MethDB--a public database for DNA methylation data.

Methylation of cytosine in the 5 position of the pyrimidine ring is a major modification of the DNA in most organisms. In eukaryotes, the distribution and number of 5-methylcytosines (5mC) along the DNA is heritable but can also change with the developmental state of the cell and as a response to modifications of the environment. While DNA methylation probably has a number of functions, scientific interest has recently focused on the gene silencing effect methylation can have in eukaryotic cells. In particular, the discovery of changes in the methylation level during cancer development has increased the interest in this field. In the past, a vast amount of data has been generated with different levels of resolution ranging from 5mC content of total DNA to the methylation status of single nucleotides. We present here a database for DNA methylation data that attempts to unify these results in a common resource. The database is accessible via WWW (http://www.methdb.de). It stores information about the origin of the investigated sample and the experimental procedure, and contains the DNA methylation data. Query masks allow for searching for 5mC content, species, tissue, gene, sex, phenotype, sequence ID and DNA type. The output lists all available information including the relative gene expression level. DNA methylation patterns and methylation profiles are shown both as a graphical representation and as G/A/T/C/5mC-sequences or tables with sequence positions and methylation levels, respectively.

Lack of association between polymorphisms of eight candidate genes and idiopathic dilated cardiomyopathy: the CARDIGENE study.

OBJECTIVES: The study investigated the potential role of eight candidate genes in the susceptibility to idiopathic dilated cardiomyopathy (IDC). BACKGROUND: Idiopathic dilated cardiomyopathy has a familial origin in 20% to 25% of cases, and several genetic loci have been identified in rare monogenic forms of the disease. These findings led to the hypothesis that genetic factors might also be involved in sporadic forms of the disease. In complex diseases that do not exhibit a clear pattern of familial aggregation, the candidate gene approach is a strategy widely used to identify susceptibility genes. All genes coding for proteins involved in biochemical or physiological abnormalities of cardiac function are potential candidates for IDC. METHODS: We studied 433 patients with IDC and 401 gender- and age-matched controls. Polymorphisms investigated were the I/D polymorphism of the angiotensin I-converting enzyme (ACE) gene, the T174M and M235T polymorphisms of the angiotensinogen (AGT) gene, the A-153G and A+39C polymorphisms of the angiotensin-II type 1 receptor (AGTR1) gene, the T-344C polymorphism of the aldosterone synthase (CYP11B2) gene, the G-308A polymorphism of the tumor necrosis factor-alpha (TNF) gene, the R25P polymorphism of the transforming growth factor beta1 (TGFB1) gene, the G+11/in23T polymorphism of the endothelial nitric oxide synthase (NOS3) gene and the C-1563T polymorphism of the brain natriuretic peptide (BNP) gene. RESULTS: None of the polymorphisms were significantly associated with the risk or the severity of the disease. CONCLUSIONS: We did not find evidence for an involvement of any of the 10 investigated polymorphisms in the susceptibility to IDC.

Nature and organization of the sequence variations in the long-range periodicity calf satellite DNA I.

The nucleotide sequences of a series of cloned repeats of the bovine satellite DNA I have been established and compared to the average sequence already determined by the other workers. Variations, which are essentially single base changes, deletions or additions, are found within clustered copies and, thus, define subfractions and domains of the satellite DNA. These results are confirmed by restriction enzyme analysis of the cloned repeats or of the total purified satellite DNA. These subfractions are essentially overlapping. Certain short regions of the DNA repeat do not appear to be involved in the changes, which are spread in a concerted way through the whole bovine karyotype. The significance of these results is discussed in the light of the recent suggestion made that gene conversion-like events are susceptible to introduce homogeneity of polymorphism within the elements of repeated families of DNA sequences.

Comparative study of the L1 family in the genus Mus. Possible role of retroposition and conversion events in its concerted evolution.

The long interspersed repetitive family L1 was analysed in different species belonging to the genus Mus. It is shown to be highly conserved even in M.n. setulosus, which diverged from the other species around ten million years ago. The study of the linkage between diagnostic restriction sites in the various species and the sequence variations of different regions of the L1Md repeat shows that the L1 family undergoes concerted changes involving subsets of repeats. The rate at which this homogenization process occurs does not appear to be the same for all the subfamilies detected. The L1Md repeat in the twelfth intron of the serum albumin gene of Balb/c mice is shown to be a recent insertion. The role retroposon- and gene conversion-like events may play in the concerted evolution of the L1 family is discussed.

