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Biomedical subjects

L M Roberts

Publications and source records attributed to L M Roberts.

At least 109 records · Page 6Linked to original sources
PubMed1985

Nucleotide sequence of cloned cDNA coding for preproricin.

The primary structure of a precursor protein that contains the toxic (A) and galactose-binding (B) chains of the castor bean lectin, ricin, has been deduced from the nucleotide sequence of cloned DNA complementary to preproricin mRNA. A cDNA library was constructed using maturing castor bean endosperm poly(A)-rich RNA enriched for lectin precursor mRNA by size fractionation. Clones containing lectin mRNA sequences were isolated by hybridization using as a probe a mixture of synthetic oligonucleotides representing all possible sequences for a peptide of the ricin B chain. The entire coding sequence of preproricin was deduced from two overlapping cDNA clones having inserts of 1614 and 1049 base pairs. The coding region (1695 base pairs) consists of a 24-amino-acid N-terminal signal sequence (molecular mass 2836 Da) preceding the A chain 267 amino acids, molecular mass 29 399 Da), which is joined to the B chain (262 amino acids, molecular mass 28 517) by a 12-amino-acid linking region (molecular mass 1385 Da).

Amino Acids↗
PubMed1984

Molecular cloning of the yeast fatty acid synthetase genes, FAS1 and FAS2: illustrating the structure of the FAS1 cluster gene by transcript mapping and transformation studies.

From a Saccharomyces cerevisiae gene bank contained in the novel yeast cosmid shuttle vector pMS201 the fatty acid synthetase (FAS) genes FAS1 and FAS2 were isolated. FAS clones were identified by in situ colony hybridization using two yeast DNA probes apparently capable of producing avian FAS cross-reacting material (J. Carbon, personal communication). Classification as FAS1 or FAS2 clones was achieved by their specific transformation of fas1 and fas2 yeast mutants. By transcription mapping FAS1 was assigned to about 5.3 kb within 14.8 kb of chromosomal DNA covered by two genomically adjacent BamHI fragments. The FAS2 gene was localized on a single BamHI fragment of 25 kb. One of the FAS clones ( FAS2 ) produces immunologically cross-reacting material in Escherichia coli. High frequency transformation of fas1 mutants was only observed with one subclone, pMS3021 , containing the intact FAS1 locus. Other DNA segments cloned in the same self-replicating vector but representing only part of FAS1 exhibited drastically lower transformation rates. As evident from this and from FAS1 /TRP1-cotransformation rates only the intact FAS1 gene in pMS3021 is capable of fas1 -mutant complementation. With partial FAS1 genes, even when coding for an intact equivalent of the mutated domain, their chromosomal integration is necessary for the expression of FAS. In integrative transformants the coexistence of integrated and autonomously replicating plasmid DNA was demonstrated. Both, the extrachromosomal and chromosomally integrated FAS DNA was mitotically unstable. Transformation studies using subcloned FAS1 DNA segments revealed the relative locations of the enoyl reductase and dehydratase domains within this pentafunctional cluster gene.

Cloning, Molecular↗
PubMed1981

The synthesis of Ricinus communis agglutinin, cotranslational and posttranslational modification of agglutinin polypeptides.

Polyadenylated RNA isolated from the endosperm tissue of maturing castor bean seeds was translated in a cell-free rabbit reticulocyte lysate system. Rabbit antibodies raised against Ricinus communis agglutinin were used to identify nascent agglutinin chains. In contrast to the authentic agglutinin polypeptides with molecular weights of 31000 (A chains) and 37000 (glycosylated B chains), immunoreactive translational products of Mr 33500 and 59000 were observed. The inclusion of canine pancreatic microsomes in the translational system resulted in the cotranslational segregation of these immunoreactive products into the lumen of the vesicles and their modification, to molecular weights of 32000 and 66000--69000 respectively. These cotranslational size modifications resulted from the cleavage of leader sequences and, in the case of the larger product, concomittant core glycosylation, 32000-Mr and 66000--69000-Mr proteins were also observed amongst the immunoreactive products initially formed during the labelling of intact endosperm tissue in vivo, together with 37000-Mr and 39000-Mr proteins. Pulse-chase experiments showed that 66000--69000-Mr proteins slowly disappeared while the smaller proteins were further cleaved to chains of Mr 31000 (authentic A chain), 34000 and 37000 (authentic glycosylated B chains). It was concluded that R. communis agglutinin polypeptides were synthesized in precursor form, possibly as a 'giant' precursor in the case of the B chain, on membrane-bound polysomes. Cotranslational translocation across the endoplasmic reticulum membrane was accompanied by proteolysis to remove leader sequences and, where appropriate, core glycosylation. Even after cotranslational processing agglutinin polypeptides were still in precursor form. Processing to authentic size appeared to occur posttranslationally.

Animals↗
PubMed1981

Synthesis and posttranslational segregation of glyoxysomal isocitrate lyase from castor bean endosperm.

