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M E MacDonald

Publications and source records attributed to M E MacDonald.

106 records · Page 6Linked to original sources
PubMed1987

Physical and genetic localization of quinonoid dihydropteridine reductase gene (QDPR) on short arm of chromosome 4.

A portion of a cDNA clone corresponding to the 3' end of the human quinonoid dihydropteridine reductase (QDPR) mRNA was used as a probe to physically map the QDPR gene by analysis of somatic cell hybrid lines. The provisional assignment of QDPR to chromosome 4, based on expression of the human enzyme in hybrids, was confirmed. The gene was further regionally localized on the short arm to 4p16.1----4p15.1. This physical localization places QDPR in the same area of the genome that contains the defect causing Huntington's disease (HD). The QDPR probe revealed a restriction fragment length polymorphism with the enzyme BanII, permitting determination of its genetic proximity to D4S10, an anonymous DNA marker tightly linked to HD. QDPR is only loosely linked to D4S10, excluding any primary role for the gene in HD.

Animals↗
PubMed1987

A somatic cell hybrid panel for localizing DNA segments near the Huntington's disease gene.

Thirty-four random DNA probes from the terminal half of the human chromosome 4 short arm were further localized within 4pter----p15.1. A panel of somatic cell hybrid lines defining six chromosomal regions within 4pter----p15.1 was constructed using human cell lines containing translocation or deletion chromosomes. The vast majority of the DNA sequences, 32 of 34 or 94%, mapped to the three most proximal regions comprising 4p16.1----4p15.1. Only two probes were localized distal to 4p16.1: one in the region 4p16.3----4p16.1 and one in 4p16.3. D4S10, a polymorphic DNA marker linked to the Huntington's disease defect, has previously been mapped to the terminal region of 4p with conflicting assignments to 4p16.1 and 4p16.3. Analysis of restriction fragment length polymorphisms demonstrated hemizygosity for D4S10 in a patient with Wolf-Hirschhorn syndrome resulting from an unbalanced translocation t(4;8)(p16.3;p23.1), supporting the 4p16.3 localization. Our panel of somatic cell hybrids provides a rapid method for mapping new probes to the same vicinity as that of D4S10. However, the relative paucity of such DNA segments identified here suggests that a more directed approach may be required to generate additional markers near the HD gene.

Animals↗
PubMed1987

Two domains within the early coding region of SV40 involved in the transformation of human fibroblasts.

The early gene products of simian virus 40 (SV40) transform human fibroblasts, whereas those of the human papovavirus BK (BKV) do not. The early gene products of both SV40 and BKV transform baby rat kidney cells. SV40-BKV chimerics were constructed which were able to transform baby rat kidney cells. Using the SV40-BKV chimerics, two domains within the SV40 large T protein were identified which are involved in the transformation of human fibroblasts.

Animals↗
PubMed1986

Structure and function of human tissue-type plasminogen activator (t-PA).

Full-length tissue-type plasminogen activator (t-PA) cDNA served to construct deletion mutants within the N-terminal "heavy" (H)-chain of the t-PA molecule. The H-chain cDNA consists of an array of structural domains homologous to domains present on other plasma proteins ("finger," "epidermal growth factor," "kringles"). These structural domains have been located on an exon or a set of exons. The endpoints of the deletions nearly coincide with exon-intron junctions of the chromosomal t-PA gene. Recombinant t-PA deletion mutant proteins were obtained after transient expression in mouse Ltk- cells, transfected with SV40-pBR322-derived t-PA cDNA plasmids. It is demonstrated that the serine protease moiety of t-PA and its substrate specificity for plasminogen is entirely contained within the C-terminal "light" (L)-chain of the protein. The presence of cDNA, encoding the t-PA signal peptide preceding the remaining portion of t-PA, suffices to achieve secretion of (mutant) t-PA into the medium. The stimulatory effect of fibrin on the plasminogen activator activity of t-PA was shown to be mediated by the kringle K2 domain and, to a lesser extent, by the finger domain. The other domains on the H-chain, kringle K1, and the epidermal growth-factor-like domain, do not contribute to this property of t-PA. These findings correlate well with the fibrin-binding properties of the rt-PA deletion-mutant proteins, indicating that stimulation of the activity is based on aligning of the substrate plasminogen and its enzyme t-PA on the fibrin matrix. The primary target for endothelial plasminogen activator inhibitor (PAI) is located within the L-chain of t-PA. Deleting specific segments of t-PA H-chain cDNA and subsequent transient expression in mouse Ltk- cells of t-PA deletion-mutant proteins did not affect the formation of a stable complex between mutant t-PA and PAI.