Characterization of the human tubulin tyrosine ligase-like 1 gene (TTLL1) mapping to 22q13.1.

This paper reports the characterization of the human tubulin tyrosine ligase-like 1 gene (TTLL1), which maps to the chromosome region 22q13.1 and has been partially duplicated on three other acrocentric chromosomes: 13, 15 and 21. We describe the complete cDNA, TTLL1a, coding for the putative 423 amino acid long TTLL1 and alternative transcripts coding for truncated TTLL1. Likely TTLL1a corresponds to the 1.8 kb transcript that was detected in a wide range of tissues and has a stronger expression in heart, brain and testis. A 4.8 kb transcript was found only in brain tissues. We present an interspecies sequence comparison, revealing three conserved domains, named TTLD1, TTLD2 and TTLD3, that are specific to the TTLs and TTL-like proteins.

A compositional map of the cen-q21 region of human chromosome 21.

A compositional map of the centromere and of the subcentromeric region of the long arm of human chromosome 21 was established by determining the GC levels (GC is the molar fraction of guanine+cytosine in DNA) of 11 YACs (yeast artificial chromosomes) covering this 13-14 Mb region which extends from the alpha-satellite sequences of the C(entromeric) band q11.1, through R(everse) band q11.2, to the proximal part of G(iemsa) band q21. The entire region is made up of GC-poor, or L, isochores with only one GC-rich H1 isochore, at least 2 Mb in size, located in band q21. The almost identical GC levels of the centromeric alpha-satellite repeats (38.5%), of R band q11.2 (39%), and of G bands (38-40%) provide a direct demonstration that base composition cannot be the only cause of the cytogenetic differences between C, G, and the majority of R bands, namely the H3- R bands (which do not contain the GC-richest H3 isochores). The results obtained also show that isochores may be as long as 6 Mb, at least in the GC-poor regions of the genome, and support previous observations suggesting that YACs from isochore borders are unstable and/or difficult to clone. Genes and CpG islands are very rare in the GC-poor region investigated, as expected from the fact that their concentration is proportional to the GC levels of the isochores in which they are contained.

A new site-specific endonuclease showing phenotypical crypticity in a tumorigenic strain of Agrobacterium tumefaciens.

AtuBVI, an endonuclease showing new site-specificity, has been isolated from the tumorigenic strain IIBV7 of Agrobacterium tumefaciens, and is undetectable in the non-tumorigenic sister strain IIBNV6. AtuBVI degrades IIBV7 DNA in vitro and should, therefore, be regarded as being phenotypically cryptic in the bacterial cell; it also shows anomalous behavior under cerain incubation conditions. These properties point to a possible role for this enzyme in the insertion of exogenous Ti-plasmid DNA into plant tissues during tumorigenesis.

Construction of a human BcgI DNA fragment library.

A library made up of 36bp DNA fragments generated by digestion of human DNA with the restriction endonuclease Bcg I has been constructed. It contains 2.5x106 independent clones, representing several times the total human genome which should contain about 400000 such fragments. It is proposed to make use of these BcgI fragments to clone part of the coding sequences contained in the minor H3 isochore which represents 3% of the human genomic DNA and a quarter of all genes.

Juxta-centromeric region of human chromosome 21 is enriched for pseudogenes and gene fragments.

