Biomedical subjects

B E Roberts

Publications and source records attributed to B E Roberts.

At least 73 records · Page 4Linked to original sources

PubMed1984

Transcriptional mapping of the DNA polymerase gene of vaccinia virus.

Vaccinia virus DNA polymerase, a single-subunit enzyme of 110,000 molecular weight, is induced early after infection. Genetic analysis suggests that the gene encoding the enzyme maps within a 15-kilobase HindIII fragment located 45 kilobases from the left-hand end of the genome. We identified the in vitro translation product with these properties and mapped the transcript by hybrid selection, RNA filter hybridization, and S1 nuclease mapping. Two mRNAs from this region, 3.4 and 3.9 kilobases in size, could be translated in vitro to yield a 110K polypeptide. The two RNAs shared a common 5' terminus and had staggered 3' ends. Sequences mapping entirely within this gene were shown to be biologically active in rescuing mutants with temperature-sensitive or drug-resistant polymerase activity to the wild-type phenotype.

Chromosome Mapping↗

PubMed1984

Organization of six early transcripts synthesized from a vaccinia virus EcoRI DNA fragment.

Four early transcripts and the polypeptides they encode have been mapped to the vaccinia virus EcoRI F DNA fragment, which spans the vaccinia HindIII J and H fragments. In addition, two transcripts for which no encoded polypeptides have been identified have also been mapped. Elucidation of the organization of these six transcripts by hybrid selection, S1 nuclease mapping, translation of size-fractionated RNA, and by filter hybridization demonstrates that approximately 90% of this DNA fragment is transcribed during early infection. All but one of these RNAs (1.35 kilobases [kb]) are transcribed in a rightward direction. The leftmost transcript of 0.6 to 0.7 kb encodes a 19,000-dalton (19K) polypeptide that has been determined to be thymidine kinase (D.E. Hruby and L.A. Ball, J. Virol. 43:403-409, 1982; G. Bajszar et al., J. Virol. 45:62-72, 1983). Immediately following the 3' end of this RNA is the 1.7-kb transcript encoding a 36K polypeptide. The 5' end of a 1.25-kb RNA encoding a 22K polypeptide is downstream of the 5' end of the 1.7-kb RNA, and its 3' end may be coterminal with the 3' end of the 1.7-kb RNA. The 2.45-kb transcript is coterminal at its 5' end with the message encoding thymidine kinase and is coterminal at its 3' end with the adjacent 1.7-kb mRNA. This RNA was not demonstrated to encode a polypeptide. Approximately 300 nucleotides from the 3' end of the 1.7-kb RNA is a 3.6-kb transcript encoding a 110K polypeptide. The 3' end of this large RNA lies several hundred nucleotides from the 3' end of the 1.35-kb RNA which is transcribed in the opposite direction. No splicing of RNA has been detected with the S1 nuclease mapping technique of Berk and Sharp. The organization of three late messages, which overlap these early transcripts, has been determined, and these results are discussed in the accompanying paper.

Animals↗

PubMed1984

Arrangement of late RNAs transcribed from a 7.1-kilobase EcoRI vaccinia virus DNA fragment.

Three late transcripts have been mapped to the vaccinia virus 7.1-kilobase (kb) EcoRI F fragment, which is located approximately 85 kb from the left end of the viral genome. Hybrid selection and translation of RNA have demonstrated that these mRNAs encode three polypeptides with apparent molecular weights of 32,000 (32K), 26K, and 19K. The transcripts encoding the 32K and 19K polypeptides are synthesized in a leftward direction and overlap each other as well as the RNA encoding the early 110K polypeptide. In addition, the RNA encoding the 32K polypeptide also overlaps an early transcript of 1.35 kb (see accompanying paper). The transcript encoding the 26K polypeptide overlaps the early 2.45-, 0.6- to 0.7-, and 1.7-kb RNAs, and all four mRNAs are transcribed from left to right. The protein coding sequences for the 26K and 32K polypeptides lie outside the 7.1-kb DNA fragment. Due to their heterogeneity in size, each of the three late transcripts is undetectable as a distinct size by filter hybridization. In addition, although S1 mapping has detected the 5' terminus of the late RNA which maps entirely within this fragment, it has been unable to size and locate the 3' ends of all three transcripts, and this result indicates that the heterodisperse size of the RNAs is due to heterogeneity at the 3' ends of these transcripts. The cause of this heterogeneity in termination of transcription is not known.

