A simple procedure for the isolation and purification of protamine messenger ribonucleic acid from trout testis.

Preparation of milligram quantities of purified poly(A)+ (polyadenylated) protamine mRNA from trout testis tissue was accomplished by a simple procedure using gentle conditions. This involves chromatography of the total nucleic acids isolated by dissociation of polyribosomes with 25 mM-EDTA to release messenger ribonucleoprotein particles and deproteinization of the total postmitochondrial supernatant with 0.5% sodium dodecyl sulphate in 0.25 M-NaCl by binding it to a DEAE-cellulose column. Total RNA was bound under these conditions, and low-molecular-weight RNA, lacking 18S and 28S RNA, could be eluted with 0.5 M-NaCl and chromatographed on oligo(dT)-cellulose columns to select for poly(A)+ RNA. Further purification of both the unbound poly(A)- RNA and the bound poly(A)+ mRNA on sucrose density gradients showed that both 18S and 28S rRNA were absent, being removed during the DEAE-cellulose chromatography step. Poly(A)- RNA sedimented in the 4S region whereas the bound poly(A)+ RNA fraction showed a main peak at 6S [poly(A+) protamine mRNA] and a shoulder in the 3-4S region. Analysis of the main peak and the shoulder on a second gradient showed that most of the main peak sedimented at 6S, whereas the shoulder sedimented slower than 4S. The identity of the poly(A)+ protamine mRNA was established by the following criteria: (1) purified protamine mRNA migrated as a set of four bands on urea/polyacrylamide-gel electrophoresis; (2) analysis of the polypeptides synthesized in the wheat-germ extract by starch-gel electrophoresis showed a single band of radioactivity which co-migrated exactly with the carrier trout testis protamine standard; and (3) chromatography of the polypeptide products on CM-cellulose (CM-52) showed the presence of three or four radioactively labelled protamine components that were co-eluted with the unlabelled trout testis protamine components added as carrier. The availability of large quantities of purified protamine mRNA should now permit a more thorough analysis of its physical and chemical properties.     

Isolation and characterization of trout testis protamine mRNAs lacking poly (A)

Poly(A)+ protamine mRNA was isolated from trout testis cells in a very pure form, and artificial poly(A)- protamine mRNA molecules were derived from it by enzymatic deadenylation with RNAase H from calf thymus after hybridization with oligo(dT). The deadenylated protamine mRNA was found to be active in a wheat germ cell-free system and yielded a labeled product which co-migrated with authentic protamine. These deadenylated mRNA molecules were subsequently used as markers on denaturing polyacrylamide gels to identify and allow the purification of the poly(A)- protamine components known to exist in vivo in the total cellular poly(A)- RNA. RNA species of molecular weights similar to the enzymatically deadenylated subcomponents of protamine mRNA were observed in the natural poly(A)-RNA population of the testis cells. These naturally occurring poly(A)- protamine mRNAs were isolated by preparative gel electrophoresis and further characterized by 3H-poly(U) hybridization assay, by hybridization to complementary DNA made against highly purified poly(A)+ protamine mRNA, and by their ability to direct protamine synthesis in a cell-free system.     

Studies of the injection of poly(A) protamine mRNA into Xenopus laevis oocytes

When poly(A)⁺ protamine mRNA from trout testes polysomes was injected into living Xenopus oocytes and the latter labelled with [¹⁴C] or [³H]arginine during subsequent incubation, a highly basic, labelled protein fraction was synthesized and could be extracted with 0.5 M H₂SO₄. In the acid extract, a major polypeptide, indistinguishable from trout protamine by several criteria: polyacrylamide and starch gel electrophoreses, carboxymethylcellulose column chromatography, lack of incorporation of [³H]histidine, and autoradiography of tryptic peptides after two-dimensional paper electrophoresis, could be demonstrated. Since no such protein is found in control oocytes injected with saline, it is concluded that poly(A)⁺ protamine mRNA programs the synthesis of trout protamine within Xenopus oocytes. This confirms our previous reports [1–3] that trout testis poly(A)⁺ protamine mRNA can direct the in vitro synthesis of protamine in Krebs II ascites, rabbit reticulocytes and wheat germ cell-free systems. The protamine synthesized upon injection of poly(A)⁺ protamine mRNA into Xenopus oocytes appears to be partially phosphorylated. Injection of increasing amounts of poly(A)⁺ protamine mRNA led to a linear increase in protamine synthesis. The sensitivity of detection was such that less than 1 ng of poly(A)⁺ protamine mRNA gave a significant response. The translational stability of protamine mRNA appeared to be less than that of globin mRNA.     

Assay of protamine messenger RNA from rainbow trout testis.

