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Differential DNA methylation of the parental alleles has been implicated in the establishment and maintenance of the monoallelic expression of imprinted genes. H19 and IGF2 are oppositely imprinted with only the maternal and the paternal alleles expressed, respectively. In Wilms tumor, a childhood renal neoplasm, loss of the H19/IGF2 imprinted expression pattern results in silencing of H19 and biallelic expression of IGF2. This was shown to be associated with biallelic methylation of the H19 promoter in the tumor and the adjacent kidney tissue suggesting that epigenetic H19 silencing is an early event in Wilms tumorigenesis. An imprinting mark region characterized by paternal allele-specific methylation has been suggested to reside in a GC-rich region of 400-base pair direct repeats starting at ؊2 kilobase pairs (kb) relative to the H19 transcription start and extending upstream. The upstream boundary of the potential paternal methylation imprint of the H19 gene has yet to be defined. We sought to define this upstream imprint boundary and investigate whether Wilms tumors with loss of imprinting are biallelically methylated in this imprinting mark region. The analysis of 6.6 kb of new upstream H19 sequence determined in this study identified a series of the direct 400-base pair repeats that extends to approximately ؊5.3 kb relative to the transcription start. DNA methylation analyses indicated that the upstream boundary of the potential imprint may coincide with the 5 end of the direct repeats. We found that Wilms tumors with loss of imprinting are biallelically methylated in the H19 upstream repeat region, and we suggest that pathological methylation in this region is the epigenetic error that initiates H19 silencing.Genomic imprinting describes the phenomenon of heritable parent-of-origin-specific expression of genes. The molecular mechanisms that determine the monoallelic expression of imprinted genes are to date not fully understood. However, it is a widely accepted concept that parental allele-specific DNA methylation plays an important role in the process.The insulin-like growth factor 2 (IGF2) and H19 genes are located in a cluster of imprinted genes on human chromosome 11p15.5 that is syntenic with the mouse distal chromosome 7. The maternally imprinted IGF2 gene is transcribed only from the paternal allele in most normal human tissues except for adult liver, choroid plexus, and leptomeninges (1, 2). In contrast, the H19 gene is paternally imprinted hence maternally transcribed (3-5). This reciprocal expression pattern is frequently lost in Wilms tumor, a childhood renal neoplasm, and in the overgrowth syndrome, Beckwith-Wiedemann syndrome, that predisposes to Wilms tumor either by maternal loss of heterozygosity at chromosome 11p15 or by loss of imprinting (LOI) 1 (6). LOI of IGF2 in Wilms tumor was first described by Rainier et al. (7)and Ogawa et al. (8). Since then several groups have shown that LOI of IGF2 in Wilms tumor and BeckwithWiedemann syndrome is associated with transcriptional repression and...

The sequence of nucleotides comprising genes V, VII, and VIII of bacteriophage fl was determined. The sequence was found to differ from that of the corresponding region of the related fd genome by eight base substitutions in gene V and one in gene VIII. The structure of gene VII was completely conserved between these two viruses and was identical to that of bacteriophage M13. Both transitions and transversions were found in cases where bases were substituted, but all substitutions were in the third codon position and had no effect on the structure of the corresponding protein product. The gene V protein product could thus be deduced to be identical to that of the corresponding proteins from bacteriophages fd and M13. A potential EcoRII cleavage site was formed by nucleotides 172 to 176 of gene V. Replicative form DNA from bacteriophage fl is normally resistant to this enzyme, and evidence is presented to suggest that the sequence was modified through methylation of cytosine 173. The probable locations of other modified nucleotides in the sequence are discussed. HapII-11 and HapII-12. The positions occupied by nucleotides in the sequence under discussion are numbered in the 5'-3' direction of the plus (sense) strand of the RFI DNA, with the "A" of the initiating ATG codon of gene V taken as nucleotide 1. The 40 on September 30, 2020 by guest http://jvi.asm.org/ Downloaded from NUCLEOTIDE SEQUENCES IN PHAGE fl DNA nucleotides identifying appropriate bands in the photographs of gel patterns have been numbered according to this convention. Nucleotides of minus-strand sequences are, however, identified on these figures by a prime. Thus, T'20 is the nucleotide complementary to A20 ofthe plus strand. (Note that the nucleotides of the minus strand are thus numbered in the 3'-) 5' direction.) We have now determined the complete sequence of the fl genome. If this sequence is numbered from the unique HindII cleavage site,,as used by Beck et al. (4) in numbering the phage fd nucleotide sequence, nucleotide 1 of the present sequence corresponds to nucleotide 843 of the total sequence. MATERIALS AND METHODS Bacteriophage fl and its host, E. coli K38, were generous gifts from N.