OBJECTIVETo investigate a 272 base pair section of the 5'-non-coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (HCV)-negative human subjects (not patients).

METHODSThis sequence section bears interest because (1) it harbors several potential methylation (Cp-rich) sites, and (2) it represents the largest part of its internal ribosomal entry site. A pre-PCR digestion protocol was established making consistent use of four restriction endonucleases selected for certain features: SmaI, XmaCI, MspI, and HpaII are inhibited if methylation(s) are present at certain cytosines within their cutting sequences.

RESULTSThe suspected HCV-specific sequence was found in the DNA of each subject tested. The pre-PCR digestion assay reveals individual differences in their pattern of methylation, which may be due to possible epigenetic phenomena.

CONCLUSIONSThe results provide formal proof that these HCV-specific sequences are contained in the genomic or extra chromosomal target DNA, and probably belong to a new class of endogenous sequences.

DNA Methylation ; Hepacivirus ; genetics ; Humans ; Restriction Mapping ; Sequence Homology, Nucleic Acid

Extrachromosomal (ec) DNA in eukaryotic cells has been known for decades. Thestructures described range from linear double stranded (ds) DNA to circular dsDNA, distinct frommitochondrial (mt) DNA. The sizes of circular forms are described from some hundred base pairs(bp) up to more than 150 kbp. The number of molecules per cell ranges from several hundred to athousand. Semi-quantitative determinations of circular dsDNA show proportions as high as severalpercentages of the total DNA per cell. These ecDNA fractions harbor sequences that are known tobe present in chromosomal DNA (chrDNA) too. Sequencing projects on, for example the humangenome, have to take into account the ecDNA sequences which are simultaneously ascertained;corrections cannot be performed retrospectively. Concerning the results of sequencings derivedfrom extracted whole DNA: if the ecDNA fractions contained therein are not taken into account,erroneous conclusions at the chromosomal level may result.

Objective: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5′-noncoding region (5′-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA. Methods: Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed. Results: The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5′-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5′-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5′-NCR revealed a broad diversity of individual patterns of methylation. Conclusions: The experimental results confirm our assumption that parts of the HCV 5′-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.

OBJECTIVE: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5'-noncoding region (5'-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA. METHODS: Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed. RESULTS: The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5'-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5'-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5'-NCR revealed a broad diversity of individual patterns of methylation. CONCLUSIONS The experimental results confirm our assumption that parts of the HCV 5'-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.
Computational Biology ; DNA Methylation ; DNA, Circular/genetics* ; DNA, Viral/genetics* ; Genome, Human ; Genomics ; Genotype ; Hepacivirus/genetics* ; Hepatitis C/virology* ; Humans ; Leukocytes, Mononuclear/virology* ; Polymerase Chain Reaction ; RNA, Viral/genetics* ; Sequence Alignment