A low molecular weight RNA fraction possessing protamine mRNA activity was prepared from rainbow trout testis polysomes. Addition of low molecular weight RNA to a Krebs II ascites S-30 cell-free protein synthesis system strongly stimulated [14C]arginine incorporation into acid-insoluble material. This stimulation was completely abolished by 10-4 M aurintricarboxylic acid, an inhibitor of eukaryotic protein synthesis at the level of initiation. Starch gel electrophoresis showed that labeled arginine was incorporated in vitro into products identical with both authentic protamine and histones as found previously (Gilmour, R. S., and Dixon, G. H. (1972) J. Biol. Chem. 247, 4621-4627). The 4 to 6 S RNA fraction, isolated from the polysomal low molecular weight RNA by sucrose gradient fractionation, enhanced the incorporation of [14C]arginine into acid-insoluble material and when this product was examined by starch gel electrophoresis, it co-migrated with authentic rainbow trout protamine.     

Red cell ghost-mediated microinjection of RNA into HeLa cells: I. A comparison of two techniques for the entrapment and microinjection of tRNA and mRNA

We have compared two techniques for introducing RNA into red blood cell ghosts. In the pre-swell technique, RNA is introduced into red cells without prior removal of endogenous contents. In the multiple lysis technique, the red cells are subjected to two or three cycles of reversible lysis, prior to introducing the RNA, in order to first remove the normal red cell constituents. The pre-swell technique offers much greater entrapment of both tRNA and protamine messenger RNA (mRNA), but the RNA appears to be degraded during the procedure. This may be due either to nuoleolytic degradation or oxidation by the high concentration of endogenous hemoglobin. The multiple lysis technique offers much lower entrapment but also results in diminished degradation of the entrapped RNA. Although some degradation is apparent, a significant portion of the biological activity of the entrapped protamine mRNA is retained. We have also fused red cells loaded with protamine mRNA by the multiple lysis technique to HeLa cells using polyethylene glycol 6000. The recipient HeLa cells are capable of translating this heterologous message into protamine, a trout testis chromosomal protein.     

Alteration in cellular RNAs during the in vitro lifespan of cultured human diploid fibroblasts.

An abrupt concommitant increase in total cellular RNA and protein was observed as cultured human diploid fibroblasts entered the senescent phase of their in vitro lifespan. DNA content remained stable from early to final passages. Fractionation of cellular RNAs by polyacrylamide gel electrophoresis demonstrated an increase in both 28S and 18S ribosomal and 4S transfer RNAs in these senescent cells. Separation of poly(A) RNA (mRNA) by oligo(dT)-cellulose chromatography suggests an increase in this group of RNAs. However, the ratios of 28S to 18S rRNAs, tRNA to rRNA, and mRNA to total cellular RNA were not significantly different in cells before and after senescence, indicating that the overall increases in total cellular RNA was not due to an accumulation of a single RNA class.     

Sequences of large T1 ribonuclease-resistant oligoribonucleotides from protamine mRNA: the overall architecture of protamine mRNA.

Limited T1 ribonuclease digestion of the family of protamine mRNA's purified from rainbow trout testis yields several large oligoribonucleotide fragments ranging in size from 12--54 nucleotides in length. Several of these fragments purified by two dimensional gel electrophoresis contain several G residues and must represent nuclease-resistant, base-paired regions of the mRNA. Sequence analysis of these oligonucleotides by the method of Simoncsits, A., Brownlee, G.G., Brown, R.S., Rubin, J.R. and Guilley, H. (1977) Nature 269: 833-836, shows that these oligoribonucleotides arise from the 5'- and 3'-non-coding regions of the mRNA. Comparisons of the sequences of the large RNA fragment with DNA sequences obtained after cloning double-stranded protamine cDNA in the plasmids pBr322 and pmB9 show precise correspondence of a 54 nucleotide RNA fragment with positions 49--100 from the 3'-poly(A) tract and extending to within 5 nucleotides of the termination codon. Two other RNA fragments of 21 and 25 nucleotides in length arise from the 5'-non-coding region of the message and possess an AUG-sequence at their 3'-termini which is the initiation codon. The presence of distinct by homologous sequences in several sets of large RNA fragments is consistent with the presence of several closely related protamine mRNA's.     

Histone messenger RNAs of the mouse testis

A 6-12S RNA fraction has been isolated following sucrose gradient fractionation of mouse testis RNA, and further resolved into poly A+ and poly A- RNA fractions by oligo-(dt)-cellulose chromatography. Polyacrylamide gel electrophoresis of products formed in a reticulocyte lysate-dependent cell-free translation system has enabled identification of histone variants, H1t, H2S, H2A . X, an H4-like protein and a low Mr protein (presumably TP and/or protamine). Cell-free synthesis of a number of these histone variants appears to be directed by poly A+ mRNAs.     