A physical map including four pseudogenes and 10 gene fragments and spanning 500 kb in the juxta-centromeric region of the long arm of human chromosome 21 is presented. cDNA fragments isolated from a selected cDNA library were characterized and mapped to the 831B6 YAC and to two BAC contigs that cover 250 kb of the region. An 85 kb genomic sequence located in the proximal region of the map was analyzed for putative exons. Four pseudogenes were found, including psiIGSF3, psiEIF3, psiGCT-rel whose functional copies map to chromosome 1p13, chromosome 2 and chromosome 22q11, respectively. The TTLL1 pseudogene corresponds to a new gene whose functional copy maps to chromosome 22q13. Ten gene fragments represent novel sequences that have related sequences on different human chromosomes and show 97-100% nucleotide identity to chromosome 21. These may correspond to pseudogenes on chromosome 21 and to functional genes in other chromosomes. The 85 kb genomic sequence was analyzed also for GC content, CpG islands, and repetitive sequence distribution. A GC-poor L isochore spanning 40 kb from satellite 1 was observed in the most centromeric region, next to a GC-rich H isochore that is a candidate region for the presence of functional genes. The pericentric duplication of a 7.8 kb region that is derived from the 22q13 chromosome band is described. We showed that the juxta-centromeric region of human chromosome 21 is enriched for retrotransposed pseudogenes and gene fragments transferred by interchromosome duplications, but we do not rule out the possibility that the region harbors functional genes also.

A method for selecting the coding DNA for a protein of known sequence. II. Use of obligatory restriction sites.

A method for selecting the coding DNA of a protein of known sequence in a library of chimeric plasmids constructed with cDNA is described. It is based on the prediction of obligatory restriction sites within the searched cDNA. A partially purified probe can be obtained by taking advantage of the occurrence of two such obligatory sites. Another suggested possibility is to combine one obligatory restriction site and a specific synthetic oligodeoxynucleotide used as primer in classical reverse transcription methods.

Characterization of a Trypanosoma cruzi specific nuclear repeated sequence.

We report the isolation of three different clones from a Trypanosoma cruzi genomic library bearing a common repeated sequence. This sequence is not tandemly repeated, and is dispersed on many chromosomes. All of the T. cruzi strains tested share this element. On the other hand, it is absent from the genome of other Kinetoplastida. The size of this element is about 10-12 kb, and its copy number is 220 in the T. cruzi Dm 28c genome. A transcript homologous to this sequence is detected in epimastigote forms of the parasite.

Long-range restriction maps of size-variable homologous chromosomes in Leishmania infantum.

In order to clarify the interpretation of molecular karyotype polymorphisms in Leishmania, the three smallest chromosomes from five cloned strains of Leishmania infantum were identified by chromosome-specific anonymous DNA probes. The presence in the same clone of homologous chromosomes of a different size was demonstrated. The chromosome size polymorphism appeared even more dramatic, with size variations affecting up to 20% of the chromosome, and the two smallest bands in one strain being equivalent to six bands in another strain. Long-range restriction maps of five different-sized homologues of chromosome I showed the size-variation to be located to a terminal fragment in 4 out of 5 cases, and to a central fragment in one. The size-variable sequence was present on at least three other chromosomes as determined by hybridisation analysis. This suggests an instability of the subtelomeric regions such as that in Plasmodium falciparum. Lastly, the finding of several pairs of distinct-sized homologous chromosomes, together with other studies, strongly suggest that Leishmania is diploid in at least part of its chromosomal complement.

Interclonal variations in molecular karyotype in Leishmania infantum imply a 'mosaic' strain structure.

The molecular karyotypes of 36 clones derived from 8 strains of Leishmania infantum were examined by pulsed-field gel electrophoresis. Although there appeared to be a high degree of genetic relatedness between the clones and the parent strain, a limited degree of polymorphism was noted in 50% of the clones, expressed mainly as the presence of an additional chromosome or as a chromosome size modification. Repeated subcloning in one strain showed that chromosomal rearrangements could occur during the cloning process. Chromosome homologies were examined by Southern analysis with chromosome-specific DNA probes. The results suggest a disomy for some chromosomes, but cannot exclude aneuploidy. The mechanisms possibly leading to such heterogeneity are discussed: they could involve frequent DNA amplification/deletion, and imply a 'mosaic' structure of the cultured strains or clones, with different individuals possessing differently sized versions of the same chromosomes.

Chromosome size and number polymorphisms in Leishmania infantum suggest amplification/deletion and possible genetic exchange.