Animals↗

PubMed1984

Organization of RNA transcripts from a vaccinia virus early gene cluster.

The detailed organization of the RNAs transcribed from an early gene cluster encoded by vaccinia virus has been determined from the information derived from several complementary techniques. These include hybrid selection coupled with cell-free translation to locate DNA sequences complementary to mRNAs encoding specific polypeptides; RNA filter hybridization to size and locate on the DNA mature RNAs as well as higher-molecular-weight RNAs; S1 nuclease mapping to precisely locate the 5' and 3' ends of the RNAs; S1 nuclease mapping to precisely locate the 5' and 3' ends of the RNAs; and fractionation of hybrid-selected mRNAs in an agarose gel containing methyl mercury hydroxide followed by the cell-free translation of these mRNAs to definitively ascertain the size of the mRNA encoding each polypeptide. The early gene cluster is located between 21 and 26 kilobases from the left end of the vaccinia virus genome and is encoded by a 5.0-kilobase EcoRI fragment which spans the HindIII-N, -M, and -K fragments. Transcribed towards the left terminus are four mature mRNAs, 1,450, 950, 780, and 400 nucleotides in size, encoding polypeptides of 55, 30, 20, and 10 kilodaltons, respectively. These mRNAs are colinear with the DNA template and are closely spaced such that the 5' terminus of one mRNA is within 50 base pairs of the 3' terminus of the adjacent RNA. In addition to the mature size mRNAs, there are higher-molecular-weight RNAs, 5,000, 3,300, 2,350, 2,300, 1,800, 1,700, and 1,350 nucleotides in size. The 5' and 3' termini of the high-molecular-weight RNAs are coterminal with the 5' and 3' termini of the mature size mRNA. The implications of this arrangement and the biogenesis of these early mRNAs are discussed.

Animals↗

PubMed1984

Identification of the DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus.

The DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus were located on the viral genome. The formation of an enzyme-guanylate covalent intermediate labeled with [alpha-32P]GTP allowed the identification of the large subunit of the capping enzyme and was used to monitor the appearance of the enzyme during the infectious cycle. This assay confirmed that after vaccinia infection, a novel 84,000-molecular-weight polypeptide corresponding to the large subunit was rapidly synthesized before viral DNA replication. Hybrid-selected cell-free translation of early viral mRNA established that vaccinia virus encoded a polypeptide identical in molecular weight with the 32P-labeled 84,000-molecular-weight polypeptide found in vaccinia virions. Like the authentic capping enzyme, this virus-encoded cell-free translation product bound specifically to DNA-cellulose. A comparison of the partial proteolytic digestion fragments generated by V8 protease, chymotrypsin, and trypsin demonstrated that the 32P-labeled large subunit and the [35S]methionine-labeled cell-free translation product were identical. The mRNA encoding the large subunit of the capping enzyme was located 3.1 kilobase pairs to the left of the HindIII D restriction fragment of the vaccinia genome. Furthermore, the mRNA was determined to be 3.0 kilobases in size, and its 5' and 3' termini were precisely located by S1 nuclease analysis.

Animals↗

PubMed1984

Structure and arrangement of the beta-tubulin genes of Leishmania tropica.

We studied the organization and arrangement of the genes encoding beta-tubulin in the protozoan parasite Leishmania tropica and examined the structure and orientation of the beta-tubulin mRNA relative to the gene. There were found to be eight to nine beta-tubulin genes arranged in an array of direct tandem repeat units with a length of 3.8 kilobase pairs, and they were extremely homologous, if not identical, in sequence. These repeat units did not contain the alpha-tubulin genes. The transcribed sequences within the beta-tubulin genes were localized, and the orientation of the major alpha-tubulin mRNA was mapped on the gene by S1 nuclease analysis.

