Robert P. Perry

On the lability of poly(A) sequences during extraction of messenger RNA from polyribosomes

Extraction of mRNA from polyribosomes with sodium dodecyl sulfate and phenol at near neutral pH results in a considerable loss of the poly(A)-containing mRNA from the aqueous phase, and also in cleavage of poly(A) from the mRNA. The loss and cleavage are associated with some specific proteins in the polyribosomes, and can be avoided if these proteins are destroyed before extraction or if extractions are done with chloroform plus phenol rather than with phenol alone.

Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency

A process unique to lymphocyte differentiation is the rearrangement of genes encoding antigen-specific receptors on B and T cells. A mouse mutant (C.B-17scid) with severe combined immune deficiency, i.e., that lacks functional B and T cells, shows no evidence of such gene rearrangements. However, rearrangements were detected in Abelson murine leukemia virus-transformed bone marrow cells and in spontaneous thymic lymphomas from C.B-17scid mice. Most of these rearrangements were abnormal: approximately 80% of Igh rearrangements deleted the entire Jh region, and approximately 60% of TCR beta rearrangements deleted the entire J beta 2 region. The deletions appeared to result from faulty D-to-J recombination. No such abnormal rearrangements were detected in transformed tissues from control mice. The scid mutation may adversely affect the recombinase system catalyzing the assembly of antigen receptor genes in developing B and T lymphocytes.

Messenger RNA-protein complexes and newly synthesized ribosomal subunits: Analysis of free particles and components of polyribosomes*

As a prerequisite for a study of the transport and functional forms of messenger RNA and ribosomes in cultured L cells, a method has been developed for preparing cytoplasmic particles rid of the nuclear contaminants routinely found with conventional procedures. This method, which uses a low concentration of nonionic detergent and avoids mechanical disruption, was used to obtain polyribosomes that were demonstrably free of co-sedimenting ribonucleoprotein. Polyribosomes from cells pulse-labeled with [ 3 H]uridine were analyzed by sucrose gradient sedimentation and equilibrium banding in CsCl before and after dissociation with EDTA. Newly synthesized messenger RNAs were released from the polyribosomes as a polydisperse array of ribonucleoprotein complexes (mRNP) with a relatively uniform RNA to protein ratio. The protein, termed m-protein, comprises about 60% of the weight of the complex. The mRNP released from polyribosomes was demonstrated to be similar in several respects to other RNP complexes in the cytoplasmic extracts which exist free of attached ribosomes. A more detailed examination of these latter structures indicated that roughly three-quarters of their protein could be reversibly removed by exposure to 0·55 m -LiCl-0·01 m -Mg 2+ , a condition which removes very little protein from the ribosomal subunits. Previously we have demonstrated that new ribosomal subunits entering the cytoplasm contain more protein than the subunits in the steady-state pool. The present experiments indicate that this extra protein, termed accessory protein, is released from the subunits when they are incorporated into polyribosomes.

The synthesis and processing of the messenger RNAs specifying heavy and light chain immunoglobulins in MPC-11 cells

The nuclear precursors of the immunoglobulin messenger RNAs of MPC-11 cells were characterized with respect to size, amount per cell and extent of polyadenylation. These cells produce three Ig mRNAs: a 1.8 kb component coding for a gamma2b heavy chain (H mRNA), a 1.2 kb mRNA coding for a k light chain (L mRNA) and a 0.8 kb mRNA coding for the constant region portion of the k light chain (Lf mRNA). To identify the pre-mRNAs without ambiguity, we constructed recombinant DNA plasmids containing H and L cDNA sequences, and used the cloned cDNAs as hybridization probes for analysis of steady state nuclear RNA and in DNA excess hybridization experiments with pulse-labeled nuclear RNA. The nuclear molecules containing Ig sequences consist of an 11 kb component (H1), which we believe to be the primary transcript of the H gene, 5.3 kb (L1), and 3.3 kb (L2) components, which seem to be primary transcripts of the L and L1 genes, components corresponding to mature size H, L and Lf mRNAs, and several intermediate-sized components which include the processing derivatives. The precursor role of these nuclear molecules was established by studies of their labeling kinetics and by appropriate pulse-chase experiments. All the pre-mRNA species including H1, L1 and L2 contain poly(A), thus suggesting that polyadenylation is an early event in the processing of these mRNAs. The MPC-11 cell contains about 30,000 and 40,000 cytoplasmic H and L mRNA molecules, respectively, which must be produced within one cell generation (approximately 24 hr). In comparison, the nucleus contains about 100-150 molecules of total pre-mRNA and only about 10-15 molecules of presumptive primary transcripts for each of these Ig species. These values indicate very rapid transcription rates (greater than 20 transcripts per min) and exceptionally fast processing rates (approximately 0.5 min for the primary transcripts and approximately 5 min for overall nuclear processing) for the Ig mRNAs. Thus rapid transcription and processing, together with high cytoplasmic stability, account for the high abundance of Ig mRNAs in the myeloma cell.

Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes

Individual B lymphocytes normally express immunoglobulin (Ig) proteins derived from single Ig heavy chain (H) and light chain (L) alleles. Allelic exclusion ensures monoallelic expression of Ig genes by each B cell to maintain single receptor specificity. Here we provide evidence that at later stages of B cell development, additional mechanisms may contribute to prioritizing expression of single IgH and IgL alleles. Fluorescent in situ hybridization analysis of primary splenic B cells isolated from normal and genetically manipulated mice showed that endogenous IgH, κ and λ alleles localized to different subnuclear environments after activation and had differential expression patterns. However, this differential recruitment and expression of Ig alleles was not typically seen among transformed B cell lines. These data raise the possibility that epigenetic factors help maintain the monoallelic expression of Ig.

Existence of methylated messenger RNA in mouse L cells

Abstract Messenger RNA of mouse L cells is methylated in both base and ribose moieties. On the average there are about 2.2 methyl groups per 1000 nucleotides in mRNA, a proportion which is about one-sixth that of mammalian ribosomal RNA. The variety of methylated bases in mRNA is more limited than in ribosomal RNA. A very low level of methylation is detected in heterogeneous nuclear RNA, suggesting that methylation, like polyadenylation, may constitute a post-transcriptional modification of messenger RNA precursor in eucaryotic cells.

Buoyant densities of cytoplasmic ribonucleoprotein particles of mammalian cells: distinctive character of ribosome subunits and the rapidly labeled components.

The buoyant densities of the cytoplasmic ribonucleoprotein particles of strain L cells were determined by banding in CsCl gradients. Stability of the particles at the high salt concentrations was achieved by means of fixation with formaldehyde. Under these conditions, the bulk of the material in monoribosome, 60 s and 40 s particles banded at characteristic densities, ρ = 1·55, 1·57 and 1·49 g/cm 3 , respectively. Preparations of 60 s and 40 s particles also yielded minor components of lower density which apparently possess a lower proportion of RNA to protein. The rapidly labeled 18 s RNA associated with the 40 s particles was found to exist in structures which band at ρ = 1·43, a density considerably lower than that characteristic of the bulk of the 40 s material. The results of pulse-chase and double-labeling experiments are consistent with the idea that the ρ = 1·43 components are lower-density precursors of the small ribosome subunits. No significant amount of labeled 40 s material banded at a density higher than the subunit itself. Such a higher density would be expected of complexes of messenger RNA and intact subunits, and therefore their presence is not indicated.

Relative occurrence of polyadenylic acid sequences in messenger and heterogeneous nuclear RNA of L cells as determined by poly (U)-hydroxylapatite chromatography.

Abstract A method, consisting of hybridization with polyuridylic acid followed by chromatography on hydroxylapatite, was developed for separating the fractions of messenger RNA and heterogeneous nuclear RNA molecules which contain polyadenylic acid. Utilizing this method it was found that essentially all of the messenger RNAs of mouse L cells, with the exception of those coding for histones, contain poly(A). A similar analysis of the heterogeneous nuclear RNA indicated that only one-fifth of these molecules contain poly(A).

Cμ-containing transcripts initiate heterogeneously within the IgH enhancer region and contain a novel 5′-nontranslatable exon

Transcriptional competence of the immunoglobulin heavy-chain locus (IgH) is established at an early stage of lymphoid cell development, leading to the appearance of RNA components, previously called Cμ RNA1 or sterile-μ RNA2, which contain constant-region sequences but lack variable-region sequences. These components are of two types: those which initiate in the D region of alleles that have undergone DJH (diversity–joining region) rearrangement (Dμ transcripts) and those which initiate within the JH–Cμ intron (hereafter termed Iμ transcripts)3,4. In pre-B and early B cells, Dμ and lμ transcripts are nearly as abundant as the messenger RNA encoding μ heavy chain2,3,5. The Dμ transcripts are spliced into RNAs containing D, JH and Cμ sequences, and in some, but not all, cases these RNAs are translated into Dμ proteins4. To establish whether the Iμ transcripts have any translational potential and to elucidate the structure of their promoter region, we have determined their transcription initiation sites and their mode of splicing. As reported here, by using sequence analysis of cloned Iμ complementary DNAs, primer extension and S1, nuclease mapping, we have found that these transcripts have remarkable 5′ heterogeneity: ther e are more than five distinct start sites spanning a region of 44 nucleotides that is located downstream of an octanucleotide found in all variable-region promoters. Such imprecise initiation may result from the lack of a well-defined T A T AA motif and the unusual proximity of the octanucleotide to the enhancer region. Approximately 700 nucleotides downstream from these initiation sites, a cryptic splice site is used to create a nontranslatable exon (‘nontron’) which is joined to the Cμ1 domain. The properties of the nontron may be important for the mechanism of allelic exclusion.

The Nucleolus and the Synthesis of Ribosomes

Publisher Summary The chapter discusses the nucleolus and the synthesis of ribosomes. It also briefly discusses some of the various lines of evidence that have implicated the nucleolus, with ribosome synthesis, trying, where possible, to indicate the limitations of current concepts, as well as the mechanisms that appear to be firmly established. The synthesis of the precursor of ribosomal RNA (rRNA) is also discussed in this chapter. Some attention is given to other metabolic activities that might be associated, with the nucleolus, but in these areas no attempt at completeness is made. In addition, some aspects of ribosome formation are discussed that, at the moment, do not seem to involve the nucleolus, but that will surely be necessary for a full understanding of the functional role of the ribosome in the cell. The chapter discusses various characterizations of the genes, coding for rRNA. The chapter concludes with the appearance of ribosomes in the cytoplasm.