Total polyadenylated RNA isolated from 3-day-old germinating castor bean endosperm was translated in the rabbit reticulocyte lyase cell-free protein-synthesizing system in either the presence or absence of canine pancreatic microsomal vesicles. Isocitrate lyase was immunoprecipitated from the translational products using rabbit antibodies raised against the purified glyoxysomal enzyme. Isocitrate lyase synthesized in vitro had the same apparent molecular weight as the authentic glyoxysomal enzyme irrespective of whether synthesis occurred in the presence or absence of microsomes. Furthermore, in contrast to several proteins encoded by mRNA isolated from maturing seed endosperm tissue, isocitrate lyase was not cotranslationally segregated into the lumen of the microsomal vesicles. Intact 3-day-old endosperm tissue was labelled in vivo by adding [35S]methionine and at various times the tissue was homogenized and the cellular organelles fractionated. Separated fractions were assayed for newly synthesized isocitrate lyase. Immunoreactive product was initially detected in the cytosolic fraction and subsequently began to accumulate in glyoxysomes. The results indicate that glyoxysomal isocitrate lyase is synthesized on free polysomes and released into the cytosol. Transfer to the matrix of the glyoxysome involves a posttranslational membrane translocation step which is not accompanied by proteolytic cleavage of the polypeptide chain.

Animals↗
PubMed1979

Developmental changes in the activity of messenger RNA isolated from germinating castor bean endosperm.

The capacity of polyadenylated RNA from developing castor bean endosperm to program protein synthesis in a wheat germ cell-free translational system has been examined. Although the use of micrococcal nuclease-treated wheat germ extracts demonstrated a low but significant content of translatable mRNA in dry seeds, a large scale increase in total translational capacity was observed during germination. The cellular content of translatable mRNA peaked on the 4th day of germination and subsequently declined. It is concluded that protein synthesis in castor bean endosperm cells during germination is directed by newly transcribed mRNA.

Journal Article↗
PubMed1977

Modification by soluble antigen of the immune response to mycobacterial infection.

After inoculation with viable BCG organisms, spleen cells from mice developed cellular immune reactivity to purified protein derivative antigen by day 10, which declined by day 25; after rechallenge on day 25, a vigorous secondary response rapidly developed (by day 29). The inclusion of autochthonous serum (from the BCG-infected mice) in the in vitro cellular immune reactivity tests altered this pattern considerably: the primary response was shortened, and the secondary response totally abated, suggesting the presence of blocking factors active against BCG-specific cellular immune reactivity in the sera of infected animals. Subsequent experiments indicated that BCG-specific serum blocking factor activity could be generated in the serum of control mice by the intravenous inoculation of purified protein derivative.

Animals↗
PubMed1976

Serum modulation of in vitro cellular immune reactivity to Epstein-Barr virus-associated antigen.

The reactivity of peripheral blood leukocytes to Epstein-Barr virus-associated antigen was assessed employing two in vitro tests, leukocyte adherence inhibition (LAI) and leukocyte migration inhibition (LMI). In both tests, autologous serum from some normal subjects was shown to confer strong reactivity on weak or non-responder cells. Reactivity to purified protein derivative of tuberculin (PPD) assessed in parallel was shown to be unaffected by autologous serum. Employing the LAI system, variations in the effects of autologous serum samples collected on consecutive days was shown to be dependent on the reactivity of the leukocytes themselves, rather than changes in the activity or concentration of factors present in the serum.

Antigens, Viral↗
PubMed1976

Dissociation of correlates of cellular immunity in man: functional heterogeneity within the antigen-reactive cell population?

Cellular immune responsiveness (CMI) to ubiquitous bacterial antigens was assessed in serial peripheral blood samples collected from normal subjects, employing three in vitro correlates of CMI in parallel. Delayed cutaneous hypersensitivity (DCH) was also assessed on a number of occasions in these subjects. These experiments showed that the reactivity of peripheral blood leukocytes from normal individuals as measured in each of the in vitro tests fluctuated from day to day, and that the reactivity of cells functioning in each of the tests fluctuated independently of one another. In contrast, DCH reactivity in these individuals was not subject to such fluctuations. The results are discussed in terms of the periodic appearance of functionally distinct subpopulations of T lymphocytes in the peripheral circulation.

Antigens, Bacterial↗
PubMed1975

Early onset of serum blocking in a murine melanoma model.

The early onset of serum blocking of host cell-mediated immunity was investigated in a transplantable murine melanoma employing two in vitro assays. One of these was based on the Takasugi and Klein cytotoxicity test; the other was a new test based on the inhibition of adherence of leukocytes to a plastic surface in the presence of tumor antigen. Both tests yielded similar results, and the adherence inhibition test was more sensitive. Blocking could be detected as early as 1 h after the inoculation of 1 times 10(7) melanoma cells. Early onset of serum blocking was also demonstrable if homogenized of tissue-cultured tumor cells were inoculated. Serum blocking under these circumstances was initially transient, declining at 24 to 48 h, but reappearing by the 4th day after inoculation. Furthermore, a functional immune system was not required for blocking factor development in this system, as evidenced by the onset of a similar pattern of blocking activity in heavily irradiated animals.

Animals↗
PubMed1975

The L.A.I. microtest: a rapid and sensitive precedure for the demonstration of cell-mediated immunity in vitro.

A new microtest for cell-mediated immunity is described, which is based on the elaboration of a diffusible factor from sensitized lymphocytes following contact with specific antigen. The factor inhibits the adherence of neighbouring leucocytes to plastic surfaces. Production of the factor is ablated by pretreatment of lymphocytes with Theta-antiserum. It is suggested that the factor may be a new lymphokine, leucocyte adherence inhibition factor (LAIF).

Animals↗