Amino Acid Sequence↗
PubMed1986

Functional analysis of the human tissue-type plasminogen activator protein: the light chain.

A pBR322::Rous sarcoma virus(RSV)-based shuttle vector was used to insert fused genes, composed of the amino-terminal portion of the bacterial chloramphenicol-acetyltransferase gene (cat) and the entire coding region for the C-terminally derived light (L) chain of human tissue-type plasminogen activator (t-PA) cDNA. Cotransfection of rat 3Y1 cells with pRSVneo DNA and pRSVcat/t-PA DNA yielded stably integrated G418-resistant transfectants which contain unrearranged copies of pRSVcat/t-PA DNA. These transfectants synthesize cat/t-PA L-chain mRNA, apparently correctly initiated and terminated. With the help of an enzyme-linked immunosorbent assay (ELISA), it is demonstrated that these cells produce human t-PA antigen. Furthermore, pRSVcat/t-PA L-chain cDNA-containing rat 3Y1 cells synthesize a plasminogen-dependent amidolytic activity which is suppressed by specific anti-human t-PA antibodies. This activity cannot be stimulated by fibrin, a property displayed by native t-PA. It is concluded that the t-PA L-chain cDNA contains the complete genetic information for the plasminogen activator activity.

Amino Acid Sequence↗
PubMed1986

Molecular genetics of Huntington's disease.

The discovery of a DNA marker linked to the HD gene has provided new avenues into the investigation of this devastating disorder. Genetic investigations have determined that in most and possibly all HD families, the disease is caused by a defect that maps near the telomere on the short arm of chromosome 4. DNA markers will soon provide presymptomatic diagnosis for this disorder, but this increased capability may be a mixed blessing in the absence of effective treatment. The most hopeful route to developing such treatment lies in cloning and characterization of the primary defect. Precise genetic and physical mapping using DNA markers and improvements in techniques for analyzing large segments of DNA have set the stage for cloning of the disease gene in the near future. It will undoubtedly reveal an interesting mechanism for complete phenotypic dominance in man for comparison with completely dominant mutations in other species, particularly Drosophila. The nature of the defect may provide new insights into the functional organization of the central nervous system. For the sake of the many individuals who are afflicted by HD or who are asymptomatic gene carriers, it is to be hoped that cloning and characterizing the disease gene will also yield the necessary information to develop an effective therapy.

Chromosome Mapping↗
PubMed1986

Molecular genetics of human chromosome 4.

The recent discovery that the gene causing Huntington's disease (HD) resides on chromosome 4 has generated increased interest in this autosome. Chromosome 4 contains two of the more informative conventional genetic markers, GC and MNS, but most loci have been assigned to it by recombinant DNA techniques. There are currently more anonymous DNA fragments detecting restriction fragment length polymorphisms (RFLPs) on chromosome 4 than on any other autosome. In addition, most of the cloned genes from this chromosome detect useful RFLPs. A genetic linkage map including both conventional and DNA markers should soon span the entire chromosome and will undoubtedly lead to the localisation of other inherited disorders.

Chromosome Mapping↗
PubMed1981

Clonal analysis of early and late stages of erythroleukemia induced by molecular clones of integrated spleen focus-forming virus.

