We have cloned a series of overlapping cDNA clones encoding a 5194 bp transcript for human DNA methyltransferase (DNA MTase). This sequence potentially codes for a protein of 1495 amino acids with a predicted molecular weight of 169 kDa. The human DNA MTase cDNA has eighty percent homology at the nucleotide level, and the predicted protein has seventy-four percent identity at the amino acid level, to the DNA MTase cDNA cloned from mouse cells. Like the murine DNA MTase, the amino terminal two-thirds of the human protein contains a cysteine-rich region suggestive of a metal-binding domain. The carboxy terminal one-third of the protein shows considerable similarity to prokaryotic (cytosine-5)-methyltransferases. The arrangement of multiple motifs conserved in the prokaryotic genes is preserved in the human DNA MTase, including the relative position of a proline-cysteine dipeptide thought to be an essential catalytic site in all (cytosine-5)-methyltransferases. A single 5.2 kb transcript was detected in all human tissues tested, with the highest levels of expression observed in RNA from placenta, brain, heart and lung. DNA MTase cDNA clones were used to screen a chromosome 19 genomic cosmid library. The DNA MTase-positive cosmids which are estimated to span a genomic distance of 93 kb have been localized to 19p13.2-p13.3 by fluorescence in situ hybridization. Isolation of the cDNA for human DNA MTase will allow further study of the regulation of DNA MTase expression, and of the role of this enzyme in establishing DNA methylation patterns in both normal and neoplastic cells.

Basic helix-oop-helix transcription factors of the achaete-scute family are instrumental in Drosophl!a neurosensory development and are candidate regulators of development in the mammalian central nervous system and neural crest. We report the isolation and initial characterization of a human achaete-scute homolog that is highly expressed in two neuroendocrine cancers, meduilary thyroid cancer (MTC) and small cell lung cancer (SCLC). The human gene, which we have termed human achaete-scute homolog 1 (hASH1), was cloned from a human MTC cDNA library. It encodes a predicted protein of238 aa that is 95% homologous to mammalian achaete-scute homolog (MASH) 1, a rodent basic helix4oop-helix factor. The 57-residue basic helix-oop-helix domain is identical to that in the rodent gene, and the basic and helical regions, exduding the loop, are 72-80% identical to Drosophila achaete-scute family members. The proximal coding region of the hASHl cDNA contains a striking 14-copy repeat of the triplet CAG that exhibits polymorphism in human genomic DNA. Thus, hASHl is a candidate locus for disease-causing mutations via triplet repeat amplification. Analysis of rodent-human somatic cell hybrids permitted assignment of hASHl to human chromosome 12. Northern blots revealed hASHl transcripts in RNA from a human MTC cell line, two fresh MTC tumors, fetal brain, and three lines of human SCLC. In contrast, cultured lines of non-SCLC lung cancers and a panel of normal adult human tissues showed no detectable hASHl transcripts. Expression of hASHl may provide a useful marker for cancers with neuroendocrine features and may contribute to the differentiation and growth regulation of these cells.Cancers with neuroendocrine features such as small cell lung cancer (SCLC) and the calcitonin-secreting tumor, medullary thyroid carcinoma (MTC), frequently lose their characteristic endocrine phenotype as tumor progression occurs (1-3). To understand the evolution ofendocrine cancers, it is important to define, at a molecular level, both the regulation of neuroendocrine phenotypic features in the normal cellular precursors of these tumors and the alterations in these processes that occur during tumor progression.In the complex regulation of neuroendocrine phenotypic expression at a transcriptional level, there is increasing evidence for involvement of basic helix-loop-helix (bHLH) transcriptional enhancer factors. bHLH proteins may have particular importance in the control of polypeptide hormone synthesis and secretion. Our laboratory (4) and others (5) have demonstrated that bHLH recognition elements form a constitutive enhancer in the human calcitonin gene. Other polypeptide hormones including insulin, gastrin, and secretin also appear to utilize bHLH enhancer factors that are reThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. stricted to their differentiated host tissues (6-8)....

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We have used High Performance Liquid Chromatography to determine metabolite characteristics of three recent isolates of Acanthamoeba which exhibit cultural characteristics consistent with those of established potential pathogens. Growing amoebae and dormant cysts of these isolates were explored in regard to their qualitative and quantitative intracellular levels of polyamine and S-adenosylmethionine metabolites. The polyamine found in the greatest concentration in the growing cells was 1,3-diaminopropane (DAP), followed by spermidine (SPD). A low level of putrescine was also found in the growing cells. These polyamines significantly decreased in concentration as the amoebae differentiated to cysts. N8-acetylspermidine and acetylspermine were found in both developmental stages while acetylcadaverine was found only in growing amoebae and N1-acetylspermidine only in cysts. Acetylputrescine was present in both stages of two isolates but only in the growing amoebae of the third isolate. Spermine was not detected in any of the isolates. S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were present in growing amoebae but SAM was undetectable or barely detectable in cysts. SAH also decreased in concentration during encystation of two of the isolates to a level comparable to that of the other isolate. The developmental transition from growing amoebae to dormant cysts is characterized metabolically by a threshold adjustment in concentration of SAM, SAH and of the polyamines (esp., DAP and SPD).