Animals↗

PubMed1984

Fractionation and further characterization of granulocytic and monocytic alpha-naphthyl acetate (ANAE) esterases.

Following characterization of myeloid nonspecific esterases by isoelectric focusing (IEF), two main groups of alpha-naphthyl acetate (ANAE) esterase isoenzymes were defined and fractionated from cytoplasmic extracts by chromato focusing techniques according to differences in their isoelectric points (pI). The first of these ANAE enzyme groups was common to leukocytes of both granulocytic and monocytic lineage, while the other, which characteristically comprised a group of isoenzymes within the pI range 5.5-6.1, was specifically associated with monocytic differentiation. The properties of the two purified ANAE enzyme fractions were compared by inhibition (heat and sodium fluoride) and further electrophoretic studies, and the results discussed in relation to the cytochemical characterization of these enzymes as markers of specific myeloid cell differentiation.

Carboxylic Ester Hydrolases↗

PubMed1984

Granulocyte Fc-IgG and C3b receptor expression in the primary myelodysplastic syndromes (MDS): relationship with dysgranulopoiesis and evidence for heterogeneity of morphological subgroups.

The expression of Fc-IgG and C3b membrane receptors by granulocytes and their precursors was examined in 78 patients with primary myeloid dysplasia (MDS). The marrows were categorized into five morphological groups, broadly corresponding to those described by the FAB group, and further graded into mild and severe according to the severity of dysgranulopoiesis. Fractionated bone marrow and peripheral blood granulocyte receptors were assessed by rosette formation with optimally sensitized ox erythrocytes and the results compared with those found in 17 normal marrows and with those previously reported in megaloblastic anaemia. Fc-IgG and C3b receptor expression was generally increased in maturing granulocytes in all MDS diagnostic groups but premature expression was particularly marked in chronic myelomonocytic leukaemia and refractory cytopenia. Receptor patterns showed a better correlation with the severity of dysgranulopoiesis than with the morphological types. It is suggested that, as in megaloblastic anaemia, changes in membrane receptor expression reflect nuclear-cytoplasmic asynchrony. The results further indicate considerable immunological heterogeneity of granulocytes within individual MDS categories.

Granulocytes↗

PubMed1984

Electrophoretic and cytochemical characterization of alpha-naphthyl acetate esterases in acute myeloid leukemia: relationships with membrane receptor and monocyte-specific antigen expression.

Alpha-naphthyl acetate esterases (ANAE) were examined by cytochemical and isoelectric focusing (IEF) techniques in 48 cases of acute myeloid leukemia that were classified by conventional morphological criteria. Four main types of ANAE isoenzyme patterns were found by IEF, and comparisons with the expression of membrane receptors (Fc-IgG and C3b) and monocyte-specific antigens (UCHM1, UCHALF, and E11) suggest relationships between ANAE isoenzyme synthesis and distinct myeloid maturational stages. The results further indicate that the blast cells of acute myelomonocytic leukemia (AMML) may represent an immature variant of monocytic leukemia (AMoL) and that morphological examination alone is inadequate in the assessment of monocytic differentiation in acute myeloid leukemias. Inhibition studies of cytochemical ANAE activity with sodium fluoride (NaF) show that the presence of NaF-sensitive or NaF-resistant ANAE enzymes is often unrelated to the diagnostic category of acute leukemia. The results of this study are examined in relation to current concepts of myeloid differentiation, and the application of these findings to the subclassification of acute myeloid leukemias is discussed.

Antibodies, Monoclonal↗

PubMed1984

C3b receptor-negative peripheral blood neutrophils. A study of normal and haematologically abnormal disorders.

