HhaI Site Research Articles

TLR2 and TLR4 gene promoter methylation status during chronic periodontitis

The objective of this study was to analyse the status of DNA methylation in the promoter region of the toll-like receptor (TLR)2 and TLR4 genes in gingival tissue samples from healthy subjects, smokers and non-smokers affected by chronic periodontitis. Genomic DNA and total RNA were purified from gingival tissue using the TRIZOL reagent protocol. Genomic DNA was then digested by methylation-sensitive restriction enzymes, amplified by polymerase chain reaction (PCR), electrophoresed on a 10% polyacrylamide gel and stained using SYBR Gold. Real-time PCR was also performed to verify the transcript levels. The CpG dinucleotides analysed were observed to be unmethylated in the majority of DNA samples of the three groups and statistical differences were not found among groups (p>0.05). However, a trend towards methylation was observed in the TLR2 HhaI site in the samples of the periodontitis non-smoker groups. In fact, the analysis of all CpG sites together shows which complete methylation is observed in the shortest level in the samples of periodontitis non-smoker group. The analysis of transcript levels demonstrated no difference among groups (p>0.05). The results demonstrated major unmethylation of the TLR4 gene promoter in all groups. However, the results for the TLR2 gene promoter are inconclusive; this gene was found as a mosaic of methylated and unmethylated DNA in the majority of samples of the three groups and we also observed a trend towards the DNA methylation of CpG sites recognized by the HhaI enzyme.

First Report of 'Candidatus Phytoplasma trifolii'-Related Strain Associated with Safflower Phyllody Disease in Iran.

During a survey in 2003, safflower plants (Carthamus tinctorius) with phyllody symptoms were observed in production fields in several districts of Fars and Yazd provinces in Iran. Affected plants showed floral virescence, phyllody, proliferation of axillary buds, and little leaf symptoms. Incidence of the disease was less than 10%. Direct and nested PCR assays were used to verify association of phytoplasma with the disease. Total DNA was extracted from fresh, fine roots of eight phyllody-affected safflower plants and one symptomless plant. With phytoplasma universal primer pair P1/P7 (5'-AAGAGTTTGATCCTGGCTCAGGATT-3'/5'-CGTCCTTCATCGGCTCTT-3'), target DNA fragments of approximately 1.8 kb were amplified by direct PCR from phyllody-affected plants and Iranian cabbage yellows (ICY) phytoplasma used as a positive control. Reamplification of P1/P7 products with 16S rRNA gene primer pair R16F2n/R16R2 (5'-GAAACGACTGCTAAGACTGG-3'/5'-TGACGGGCGGTGTGTACAAACCCCG-3') yielded fragments of the expected size (1.2 kb) from all eight diseased plants and the ICY-positive control. No products were amplified from the symptomless plant by either assay. R16F2n/R16R2 products were subjected to restriction fragment length polymorphism (RFLP) analysis by separate digestion with AluI, HaeIII, HhaI, HinfI, HpaII, MseI, RsaI, Sau3AI, or TaqI endonuclease. Comparison of resulting RFLP patterns with published patterns of other phytoplasmas (2) tentatively identified safflower phyllody (SP) phytoplasma as a member of clover proliferation group 16SrVI, subgroup C. HhaI digests also differentiated SP from ICY phytoplasma, a previously reported subgroup 16SrVI-A strain (3). After sequencing of the 16S rDNA fragment (GenBank Accession No. DQ88948), a BLAST search determined that SP phytoplasma shared closest homology with 16SrVI group members ('Candidatus Phytoplasma trifolii') and related strains (4). Furthermore, phylogenetic analysis of 16S rDNA sequences revealed SP phytoplasma to be most similar (99.7%) to brinjal little leaf (BLL) phytoplasma (GenBank Accession No. X83431). Analysis of putative restriction sites in 16S rRNA gene sequences revealed that SP and BLL shared identical restriction profiles and that both differed from the 'Ca. Phytoplasma trifolii' reference strain (GenBank Accession No. AY390261) because of the absence of a single HhaI site and the presence of an additional MseI site. Although safflower phyllody disease has been previously reported in Israel, the associated phytoplasma was classified as a strain of the aster yellows subgroup 16SrI-B (1). To our knowledge, this is the first report of safflower as a host of a 'Ca. Phytoplasma trifolii'-related strain.

