CpG Doublets Research Articles

CpG and TpA frequencies in the plant system.

Higher plant nuclear sequences reveal avoidance of CpG and TpA doublets. Chloroplast sequences avoid the TpA doublet in all codon positions. The chloroplast genome is not methylated but codon positions II-III and untranslated regions avoid CpG. The mitochondrial genome, also unmethylated, avoids CpG in all codon positions. We therefore deduce that methylation is not sufficient to explain CpG avoidance in the higher plant systems. Other factors must be taken into account such as amino acid composition, codon choices and perhaps stability of the DNA helix.

Long range genome structure around the human alpha-globin complex analysed by PFGE.

A map encompassing 300 kilobases (kb) in and around the human alpha-globin gene complex shows features with important implications for understanding the structure and function of the human genome. In contrast to other segments of the mammalian genome that have been analysed by pulsed field gradient electrophoresis (PFGE), this region contains an unusually high density of sites for infrequently cutting restriction enzymes that recognise GC rich motifs including the under-represented CpG doublet. This suggests that the 26 kilobase (kb) stretch of DNA containing the alpha-globin gene family, which is known from sequence analysis to be 60% GC rich, is itself embedded within a region of high GC content. This long-range structure, identified by PFGE, corresponds to a class of GC rich isochores that are thought to represent early replicating DNA present in Giemsa negative chromosomal bands. The identification of such regions by PFGE will be of value in understanding the organisation of human chromosomes and will influence the strategies used to construct a physical map of the genome.

The chromatin structure of Rous sarcoma proviruses is changed by factors that act in trans in cell hybrids.

In several lines of Rous sarcoma virus (RSV)-transformed rat cells the proviruses are in a configuration typical of active eukaryotic genes. They are sensitive to pancreatic DNase I, with sites hypersensitive to nuclease near the 5' end of the genome, they are close to the nuclear 'cage' and they show a low level of cytosine methylation in CpG doublets. In contrast, in phenotypically untransformed hybrids between these cells and uninfected rat or mouse cells, RSV inactivity is associated with hypermethylation of the provirus, reduced DNase I sensitivity (in two out of three examples) and, where examined, relative remoteness from the nuclear cage. These changes in proviral configuration, which occur rarely in spontaneous reversion of transformed cells, can thus be induced at high frequency and stability in cell hybrids by trans-acting influences of the uninfected parents.

DNA methylation in the fungi.

A systematic study on the incidence and patterns of cytosine methylation in the fungi has been carried out by restriction and nearest-neighbor analysis of DNAs isolated from undifferentiated cells of several fungal species. With respect to DNA modification, the fungi appear to be a heterogeneous group, with a 5-methylcytosine content ranging from undetectable levels (less than or equal to 0.1% of cytosine residues methylated in 18 out of 20 species tested) to low but detectable levels (e.g. congruent to 0.2 and congruent to 0.5% of the total cytosines methylated in Sporotrichum dimorphosporum and Phycomyces blakesleeanus, respectively). In the species where it has been detected, 5-methylcytosine is located mostly at CpG doublets, and the methylated sites are clustered in long tracts (10-30 kilobase pairs) separated from essentially unmethylated regions. This methylated compartment, which comprises a small fraction (1-11%) of the total DNA, contains at least a specific set of repetitive sequences. These results contrast with the higher 5-methylcytosine content found in the fungus Physarum polycephalum and in vertebrates and higher plants.

The changes in proviral chromatin that accompany morphological variation in avian sarcoma virus-infected rat cells.

The clone All of avian sarcoma virus B77-infected Rat-1 cells comprises both morphologically normal and morphologically transformed derivatives. Transformed subclones, in which virus-specific RNA is readily detectable, contain a provirus that is very sensitive to DNase 1 digestion of chromatin, and show DNase 1 hypersensitive sites at the 5' end of the provirus and in 5' flanking cell DNA. Normal subclones with no detectable virus-specific RNA, whether infected cells that have never been transformed or revertants derived from transformed cells, contain a provirus that is far more resistant to DNase 1 digestion. Moreover this provirus lacks hypersensitive sites at its 5' end, although DNase 1 hypersensitive sites were detected at the 3' end of the provirus in either normal or transformed clones. The pattern of cytosine methylation in the proviral restriction sites of the isoschizomers Msp I and Hpa II differed between transformed and revertant clones; the revertants show additional methylation at some CpG doublets.

Sequence organisation in nuclear DNA from Physarum polycephalum: methylation of repetitive sequences

Nuclear DNA from the slime mould Physarum polycephalum is digested by the restriction endonuclease HpaII to generate a high molecular weight and a low molecular weight component. These are referred to as the M+ and the M- compartment, respectively. Sequences that are present in the M+ compartment are cleaved by MspI, the restriction enzyme isoschizomer of HpaII, thus showing that the recognition sequences for these enzymes in M+ DNA contain methylated CpG doublets. The distribution of repetitive sequences in the M+ and M- DNA compartments was investigated by comparison of the 'fingerprint' patterns of total Physarum DNA and isolated M+ DNA after digestion using different restriction endonucleases, and by probing for the presence of specific repetitive sequences in Southern blots of M+ and M- DNA by the use of cloned DNA segments. Both types of experiment indicate that many repetitive sequences are shared by both compartments, though some repetitive sequences appear to be considerably enriched, or are present exclusively, either in M+ DNA or in M- DNA.

Sequence analysis of protamine mRNA from the rainbow trout. Depurination and nearest neighbor analysis of protamine cDNA.

Protamine cDNA, which was a full length copy of protamine mRNA was labeled during its synthesis by using deoxynucleoside [alpha-32P]triphosphates. Depurination analysis showed that there were 19 different pyrimidine oligonucleotides in protamine cDNA, some of which contained isomeric sequences. The stoichiometry of the pyrimidine oligonucleotides indicated that, while some sequences probably occur in each of the protamine mRNA components, other sequences are clearly absent from one or more of the components. Several of the pyrimidine oligonucleotides had sequences consistent with the amino acid sequences of the rainbow trout protamines. The longest oligopyrimidine tract, C7T4, had a complementary RNA sequence of AGGAGAGGAGG, a stoichiometry of close to 1, and fitted the amino acid sequence Arg-Arg-Gly-Gly which occurs near the COOH terminus of each of three major protamine components. Other pyrimidine oligonucleotides analyzed were complementary to RNA sequences from the noncoding region of protamine mRNA. There appears to be no preferential use of one particular arginine codon or set of codons. Of the 21 to 22 arginine codons in protamine mRNA no less than 7 and no more than 12 are of the CGX series. The other two codons, AGA and AGG, both occur but not in a series of more than two together. This indicates that the RNA sequences coding for the arginine tracts tend to contain a mixture of arginine codons. Nearest neighbor frequency analysis of protamine cDNA gives a low value for the frequency of the CpG doublet, despite its occurrence in four out of the six arginine codons. This is in accordance with the observation that the sequence CpG is surprisingly rare in vertebrate DNA and in the RNA transcribed from it.