The integrated proviral DNA of the polycythemia-inducing isolate of Friend spleen focus-forming virus (SFFVp) has been identified in rat cell clones nonproductively infected with this replication-defective erythroleukemia virus and cloned in phage lambda vectors. These lambda SFFVp recombinants, lambda SFFVp502 and lambda SFFVp542, contain endonuclease EcoRI inserts of size 7.4 and 8.2 kilobases, respectively, and include full copies of the SFFVp genome, along with host flanking sequences. Infectivity of the cloned SFFVp genomes was tested by a two-step DNA transfer procedure involving transfection of the cloned DNA into 3T3 mouse fibroblasts or cotransfer of the cloned DNA into thymidine kinase-deficient 3T3 cells together with the cloned thymidine kinase gene of herpes simplex virus, followed by rescue of the transferred DNA by superinfection with a helper virus. Inoculation of the rescued virus into adult mice resulted in the appearance of spleen foci, rapid splenomegaly, and polycythemia. Early after infection, spleen cell populations contained large numbers of cells capable of forming small erythroid colonies in vitro (CFU-E) in the absence of erythropoietin. Late after infection, these mice contained cells capable of forming macroscopic colonies (CFU-FV) in vitro. These data indicate that molecular clones of SFFVp, in conjunction with a helper virus, induce the appearance of hemopoietic colony-forming cells characteristic of both the early and late stages of Friend leukemia.

Animals↗
PubMed1981

Clonal analysis of the late stages of erythroleukemia induced by two distinct strains of Friend leukemia virus.

We observed striking differences between the tumorigenic colony-forming cells present in the spleens of mice late after infection with the anemia-inducing strain of Friend leukemia virus (strain FV-A) and those present after infection with the polycythemia-inducing strain (strain FV-P). Cells within primary colonies derived from FV-A- and FV-P-transformed cells (CFU-FV-A and CFU-FV-P, respectively) contained hemoglobin and spectrin, indicating that the CFU-FV-A and CFU-FV-P were transformed erythroid progenitor cells. The proportion of cells containing hemoglobin was relatively high (> 25%) in newly isolated cell lines derived from CFU-FV-P colonies, whereas cell lines derived from CFU-FV-A colonies had only low levels (0 to 2%) of hemoglobin-containing cells. A high proportion of the cell lines derived from CFU-FV-A colonies responded to pure erythropoietin and accumulated spectrin and hemoglobin, whereas the cell lines derived from CFU-FV-P colonies did not. A cytogenetic analysis indicated that primary CFU-FV-P colony cells were diploid, whereas chromosomal aberrations were observed in the immediate progeny of CFU-FV-A. The presence of unique chromosomal markers in the majority of the cells within individual colonies derived from CFU-FV-A suggested that these colonies originated from single cells. Finally, leukemic progenitor cells transformed by strain FV-A appeared to have an extensive capacity to self-renew (i.e., form secondary colonies in methylcellulose), whereas a significant proportion of the corresponding cells transformed by strain FV-P did not. In addition, the self-renewal capacity of both CFU-FV-A and CFU-FV-P increased as the disease progressed. From these observations, we propose a model for the multistage nature of Friend disease; this model involves clonal evolution and expansion from a differentiating population with limited proliferative capacity to a population with a high capacity for self-renewal and proliferation.

Animals↗
PubMed1980

Anemia- and polycythemia-inducing isolates of Friend spleen focus-forming virus. Biological and molecular evidence for two distinct viral genomes.