Expression of peripheral blood neutrophil (PBN) C3b receptors, as assessed by rosette formation with C3b-coated ox erythrocytes, was examined and compared with neutrophil alkaline phosphatase (NAP) activities in both normal and haematologically abnormal conditions. The results indicate that a small percentage of normal PBN are apparently C3b receptor-negative and that these neutrophils do not appear to differ with respect to age from those with detectable C3b receptors. Examination of PBN C3b receptors from 154 cases of various haematological disorders revealed a significant proportion of cases with increased numbers of C3b receptor-negative neutrophils. These abnormalities did not appear to be related to peripheral leucocyte counts, NAP activities or serum lysozyme concentrations and it is suggested that the increased numbers of C3b receptor-negative PBN may be related to intravascular factors such as immune complexes.

Alkaline Phosphatase↗

PubMed1983

Methods utilizing cell-free protein-synthesizing systems for the identification of recombinant DNA molecules.

Herein we outline three methods that, when coupled with cell-free protein synthesis, permit the identification of recombinant DNA molecules encoding specific polypeptides. RNAs enriched by fractionation on methylmercury hydroxide agarose gels are used to prepare sequence-specific probes. Recombinant DNA clones thus identified are further characterized as to their encoded polypeptides by either hybrid selection or hybrid arrest of translation.

Actins↗

PubMed1983

Plasma fibronectin in myeloproliferative disorders and chronic granulocytic leukaemia.

A significant reduction of plasma fibronectin levels was found in polycythaemia vera and myelofibrosis, the lowest levels being found in patients with marked splenomegaly. Plasma fibronectin concentration was normal in essential thrombocythaemia, and only modest reduction was seen in chronic granulocytic leukaemia in either controlled chronic phase or blast cell crisis. In a patient with myelofibrosis, the plasma fibronectin rose from less than 100 mg/l to 177 mg/l after splenectomy. Possible explanations include increased consumption of plasma fibronectin in the expanded mononuclear phagocyte system present in the liver and spleen, reduced hepatic synthesis, and the clearance of circulating immune complexes. Low plasma fibronectin concentrations may increase susceptibility to infection.

Adult↗

PubMed1983

Decreased T helper cells in the myelodysplastic syndromes.

The myelodysplastic syndromes (MDS) are disorders in which the abnormalities are thought to be confined to cells of the myeloid series. However, examination of peripheral blood from 56 patients with MDS showed that the majority had low lymphocyte counts. In a detailed study of 25 patients, using OKT3, OKT4 and OKT8 monoclonal antisera together with SRBC rosettes, it was shown that there is a consistent and significant reduction in peripheral blood T lymphocytes. This decrease was primarily confined to the OKT4-defined helper subpopulation. There is a consequent relative increase in the OKT8-defined subset but in absolute numbers however, suppressor cells are reduced compared to normal particularly in those patients with lymphopenia. No correlations between T cell abnormalities and the different morphological groups of MDS were found. The possible implications of the reversed helper/suppressor ratio in the pathogenesis of MDS are discussed.

Antibodies, Monoclonal↗

PubMed1983

Esterase cytochemistry in primary myelodysplastic syndromes and megaloblastic anaemias: demonstration of abnormal staining patterns associated with dysmyelopoiesis.

Acid alpha naphthyl acetate esterase (ANAE) and combined ANAE-chloroacetate esterase cytochemistry was performed on 121 bone marrow aspirates from primary myelodysplastic syndromes (MDS) and a secondary dysplasia-megaloblastic anaemia (MA). The investigation demonstrated the presence of abnormal ANAE positive granulocyte populations in a significant proportion of cases. These cells, in which the staining patterns were characterized by atypical granular ANAE positivity and double ANAE-chloroacetate reactions, were shown immunologically to lack the receptor and antigenic characteristics of monocytes and morphologically to be granulocytes. Isoelectric focusing, however, indicated that the atypical esterase cytochemistry of these granulocytes was due to the presence of markedly increased concentrations of ANAE isoenzymes usually found in monocytes. Atypical ANAE-staining granulocytes were particularly evident in MDS marrows showing sideroblastic erythroid changes, whilst in MA they were mainly seen in cases of intermediate severity. It is suggested that these cells are associated with dysmyelopoietic changes in both malignant and non-malignant conditions.

