DNA methylation contributes to tissue- and allele-specific expression of the T-cell differentiation marker RT6

We investigated the role of DNA methylation in gene regulation of the rat T-cell differentiation marker RT6. Analysis of the methylation status of various tissues revealed that the RT6 promoter was hypomethylated in RT6-expressing tissues, and methylated in nonexpressing ones. Remarkably, among RT6-nonexpressing tissues, the extent of methylated regions varied greatly between lymphatic tissues, where regions larger than 23 kb were methylated, and nonlymphatic tissues, where methylation was restricted to a 3- to 4-kb region surrounding the promoter. We have previously shown that cis-regulatory elements determine differential expression of the two RT6 alleles in a subpopulation of T cells. We now show that the RT6 alleles in these cells differed in their methylation status. The promoter region of the silent allele was methylated, while that of the transcribed allele was not. Upon treatment of RT6-nonexpressing thymoma cells with the methyltransferase inhibitor 5-azacytidine, RT6 expression was induced. In RT6 heterozygous hybridoma cells, expressing only one RT6 allele, induction of the silent, methylated RT6 allele was observed. Sensitivity of the RT6 promoter to DNA methylation was demonstrated by promoter-specific in vitro methylation, which inhibited RT6 promoter activity, while that of the SV40 promoter was not influenced. Our findings indicate that DNA methylation plays an important role in the control of monoallelic and tissue-specific RT6 expression.     

An investigation of playful behaviour in Rattus norvegicus and Mus musculus (Mammalia)

The playful behaviour of laboratory rats ( Rattus norvegicus ) was investigated in litters of five individuals with the mother present; parallel observations were made on mice ( Mus musculus ). Seven mixed litters containing four young rats and a young mouse fostered at birth were also observed.
Solitary play was recorded in both species and took a similar form but social play was only observed in rats. In rats, solitary play frequently preceeded social play.
The behavioural elements involved in the social play of Rattus norvegicus were described, and the majority of these were the playful equivalent of adult agonistic behaviour elements. These social play elements were found to be organized into definite sequences which differed from those of adult aggression. Each behavioural element was found to act as a social releaser.
Young mice did not respond playfully to social play from a rat litter mate; mice were less attractive to rats as playmates in comparison with fellow rats.     

Evolutionary lineages of RT1.Ba in the Australian Rattus

In this study, the evolutionary history of the variable second exon of RT1.Ba and its adjoining intron b are compared across a number of species and subspecies of the Australian RATTUS: Three lineages are identified in the second intron across a range of Rattus species. Two of these lineages, separated by the insertion of a probable rodent short interspersed nucleotide element and by point mutations outside the indel region, are both found in each of the major clades of the endemic Australian RATTUS: This pattern of ancestral polymorphism is reflected in the adjoining exon 2 sequences, although phylogenetic constraints confirm that the clustering is not identical to that of the associated intron sequences. In addition, the coding sequences show evidence of the retention of ancestral polymorphism, with identical exon sequences found in two divergent species, and some indication of gene conversion detected for the exon sequences.     

ID sequences in the genes of three brain-specific proteins

We characterized the brain-specific gene coding for rat S-100 protein beta-subunit and found three "brain identifier (ID)" elements, which have been proposed to regulate the gene expression in rat brain. The nucleotide sequences of these elements corresponded well with that of the consensus ID element and were clearly different from those of "ID-like" elements in rat beta B1-crystallin gene, etc. ID elements were also observed in the flanking regions of rat neuron-specific enolase and cholecystokinin genes, which were expressed in the neuronal cells. Direct repeats were observed in the regions flanking ID elements.     

