication or repair serve as "nucleation" sites for de novo methylation of adjacent DNA regions.

Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3′ end target sequence

Unique, small sequences (sequence tag sites) have been identified at the 3′ ends of most human genes that serve as landmarks in genome mapping. We investigated whether a single copy gene could be isolated directly from total human DNA by transformation-associated recombination (TAR) cloning in yeast using a short, 3′ unique target. A TAR cloning vector was constructed that, when linearized, contained a small amount (381 bp) of 3′ hypoxanthine phosphoribosyltransferase (HPRT) sequence at one end and an 189-bp Alu repeat at the other end. Transformation with this vector along with human DNA led to selective isolations of the entire HPRT gene as yeast artificial chromosomes (YACs) that extended from the 3′ end sequence to various Alu positions as much as 600 kb upstream. These YACs were retrofitted with a NeoR and a bacterial artificial chromosome (BAC) sequence to transfer the YACs to bacteria and subsequently the BACs to mouse cells by using a Neo selection. Most of the HPRT isolates were functional, demonstrating that TAR cloning retains the functional integrity of the isolated material. Thus, this modified version of TAR cloning, which we refer to as radial TAR cloning, can be used to isolate large segments of the human genome accurately and directly with only a small amount of sequence information.