Terence E. Martin

Dissociation and reassociation of skeletal muscle ribosomes

Abstract The dissociation of skeletal muscle ribosomes by chelation of magnesium ions with EDTA led to the formation of 30 and 53 s particles. Active ribosome monomers could not be formed from these subunits. Exposure of rat muscle ribosomes to potassium ion concentrations greater than 0.5 m caused the formation of ribosomal particles of 90 and 105 s when the procedure was carried out at 4 °C. However, at 28 °C the ribosomes dissociated to 40 and 60 s subunits and a small amount (20 to 25%) of a 75 s component. The 75 s fraction contained 18 and 28 s RNA and also retained the nascent polypeptide. Ribosomes pre-incubated with puromycin did not form a 75 s component; moreover, the yield of the 40 and 60 s subunits was increased. It is postulated that the nascent protein and perhaps messenger RNA protect the ribosome from dissociation by high concentrations of potassium. When ribosomes were dissociated with potassium chloride in the presence of an antioxidant, the subunits so formed could be recombined by removal of the excess potassium chloride, and the reassociated 80 s ribosomes catalyzed protein synthesis, but only in the presence of added template RNA. The isolated 60 s subunit formed dimers (90 s) at 4 °C. The 75 s component reverted to an 80 s particle when the potassium chloride concentration was adjusted and was active in protein synthesis in the absence of added template.

Active hybrid 80 s particles formed from subunits of rat, rabbit and protozoan (Tetrahymena pyriformis) ribosomes

Abstract Ribosomes of rat skeletal muscle and rat liver were dissociated in 880 m m -potassium chloride at 28 °C in the presence of magnesium ions (12.5 m m ); at 4 °C the subunits formed aggregates. In contrast, the subunita formed from rabbit muscle and Tetrahymena pyriformis ribosomes in the same ionic circumstances did not aggregate at 4 °C. The ability of rat muscle ribosomal subunits to combine with subunits of other 80 s ribosomes was determined by density-gradient analysis and by estimation of polyuridylic acid-directed polypeptide synthesis. Both hybrids formed and were active when rat muscle and rat liver subunits were combined. Subunits from rat and rabbit skeletal muscle ribosomes also formed active hybrids, despite a difference in the 60 s particles of the two species; the rat 60 s subunit has a far greater tendency to form 90 s dimers at low temperatures. However, only one hybrid was detected by density-gradient analysis when rat muscle and Tetrahymena subunits were mixed. The small subunits of the protozoan and the large subunits of rat formed 80 s particles, which synthesized polyphenylalanine at the same rate as re-associated Tetrahymena ribosomes. The 40 s of rat and the 60 s of the protozoan did not appear to form 80 s particles, but the mixture had some protein synthetic activity in the presence of polyuridylic acid. The ribosomal proteins contained in the subunits of rat muscle and Tetrahymena ribosomes were markedly different.

Cis-acting sequences that affect somatic hypermutation of Ig genes

Summary: We review our studies on the mechanism of somatic hypermutation of immunoglobulin genes. Most experiments were carried out using Ig transgenes. We showed in these experiments that all required cisacting elements are present within the 10–16 kb of a cransgene. Only the Ig variable region and its proximate flanks are mutated, not the constant region. Several Ig gene enhancers are permissive for somatic mutation. Association of the enhancer with its natural Ig promoter is not necessary. However, the mutation process seems specific for Ig genes. No mutations were found in housekeeping genes from cells with high levels of somatic hypermutation of their Ig genes. The Ig enhancers may provide the Ig gene specificity. An exception may he the BCL6 gene, which was mutated in but not hut not in mouse B cells

Somatic Hypermutation of Immunoglobulin Genes is Linked to Transcription

Immunoglobulin (Ig) genes are rearranged in pre-B cells. Pre-B cells that express Ig heavy (H) and light (L) chain genes whose V(D)J recombination results in a functional reading frame mature into B cells that exit the bone marrow. The V(D)J recombination process creates a large repertoire of different variable regions from a restricted pool of germline genes. Additional variablity arises during the process of somatic hypermutation in mature B cells proliferating in germinal centers of lymphoid organs (reviewed in French et al. 1989). B cells that have mutated to express high-affinity antibodies are selected and develop into plasma cells or memory cells. B cells with mutations that decrease the affinity of the expressed Igs or that prevent Ig expression die by apoptosis. The somatic point mutations are located within the variable region and their proximate upstream and downstream flanks, but not generally within the constant region.

Effects of Sequence and Structure on the Hypermutability of Immunoglobulin Genes

Somatic hypermutation (SHM) is investigated in related immunoglobulin transgenes that differ in a short artificial sequence designed to vary the content of hotspot motifs and the potential to form RNA or DNA secondary structures. Mutability depends on hotspots, not secondary structure. Hotspot motifs predict about 50% of the mutations; the rest are in neutral and coldspots. Clusters of mutations and the sequential addition of mutations found in cell pedigrees suggest epigenetic attributes of SHM. Sometime in SHM, an essential factor seems to become limiting. Particular error-prone DNA polymerases appear to create mutations in hotspots on the top and bottom DNA strands throughout the target and the SHM process. One transgene is superhypermutable in all regions, suggesting the presence of a cis-element that enhances SHM.

Ultrastructural analysis of testes from mice fed on genetically modified soybean.