Physical aspects and cytoplasmic distribution of messenger RNA in mouse kidney.

As a prerequisite to examining mRNA metabolism in compensatory renal hypertrophy, polyadenylated RNA has been purified from normal mouse kidney polysomal RNA by selection on oligo(dT)-cellulose. Poly(A)-containing RNA dissociated from polysomes by treatment with 10 mM EDTA and sedimented heterogeneously in dodecyl sulfate-containing sucrose density gradients with a mean sedimentation coefficient of 20 S. Poly(A) derived from this RNA migrated at the rate of 6-7 S RNA in dodecyl sulfate-containing 10% polyacrylamide gels. Coelectrophoresis of poly(A) labeled for 90 min with poly(A) labeled for 24 h indicated the long-term labeled poly(A) migrated faster than pulse-labeled material. Twenty percent of the cytoplasmic poly(A)-containing mRNA was not associated with the polysomes, but sedimented in the 40-80 S region (post-polysomal). Messenger RNA from the post-polysomal region had sedimentation properties similar to those of mRNA prepared from polysomes indicating post-polysomal mRNA was not degraded polysomal mRNA. Preliminary labeling experiments indicated a rapid equilibration of radioactivity between the polysomal and post-polysomal mRNA populations, suggesting the post-polysomal mRNA may consist of mRNA in transit to the polysomes.     

Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNAPhe binding

The effects of P/P- and P/E-site tRNA(Phe) binding on the 16S rRNA structure in the Escherichia coli 70S ribosome were investigated using UV cross-linking. The identity and frequency of 16S rRNA intramolecular cross-links were determined in the presence of deacyl-tRNA(Phe) or N-acetyl-Phe-tRNA(Phe) using poly(U) or an mRNA analogue containing a single Phe codon. For N-acetyl-Phe-tRNA(Phe) with either poly(U) or the mRNA analogue, the frequency of an intramolecular cross-link C967 x C1400 in the 16S rRNA was decreased in proportion to the binding stoichiometry of the tRNA. A proportional effect was true also for deacyl-tRNA(Phe) with poly(U), but the decrease in the C967 x C1400 frequency was less than the tRNA binding stoichiometry with the mRNA analogue. The inhibition of the C967 x C1400 cross-link was similar in buffers with, or without, polyamines. The exclusive participation of C967 with C1400 in the cross-link was confirmed by RNA sequencing. One intermolecular cross-link, 16S rRNA (C1400) to tRNA(Phe)(U33), was made with either poly(U) or the mRNA analogue. These results indicate a limited structural change in the small subunit around C967 and C1400 during tRNA P-site binding sensitive to the type of mRNA that is used. The absence of the C967 x C1400 cross-link in 70S ribosome complexes with tRNA is consistent with the 30S and 70S crystal structures, which contain tRNA or tRNA analogues; the occurrence of the cross-link indicates an alternative arrangement in this region in empty ribosomes.     

Identification of proteins functionally altered by chemical modification of the transfer RNA and polyuridylic acid binding sites of 30 S ribosomal subunits

Previous studies have shown that Rose Bengal-sensitized photo-oxidation of 30 S ribosomal subunits causes inactivation of tRNA binding and partial loss of poly(U) binding activities (Noller et al., 1971). The present studies, reconstitution of 30 S subunits from 16 S RNA, total protein from modified subunits, and purified proteins from untreated subunits, show that proteins S2 and S3 together completely restore these activities to the reconstituted subunits. The modified proteins are capable of in vitro assembly, and give rise to particles with normal sedimentation constants, showing that restoration of activity is not simply due to correction of an assembly defect.Protein S3 restores poly(U) binding and tRNA binding to the same extent, accounting for the lowered mRNA binding activity of the modified particles as well as a corresponding fraction of the tRNA binding activity. Protein S2 restores the remaining fraction of the tRNA binding activity, but has no effect on poly (U) binding. In 50 S-stimulated tRNA binding, proteins S1 and S5 are required in addition to S2 and S3 for full activity.     

Hyperacetylation of histone H4 promotes chromatin decondensation prior to histone replacement by protamines during spermatogenesis in rainbow trout

During the final stages of spermatogenesis in rainbow trout a dramatic increase in the level of histone H4 hyperacetylation is observed which is closely correlated with the replacement of histones by protamines. In order to understand further how H4 hyperacetylation might assist in protamine replacement of the histones, we have investigated the effect of H4 hyperacetylation on chromatin structure in trout testes actively undergoing the replacement process. Long chromatin fragments enriched in hyperacetylated H4 have been isolated and characterized. Evidence is presented that hyperacetylated H4 is clustered in certain regions (domains) of late stage testis chromatin and within these domains the chromatin exhibits an altered, highly relaxed structure which is believed to be the result of the extensive hyperacetylation. These domains, which are nearly devoid of protamine, are postulated to represent an initial structural transition which is necessary for the proper histone removal and protamine replacement process to take place.     

