Molecular cloning and expression of human trophoblast antigen FDO161G and its identification as 3 beta-hydroxy-5-ene steroid dehydrogenase

The monoclonal antibody FDO161G reacts with a 43-kDa protein found in human extravillous trophoblast, syncytiotrophoblast, adrenal cortex, interstitial cells of the testis and ovarian follicle cumulus cells. cDNAs for this protein have been isolated from the lambda gt11 library, sequenced, and expressed in COS-7 cells. The protein was identified as 3 beta-hydroxy-5-ene steroid dehydrogenase (HSD). The sequence of the HSD protein raises questions about its association with cell membrane systems. The lack of reactivity of FDO161G with other tissues suggests that HSD has a limited tissue distribution and that other enzymes may exist in peripheral tissues, which can convert delta 5 3-hydroxysteroids to delta 4 3-ketosteroids.     

The association between spontaneous pyelonephritis and maturity-onset diabetes mellitus in male MM mice

Blood glucose and glucose tolerance tests demonstrated that many male MM mice are diabetic. Serial urine sampling showed that the diabetes occurred only in mature MM males and consisted of a single self-limiting episode. Histological examination of the pancreas, together with measurements of body weight, glycosylated haemoglobin and plasma insulin, revealed that the diabetes was of the maturity-onset insulin-resistant type. Bacteriological examination of the urine samples showed that urinary tract infection, a known feature of male MM mice, occurred in the diabetics but only after the onset of hyperglucosuria. It was concluded that the high urinary glucose levels of diabetic MM males are of prime importance in the aetiology of the renal infection which occurs rarely in non-diabetic MM males or in other strains in the colony. An infectious aetiology for the diabetes per se was excluded by the existence of diabetes in germfree MM males.     

Analysis of the interaction of antibodies with immunoglobulin idiotypes on neoplastic B lymphocytes: implications for immunotherapy

We have examined the reactions of a panel of nine monoclonal anti-idiotype antibodies with the surface immunoglobulin in situ on guinea pig L2C leukemic lymphocytes. Equilibrium binding constants were shown to range between 10(7) and 10(8) M-1 for univalent Fab' gamma fragments and between 10(8) and 10(9) M-1 for intact IgG. Saturation of the cell surface binding sites was achieved with 2.9 X 10(5) Fab' gamma molecules/cell and 1.2 X 10(5) IgG molecules/cell for each antibody, a result that is consistent with a bivalent mode of interaction for the IgG. Despite these overall similarities in binding characteristics antibodies showed striking differences in their ability to clear Ig from the cell surface by antigenic modulation in vitro. This suggested differences in the readiness with which the antibodies cross-linked neighboring surface Ig molecules. Such an interpretation was supported by differences in the times required to achieve bivalent binding at 0 degree C, and in the rates at which labeled antibody dissociated from the cell surface in the presence or absence of an excess of unlabeled antibody. The data are consistent with there being two functionally distinct types of anti-idiotype antibody: those that form predominantly intra-Ig bridges, with each antibody Fab being linked to an Fab on one target molecule ("monogamous" binding) and not favoring modulation; and those that form predominantly inter-Ig bridges ("bigamous" binding) and favor modulation. The nature of interaction is presumably dictated by the orientation of the particular idiotope concerned. This distinction could be of great importance in the therapeutic use of anti-idiotype to ablate B cell neoplasms.     

Human debrisoquine 4-hydroxylase (P450IID1): cDNA and deduced amino acid sequence and assignment of the CYP2D locus to chromosome 22

The enzyme P450db1 (db1) is responsible for the common human defect in drug oxidation known as the "debrisoquine/sparteine polymorphism." Polyclonal antibody against the rat db1 protein was used to screen a human liver lambda gt11 library for the db1 cDNA clone. A cDNA containing the full protein coding sequence was isolated; the deduced NH2-terminal sequence of this cDNA was identical to that derived from direct sequencing of the purified human db1 protein. Comparison of the human db1 with rat db1 revealed 71 and 73% similarities of nucleotides and amino acids, respectively. By use of human-rodent somatic cell hybrids the db1 gene was localized to human chromosome 22 (CYP2D locus).     

Application of automated DNA sizing technology for genotyping microsatellite loci.

Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, we report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. We capitalize on the availability of three distinct fluorescent dyes to label uniquely loci that overlap in size, and this innovation increases by threefold the number of loci that can be analyzed simultaneously. We label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy.     

Induction by ionizing radiation of the gadd45 gene in cultured human cells: lack of mediation by protein kinase C.

The effect of ionizing radiation on the expression of two DNA-damage-inducible genes, designated gadd45 and gadd153, was examined in cultured human cells. These genes have previously been shown to be strongly and coordinately induced by UV radiation and alkylating agents in human and hamster cells. We found that the gadd45 but not the gadd153 gene is strongly induced by X rays in human cells. The level of gadd45 mRNA increased rapidly after X rays at doses as low as 2 Gy. After 20 Gy of X rays, gadd45 induction, as measured by increased amounts of mRNA, was similar to that produced by the most effective dose of the alkylating agent methyl methanesulfonate. No induction was seen after treatment of either human or hamster cells with 12-O-tetradecanoylphorbol-13-acetate, a known activator of protein kinase C (PKC). Therefore, gadd45 represents the only known mammalian X-ray-responsive gene whose induction is not mediated by PKC. However, induction was blocked by the protein kinase inhibitor H7, indicating that induction is mediated by some other kinase(s). Sequence analysis of human and hamster cDNA clones demonstrated that this gene has been highly conserved and encodes a novel 165-amino-acid polypeptide which is 96% identical in the two species. This gene was localized to the short arm of human chromosome 1 between p12 and p34. When induction in lymphoblast lines from four normal individuals was compared with that in lines from four patients with ataxia telangiectasia, induction by X rays of gadd45 mRNA was less in the cell lines from this cancer-prone radiosensitive disorder. Our results provide evidence for the existence of an X-ray stress response in human cells which is independent of PKC and which is abnormal in taxia telangiectasia.     

Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

Summary A collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.     

Formation of transient polykaryons by fusion of erythrocytes of different developmental programs

We report here two methods of fusing erythroid cells from bullfrogs (Rana catesbeiana), using polyethylene glycol or calcium phosphate, which yield masses of polykaryons in which the cytoplasms and nuclei of tadpole and adult frog erythroid cells are intermixed. The masses of fused cells carry out protein synthesis in culture, including the assembly of normal hemoglobin (Hb) tetramers. In these polykaryons there is reactivation of the expression of specific Hbs that have previously been "turned off" in vivo as the result of either a developmental Hb switch or normal cellular differentiation and RBC maturation. For example, the products of fusion of tadpole erythroblasts with adult frog mature RBCs synthesize adult Hb, whereas neither cell population alone does so. Recent experiments have taken advantage of a Hb-expression polymorphism that we discovered in this species, such that some tadpoles have greatly reduced expression of one of the larval Hbs (Hb Td-4). Fusion of erythroblasts from such tadpoles with RBC from frogs that had expressed Hb Td-4 when they were tadpoles produces polykaryons that synthesize Hb Td-4, indicating there is a trans factor that stimulates Td-4 expression. Heterospecific erythroid cell polykaryons can be constructed in an analogous manner, facilitating the study of trans-acting factors that regulate specific globin gene expression during development.