It is likely that most vertebrate genes are associated with 'HTF islands'--DNA sequences in which CpG is abundant and non-methylated. Highly tissue-specific genes, though, usually lack islands. The contrast between islands and the remainder of the genome may identify sequences that are to be constantly available in the nucleus. DNA methylation appears to be involved in this function, rather than with activation of tissue specific genes.

CpG-rich islands and the function of DNA methylation

CpG islands in vertebrate genomes.

Two different human genomic DNA libraries were screened for the gene for blood coagulation factor IX by employing a cDNA for the human protein as a hybridization probe. Five overlapping lambda phages were identified that contained the gene for factor IX. The complete DNA sequence of about 38 kilobases for the gene and the adjacent 5' and 3' flanking regions was established by the dideoxy chain termination and chemical degradation methods. The gene contained about 33.5 kilobases of DNA, including seven introns and eight exons within the coding and 3' noncoding regions of the gene. The eight exons code for a prepro leader sequence and 415 amino acids that make up the mature protein circulating in plasma. The intervening sequences range in size from 188 to 9473 nucleotides and contain four Alu repetitive sequences, including one in intron A and three in intron F. A fifth Alu repetitive sequence was found immediately flanking the 3' end of the gene. A 50 base pair insert in intron A was found in a clone from one of the genomic libraries but was absent in clones from the other library. Intron A as well as the 3' noncoding region of the gene also contained alternating purine-pyrimidine sequences that provide potential left-handed helical DNA or Z-DNA structures for the gene. KpnI repetitive sequences were identified in intron D and the region flanking the 5' end of the gene. The 5' flanking region also contained a 1.9-kb HindIII subfamily repeat. The seven introns in the gene for factor IX were located in essentially the same position as the seven introns in the gene for human protein C, while the first three were found in positions identical with those in the gene for human prothrombin.

Nucleotide sequence of the gene for human factor IX (antihemophilic factor B).

Automated DNA sequencing of the human HPRT locus

Cloning and expression of steroid sulfatase cDNA and the frequent occurrence of deletions in STS deficiency: Implications for X-Y interchange

During detailed restriction enzyme mapping of the human X-linked gene Gd, specifying the enzyme glucose 6-phosphate dehydrogenase (G6PD), we have observed the presence, over a 14-kb DNA region spanning across the 3' end of the G6PD transcript, of a large number of methylatable sites These include 60 HpaII sites, 13 SmaI sites, 22 AvaI sites and 46 HhaI sites In male leukocyte DNA the majority of HpaII sites are resistant to digestion, indicating that they are in the Cm5CGG form However, a few sites are found reproducibly unmethylated in 24 samples analyzed By double and triple digestions we have mapped five unmethylated sites, four of which are within the gene transcript and one distal to the end of transcription We have also identified a number of sites which are fully methylated, whereas for others the methylation status could not be positively assessed Thus, in a housekeeping gene expressed in leukocytes, the 3' end is extensively methylated, but some specific sites are unmethylated In female leukocyte DNA, we found that all sites methylated in males were also methylated However, of the five sites that are unmethylated in males two are partly methylated in females This additional site-specific methylation involves approximately 50% of the female leukocyte DNA, and we show evidence that it is associated with the inactive X-chromosome

Specific methylation pattern at the 3' end of the human housekeeping gene for glucose 6-phosphate dehydrogenase.

Glucose-6-phosphate dehydrogenase (G6PD) is a household enzyme that accounts, in many cells, for about 0.03% of cellular protein. We have developed an assay for G6PD-specific mRNA based on in vitro translation of RNA from human fibroblasts and immunoprecipitation of the translation products with an anti-G6PD antiserum. By making use of this assay, G6PD mRNA has been purified 50 to 100-fold in three steps. We estimate that the mRNA encoding G6PD constitutes less than 0.02% of total poly(A)+ RNA in human fibroblasts. The size of the G6PD mRNA has been established in denaturing conditions as being in the range between 2800 and 3200 nucleotides. This has been confirmed by Northern blot analysis. Since the G6PD coding sequence is estimated to be about 1491 nucleotides, the G6PD mRNA has long untranslated sequences, most of which is at the 3' end and which may be heterogeneous in length. The sequence of the last 608 nucleotides of this mRNA has been determined.

Partial purification and characterization of the messenger RNA for human glucose-6-phosphate dehydrogenase

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Specific methylation pattern at the 3' end of the human housekeeping gene for glucose 6-phosphate dehydrogenase.

Partial purification and characterization of the messenger RNA for human glucose-6-phosphate dehydrogenase