Isolation of cDNA for a Xenopus sperm-specific basic nuclear protein (SP4) and evidence for expression of SP4 mRNA in primary spermatocytes

A cDNA library was prepared in lambda gt 11 from poly(A)+ mRNA isolated from a pure population of Xenopus round spermatids and screened with an antibody against SP3-5 (sperm-specific proteins) of Xenopus sperm. Positive clones were sequenced and an arginine-rich clone, designated pXSP531, was obtained. The 473-nucleotide sequence of pXSP531 contained an open reading frame of 237 nucleotides which was preceded by a 5' untranslated region of 67 nucleotides. The 3' untranslated region contained 149 nucleotides, including a consensus polyadenylation signal (AAATAAAA). Twenty nucleotides of a poly(A) tail was contained in the pXSP531. SP3-5 were separated from each other by reverse-phase chromatography and sequenced. The amino acid sequence of the peptide fragments which were obtained by digestion of SP4 with V8 protease and separated by reverse-phase chromatography was identical to the sequence of the N-terminal 43 and C-terminal 15 amino acids deduced from the nucleotide sequence of pXSP531. This result demonstrates that pXSP531 encodes SP4. Northern hybridization of RNA extracted from primary spermatocytes and round spermatids on Days 0 and 6 with SP4 cDNA probe (pXSP531) showed that SP4 mRNA is present both in primary spermatocytes and in round spermatids as is protamine mRNA in the rainbow trout. The size of the SP4 mRNA in round spermatids on Day 0 was longer by 60 nucleotides compared to that in primary spermatocytes and that in spermatids on Day 6 was shorter by 30 nucleotides compared to that on Day 0. These size differences were due to differences in the length of the poly(A) tracts because digestion of poly(A) with ribonuclease H resulted in the shortening of mRNA to the same size for three stages.     

Translation of partially purified poly(A)+ protamine messenger RNA components in wheat germ and rabbit reticulocyte cell-free systems. Evidence for translational control mechanisms.

The coding properties of individual poly(A)+ protamine mRNA subcomponents have been explored by analysis of their translation products in two different cell-free protein synthesis systems, the rabbit reticulocyte lysate and the wheat germ S-30, both of which can translate total protamine mRNA. The products synthesized in the reticulocyte lysate in the presence of total poly(A)+ PmRNA consisted mainly of protamine components CII and CIII with component CI only a minor product. However, in the wheat germ S-30, the same mRNA preparation supported the synthesis of all three protamine components, in approximately equal amounts. In addition a new polypeptide, a putative fourth protamine component, labelled CO, was also synthesized. The translation products of subcomponents of poly(A)+ PmRNA separated as individual bands on polyacrylamide gels were similarly analyzed and it was shown that each of the isolated poly(A)+ PmRNA species could stimulate the incorporation of [3H]arginine into protamines in both translational systems. Although each mRNA band stimulated the synthesis of one particular protamine polypeptide predominantly in a given cell-free system, the same RNA preparation was found to direct preferentially the synthesis of a different protamine component in the second cell-free system. The products synthesized in the rabbit reticulocyte lysate in the presence of the individual mRNA species still showed component CI present as a minor product.     

Identification and partial characterization of multiple discrete polyadenylic acid containing RNA components coded for by HeLa cell mitochondrial DNA

Poly(A)-containing RNA isolated from the components of a HeLa cell mitochondrial lysate which sediment in the polysome region of a sucrose gradient have been analyzed for the presence of discrete species. Eight distinct components have been identified by polyacrylamide gel electrophoresis after formaldehyde treatment. These components, which are highly reproducible in their occurrence and relative amounts under widely varying conditions of isolation, have been characterized as to their sedimentation behavior under denaturing conditions, poly(A) content and homology to separated strands of mitochondrial DNA.One of the discrete components was previously shown to have a sedimentation coefficient of about 7 S in the native state and a molecular weight of about 9.0 × 10⁴, as estimated from its sedimentation rate in formaldehyde. The molecular weights of the other seven components, as derived from sedimentation data, range between 2.6 and 5.3 × 10⁵.The 7 S RNA is complementary to the light mitochondrial DNA strand, while the other seven components are complementary to the heavy strand. Together with the two mitochondrial rRNA species and with mitochondrial 4 S RNA, the eight poly(A)-containing RNA components, if distinct in sequence, would account for about 70% of the single-strand informational content of HeLa mitochondrial DNA.