Esterase-13 of the rat (Rattus norvegicus) and its homology with esterase-3 of the house mouse (Mus musculus)

A new allele of esterase-13 was detected in various laboratory inbred strains of Rattus norvegicus and designated Es-13c. The activity of ES-13 towards a range of chromogenic substrates, inhibitor profile, isoelectric points and retardation coefficients on polyacrylamide gel electrophoresis were determined. The organ specific expression of ES-13 alleles was investigated and it was shown that kidney homogenates contained a factor which modified the liver enzyme banding pattern in vitro. The features of ES-13 from the rat indicated homology between this esterase and ES-3 from the house mouse, Mus musculus domesticus.     

Origin of agouti-melanistic polymorphism in Wild Black Rats (Rattus rattus) inferred from Mc1r gene sequences

We examined nucleotide changes that underlie coat color variation in Black Rats (the Rattus rattus species complex), which show polymorphism in dorsal fur color, including either grayish brown (agouti) or black (melanistic) forms. We examined the full coding sequence of a gene known to produce melanism in other vertebrates-melanocortin-1-receptor gene Mc1r (954 bp) -using samples of both R. rattus (with 2n = 38) and its close relative Asian Black Rat (R. tanezumi; 2n = 42). We used 61 specimens from Japan with karyotype-known individuals and four samples from Pakistan. We found 11 allele sequences and constructed a network tree that shows two distinct clusters, with allelic segregation according to karyotype and by inference, representing the two species. We found that a nucleotide substitution from G to A at site 280, producing an amino acid change from glutamic acid to lysine, was associated with the dominant trait of the melanistic form of the coat color in R. rattus. Notably, the derived SNP 280A was found in a single allele, with the ancestral SNP 280G present in seven alleles. By contrast, all three alleles for R. tanezumi retain the ancestral SNP 280G. These results suggest a possible recent origin of melanism in R. rattus.     

Genetic and molecular analysis of the Spm-dependent a-m2 alleles of the maize a locus

The Suppressor-mutator (Spm) transposable element family of maize consists of the fully functional standard Spm (Spm-s) and many mutant elements. Insertion of an Spm element in or near a gene can markedly alter its expression, in some cases bringing the gene under the control of the mechanisms that regulate expression of the element. To gain insight into such mechanisms, as well as to enlarge our understanding of the Spm element's genetic organization, we have analyzed derivatives of a unique Spm insertion at the maize a locus in which the gene is co-expressed and co-regulated with the element. We describe the genetic properties and the structure of the a locus and Spm element in 9 strains (collectively designated the a-m2 alleles) selected by McClintock from the original a-m2 allele for heritable changes affecting either the Spm element or expression of the a gene. Most of the mutations are intra-element deletions within the 8.3-kb Spm element; many alter both Spm function and expression of the gene. Spm controls a gene expression in alleles with internally deleted, transposition-defective Spm elements and element ends contain the target sequences that mediate Spm's ability to activate expression of the gene. We argue that the properties of the a-m2 alleles reflect the operation of an element-encoded positive regulatory mechanism, as well as a negative regulatory mechanism that affects expression of the element, but appears not to be mediated by an element-encoded gene product.     

Predicting mammalian SINE subfamily activity from A-tail length

Based on previous observations that newly inserted LINEs and SINEs have particularly long 3' A-tails, which shorten rapidly during evolutionary time, we have analyzed the rat and mouse genomes for evidence of recently inserted SINEs and LINEs. We find that the youngest predicted subfamilies of rodent identifier (ID) elements, a rodent-specific SINE derived from tRNA(Ala), are preferentially associated with A-tails over 50 bases in the rat genome, as predicted. Furthermore, these studies detected a subfamily of ID elements that has made over 15,000 copies that is younger than any previously reported ID subfamily. We use PCR analysis of genomic loci to demonstrate that all subfamily members tested inserted after the divergence of Rattus norvegicus from Rattus rattus. We also found evidence that the rodent B1 family of elements is much more active currently in mouse than in rat. These data provide useful estimates of recent activity from all of the mammalian retrotransposons, as well as allowing identification of the most recent insertions for use as population and speciation markers in those species. Both the current rat ID and mouse B1 elements that are active have small, specific interruptions in their 3' A-tail sequences. We suggest that these interruptions stabilize the length of the A-tails and contribute to the activity of these subfamilies. We present a model in which the dynamics of the 3' A-tail may be a central controlling factor in SINE activity.     

