Biomedical subjects
Publications and source records attributed to R Williamson.
We report a case of subacute sclerosing panencephalitis (SSPE) with severe peripheral chorioretinal pigment changes in addition to the more typical pigment clumping in the macula. The differential diagnosis of peripheral inactive chorioretinitis, the typical ocular findings, and clinical course of SSPE are discussed.
Using cDNA hybridization, mouse globin RNA sequences can be detected in the nuclear and cytoplasmic RNA not only from erythroid cells (fetal liver and reticulocytes), but also in low amounts in non-erythroid tissues (adult brain and liver, and cultured lymphoma, untransformed, and transformed fibroblast cell lines). For the nuclear RNAs, melting curve, density, and size determinations on the hybrids confirmed the presence of an RNA species indistinguishable by cDNA hybridization from reticulocyte globin mRNA. Thus the genes coding for alpha- and beta-globin, proteins thought to be found only in a restricted range of differentiated cell types, may nonetheless be transcribed at very low levels in all mouse cells. The proportion of the globin RNA sequences in the nuclear RNA that are found associated with poly(A) sequences does not vary markedly from one tissue or cell line to another. In erythroid cells, however, the proportion of the cellular globin RNA sequences that are found in the cytoplasm is very much higher than in non-erythroid cells. This suggests the existence of messenger-specific nuclear post-transcriptional control mechanisms which do not use poly(A) as a signal, and which allow the cytoplasmic accumulation of globin mRNA only in erythroid tissues.
DNA has been prepared from peripheral blood or cultured skin fibroblasts obtained from three Sicilian and one Greed deltabeta-thalassemia homozygotes. Globin-gene analysis was carried out using a cDNAbeta probe, and the results indicate that deltabeta-thalassemia has arisen from a deletion of the beta-globin genes. A similar result was obtained using DNA prepared from cultured skin fibroblasts from an individual homozygous for the Negro form of hereditary persistence of fetal hemoglobin (HPFH). In both cases, the deletion has spared the Ggamma and Agamma loci directing the gamma chains of hemoglobin F, but it has not been possible to demonstrate any difference between the size of the deletion involved in the production of delta-beta-thalassemia and that which gave rise to HPFH. These experiments provide further direct evidence that deletions of critical areas of the gamma-delta-beta gene cluster result in persistent gamma chain synthesis in adult life.
The number of genes specifying human gamma-globin has been determined directly by hybridization of complementary DNA to total human DNA. The complementary DNA was enriched in sequences specific for gamma-globin genes by transcribing globin mRNA isolated from fetal reticulocytes with viral reverse transcriptase, and collecting the material which does not back-hybridize to adult globin mRNA. When hybridized in cDNA excess to DNA, very similar values are found for gamma-gene number as for beta-gene number, suggesting two or at most three gamma-globin genes per haploid human genome. This indicates that the non-Mendelian ratios of gamma-chain mutants found in heterozygotes are due to transcriptional or post-transcriptional regulation rather than to gene dosage. The number of each major human globin gene has now been determined directly by molecular methods.
Using complementary DNA prepared from adult chicken globin messenger RNA, we show the existence of 2-3 non-cross-hybridising globin sequences in the chicken genome, each of which is present in only one copy per haploid genome. This was done by solution hybridization to total DNA under conditions of cDNA excess. The data agreses with results of RNA-driven hybridisation of globin complementary DNA, and with protein data.
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The molecular weight of fibroin messenger RNA has been measured by its contour length in the electron microscope and its electrophoretic mobility under conditions of complete denaturation. These methods give values of 5.6 times 10-6 and 6.0 times 10-6 daltons, respectively. The average molecular weight of 5.8 times 10-6 (1.6 times 10-4 base residues) would be expected to code for a protein of 414,000 daltons, a value about 12 percent larger than the most recently published size for fibroin. When the mRNA is prepared carefully to avoid shear breakage, it is as homogeneous in size as ribosomal RNA. Between 43 percent and 81 percent of the mRNA molecules bind to oligo(dT)-cellulose and are presumed to contain a poly(A) sequence. This sequence, isolated after ribonuclease digestion, comprises about 0.6 percent of the molecule, or an estimated 100 nucleotides. The poly(A) has been localized at the end of the molecule by hybridizing with a small poly (dT:dA) piece and subsequent visualization by electron microscopy. This observation, along with the ability of reverse transcriptase to transcribe fibroin mRNA with an oligo(dT) primer, demonstrates that the poly(A) is at the 3' end of the mRNA.
During embryonic development there is a transition from embryonic and fetal to adult beta-type globin chains. The high-molecular-weight RNA found in nuclei from embryonic and adult human erythropoietic tissues, fetal liver, and bone marrow, have been investigated for the presence of gamma(fetal)- and beta(adult)-globin messenger RNA sequences by molecular hybridization. Unlike alpha- and beta-globin mRNA sequences, gamma-globin mRNA sequences are absent from both total and high-molecular-weight nuclear RNA isolated from adult bone marrow. The amount of cytoplasmic gamma-globin mRNA is proportional to the level of gamma-chain synthesis, demonstrating that translational control is not a major control mechanism in the expression of globin genes. Since the gamma-, delta-, and beta-globin genes are known to be closely linked genetically, transcriptional control can discriminate between similar gene sequences that are spatially adjacent to one another.
Complementary DNA (cDNA) was prepared with RNA-dependent DNA polymerase from human globin messenger RNA (mRNA). Annealing and translation experimenta with total mRNA from circulating cells from a patient with heterozygous beta/heterozygous beta-delta-o thalassemia (beta-o/delta beta-o-thalassemia) demonstrated no detectable mRNA for beta-globin. cDNA enriched in sequences homologous to beta-globin mRNA was prepared by hydroxylapatite fractionation of hybrids formed between beta-o/delta beta-o-thalassemic mRNA and cDNA made from mRNA from a patient with alpha-thalassemia (hemoglobin H disease). The rate of annealing of this beta-enriched cDNA to normal human nuclear DNA was that of a sequence present as only a single copy per haploid genome. The beta-enriched cDNA annealed to the beta-o-delta beta-o-thalassemia total DNA with approximately the same kinetics as to normal DNA, indicating that no total gene deletion of beta-globin genes from the diploid genome has occurred, although the accuracy of the technique could not exclude with certainty a partial deletion or a deletion of a beta-globin gene from only one of the haploid genomes. This demonstrates that at least one of the beta-o- or the delta beta-o-thalassemia haploid genomes in this case contains a substantially intact beta-globin gene.
Mouse globin messenger ribonucleoprotein (mRNP) has been isolated from reticulocyte polysomes by affinity chromatography to oligo(T)-cellulose, using a procedure modified from that of Lindberg and Sundquist. The messenger RNA and protein moieties fo the mRNP are indistinguishable from those isolated by less rapid techniques, such as zonal ultracentrifugation.
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After combined pancreatic and T(1) ribonuclease treatment of RNA, a characteristic series of products of the type A(n)N are obtained, where N can be any of the four ribonucleosides. Depending on whether phosphatase treatment is used before the addition of labelled phosphate at the 5'-terminus with bacteriophage phosphokinase, the labelled oligonucleotides obtained may or may not possess a phosphate group at the 3'- as well as the 5'-end. The behaviour of these characteristic products after electrophoresis on cellulose acetate strips followed by chromatography on polyethyleneimine-cellulose thin-layer plates was examined.