We have studied the molecular karyotypes of 21 strains and 14 clones of Leishmania infantum using pulsed field gel electrophoresis (PFGE). We detected a high degree of polymorphism within this species, with 'strain-specific' patterns for most isolates, even within a restricted endemic area. Variations relate to both the size of chromosomes (270-2600 kb) and their number, which can vary from 24 to 31 between closely related isolates. This polymorphism does not correlate with isoenzyme analysis. Small size variations between homologous chromosomes of different strains are suggestive of DNA amplification/deletion events. Strains are also shown to be multiclonal, with slight differences between most clones, but with a predominant clone concealing the others in PFGE analysis. The analysis of these data leads to the hypothesis of occasional genetic exchange by nuclear fusion in Leishmania, as recently shown in the related protozoan Trypanosoma brucei.

Construction and characterization of a partial library of yeast artificial chromosomes from human chromosome 21.

We report a protocol for cloning large DNA fragments in yeast artificial chromosomes (YAC). A partial library has been constructed from a somatic hybrid containing chromosome 21 as the single source of human DNA. About 4.0 Mb of human DNA was recovered in 17 YAC clones. Three clones were analyzed by in situ hybridization and mapped on chromosome 21. One clone hybridized with the chromosome 21 centromeric region and may provide new insight both on the molecular structure of centromere and on the localization of Alzheimer disease gene.

Methylation patterns of long interspersed repeated DNA and alphoid repetitive DNA from human cell lines and tumors.

The methylation status of CCGG sites has been determined in long interspersed repeated (L1Hs sequences) DNA and in alphoid satellite DNA extracted from human cell lines and also from pathological specimens. Southern blot experiments were performed using a cloned 1.2 kb KpnI fragment of L1Hs DNA and a cloned 680 bp tetrameric unit of alphoid satellite. DNA as probes for these sequences. In vitro transformation of human lymphocytes by Esptein-Barr virus is correlated with a progressive hypomethylation of L1 Hs DNA sequences. This loss of modification of L1 sequences is also observed in DNA isolated directly from colon adenocarcinoma and in chronic lymphocytic leukemia. Therefore, alteration of the level of methylation of L1 sequences seems not to be due solely to in vitro cultivation of human cells but is associated with the immortalization of these cells. In addition, these sequences are preferentially hypomethylated when compared to alpha-satellite DNA in several lymphoblastoid cell lines and pathological specimens.

Structure and function of alleles in the 3' end region of human apoB gene.

OBJECTIVE: To study the structure of alleles in the 3' end of the apoB gene in Han, Mongolian and Tibetan populations in China as well as the roles in the regulation of gene expression. METHODS: DNA were obtained from human leukocytes by phenol-chloroform extraction and ethanol precipitation. PCR were carried out in a 50 microliters volume containing 50 ng genomic DNA as template. The Ssp1-digested products were loaded on a gradient acrylamide gel and run for 3 hours. The constructs containing alleles were tested in cultured HepG2 and HeLa cells using transient assays. RESULTS: Sixteen alleles with different repeat number were characterized. All of the alleles varying from HVE22 to HVE52, allele HVE34 was the most common (58.4%), followed by allele HVE36 (13.8%) and HVE32 (10.5%). 258 PCR products were digested with Ssp1 and run in 4-12% PAGE. We detected the fragments of 266bp, 91 bp, 61 bp and 39 bp in almost all samples. The small alleles (including HVE22, HVE24, HVE26 and HVE36) decreased the expressive activity of the luciferase reporter, in contrary, the large alleles (including HVE44, HVE46 and HVE48) elevated obviously the expressive activity of the luciferase reporter. CONCLUSIONS: More alleles with different number of tandem repeats in 3' end of apoB gene exist in the Chinese populations. The alleles in 3' end minisatellite of human apoB gene could control the expression of the gene itself.

[Study of alpha-satellite DNA in cosmid libraries, specific for chromosomes 13, 21, and 22, using fluorescence in situ hybridization]. / Issledovanie al'fa-satellitnykh DNK v sostave kosmidnykh bibliotek, spetsifichnykh dlia khromosome 13, 21, i 22, s pomoshch'iu fliuorestsentnoi gibridizatsii in situ.