Two distinct clones of Friend spleen focus-forming virus (SFFV), differing in their erythroleukemic potential, are described. These isolates have been cloned free of their associated helper viruses and shown to be replication-defective. Both SFFV isolates have been rescued from rat fibroblast nonproducer cell clones with cloned replication-competent viruses, F-MuLVA and F-MuLVP, obtained from the anemia- or polycythemia-inducing isolates of Friend virus complex, respectively. These rescued viruses induce a rapid proliferative disease associated with the appearance of macroscopic spleen foci and splenomegaly. In addition, each is subject to regulation by the W, Steel (Sl), and Fv-2 host gene loci. These two isolates of SFFV can, however, be distinguished by both biological and molecular criteria. Friend SFFVP induces a rapid polycythemia associated with the appearance of large numbers of erythropoietin (EPO)-independent erythroid colony-forming cells in the marrow and spleen. In contrast, SFFVA induces a rapid anemia associated with a progressive decrease in the number of EPO-dependent erythroid colony-forming cells in marrow, and a rapid increase in the number of EPO-dependent erythroid colony-forming cells in spleen. Furthermore, the nature of the disease induced by the two isolates of SFFV is independent of the Friend helper virus: SFFVP, rescued from a nonproducer cell clone with either F-MuLVA or F0MuLVP, induced a polycythemic transformation, whereas SFFVA, rescued with either F-MuLVA or F-MuLVP, induced an anemic transformation. The two Friend SFFV isolates can also be discriminated on the basis of translational products encoded by their gag and env genes: SFFVP encodes the amino-terminal gag-gene protein p15, whereas SFFVA encodes the gag-gene proteins p15, p12, and p30. In addition, the SFFV isolates encode nonidentical 55,000-mol wt env gene-related proteins that can be distinguished by analysis of their methionine-containing tryptic peptides.

Anemia↗
PubMed1980

Erythroleukemia induction by replication-competent type C viruses cloned from the anemia- and polycythemia-inducing isolates of Friend leukemia virus.

In this study, the biological properties of the replication-competent viruses, F-MuLVA, present in the anemia-inducing isolate of Friend leukemia virus complex (FV-A); and F-MuLVP, present in the polycythemia-inducing isolate of Friend leukemia virus complex (FV-P) have been examined. BALB/c mice infected as newborns with clonal isolates of F-MuLVA or F-MuLVP become anemic and show splenic enlargement characterized by an increased proportion of cells that resemble immature nucleated erythroid cells. In addition, the spleens of these F-MuLVA- or F-MuLVP-infected mice contain a markedly increased proportion of both erythropoietin-dependent erythroid progenitor cells and spectrin-containing erythroid cells. These results suggest that Friend murine leukemia virus (F-MuLV) by itself can induce an erythroleukemic transformation in newborn BALB/c mice similar to that induced by the anemia-inducing spleen focus-forming virus (SFFVA) in newborn or adult mice. Kinetic studies indicated that the alterations in hemopoietic cell populations induced by F-MuLVA or F-MuLVP in newborn BALB/c mice occurred more slowly than the rapid changes observed after infection with FV-A. In addition, adult BALB/c mice were fully susceptible to the erythroleukemic transformation induced by either SFFVA or SFFVP, whereas only newborn mice were susceptible to F-MuLV. Taken together, these results suggest that, although the replication-defective Friend spleen focus-forming viruses appear to be the major determinant of erythroleukemia induction in adults, the replication-competent helper F-MuLV also have erythroleukemic potential when assayed in newborn animals.

Anemia↗
PubMed1980

Demonstration with monoclonal antibody of the glycoprotein nature of Thy-1.2 alloantigen.

Thy-1 antigen present in large amounts on the brain and thymus of mice, exists in two allelic forms, either Thy-1.1 or Thy-1.2. Previous results have shown that Thy-1.1 alloantigen is expressed on a glycoprotein of molecular weight 25 000, therefore referred to as the Thy-1 glycoprotein. However the presence of Thy-1.2 alloantigen on the purified Thy-1 glycoprotein could not be established unequivocally. The present paper demonstrates that Thy-1.2 alloantigen, identified by F7D5 monoclonal antibody, is localized on the Thy-1 glycoprotein. The limited neutralization by the glycoprotein of the reactivity of AKR anti-C3H allosera to thymocytes is explained by the fact that the antiserum contains antibodies to determinants other than Thy-1.2. The alloantisera are shown to react with viral proteins encoded for by endogenous murine leukemia virus and expressed at the surface of Friend cells and thymocytes.

Animals↗