We have considered the possible effects of a diet containing genetically modified (GM) soybean on mouse testis. This organ, in fact, is a well known bioindicator and it has already been utilized, for instance, to monitor pollution by heavy metals. In this preliminary study, we have focussed our attention on Sertoli cells, spermatogonia and spermatocytes by means of immunoelectron microscopy. Our results point out that the immunolabelling for Sm antigen, hnRNPs, SC35 and RNA Polymerase II is decreased in 2 and 5 month-old GM-fed mice, and is restored to normal at 8 months. In GM-fed mice of all ages considered, the number of perichromatin granules is higher and the nuclear pore density lower. Moreover, we found enlargements in the smooth endoplasmic reticulum in GM-fed mice Sertoli cells. A possible role played by traces of the herbicide to which the soybean is resistant is discussed.

Immunoelectron microscope localization of snRNPs in the polytene nucleus of salivary glands ofChironomus thummi

The ultrastructural distribution of small nuclear ribonucleoproteins (U1-snRNP and Sm antigen) in the nucleus ofChironomus salivary gland cells was investigated by means of specific antibodies and immunocytochemistry using colloidal gold complexes as markers. Particular attention was paid to the structural relationships of snRNPs with transcriptionally active areas in polytene chromosomes and with Balbiani ring granules. Our results demonstrate strong binding of anti-snRNP antibodies to RNP fibrils or the fibrillar network occurring within nuclear regions active in transcription (Balbiani rings or minor puffs). This confirms data reported previously on mammalian cells showing early association of snRNPs with nuclear fibrillar constituents containing newly synthesized pre-mRNA. In addition, Balbiani ring granules in the process of formation at the transcriptionally active sites were sometimes labeled on their periphery or on the transitory part between the granule and its precursor RNP fibril, while free granules observed in the nucleoplasm were virtually devoid of label. These findings suggest that mRNA processing, including splicing, takes place prior to the formation of mature Balbiani ring granules.

A simple general method to determine the proportion of active ribosomes in eukaryotic cells

Abstract The resistance of 80S ribosomes derived from active polyribosomes to dissociation in high KCl cone, can conveniently be used to assay the proportion of active ribosomes in eukaryotic cells. The method described has been useful in studies of organisms as diverse as fungi (yeast), protozoa ( Tetrahymena ), higher plants (barley), echinoderms (sea urchin), insects ( Musca ), birds (chick) and mammals (mouse, rat). The technique is relatively insensitive to the use of harsh mechanical treatments for cell disruption or the presence of endogenous ribonuclease activity.

Fine structural cytochemical and immunocytochemical analysis of nucleic acids and ribonucleoprotein distribution in nuclei of pig oocytes and early preimplantation embryos

The fine structure of pig oocytes at the germinal vesicle (GV) stage and early preimplantation embryos (one to four blastomeres) isolated at slaughter was investigated by cytochemical and immunocytochemical methods. The distribution of nucleic acids and ribonucleoproteins (RNPs) in “compact nucleoli” [denominated nucleolus-like bodies (NLB) in oocytes and nucleolus precursor bodies (NPB) in early embryos] and in intranuclear bodies or granules was investigated by staining methods preferential for nuclear RNPs or using the osmium ammine or ethidium bromide-phosphotungstic acid (EB-PTA) reactions for nucleic acids. The distributions of the Sm antigen of nucleoplasmic small nuclear RNPs (snRNPs), the methyl-3 guanosine (m3G) cap of snRNAs and the splicing factor SC-35 were detected by immunoelectron microscopy using specific antibodies. The RNP nature of both NLBs and NPBs, and of nuclear granules in oocytes and embryos, and of fibrillar strands radially projecting from NLBs was revealed. Cytochemical evidence for RNA as a component of NLBs was further provided by EB-PTA staining in combination with the enzymatic removal of RNA, or by osmium-ammine staining without previous acid hydrolysis, while the absence of DNA in NLBs was established by Feulgen-like osmium-ammine staining. In addition, autoradiography demonstrated the absence of [6-3H]thymidine incorporation into NPBs. Other autoradiographic evidence attested the accumulation of RNA in NLBs of oocytes after a 60 min in vitro pulse of [5-3H]uridine. Immunoelectron microscopy using specific antibodies revealed the occurrence of nucleoplasmic snRNPs in both NLBs and NPBs. The presence of snRNA in NLB was confirmed by means of an antibody recognizing the m3G-cap structure. Another spliceosomal component, the protein SC-35 was also detected in NLBs. Among the numerous and variable intranuclear granules occurring mostly in aggregates, the Sm antigen was clearly detected only in the interchromatin granule-type component. Some Sm labeling was occasionally seen in other categories of larger granules. No reaction was detected over any granules when using the anti-m3G-cap antibody. The aggregates consisting of large granules and a finely fibrillar component were intensely immunolabeled by the anti-SC-35 splicing factor probe. Our observations suggest that the compact nucleoli, known to be present before and after fertilization in mammals (NLBs of oocytes and NPBs of early embryos), represent nuclear structural elements containing nonnucleolar, spliceosomal components.

DADLE induces a reversible hibernation-like state in HeLa cells.

Abstract[d-Ala(2)-d-Leu(5)-Enkephalin] (DADLE) can induce hibernation when injected into ground squirrels in summer and is able to increase the survival time of explanted organs such as liver and lung. Since cell metabolism is a target of this peptide, we have treated HeLa cells with DADLE and investigated its possible effect on transcription and proliferation as well as the resumption of metabolic activity after treatment. The labelling for Pol I, Pol II and for splicing factors such as snRNPs and SC-35 decreased after treatment as did the nucleolar labelling for UBF. In treated cells, several spherical nuclear bodies were found to be labelled for hnRNPs. In parallel, the number of proliferating cells decreased after treatment with DADLE. After recovery, there was a gradual resumption of cell function: transcription and splicing factors had a distribution similar to that of controls; proliferation resumed; nuclear bodies, representing storage sites for RNPs, disappeared.