Transferrin (Tf) is the major iron binding protein in vertebrate serum. It shares homologous amino acid sequences with four other proteins: lactotransferrin, ovotransferrin, melanoma antigen p97, and HuBlym-l. Antigen p97 and the Tf receptor genes have been mapped on human chromosome 3. The goal of the study described here was to initiate the characterization of the Tf gene by identifying and characterizing its cDNA and mapping its chromosomal location. Recombinant plasmids containing human cDNA encoding Tf have been isolated by screening an adult human liver library with a mixed oligonucleotide probe. Within the 2.3 kilobase pairs of TfcDNA analyzed, there is a probable leader sequence encoded by 57 nucleotides followed by 2037 nucleotides that encode the homologous amino and carboxyl domains. During evolution, three areas of the homologous amino and carboxyl domains have been strongly conserved, possibly reflecting functional constraints associated with iron binding. Chromosomal mapping by in situ hybridization and somatic cell hybrid analysis indicate that the Tfgene is located at q21-25 on human chromosome 3, consistent with linkage of the Tf, Tf receptor, and melanoma p97 loci.Transferrin (Tf) carries ferric iron from the intestine, reticuloendothelial system, and liver parenchymal cells to all proliferating cells in the body. The family of Tf-like proteins represents the product of an intragenic duplication followed by a series of independent gene duplications (1-4). Serum Tf (1), hen ovotransferrin (2, 3), lactotransferrin (4), melanoma antigen p97 (5), and a transforming protein from chicken lymphoma ChBlym-1 (6) share strong amino acid sequence homologies. A transforming protein from Burkitt lymphomas recently described (7) may also belong to the Tf family. There is also significant internal homology in the amino-terminal (NH2) and carboxyl-terminal (COOH) domains of Tf, lactotransferrin, and ovotransferrin (1-4). For example, the NH2 and COOH domains of human Tf reveal 40% identity when the NH2 domain (residues 1-336) and the COOH domain (residues 337-678) are compared (1). In the study described Screening of cDNA Clones. The cDNA library, kindly provided by Stuart H. Orkin (Harvard Medical School, Boston) was constructed from human liver RNA as described (10). The cDNA library was incubated overnight on L agar plates containing 10 jig of tetracycline per ml and was transferred to nitrocellulose filters. The plasmids were amplified with 250 ,tg of chloramphenicol per ml and the filters were prepared for hybridization as described by Grunstein and Hogness (11). The filters were hybridized at 37°C with 32P-labeled oligonucleotide mixed probes as described by Wallace, et al. (12). The hybridization mixture contained 0.9 M NaCl, 0.09 M Tris HCl (pH 7.5), 0.006 M EDTA, 0.5% NaDodSO4, Denhardt's (13) solution (5 x strength), 100 ,ug of denatured Escherichia coli DNA per ml, and 6.4 ng of 5' end-labeled oligonucleotide mixed probes per ml having a specific activity of -7 x 108 cpm/,ug.Preparation...

Oncogene amplification has been observed in a broad spectrum of human tumors and has been associated with a poor prognosis for patients with several different types of malancies. Importantly, at biopsy, the amplified genes localize to acentric extrachromosomal elements such as doubleminute chromosomes (DMs) in the vast majority of cases. We show here that treatment of several human tumor cell lines with low concentrations of hydroxyurea accelerates the loss of their extrachromosomally amplified oncogenes. The decreases in MYC copy number in a human tumor cell line correlated with a dramatic reduction in cloning efficiency in soft agar and tumorigenicity in nude mice. No effect on gene copy number or tumorigenicity was observed for a closely related cell line containing the same number of chromosomally amplified MYC genes. One step involved in the accelerated loss of extrachromosomal elements is shown to involve their preferential entrapment of DMs within micronuclei. The data suggest that agents that accelerate the loss of extrachromosomally amplified genes could provide valuable tools for moderating the growth of a large number of human neoplasms.

Analyses of 0.8 and 1 GeV p + ' 'Pb elastic angular distribution data have obtained neutron-proton rootmean-square radius differences (hr") which are not consistent. Therefore, the 0.8 GeV experiment was repeated using a high resolution spectrometer. The new higher precision data are consistent with the older data, apart from a 15% overall normalization difference. A second order Kerman-McManus-Thaler optical model analysis of the new data, using a model-independent neutron density, yields Ar"~= 0, 14+0.04 fm, in good agreement with the most recent result obtained (0.16+0.05 fm} from a similar analysis of the older 0.8 GeV data. In addition, the elastic angular distribution was extended to 42. 5' center of mass in order to explore the momentum transfer region from 3.5 to 5.3 fm '. Although the familiar diffraction pattern persists to 42.5', it was not possible within the framework of our application of the Kerman-McManus-Thaler optical model to fit the data even qualitatively at the larger momentum transfers. NUCLEAR REACTIONSPb{p,p), E=0.8 GeV, measured g(0); enriched target; resolution = 100 keV, 6~m = 2. 5' to 42.5 . Microscopic optical model analysis using KMT potential; deduced Ar, error analysis and comparison with other results.

Ceruloplasmin (CP) is a copper-binding protein in vertebrate plasma. It is the product of an intragenic triplication and is composed of three homologous domains. Oligonucleotide probes constructed according to published amino acid sequences were used to identify cDNA clones encoding human CP. Two clones, CP-1 and CP-2, differed from each other by the presence or absence, respectively, of a deduced sequence of four amino acids. The two clones provided 81% of the sequence encoding CP. Comparison of the nucleotides of the three domains of the CP coding sequence revealed internal domain homology with identity of sequences ranging from 50.1% to 56%. The nucleotide sequence of CP-2 cDNA was compared to that of a homologous human protein, clotting factor VIII, and was found to be 48% identical overall. The CP gene was mapped to human chromosome 3 by somatic-cellhybrid analysis and to 3q25 by in situ hybridization; however, sites of hybridization to DNA on other chromosomal sites suggested additional CP-like DNA sequences in the human genome. A DNA polymorphism was detected with CP cDNA after endonuclease digestion of human DNA by Pst I. CP mRNA was detected in human liver, macrophages, and lymphocytes by in situ histohybridization.

Acute promyelocytic leukemia (APL) is a highly aggressive disease requiring prompt diagnosis and specific early intervention. Immunophenotyping by flow cytometry (FCM) facilitates a rapid diagnosis, but commonly used criteria are neither sufficiently sensitive nor specific. With an antibody panel for diagnostic screening in routine practice, we found all 149 APL cases in this study exhibited a unique immunophenotypic profile, ie, a characteristic CD11b- myeloid population and absent CD11c expression in all myeloid populations; 96.6% of cases also lacked HLA-DR expression. These distinctive features allowed recognition of all unusual cases phenotypically resembling the regular myeloblasts (CD34+/HLA-DR+) or granulocytes (CD117-/CD34-/HLA-DR-). FCM effectively identified all 19 APL cases with variant translocations, including cases with a normal karyotype due to a cryptic submicroscopic t(15;17)(q22;q21), t(11;17)(q23;q21) that escaped the detection by fluorescence in situ hybridization for t(15;17) and der(15)ider(17)(q10) that lacked a simple reciprocal t(15;17). When APL-associated profiles were validated against 107 AML cases of non-APL subtypes, including 51 HLA-DR- cases, the diagnostic specificity and positive predictive value were 98%. FCM effectively provides independent detection of APL during diagnostic workup and harmonizes with the subsequent molecular cytogenetic diagnosis.