Intra-isolate heterogeneity of the ITS region of rDNA in Pythium helicoides

Heterogeneity of the rDNA ITS region in Pythium helicoides and the phylogenetic relationship between P. helicoides and closely related species were investigated. In PCR-RFLP analysis of the rDNA ITS region of six P. helicoides isolates investigated, including the type culture, intraspecific variation was found at the HhaI site. The total length of fragments was longer than before cutting, indicating sequence heterogeneity within isolates. Digestion of the cloned rDNA ITS region derived from seven isolates with HhaI revealed polymorphisms among and within single zoospore isolates, and variability of the region was also present among the clones derived from the same isolate. To test whether the rDNA ITS region of closely related species and other regions in the genome of P. helicoides are also variable, the rDNA ITS region of P. ultimum and the cytochrome oxydase II ( cox II) gene encoded in mitochondria were sequenced. P. ultimum had little variation in the rDNA ITS region. The cox II gene sequences of both species revealed only a low intraspecific variability and no intra-isolate variation. In the phylogenic tree based on the rDNA ITS sequences, all clones of P. helicoides formed one large clade that was distinct from the clades comprising morphologically similar species, such as P. oedochilum and P. ostracodes, and was closely related to P. chamaehyphon rather than the other species.

Tissue-specific characterisation of DNA methylation in the gonad-specific proto-oncogene, c-mos, in the male laboratory mouse.

The proto-oncogene, c-mos, which is expressed only in the germ cells of both testis and ovary, plays an important role in meiotic maturation of these cells. In this research, the methylation status of several CpG sites, present both upstream and within the coding region of the c-mos gene, has been studied. The HpaII and HhaI sites examined in the 5' half of the coding region were unmethylated in both the c-mos expressing and non-expressing tissues. A HhaI site, h3, present 380bp downstream of the transcription start site, was unmethylated in germ cells, but was partially methylated in the somatic tissues, inversely correlating with the expression status of the gene. In contrast to these tissues, in the mouse fibroblast cell line L929, all the analysed sites were completely methylated.

Mechanism and clinical significance of cyclooxygenase-2 expression in gastric cancer

To determine the correlation between methylation status of 5' CpG island of cyclooxygenase-2 (COX-2) gene and protein expression in gastric cancer tissues for distinguishing the molecular characters of gastric cancers. Methylation status of 5' CpG island of COX-2 gene was studied by PCR amplification after HpaII and Hha I restrictive enzyme digestion; COX-2 expression was evaluated by immunohistochemical method. Hpa II and HhaI site were all methylated in 12 normal gastric mucosa tissues, whereas they were demethylated in 77.27% (34/44) and 84.09% (37/44) gastric cancer tissues, respectively. Expression of COX-2 was detected in 68.18% (30/44) gastric cancer tissues, but no expression was found in normal gastric mucosa tissues. In gastric cancer tissues, COX-2 expression was correlated significantly with HpaII site demethylation (29/30 vs 5/14, P<0.001 and HhaI site demethylation (28/30 vs 9/14, P<0.05). The demethylation of 5' CpG island of gene is necessary for COX-2 expression in human gastric cancer. The expression status of COX-2 may provide theoretical basis for COX-2 targeting gastric cancer treatments.

Differentiation of pathogenic Bartonella species by infrequent restriction site PCR.

Infrequent restriction site PCR (IRS-PCR) is a recently described DNA fingerprinting technique based on selective amplification of restriction endonuclease-cleaved fragments. Bartonella isolates associated with human disease and related nonhuman isolates were analyzed by IRS-PCR genomic fingerprinting. Preparation of DNA templates began with double digestion using three different restriction endonuclease combinations. Combinations included the frequently cutting endonuclease HhaI in conjunction with an infrequently cutting endonuclease, EagI, SmaI, or XbaI. Digestion was followed by ligation of oligonucleotide adapters designed with ends complementary to the restriction endonuclease sites. Amplification of fragments flanked with an EagI, SmaI, or XbaI site in combination with an HhaI site produced a series of different-sized amplicons resolvable into patterns by polyacrylamide gel electrophoresis (PAGE). The pattern complexity was varied by the addition of selective nucleotides to the 3' ends of the EagI-, SmaI-, or XbaI-specific primers. Amplicons were also generated with fluorescently labeled primers and were subsequently resolved and detected by capillary electrophoresis. Analysis by traditional slab PAGE and capillary electrophoresis provided suitable resolution of patterns produced with the enzyme combinations EagI-HhaI and SmaI-HhaI. However, the combination of XbaI-HhaI produced too many fragments for sufficient resolution by traditional PAGE, thus requiring the better resolving properties of capillary electrophoresis. Due to the flexibility in modulating the pattern complexity and electrophoresis methods, these techniques allow for a high level of experimental optimization. The results provide evidence of the discriminatory power, ease of use, and flexibility of the IRS-PCR method as it applies to the identification of human-pathogenic Bartonella species.