Anemia↗

PubMed1983

T lymphocyte subpopulations in idiopathic thrombocytopenic purpura (ITP).

Absolute T cell counts and the relative distributions of antibody-defined helper and cytotoxic/suppressor T lymphocytes are reported in 26 patients with chronic idiopathic thrombocytopenic purpura (ITP). T cell numbers are generally normal in ITP and alterations in relative helper (OKT4 positive) and suppressor (OKT8 positive) T cell numbers show no consistent patterns, although 35% of the cases examined showed inverted distributions. The combined percentages of helper and suppressor T cells consistently exceeded 100% in ITP indicating the presence of increased OKT4+8+ cells in these patients. A cell population, previously defined as SRBC positive and OKT3 negative, was found in the normal group and in ITP patients following splenectomy but was absent in those patients with intact spleens. The role of these changes in the pathogenesis of ITP is discussed.

Adolescent↗

PubMed1983

Vaccinia virus induces cellular mRNA degradation.

The infection of mouse L cells with vaccinia virus induced a rapid inhibition of cellular polypeptide synthesis and a diversion of protein synthesis to the exclusive production of viral polypeptides. This shutoff of cell-specific protein synthesis was achieved by a novel mechanism by which the virus induced the rapid degradation of cellular mRNAs. Concurrent with the degradation of cellular mRNA, the virus proceeds in the orderly temporal expression of its own genetic information. The effect of vaccinia virus infection upon two abundant L-cell mRNAs was assessed by using the highly conserved cDNA sequences that encode chicken beta-actin and rat alpha-tubulin. Hybridization analyses demonstrated that throughout infection there is a rapid and progressive degradation of both of these mRNAs. In fact, after 3 h of infection they are reduced to less than 50% of their concentration in uninfected L cells, and between 8 to 10 h they are almost entirely degraded. This observation explains in part the mechanism by which vaccinia virus inhibits host cell protein synthesis.

Actins↗

PubMed1983

Membrane Fc-IgG and C3b receptors on myeloid leukaemia cells: a comparison with cytoplasmic acid naphthyl acetate esterase cytochemistry.

Membrane receptors for IgG and C3b were examined on blast cells from 57 cases of acute myeloid leukaemia. These acute leukaemias were classified as myeloblastic, myelomonocytic or monocytic following morphological, cytochemical, and immunological investigations. The membrane receptors of leukaemic blast cells appear to be directly related to the degree of monocytic differentiation with the lowest receptor activities found in acute myeloblastic leukaemia. A comparison was also made between receptor and cytoplasmic acid naphthyl acetate esterase (ANAE) activities in 29 morphologically and immunologically-defined myelomonocytic and monocytic leukaemias. This study revealed that the receptor-positive "monocytic component" in a significant proportion of cases showed unexpectedly weak or negative ANAE reactions suggesting a more cautious approach to the interpretation of ANAE cytochemistry in acute leukaemias. The normal development of cytoplasmic ANAE and membrane receptors is also discussed and compared with their abnormal patterns of expression associated with leukaemic transformation.

Carboxylic Ester Hydrolases↗

PubMed1983

Serum beta-2-microglobulin in disorders of myeloid proliferation.

Serum beta-2-microglobulin (beta 2-m) was measured on 182 samples from 98 patients with diseases characterized by proliferation of predominantly non-lymphoid marrow elements. Raised levels were found in myeloproliferative disorders, chronic granulocytic leukaemia, acute myeloid leukaemia and in many patients with myelodysplastic syndromes. The most striking elevation was seen in acute and chronic myelomonocytic leukaemia. These raised serum levels are probably derived from increased cell turnover, and it is suggested that cells of monocyte-macrophage series are particularly high producers of beta 2-m.

Anemia, Aplastic↗