Isolation and characterization of microsatellites in Rattus rattus

We isolated and characterized 10 microsatellite loci in the black rat Rattus rattus (Muridae, Rodentia), a widespread invasive species largely known to cause serious problems in agriculture and human health. Polymorphism was studied in two populations, one from Madagascar and one from Senegal. It ranged from three to 12 alleles in Madagascar, and from two to five alleles in Senegal. Together with the loci previously adapted from Rattus norvegicus, this set of markers should allow the conduct of thorough studies on the genetic structure of natural populations of R. rattus.     

Epigenetic Control of a Transposon-Inactivated Gene in Neurospora Is Dependent on DNA Methylation

An unstable allele of the Neurospora am (GDH) gene resulting from integration of the retrotransposon Tad3-2 into 5'' noncoding sequences was found in previous work. We report that reversion to Am(+) depends on DNA methylation within and upstream of Tad. Levels of methylation were correlated with the proportion of Am(+) conidia, whether the cultures were derived from Am(-) or Am(+) isolates. Reversion to Am(+) did not occur when conidia were plated on 5-azacytidine, which reduces DNA methylation. The mutation dim-2, which appears to abolish DNA methylation, also prevented reversion to Am(+). The native am allele, in a strain that lacked Tad elements, was replaced with am::Tad3-2 or with a deletion derivative that prevents transposition of Tad. Transformants of both classes showed instability comparable with that of the original isolates, which contain multiple Tad elements. Deletion of the upstream enhancer-like sequences, URSamα and β, did not prevent the instability of am::Tad3-2. The results suggest that am expression is dependent on DNA methylation but not on proliferation or transposition of the Tad element and that the instability does not require the upstream sequences of am.     

Rat kappa-chain allotypes. III. Serological and genetic analysis of RI-1b cross-reactivity in Australian Rattus

The presence of rat kappa-chain allotype specificities (RI-1a and b) has been studied in 13 subspecies of the seven native Australian species of Rattus. RI-1a reactivity was not detected among these rats. On the other hand, extensive cross-reactivity was seen with RI-1b, some sera cross-reacting totally (R. leucopus cooktownensis), some not at all (R. colletti), and the remainder showing at least two distinct levels of partial cross-reactivity, confirming the existence of multiple specificities for RI-1b. Three subspecies show polymorphism with respect to RI-1b cross-reactivity (R. sordidus, R. colletti, and R. l. leucopus) and in one case (leucopus) breeding studies have indicated that there is allelic inheritance of this trait. The segregation of RI-1b reactivity has been studied in crosses and backcrosses made between species differing in their RI-1b reactivity, and the results are consistent with the existence of codominant alleles at a single locus. The fact that these species differ extensively in their karyotype opens the door to possible chromosomal localization of this and other genetic traits.     

Differential methylation persists at the mouse Rasgrf1 DMR in tissues displaying monoallelic and biallelic expression

A subset of mammalian genes exhibits genomic imprinting, whereby one parental allele is preferentially expressed. Differential DNA methylation at imprinted loci serves both to mark the parental origin of the alleles and to regulate their expression. In mouse, the imprinted gene Rasgrf1 is associated with a paternally methylated imprinting control region which functions as an enhancer blocker in its unmethylated state. Because Rasgrf1 is imprinted in a tissue-specific manner, we investigated the methylation pattern in monoallelic and biallelic tissues to determine if methylation of this region is required for both imprinted and non-imprinted expression. Our analysis indicates that DNA methylation is restricted to the paternal allele in both monoallelic and biallelic tissues of somatic and extraembryonic lineages. Therefore, methylation serves to mark the paternal Rasgrf1 allele throughout development, but additional factors are required for appropriate tissue-specific regulation of expression at this locus.