Genetic association analysis between CYP2D6*2 allele and tardive dyskinesia in schizophrenic patients

Previous studies have shown a possible association between tardive dyskinesia (TD) and debrisoquine 4-hydroxylase (CYP2D6) polymorphisms, which result in absent enzyme activity. We have recently found a positive association between TD and the CYP2D6*10 allele, which codes for the intermediate metabolizer (IM) phenotype and is characterized by decreased but not absent CYP2D6 activity in Japanese schizophrenic patients. In addition, the CYP2D6*2 allele with the HhaI site mutation in exon 6 has also been reported to be an IM allele and a risk factor for Parkinson’s disease (PD) in the Japanese population. In the present study, we investigated potential contributions of the CYP2D6*2 allele to TD using case-control and regression analysis in 99 schizophrenic patients. No significant differences in genotypic and allelic frequencies were found between patients with and without TD. Even after using regression analysis to adjust for the confounding variables, there was no significant association of the CYP2D6*2 genotype with either outcome variable, the occurrence of TD or the total AIMS score. These results suggest that the CYP2D6*2 allele may not contribute to the pathogenesis of TD.

CYP2D6 HhaI Genotype and the Neuroleptic Malignant Syndrome

To investigate the relationship between CYP2D6 genotypes (reported to be associated with the susceptibilities to Parkinson’s disease and multisystem atrophy) and the possible susceptibility to neuroleptic malignant syndrome (NMS) and subacute myelo-optico-neuropathy (SMON), we analyzed the CYP2D6 gene by polymerase chain reaction and restriction fragment length polymorphism in Japanese schizophrenia patients with a history of NMS. There was no significant difference in the frequency of the poor metabolizer genotype of CYP2D6 between the cases with a history of NMS and controls (p > 0.05). The frequency of the mutation located at the HhaI site in exon 6 of CYP2D6 in the cases was higher, but not significantly (p > 0.05; the mutated allele frequency was 0.25), than that in the controls, schizophrenia patients without NMS (0.11) and healthy controls (0.09). The frequency (0.10) of the HhaI mutation type in patients with a diagnosis of SMON was also not significantly higher than in healthy controls. These results suggest that the poor metabolizer and HhaI polymorphism of CYP2D6 may not be a useful molecular marker for predicting the onset of NMS and SMON.

Genetic analysis of the CYP2D6 gene in patients with parkinson' disease

To further investigate the association between Parkinson's disease (PD) and genetic polymorphsim of the CYP2D6 gene, a mutant allele (CYP2D6J) frequently observed in the Japanese population and related to EM/PM polymorphism (phenotypically, individuals are either extensive metabolizers [EM] or poor metabolizers [PM] of debrisoquine) was investigated. The CYP2D6J gene with a nucleotide substitution from C to T at position 188 (the Hphl site in exon 1), which reduces CYP2D2 enzyme activity, was analyzed by polymerase chain reaction (PCR) and by digestion with HphI. No significant relationship was observed between PD patients and controls for this mutation. This suggests that the EM/PM polymoprhism of CYP2D6 contributes little to the pathogenesis of PD. To further study the molecular basis for the relationship between PD and CYP2D6, the heterogeneity of CYP2D6 was investigated by combined genotype analysis of the two mutant CYP2D6 genes (ie, CYP26J, the HphI site mutation in exon 1, and CYP2D6L, the HhaI site mutation in exon 6). Although some characteristic patterns of the combined genotypes were observed in both PD patients and controls, a strong association between the heterogeneity of the CYP2D6 gene and PD was not shown by combined genotype analysis.

PCR amplification of a locus with RFLP alleles specific to African honey bees.