Fluorescent in situ hybridization (FISH) was employed in mapping the alpha-satellite DNA that was revealed in the cosmid libraries specific for human chromosomes 13, 21, and 22. In total, 131 clones were revealed. They contained various elements of centromeric alphoid DNA sequences of acrocentric chromosomes, including those located close to SINEs, LINEs, and classical satellite sequences. The heterochromatin of acrocentric chromosomes was shown to contain two different groups of alphoid sequences: (1) those immediately adjacent to the centromeric regions (alpha 13-1, alpha 21-1, and alpha 22-1 loci) and (2) those located in the short arm of acrocentric chromosomes (alpha 13-2, alpha 21-2, and alpha 22-2 loci). Alphoid DNA sequences from the alpha 13-2, alpha 21-2, and alpha 22-2 loci are apparently not involved in the formation of centromeres and are absent from mitotically stable marker chromosomes with a deleted short arm. Robertsonian translocations t(13q; 21q) and t(14q; 22q), and chromosome 21p-. The heterochromatic regions of chromosomes 13, 21, and 22 were also shown to contain relatively chromosome-specific repetitive sequences of various alphoid DNA families, whose numerous copies occur in other chromosomes. Pools of centromeric alphoid cosmids can be of use in further studies of the structural and functional properties of heterochromatic DNA and the identification of centromeric sequences. Moreover, these clones can be employed in high-resolution mapping and in sequencing the heterochromatic regions of the human genome. The detailed FISH analysis of numerous alphoid cosmid clones allowed the identification of several new, highly specific DNA probes of molecular cytogenetic studies--in particular, the interphase and metaphase analyses of chromosomes 2, 9, 11, 14, 15, 16, 18, 20, 21-13, 22-14, and X.

Analysis of alphoid DNA variation and kinetochore size in human chromosome 21: evidence against pathological significance of alphoid satellite DNA diminutions.

Centromeric alpha satellite DNA sequences are linked to the kinetochore CENP-B proteins and therefore may be involved in the centromeric function. The high heterogeneity of size of the alphoid blocks raises the question of whether small amount of alphoid DNA or "deletion" of this block may have a pathological significance in the human centromere. In the present study, we analysed the correlation between size variations of alphoid DNA and kinetochore sizes in human chromosome 21 by molecular cytogenetic and immunochemical techniques. FISH analyses of alpha satellite DNA sizes in chromosome 21 homologues correlated well with the variation of their physical size as determined by pulsed field gel electrophoresis (PFGE). By contrast, the immunostaining study of the same homologous chromosomes with antikinetochore antibodies suggested that there is no positive correlation between the alpha satellite DNA block and kinetochore sizes. FISH analysis of chromosome 21-specific alphoid DNA and immunostaining of kinetochore extended interphase chromatin fibers indicate that centromeric kinetochore-specific proteins bind to restricted areas of centromeric DNA arrays. Thus, probably, restricted regions of centromeric DNA play an important role in kinetochore formation, centromeric function and abnormal chromosome segregation leading to non-disjunction.

Two new cases of the Christchurch (Ch1) chromosome 21: evidence for clinical consequences of de novo deletion 21P-.

We performed an investigation of two unrelated cases with extremal variants of chromosome 21 without visible materials of the short arms (Christchurch or Ch1 chromosome). In the first case chromosome 21p- was initially detected during routine cytogenetic amniocentesis. Chromosomal variant was inherited from phenotypically normal father to phenotypically normal fetus (phenotypically normal boy after the birth). The second case of chromosome 21p- was detected in 7 years old boy, referred to cytogenetic analysis due to mental retardation and mild congenital malformation, including prenatal hypoplasia, microcephaly, low-set dysplastic ears, short nose, micrognatia, short neck. Molecular characterization of 21p-variant chromosomes was performed by the use of FISH with DNA probes specific to the short arm and centromeric region of chromosome 21 (telomeric, beta-satellite, ribosomal, classical satellite and alphoid DNA probes). Chromosomes 21p-hybridized positively only with telomeric DNA at both chromosomal ends and alphoid DNA probes at centromeric region of the first patient. In second case (de novo deletion of 21p), the Ch1 was associated with clinical phenotype and loss of telomeric and subtelomeric DNA in the p-arm of chromosome 21. Therefore, the complete absent of the short arm of chromosome 21 may be considered as abnormal. We propose that de novo deletion 21p- could have negative consequences due to absence of large portion of chromosomal DNA from the p-arm (telomeric, satellite or ribosomal DNAs) and following imbalance in organization and functioning of genome.