An anonymous honey bee locus, detected previously with a cloned probe, has HhaI RFLP alleles specific to African bees or common to both African and European bees. To facilitate identification of these alleles, this region, 1231, was made analyzable with the PCR. The two halves of the region, excluding the termini, were amplified as two overlapping segments. Restriction sites were mapped, and the site differences responsible for the allelic RFLP patterns were determined. In the first half of the region, two polymorphic HhaI sites are present in the common alleles, whereas one, the other, or both of the sites are absent in the African alleles. In the second half, a third polymorphic HhaI site is present or absent in both common and African alleles. A short part of the second half of the region, including more of the terminus, was amplified as a third segment. Within this segment, close to this terminus, a fourth polymorphic HhaI site is absent in some African alleles.

Methylation sensitive protein binding to an intragenic active X-specific methylated region in theM. robustus HPRT gene

An intragenic region of the wallaroo (Macropus robustus) hypoxanthine phosphoribosyltransferase gene which contains three active X-specific methylated cytosines was examined for protein(s) binding. In vitro DNase I footprinting of unmethylated DNA identified three footprints, one of which (footprint I) contained two of the known differentially methylated sites (a HpaII and a HhaI site). Methylation of the footprint I HpaII site only, abolished the formation of several, specific DNA-protein complexes in mobility shift assays. UV cross-linking experiments indicated that polypeptides involved in the methylation sensitive interactions with footprint I had molecular weights ranging from 72 to 48 kDa. Analogous results were obtained with nuclear extracts from both eutherian and metatherian cells, indicating that these proteins are conserved. We suggest that the binding of these proteins to the inactive X may play some role in gene silencing, with the active gene being protected from this effect by methylation of the binding site.

Physical calibration of yeast artificial chromosome contig maps by RecA-assisted restriction endonuclease (RARE) cleavage.

Clone-based genome maps can be constructed by determining the presence or absence of sequence-tagged sites (STSs) in a redundant collection of yeast artificial chromosome clones (YACs). While STS-content mapping has proven to be an effective means of ordering clone ends and STSs along chromosomes, the exact physical map positions of these landmarks are not determined. This fundamental weakness can be overcome by RecA-assisted restriction endonuclease (RARE) cleavage, a method that exploits the binding specificity on duplex DNA of a RecA-protein-oligodeoxynucleotide complex to enhance the cleavage specificity of a restriction endonuclease. This technique allows selective cleavage at individual members of a large set of restriction sites. RARE-cleavage mapping was applied to a contig comprising 5 overlapping YACs spanning 580 kb on human chromosome 14. An STS-content map comprising 10 YAC-end specific STSs and one internal STS was constructed. RARE cleavage was performed on 2 YACs that span the entire contig at the EcoRI sites defining the vector-insert junctions of all 5 YACs, as well as at a HhaI site within the STS that was initially used to screen the YAC library for the clones in the contig. The sizes of the RARE-cleavage fragments were measured by pulsed-field gel electrophoresis and used to convert the STS-content map into a true physical map that indicates precise positions of clone ends and STSs.

Binding of the transcription factor EBP-80 mediates the methylation response of an intracisternal A-particle long terminal repeat promoter.

Intracisternal A-particle (IAP) expression in mouse cells has been correlated with hypomethylation of HhaI and HpaII sites in proviral long terminal repeats (LTRs). In a previous study, in vitro methylation of three HhaI sites in the U3 region of the LTR from the cloned genomic IAP element, MIA14, was shown to inhibit promoter activity in vivo. In this study, we found by site-directed mutagenesis that the two more downstream HhaI sites within this LTR were responsible for the methylation effects on promoter activity in vivo; methylation of the other (5') HhaI site, which lies within a putative SP1 binding domain, did not affect promoter activity. Methylation of the HhaI sites also inhibited promoter activity of the LTR in a cell-free transcription system. Exonuclease III footprinting demonstrated methylation-induced changes in protein binding over the region encompassing the downstream HhaI site, designated the Enh2 domain. The protein that interacts specifically with this domain, EBP-80, was characterized in a previous study (M. Falzon and E. L. Kuff, J. Biol. Chem. 264:21915-21922, 1989). We show here that the presence of methylcytosine in the HhaI site within the Enh2 domain inhibited binding of EBP-80 in vitro. The methylated MIA14 LTR construct was much less responsive to added EBP-80 in an in vitro transcription system than was the unmethylated construct. These data suggest that CpG methylation within the Enh2 domain may exert its effect on transcription in vivo by altering the interaction between EBP-80 and its cognate DNA sequence.

Binding of the Transcription Factor EBP-80 Mediates the Methylation Response of an Intracistemal A-Particle Long Terminal Repeat Promoter

Intracisternal A-particle (IAP) expression in mouse cells has been correlated with hypomethylation of HhaI and HpaII sites in proviral long terminal repeats (LTRs). In a previous study, in vitro methylation of three HhaI sites in the U3 region of the LTR from the cloned genomic IAP element, MIA14, was shown to inhibit promoter activity in vivo. In this study, we found by site-directed mutagenesis that the two more downstream HhaI sites within this LTR were responsible for the methylation effects on promoter activity in vivo; methylation of the other (5') HhaI site, which lies within a putative SP1 binding domain, did not affect promoter activity. Methylation of the HhaI sites also inhibited promoter activity of the LTR in a cell-free transcription system. Exonuclease III footprinting demonstrated methylation-induced changes in protein binding over the region encompassing the downstream HhaI site, designated the Enh2 domain. The protein that interacts specifically with this domain, EBP-80, was characterized in a previous study (M. Falzon and E. L. Kuff, J. Biol. Chem. 264:21915-21922, 1989). We show here that the presence of methylcytosine in the HhaI site within the Enh2 domain inhibited binding of EBP-80 in vitro. The methylated MIA14 LTR construct was much less responsive to added EBP-80 in an in vitro transcription system than was the unmethylated construct. These data suggest that CpG methylation within the Enh2 domain may exert its effect on transcription in vivo by altering the interaction between EBP-80 and its cognate DNA sequence.

Evidence for tissue-specific cytosine-methylation of plastid DNA from Zea mays

Total DNA isolated from leaves, etiolated seedlings, roots, endosperm or embryos of Zea mays was digested separately with each of the restriction enzymes HpaII, MspI and HhaI, and the resulting fragment patterns, which were specific for the plastid rRNA operon, were analyzed by Southern hybridization. While most of the fragment patterns were consistent with previously established physical maps, the partial resistance shown by one HpaII site and one HhaI site, both of which reside in the 16S/23S rDNA spacer region, was observed in DNA isolated from embryo, root tissue and endosperm. The partially resistant HpaII site was susceptible to cleavage with restriction enzyme MspI. From this and from the known inhibition of restriction enzyme Hhal at methylated HhaI sites, we conclude that the partial resistance of the two sites is caused by C-specific methylation of plastid DNA in the respective tissues. The tissue specificity of this DNA methylation is likely to reflect a differential expression of plastome encoded genes.

DNA methylation and genetic inactivation at thymidine kinase locus: two different mechanisms for silencing autosomal genes.

Patterns of methylation of CpG dinucleotides in the promoter region of the thymidine kinase (TK) gene in wild-type and TK-deficient Chinese hamster cell lines were studied. Whereas wild-type cells were unmethylated, three conventionally derived TK-deficient cell lines were all almost completely methylated in the promoter region. Demethylation at a number of different CpG sites was observed upon selection for reexpression of the TK gene. Of thirteen HhaI (GCGC) or HpaII (CCGG) sites studied, the highest correlation between absence of methylation and at least partial TK activity was obtained at one HhaI site within 20 bp of the putative cap site. Silencing in the three conventionally derived mutants is therefore accompanied by hypermethylation of the promoter-associated CpG-rich island. We contrast this situation with another type of silencing event, in which TK was coordinately inactivated at a high frequency with at least one other linked allele. Methylation of the promoter region of TK was not associated with this event, but two lines of evidence suggested a role for methylation at sites other than in the promoter region of TK.

The 5-methylcytosine content of highly repeated sequences in human DNA.

Previously, we found much tissue- or cell-specificity in the levels of 5-methylcytosine (m5C) in the total human genome as well as in DNA fractions resolved by reassociation kinetics. We now report that there were even greater differences in the m5C content of the highly repeated, tandem EcoRI family of DNA sequences from different human organs or cell populations. The ratio of m5C levels in this DNA fraction from brain, placenta, and sperm was 2.0:1.2:1.0. At a HhaI site in this repeat family, sperm DNA was 5-10 fold less methylated than somatic DNAs. In contrast, the highly repeated Alu family, which is approximately 5% of the genome, had almost the same high m5C content in brain and placenta despite marked tissue-specific differences in m5C levels of the single copy sequences with which these repeats are interspersed. These data show that very different degrees of change in methylation levels of various highly repeated DNA sequences accompany differentiation.